SERIES EDITORS

STEPHEN G. WAXMAN
Bridget Marie Flaherty Professor of Neurology
Neurobiology, and Pharmacology;
Director, Center for Neuroscience & Regeneration/Neurorehabilitation Research
Yale University School of Medicine
New Haven, Connecticut
USA

DONALD G. STEIN
Asa G. Candler Professor
Department of Emergency Medicine
Emory University
Atlanta, Georgia
USA

DICK F. SWAAB
Professor of Neurobiology
Medical Faculty, University of Amsterdam;
Leader Research Team Neuropsychiatric Disorders
Netherlands Institute for Neuroscience
Amsterdam
The Netherlands

HOWARD L. FIELDS
Professor of Neurology
Endowed Chair in Pharmacology of Addiction
Director, Wheeler Center for the Neurobiology of Addiction
University of California
San Francisco, California
USA
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 List of Contributors

U. Albrecht, Department of Biology, Unit of Biochemistry, University of Fribourg, Fribourg, Switzerland

A.E. Allen, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
S.N. Anand, Neurobehavioural Genetics, MRC Harwell, Harwell Science and Innovation Campus,
Oxfordshire, United Kingdom
C. Ancel, Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et
Integratives, Strasbourg, France
L. Ansel, Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et
Integratives, Strasbourg, France
J.L. Barclay, Circadian Rhythms Group, Max Planck Institute for Biophysical Chemistry, Göttingen,
Germany
P. Battley, Ecology Group, Institute of Natural Resources, Massey University, Private Bag, Palmerston
North, New Zealand
C. Bertolucci, Department of Biology and Evolution, University of Ferrara, Ferrara, Italy
P.H.L.T. Bisschop, Department of Endocrinology and Metabolism, Academic Medical Center (AMC),
University of Amsterdam, Amsterdam, The Netherlands
T.M. Brown, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
I. Bur, Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et
Integratives, Strasbourg, France
A. Castillo-Ruiz, Department of Neurology, University of Massachusetts Medical School, Worcester,
MA, USA
E. Christ, Dr. Senckenbergische Anatomie II, Fachbereich Medizin, Goethe-Universität Frankfurt,
Frankfurt am Main, Germany
C.S. Colwell, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden Univer-
sity Medical Center, Leiden, The Netherlands, and Laboratory of Circadian and Sleep Medicine,
Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University
of California, Los Angeles, CA, USA
F. De Carli, Institute of Bioimaging and Molecular Physiology, Genoa Unit, National Research Council,
Genoa, Italy
L. De Gennaro, Department of Psychology, University of Rome “Sapienza,” Roma, Italy
T. Deboer, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University
Medical Center, Leiden, The Netherlands
A. Dekinga, Department of Marine Ecology, Royal Netherlands Institute for Sea Research (NIOZ), Den
Burg, Texel, The Netherlands
H. De la Iglesia, Department of Biology and Program of Neurobiology and Behavior, University of
Washington, Seattle, USA
D.F. Dinges, Division of Sleep and Chronobiology, Department of Psychiatry, Perelman School of Med-
icine, University of Pennsylvania, Philadelphia, PA, USA

v
vi

J.K. Edwards, Neurobehavioural Genetics, MRC Harwell, Harwell Science and Innovation Campus,
Oxfordshire, United Kingdom
M. Ferrara, Department of Health Sciences, University of L’Aquila, L’Aquila, Italy
E. Fliers, Department of Endocrinology and Metabolism, Academic Medical Center (AMC), University
of Amsterdam (UvA), Amsterdam, The Netherlands
R.G. Foster, Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology,
John Radcliffe Hospital, Headley Way, Oxford, United Kingdom
N.S. Foulkes, Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-von-
Helmholtz-Platz, Eggenstein-Leopoldshafen, Germany
N. Goel, Division of Sleep and Chronobiology, Department of Psychiatry, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Y. Gothilf, Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv, Israel
E. Gwinnerw, Max-Planck-Institut für Ornithologie, Andechs, Germany
M.W. Hankins, Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology,
John Radcliffe Hospital, Headley Way, Oxford, United Kingdom
D. Hazlerigg, Institute of Biological & Environmental Sciences, University of Aberdeen, Aberdeen,
Scotland, United Kingdom
C. Helfrich-Förster, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany
B. Helm, Max-Planck-Institut für Ornithologie, Andechs; Fachbereich Biologie, Universität Konstanz,
Konstanz, Germany, and Institute of Biodiversity, Animal Health and Comparative Medicine, Univer-
sity of Glasgow, Glasgow, United Kingdom
K. Honma, Department of Chronomedicine, Hokkaido University Graduate School of Medicine,
Sapporo, Japan
S. Honma, Department of Physiology, and Department of Chronomedicine, Hokkaido University Grad-
uate School of Medicine, Sapporo, Japan
T. Houben, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University
Medical Center, Leiden, The Netherlands
P. Houdek, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
W.-P. Hu, Department of Pharmacology, Hubei University of Science and Technology, Xianning, Hubei,
PR China
A.T.L. Hughes, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
S. Hughes, Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology, John
Radcliffe Hospital, Headley Way, Oxford, United Kingdom
R.A. Hut, Chronobiology unit, Center for Behaviour and Neurosciences, University of Groningen,
Groningen, The Netherlands
M.L. Idda, Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-von-
Helmholtz-Platz, Eggenstein-Leopoldshafen, Germany
N. Inagaki, Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo,
Japan
C.F. Jonkers, Department of Nutrition, Academic Medical Center (AMC), University of Amsterdam,
Amsterdam, The Netherlands

w
Deceased
 vii

A. Kalsbeek, Department of Endocrinology and Metabolism, Academic Medical Center (AMC), Univer-
sity of Amsterdam, The Netherlands and Hypothalamic Integration Mechanisms, Netherlands Institute
for Neuroscience (NIN), An Institute of the Royal Netherlands Academy of Arts and Sciences
(KNAW), Amsterdam, The Netherlands
T. Kantermann, Centre for Chronobiology, Faculty of Health and Medical Sciences, University of Sur-
rey, Guildford, Surrey, United Kingdom
P. Klosen, Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et
Integratives, Strasbourg, France
A. Koolhaas, Department of Marine Ecology, Royal Netherlands Institute for Sea Research (NIOZ),
Den Burg, Texel, The Netherlands
H.-W. Korf, Dr. Senckenbergische Anatomie II, Fachbereich Medizin, and Dr. Senckenbergisches
Chronomedizinisches Institut, Goethe-Universität Frankfurt, Frankfurt am Main, Germany
F. Kreier, Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience, an Institute of
the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands
N. Kronfeld-Schor, Department of Zoology, Tel Aviv University, Tel Aviv, Israel
G.S. Lall, Medway School of Pharmacy, University of Kent, Chatham, United Kingdom
J.-D. Li, The State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan,
PR China
R.J. Lucas, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
J.H. Meijer, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University
Medical Center, Leiden, The Netherlands
S. Michel, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University
Medical Center, Leiden, The Netherlands
Y. Miyazaki, Faculty of Clinical Education, Ashiya University, Hyogo, Japan
C. Moreno, University of São Paulo, School of Public Health, São Paulo, Brazil
F. Moroni, Department of Psychology, University of Rome “Sapienza,” Roma, and Department of Psy-
chology, University of Bologna, Bologna, Italy
C.J. Morris, Division of Sleep Medicine, Brigham and Women’s Hospital, and Harvard Medical School,
Boston, MA, USA
W. Nakamura, Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo, and
Department of Chronodentistry, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan
T. Nisimura, College of Bioresource Sciences, Nihon University, Fujisawa, Japan
L. Nobili, Centre of Epilepsy Surgery “C. Munari,” Center of Sleep Medicine, Niguarda Hospital, Milan,
and Institute of Bioimaging and Molecular Physiology, Genoa Unit, National Research Council,
Genoa, Italy
P.M. Nolan, Neurobehavioural Genetics, MRC Harwell, Harwell Science and Innovation Campus,
Oxfordshire, United Kingdom
M. Novakova, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech
Republic
H. Numata, Graduate School of Science, Kyoto University, Kyoto, Japan
D. Ono, Department of Physiology, and Advanced Photonic Bioimaging Section, Research Center for
Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, Japan
H. Oster, Circadian Rhythms Group, Max Planck Institute for Biophysical Chemistry, Göttingen, and
Medical Department I, University of Lübeck, Lübeck, Germany
M.J. Paul, Department of Psychology, University of Massachusetts Amherst, Amherst, MA, USA
viii

S.N. Peirson, Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology,

John Radcliffe Hospital, Headley Way, Oxford, United Kingdom
T. Piersma, Department of Marine Ecology, Royal Netherlands Institute for Sea Research (NIOZ), Den
Burg, Texel, The Netherlands
H.D. Piggins, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
A. Pigorini, Department of Clinical Sciences L. Sacco Università degli Studi di Milano, Milan, Italy
L. Polidarova, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech
Republic
P. Proserpio, Centre of Epilepsy Surgery “C. Munari,” Center of Sleep Medicine, Niguarda Hospital,
Milan, Italy
D. Rieger, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany
J. Ripperger, Department of Biology, Unit of Biochemistry, University of Fribourg, Fribourg,
Switzerland
J.H.T. Rohling, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden Univer-
sity Medical Center, Leiden, The Netherlands
F.J. Sánchez-Vázquez, Department of Physiology, Faculty of Biology, University of Murcia, Murcia,
Spain
S. Sarasso, Department of Clinical Sciences L. Sacco Università degli Studi di Milano, Milan, Italy
F.A.J.L. Scheer, Division of Sleep Medicine, Brigham and Women’s Hospital, and Harvard Medical
School, Boston, MA, USA
I. Schwabl, Max-Planck-Institut für Ornithologie, Andechs, Germany
W.J. Schwartz, Department of Neurology, University of Massachusetts Medical School, Worcester, MA,
USA
V. Simonneaux, Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et
Integratives, Strasbourg, France
D.J. Skene, Centre for Chronobiology, Faculty of Health and Medical Sciences, University of Surrey,
Guildford, Surrey, United Kingdom
M. Sladek, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
A.M. Spaeth, Department of Psychology, School of Arts and Sciences, University of Pennsylvania,
Philadelphia, PA, USA
D.J. Stenvers, Department of Endocrinology and Metabolism, Academic Medical Center (AMC),
University of Amsterdam, Amsterdam, The Netherlands
A. Sumova, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Y. Suzuki, Advanced Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido
University Graduate School of Medicine, Sapporo, Japan
A.H. Tsang, Circadian Rhythms Group, Max Planck Institute for Biophysical Chemistry, Göttingen,
Germany
M.A. Ulhôa, University of São Paulo, School of Public Health, São Paulo, Brazil
D. Vallone, Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-von-
Helmholtz-Platz, Eggenstein-Leopoldshafen, Germany
R. van der Spek, Department of Endocrinology and Metabolism, Academic Medical Center (AMC),
University of Amsterdam (UvA), Amsterdam, The Netherlands
V. van der Vinne, Chronobiology unit, Center for Behaviour and Neurosciences, University of
Groningen, Groningen, The Netherlands
 ix

C. von Gall, Dr. Senckenbergische Anatomie II, Fachbereich Medizin, and Dr. Senckenbergisches
Chronomedizinisches Institut, Goethe-Universität Frankfurt, Frankfurt am Main, Germany
S.M.T. Wehrens, Centre for Chronobiology, Faculty of Health and Medical Sciences, University of
Surrey, Guildford, Surrey, United Kingdom
J.N. Yang, Division of Sleep Medicine, Brigham and Women’s Hospital, Boston, MA, USA
R. Yasenkov, Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden Univer-
sity Medical Center, Leiden, The Netherlands
T. Yoshii, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
T. Yoshikawa, Advanced Photonic Bioimaging Section, Research Center for Cooperative Projects,
Hokkaido University Graduate School of Medicine, Sapporo, Japan
Q.-Y. Zhou, Department of Pharmacology, University of California Irvine, CA, USA
 Preface

The XII Congress of the European Biological Rhythms Society (EBRS), held at the University of Oxford
Examination Schools in Oxford, UK, from August 20–26, 2011, dealt with a broad range of chronobio-
logical disciplines, in an attempt to discuss and further the knowledge in this field.
The research aims of many branches of biology and biomedicine, including neuroscience, are to
deduce fundamental mechanisms and to understand how they are compromised under abnormal or path-
ological conditions. In this regard, the field of chronobiology—the study of periodic (cyclic) phenomena
in living organisms and their adaptation to environmentally generated rhythms—represents one of the
great success stories in biology. The best understood of these periodic phenomena are circadian
rhythms—the rhythms that organize 24-h daily activity and are often referred to as “biological rhythms”
or “the body clock.” Circadian researchers have generated a real understanding, in multiple groups
of organisms, of how genes and their protein products interact to generate stable 24-h cycles of cellular
physiology and behavior. We now also understand some of the mechanisms whereby the environment
interacts with these clocks so that internal time and solar time are appropriately coupled and aligned.
In addition, considerable insight has been gained regarding the central role of internal time in the
regulation of broad areas of physiology and behavior, including sleep and seasonal rhythms. In a little
over 40 years, we have moved from “black box” speculation to real mechanistic insight and clinical
application.
In mammals, including humans, the central biological clock resides in the central nervous system in
the bilateral hypothalamic suprachiasmatic nuclei (SCN). The SCN generates an autonomic rhythm
of electrical activity with a period of approximately (but not exactly) 24 h. This rhythm continues to
oscillate even when SCN cells are removed from a living organism and brought into culture. Approxi-
mately 20 years ago, it was discovered that the endogenous SCN rhythm is based on a molecular
clock mechanism. The core clock genes (Clock, BMAL1, Period, and Cryptochrome) are expressed in
a transcriptional–translational feedback loop with a duration of approximately 24 h. Soon after the dis-
covery of the clock genes, it became clear that not only SCN neurons but virtually every mammalian
cell contains such a molecular clock. These so-called peripheral clocks also have an autonomic cycle of
approximately 24 h that requires synchronization with the external light–dark cycle. Since most peripheral
clocks do not receive light information, the SCN communicates its rhythm to the peripheral clocks.
In addition to the direct hormonal and neuronal signals from the SCN, peripheral clocks are synchronized
by several indirect signals and are probably most strongly influenced by daily changes in energy metabolism.
It is becoming increasingly clear that the different sensitivities of the central and peripheral clocks may lie at
the heart of the discrepancy that often arises between our circadian and social clocks and all of the associated
pathologies.
As the many and diverse contributions to this special edition of Progress in Brain Research on the
“Neurobiology of Circadian Timing” prove, the field of chronobiology has come of age. Of our own spe-
cies, we now know that disruption of our internal timing may lead to major disorders and diseases, from

xi
xii

mental health issues to cancer. The medical world benefits greatly from this knowledge, not least in the
form of the delivery of therapeutics at the most opportune time in terms of good clinical outcome.
Unfortunately, however, many key questions still remain unanswered and many more links with the
clinical practice remain to be established.
Andries Kalsbeek
Martha Merrow
Till Roenneberg
Russell G. Foster
A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 1

How rod, cone, and melanopsin photoreceptors

come together to enlighten the mammalian
circadian clock

Robert J. Lucas{,*, Gurprit S. Lall{, Annette E. Allen{ and Timothy M. Brown{

{
Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
{
Medway School of Pharmacy, University of Kent, Chatham, United Kingdom

Abstract: In mammals, a small number of retinal ganglion cells express melanopsin, an opsin
photopigment, allowing them to be directly photoreceptive. A major function of these so-called
intrinsically photosensitive retinal ganglion cells (ipRGCs) is to synchronize (entrain) endogenous
circadian clocks to the external light:dark cycle. Thanks to their intrinsic light response, ipRGCs can
support photoentrainment even when the other retinal photoreceptors (rods and cones) are absent or
inactive. However, in the intact retina the ipRGC light response is a composite of extrinsic (rod/cone)
and intrinsic (melanopsin) influences. As a result all three photoreceptor classes contribute to the retinal
pathways providing light information to the clock. Here, we consider what each photoreceptor type
contributes to the clock light response. We review electrophysiological and behavioral data pertinent to
this question, primarily from laboratory rodents, drawing them together to provide a conceptual model
in which each photoreceptor class plays a distinct role in encoding the light environment. We finally use
this model to highlight some of the important outstanding questions in this field.

Keywords: circadian; photoentrainment; melanopsin; ipRGCs; photoreceptor; rods; cones;

suprachiasmatic nuclei; retinohypothalamic tract.

Organisms throughout the living world use
endogenous circadian clocks to modulate their
behavior and/or physiology according to the vary-
ing demands of the celestial day. However, the
*Corresponding author.
Tel.: þ44 161 2755251
E-mail: robert.lucas@manchester.ac.uk
internal representation of time provided by these
biological clocks is only useful insofar as it reflects
external time. As a result, all circadian clocks
have sensory input pathways that report diurnal
variations in the physical environment in order
to synchronize (entrain) to local time.
No environmental variable provides a more
reliable indication of time of day than ambient

http://dx.doi.org/10.1016/B978-0-444-59427-3.00001-0 1
2
Rods and cones
M1 class ipRGC
Light
SCN
EYE
Fig. 1. A schematic depiction of the mammalian
photoentrainment pathway. The master circadian clock in the
hypothalamic suprachiasmatic nuclei (SCN) is connected to
the retina via a specific component of the optic nerve (the
RHT). The RHT is dominated by fibers from the M1 class of
ipRGCs (shown in magnified version of the retina), which are
directly photosensitive, but also receive signals from rod and
cone photoreceptors via multiple types of bipolar and
amacrine cell.
illumination, with light levels varying over up to
nine decades between midnight and midday. It is
unsurprising therefore that light is the most
important cue for circadian entrainment and that
photoreception is intimately associated with circa-
dian clocks throughout the living world. In the
case of mammals, entrainment to the diurnal light
cycle (photoentrainment) originates in the retina,
with a distinct projection of the optic nerve,
known as the retinohypothalamic tract (RHT),
targeting the master circadian clock in the
suprachiasmatic nuclei (SCN; Fig. 1).
The conventional view of retinal function is
that all visual information originates with rod
and/or cone photoreceptors. These specialized
cells absorb light and translate that event into
neurochemical signals, which are passed through
the retinal circuitry (being sculpted along the
way) before reaching the retinal ganglion cells.
According to this scheme, retinal ganglion cells
act principally as relays, receiving information
from the outer retina and communicating it to
the brain in the form of action potentials
propagated along their axons (which form the
optic nerve). The past decade, however, has seen
the acceptance that a small proportion of retinal
ganglion cells are themselves photoreceptive,
capable of responding directly to light even when
isolated from the rest of the retina (Berson et al.,
2002). The photoreceptive capacity of these so-
called intrinsically photosensitive retinal ganglion
cells (ipRGCs) relies upon an opsin-based photo-
pigment called melanopsin (Hattar et al., 2002;
Lucas et al., 2003; Melyan et al., 2005; Panda
et al., 2005; Provencio et al., 2000; Qiu et al.,
2005). Light absorption by melanopsin activates
a G-protein signaling cascade, which causes cellu-
lar depolarization and increased firing rate.
Importantly for the purposes of this review, the
SCN is a primary central target of ipRGCs. In
fact, despite their relative scarcity in the retina,
ipRGCs dominate the RHT, with this ganglion
cell class providing the vast majority of fibers
reaching the SCN (Morin et al., 2003; Sollars
et al., 2003). The functional significance of this
anatomical arrangement is confirmed by the
observation that photoentrainment is lost follow-
ing selective lesion of ipRGCs (Goz et al., 2008;
Guler et al., 2008; Hatori et al., 2008). It seems
then, that ipRGCs represent the main route via
which light information reaches the clock.
As ipRGCs contain mechanisms both for
detecting light and transmitting that information
to the brain, one could envisage a scenario in
which they act virtually autonomously to entrain
the clock. However, ipRGCs do not sit in the ret-
ina as islands entire of themselves, but receive
synaptic input from several elements of the con-
ventional rod/cone circuitry within the retina
(Belenky et al., 2003; Dumitrescu et al., 2009;
Hoshi et al., 2009; Ostergaard et al., 2007; Perez-
Leon et al., 2006; Viney et al., 2007). As a result,
their physiological light response (a strong and
sustained increase in firing) is a product both of
their intrinsic phototransduction mechanism and
extrinsic influences originating in rods and cones
(Dacey et al., 2005; Schmidt et al., 2008; Tu
et al., 2005; Viney et al., 2007; Wong et al., 2007).
Physiological and anatomical data therefore
confirm that the afferent limb of the photo-
entrainment pathway extends to all three

photoreceptor types (rod, cone, and melanopsin).
Importantly, mice carrying near complete lesions
of all three types of photoreception lack
photoentrainment (Hattar et al., 2003; Panda
et al., 2003), confirming that no other, as yet
unidentified, photoreceptor can influence the
clock. However, this still leaves the question of
what role (if any) each of the known photo-
receptors plays in photoentrainment. Here, we
aim to summarize experimental data pertinent
to this question, concentrating mainly on
findings in mice and related laboratory rodent
species because they provide the most compre-
hensive picture. We then draw this together
around a simple conceptual model.
Methods of study
A number of studies have now addressed the ques-
tion of under what circumstances, and to what
extent, photoentrainment relies upon each photore-
ceptor class. They have achieved this using a variety
of quantitative measures of the clock’s light
response. In some cases, photoentrainment of loco-
motor activity rhythms (an output of the SCN clock)
has been studied directly, with the proportion of
animals entrained under a particular light regime
reported, or the nature of that entrainment
quantified, for example, in terms of its phasing with
respect to the light cycle (e.g., Mrosovsky, 2003).
Other studies have quantified the effect of light on
this behavioral rhythm by measuring the
adjustments in phase or period that presumably
underlie entrainment. These have assessed either
the period lengthening effects of constant light (LL
paradigm), or light induced adjustments in the
phase of rhythms free-running in constant darkness
(phase shifting paradigm) (e.g., Mrosovsky and
Hattar, 2005). Such behavioral analyses have the
advantage of reporting phenomena with a fairly
obvious relevance for entrainment. However,
because these assays rely upon end point measures
they do not allow a real-time readout of the clock
response to light. Electrophysiological recordings
3
of light-evoked changes in neuronal firing in the
rodent SCN have been used to address this latter
objective. Such experiments reveal the hypotha-
lamic light response in real time and therefore
describe the dynamic nature of light information
reaching the SCN (e.g., Meijer et al., 1998). On the
other hand, their relevance for entrainment is less
obvious, as the mechanistic relationship between
changes in firing of neurons within the SCN and
adjustments in phase or period of the circadian clock
remains unclear.
Several strategies have also been employed to
determine the contribution made by each photore-
ceptor class to these assays of circadian photosen-
sitivity. The most conceptually straightforward
has been to study the effects of genetic lesions of
candidate receptor types. The earliest experi-
ments of this kind employed naturally occurring
mutations causing rod and cone degeneration.
By demonstrating that entrainment survived mas-
sive loss of these conventional photoreceptors
(Ebihara and Tsuji, 1980; Foster et al., 1991), those
efforts provided justification for the search for non-
rod, non-cone photoreceptors, and can be regarded
as one of the foundations of this field. More
recently, increasingly sophisticated methods have
been developed to lesion each of the photorecep-
tive classes either singly or in combination, and
without associated cell death (Altimus et al., 2010).
Retinally degenerate and knockout mice pro-
vide a powerful method for asking whether each
photoreceptor class is necessary and sufficient to
account for aspects of the clock light response.
However, they may provide an incomplete picture
of what occurs in the intact organism. Leaving
aside the possibility of compensatory/disruptive
reorganization, such reduced preparations do not
allow for emergent properties of the intact system
as, for example, the behavior of one photoreceptor
system is influenced by activity of another. Such
interactions are in fact well documented in the
retina (Barnard et al., 2006; Cameron and Lucas,
2009; Cao et al., 2006; Coletta and Adams, 1984;
Frumkes et al., 1992; Hankins and Lucas, 2002;
Peachey et al., 1992).
4
To define the function of each photoreceptor in
the context of an intact visual system then, it has
been necessary to define additional strategies. Sev-
eral researchers have achieved this by assessing
the spectral sensitivity of response components
(Fig. 2). The efficiency with which photopigments
absorb photons is a function of wavelength. This
relationship can be described with high precision
and expressed as a spectral sensitivity profile
(Fig. 2a). Comparing such photoreceptor spectral
sensitivity functions with assessments of the relative
sensitivity of evoked responses to different
wavelengths can therefore be a powerful method
for linking candidate photoreceptors to specific
clock responses (Fig. 2b). This strategy has been
employed to identify rod and cone influences on
light induced firing in the rat SCN (Aggelopoulos
and Meissl, 2000); to confirm that photoentrainment
in rodless þ coneless mice relies upon melanopsin
(Hattar et al., 2003; Fig. 2b); and also to demonstrate
that cones alone cannot account for melatonin sup-
pression in humans (Brainard et al., 2001; Thapan
et al., 2001).
A challenge for adopting the spectral sensitivity
approach in rodents is that there are only slight
differences in this parameter between melanopsin,
rod opsin and M-cone opsin (Fig. 2a). As a result,
very precise estimates of the spectral sensitivity
of evoked responses are required in order to
distinguish between the influence of photorecep-
tors expressing these three photopigments.
Descriptions of circadian spectral sensitivity in
wild-type mice (and related rodents) thus have
rarely been able to assign responses unambigu-
ously to a single photoreceptor class (Provencio
and Foster, 1995; Takahashi et al., 1984;
Yoshimura and Ebihara, 1996). This limitation
can, however, be overcome using genetic tec-
hniques to change the spectral sensitivity of mouse
photoreceptors. Dkhissi-Benyahya et al. (2007)
achieved this using thyroid hormone receptor
(TRß2) knockout mice. TRß2 regulates photore-
ceptor gene expression and M-cone opsin is not
expressed in TRß2/ mice, leaving all cones to rely
upon S-cone opsin for their photosensitivity.
Aspects of the clock response that are impaired at
longer, but not shorter, wavelengths in TRß2/
compared to wild types have thus been attributed
to cones (see below). Our own approach has been
to use mice (Opn1mwR) in which the coding
sequence for human red-cone opsin (whose spec-
tral sensitivity function is shifted to much longer
wavelengths than rod opsin or melanopsin) has
been “knocked-in” to the mouse M-cone opsin
locus (Smallwood et al., 2003). The effect is to pro-
duce an intact and fully functional retina in which
cones that would ordinarily express M-cone opsin
instead express the longer wavelength human pig-
ment (Fig. 3a). By carefully describing the sensitiv-
ity of various response components to medium and
long wavelength stimuli, it has been possible to
identify aspects of the clock response attributable
to each of the photoreceptor classes (Fig. 3b and
c; Brown et al., 2011; Lall et al., 2010).
Relying on rods
Measured under the right circumstances, circa-
dian clocks can be extremely sensitive to light
(Evans et al., 2009). As rods are the most sensi-
tive of all retinal photoreceptors it would make
sense if they were to provide this high sensitivity
input to the clock. We have tested this predic-
tion by assessing the spectral sensitivity of the
Opn1mwR clock in the LL paradigm (Lall et al.,
2010). We found that even very dim light (near
threshold for conventional vision) influenced
the clock in this LL paradigm, and that the
spectral sensitivity of this effect did indeed match
that of rod, but not cone or melanopsin
photoreceptors.
The clock’s reliance on rod photoreceptors is,
however, not restricted to very dim light. “Rod
only” mice, genetically engineered to lack cone
and melanopsin photoreception, entrain well
under a light:dark (LD) cycle in which the light
portion is set at 500 photopic lux (roughly equiv-
alent to bright indoor lighting) (Altimus et al.,
2010). This confirms that the clock can employ
 5

(a) (b)

100

10–1
Relative sensitivity

10–2

10–3

S cone opsin
10–4 Melanopsin
Rod opsin
M cone opsin
10–5
400 450 500 550 600 650 700
Wavelength (nm)

(c) (d)
100

10–1
420 nm
Relative sensitivity
Response amplitude

480 nm
500 nm 10–2
550 nm
580 nm
10–3

10–4 Melanopsin
Circadian Response
10–5
400 450 500 550 600 650
Log (irradiance)
Wavelength (nm)

Fig. 2. Assigning response elements to particular photoreceptors on the basis of their spectral sensitivity. (a) The four mouse retinal
photopigments have more or less well-defined spectral sensitivity profiles, whose forms match the standard template for opsin:
vitamin A based photopigments with peak sensitivities at 360 nm (S-cone opsin), 480 nm (melanopsin), 498 nm (rod opsin), and
508 nm (M-cone opsin). Comparison of these profiles with the spectral sensitivity of specific clock responses can be used to
determine the contribution of each pigment to entrainment. Here, we illustrate this process using mock data for circadian phase
shifts in rodless þ coneless mice (Hattar et al., 2003). (b) The process relies upon measuring phase shifts induced by a discrete
15-min light pulse presented 4 h after activity onset to mice free-running in constant darkness. A typical actogram for a single
mouse exposed to such a treatment, in which bouts of wheel running activity are shown as columns (height indicating number of
wheel revolutions in each 5 min bin) and each line represents 24 h. After entraining the mouse to a light–dark cycle (phasing
shown as black:white boxes at top) for several days, mice are released into constant darkness. Seven to ten days later they
receive a single 15-min light pulse at time indicated by star. By tracing a line through activity onsets before and after the pulse, it
is possible to calculate the associated adjustment in phase. (c) Expressing phase shift magnitude as a function of stimulus
irradiance for several wavelengths of light provides a family of irradiance response curves. The relative sensitivity of the phase
shifting response at each wavelength can then be estimated by the irradiance required to drive a half saturating shift and plotted
as a so-called action spectrum to compare against the absorbance spectrum of candidate photoreceptors. (d) The example here is
for a rodless þ coneless mouse and, as a result, the spectral sensitivity of the phase shifting response closely matches that of
melanopsin, confirming that, in the absence of rods and cones it is melanopsin that supports circadian photosensitivity.
6

(a) Opn1mwR Mouse

(b)
Irradiance log (photons/cm2/s)
100
8 9 10 11 12 13 14
0
10–1

Phase shift (min)

Relative sensitivity
–25
10–2
–50
10–3
–75
500 nm
S cone opsin 650 nm
10–4 Melanopsin –100
Rod opsin
Red cone opsin
10–5
400 450 500 550 600 650 700
Wavelength (nm)

(c) Effective irradiance log (photons/cm2/s) (d)

7 8 9 10 11 12 13 14 650 nm
0

6 spikes/s
460 nm
500 nm
Phase shift (min)

–25 650 nm corrected for:

Cones
–50 Rods
Melanopsin 60 s
–75

–100

Fig. 3. Red-cone knock-in (Opn1mwR) mice. (a) Similarities in the spectral response profile of melanopsin, rod, and M-cone opsins
in wild-type mice (Fig 2a) make it difficult to use the spectral sensitivity of a particular circadian response to establish which of these
pigments it relies upon. We have addressed this problem using a red-cone knock-in mouse (Opn1mwR), in which the introduction of
the human red (L-) cone opsin coding sequence at the mouse M-cone opsin locus results in a long wavelength shift in spectral
sensitivity of the cone population. Using this mouse we have studied the photoreceptive origins of photoentrainment using LL
and phase shifting paradigms (Lall et al., 2010), as well as electrophysiological recordings from the SCN (Brown et al., 2011).
Rather than derive full action spectra for phase shifts in this genotype, spectral sensitivity can be inferred by describing
irradiance response curves at just two wavelengths (b; reproduced with permission from Lall et al., 2010). The phase shifting
response was much more sensitive to 500 than 650 nm stimuli. According to its known spectral sensitivity profile (Fig 2a), red-
cone opsin is 7.7 times more sensitive to 500 than 650 nm. If the phase shifting response were driven by red-cone opsin then we
would be expect the irradiance response curves to be superimposed when stimuli at 500 nm are 7.7 times dimmer than those at
650 nm. To test this prediction, the two curves are expressed in red-cone opsin “effective irradiance” by multiplying measured
irradiance at 650 nm by 0.13. (c) The clear distinction between the two irradiance response curves following this manipulation
indicates that phase shifts are not defined by red-cone opsin under these conditions. Similar corrections based upon the spectral
sensitivity of melanopsin and rod opsin (c), reveal rod opsin as the most likely origin for this response. A simplified version of
this strategy can be employed in which the irradiance of two stimuli of different wavelength (here 460 and 650 nm) are adjusted
so as to appear equally bright to a candidate photoreceptor (in this case red-cone opsin in Opn1mwR mice). A comparison of the
electrophysiological response in the SCN of mice exposed to these two stimuli (d; based upon data presented in Brown et al.,
2011) reveals elements (transient “on” excitation) that are equivalent at the two wavelengths and thus likely originate with red-
cone opsin. Other elements (sustained increase in firing), by contrast, are observed only at the shorter wavelengths and cannot
therefore be attributed to this photopigment. The sustained firing could in theory originate with any photopigment more
sensitive to shorter wavelengths, but for reasons outlined in Brown et al. (2011) probably relies largely on melanopsin.

rods to distinguish light from dark under such
conditions. Moreover, data from Opn1mwR mice
indicate that the clock also relies upon rods for
more quantitative assessments of irradiance
under relatively bright conditions, and even when
cones are fully functional. Thus, the spectral sen-
sitivity of phase shifting responses to a 15-min
light pulse in this genotype matches that of rods
(Fig. 3c) rather than cones (Lall et al., 2010), even
though the dynamic range for this response lies
 4–5 decimal orders above the threshold for
rod-based vision and well within the sensitivity
range of cones (Nathan et al., 2006).
The behavioral data thus reveal that rods are a
potent influence on the clock not only under very
dim conditions, but also at light levels experienced
well into the dawn/dusk transition. Interestingly,
however, this influence is much less apparent in
electrophysiological recordings of light responses
in the rodent SCN. Light pulses can evoke large
changes in firing (mostly excitatory) in SCN neu-
rones, however this requires relatively high irradi-
ance. Responses are much less pronounced at the
dimmer light intensities that produce large behav-
ioral responses (Brown et al., 2011; Groos and
Mason, 1978; Meijer et al., 1992, 1998). This
indicates that changes in RHT activity driven by
rods may be rather slight but nonetheless are suffi-
cient to recruit mechanisms of entrainment.
Inconstant cones
Although rods appear to define circadian responses
even at moderate light intensities, at some point
they are expected to saturate (Wyszecki and Stiles,
1982). Under such conditions, the SCN would no
longer be able to rely upon them to measure further
increases in light intensity. Cones, by contrast, can
support conventional vision even under the
brightest daylight. Could they then compensate
for rod saturation in the RHT? In fact, the role of
cones has been (and indeed remains) the hardest
to define.
Photoentrainment is superficially intact follow-
ing substantial cone degeneration (Freedman
7
et al., 1999), while “cone only” mice (lacking both
rods and melanopsin) fail to entrain reliably to
standard (at least in the laboratory) LD cycles
(Lall et al., 2010; Mrosovsky and Hattar, 2005).
Those data then indicate that cones are neither
necessary nor sufficient for photoentrainment.
Indeed, our observation that it is rods that deter-
mine the clock response to a 15-min light pulse even
at irradiances well within the cone sensitivity range
(Lall et al., 2010) represents further evidence that
cones have little influence on mechanisms of
entrainment. Nonetheless, anatomical and electro-
physiological studies indicate that cone signals
reach the M1 class of ipRGCs responsible for
entrainment (Belenky et al., 2003; Dumitrescu
et al., 2009; Hoshi et al., 2009; Ostergaard et al.,
2007; Perez-Leon et al., 2006; Schmidt et al., 2008;
Viney et al., 2007). Moreover, aspects of the
electrophysiological light response of the SCN itself
can be attributed to cones on the basis of their spec-
tral sensitivity (Aggelopoulos and Meissl, 2000;
Brown et al., 2011), indicating that cones are indeed
functional components of the light input pathway.
What could explain the apparent contradiction
between the ability of cones to influence behavioral
versus electrophysiological endpoints? A closer
look at the electrophysiological light responses
suggests a possible answer. When presented with a
simple light pulse, both ipRGCs (Berson et al.,
2002; Dacey et al., 2005; Schmidt et al., 2008;
Tu et al., 2005; Wong et al., 2007) and most light
responsive SCN neurons (Brown et al., 2011;
Meijer et al., 1998) show a transient large increase
in firing that relaxes over a few seconds to a lower
level of excitation that is sustained over at least tens
of seconds of light exposure (Fig. 3d). Pharmaco-
logical and genetic manipulations suggest that the
acute phase of the ipRGC response originates in
the outer retina implicating rods and/or cones
(Berson et al., 2002; Dacey et al., 2005; Schmidt
et al., 2008; Tu et al., 2005; Wong et al., 2007). We
studied this aspect of the response in the SCN of
Op1mwR mice (Brown et al., 2011) and observed
that wavelengths capable of activating cones, but
not melanopsin, elicited strong increases in firing
8
at lights on, but drove very little sustained activity
under continuous light exposure (Fig. 3d). These
data indicate that cone-dependent excitation is
largely restricted to the first few seconds of light
exposure.
If cones activate the RHT only briefly at light
onset, that could explain why this photoreceptor
class is ill-suited to supporting entrainment to con-
tinuous long duration ( 15 min) light pulses (Lall
et al., 2010; Mrosovsky and Hattar, 2005). In this
case, one might expect cones to make a larger con-
tribution to responses driven by shorter duration
pulses (in which the transient cone signal accounts
for a greater proportion of the total light-evoked
activity of the RHT) and/or a series of light pulses
(in which the cones signal multiple light onsets).
There is evidence to support both those pre-
dictions. Dkhissi-Benyahya et al. (2007) reported
that phase shifts of cone-deficient TRß2/ mice
are equivalent to those of wild types when elicited
by a 15-min light pulse, but deficient for 1 min
stimuli. Similarly, working with Opn1mwR mice
we found that cone-activating long wavelength
stimuli that drove no phase shift when given as a
single continuous 15 min pulse elicited large
responses when presented as 15 separate 1 min
pulses presented over 43 min (Lall et al., 2010).
The available evidence from laboratory rodents
therefore indicates that while cones are a less reli-
able mediator of entrainment than rods, they can
be an important influence on the clock. They fur-
ther show that cone influence is especially appar-
ent in clock responses elicited by one or more
sudden increases in irradiance rather than steady,
extended light exposure.
Melanopsin
If cones provide such an unreliable influence on
the clock, which photoreceptor tells the SCN how
bright the world is under the daylight conditions
at which rods are saturated? Melanopsin is the
least sensitive of the three photoreceptors and
would appear well set to perform this function.
In fact, there is little direct evidence that any
aspect of mouse photoentrainment relies upon
melanopsin. Melanopsin knockout mice entrain
well to full light cycles over a range of irradiances
(Altimus et al., 2010; Morin and Studholme, 2011;
Panda et al., 2005; Ruby et al., 2002). These
animals do show reduced responsiveness in both
phase shifting and LL paradigms (Altimus et al.,
2010; Morin and Studholme, 2011; Panda et al.,
2005; Ruby et al., 2002). However, the simple
explanation for these latter data—that they indi-
cate a strong melanopsin contribution—is refuted
by evidence that in animals with an intact retina
both such assays of circadian light response can
be explained in terms of rod and/or cone activity
(Lall et al., 2010).
The likely explanation for failures to unam-
biguously attribute aspects of behavioral pho-
toentrainment to melanopsin is that all such
response parameters saturate at relatively low light
levels. In view of the high threshold for melanopsin
phototransduction (Do et al., 2009) one might
expect that melanopsin’s contribution to entrain-
ment would be more apparent in paradigms better
able to measure low-sensitivity aspects of the clock
response. This prediction has so far not been tested
in mouse behavioral studies (although rod/cone
degeneration has been shown to impair entrain-
ment at low, but not high, irradiances; Morin and
Studholme, 2011; Mrosovsky, 2003). The clock’s
reliance upon melanopsin at higher irradiances is,
however, very apparent in electrophysiological data
from the mouse SCN. SCN light responses build up
at irradiances within the predicted melanopsin sen-
sitivity range (and significantly above those
saturating clock responses in most entrainment
and phase shifting studies). Under such conditions,
stimuli targeting cones drive only transient light
responses in the Opn1mwR SCN, while much more
sustained excitation is evoked by melanopsin-
activating shorter wavelengths (Fig. 3d; Brown
et al., 2011). The implication that melanopsin
makes a strong contribution to such sustained activ-
ity is supported by data from melanopsin knockout
mice (Mure et al., 2007) and from physiological

recordings of ipRGCs themselves (Berson et al.,
2002; Dacey et al., 2005; Schmidt et al., 2008; Tu
et al., 2005; Wong et al., 2007).
A conceptual model
The behavioral and physiological data reviewed
above suggest a relatively simple separation in
the contributions of rods, cones, and melanopsin
to photoentrainment. We describe it here in terms
of light information reaching the clock under a
putative “naturalistic” pattern of light exposure.
For our purposes, we assume that superimposed
upon the gradual, diurnal, change in light inten-
sity are higher frequency modulations in the
amount of light reaching an animal’s eye as, for
example, clouds obscure the sun or the animal
moves in and out of shade (including its burrow).
A highly stylized depiction of such a light expo-
sure pattern is provided in Fig. 4a. We can use
the data summarized above to consider how the
RHT encodes such a profile, and the contribution
of each photoreceptor class to that process.
Our most direct available measure of retinal
influences on the circadian clock comes from
electrophysiological recordings of the SCN. These
provide a clear prediction for the separate con-
tributions made by cones and melanopsin to
encoding the dynamic light exposure profile
presented in cartoon form in Fig. 4a. Thus, the
combination of a cone-derived transient and a
melanopsin-dependent sustained SCN response
implies that cones would provide information
about higher, and melanopsin lower, frequency
changes in irradiance. According to this view, the
signal reaching the SCN in an animal exposed to
the irradiance profile in Fig. 4a would comprise a
series of transient cone-derived excitations super-
imposed upon a gradual, melanopsin-dependent,
build up in activity. This prediction, based upon
electrophysiological data (Brown et al., 2011;
Mure et al., 2007), is also qualitatively consistent
with the behavioral data showing that light expo-
sure paradigms skewed towards rapid increases
9
in irradiance maximize cone-dependent responses
(Dkhissi-Benyahya et al., 2007; Lall et al., 2010),
while (at least in rodless þ coneless preparations)
melanopsin can support irradiance dependent
responses to extended light exposure (Freedman
et al., 1999; Mrosovsky, 2003).
Sadly, it has proven difficult to record detect-
able SCN electrophysiological responses to dim
stimuli (below estimates of melanopsin threshold)
and thus to determine whether rods and/or cones
can support transient/sustained responses under
such conditions. Aggelopoulos and Meissl (2000)
did record rod-derived responses to dim light in
the rat SCN, but used only 1 s pulses, leaving
the question of their temporal profile under lon-
ger term illumination unanswered. Nonetheless,
the evidence that rods can support irradiance
dependent adjustments in circadian phase under
extended light exposure (Altimus et al., 2010; Lall
et al., 2010), argues that these receptors do allow
the SCN to track low frequency changes in irradi-
ance. Given the known ability of rods to also
track higher frequency events, it seems reason-
able to assume that the SCN could rely on a com-
bination of rods and cones to encode all salient
features of the dynamic light environment at
dim-moderate irradiances (Fig. 4b).
In summary then, according to the conceptual
model each photoreceptor class contributes a dif-
ferent quality of information about the dynamic
light environment. Cones encode rapid changes
in light intensity, leaving rods and melanopsin
to track more gradual modulations (including
the diurnal variation) at dim and brighter irra-
diances respectively. Combining these informa-
tion sources allows the SCN to monitor light
falling on the eye over a wide range of intensities
and temporal frequencies.
Assumptions, implications, and uncertainties
The cartoon depiction in Fig. 4b represents a best
guess of how the light input pathway would encode
“naturalistic” patterns of light exposure. It has the
advantage of relative simplicity and of accounting
10

(a) (b) ~

Input to clock (A.U.)

log (irradiance)
§

Cones

* Rods

Melanopsin

Time
Time
* Around 106 photons/cm2/s
§ Around 1011 photons/cm2/s
~ Around 1017 photons/cm2/s
(c) (d)
High pass filter Low pass filter
Activity (A.U.)

Activity (A.U.)

Time Time

Fig. 4. A conceptual model of rod, cone, and melanopsin contributions to encoding dynamic patterns of light exposure for the
circadian clock. (a) A stylized depiction of the change in corneal irradiance (arbitrary units) over the dawn transition, that
include a gradual (diurnal) increase, as well as more abrupt changes reflecting alterations in the animal’s exposure to solar
radiation as, for example, it moves into/out of shade. (b) The predicted activity (arbitrary units) of the afferent limb of the
photoentrainment pathway to the light exposure profile in (a). The diurnal increase in irradiance elicits a gradual enhancement
of activity dependent on rod (black portion of the curve) and melanopsin (blue) photoreception. More sudden increases are
encoded by rods and/or cones (in red) and appear as spikes in activity of the photoentrainment pathway. Estimates for threshold
and saturation irradiance of rod and melanopsin photoreception are provided (see text for justification). Although information
about the light environment reaching the clock thus has contributions from all three photoreceptor classes, downstream
processing employing either high (c) or low (d) pass temporal filters could exclude melanopsin or cone elements of this signal.

for the available experimental data. However, it is changes in irradiance to occur at only two rates
at best semiquantitative. A number of areas of (“fast” and “slow”), and by assuming that all fast
uncertainty are worth especial mention. changes have sufficient magnitude to evoke a
cone-dependent response. In fact, under dynamic
The role of cones conditions irradiance is likely to vary over a wide
variety of timescales and amplitudes. If we are
Figure 4b masks great uncertainty regarding the to predict the importance of cones for photo-
cone contribution to entrainment, by allowing entrainment under such conditions we therefore

need to know much more about the sensory cha-
racteristics of this cone-dependent component of
the SCN response. What is its temporal frequency
tuning, that is, what is the relationship between its
activity and the speed/size of changes in irradi-
ance? To what extent is the sensitivity of this sig-
nal adjusted according to the gradual, diurnal,
change in irradiance? Does a sudden decrease
in illumination induce a concomitant transient
cone-dependent inhibition of SCN firing?
A useful question is whether these properties
can simply be inferred from the fundamental sen-
sory characteristics of cone photoreceptors. It is
our view that in fact they are likely defined down-
stream of the photoreceptors. The transience of
the cone-derived SCN response to an extended
light pulse mirrors to some extent that of the cone
itself. Thus, cones respond to such stimuli with an
acute high amplitude hyperpolarization that
relaxes over time. However, under extended light
exposure, cone polarization reaches a “steady
state” level that is itself dependent upon irradi-
ance (at least at nonbleaching levels; Burkhardt,
1994; Normann and Perlman, 1979; Schnapf
et al., 1990; Valeton and van Norren, 1983). In
this way, cones can encode both the contrast
and irradiance of a light step in transient and
steady state phases of their response. The behav-
ioral and electrophysiological evidence reviewed
above showing that the clock relies on cones
solely to track high frequency changes in irradi-
ance argues that the latter, steady state compo-
nent of the cone response is actively excluded
from the photoentrainment pathway. Inclusion
of further adaptation in the neural network bring-
ing cone signals to the clock, as described for
other retinal pathways (Dunn et al., 2007), could
achieve this goal.
We propose therefore that the inability of
cones to support sustained aspects of the SCN
response is defined by the neural pathway bring-
ing cone signals to the clock. It follows that the
temporal frequency tuning characteristics of cone
input to the clock must be determined em-
pirically, rather than inferred from known
11
photoreceptor physiology. More excitingly, slight
changes in the characteristics of this neural path-
way could fundamentally alter the range of infor-
mation cones provide the clock. In particular, less
active adaptation in this pathway could allow the
steady state (irradiance coding) phase of the cone
response to reach the clock. In this situation,
cones could contribute to the SCN’s ability to
track lower frequency (diurnal) changes in irradi-
ance. This could explain evidence that the clock is
more reliant on cones in humans than in mice
(Gooley et al., 2010). Moreover, recent reports
suggest that this could be the case even for differ-
ent cone classes in mice. The data reviewed
above (and upon which the model is based)
describe responses elicited by cones expressing
M-cone opsin (> 95% of the total in mice). Mice
also have a small population of uniquely short
wavelength sensitive cones (Haverkamp et al.,
2005). We have recently reported that these
S-cones encode more gradual changes in irradi-
ance for a different ipRGC related response, the
pupil light reflex (Allen et al., 2011). If that were
also the case for the SCN, it could explain
older data that the rodent clock is especially sen-
sitive to UV light (Provencio and Foster, 1995).
Although a caveat for such a simple explanation
for that latter data is that Syrian hamsters share
the enhanced circadian sensitivity to UV
(Calderone and Jacobs, 1999; von Schantz et al.,
1997), despite lacking S-cone opsin (Calderone
and Jacobs, 1999; von Schantz et al., 1997).
Of course it is equally possible that there are
circumstances in which the schematic in Fig. 4b
greatly overestimates the ability of cones to influ-
ence the clock. Indeed, electrophysiological data
indicate that cone-derived SCN responses are
much reduced during the day and under light
adapted conditions (Brown et al., 2011).
Temporal frequency tuning of melanopsin
Melanopsin’s ability to track dynamic patterns of
light intensity remains largely unexplored. In the
12
absence of rod/cone input, changes in ipRGC fir-
ing lag the onset and offset of light pulses by up to
tens of seconds (Berson et al., 2002). It will be
important to determine whether melanopsin
influences in the intact retina (in which ipRGC
firing is also defined by rod/cone signals), and
under more naturalistic modulations in irradi-
ance, show similarly poor temporal fidelity. None-
theless, the long integration time for melanopsin
phototransduction (Do et al., 2009) is consistent
with other evidence (Wong et al., 2007) that it
integrates across most high frequency events.
A more pertinent issue for the clock is whether
melanopsin responds preferentially to modulations
in irradiance occurring at particular frequencies.
According to the conceptual model in Fig. 4,
melanopsin acts as a simple “photon counter”
tracking the slow, diurnal, variation in irradiance,
irrespective of higher frequency events. In
fact, sudden increases in irradiance have been
reported to induce anomalously large melanopsin
responses, presumably reflecting light adaptation
under continuous exposure (Wong et al., 2005).
According to the conceptual model in Fig. 4, the
inclusion of cones in the entrainment pathway
would make the clock especially responsive to
such an abrupt change in light exposure. Do
melanopsin’s light adaptation characteristics aug-
ment this effect? An answer to that question awaits
further study of melanopsin light adaptation and its
significance under “naturalistic” light exposure
profiles. However, the importance of any such
effect is questioned by evidence that the photo-
entrainment pathway has an impressive ability to
count photons irrespective of the temporal pattern
with which they are presented. Thus, Nelson and
Takahashi (1999) showed that the magnitude of
hamster phase shifts were defined by the number
of photons encountered, irrespective of whether
they appeared in a single long, or up to 100
shorter light pulses. Such temporal integration is
lost for very short, very bright, pulses (Vidal and
Morin, 2007), and melanopsin light adaptation
could contribute to that process. On balance, how-
ever, this behavioral data favors the conclusion
that, under most circumstances, melanopsin light
adaptation does not dramatically skew the clock’s
response towards higher frequency changes in illu-
minance. According to this view, the most impor-
tant consequence of adaptation is extension of
the melanopsin sensitivity range (Wong et al.,
2005). This feature is discussed below.
Photoreceptor sensitivity ranges
The depiction of photoentrainment in Fig. 4 is
semiquantitative by design. However, the relative
sensitivities of rod, cone, and melanopsin inputs
remain an important detail. We have based these
on a combination of known photoreceptor physi-
ology and more direct studies of circadian pho-
tosensitivity. As outlined below, a particular
challenge in reviewing this data is that descriptions
of photoreceptor sensitivity in radiometric units
(energy or quanta) will vary according to the spec-
tral quality of the light used in their estimation.
This makes it difficult to extract quantitative infor-
mation from studies using “white” light. We have
based our estimates on data from studies using
near monochromatic light at around 480–500 nm,
and all irradiances in the following discussion refer
to such conditions.
We have estimated the threshold for melanopsin
input to the clock by the minimum corneal irradi-
ance required for phase shifts in mice lacking rods
and cones (a little below 1011 photons/cm2/s;
Hattar et al., 2003; Yoshimura and Ebihara,
1996). In comparing this figure with data from
in vitro recordings from ipRGCs it is important
to account for the distinction between retinal
and corneal irradiance. Light measures from
in vitro studies reflect the former, while it is the
latter that is most relevant for behavioral and
other in vivo studies. The relationship between
retinal and corneal irradiance is defined by the
ratio of pupil to retinal area. If we assume that
the mouse retina has an area of  18 mm2, and
the pupil area varies from 9 to 0.1 mm2
(Lyubarsky et al., 2004), this implies a correction

factor of between 0.5 (fully dilated pupil) and
0.005 (fully constricted pupil) in transforming cor-
neal to retinal irradiance. On this basis, the ability
of Schmidt et al. (2008) to record melanopsin
responses from ipRGCs at irradiances  1010 pho-
tons/cm2/s is broadly consistent with the estimate
from behavioral data (Hattar et al., 2003;
Yoshimura and Ebihara, 1996). Although, note
that for most in vitro studies brighter lights have
been required to elicit measurable responses
(Berson et al., 2002; Dacey et al., 2005; Sekaran
et al., 2003; Tu et al., 2005).
There is more uncertainty regarding
melanopsin’s saturation point. Phase shifting
responses in rodless þ coneless mice saturate at
corneal irradiance  1013.5 photons/cm2/s (Hattar
et al., 2003). However, this is unlikely to reflect
saturation in melanopsin phototransduction,
because other responses in animals with this phe-
notype saturate at > 1014 photons/cm2/s even with
fully dilated pupils (Brown et al., 2011; Lucas
et al., 2003; Panda et al., 2003). In vitro studies
report saturation in aspects of the melanopsin-
driven light response at anything from 1013.5
(Schmidt et al., 2008) to > 1015 photons/cm2/s
(Sekaran et al., 2003). In view of the evidence
for multiple classes of ipRGC with divergent sen-
sory capacity (Ecker et al., 2010; Schmidt and
Kofuji, 2010), we have based our estimate of the
saturation point for melanopsin signals to the
clock upon Berson and colleagues’ original data
for the ipRGCs innervating the rat SCN (Berson
et al., 2002). This suggests saturation at a retinal
irradiance  1014–1015 photons/cm2/s. As the pupil
will presumably be fully constricted under such
conditions (Lucas et al., 2001), this translates to
a corneal irradiance closer to 1017 photons/cm2/s.
Rod-dependent clock responses are evident at
very low corneal irradiance (> 107 photons/cm2/s),
while rods also define phase shifting responses
in the range 109–1011 photons/cm2/s (Lall et al.,
2010). Rodent rods in vitro saturate around
1011 photons/cm2/s (equivalent corneal irradiance
 1012 photons/cm2/s; Demontis et al., 1993;
Matthews, 1991). Based upon these data a
13
conservative estimate is that the rod phase of
the clock light response lies between 107 and
1011 photons/cm2/s. Given that the equivalent esti-
mate for melanopsin is 1011–1017 photons/cm2/s,
this implies that the clock can track irradiance over
a most impressive range (up to ten decades) using
just these two photoreceptor types.
Cones adjust their sensitivity to any background
illuminance, and so could encode high frequency
modulations in irradiance at any light level above
threshold (>1010 photons/cm2/s; Nathan et al.,
2006). Although the schematic in Fig. 4b does not
allow the steady state, irradiance coding, compo-
nent of the cone response to reach the clock, it is
worth considering also its range in view of the pos-
sibility that it could influence entrainment under
different circumstances (see above). Data from
in vitro recordings of cone photoreceptors
indicates that this aspect of their response
saturates at irradiances around  1000 above
threshold (Burkhardt, 1994; Normann and Per-
lman, 1979; Valeton and van Norren, 1983). On
this basis, we estimate the dynamic range for any
putative cone-dependent low frequency signal to
lie between  1010 and 1013 photons/cm2/s. As
melanopsin is capable of measuring significantly
higher irradiances, this implies that melanopsin is
unique among mammalian photoreceptors in
being able to encode low frequency changes in
irradiance under brighter (daylight) conditions.
Before leaving the issue of photoreceptor sensi-
tivity ranges it is worth making a couple of further
points regarding the wider applicability of these
estimates. Firstly, they reflect the situation with
respect to corneal irradiance in mice. In the
absence of evidence to the contrary, it seems rea-
sonable to assume that the fundamental sensitiv-
ity range of the various photoreceptor classes is
retained across mammalian species. However,
differences in the ratio of pupil to retinal area
influence the corneal irradiances required to
achieve comparable light levels at the retina.
In the case of humans, this would likely push all
of the estimates described above for mouse sensi-
tivity ranges to slightly brighter light. Secondly,
14
while it has been helpful here to discuss sensitivity
ranges in terms of photon flux for monochromatic
light, outside of the laboratory almost all light is
polychromatic. It is possible to express these
ranges for each photoreceptor system in terms
of the photopic units relevant for measuring poly-
chromatic light (for discussion see Enezi et al.,
2011). Thus, dynamic ranges for rods correspond
to 10 4–100 scotopic lux, the “steady state” cone
response to 10 2–101 photopic lux, and melanopsin
to 100–106 m-lux; (Enezi et al., 2011). Nonetheless,
the relative sensitivity of the various photoreceptor
types will, of course, be dependent upon the spec-
tral composition of light, especially in species in
which cones have very different spectral sensitivity
profiles from rods and melanopsin.
Moving from input to output
The contribution of each photoreceptor class to
light information reaching the clock summarized
in Fig. 4b has been inferred from the recorded
impact of light on electrophysiological activity in
the SCN and/or circadian locomotor activity
rhythms. However, it does not follow that all
aspects of circadian photosensitivity will be
influenced by all three photoreceptor types. It is
clear that, for example, an aspect of the clock
response that has high sensitivity and a relatively
low saturation point (e.g., the phase shift induced
in mice by a 15-min light pulse) would, in effect,
rely entirely on rods (Lall et al., 2010). By con-
trast, it would be surprising if suppression of
pineal melatonin in humans, which has a high
threshold, were especially reliant upon rods
(Brainard et al., 2001; Thapan et al., 2001).
A less obvious way in which the relative signif-
icance of each photoreceptor for a given response
parameter could be modulated is through applica-
tion of a temporal filter (Fig. 4c). The potential
effect of such a process is apparent in electrophys-
iological recordings in the mouse hypothalamus
(Brown et al., 2011). Thus, outside of the SCN
we found large numbers of cells that lacked any
transient response to lights on/off but showed
sustained excitation/inhibition by light. As a
result their activity was strongly influenced by
melanopsin but not by cones. As cone signals
are a fundamental feature of the ipRGC light
response, their absence in these hypothalamic
cells implies the presence of a low pass temporal
filter somewhere in the network linking these
cells to the RHT. Conversely, a minority of light
responsive SCN units show only transient excita-
tion at lights on/off and lack any sustained
response during extended light exposure. As a
result, their activity appears not to be influenced
by melanopsin at all. This response phenotype
might be expected if such units were downstream
of melanopsin-negative non-photoreceptive reti-
nal ganglion cells. However, it would be equally
possible to produce such a phenotype by includ-
ing a high pass temporal filter on the information
provided by the RHT.
Summary and conclusions
Data from laboratory rodents indicate that each
of the three known retinal photoreceptors (rods,
cones, and melanopsin) provides a distinct qual-
ity of information for the clock. Rods and
melanopsin acting, respectively, at “low” and
“high” irradiance allow the clock to respond to
gradual changes in irradiance over a very wide
range (up to ten decimal orders). Cones, on the
other hand, provide information about more sud-
den increases in light intensity. We have used
these characteristics to provide a qualitative pre-
diction of the RHT’s response to dynamic
patterns of light exposure.
There is an obvious requirement to test the con-
ceptual model summarized in Fig. 4 by recording
the activity of the entrainment pathway under such
a dynamic pattern of light exposure. Moreover, it
would be useful to replace the highly stylized ver-
sion of the dawn transition depicted in Fig. 4a with
more “natural” versions defined by recording
light exposure under “field” conditions. Leaving

aside these important issues, future work could
concentrate on addressing several fundamental
aspects of the conceptual model that remain
poorly understood. In particular, the temporal
frequency tuning of cone input to the clock needs
to be defined. We predict that, in fact, this charac-
teristic could be labile; with differences across
species and/or cone classes in the degree to which
these photoreceptors could be used to track more
gradual (including perhaps even diurnal) changes
in irradiance. Resolving these uncertainties will
be not merely of academic interest. Understanding
the quality of information about the light environ-
ment provided by each photoreceptor class is a
first step to designing light environments that
either maximize or minimize clock responses. As
such, they represent a most promising strategy
for controlling circadian phase (and potentially
amplitude) for the benefit of human health.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 2

Melanopsin phototransduction: Slowly

emerging from the dark

Steven Hughes, Mark W. Hankins, Russell G. Foster* and Stuart N. Peirson*

Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology,

John Radcliffe Hospital, Headley Way, Oxford, United Kingdom

Abstract: Melanopsin expressing retinal ganglion cells represent a third class of ocular photoreceptors
and are involved in irradiance detection and non-image-forming responses to light including pupil
constriction, circadian entrainment, and regulation of sleep. Over recent years, there has been a rapid
increase in our understanding of the anatomical variety of pRGC subtypes, the regions of the brain which
they innervate, and the behavioral responses of melanopsin-based light detection. However, by contrast,
our understanding of the intracellular signaling cascade initiated following activation of melanopsin has,
until recently, remained poorly characterized. This chapter focus on the melanopsin signaling pathway,
detailing the cellular mechanisms of phototransduction that occur within pRGCs, highlighting recent
advances, but also the gaps in our understanding of this important light detecting system.

Keywords: melanopsin; Opn4; phototransduction; pRGC; retina; TRPC.

Introduction the ventrolateral preoptic areas (VLPO) (Baver

In addition to the rods and cones, a third class of
photoreceptor has been unequivocally identified
within the mammalian retina, the melanopsin-
expressing photosensitive retinal ganglion cell
(pRGC) (for review see Do and Yau, 2010;
Hankins et al., 2008). pRGCs innervate numerous
regions of the brain, including the suprachiasmatic
nuclei (SCN), olivary pretectal nucleus (OPN), and
*Corresponding authors.
Tel.: þ44 (01865) 234782; Fax: þ 44 (01865) 234795
E-mail: russell.foster@eye.ox.ac.uk; stuart.peirson@eye.ox.ac.uk
et al., 2008; Chen et al., 2011; Gooley et al.,
2003; Hannibal and Fahrenkrug, 2004; Hattar
et al., 2002, 2006) and are primarily involved in
non-image forming responses to light, including
circadian entrainment (phase shifting), suppression
of melatonin release, pupil constriction, masking
and induction of sleep, and negative phototaxis
(Freedman et al., 1999; Johnson et al., 2010; Lucas
and Foster, 1999; Lucas et al., 2001, 2003; Lupi
et al., 2008; Mrosovsky et al., 2001; Panda et al.,
2002; Ruby et al., 2002; Semo et al., 2010). How-
ever, more recently, it has become clear that
pRGCs also innerve regions of the brain tasked

http://dx.doi.org/10.1016/B978-0-444-59427-3.00002-2 19
20

with processing visual information, and it would
seem that melanopsin may perform roles in visual-
processing and image-forming vision (Brown et al.,
2010; Ecker et al., 2010).
pRGC subtypes
Since their original description, it has become clear
that pRGCs are not a homogeneous population of
cells, but instead consist of numerous functionally
distinct cell types (Fig. 1). To date, the classification
of pRGC subtype has extended to include five clas-
ses of pRGC, which are distinguished based on
levels of melanopsin expression and the
stratification of their dendrites within specific sub-
laminae of the inner plexiform layer (IPL) (for
review see Do and Yau, 2010 and Schmidt et al.,
2011). The first melanopsin cells to be identified
were the M1-type pRGCs (Berson et al., 2002;
Hattar et al., 2002, 2006; Provencio et al., 2002).
M1 cells express high levels of melanopsin protein
and have sparsely branching dendritic fields that
stratify exclusively in the OFF layer of the IPL.
The majority of M1-type cells have cell bodies
located in the GCL although a subset of M1 cells
have their cell bodies located in the INL, termed
displaced M1 pRGCs. Soon after the characteriza-
tion of M1-type cells, a second class of melanopsin

(a) (c)

OS

ONL

OPL
INL
OFF
IPL
ON M1 25 mm M2 25 mm
GCL

M1 M2 M3
(b)
OFF
ON

M3 50 mm M4, M5 50 mm

M1 M2 M3 M4 M5

Fig. 1. Subtype of melanopsin-expressing pRGC. (a) Schematic showing the layers and cell types of the mammalian retina,
including the melanopsin-expressing photosensitive ganglion cells (pRGCs). (b) Schematic showing the different levels of
melanopsin expressed in pRGC subtypes (denoted by intensity of red coloring) and differing patterns of stratification within the
inner plexiform layer. (c) Images showing the pRGC subtypes indentified in the mouse retina to date. Panels show examples of
M1-, M2-, and M3-type pRGCs in wild-type mouse retina stained with an N-terminal melanopsin antibody (shown in red, UF006
antibody) and also M4- and M5-type cells that are only identified via detection of EYFP expression in Opn4-Cre mice (shown in
green). In the panel showing M4-and M5-type cells, a melanopsin-immunoreactive M1-type cell is included for comparison
(Opn4-Cre mice (Ecker et al., 2010) were kindly provided by Sammar Hattar, John Hopkins University). Outer segments (OS),
outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell
layer (GCL). OFF and ON denote the OFF and ON sublaminae of the IPL, respectively.

cell was identified. M2-type pRGCs express lower
levels of melanopsin compared to M1-type cells,
and have processes that stratify in the ON layer
of the IPL. M2 cells also have larger cell bodies
and larger and more complex dendritic fields
(Baver et al., 2008; Berson et al., 2010; Ecker
et al., 2010; Hattar et al., 2006; Schmidt and
Kofuji, 2009; Viney et al., 2007). M3-type
pRGCs are bistratified and have dendrites
located in both the OFF and ON layers of the
IPL (Berson et al., 2010; Schmidt and Kofuji,
2011; Schmidt et al., 2008; Viney et al., 2007).
These cells are anatomically diverse and rela-
tively rare, failing to tile the entire retina. It
has been suggested that these cells may repre-
sent a developmental anomaly and may not rep-
resent a true class of pRGC (Berson et al.,
2010), although they do seem to have distinct
functional properties (Schmidt and Kofuji,
2011). Most recently, M4- and M5-type pRGCs
have been identified. M4- and M5-type pRGCs
are broadly similar in morphology to M2-type
pRGCs with dendrites located in the ON layers
of the IPL, yet are distinguished based on the
size and complexity of their dendritic fields
(Ecker et al., 2010). Levels of melanopsin
expression in M4 and M5 cells are seemingly
very low, and below that which can be detected
reliably with melanopsin antibodies (Berson
et al., 2010; Ecker et al., 2010). Most recently,
it has become clear that the M1 subclass of
pRGCs is further subdivided into cells that
express the Brn3b transcription factor and cells
that are Brn3b negative (Chen et al., 2011).
Thus, the anatomical diversity of pRGCs
subtypes continues to be refined.
Functional differences between pRGC subtypes
From early studies, it was clear that heterogeneity
exists in the responses of individual pRGCs. At
least three distinct types of responses have been
observed from pRGCs using calcium imaging of
retinal explants, classified as transient, sustained,
and repetitive responses (Sekaran et al., 2005)
21
and at least two distinct types of responses have
been observed from adult pRGCs using multiple
electrode arrays (MEAs), based on sensitivity
and response latency (Tu et al., 2005). Although
these studies did not correlate these responses
with the different anatomical subtypes of pRGC,
subsequent reports have indicated that a range
of functional differences do exist between these
cells. These include differences in membrane pro-
perties and resting membrane potentials, as well
as their overall sensitivity to light and kinetics of
photoresponses (Ecker et al., 2010; Schmidt and
Kofuji, 2009, 2010, 2011).
For M1 cells, there are two clear phases of the
light-induced photoresponse. An initial fast-acting
transient followed by a smaller more sustained
component. The initial large transient component
to the light response is absent from M2-type
pRGCs (and M3-, M4-, and M5-type pRGCs)
that show small but sustained responses (Ecker
et al., 2010; Schmidt and Kofuji, 2009, 2011).
M1 cells increase spike-firing rate with increasing
intensities of light (Do et al., 2009), yet M1 cells
are prone to depolarization block and are only
able to fire action potentials at relatively low rates
(Schmidt and Kofuji, 2009; Wong et al., 2007).
In contrast, M2 cells are 10-fold less sensitive to
light than M1 cells, produce a 10-fold smaller
maximum photocurrent and can fire action
potentials at higher frequencies than M1-type
cells (Ecker et al., 2010; Schmidt and Kofuji,
2009). M3-type cells share many properties of
M2-type cells, including more hyperpolarized
membrane potentials and reduced input resis-
tance compared to M1-type cells, yet their overall
sensitivity to light and their propensity to achieve
depolarization block is increased compared to M2
cells, but is less than that observed for M1 cells
(Schmidt and Kofuji, 2011). In keeping with the
low levels of melanopsin expression, the intrinsic
photoresponses recorded from M4 and M5 cells
are small, even smaller than those observed in
M2-type cells, with properties similar to that
observed for M2-type cells (Ecker et al., 2010).
Although it is clear that the biophysical properties
22
of the responses vary between pRGC subtypes,
the properties of responses elicited by each sub-
class have yet to be fully determined.
Retinal connections
In addition to their innate photosensitivity,
melanopsin-expressing pRGCs also receive syn-
aptic inputs from the outer retina, via connections
with bipolar cells and amacrine cells, and act as
the principle conduit for delivering rod cone
driven information to non-image forming areas
of the brain (Belenky et al., 2003; Guler et al.,
2008; Hatori et al., 2008; Ostergaard et al., 2007;
Vugler et al., 2007; Wong et al., 2007). As a result,
the photoresponses elicited by individual pRGCs
are ultimately a fusion of signals generated from
within rods, cones, and also the pRGCs them-
selves. As indicated by the differing location of
their dendrites in either the ON or OFF layers
of the IPL, it is now clear that the pRGC subtypes
receive different inputs from the outer retina. M2-
type pRGCs receive inputs from ON bipolar cells,
as might be expected. Interestingly, M1-type
pRGCs receive not only OFF inputs but also
anomalous ON inputs from synaptic connections
with cone ON bipolar cells as their dendrites pass
through the ON layer en route to the OFF layer
of the IPL and also within the OFF layer of the
IPL itself (Dumitrescu et al., 2009; Hoshi et al.,
2009; Wong et al., 2007). Indeed, it would appear
that despite the pattern of stratification, the ON
pathway exerts larger effects on M1 cells than
the OFF pathway (Schmidt and Kofuji, 2010;
Wong et al., 2007). In addition to contacts with
bipolar cells, pRGCs are also known to receive
inhibitory inputs from amacrine cells, yet again
there are cell type-specific differences. M1 cells
synapse with dopaminergic amacrine cells in the
OFF layer of the IPL (Ostergaard et al., 2007;
Viney et al., 2007; Vugler et al., 2007), whereas
M2-type cells appear to synapse with a mono-
stratified amacrine cell type (Viney et al., 2007).
It is now clear that signals from the outer retina
exert larger influence on M2-type cells than M1-
type cells. Photoresponses of M1 cells are driven
primarily by melanopsin function with little influ-
ence from synaptic inputs (Schmidt and Kofuji,
2009, 2010). By contrast, inputs from cone ON
bipolar cells represent the major driving force
behind the generation of photoresponses in M2-
type pRGCs, with similar photoresponses observed
from these cells in the absence of melanopsin
expression (Schmidt and Kofuji, 2010). Similar
observations have been reported for M3-type
pRGCs (Schmidt et al., 2011) and are likely also
true for M4- and M5-type pRGCs which have even
lower levels of melanopsin expression and elicit
even smaller photoresponses than M2-type cells.
Given the lower levels of innate photosensitivity
compared to M1-type cells, it would appear that
the functioning of M2-type cells, M3-type cells,
and presumably M4- and M5-type cells are more
closely linked to that of the classical photoreceptors
compared to M1-type cells.
pRGC subtypes mediate different physiological
responses to light
In addition to differences in cell morphology and
the nature of retinal connections, there is also evi-
dence that pRGC subtypes differentially innervate
specific retino-recipient brain areas (Baver et al.,
2008; Chen et al., 2011; Hattar et al., 2002, 2006;
McNeill et al., 2011) and would therefore seem to
be tasked with performing different physiological
roles (Chen et al., 2011: for review see Do and
Yau, 2010; Schmidt et al., 2011). M1 cells predomi-
nately innervate the SCN and shell of OPN (Hattar
et al., 2006; McNeill et al., 2011), with these
structures each innervated by distinct subtypes of
M1-type pRGCs (Chen et al., 2011). Brn3b nega-
tive M1-type pRGCs comprise the primary input
to the SCN and contribute to circadian entrainment
whereas Brn3b positive M1-type pRGCs innervate
the core of the OPN and contribute to the pupillary
light response (Chen et al., 2011). By comparison,
non-M1-type pRGCs (collectively) show little

innervation of the SCN, but do innervate the core
region of the OPN, the SC, and the dLGN (Baver
et al., 2008; Brown et al., 2010; Chen et al., 2011;
Ecker et al., 2010; McNeill et al., 2011). However,
by using currently available transgenic models, it
is not possible to selectively label the different
non-M1 classes of pRGCs, and little is known
regarding the specific regions of the brain innerva-
ted by M2-, M3-, M4-, or M5-type pRGCs or the
specific functions associated with these cell types.
Data from retrograde labeling-based experiments
indicate that M2-type pRGCs (detected by
melanopsin antibodies but not b-gal antibodies in
the tau-lacZ-Opn4 mouse that reports only M1-
type cells) innervate the core of the OPN and the
SC (Baver et al., 2008). Based on their innervation
of the SC, it would seem that non-M1 cells, includ-
ing M2-type pRGCs contribute to melanopsin-
dependent light avoidance and photophobia
(Johnson et al., 2010; Semo et al., 2010) and recent
studies have suggested a role for non-M1-type
pRGCs in image-forming pathways and visual pro-
cessing (Brown et al., 2010). Further work will
be needed to establish the precise pattern of
innervations associated with each pRGC subtype,
and in turn, this information will undoubtedly
provide invaluable insight into the physiological
functions performed by each class of pRGC.
Melanopsin phototransduction
The phototransduction signaling pathway of rods
and cones has been characterized extensively.
Absorption of light by an opsin/vitamin A-based
visual pigment leads to activation of the
G-protein transducin (a member of the Gi/o class
of G-proteins), resulting in phosphodiesterase
activation and hydrolysis of cGMP to GMP
(Lamb, 1996; Luo et al., 2008). Falling levels of
cGMP lead to closure of cyclic nucleotide-gated
(CNG) cation channels and result in a membrane
hyperpolarization.
By contrast, the phototransduction signaling
pathway initiated within pRGCs following
23
activation of melanopsin has received far less
attention, and to date the precise cellular
mechanisms of phototransduction in pRGCs have
not been fully characterized. Until recently, our
understanding of the melanopsin signaling
pathway had been informed largely by pharmaco-
logical studies performed in retinal explants,
disassociated pRGCs, and cellular expression sys-
tems. Overall, the data from these studies suggest
a melanopsin signaling pathway starting with the
activation of a Gq/11-type G-protein, leading
to PLC activation and ultimately the influx of
Ca2 þ through TRP-type ion channels in the cell
membrane, leading to the generation of action
potentials (for review see Do and Yau, 2010 and
Hankins et al., 2008) (Fig. 2). Interestingly, all of
the components necessary for generating light
responses within pRGCs appear to be localized
to a membrane-bound signaling complex, and
the classical endpoint of the Gq/11 signaling path-
way, namely, inositol 1,4,5-triphosphate (IP3)-
dependent release of calcium from intracellular
stores, does not seem necessary for transduction
of light stimuli by pRGCs (Graham et al., 2008;
Hartwick et al., 2007; Sekaran et al., 2003).
From these studies, it is clear that there are
striking differences in the phototransduction cas-
cade employed by melanopsin-expressing pRGCs
and rod and cone photoreceptors. Melanopsin
employs a different G-protein signaling mec-
hanism to the rods and cones and melanopsin
activation ultimately results in membrane depo-
larization rather than cellular hyperpolarization.
It has been suggested that the melanopsin signaling
cascade may be analogous to the invertebrate pho-
totransduction pathway present in rhabdomeric
photoreceptors (Berson, 2007; Contin et al., 2006;
Graham et al., 2008; Hankins et al., 2008; Isoldi
et al., 2005).
Although these initial studies provided an
important framework to consider melanopsin
phototransduction, the specific identity of almost
all, if not all, the components involved remained
to be determined beyond doubt. More recently,
transgenic approaches have been used to identify
24

TRPC6/7 VOCC
4 5
1

PIP2
Na+
Gq/11 Ca2+

2 β γ
PLCb4 X
3 IP3 + DAG
X

ER Ca2+
release

Fig. 2. The melanopsin signaling pathway. Schematic representation of the melanopsin signaling pathway based on available
evidence. Melanopsin activation (Step 1) leads to activation of a Gq/11-type G-protein (Step 2) followed by activation of PLCb4
(Step 3) and ultimately the downstream opening of TRPC6/7-type channels (Step 4) followed by VOCC activation and
action potential generation (Step 5). The effectors of PLCb4 are not clear, but seem not to involve IP3-mediated release of Ca2 þ
from internal stores or the direct activity of DAG (as indicated by dashed blue lines). PLC, phospholipase C; PI3K,
phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; ER,
endoplasmic reticulem; VOCC, voltage-operated calcium channel.

a number of key components in the melanopsin
signaling pathway, and have shown further
similarities between invertebrate and melanopsin
phototransduction. In the following sections, we
will consider, in turn, the evidence for each stage
of the signaling cascade and highlight recent
advances and also areas where clear omissions
in our understanding remain.
Step 1: Light absorption by melanopsin
photopigment
Melanopsin is a seven transmembrane G-protein
coupled receptor (GPCR) with all the key struc-
tural features of an opsin (Provencio et al.,
1998). Phylogenetic analysis suggests that mam-
malian melanopsin is more closely related to the
invertebrate opsins than the classical vertebrate
visual opsins (Koyanagi and Terakita, 2008;
Provencio et al., 1998). The deduced structure
of melanopsin shows several features that are
reminiscent of invertebrate opsins, including the
presence of a tyrosine instead of a glutamate
counterion (E113), an elongation of the third
cytoplasmic loop, and an extended intracellular
C terminus (Provencio et al., 1998). In vivo
melanopsin binds 11-cis retinal as its chromo-
phore and converts this to all-trans retinal follow-
ing absorption of a photon (Walker et al., 2008).
Interestingly, there is strong evidence that
melanopsin is capable of acting as a bistable pho-
topigment (Melyan et al., 2005; Mure et al., 2007;
Panda et al., 2005), and is capable of regenerating
11-cis chromophore from all-trans retinal. How-
ever, recent studies of the zebrafish melanopsin

family suggest that bistability is not a universal
property of vertebrate melanopsins (Davies
et al., 2011). The photobiology and visual cycle
of melanopsin has been reviewed in detail
elsewhere (Do and Yau, 2010) and shall not be
considered here.
The general consensus is that the peak spectral
sensitivity of mammalian melanopsin is in the
region of 480 nm. There is strong evidence for
this from studies of melanopsin-driven light
responses in cell line models (Panda et al., 2005;
Qiu et al., 2005), and also pRGCs (Berson
et al., 2002; Dacey et al., 2005; Tu et al., 2005),
behavioral responses in animal models (Gamlin
et al., 2007; Hattar et al., 2003; Lucas et al., 2001)
and physiological measurements from human
patients (Brainard et al., 2001; Hankins and Lucas,
2002; Thapan et al., 2001; Zaidi et al., 2007).
However, there are some inconsistencies in the
literature with reports of peak sensitivity for
melanopsin ranging from 420 to 500 nm. Some of
these results originate from in vitro cell expression
systems that may potentially be sensitive to
artifacts (Melyan et al., 2005; Newman et al.,
2003). The first successful absorption spectra of
mouse melanopsin suggested a peak absorbance
of 424 nm (Newman et al., 2003), while other
reports have indicated values of around 500 nm
(Walker et al., 2008). Spectrophotometry analysis
of melanopsin photopigments from other species
have indicated a peak sensitivity in the region of
480 nm, including cephalochordate (Koyanagi
et al., 2005) and zebrafish (Davies et al., 2011).
Collectively, it seems likely that the peak sensitiv-
ity of melanopsin is close to 480 nm, but direct
confirmation of this fact is hindered by the techni-
cal difficulties in obtaining high-quality spectro-
photometry data for this class of opsin, most
likely related to issues of protein misfolding in
overexpression systems.
In nonmammalian species, numerous distinct
forms of melanopsin have been reported,
encoded not only by separate genes, but also
alternate splicing (Bellingham et al., 2006; Davies
et al., 2011; Drivenes et al., 2003; Torii et al.,
25
2007). In mammals, only one melanopsin gene
has been identified, but again splice variants have
been reported. Two distinct isoforms of mouse
melanopsin, Opn4L and Opn4S, are generated
by alternative splicing of the murine Opn4 gene
(Pires et al., 2009). These two isoforms of
melanopsin differ only at the distal C-terminal
regions and are differentially expressed in M1-
and M2-type pRGCs of the adult mouse retina.
M1-type cells express both Opn4L and Opn4S
whereas only Opn4L can be detected in M2-type
cells. However, the functional relevance of these
distinct isoforms is currently unclear.
Step 2: Activation of a G-protein signaling
pathway
Once activated by light, melanopsin, like other
GPCRs, binds to specific G-proteins to activate
intracellular signaling cascades. To date, approx-
imately 20 Ga subunits have been identified.
They are grouped into four functionally distinct
subclasses: Gs, Gi/o, Gq/11, and G12/13, based on
the nature of the signaling pathway initiated
(Hubbard and Hepler, 2006; Mizuno and Itoh,
2009). By analogy to rhabdomeric transduction,
it was proposed that melanopsin might be specif-
ically coupled to the Gq/11 class of G-proteins and
downstream activation of PLC (Provencio et al.,
1998; Warren et al., 2003). Since then, several
studies have utilized retinal explants or disso-
ciated ganglion cell cultures to study the nature
of melanopsin G-protein interactions within
pRGCs (Graham et al., 2008; Hartwick et al.,
2007; Sekaran et al., 2003, 2005, 2007; Tu et al.,
2005; Warren et al., 2006). Current pharmacolog-
ical evidence does indeed suggest a melanopsin
signaling pathway involving the activation of a
Gq/11-type G-protein (Table 1). Most striking is
the finding that application of GPant-2a, a spe-
cific inhibitor of the Gq/11 class of G-proteins,
completely abolishes melanopsin light responses
in pRGCs whereas the related peptide GPant-2,
a specific inhibitor of the Gi/o class, has no effect
26

Table 1. Summary of evidence that melanopsin phototransduction involves Gq/11-type G-proteins

Reference Cell type Assay Agent/targets Effect

Warren et al. pRGCs/retina Patch clamping GTPgS (stimulatory G-protein) Blocked response
(2006) explant GDPbS (inhibitor of GTP Blocked response
binding)
IBMX and cAMP or cGMP No effect
Hartwick et al. Isolated pRGCs Patch clamping Ca2 þ Removal of external Ca2 þ Blocked response
(2007) imaging Thapsigargin No effect
Graham et al. Isolated pRGCs Patch clamping GDPbS (inhibitor of GTP Blocked response
(2008) binding)
GPAnt-2a (inhibits Gq/11 class) Blocked response
U73122 (PLC antagonist) Blocked response
Thapsigargin No effect
GPAnt-2 (inhibits Gi/o class) No effect
PTX (inhibits Gi/o class) No effect
Cholera toxin (inhibits Gs class) No effect
OAG (active DAG analogue) No effect
Panda et al. hOpn4 in Xenopus Patch clamping Gnaq/11 antibodies Blocked response
(2005) oocytes U73122 (PLC antagonist) Blocked response
PTX (inhibits Gi/o class) No effect
Gnas, Gnai antibodies No effect
Qiu et al. (2005) mOpn4 in HEK293 Patch clamping Ca2 þ GDPbS (inhibits GTP binding) Blocked response
imaging GPant-2a (inhibits Gnaq/11 class) Blocked response
U73122 (PLC antagonist) Blocked response
PTX (inhibits Gi/o class) No effect
Melyan et al. hOpn4 in Neuro-2A Patch clamping Suramin (G-protein inhibitor) Blocked response
(2005) Thapsigargine Blocked response
8-Br-cGMP Blocked response
GTPgS (stimulatory G-protein) Induced response
U73122 (PLC antagonist) No effect
NF023 (inhibits Gi/o class) No effect

(Graham et al., 2008). In addition to studies of
native pRGCs, melanopsin G-protein signaling
has also been investigated using heterologous
expression in a variety of cell lines (Giesbers
et al., 2008; Kumbalasiri et al., 2007; Melyan
et al., 2005; Panda et al., 2005; Qiu et al., 2005).
In general, the pharmacological data obtained
from these in vitro models again indicate that
melanopsin couples to Gq/11-type G-proteins
(Table 1). For example, melanopsin responses
are greatly attenuated by antibodies against
Gq/11-type G-proteins but not by antibodies
against Gi/o G-proteins (Panda et al., 2003).
Gq/11 agonists fully blocked the melanopsin-
dependent light responses in HEK293–TRPC3
cells (Qiu et al., 2005), whereas blockers of Gi/o
fail to inhibit melanopsin-dependent light
responses in Neuro-2A cells (Melyan et al.,
2005). By contrast, one study has reported that
melanopsin can couple to transducin in a bio-
chemical assay (Newman et al., 2003), yet there
is no evidence of transducin expression in
pRGCs and melanopsin-based signaling is nor-
mal in Gnat/ mice (Hattar et al., 2003). Collec-
tively, there is relatively strong evidence that
melanopsin couples to Gq/11-type G-proteins
in vitro and in vivo, and a general consensus has
formed that this is indeed the case. Yet despite
the growing evidence most, if not all, previous
studies have utilized pharmacological tools that

fail to distinguish between specific members of
the Gq/11 family (Table 1). As a result, the precise
identity of the G-protein subunit(s) involved in
melanopsin phototransduction remains to be
determined. The Gq/11 family itself contains four
members: Gaq, Ga11, Ga14, and Ga15 (Ga16 in
humans) (Davignon et al., 1996; Wilkie et al.,
1992). All members of the Gq/11 subclass share a
high degree of sequence homology and all exert
their effect via the activation of the effector
enzyme PLCb (Hubbard and Hepler, 2006; Rhee,
2001; Weschler et al., 2006). Gaq and Ga11 are
expressed in almost all cell types (Wilkie et al.,
1991) and share a near complete functional over-
lap with few, if any, receptors discriminating
between them (Hubbard and Hepler, 2006;
Wettschureck and Offermanns, 2005). In com-
parison, the expression profile of Ga14 is far
more restricted (Wilkie et al., 1991) and typically
observed only in specialized cell types (Shindo
et al., 2008; Tizzano et al., 2008; Yule et al.,
1999). The pattern of expression observed for
Ga14 seems to suggest specific roles in cell signal-
ing, however, to date there is limited information
regarding the functional differences between
Ga14 and Gaq/11 (Nakamura et al., 1996). Ga15
is a highly promiscuous G-protein subunit whose
expression is almost exclusively restricted to
hematopoietic cell types (Amatruda et al., 1991;
Davignon et al., 1996; Kostenis et al., 2005;
Wilkie et al., 1991). Our own qPCR and micro-
array data (Steven Hughes, unpublished data), as
well as several previous studies have shown that
Gaq, Ga11, and Ga14 but not Ga15 are expressed
in the mouse retina (Graham et al., 2008; Peirson
et al., 2007) and single-cell PCR studies have
confirmed the expression of mRNA for these
G-proteins in pRGCs (Graham et al., 2008), with
Ga14 the most commonly detected. As a result, it
is theoretically possible that melanopsin may
couple to Gaq, Ga11, or Ga14 within pRGCs, or a
combination of these G-protein subunits. Further
work is required to determine the precise identity
of the G-protein subunit(s) involved in melanopsin
phototransduction.
27
Step 3: Phospholipase C activation
The Gq/11 signaling pathway classically leads to
the activation of PLCb isoforms and the genera-
tion of 1,2-diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3) following the hydrolysis
of phosphatidylinositol 4,5-bisphosphate (PIP2)
(Hubbard and Hepler, 2006; Mizuno and Itoh,
2009). Pharmacological evidence supports a role
for PLC activity in melanopsin-driven light
responses, both in pRGCs and cell line models
(Table 1). Most notable is the observation that
pRGC responses are blocked by the PLC inhibi-
tor U73122, but not the inactive homolog
U73343 (Graham et al., 2008). In addition, PLC
activity has been shown to be an important
step in melanopsin signaling in a range of non-
mammalian species, including chicken (Contin
et al., 2006), Xenopus (Isoldi et al., 2005), and
Amphioxus (Nasi and del Pilar Gomez, 2009).
However, the pharmacological evidence supports
only the involvement of a PLC-type enzyme,
and does not provide information regarding the
subclass of PLC involved, but by inference and
logical assumption it is predicted to be a member
of the PLCb subclass.
To date, four isoforms of PLCb have been
reported. PLCb1 and PLCb3 are widely
expressed, whereas the expression of PLCb2 is
largely restricted to hematopoietic cells. Interest-
ingly, expression of PLCb4 is highest in the
retina, with expression highest in cones and gan-
glion cells (Adamski et al., 1999; Ferreira and
Pak, 1994; Ferreira et al., 1993). The profile
and efficiencies with which Gaq, Ga11, and Ga14
activate PLCb isoforms are very similar. All are
potent activators of PLCb1, PLCb3, and PLCb4,
and all show relatively weak activation of PLCb2
(Hubbard and Hepler, 2006). Graham et al.
(2008) confirmed the expression of specific PLCb
isoforms in pRGCs using single-cell RT-PCR, and
indicated that levels of expression are highest for
PLCb4 compared to other isoforms (Graham
et al., 2008). It is interesting to note that PLCb4
is 50% identical to the Drosophila PLCb norpA
28
protein, a value higher than that shared with the
other mammalian PLCb isoforms (35–40%)
(Hubbard and Hepler, 2006; Rhee, 2001). Thus,
based on comparison with the invertebrate signal-
ing cascade, PLCb4 is predicted to be the media-
tor of melanopsin signaling. These assumptions
have been confirmed recently. Endogenous
photoresponses are almost entirely abolished in
M1-type pRGCs from PLCb4/ mice, with only
a small residual response remaining representing
< 1% of the wild-type response (Xue et al., 2011).
The downstream effectors of PLCb activity in
pRGCs are unclear, and little to nothing is known
concerning the mechanisms by which second
messengers influence the activity of the light-
sensitive ion channels. Typically, PLCb activation
leads to the hydrolysis of PIP2 to form IP3 and
DAG. However, application of DAG analogues
to membrane patches excised from pRGCs fails
to induce or modify responses from pRGCs
suggesting that DAG itself is not required for
melanopsin phototransduction (Graham et al.,
2008; Warren et al., 2006). In addition, intracellu-
lar application of IP3 analogues neither induces
nor blocks photoresponses in pRGCs (Graham
et al., 2008; Hartwick et al., 2007), suggesting that
IP3 is also not required. However, the generation
of IP3 and DAG results in a depletion of PIP2
from the cell membrane. It is possible that the
light-sensitive ion channels in pRGCs are gated
directly by interactions with PIP2 itself. PIP2 is
known to modulate the gating of several classes
of ion channels (Hardie, 2007; Suh and Hille,
2008), including the light-gated channels in Dro-
sophila (Hardie, 2003; Raghu, 2006; Suh et al.,
2006). There is some evidence that pRGCs light
responses may indeed be directly gated by PIP2.
Pharmacological inhibition of PIP2 synthesis (using
wortmannin) leads to prolonged light responses in
pRGCs (Graham et al., 2008). It is possible that
the prolonged responses are caused by a delay in
response termination due to a delay in restoring
levels of PIP2 at the cell membrane and a failure
to close the activated ion channels. There is
also evidence that the light-gated channels in
Drosophila can be influenced directly by polyun-
saturated fatty acids (PUFAs), produced by the
breakdown of DAG (Chyb et al., 1999b). By anal-
ogy to the invertebrate cascade, it is possible that
the activity of the light-gated channels in pRGCs
may be influenced by PLCb4 activity via numerous
mechanisms, including the depletion of PIP2
and the generation of PUFAs. Further work is
required to determine the mechanisms by which
PLCb4 influences the activity of ion channels posi-
tioned downstream in the melanopsin signaling
cascade.
Step 4: TRP channel activation
Electrophysiological recordings from pRGCs
(both whole cell and excised membrane patches)
have provided good evidence for the involvement
of a TRP-like channel mediating the initial cellular
depolarization of pRGCs following light stimula-
tion. For example, the light-induced photocurrent
reverses close to 0 mV, shows inward and outward
rectification, and has a small single-channel con-
ductance ( 1 pA) (Do et al., 2009; Schmidt and
Kofuji, 2009), and calcium seems to be the signifi-
cant charge carrier through the light-sensitive
channels (Warren et al., 2006). These features are
all consistent with involvement of a TRP-like ion
channel.
Again, by analogy to the invertebrate cascade,
and the biophysical characteristics of the light
currents recorded from pRGCs, initial
investigations focused on members of the TRPC
subfamily of TRP channels. Data from pharma-
cology-based studies suggested that the most
likely candidates for this TRP-like channel are
indeed members of the TRPC channel subfamily,
potentially TRPC3, TRPC6, or TRPC7 (Hartwick
et al., 2007; Sekaran et al., 2007; Warren et al.,
2006). Light-evoked responses in pRGCs are
suppressed by lanthanides, ruthenium red, and
SKF-96365, and are enhanced by flufenamic
acid and 1-oleoyl-2-acetyl-sn-glycerol (Hartwick
et al., 2007; Warren et al., 2006). Further,

2-aminoethoxydiphenyl borate (2-APB), an
inhibitor of the TRPC subclass of TRP channels,
is an extremely potent inhibitor of mouse pRGC
light responses in vitro (Hartwick et al., 2007;
Sekaran et al., 2007), and also in vivo where
2-APB induces an acute reduction of the pupil-
lary light reflex (Sekaran et al., 2007).
Immunolocalization studies indicate the expres-
sion of TRPC6 and TRPC7 but not TRPC3 in
pRGCs (Sekaran et al., 2007; Warren et al.,
2006), and so these channels represent the prime
candidates for the depolarizing current observed
in pRGCs. Despite these early conclusions,
subsequent work suggested that the light-induced
channels may not be members of the TRPC chan-
nel subfamily. pRGC photoresponses were found
to be largely unaltered in mice lacking TRPC3,
TRPC6, and TRPC7, suggesting that these
channels are not required for the generation of
light responses in pRGCs (Perez-Leighton et al.,
2011). However, this study only reported the effect
of removing each of these channels in isolation,
and could not exclude the possibility that multiple
TRPC-like channels are involved in melanopsin
phototransduction, potentially as functional het-
eromeric channels (Schaefer, 2005). Recently,
the identity of the ion channels responsible for
passing the light-sensitive current in pRGCs has
been determined, at least for M1-type pRGCs.
Photoresponses in these cells are abolished in
double knockout mice lacking both TRPC6 and
TRPC7, but were again unaltered in single knock-
out mice lacking TRPC3, TRPC6, or TRPC7 or
various other members of the TRPC subfamily in
isolation or combination. Overall, these results
indicate that elimination of both TRPC6 and
TRPC7 is required to eliminate photoresponses
in M1-type pRGCs (Xue et al., 2011), although it
remains to be shown conclusively whether these
channels are performing redundant roles or acting
as functional heteromers. The definitive identifica-
tion of the light-sensitive channels in pRGCs will
no doubt prove to be an important step in our
understanding of melanopsin phototransduction,
and will allow more focused studies of the
29
mechanisms by which PLCb4 activation drives
the opening of these channels and transduction of
the melanopsin signal.
Step 5: Activation of voltage-gated ion channels
and action potential firing
The initial calcium influx through the TRPC
channels results in cellular depolarization and
ultimately leads to the activation of voltage-gated
ion channels and the generation of action
potentials (Graham et al., 2008; Hartwick et al.,
2007; Warren et al., 2006). Initial pharmacology
suggests the involvement of L-type VOCCs acting
downstream of TRP channel activation, as verap-
amil and cis-diltiazem significantly reduced light
responses recorded from pRGCs (Hartwick
et al., 2007). However, it is clear that the genera-
tion of action potentials requires the cooperative
action of numerous classes of ion channel, includ-
ing voltage-gated calcium, sodium, and potassium
channels, as well as calcium-sensitive channels,
inward rectifiers, and background leak-type
channels. The precise identity of the ion channels
that contribute to the generation and regulation
of action potentials in pRGCs has not been fully
determined.
Membrane potential is, by definition, a key fac-
tor in determining the activation of voltage-gated
channels and the progression to action potential
firing, and as such plays a key role in regulating
the excitability and sensitivity of neuronal cells.
Single photon responses, that generate small
currents of only few pA, are sufficient to induce
spike activity in pRGCs (Do et al., 2009) and
spontaneous firing of pRGCs is observed in
the dark (Berson et al., 2002; Do et al., 2009;
Schmidt et al., 2008; Tu et al., 2005). From these
observations, it appears that the resting mem-
brane potential of pRGCs is tightly regulated
and held very close to the threshold for action
potential firing. However, the mechanisms that
control resting membrane potential and cellular
excitability in pRGCs remain poorly understood.
30
pRGCs are known to receive excitatory synap-
tic inputs from bipolar cells and inhibitory inputs
from amacrine cells (Schmidt and Kofuji, 2009,
2010, 2011; Wong et al., 2007). These inputs influ-
ence the membrane potential of pRGCs by the
generation of cation- and chloride-based con-
ductances, respectively (Wong et al., 2007).
In darkness, inhibitory inputs dominate over
excitatory inputs and contribute to setting the
resting membrane potential of pRGCs below the
threshold for action potential firing (Wong et al.,
2007). However, the nature and influence of these
inputs varies between pRGC subtypes (Schmidt
and Kofuji, 2009, 2010, 2011) and would seem to
explain in part the differences in resting mem-
brane potential and excitability observed between
these cells. However, pRGC subtypes continue to
show differences in resting membrane potential
under conditions where all synaptic inputs are
blocked (Schmidt and Kofuji, 2009, 2010, 2011).
Under these conditions, M1-type cells remain sig-
nificantly more depolarized than M2-type cells
(and M3- and M4-type cells) and therefore sit
closer to the threshold for action potential firing.
These differences are seemingly the result of
intrinsic differences in levels of baseline ion-
channel activity in these cells. However, the
mechanisms which contribute to the intrinsic rest-
ing membrane potential of the pRGC subtypes
remain largely unexplored.
Gaps in the current model of melanopsin
phototransduction
With recent discoveries, our understanding of
the melanopsin signaling pathway has improved.
There is now good evidence that melanopsin
couples to Gnaq/11-type G-proteins, leading to
the activation of PLCb4 and ultimately the influx
of calcium through TRPC6 and TRPC7 channels.
These results confirm the assumption that
melanopsin uses an invertebrate-like signaling
cascade. The G-proteins involved in both pat-
hways belong to the same class of G-proteins
and the PLC isoform and downstream TRP
channels involved in melanopsin signaling are
the closest homologues of the respective com-
ponents in the Drosophila cascade. However,
we still only have a very basic model of the
melanopsin signaling pathway naive to the
pRGCs. The precise identity of the Ga subunit
(s) involved still remains unknown, as does
the identity and function of the Gb subunits.
The intermediary step(s) in the signaling path-
way, particularly those linking PLCb4 activation
and ion-channel opening, remain to be deter-
mined. In addition, based on our knowledge of
G-protein-signaling events, and also by more
specific comparison to the invertebrate signaling
cascade, it is clear that there are a number of
important gaps in the current model. Most nota-
ble is the lack of data concerning the regulation
and termination of the melanopsin signaling path-
way (Fig. 3).
Role of protein kinases: Desensitization,
adaptation, and termination of melanopsin
signaling
The most striking gap in our knowledge of the
melanopsin signaling pathway is the lack of any
description of protein kinase activity. Many clas-
ses of protein kinase exist, and these have wide-
spread functions and are key to regulating the
activity of second messengers, membrane-bound
proteins and ion channels and ultimately act to
fine tune the properties of cellular responses. Pro-
tein kinases may potentially exert an influence
over every stage of the melanopsin signaling path-
way. PKA and PKC represent two of the major
classes of protein kinases, and form core compo-
nents of cell signaling pathways that are typically
involved in the regulation of GPCR signal adap-
tation and desensitization, however little data
exists concerning the role of PKA or PKC in
melanopsin signaling.
PKC activation is driven by DAG in a process
requiring Ca2 þ, and is a key feature of Gq/11-type
 31

TRPC6/7 VOCC
4 5
1

Arr PIP2 PIP2

? Na
+
β γ
Gq/11 2+
GRK Ca
β γ ?
2
PLCb4 X
Gq
? IP3 + DAG
β γ 3
β γ
?
X
PKC
G11
?
β γ PLC AC PI3K ER Ca2+
? release PUFA Confirmed components and interactions
Interactions not involved
G14 PKA cAMP ? Predicted by analogy
?
β γ Common to Gq/11 signalling cascades
? ? PKCz ? Potential interactions

Scaffold proteins

Fig. 3. A potential melanopsin signaling pathway. Representation of a potential model of melanopsin phototransduction.
Components and interactions highlighted in solid blue represent confirmed components of the melanopsin signaling pathway in
pRGCs. Blue dashed lines indicate interactions or events that are known not to be involved. Items and interactions highlighted
in red represent components of the pathway that are missing or as yet unidentified, based on existing knowledge of G-protein
signaling events (i.e., arrestin and GRK activity) and also based on a direct comparison to the invertebrate signaling cascade
(i.e., involvement of PKC- and scaffold-type proteins). Items outlined in black represent signaling interactions known to occur
in classical Gq/11-type cascades and may therefore potentially perform a role in melanopsin signaling, but for which there
is currently no direct evidence. Dashed black lines represent potential interactions. Arr, arrestin; PLC, phospholipase C; P13K,
phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; ER, endoplasmic
reticulem, PKA, protein kinase A; PKC, protein kinase C; PKCz, protein kinase C zeta (atypical PKC); AC, adenylate cyclase; VOCC,
voltage-operated calcium channel.

signaling cascades. In invertebrate photoreceptors,
PKC activity influences response inactivation and
adaptation but not excitation of the light response
(Yau and Hardie, 2009). Interestingly, adaptation
and desensitization of pRGC responses have
also been reported. Adaptation is evident by
shortening of time to peak response in response
to increasing intensities of light (Do et al., 2009),
a reduction in amplitude of response following
repeated light stimulation (Wong et al., 2005),
and increase in incremental responses in the pres-
ence of steady background illumination (Wong
et al., 2005). However, the mechanisms, if any,
by which PKC regulate the desensitization and
adaptation of the melanopsin signaling pathway,
and the stage in the signaling pathway where
these regulatory mechanisms act are unknown.
The identification of TRPC6 and TRPC7 as the
light-sensitive channels in pRGCs will no doubt
accelerate our understanding of how PKC-based
32
signaling influences the activity of these channels
and modulates the nature of light responses in
pRGCs.
Activation of PKA is driven by increased
cAMP, which in turn is driven by adenylate
cyclase activity. cAMP is not normally associated
with the activation of Gq/11-type signaling pat-
hways, but instead is the classical mediator of
Gs- and Gi-type G-proteins. However, it is now
clear that various isoforms of adenylate cyclase
are activated by direct interactions with free Gb
subunits released from activated G-protein
complexes (see below), and thus a direct role
for cAMP and PKA in the melanopsin signaling
pathway cannot be excluded. However, it is
more likely that the cAMP–PKA pathway
may represent a mechanism of external regula-
tion. Dopamine-mediated activation of the
cAMP–PKA pathway, via the Gs-coupled Drd1a
receptor, leads to an inhibition of spike firing
in RGCs (Hayashida et al., 2009). Given the
known interactions between M1-type pRGCs
and dopaminergic amacrine cells (Vugler et al.,
2007; Zhang et al., 2008), dopamine and the
cAMP–PKA pathway may represent an impor-
tant mechanism for regulating pRGC function.
Interestingly, there is evidence that cAMP and
PKA activity modulate the properties of light
responses generated in Drosophila photo-
receptors (Chyb et al., 1999a; Hardie and Raghu,
2001), However, currently there is no direct evi-
dence for PKA-based modulation of melanopsin
signaling in pRGCs.
There is evidence for at least one class of pro-
tein kinase acting in the melanopsin signaling
pathway; PKCz. The potential role for PKCz
was first highlighted by microarray-based exper-
iments that investigated changes in gene tran-
scription within the eyes of mice lacking rod and
cone photoreceptors following acute light stimula-
tion. Remarkably, Prkcz/ mice were found to
precisely phenocopy Opn4/ mice. They show
attenuated circadian phase-shifting responses to
light, reduced period lengthening under constant
light, and attenuated pupillary responses at high
irradiances, as well as impaired light-induced
gene expression in the SCN. PKCz is an atypical
PKC and lacks binding domains for DAG and
Ca2 þ. The involvement of PKCz is therefore com-
patible with these second messengers exerting
little to no effect on melanopsin signaling. How-
ever, there is no direct evidence for a role of
PKCz in pRGC light responses and further stud-
ies are required to confirm the role of PKCz in
melanopsin phototransduction. By analogy to
the Drosophila phototransduction cascade, PKCz
may influence TRP ion-channel activity via
participation in an INAD-like signaling complex
that potentially also includes PLC, PKC, and
PDZ-domain-containing scaffolding proteins
(Hardie, 2001).
To date, research on melanopsin signaling has
focused on activation of the signaling pathway.
There are no data concerning the deactivation
or termination of the melanopsin signaling cas-
cade within pRGCs. G-protein-coupled receptor
kinases (GRKs) are a special class of protein
kinases (serine/threonine kinases) involved in
modulation (desensitization) and termination of
GPCR signaling events. GRK phosphorylation
prepares the activated receptor for arrestin bind-
ing, and ultimately termination of G-protein
signaling activity. Subtypes of GRKs and arrestins
are differentially expressed in rod and cone pho-
toreceptors, where they perform essential roles
in visual phototransduction and influence the
nature of light responses (Makino et al., 2003).
Arrestins are known to perform important roles
in shaping the properties of light responses
in Drosophila (Hardie, 2001), and overexpression
of arrestins (mouse b1 and b2 arrestin and
also Drosophila arrestin-2) has been shown to
enhance photocurrents recorded from Xenopus
oocytes expressing melanopsin (Panda et al.,
2005). To date, the functional roles of GRKs
and arrestin molecules in melanopsin photo-
transduction within pRGCs remain undeter-
mined. These molecules represent obvious
candidates as regulators of melanopsin signaling
and pRGC function.

Scaffold proteins
Patch clamp experiments from excised membrane
patches demonstrate that all the components
required to initiate light responses within pRGCs
are tightly bound to the plasma membrane.
However, the nature of the signaling complex pro-
tein present in pRGCs, and how scaffold-type
proteins aid the assembly and function of this com-
plex remains unclear. Based on analogy to the
invertebrate signaling cascade, we might expect
the involvement of an Inad-like scaffold protein
(Hardie and Raghu, 2001; Raghu, 2006), and
indeed previous microarray analysis has suggested
that the mammalian homologue INADL, and also
RACK-1 and NHERF-1 scaffold-type proteins
may be candidates for this role (Peirson et al.,
2007). Further work will be required to determine
the identity and role of the scaffold protein(s) that
participate in melanopsin phototransduction.
Gb subunits
Functional G-protein complexes contain Ga, Gb,
and Gg subunits. In addition to the activation of
Ga subunits, it is now clear that the Gb subunits
also perform functional roles, and represent a
mechanism for generating secondary signaling pat-
hways within G-protein-signaling cascades. Gb
subunits can influence directly the activity of ion
channels, certain isoforms of adenylate cyclase
and multiple classes and isoforms of PLC, includ-
ing PLCb1, PLCb 2, and PLCb 3 but not PLCb4
(Hubbard and Hepler, 2006; Mizuno and Itoh,
2009; Rhee, 2001). It is possible therefore that
Gb subunits may themselves participate in
melanopsin signaling, potentially activating sec-
ondary signaling pathways that are independent
of PLCb4 activity.
Five different Gb subunits have been identified,
Gb1–5, (and 12 Gg subunits) (Cabrera-Vera et al.,
2003) and evidence from transgenic mouse models
suggests that the different Gb subunits perform
specific physiological roles (Dupre et al., 2009).
33
Current evidence and data from our own gene
array studies (Hughes, S. & Butler, R., unpub-
lished data) indicate that all five Gb subunits are
expressed in the mouse retina. Significantly, these
different Gb subunits show distinct patterns of
expression within cell types of the retina. Gb1 is
expressed in rods and amacrine cells but not cones,
and Gb3 is expressed in all classes of cones, and a
subset of bipolar cells but is absent from rods (Lee
et al., 1992; Peng et al., 1992). Deletions and
mutations of Gb1 and Gb3 have been associated
with retinal degeneration (Kitamura et al., 2006;
Tummala et al., 2006). Expression of Gb5 is
restricted to ON bipolar cells and performs an
important role in bipolar light responses (Rao
et al., 2007) and the long isoform splice variant of
Gb5 (Gb5L) is restricted to rods where again it
contributes to the nature of light responses
(Krispel et al., 2003; Watson et al., 1996). Gb
subunits also contribute to light responses in Dro-
sophila photoreceptors, acting to regulate levels
of spontaneous firing (Elia et al., 2005). However,
the data concerning the localization of Gb subunits
in the retina is limited and currently there are no
data concerning the identity of the Gb subunits
expressed within pRGCs, nor is there any data
concerning the roles that these subunits perform
in melanopsin phototrans-duction.
Interestingly, recent studies have reported the
presence of a secondary “minor” signaling path-
way within M1-type pRGCs that is dependent
on melanopsin but independent of PLCb4 and
TRPC6/7 function. Small photoresponses persist
in M1-type pRGCs from both PLCb4/ and
TRPC6/7/ mice. These responses represent
< 1% of the wild-type response, but interestingly
are completely absent in OPN4/ mice (Xue
et al., 2011). The physiological relevance of this
minor response, if any, is unknown. It is also
unclear whether this minor pathway is functional
in other pRGC subtypes, where potentially it
may exert more influence. It is possible that free
G-protein beta gamma subunits may contribute
to this secondary pathway, yet further work will
be required to confirm this possibility.
34
Variable responses in pRGCs
In addition to the obvious gaps in our understand-
ing of pRGC phototransduction, it is also clear
that the existing model fails to account for all
experimental observations, most notably those
describing numerous distinct types of light
response from individual pRGCs. As discussed
earlier, multiple distinct response types have been
recorded from pRGCs. Multiple response profiles
are observed not only in adult tissue but also in
early postnatal development prior to the emer-
gence of rod and cone photoreceptors (Sekaran
et al., 2005; Tu et al., 2005). Further, distinct
response profiles are recorded from pRGCs fol-
lowing blockade of all synaptic inputs. Thus, it
would seem that the mechanism for generating
diversity is inherent to the pRGCs themselves,
and not dependent, at least not entirely, on the
differential inputs from the outer retina. How-
ever, the cellular mechanisms responsible for
generating this diversity are unknown. At pres-
ent, we have one (incomplete) signaling model
and as many as three distinct response types. Per-
haps, multiple models of melanopsin signal trans-
duction are needed to explain these observations,
or alternatively, we need to identify the cellular
mechanisms that allow melanopsin signaling
events to be modified in such a manner as to pro-
duce multiple response profiles. The obvious and
intriguing question is whether or not melanopsin
couples to distinct and specialized signaling pat-
hways within the different pRGC subtypes.
Specific components of the signaling pathway
have been identified for M1-type pRGCs, but it
is yet to be determined whether the same photo-
transduction pathway occurs in other pRGCs
subtypes. It is clear from recent evidence that dif-
ferent classes of pRGC express different profiles
of transcription factors (Chen et al., 2011), and
so it seems likely that different signaling compo-
nents may be available for participation in
melanopsin-dependent signaling pathways.
Levels of melanopsin expression and
differences in resting membrane potential may
account, at least in part, for the differences
in levels of photosensitivity observed between
pRGC subtypes (Schmidt and Kofuji, 2009).
However, different response profiles observed
between pRGC subtypes are not so easily
explained. The large fast-acting transient compo-
nent of the response observed from M1-type cells
is likely the result of adaptation or saturation of
some component(s) of the phototransduction
pathway in these cells (Warren et al., 2003; Wong
et al., 2005), but the mechanisms involved have
not been determined.
One potential explanation for the difference
in light responses recorded from pRGCs is the
differential expression of ion channels that act
downstream in the signaling pathway, but ulti-
mately act to shape the properties of the mem-
brane currents and changes in membrane
voltage. Interestingly, existing data suggest the
involvement of L-type VOCC channels in
melanopsin phototransduction, preceding the
initial TRP channel-mediated depolarization
(Hartwick et al., 2007). However, L-type VOCCs
(but not T-type VOCCs) are reportedly
expressed only in OFF-type RGCs and are absent
from ON-type RGCs (Margolis et al., 2010), and
therefore presumably absent from M2, M4, and
M5 pRGCs. This observation could explain, in
part, the differences in photoresponses observed
between these pRGC subtypes. Given the
differences in resting potential of pRGC subtypes
and their different spike-firing capabilities it
would seem likely that these cells express differ-
ent profiles of ion channels, or at least the func-
tioning of ion channels present in these cells are
differentially regulated.
Another, more intriguing possibility, is that the
variant responses observed for M1- and M2-type
cells are not entirely the result of differences in
downstream propagation of the melanopsin signal-
ing cascade, but instead are driven by differences
in the function of melanopsin itself, perhaps due to
the expression of different isoforms of melanopsin
(Pires et al., 2009) or due to functional modi-
fication of melanopsin proteins by glycosylation

(Fahrenkrug et al., 2009) and or phosphorylation
(Pires et al., 2009; Provencio et al., 1998, 2000).
The Opn4L and Opn4S isoforms of melanopsin dif-
fer only in their proximal C-terminal domain with
the extended C-terminus of Opn4L containing four
additional predicted PKC phosphorylation sites
compared to the shorter tail of Opn4S (Pires et al.,
2009). It seems likely that any functional differences
between these isoforms may reside in their differen-
tial sensitivity to PKC-based signaling, and this
may provide a mechanism to differentially regulate
melanopsin-based signaling within the pRGC
subtypes. Further work will be needed to address
these possibilities.
Conclusions
Since the discovery of melanopsin-expressing
pRGCs, there has been a rapid increase in our
understanding of the anatomical variety of pRGC
subtypes; the brain regions that they innervate;
and the behavioral responses they regulate.
A growing body of evidence suggests that the
pRGC subtypes are morphologically, anatomi-
cally, and functionally distinct with specific physi-
ological roles. Although the precise functions
performed by each cell type remain to be
fully determined. Our understanding of the
melanopsin signaling pathway has also seen prog-
ress, but it is only very recently that the precise
identity of the cellular components has begun to
emerge. Although there have been important
recent advances, there remain clear gaps in our
understanding of melanopsin phototransduction,
most notably the identity of the Ga subunit(s)
involved remains to be confirmed, as does the
mechanisms by which PLCb activity leads to gat-
ing of the downstream ion channels, seemingly
TRPC6 and TRPC7. It is also clear that the cur-
rent model fails to describe any aspects of the
regulatory pathways that undoubtedly exist, or
the mechanisms that regulate inactivation of
melanopsin activity. Further, the existing model
fails to account for all experimental observations,
35
most notably those describing the numerous dis-
tinct light responses observed from individual
pRGCs. Overall, it is likely that many factors
contribute to the different functional responses
observed from pRGC subtypes, including external
synaptic influences, differences in levels of
melanopsin expression, and differences in resting
membrane potential. It remains to be seen whether
differences also exist in the components of the sig-
naling cascades themselves, or whether differential
modulation of these components contributes to
the functional differences observed. Future work
must focus on the role of protein kinases in regula-
tion of melanopsin-driven signaling events, both
in response to external signals but also as part of
feedback mechanisms that are no doubt intrinsic to
the melanopsin signaling pathway.
The melanopsin signaling pathway is broadly
similar to that observed in invertebrate photo-
receptors. However, it is worth noting that
although there are clear similarities, the func-
tional properties of invertebrate photoreceptors
and pRGCs are markedly different. Invertebrate
photoreceptors are tasked with visual image pro-
cessing and show the fastest signaling event of
any known GPCR, with arrestins performing an
essential role in accelerating inactivation (Hardie,
2001). Such properties are not required of the
melanopsin signaling pathway within pRGCs
tasked with providing background luminance
information. It is possible that the signaling cas-
cade of these two classes of photoreceptors has
diverged significantly, as has their function.
In summary, it is clear that, despite recent
advances, our understanding of the melanopsin
signaling cascade remains a work in progress.
Microarray-based techniques have generated a
number of potentially important candidates
(Peirson et al., 2007), and the confirmation that
the melanopsin signaling cascade shows many
similarities to invertebrate signaling cascades will
surely accelerate the identification of other compo-
nents of this pathway based on analogy and func-
tional comparisons. However, validating these
candidates in the absence of robust pharmacological
36
tools remains a serious challenge. Although these
issues can be partially approached with transgenic
models and gene ablation, the study of this complex
system will surely benefit from the application of
conditional and inducible transgenic models in addi-
tion to use of techniques such as in vivo RNA
interference.
Acknowledgments
The authors would like to thank the EBRS for
the opportunity to contribute to this volume.
The author’s work is supported by the Wellcome
Trust and BBSRC.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 3

Circadian clocks: Lessons from fish

M. Laura Idda{, Cristiano Bertolucci{, Daniela Vallone{, Yoav Gothilf},

Francisco Javier Sánchez-Vázquez} and Nicholas S. Foulkes{,*

{
Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-von-Helmholtz-Platz,
Eggenstein-Leopoldshafen, Germany
{
Department of Biology and Evolution, University of Ferrara, Ferrara, Italy
}
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
}
Department of Physiology, Faculty of Biology, University of Murcia, Murcia, Spain

Abstract: Our understanding of the molecular and cellular organization of the circadian timing system
in vertebrates has increased enormously over the past decade. In large part, progress has been based on
genetic studies in the mouse as well as on fundamental similarities between vertebrate and Drosophila
clocks. The zebrafish was initially considered as a potentially attractive genetic model for identifying
vertebrate clock genes. However, instead, fish have ultimately proven to be valuable complementary
models for studying various aspects of clock biology. For example, many fish can shift from diurnal to
nocturnal activity implying specific flexibility in their clock function. We have learned much about the
function of light input pathways, and the ontogeny and function of the pineal organ, the fish central
pacemaker. Finally, blind cavefish have also provided new insight into the evolution of the circadian
clock under extreme environmental conditions.

Keywords: zebrafish; cavefish; genetics; clock mutants; peripheral clocks; pineal gland; clock ontogeny;
cell lines; blind clocks.

Introduction progress has been based on detailed studies of

Our understanding of the circadian clock, how
it is regulated by zeitgebers, and also how it in
turn regulates many physiological processes has
increased enormously over the past years. This
*Corresponding author.
Tel.: þ49-721-60823394; Fax: þ49-721-60823354
E-mail: nicholas.foulkes@kit.edu
the clock mechanism in a range of model
organisms amenable to detailed genetic analysis,
most notably Drosophila, cyanobacteria, Neuros-
pora, Arabidopsis, and the mouse. Further, in
the case of the vertebrate clock, the surprising
discovery of functional circadian clocks in com-
monly used cell lines enabled detailed biochemi-
cal analysis that has taught us much about how
core clock mechanisms work. However, a focus

http://dx.doi.org/10.1016/B978-0-444-59427-3.00003-4 41
42
on a limited number of model species inevitably
constrains the scope of biological problems that
can be tackled. With the advent of powerful tools
for molecular analysis (such as complete genome
or transcriptome sequencing) that can theoretically
be applied to any species, it is now easier to
explore aspects of clock biology, not necessarily
addressable in the classic genetic model organisms.
Teleosts represent one of the most successful
groups of vertebrates, showing diverse anatomical
and physiological adaptations to a wide range of
habitats. For this reason, studying these animals
provides a fascinating opportunity to explore the
plasticity of circadian clocks and how they adapt
to various environmental conditions during evolu-
tion. For example, in contrast to mammals, the
circadian system of fish shows impressive flexibil-
ity, as the same fish species can exhibit diurnal
or nocturnal behavior and shift from one type of
phasing behavior to another depending on the sea-
son or during ontogeny.
Detailed information on the molecular and
cellular basis of the clock in fish has stemmed
primarily from studies of the genetic model spe-
cies, the zebrafish. In this review, we will over-
view our current understanding of the regulation
and mechanism of the zebrafish circadian clock
and then describe how more recent work compar-
ing the zebrafish clock with that of blind cavefish
provides a powerful route to exploring clock
evolution.
Zebrafish: A genetic model species
A model for studying embryonic development
The zebrafish was originally developed as model
for studying the genetics of embryology and
developmental biology in vertebrates (Haffter
et al., 1996; Mullins et al., 1994). Several features
made it particularly attractive for exploring early
developmental processes. Firstly, it has transpar-
ent embryos that develop rapidly in a transparent
egg–shell outside of the mother. This basic prop-
erty, combined with the establishment of many
tools for generating stable and transient trans-
genic lines, has made the zebrafish a favorite
model for in vivo imaging of gene expression dur-
ing early embryogenesis (Nüsslein-Volhard and
Dahm, 2002). Another major advantage of the
zebrafish is its amenability to large-scale forward
genetic screens (Haffter et al., 1996; Mullins
et al., 1994). A single female zebrafish can nor-
mally produce hundreds of offspring each week.
Further, robust methods now exist for generating
large panels of mutagenized zebrafish. By screen-
ing these panels, the consequent identification
of specific mutants, and then the mapping of
mutated genetic loci, we have learned much
about basic developmental processes (Nüsslein-
Volhard and Dahm, 2002). The past few years
have seen the use of zebrafish expanding from
its traditional user base and being applied to
study diverse aspects of biology including behav-
ior and various aspects of physiology (Ingham,
2009; Petzold et al., 2009). Notably, the impres-
sive capacity of most zebrafish adult tissues to
regenerate following injury has attracted consid-
erable attention from research aimed at under-
standing and treating certain human diseases
such as heart and neurodegenerative diseases
and cancer (Brittijn et al., 2009; Ingham, 2009).
Further, the completion of the zebrafish genome
sequence has greatly facilitated the process of
mapping and identification of mutated loci and is
now enabling accurate analysis of transcriptomes
by next generation sequencing technology. Thus,
today the zebrafish has become an important com-
plementary model species alongside the mouse for
studying the molecular genetics of many aspects of
vertebrate biology and pathology.
Chronobiology and the zebrafish
The attention of chronobiologists originally
turned to zebrafish many years ago (Cahill,
1996, 2002) during the “dark ages” of our knowl-
edge of the molecular workings of the vertebrate
clock. No vertebrate circadian clock genes had

been cloned and so the molecular mechanism
of the circadian clock in vertebrates was a com-
plete mystery. At a stage, when large-scale
genetic analysis of mice for complex behavioral
phenotypes was prohibitive in terms of cost and
infrastructure, zebrafish offered the attractive
possibility to perform a low cost screen for clock
mutants and thereby a route to identify verte-
brate clock genes. Further, zebrafish also showed
great potential to trace the origin of the circadian
clock during embryogenesis. In an unexpected
turn, following the identification of the first clock
gene, clock, from the mouse and then the cloning
and characterization of the zebrafish homolog
of this gene, it soon became apparent that
the organization of the zebrafish circadian clock
mechanism and its regulation by light was fun-
damentally different from mammals. Specifically,
zebrafish peripheral clocks are directly entrainable
by light (Whitmore et al., 2000), actually closely
resembling the situation in Drosophila (Plautz
et al., 1997). This direct light-sensing property of
peripheral clocks was also encountered in cell lines
derived from zebrafish embryos such as Pac-2
cells (Whitmore et al., 2000). This observation
has raised many basic questions, most notably
which molecules serve as the photoreceptors in
peripheral tissues in fish?
Another element of the circadian timing system
in zebrafish that has received considerable att-
ention is the pineal organ. In nonmammalian
vertebrates, including zebrafish, the pineal con-
tains all the elements required for photic entrain-
ment and circadian rhythm generation: it is
photoreceptive and contains an intrinsic circadian
oscillator (Falcon et al., 2010; Korf et al., 1998). In
this regard, the pineal resembles peripheral tissue
clocks in zebrafish. However, importantly, the
pineal organ is considered to serve as the central
pacemaker in fish. Further, fish pineal cells are
specialized photoreceptor cells sharing various
structural and functional properties with retinal
photoreceptors. Indeed, pineal and retinal photo-
receptor cells express a similar set of genes, or,
in certain cases, paralogs. In the fish pineal, the
43
circadian clock drives rhythmic synthesis of the
hormone melatonin. Melatonin levels are high at
night and low during the day as a result of
regulated transcription and stability of serotonin-
N-acetyl-transferase (AANAT), a key enzyme
in melatonin synthesis. In common with other
teleosts, zebrafish possess two aanat genes:
aanat1, which is expressed predominantly in
the retina, and aanat2, which is expressed in the
pineal and at lower levels in the retina (Falcon
et al., 2003; Gothilf et al., 1999). Expression of
this enzyme is regulated by the circadian clock
and its activity is rapidly suppressed in response
to illumination during the night (Appelbaum
et al., 2006; Ziv et al., 2007). In addition to the
melatonin-producing photoreceptors, the pineal
contains neurons (Masai et al., 1997) that inner-
vate a variety of brain regions, a fact that has
received little attention until recently (Yáñez
et al., 2009). Thus, the pineal gland transduces
environmental light information into a neural
and a neuroendocrine signal. Many studies now
focus on identifying the control mechanisms
directing the first appearance of rhythmic melato-
nin synthesis during development, its regulation
by the clock and light as well as pineal-specific
patterns of gene expression.
Promoter studies using a transient transgenic
reporter assay as well as transfection of the Pac-2
cell line have identified a cluster of enhancer
elements lying downstream of the aanat2 gene
that is termed the pineal-restrictive downstream
module (Appelbaum et al., 2004). This includes
three photoreceptor conserved elements (PCEs)
and an E box. The E box confers clock regulation
by interacting with Clock:Bmal heterodimers (see
next section), while the PCEs are a target for the
homeobox gene, otx5, and thereby determine
pineal-specific expression (Appelbaum et al.,
2006). These factors appear to interact synergisti-
cally (Appelbaum et al., 2005) demonstrating how
the clock mechanism may interact with
other developmental and cellular processes. More
recently, bioinformatic analysis of the promoters
of pineal-specific genes that were identified
44
using DNA microarrays indicates that additional
mechanisms and enhancer sequences regulate
the expression of pineal-specific genes (Alon
et al., 2009).
Zebrafish and the vertebrate core clock
mechanism
Searching for new clock genes using zebrafish
The very first studies of circadian biology in the
zebrafish aimed at large-scale screens to identify
clock mutants. Initial attention focused on moni-
toring behavioral rhythms (locomotor activity)
that had been so successfully exploited as a circa-
dian clock assay in Drosophila and rodents. How-
ever, it soon became apparent that for isolated
adult fish, these rhythms were far from robust
and there was significant variability even among
populations of wild-type fish (Cahill, 2002;
Hurd et al., 1998). An alternative, far more reli-
able strategy was to monitor the movement of
zebrafish larvae in 24-well plates using an
automated video analysis system (Cahill et al.,
1998; Hurd et al., 1998). This assay was indeed
successfully used in a large-scale screen for clock
mutant zebrafish (DeBruyne et al., 2004). More
recently, studies of locomotor activity in zebrafish
have also taught us about key features of the
organization of the circadian timing system. Thus,
independent phasing between locomotor (mostly
diurnal) and feeding (nocturnal) rhythms was
found in zebrafish, providing evidence for the
flexibility of their circadian system and suggesting
it possesses an underlying multioscillatory control
(del Pozo et al., 2011).
An alternative clock output that was exten-
sively tested as a screening assay is the rhythmic
release of melatonin either from individual
embryos or from explanted adult pineal organs.
In developing embryos raised under a light–dark
(LD) cycle, melatonin production is first detected
around 24 h postfertilization (hpf), coinciding
with the expression of aanat (Gothilf et al., 1999;
Kazimi and Cahill, 1999). A flow-through culture
system was successfully used to accurately mea-
sure circadian rhythms of melatonin release from
individual adult zebrafish pineal organs (Cahill,
1996, 1997). Although not suitable for large-scale
analysis, this approach was used to validate
clock mutant phenotypes identified in locomotor
activity screens.
Finally, as in the case of many other genetic
models, the development of transgenic biolumi-
nescent reporter lines has provided a powerful
approach to screen for clock mutants. Specifically,
transgenic zebrafish lines were established where
expression of a luciferase (luc) reporter gene is
driven by the clock-regulated per3 gene promoter
(Kaneko and Cahill, 2005). Subsequently, when
luciferin is added to the fish water of these trans-
genics, a sufficient quantity diffuses into the
tissues and cells to permit the oxidation of lucif-
erin by the luc enzyme and so the generation of
bioluminescence that can be measured in a scintil-
lation counter- or camera-based assay. Impor-
tantly, bioluminescence can be measured from
larvae over a period of several days in small
volumes of medium without requiring the addi-
tion of extra luciferin (Kaneko and Cahill, 2005).
The only potential complication of this assay is
that while locomotor activity or melatonin pro-
duction specifically reflects central clock activity,
luc expression in a transgenic line will predomi-
nantly assay peripheral clock function.
Having identified a clock mutant phenotype, the
next stage is to pinpoint the location of the mutated
genetic locus. In zebrafish, these subsequent
“mapping” steps require crossing of the mutant
into another genetic background displaying signifi-
cant polymorphism relative to the strain where the
original mutagenesis was performed. Subsequent
segregation of the mutant phenotype with poly-
morphic, microsatellite markers is used as a way
to map the mutation to a particular subregion or
locus (Nüsslein-Volhard and Dahm, 2002). This
approach is time consuming but relatively straight-
forward in the case of easily scorable phenotypes,
for example, anatomical abnormalities in the early

embryo (Mullins et al., 1994). However, for long-
term behavioral assays and relatively subtle
mutant phenotypes that require many repeats to
achieve statistical confidence as for the identifica-
tion of circadian clock mutants, the whole mapping
process can be extremely laborious and may take
several years.
To date, one major forward genetic screen has
been performed where the equivalent of 6500
mutagenized zebrafish genomes were screened
for dominant mutations affecting circadian
rhythms of locomotor activity (DeBruyne et al.,
2004). Eight homozygous viable, semidominant
mutants were subsequently identified. In one
mutant that was identified by virtue of a
shortening of the free-running circadian period,
an isoleucine to asparagine mutation was
identified in the PAS domain of the clock gene
clock1a (see next section; Tan et al., 2008).
Another mutant termed lager and lime (lag(dg2))
was also identified by a shortening of the circadian
free-running period and by a reduction in tem-
perature compensation of the clock rhythm
(DeBruyne et al., 2004). Although the mutated
gene has yet to be characterized, it does not affect
any of the known clock genes (DeBruyne et al.,
2004). Thus, forward genetic analysis of the
zebrafish clock, although laborious, may yet prove
valuable for the identification of accessory
elements of the circadian clock mechanism.
Multiple clock genes in fish
Ultimately, following the cloning of the gene clock
in the mouse by Takahashi and colleagues (King
et al., 1997), rapid progress was made in the eluci-
dation of the core clock mechanism in vertebrates
without input from the zebrafish. It emerged that
at the core of the mammalian circadian clock is a
transcription–translation feedback loop that cycles
with a period of approximately 24 h (Albrecht,
2004; Reppert and Weaver, 2002). This regulatory
loop consists of positive elements (Clock and
Bmal) that drive the expression of negative
45
elements (Period (Per1, 2, and 3) and
Cryptochrome (Cry1 and 2)), which in turn feed-
back to downregulate their own expression and
so allow the start of a new cycle of the feedback
loop. The bHLH PAS domain transcription
activators Clock and Bmal bind as heterodimers
to E box elements, located in the promoters of
the per and cry genes and induce their transcrip-
tion. After translation, dimerization, and trans-
location to the nucleus, the Per and Cry proteins
physically interact with and thereby inhibit the
transcriptional activation driven by the Clock:
Bmal complex. Further, an additional feedback
loop directs the rhythmic expression of the bmal
transcript (Emery and Reppert, 2004). The pres-
ence of this additional loop together with the
tightly regulated stability, turn over, and subcellu-
lar localization of core clock components all con-
tribute to conferring accuracy and robustness on
the clock mechanism.
Thus, attention on the clock system in zebrafish
shifted from identifying elements of the core clock
machinery, to the characterization of zebrafish
homologs of known clock genes. A genome dupli-
cation event that occurred during the evolution of
the teleost lineage has lead to a situation where
there are extra clock gene copies in zebrafish com-
pared with the mouse (Postlethwait et al., 1998).
In some cases, duplicated gene copies have subse-
quently been lost during evolution; however, in
many cases, the extra copies persist. This may
reflect redundancy with functions being shared by
the extra genes. Alternatively, the duplicated genes
diverge in function and then multiple roles per-
formed by the original gene may be distributed
among the “more specialized” copies. Thus, in the
zebrafish clock, six cry genes have been identified:
cry1a, 1b, 2a, 2b, 3, and 4 (Kobayashi et al., 2000).
Cry1a, 1b, 2a, and 2b share most sequence homol-
ogy with mammalian mcry1 and also repress
Clock:Bmal directed transcriptional activation.
Interestingly, Cry3 and Cry4 do not repress Clock
and Bmal activation. While Cry3 does share
sequence similarity with the mammalian Crys,
Cry4 more closely resembles Drosophila Cry.
46
Given the role of Cry as a blue light photoreceptor
in Drosophila, this has led to speculation that
zebrafish Cry4 might also function as a photorecep-
tor (see later section). The temporal expression
patterns of the various cry genes vary significantly
(Kobayashi et al., 2000). Thus, cry3 peaks during
the morning, cry1a, 1b, and 4 show peaks of expres-
sion during the middle of the light phase, while
cry2a and 2b rhythms peak during the evening.
Cry1a shows a light-driven pattern of expression,
while the remaining cry genes are predominantly
clock regulated. Indeed, cry1a appears to represent
a key element of the mechanism underlying photic
entrainment and the maintenance of high ampli-
tude cycling (Tamai et al., 2007). These functions
result from the ability of Cry1a to interact with
key regions of the Clock and Bmal activators, thus
preventing their heterodimerization and hindering
their ability to transactivate from E box enhancer
elements (Tamai et al., 2007). With regard to the
positive elements of the clock mechanism, three
clock genes (clock 1a, 1b, and 2 formerly identified
as clock1, 2, and 3, respectively) and three bmal
genes (bmal1a, 1b, and 2, formerly identified as
bmal 1, 3, and 2, respectively) have been identified
and have been shown to interact pairwise in various
heterodimeric combinations (Hirayama et al., 2003;
Ishikawa et al., 2002; Wang, 2008b, 2009). These
heterodimers show different transactivation pro-
perties and are differentially inhibited by Crys such
as Cry1a. The expression pattern of the zebrafish
clock, and bmal family members also provides
indications of significant differences in the organi-
zation of the core transcription–translation feed-
back loop in zebrafish compared with mouse
(Whitmore et al., 1998). In most tissues, each of
these genes shows a robust mRNA expression
rhythm with a peak just after the LD transition
(Cermakian et al., 2000). This contrasts with the sit-
uation in mammals where bmal, but not clock,
shows rhythmic mRNA expression (Shearman
et al., 1999). Finally, extra per genes have also been
encountered in fish. Two per1 homologs (per1a and
1b) together with single per2 and per3 genes have
been cloned and characterized that exhibit different
spatial expression patterns (Wang, 2008a). Further,
while per2 is a light-driven gene, the remaining per
genes are predominantly clock regulated. Impor-
tantly, Per2 has been shown to function in combina-
tion with Cry1a as an element of the light input
pathway as well as playing an important role in
the early development of the pineal clock during
embryogenesis (Hirayama et al., 2003; Tamai
et al., 2007; Ziv and Gothilf, 2006; Ziv et al.,
2005). Thus, as in the case of the crys, combined
evidence seems to point to specialization of func-
tion for the various period genes.
Starting the clock during development
One fundamental issue addressed using the
zebrafish concerns when and how the clock is
established during development. Clock gene tran-
scripts are among the pool of maternally inherited
transcripts that are typically required for the very
first stages of embryonic development (Delaunay
et al., 2003); however, the functional significance
of these clock transcripts remains unclear. The
earliest detectable circadian rhythms in the devel-
oping embryo are rhythms of clock gene expres-
sion detectable in the entire embryo as well as
rhythmic melatonin synthesis and aanat2 expres-
sion in the pineal gland (Dekens and Whitmore,
2008; Gothilf et al., 1999; Kazimi and Cahill,
1999). It is also conceivable that clock genes
may perform non-clock-related functions at these
early developmental stages.
The establishment of circadian rhythms during
early development clearly requires exposure to
environmental zeitgebers such as light and tem-
perature changes. No circadian rhythms are
observed at the whole animal level in embryos
raised under constant temperature and constant
darkness (DD). Theoretically, this lack of rhyth-
micity could be explained by the presence of nor-
mal clocks in each cell, which are asynchronous
with respect to each other. Alternatively, this
could result from a lack of clock function even
at the single-cell level. One study has addressed

this issue directly by investigating the regulation of
per1b expression during early embryogenesis
(Dekens and Whitmore, 2008). In DD, oscillations
in per1b expression in single cells were documented
that were asynchronous in the embryo, indicating
that even in the absence of LD cycles, circadian
clocks do start ticking.
Light pulses delivered as early as the blastula
stage up to early segmentation stages (4–16 hpf),
before any classical photoreceptor organ appears,
are sufficient to set the phase of the central oscilla-
tor in the pineal gland 2–3 days later (Ziv and
Gothilf, 2006). Thus, clearly light can be detected
by nonspecialized photoreceptor cells and then
this photic information must be preserved through
the subsequent rapid cell proliferation and differ-
entiation. Interestingly, exposure to light only
following the so-called mid-blastula transition is
effective at setting the phase of these pineal organ
rhythms (Ziv and Gothilf, 2006). Given that this
represents the step where zygotic transcription is
initiated, this suggests that regulation of transcrip-
tion may well be a critical step in the light
response. Consistent with this prediction, light-
induced increases in the mRNA expression of
per2 and 6-4 DNA photolyase have been detected
as early as at the blastula and gastrula stages
(Tamai et al., 2004). Further, knockdown of per2
expression (by injection of one cell stage em-
bryos with morpholino-modified antisense oli-
gonucleotides that block either translation
initiation or correct splicing of the per2 transcript)
abolished the effects of early light exposure on the
pineal (Ziv and Gothilf, 2006). Given the key role
for the mouse per2 homolog in the entrainment of
the clock by light (Albrecht et al., 2001; Zheng
et al., 2001), this points to the light-induced tran-
scription of the zebrafish per2 gene being a critical
step in the maturation of the clock during embryo-
genesis (Ziv and Gothilf, 2006; Ziv et al., 2005).
Interestingly, while both clock and bmal genes
show rhythmic expression at the mRNA level in
adult tissues, this expression is absent in the early
embryonic stages. Rhythmic clock and bmal
mRNA expression as measured in entire embryo
47
RNA extracts first emerges around day 4/5 coinci-
dent with the appearance of other key clock out-
puts such as locomotor activity rhythms (Cahill
et al., 1998; Dekens and Whitmore, 2008; Dekens
et al., 2003). Thus, there would appear to be funda-
mental changes in the regulatory mechanisms
underlying the core clock mechanism during
development. Recent research in fish species with
a longer incubation period further supports the
concept that clock systems in fish are present, light
responsive and fully functional during embryonic
development; thus continuous illumination
abolishes rhythmic expression of clock genes in
these species (Davie et al., 2011).
Light-entrainable peripheral clocks
The cloning of the first mouse clock gene clock by
Joe Takahashi’s group was a milestone for the cir-
cadian clock field (King et al., 1997). For the
zebrafish, this was also an important step since
the zebrafish homolog of clock became the first
tool for studying the general molecular organiza-
tion of the circadian timing system (Whitmore
et al., 1998). These studies lead to the conclusion
that as in the mouse, cycling clock gene expression
was a property of most organs and tissues. Further,
these rhythms persisted in organs that were
explanted into primary cultures showing that they
were not “driven” by systemic cues—but were
actually generated by self-sustaining, independent
“peripheral” circadian oscillators. This conclusion
was entirely consistent with the first results from
the mouse and also Drosophila pointing to the
existence of peripheral clocks (Plautz et al., 1997;
Schibler and Sassone-Corsi, 2002). The big sur-
prise came when in a subsequent study, these
organ cultures were exposed to various LD cycles
(Whitmore et al., 2000). Remarkably, the periph-
eral clocks were sustained and entrained by
the LD cycle, leading to the conclusion that in this
vertebrate, as in the case of the fruit fly, tissues
such as the heart and kidney were directly photo-
sensitive. Later, directly light-entrainable clocks
48
were even observed in zebrafish cell lines
(Whitmore et al., 2000). Thus, instead of acute
changes in culture medium conditions such as
serum shocks (Balsalobre et al., 1998), clocks
could be entrained in the zebrafish cell cultures
simply and noninvasively by exposure to light
(Vallone et al., 2004). Rhythms that have been
established in zebrafish cell cultures subsequently
slowly dampen when the cells are transferred to
DD. Using single-cell imaging of bioluminescent
reporter cell lines, this dampening has been shown
to result from a progressive desynchronization of
inaccurate single-cell clocks (Carr and Whitmore,
2005). After extended periods in DD, the cells dis-
play widely distributed phases and also marked
stochastic fluctuations in free-running period.
Light exposure serves not only to reset the phase
and thereby synchronize the single-cell oscillators
but also to stabilize the free-running period (Carr
and Whitmore, 2005). The mechanism whereby
deprivation of light results in increasingly stochas-
tic clock properties remains unclear. In this regard,
evidence obtained from a study of per3::luc trans-
genic fish has revealed significant tissue-specific
differences in peripheral clock properties (Kaneko
et al., 2006). Clock parameters such as free-
running period length, phase, light response, and
temperature compensation all vary from tissue to
tissue, possibly reflecting differential expression
patterns of members of the various clock gene
families.
All these findings raise fundamental questions:
What are the widely expressed photoreceptors,
and how do they signal to the clock? Do central
pacemakers in the fish coordinate the peripheral
clocks as they do in mammals, and if so, what is
the underlying mechanism?
The identity of the peripheral photoreceptors
remains unclear. Three possible candidates have
been proposed: (1) Extra retinal opsins. In teleosts
and other nonmammalian vertebrates, a group
of opsin-like genes are expressed widely outside
the retina. For example, teleost multiple tissue
(TMT) opsin mRNA is detected in the central ner-
vous system, most peripheral tissues and even in
zebrafish cell lines (Moutsaki et al., 2003).
Recently, functional analysis has shown that
TMT opsin serves as a photoreceptor (Cavallari
et al., 2011). Also exorhodopsin appears to act as
a photoreceptor-regulating gene expression in
the pineal organ and in turn affects the production
of melatonin (Pierce et al., 2008). (2) Crys. The
argument for Crys being involved is based on sev-
eral lines of evidence: the role played by Cry in
photoreception of lateral neurons in Drosophila
(Ceriani et al., 1999), and the fact that one of the
zebrafish cry gene homologs (cry4) shares closer
sequence similarity with drosophila cry than with
the mammalian homologs (Kobayashi et al.,
2000). Moreover, blue light wavelengths via
retinol-independent photopigments were shown
to upregulate per2 mRNA expression in cell
culture (Cermakian et al., 2002). (3) A third model
implicates the production of hydrogen peroxide by
a phototransducing flavin-containing oxidase. This
hypothesis stems from the observation that light
triggers increases in intracellular levels of hydro-
gen peroxide in the zebrafish Z3 cell line
(Hirayama et al., 2007). It has been proposed that
light-activated enzymes such as flavin-containing
oxidases may trigger accumulation of H2O2 in
the Z3 cells upon exposure to near violet–blue
wavelengths of light. This then results in activation
of the MAPK signaling pathway that in turn drives
light-dependent activation of genes such as cry1a
and per2. Light was also shown to induce catalase
expression that is then predicted to serve to
downregulate the light response (Hirayama et al.,
2007). Of course, a fourth possibility is the involve-
ment of not one but a combination of separate
photoreceptor systems.
Light-inducible clock gene expression
How does the light-activated photoreceptor(s)
signal in the cell? Several reports have
documented light-dependent activation of the
MAPK signaling pathway. Transient increases in
pERK and pMEK levels have been documented

within 30 min of exposing zebrafish cell lines to
light (Cermakian et al., 2002; Hirayama et al., 2007,
2009). This result resembles the situation in the
mammalian suprachiasmatic nucleus (SCN) where
photic signals indirectly relayed by the retino-hypo-
thalamic tract trigger NMDA receptor activation
and thereby activate the MAPK pathway in SCN
neurons. In turn, phosphorylation of the trans-
criptional activator, CREB (cAMP-responsive
element binding protein), as well as chromatin
remodeling events have been proposed to repre-
sent key steps in the light-induced activation of
the per1 and 2 genes and then the subsequent
resetting of the phase of the mouse clock
(Travnickova-Bendova et al., 2002). A key obser-
vation that served as a starting point for the eluci-
dation of the light-sensing mechanisms in
zebrafish tissues has been that light exposure
directly induces expression of certain clock genes
(per2 and cry1a) even in fish cell lines.
Interestingly, one of the sites showing the
strongest induction of per2 is the pineal organ.
Further, it is now clear that light exposure not
only affects the expression of clock genes. A sys-
tematic microarray analysis has been performed
to assess the effects of light exposure on the
transcriptome of zebrafish cells and tissues; spe-
cifically cell lines, cultured adult zebrafish hearts,
and 5-days-old larvae. The results point to a
group of around 100 genes whose expression is
significantly upregulated upon light exposure.
These genes belong to diverse functional groups
including notably genes involved in repairing
DNA damage induced by exposure to the ultravi-
olet (UV) wavelengths of sunlight. Comparison of
the predicted promoter regions of these light-
induced genes revealed an enrichment of the
so-called D box enhancer elements. The impor-
tance of these enhancer elements in light-induced
gene expression has been confirmed by promoter
analysis of the per2 gene. A critical light-responsive
module within this promoter contains a D box as
well as an E box whose integrity is critical
for light-induced gene expression. This region
is also highly conserved in the per2 gene
49
promoters from other vertebrates, where interest-
ingly light exposure does not directly affect clock
function. This suggests a key, ancestral role for
D box enhancers in clock gene regulation. In
mammals, four transcription factors have shown
to directly interact with and regulate gene expres-
sion from the D box, with three of these function-
ing as activators (TEF, thyrotroph embryonic
factor; DBP, albumin D box-binding protein;
and HLF, hepatic leukemia factor) and a fourth,
E4BP4, that serves as a repressor (see Fig. 1).
These factors have been studied extensively due
to their role in clock output pathways in the
mouse (Gachon, 2007; Gachon et al., 2006;
Lavery et al., 1999). In contrast, partly as the
result of the teleost genome duplication events,
in zebrafish 12 different factors with close homol-
ogy to the mammalian D box regulatory factors
have been cloned and characterized (Ben-Moshe
et al., 2010) (Fig. 1). Interestingly, these factors
frequently show clock and light regulation. Fur-
ther, in situ hybridization analysis has revealed
very different tissue specific and temporal expres-
sion patterns, frequently with highest expression
levels in the pineal organ (Ben-Moshe et al.,
2010). Morpholino knockdown of one of the acti-
vator proteins, TEF1, has been shown to severely
attenuate light-induced per2 expression in the
pineal, pointing to a key role for this factor in
the pineal organ light transduction pathway
(Vatine et al., 2009). Clearly, an important future
goal will be to assess the relative contribution of
the various D box-binding factors to the light
response (Fig. 2).
Blind cavefish reveal circadian clock
photoreceptors
Unexpected genetic evidence for the identity of
the peripheral photoreceptors has come from
tackling a fundamental question concerning circa-
dian clock evolution. What happens when animals
evolve completely isolated from the day–night
cycle? Does the clock persist? If so, is it still
50

tef, hlf, dbp

E4BP4

ztef ztef2 zhlf1 zhlf2 zdbp1 zdbp2

P F P P
P P H P
E H
E R
E

ze4bp4-1 ze4bp4-2 ze4bp4-5 ze4bp4-6

F P P P
R
R R

Fig. 1. Zebrafish D box-binding factors. Upper panel: schematic representation of the general structure of the activator (tef, hlf, and
dbp) PAR bZip transcription factors and the repressor E4BP4 factors. Red box represents the PAR domain; a purple box, the basic
region; and a blue box denotes the leucine zipper domain. Lower panel: in situ hybridization of zebrafish embryos with the various
D box-binding factor cDNAs. A clock or light symbol above each panel indicates whether the gene’s expression is predominantly
clock regulated or light driven, respectively. The identity of key anatomical structures within the embryo is indicated by letters.
P ¼ pineal gland, E ¼ eye, R ¼ retina, F ¼ forebrain, H ¼ hypothalamus.

entrained by light? Fish represent fascinating
models to address these questions. Around 100 spe-
cies of fish worldwide inhabit subterranean caves
where they are isolated from daylight and have
experienced constant conditions in some cases
for millions of years. These cavefish species
exhibit a set of striking convergent “troglomorphic”
phenotypes including notably eye loss as well as loss
of pigment and lowering of metabolic rate. Do
cavefish still retain clocks? This question has been
recently explored by comparing zebrafish with a
species of blind cavefish at the behavioral, cellular,
and molecular levels (Cavallari et al., 2011). The
Somalian cavefish, Phreatichthys andruzzii, was
chosen due to the severity of its troglomorphic
phenotype, the consequence of complete isolation
from sunlight beneath the Somalian desert for a
predicted 2 million years.
In the case of the zebrafish under LD cycles, a
robust diurnal pattern of locomotor activity as well
as rhythmic clock gene expression is observed. In
contrast, exposure of the blind cavefish to LD
cycles resulted in arrhythmicity in terms of both
locomotor activity and clock gene expression
(Fig. 3). Likewise, a cell line derived from adult
cavefish fins and exposed to LD cycles failed to
exhibit the cycling clock gene expression exten-
sively documented in zebrafish cell lines under
identical conditions. These findings could have
two alternative explanations. Either the cavefish
possess a blind circadian clock, unable to respond
to light, or alternatively, they may have completely
lost circadian clock function. To distinguish
between these two possibilities, the effects of
exposing the cavefish to an alternative zeitgeber
were tested. A regular feeding time has been well
 51

Fig. 2. Schematic representation of the light input pathway in zebrafish cells. Light is perceived by opsins at the cell membrane and
relayed to the nucleus by various signal transduction pathways. Here, the light signal regulates the activity of a large family of D
box-binding factors that function as activators and repressors and can bind to the D box enhancer in various homo- and
heterodimeric combinations. D box-regulated genes include key clock genes such as per2 as well as other classes of gene
including those involved in the repair of UV-damaged DNA. The activation of a subset of the genes encoding the D box-binding
factors by light (TEF and E4BP4-6) serves as an additional regulatory loop that may contribute to defining the kinetics of light-
induced gene expression.

documented to entrain the circadian clock in many
animals, including zebrafish, in which feeding
cycles entrain clock gene expression in the liver,
but not in the brain (Lopez-Olmeda et al., 2010).
Thus, fish were fed regularly (once each 24 h) for
a period of 1–2 months followed by a period of
starvation. This led to increases in locomotor activ-
ity that preceded the time of feeding, so-called
food anticipatory activity (FAA) (see Fig. 3). Fur-
ther, rhythmic expression of clock genes was
established in the brain, heart, and liver that
persisted even during the subsequent starvation
period. In the case of the cavefish cell cultures,
cells were transiently treated with dexamethasone,
a treatment widely used with mammalian cell lines
to synchronize single-cell oscillators. This treat-
ment led to rhythmic expression of a clock-
regulated reporter gene, which surprisingly
exhibited a 47-h free-running period. Further,
changes in temperature led to alterations in the
length of the free-running period, suggesting a
reduction in the temperature compensation of
 (a) (b)

(c) (d)

Days
10

10

15

15

0 12 0 12 0 12 0 12
ZT ZT

(e) Feeding time

(f) Feeding time

5
5

10
10
Days

15
15

20
20

25
25

30
30
12 0 12 0 12 12 0 12 0 12
ZT ZT

Fig. 3. The pattern of zebrafish (Danio rerio, a) and cavefish (Phreatichthys andruzzii, b) locomotor activity during exposure to LD
cycles (c and d, respectively) and periods when food was delivered regularly at the same time each 24 h (e and f, respectively).
Zebrafish exhibit a robust diurnal pattern of activity under LD conditions and show increased activity just prior to the time of
feeding (so-called food anticipatory activity, FAA). In contrast, cavefish activity is arrhythmic under LD cycles, but like the
zebrafish, blind cavefish do show characteristic FAA. The number of days is indicated on the y-axis, while the time is plotted on
the x-axis as zeitgeber time (ZT) where ZT0 is defined either as “lights-on” or as feeding time.

the clock mechanism. Thus it would appear that
this species of cavefish does retain a clock, albeit
an abnormal one that is no longer entrained by
light but can still be entrained by feeding time
(Cavallari et al., 2011).
The molecular basis of the blindness of the
cavefish peripheral clocks was tracked down
by the cloning and characterization of the
melanopsin and TMT opsin gene orthologs. Both
opsins are widely expressed in fish tissues and
encode seven-transmembrane domain proteins
with the seventh membrane-spanning segment
being covalently linked with a chromophore, ret-
inaldehyde. In the cavefish, both opsin genes car-
ried mutations that lead to truncation of the
proteins between the fifth and sixth membrane-
spanning segments. The truncated protein would
be predicted not to bind to the chromophore
and therefore not to serve as a functional photo-
pigment. To test whether the mutated opsins
could be responsible for the blind peripheral
clock phenotype, cavefish cells were transfected
with expression vectors encoding the wild-type
zebrafish versions of these opsins. Light-induced
expression of a per2 promoter-luc reporter gene
was restored in these cells, a result consistent
with the opsin gene mutations contributing to
the clock blindness. Interestingly, however,
the cavefish data also point to the existence of
additional mutations affecting other photopig-
ments (Cavallari et al., 2011). While TMT and
melanopsin both react to blue and green light
wavelengths, zebrafish cells respond to a much
broader range of wavelengths extending to red
wavelengths. Thus, this predicts the existence of
additional photoreceptors that have also
accumulated mutations in the cavefish (see
Fig. 2). The loss of a functional light input path-
way but the retention of a food-entrainable oscil-
lator make this fish an extremely attractive model
for studying how feeding regulates the clock.
The ability of this blind cavefish species to
survive long periods of fasting adds to its suitabil-
ity for studying this process of feeding
53
entrainment. One unanswered question remains:
Which mutations are responsible for the
extremely long free-running period of this fish?
So far, no mutations have been encountered in
the core clock genes. This would tend to impli-
cate the existence of mutations in other regu-
latory elements that may influence the stability
of clock proteins or their subcellular location.
Thus, the cloning and characterization of the
mutations responsible for this aberrant cavefish
clock promise to teach us much about the
elements of the core circadian clock. Further,
the discovery of this aberrant clock mechanism
in a species of blind cavefish represents an impor-
tant step in studying evolution under extreme
environmental conditions. It raises many basic
questions: Does the clock phenotype confer some
selective advantage for the fish in their subterra-
nean cave environment? Alternatively, does this
reflect the absence of positive selective pressure
to maintain a normal clock and so are we wit-
nessing gradual loss of a mechanism that confers
no selective advantage? Comparative studies of
other species of cavefish that have evolved inde-
pendently in different hypogean biotopes will
hopefully provide some answers to these
questions.
Concluding remarks
Teleosts represent the largest and one of the most
remarkable groups of vertebrates with unique cha-
racteristics for chronobiological studies. With over
30,000 known species, they exhibit a considerable
degree of anatomical and physiological plasticity
that has enabled them to occupy a wide diversity
of habitats and ecological niches. In the highly
dynamic aquatic environment, one can predict that
during teleost evolution the circadian timing system
has been subjected to a range of different selective
pressures depending on a diversity of lighting and
temperatures as well as the flexible patterns of
behavior adopted. Thus, the initial studies in the
54

zebrafish as well as teaching us much about TMT teleost multiple tissue

key aspects of the vertebrate timing system also UV ultraviolet
promise to serve as a foundation for broader, com- ZT zeitgeber time
parative studies involving diverse species such as
the blind cavefish. These future studies should
tackle basic questions concerning the evolution of
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 4

Two clocks in the brain: An update of the morning

and evening oscillator model in Drosophila

Taishi Yoshii{, Dirk Rieger{ and Charlotte Helfrich-Förster{,*

{
Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
{
Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany

Abstract: Circadian clocks play an essential role in adapting the activity rhythms of animals to the day–night
cycles on earth throughout the four seasons. In many animals, including the fruit fly Drosophila melanogaster,
two separate but mutually coupled clocks in the brain —morning (M) and evening (E) oscillators— control
the activity in the morning and evening. M and E oscillators are thought to track dawn and dusk,
respectively. This alters the phase-angle between the two oscillators under different day lengths, optimally
adapting the animal’s activity pattern to colder short and warmer long days. Using excellent genetic tools,
Drosophila researchers have addressed the neural basis of the two oscillators and could partially track
these to distinct clock cells in the brain. Nevertheless, not all data are consistent with each other and many
questions remained open. So far, most studies about M and E oscillators focused on the influence of light
(photoperiod). Here, we will review the effects of light and temperature on the two oscillators, will update
the present knowledge, discuss the limitations of the model, and raise questions that have to be addressed
in the future.

Keywords: circadian clock; seasonal adaptation; dual oscillator model; temperature; light; Drosophila
melanogaster; clock neurons.

Introduction organisms living on earth have obtained time-

The rotation of the earth causes cyclical alterations
in the environment of which light and temperature
oscillations seem to be the most important. The
*Corresponding author.
Tel.: þ49-931-318-8823; Fax: þ49-931-318-4452
E-mail: charlotte.foerster@biozentrum.uni-wuerzburg.de
measuring structures in the brain, which help them
to predict the regularly occurring environmental
cycles and thus maximize fitness. Circadian clocks
and circannual clocks evolved to anticipate daily
and yearly environmental changes, respectively.
Both clocks may be linked, as circadian clocks
can provide the necessary reference for measuring
day length (Bünning, 1936). Changes in day length

http://dx.doi.org/10.1016/B978-0-444-59427-3.00027-7 59
60
together with annual temperature cycles (T-cycles)
synchronize circannual clocks to the yearly cycle,
though it is still under debate whether circadian
clocks participate in this synchronization in all
organisms (for a review, see Bradshaw and
Holzapfel, 2010). In order to survive in the seasonal
environmental changes that affect organisms,
animals have to anticipate the coming winter by pre-
paring themselves in time for hibernation or dia-
pause. In addition, seasonal changes have a strong
impact on the animal’s daily activity pattern. Espe-
cially in poikilothermic insects, the entire physiol-
ogy (including the activity) depends highly on the
ambient temperature. Under short cold days in
spring and autumn, it is favorable for them to be
active during the warmer parts of the day, whereas
it is better to shift activity to the morning, the late
evening, or even the night under long, hot summer
days (Fig. 1). There are two mechanisms that help
insects to be active at the best time of the day:
(1) They respond quickly to the acute ambient tem-
perature and (2) their circadian clocks determine
the daily activity pattern under spring, summer, or
autumn conditions. The latter mechanism implies
that the clock entrains differently (has different
waveforms) at different day lengths. Indeed, this
was observed in nature, even in vertebrates
(a) Longer yM,E
Hot day Comfortable
(b) Longer yM,E
long day LD 12:12
Fig. 1. Temperature and photoperiod influence the phases of M and E activity peaks. (A) Under hot days (left), the two peaks shift
in opposite directions giving rise to a wide trough, allowing the flies to rest during the heat of midday. In contrast, the two peaks are
close together under cold days, allowing the fly to be active during the warmer daytime. (B) Similar effects can also be seen under
long and short photoperiods.
(Aschoff, 1966): birds, fishes, and several mammals
show two activity bouts, one in the morning (M)
and one in the evening (E). M and E activity bouts
are close together under short spring days and
separated by a pronounced siesta under long sum-
mer days (Aschoff, 1966; see Fig. 1b). These two
activity bouts persist even under constant light
(LL) and temperature conditions in the lab,
indicating that they are endogenous. The effects of
temperature on the two activity bouts have not been
systematically investigated in vertebrates, but the
effects of light have been studied extensively. In
nature, light and temperature are closely linked:
more visible light (e.g., on long summer days) is
accompanied by more infrared light, resulting in
higher temperatures. Thus in the lab, the animal’s
circadian clock may predict a warm summer day
from the amount of light perceived even if no heat
is produced. Indeed, under constant conditions in
the lab, M and E activity bouts of finches shift apart
with increasing light intensity (Aschoff, 1966). The
same happens in Syrian hamsters and other noctur-
nal rodents (reviewed by Schwartz and de la Iglesia,
2003). At a certain light intensity, the two compo-
nents free-run with different periods until they
become stably coupled 180 apart, a phenomenon
called “splitting.”
Shorter yM,E
Cold day
Shorter yM,E
Short day

The dual oscillator model
Based on the hamster’s “split rhythms” in LL,
Pittendrigh and Daan (1976) proposed that the
clock consists of two separate oscillators with dif-
ferent responses to light: one oscillator accelerates
and the other decelerates upon light exposure.
Under light–dark (LD) cycles, the first oscillator
tracks dawn and is therefore called morning (M)
oscillator and the second tracks dusk and is called
evening (E) oscillator. Due to the different pro-
perties of the M and E oscillators, the two activity
bouts in the morning and evening are close
together under short days and far apart under long
days. Thus, the dual oscillator model can explain
that M and E activity bouts change their phase-
angle (CM,E), leading to different waveforms of
the activity rhythm at different photoperiods.
For many years, the dual oscillator model
remained gray theory, very useful to explain
behavioral phenomena but without having any
mechanistic background. Only in the past few years
has it become clear that the two oscillators have a
neuronal basis. This view was propelled by studies
performed in the fruit fly Drosophila melanogaster
(Grima et al., 2004; Rieger et al., 2006; Stoleru
et al., 2004; see below). In mammals, Jagota et al.
(2000) demonstrated first that the mammalian
central pacemaker, the suprachiasmatic nucleus
(SCN), can exhibit two separate peaks in electrical
activity that change C in response to photoperiod.
Consecutive studies at the molecular and electro-
physiological level unveiled that there are two or
more types of cell populations in the SCN that
respond differently to short and long photoperiods
(e.g., Brown and Piggins, 2009; Burgoon et al.,
2004; Hazlerigg et al., 2005; Inagaki et al., 2007;
Naito et al., 2008; Sosniyenko et al., 2009; reviewed
in Meijer et al., 2010). Nevertheless, it remained
difficult to trace the different oscillators to specific
clock neurons in the SCN and to reveal the
mechanisms by which they follow dawn and
dusk. One reason for this difficulty is the complex-
ity of the SCN that consists of more than 10,000
neurons. In addition, it remains hard to genetically
61
manipulate specific regions of the SCN in order
to investigate the function of distinct neuronal
populations under different photoperiods.
All this is easier to achieve in the fruit fly
D. melanogaster. While the basic neuronal func-
tion of endogenous clocks appears well con-
served between flies and mammals, the entire
brain clock of Drosophila consists of only  150
clock neurons, and there are excellent genetic
tools by which specific clock neurons can be
manipulated. Even at the behavioral level, the
activity rhythms of fruit flies resemble in many
aspects those of vertebrates: Fruit flies show
bimodal activity patterns with M and E peaks,
and these persist to some extent under constant
conditions (Helfrich-Förster, 2000). Under short
and long photoperiods, CM,E becomes smaller
or larger, respectively (Rieger et al., 2003;
2012). The effects on CM,E are enhanced when
short or long photoperiods are combined with
low or high constant temperatures, respectively
(Majercak et al., 1999). Even temperature alone
(under constant dark (DD) conditions) affects
CM,E in the same direction: CM,E is larger under
high temperatures than under low temperatures
(Majercak et al., 1999). LL alone is more diffi-
cult to test in Drosophila, because the blue-light
photopigment cryptochrome (CRY) renders flies
arrhythmic already at very low light intensities
(Emery et al., 2000a). But, in the absence of
CRY, CM,E becomes larger with increasing light
intensity and finally rhythm splitting and internal
desynchronization into two free-running compo-
nents occurs—very similar to the hamsters’ behav-
ior in LL (Helfrich-Förster et al., 2001; Yoshii
et al., 2004). Higher light intensities even increase
CM,E under 12:12 LD-cycles (Rieger et al., 2007),
indicating that the flies interpret a day with high
light intensity as a hot day.
After M and E oscillators were anatomically
traced to specific clock neurons in the Drosophila
brain by the groups of Francois Rouyer and
Michael Rosbash (Grima et al., 2004; Stoleru
et al., 2004; see below), many papers appeared
that have modified and refined the original dual
62
oscillator model whereby the results of these stud-
ies have been controversially reviewed (Dubruille
and Emery, 2008; Helfrich-Förster, 2006, 2009;
Helfrich-Förster et al., 2007b; Sheeba et al.,
2008b). In the meantime, more papers appeared
that challenged the original dual oscillator model,
and it seems high time to write an elaborate
update. We will do so, discuss new aspects as well
as the limitations of the model and put special
emphasis on the effects of natural-like T-cycles
on M and E oscillators.
The clock network in the Drosophila brain
and the possibility to manipulate selected
clock neurons
Drosophila’s 150 clock neurons are located in
the lateral and dorsal brain and are thus called
lateral and dorsal neurons (LNs and DNs). The
LNs and DNs are further divided into different
subclusters that are depicted in Fig. 2. All clock
neurons form a neuronal network that has been
described in detail and been shown in several
reviews (a still valid detailed schematic presenta-
tion of the clock network can be found in
Helfrich-Förster et al., 2007a). Here, we just
focus on the neurochemistry of the different
clock neurons that helped to manipulate them
selectively. All clock neurons express the core
clock proteins Period (PER), Timeless (TIM),
Clock (CLK), and Cycle (CYC) (Houl et al.,
2006; Kaneko and Hall, 2000). However, the
blue-light photopigment CRY that works as “circa-
dian photoreceptor” is not uniformly present in all
clock neurons (Fig. 2a; Benito et al., 2008; Yoshii
et al., 2008). A further divergence of the clock
neurons becomes evident when one regards their
neurotransmitters (neuropeptides) (Fig. 2b). The
neuropeptide pigment-dispersing factor (PDF)
was the first neurotransmitter found in eight LNs
per brain hemisphere (Fig. 2b; Helfrich-Förster,
1995). PDF and the PDF-containing LNs later
turned out to be quite important for the function
of the circadian clock under constant darkness
(Helfrich-Förster et al., 2000; Renn et al., 1999).
Other neurotransmitters followed (see Fig. 2b)
and their role in the clock is currently investigated.
Knowing the genes expressed in the clock
neurons is the first prerequisite to genetically manip-
ulate the neurons with the gal4-uas binary expres-
sion system of the yeast Saccharomyces cerevisiae
(Brand and Perrimon, 1993). GAL4 is a transcrip-
tional activator that binds to the upstream activating
sequence (uas) of other genes, thus activating their
transcription. As flies do not have the gal4 and uas
sequences in their genome, these can be used to
express genes of interest in specific fly cells (tissues).
All one has to do is to fuse (1) the promoter of inter-
est (e.g., that of the Pdf gene; Park and Hall, 1998)
with the gal4 gene and (2) the gene that one wants
to express (e.g., the apoptotic gene head involution
defect (hid); McNabb et al., 1997) with the uas
sequence, and (3) to insert both constructs into the
fly genome (reviewed in Duffy, 2002). Flies con-
taining both constructs will express hid in the Pdf-
expressing cells, and as a consequence, the PDF
neurons will die early in development. The gal4-
uas system enables not only to kill specific neurons
but also to manipulate them in many different ways,
for example, they can be electrically silenced by
expressing Kþ channels (Nitabach et al., 2002), they
can be overexcited by expressing Naþ channels
(Nitabach et al., 2006), neurotransmitter release
can be blocked by expressing tetanus toxin (e.g.,
Blanchardon et al., 2001; Kaneko et al., 2000), and
known genes (e.g., per) can be overexpressed (e.g.,
Murad et al., 2007; Yang and Sehgal, 2001). It is
also possible to “rescue” the expression of a gene
(e.g., per) in specific cells of null-mutant flies (e.g.,
per01 mutants; Grima et al., 2004). In 1999, Lee and
Luo introduced a third component—GAL80, a sup-
pressor of GAL4 activity—which can be utilized to
refine the spatial expression of gal4 with a second
promoter fused to gal80.
In the meantime, many gal4 drivers are avail-
able that drive expression in different populations
of the clock neurons (Fig. 2c). The first ones
developed are period (per)-gal4 and timeless
(tim)-gal4 lines that drive gene expression in all
clock cells (Blau and Young, 1999; Kaneko and
Hall, 2000), a Pdf-gal4 line that drives gene
 (a) (b)
DN1p

DN3
DN1a
PDF+ ITP+
DN2 PDF- ITP-

LNd
LPN
5th
S-LNv
I-LNv

s-LNv CRY+
NPF+ sNPF+
CRY- NPF- sNPF-

Cha+ IPNamide+
Cha- IPNamide-

(c)
cry-gal4 Mai179-gal4 Mz520-gal4 c929-gal4

r6-gal4 Mai179-gal4, Pdf-gal80 tim-gal4, cry-gal80 Clk4.1M-gal4

Fig. 2. Circadian clock neurons of D. melanogaster and their neurochemistry. All pictures show the right brain hemisphere. The
neurons that are located in the dorsal brain are called dorsal neurons (DN1–3 s). Among the DN1s, a pair of cells lies more anterior
and is called DN1anterior (DN1a). The remaining 15 DN1s have a more posterior position and are called DN1posterior (DN1p). The
DN2 are located slightly ventral from the DN1p. The DN3s represent the largest cluster and are composed of 30 cells, most of
which have very small somata. The lateral neurons (LNs) are located in a lateral position of the central brain. This group is also
further subdivided. One cluster of three cells, the lateral posterior neurons (LPNs), lies in a more central and posterior position.
The other clusters lie in the anterior brain. Among these, the most dorsal group consisting of six cells are called LNdorsal (LNds).
64

expression specifically in the PDF neurons (Park
et al., 2000; Renn et al., 1999), a Clk-gal4 line
(Glossop et al., 2003), as well as several cry-gal4
lines (Emery et al., 2000b; Klarsfeld et al., 2004;
Zhao et al., 2003). Not all of these drivers express
exclusively in the clock neurons. Animals possess
clocks also in most body organs (so-called periph-
eral clocks) in addition to the brain clocks
(Dibner et al., 2010; Plautz et al., 1997). Therefore,
the core clock genes (per, tim, Clk, and cyc) are
naturally expressed in many tissues—not only
in the brain. Within the head, natural expression
is found in the photoreceptor cells of the eyes
and the ocelli as well as in many glia cells of the
brain. However, the available per- and tim-gal4
lines drive expression even in cells that do not con-
tain native PER or TIM. This is because the spatial
information is not only coded by the per- and tim-
promoters but also post-transcriptionally (Kaneko
and Hall, 2000; Stanewsky et al., 1997). In addi-
tion, the expression pattern of gal4 depends on
the length of the clock gene promoter used. This
is very evident for the existing Clk-gal4 drivers.
Clk is naturally expressed in all clock neurons plus
many other cells (Houl et al., 2006). Fusing differ-
ent parts of the Clk-promoter to gal4 has also lead
to Clk-gal4 lines with more restricted expression
(Gummadova et al., 2009). In 2010, a very interest-
ing DN1p-specific Clk-gal4 line —Clk4.1 M-gal4—
came out that contains the  0.2 to 0.5 kb fragment
of the Clk gene and drives expression specifically
in the CRY-positive DN1p (Fig. 2c; Zhang et al.,
2010a,b).
CRY is found in subsets of the clock neurons
(Fig. 2a), in the compound eyes, glia cells, and ring
neurons of the ellipsoid body. Nevertheless, the
existing cry-gal4 lines are all more specific than
the per-gal4, tim-gal4 lines. But again the expres-
sion pattern depends on the length of the cry-
promoter fused to the gal4 sequence as well as on
the insertion site in the genome that strongly affects
expression strength. Some cry-gal4 lines (Emery
et al., 2000a; Hao et al., 2008; Klarsfeld et al., 2004;
Picot et al., 2007; Zhao et al., 2003) express strongly
in all clock neurons, glia cells, and photoreceptor
and ring neurons, and others mainly in the CRY-
positive clock neurons and ring neurons, whereby
there are differences in the expression strength
(Helfrich-Förster et al., 2007a,b).
In addition to the promoter-fused gal4 lines, there
are gal4 drivers gained by the enhancer trap tech-
nique (reviewed in Duffy, 2002). These may not nec-
essarily be related to known clock genes (in several
cases, it is still unknown which gene they trapped).
Nevertheless, several of these lines are very useful
because they drive gene expression in restricted
subsets of the clock neurons. These are mai179-
gal4, Mz520-gal4, c929-gal4, and r6-gal4 (Fig. 2c),
as well as gal1118 and gal1501 that drive essentially
in the PDF neurons (Blanchardon et al., 2001;
Lamaze et al., 2011; not shown) and two clock work
orange (cwo) enhancer trap lines that drive in all
neurons (Kadener et al., 2007; not shown).
Useful gal80 lines for refining expression to cer-
tain clock neurons are cry-gal80 and Pdf-gal80
(Stoleru et al., 2004). By combining tim-gal4 with
cry-gal80, one can manipulate all cells that express
tim but not cry (most of the DNs). By combining
tim-gal4 with Pdf-gal80, only the tim-expressing
cells that have no PDF can be manipulated (the
DNs and half of the LNs), and by combining cry-
gal4 with Pdf-gal80, one can manipulate half of
the LNs and few DNs. By combining mai179-gal4
with Pdf-gal80, it is even possible to restrict gene

The remaining more ventrally located clusters are classically called LNventral (LNvs). Most LNv cells express the neuropeptide pigment-
dispersing factor (b). The large LNvs (l-LNvs) have large somata and send projections to optic lobe and the contralateral side of the
brain. The small LNvs (s-LNvs) send projections to the dorsal brain where DN cells lie. One of s-LNv cells does not express PDF, is
located among the l-LNvs, and is called 5th s-LNv. (a) Cryptochrome (CRY)-positive neurons are highlighted in white (Benito et al.,
2008; Yoshii et al., 2008). (b) Neurons containing different neuropeptides/neurotransmitters are highlighted in white and marked by
arrows. PDF, pigment-dispersing factor (Helfrich-Förster, 1995); NPF, long neuropeptide F (Hermann et al., 2012; Johard et al., 2009;
Lee et al., 2006); Cha, choline acetyltransferase (Johard et al., 2009); ITP, ion transport peptide (Johard et al., 2009); sNPF, small
neuropeptide F (Johard et al., 2009); IPNamide (Shafer et al., 2006). (c) Useful gal4 lines or gal4/gal80 combinations that express only
in selected clock neurons (highlighted in white).

expression to four LNs per hemisphere (the 5th
s-LNv and three LNd; see Fig. 2c and below).
In general, it is wise to study first the exact
expression pattern of any used gal4-line and gal4/
gal80 combination by expressing a reporter gene
as the green fluorescent protein (gfp) gene before
manipulating the neurons. Unfortunately, this was
not well done in every study.
The original studies of Stoleru et al. (2004)
and Grima et al. (2004)
As mentioned earlier, fruit flies show bimodal
activity patterns with M and E peaks. Under
12 h:12 h LD-cycles (LD 12:12), M and E activity
bouts begin to increase before lights-on and
lights-off, respectively. This shows that the clock
anticipates morning and evening and the two activ-
ity bouts are not mere responses to lights-on and
lights-off. The Rosbash and Rouyer groups con-
centrated on this anticipatory activity increase
and defined the M oscillator to be present if M
anticipation is seen and the E oscillator to be pres-
ent if E anticipation is evident (Grima et al., 2004;
Stoleru et al., 2004). This simple definition allowed
them to screen for flies that lost the anticipations
after genetic manipulation.
The main strategy of Stoleru et al. (2004) was to
selectively ablate clock neurons by expressing hid.
They ablated the PDF-positive LNs (Fig. 1b) with
Pdf-gal4;uas-hid or the other LNs by combining
Pdf-gal80 with cry-gal4;uas-hid (note that all LNd,
not only the CRY-positive ones, plus the 5th
s-LNv and few DN1 are ablated by this driver
combination). Interestingly, the flies lacking the
PDF-positive neurons lost M anticipatory activity,
while the flies lacking the other LNs (plus few
DN1) lost E anticipation. This clearly suggests that
the M oscillator resides in the PDF-positive LNs
and the E oscillator in the PDF-negative ones (plus
few DN1). Thus, the two oscillators seem to reside
in distinct clock neurons (mainly LNs).
To confirm this conclusion, Stoleru et al. (2004)
also took an opposite approach, in which they
rescued per expression in per null (per0) mutants.
65
They combined a pan-neural gal4 driver (elav-
gal4) with either Pdf-gal80 or cry-gal80 to drive
uas-per in per0 mutants. In the first case, they res-
cued per in all neurons except the PDF-positive
LNs; in the second case, they rescued per in all
neurons except all LNs plus few DN1. One would
expect that in the first case, the flies will lack the M
anticipation, and in the second case, M and E antici-
pation. However, this was not the case, showing that
the LNs are not the only neurons that are responsi-
ble for M and E activity. Obviously, the DNs do also
contain M and E oscillators. This reminds of the
results gained with a classical mutant, disco, in which
all LN cells are developmentally absent but all
DN groups are intact. Most disco mutants loose
free-running rhythms in DD but exhibit rather nor-
mal M and E peaks in LD (Blanchardon et al., 2001;
Dushay et al., 1989; Hardin et al., 1992; Helfrich-
Förster, 1998; Wheeler et al., 1993), suggesting
that the DN groups are sufficient for both peaks.
The study of Grima et al. (2004), which was
published on consecutive pages in the same
Nature issue, nicely complemented the per-rescue
experiments of Stoleru et al. (2004). These
authors used different gal4-drivers to restrict per
to certain clock neurons (Fig. 1c): (1) mai179-
gal4 that is expressed in the s-LNv, 5th s-LNv,
and three to four LNd; (2) c929-gal4 that is
expressed in the l-LNv plus many neurosecretory
cells that are not clock neurons (Taghert et al.,
2001); (3) Pdf-gal4 and Mz520-gal4 that both
express in the PDF-positive LNs. When per was
rescued in most LNs (s-LNv, 5th s-LNv, and
LNd) by mai179-gal4, M and E anticipations were
restored, suggesting that these cells contain the M
and E oscillators. When per was only rescued in
the eight PDF-positive LNs by Pdf-gal4 and
Mz520-gal4, M anticipation was restored, but
when per was only rescued in the four PDF-
positive l-LNs by c929-gal4, the flies behaved as
per01 mutants and did not show any anticipatory
M activity, suggesting the M oscillator resides in
the s-LNvs. Together, these results suggest that
the M oscillator resides in the four PDF-positive
s-LNvs and the E oscillator in three to four LNds
(and the 5th s-LNv; however, the 5th s-LNv was
66
not detected in the two studies; it was recognized
as an E oscillator only later (Rieger et al., 2006)).
Coincidentally, the two independent groups
arrived at exactly the same conclusion that M
and E oscillators reside in distinct clock neurons,
which made their discovery quite solid and con-
vincing. Those exciting reports raised the dual
oscillator model by Pittendrigh and Daan to a
hot topic in Drosophila chronobiology.
Dominance of the M cells under short days
and of the E cells under long days
In order to reveal the hierarchy of the two
oscillators as well as the communication between
them, Stoleru et al. (2005) accelerated the oscilla-
tion speed of the M cells or the E cells by selec-
tively overexpressing the shaggy (sgg) gene in
either of the two cell clusters. SGG is the Dro-
sophila ortholog of the mammalian GSK3 that
seems to phosphorylate TIM and by this way
increase the speed of the clock (Martinek et al.,
2001). They found that the speed of M cells (but
not that of the E cells) is directly reflected in the
free-running activity rhythm in DD. Further, the
M cells influenced the rhythm of tim transcription
in the E cells, suggesting that the M cells are the
dominating oscillators that entrain the E cells in
DD. However, the situation was different under
LL conditions (Stoleru et al., 2007): Whereas flies
overexpressing sgg in the M cells (Pdf-gal4;uas-
sgg) became arrhythmic similarly to wild-type
flies, flies overexpressing sgg in E cells (tim-gal4;
Pdf-gal80;uas-sgg) remained rhythmic in LL.
Thus, Stoleru and coworkers assumed that M
cells are master clocks in the dark and E cells in
the light. Next, the activity rhythm of flies with
sgg overexpression in either the M cells or the E
cells was tested under short (LD 10:14) and long
photoperiods (LD 14:10) to see which oscillator
dominates at the two conditions. Indeed, under
the short photoperiod, the fast sgg-overexpressing
M cells set the phases of M and E peaks, whereas
the fast clock in the E cells set the phases of both
peaks under the long photoperiod. Stoleru et al.
(2007) concluded that the M cells dominate in
darkness and on long nights and the E cells in
light and on long days. This differential dom-
inance of M and E oscillators may also be
reflected in the amplitudes of M and E peaks in
wild-type flies under short and long photoperiods
(Rieger et al., 2003): under short days (LD 8:16),
their M peak was higher than their E peak,
whereas the opposite was true under long days
(LD 16:8).
Light activates output from the E cells and
inhibits output from the M cells
Stoleru et al. (2007) found that flies over-
expressing sgg in E cells remained rhythmic in
LL, a behavior that is quite different from wild-
type flies that become arrhythmic in LL. This is
because CRY that is activated by light interferes
with TIM and provokes its degradation; TIM in
turn is needed to stabilize its partner PER (Busza
et al., 2004; Ceriani et al., 1999; Rosato et al.,
2001). Thus, in wild-type flies, permanent degra-
dation of TIM and PER in LL finally stops the
molecular feedback loops of the clock (Myers
et al., 1996). Sgg overexpression seems to block
CRY signaling, which in turn stabilizes TIM and
PER oscillations and keeps the clock running
under LL (Stoleru et al., 2007). A similar
LL rhythmicity is observed if per or tim is
overexpressed (Murad et al., 2007; Stoleru et al.,
2007) or if cry is mutated (Emery et al., 2000a).
All these manipulations prevent the degradation
of TIM (and PER) in response to light and keep
the clock running under LL conditions. However,
if the clock is kept running only in subsets of the
clock neurons, this does not always lead to rhyth-
micity in LL. Stoleru et al. (2007) could not see
any behavioral rhythms after they overexpressed
sgg in the M cells (¼ PDF neurons). This is
very interesting because it shows that the PDF
neurons alone cannot drive rhythmicity under
LL conditions.

The Rouyer and Emery labs (Murad et al., 2007;
Picot et al., 2007) looked in more detail into the
ability of different clock neurons to drive behavioral
rhythmicity under LL. Whereas Murad et al. (2007)
took a similar approach as Stoleru et al. (2007)
(overexpression of per and another gene morgue)
to make clock neurons rhythmic in LL, Picot
et al. (2007) restricted CRY to subsets of clock
neurons. Picot and coworkers took either cry
mutant flies and rescued cry function either in
the M or in the E cells (using Pdf-gal4 or mai179-
gal4;Pdf-gal80) or they knocked cry down, either
in the M or in the E cells (using the same drivers).
As a consequence, only neurons without CRY
were oscillating in LL. Similar to Stoleru et al.
(2007), they found that a working clock in the
M cells (¼ PDF neurons) alone is not enough
to drive behavioral rhythms in LL but that a work-
ing clock in the E cells alone could do so. This
fact let Picot et al. (2007) conclude that the
output from the M cells is disturbed in LL. On the
other hand, the output from the E cells seems to
be disturbed in DD because these cells alone
could not drive behavioral rhythms in DD (Grima
et al., 2004; Picot et al., 2007; Rieger et al., 2009).
It is important to note that the definition of the E
cells is different in the studies of Stoleru et al.
(2007) and Picot et al. (2007). In the first paper,
LL rhythmicity was found when all clock neurons
except the PDF neurons were working, whereas
Picot et al. could restrict the cells responsible
for LL rhythmicity to the 5th s-LNv and three to
four LNd.
Murad et al. (2007) overexpressed per in all
clock cells (with tim-gal4) or in all clock cells
except the PDF neurons (with tim-gal4; Pdf-
gal80) and found the flies remaining rhythmic in
LL, very similar to Stoleru et al. (2007) after sgg
overexpression with the same drivers. However,
Murad et al. (2007) could trace the neurons
responsible for LL rhythmicity to a subset of the
DN1ps and not to the 5th s-LNv and three to four
LNd. These DN1ps were the only neurons that
showed rhythmic expression of PDP1, another
core clock protein for which an excellent antibody
67
exists (Cyran et al., 2003). Thus, there is a group
of DN1p neurons that can also drive rhythmicity
in LL, when all other clock neurons are not work-
ing. Most probably these DN1ps are CRY nega-
tive, because overexpression of per or sgg with
cry-gal4 could not provoke rhythmic behavior in
LL (Murad et al., 2007; Stoleru et al., 2007); but
at that time, it was not clear whether they belong
to the M or the E cells. In retro perspective, these
CRY-negative DN1ps likely belong to the E cells,
because recently the Emery lab identified a group
of CRY-positive DN1ps that unequivocally belong
to the M cells (Zhang et al., 2010a). These neurons
were detected with the Clk 4.1 M-gal4 line
(Fig. 2c), and they were not able to drive rhythmic-
ity in LL (after compromising CRY signaling)
(Zhang et al., 2010a). When per was rescued only
in these neurons, the flies exhibited a robust M
peak anticipating the lights-on in LD 12:12 and
only a tiny E peak (at a certain temperature and
light intensity; see later). When the period of these
DN1ps was shortened (by overexpressing a mutant
form of doubletime, dbt S; Preuss et al., 2004), the
M peak but not the tiny E peak phase-advanced
showing convincingly that the M peak is driven
by the CRY-positive DN1p cells.
In summary, the Drosophila clock seems to
consist of M cells (the PDF-positive s-LNvs and
CRY-positive DN1ps) that cannot drive rhythmic-
ity in LL when working alone and E cells (the 5th
s-LNv, three to four LNds, and perhaps the CRY-
negative DN1ps) that can drive rhythmicity in LL,
but not in DD, when working alone.
Light accelerates the M cells and decelerates
the E cells
According to the original dual oscillator model of
Pittendrigh and Daan (1976), M and E oscillators
should respond to light by accelerating and
decelerating their velocity, respectively. Indeed,
a detailed behavioral analysis of cryb mutants under
LL of different light intensities revealed two activity
components free-running with short and long
68

Fig. 3. Activity rhythms in constant light (LL) of per0;;cryb flies in which per was rescued only in subsets of the clock neurons.
Representative actograms (doubleplots) are shown for each genotype. All flies were first entrained to an LD 12:12 and then
transferred into LL. The bar on top of each actogram shows the light–dark (LD) schedule during the LD period. (a) Actogram
of a fly with per only in 4 M cells—the PDF-positive s-LNvs—(genotype: per0;Pdf-gal4/þ;cryb uas-per16. Note that per is also
present in the l-LNvs, but that these are neither M nor E cells); all these flies were arrhythmic in LL. (b) Actograms of three
flies with per expression in four E cells —the 5th s-LNv and three LNds— (per0;mai179-gal4/þ;Pdf-gal80;cryb uas-per). Most of
these flies show three activity components with long period (trimodal activity), some flies showed two activity components
(bimodal activity), and only few a unimodal activity pattern. (c) Actogram of a fly with per in four M and four E cells—the
PDF-positive s-LNvs, the 5th s-LNv, and three LNds (per0; mai179-gal4/þ;uas-per). Typically, the flies were bimodal and free-ran
with long period, and sometimes a weak short-period component splits off the long one (see arrow) (d) Actograms of two flies
with per in the same four M and four E cells and additionally in three to six DN1p-M cells (per0;cry-gal4/þ;cryb uas-per). These
flies show internal desynchronization in a short- and long-period component, though their activity pattern is not very clear.
(e) Actogram of a fly with per in all clock neurons (per0;tim-gal4/þ;cryb uas-per). This fly shows a clear internal desynchronization
in a short-period component that originates from the M peak and a long-period component that originates from the E peak. This
activity pattern is typical for cryb and cry0 mutants (Dolezelova et al., 2007; Rieger et al., 2006; Yoshii et al., 2004).

periods (Rieger et al., 2006; Yoshii et al., 2004; see
Fig. 3). These independent components appeared
as soon as light intensity exceeded 10 lux, whereby
the short-period component became faster and
the long-period component became slower with
increasing light intensity (Yoshii et al., 2004). This
phenomenon was present in the great majority
of flies, and —at a given light intensity— the regular
criss-cross of short- and long-period components
occurred rather simultaneously in every fly enabling
molecular and histological analyses (see below).
The reason why the two free-running components
were not found in other studies (e.g., Emery et al.,
2000a; Picot et al., 2007) seems due to the recorded
period and the genetic background. For unknown
reasons, the short-period component is often barely
visible for first few cycles but becomes apparent
after the two components crossed each other for
the first time and dissociate again, an event occur-
ring  10–20 days after transfer to LL. Most
researchers recorded the activity of the flies maxi-
mally for 10–14 days. Due to the weakness of the

short activity component that will not be revealed by
periodogram analysis of time series < 10 days, it
remained undetected. Further, Yoshii et al. (2004)
and Rieger et al. (2006) used cryb mutants with
red eyes that showed the two free-running compo-
nents more clearly than white-eyed mutants (w;cryb
or y w;cryb) that have been used by the other
researchers. Nevertheless, Dolezelova et al. (2007)
reported in the meantime the same phenomenon
for complete knock-out mutants, cry01, cry02, and
cry03, indicating that the splitting rhythms are real
and caused by clocks free-running in LL in the
absence of CRY.
As mentioned, the short component is often
not visible after the flies have been transferred
from LD to LL so that it is difficult to judge
where it originates from. But in most cases it
seems rather to originate from the E peak than
from the M peak, suggesting that the E peak is
controlled by M and E oscillators (see discussion
in Helfrich-Förster et al., 2007b). Nevertheless,
in some flies, the short period originates from
the M peak (see Fig. 3e). Thus, light seems to
shorten the period of the M oscillator and to
lengthen that of the E oscillator, in good agree-
ment with the model of Pittendrigh and Daan
(1976). Rieger et al. (2006) demonstrated that
when the two activity components were out of
phase on day 5 of LL, PER and TIM oscillations
in the clock neurons were also out of phase. The
s-LNv,  3 LNd, and some DN1s ran in parallel
to the short-period component, whereas the 5th
s-LNv,  3 LNd, and other DN1s ran in parallel
to the long-period component. These results fit
almost perfectly to the classification of the clock
neurons in M and E cells by Stoleru et al.
(2004), Grima et al. (2004), Picot et al. (2007),
Murad et al. (2007), and Zhang et al. (2010a,b)
except for the fact that the LNds seemed to be
split up in M and E cells (for further discussion,
see Helfrich-Förster et al., 2007b).
Nevertheless, there remains one caveat. We have
lined out above that neither the s-LNvs alone nor
the CRY-positive DN1ps alone can drive rhythmic
behavior under LL conditions. How it is then
69
possible that they give rise to a short-period compo-
nent that splits off the long one? To solve this ques-
tion, we have repeated our LL experiments with
per0;cryb flies in which per was rescued in different
subsets of M and E cells: (1) only in the PDF-
positive LNvs, (2) in the 5th s-LNv and three LNds,
(3) in most LNs, (4) in the LNs plus some DNs, or
(5) in all clock neurons (see exact genotypes in the
legend of Fig. 3). We found that the PDF-positive
LNvs alone are indeed not capable to produce a
short-period component but all flies are arrhythmic
in LL (Fig. 3a; only 1 out of 30 flies showed initially
a short-period component before it became arrhyth-
mic). Thus, they are very similar to the flies that have
a functional clock only in the CRY-positive DN1p-
M-oscillators (Zhang et al., 2010a). The flies with
per in the 5th s-LNv and three LNds (“E-oscillator
flies without DN1p-E-oscillators”) exhibited as
expected a long period, but the minority did show
the usual unimodal activity bout continuing from
the evening peak (13%). Most flies exhibited a tri-
(66%) or bimodal (21%) activity profile and the dif-
ferent components did not necessarily originate
from the E peak in LD (Fig. 3b). This strange activ-
ity pattern will be discussed in more detail later.
Adding functional PDF-positive LNvs to the 5th
s-LNv and three LNds (“M and E-oscillator flies
without DN1p-oscillators”) consolidated the E activ-
ity (Fig. 3c). The majority of flies showed bimodal
(75%) or unimodal (20%) activity profiles, and
the trimodal profiles almost disappeared (5%).
Again the free-running rhythm had a long period,
and only in few cases (5%) was a second weak
short-period component evident. But as soon as
the CRY-positive DN1ps were added to the per-
expressing cells (“M and E-oscillator flies with
DN1p-M-oscillators”), 43% of flies showed long
and short free-running components in their activity
pattern (Fig. 3d), although the two components
were never as clear as in flies that have PER in all
clock neurons (Fig. 3e). A possible reason for the
weakness of the short-period component could be
the fact that the cry-gal4 driver used to rescue per
in the DN1p-M-oscillators in addition to the LNv-
M-oscillators is not very strong. In the majority of
70
flies, only two to four of the six DN1p cells were PER
positive. Nevertheless, the results indicate that both
M cell groups together are able to drive rhythmicity
under LL conditions and that they do so with a short
period. That both groups of M cells do indeed com-
municate with each other was shown recently
(Zhang et al., 2010b).
In summary, it seems quite convincing that the
above defined M and E cells (1) give rise to M
and E peaks under LD 12:12 conditions, (2) have
different capabilities to control behavioral
rhythms in DD and LL, and (3) respond to light
by either accelerating or decelerating their speed.
These are exciting discoveries, as they give a first
clue about the organization of the circadian clock
in the brain that seems to be composed of two
principle types of clock cells with quite different
properties. Nevertheless, one has always to be
aware of the fact that LL rhythmicity is
completely artificial in flies. It can occur only
when photoreception via CRY is impaired or
TIM and PER are otherwise stabilized. Without
such a manipulation, M and E neurons will not
respond differently to LL conditions with light
intensities above  5 lux. Both types of clock
neurons would just stop their molecular
oscillations. In wild-type flies, even normal light
during the day will lead to complete degradation
of CRY and TIM: already before midday, no
CRY and TIM are left in any of the clock neurons
(Miyasako et al., 2007; Shafer et al., 2002; Yoshii
et al., 2008). Thus, it makes no difference for
wild-type behavior that some E and M cells can
principally drive rhythmicity under LL conditions
and others cannot, or that some cells are
accelerated and others decelerated by LL—at least
not during the highly illuminated day. To under-
stand the real responses of M and E oscillators to
natural day–night cycles and finally the adaptation
to different photoperiods, one should better not
interfere with photoreception but study flies with
normal light sensitivity. Further, it may be wise to
look at the effects of dim light that is naturally
present during dawn and dusk as well as during
star- and moonlight during the night.
M and E oscillators under moonlit nights and
constant moonlight
Bachleitner et al. (2007) subjected wild-type flies
to day–night cycles in which the night was not
completely dark but illuminated by dim light
(0.03 lux) corresponding to the intensity of quar-
ter moonlight. The flies responded strongly to
the moonlight and shifted M and E peak clearly
into the night. The M peak advanced and the E
peak delayed so that the two peaks had a larger
CM,E than under dark nights. As a consequence,
the flies become mostly nocturnal. There may be
two reasons for the nocturnal behavior. First, flies
seem to prefer generally dim light and do neither
like to stay under high illumination nor in com-
plete darkness as revealed by a video monitoring
system (Rieger et al., 2007). This behavior is inde-
pendent of a functional clock (Kempinger et al.,
2009). Second, the experiment was performed in
the lab where the temperature remained during
day and night at pleasant 20  C, allowing the flies
to become nocturnal. In nature, nocturnal activity
of fruit flies is not observed, even not in full-moon
nights (Bhutani, 2009; Vanin et al., 2012). Simi-
larly, fruit flies do not become nocturnal under
moonlit nights in the lab, when natural-like
T-cycles are simulated (Yoshii et al., 2010; see
also below). Although the nocturnal behavior of
the flies seems not natural and partly caused by
“masking” effects that do not depend on the clock,
the moonlight experiment showed that M and E
peaks responded in the way that was proposed
by Pittendrigh and Daan: They shifted into oppo-
site directions—the M oscillator advanced upon
moonlight and the E oscillators delayed. Most
important, this was also true for the molecular
PER and TIM oscillations in the above described
M and E cells (Bachleitner et al., 2007). This result
shows that M and E cells do respond in the
expected way to dim light that is naturally present,
if not at night then at least during early dawn and
dusk in the morning and evening (see also below).
The next question was what would happen to
CM,E under constant moonlight. According to
 71

Wild-type
LD ® DD LR ® RR

LD/LR
5

DD/RR
10

15

20

Fig. 4. Typical locomotor activity rhythms of wild-type flies that are subjected to different illuminations. In the left actogram, a fly
was entrained in LD 12:12 for 5 days and then was transferred into DD. The bimodal rhythm in LD does not persist in DD but
becomes unimodal (green line). In the right actogram, a fly was entrained in white-light/red-light (LR) cycles for 5 days before
constant red light (RR). This fly had a larger CM,E under LR, and M and E peaks persist in RR free-running in parallel to each
other (green lines).

Pittendrigh and Daan (1976) and Aschoff (1966),
CM,E should be larger under dim light than under
darkness. In DD, CM,E usually gets very small in
fruit flies and sometimes M and E peaks are not
anymore distinguishable as separate peaks (see
Fig. 4a). Under constant moonlight, M and E
peaks were clearly distinguishable in each single
fly, CM,E was  11 h, in contrast to  8 h in DD
(Bachleitner et al., 2007). The same effect can be
seen when flies are subjected to constant colored
light of longer wavelength (> 560 nm, Fig. 4b). In
this case, irradiance can even be increased to very
high values without the risk that the flies become
arrhythmic (Yoshii, Roidl, Wülbeck, and Char-
lotte Helfrich-Förster, unpublished results). This
is because CRY is not activated by wavelengths
above 540 nm (Berndt et al., 2007; Helfrich-
Förster et al., 2002; VanVickle-Chavez and Van
Gelder, 2007). We are currently using constant
colored light to test the effects of light on the
two oscillators in more detail. But even low white
light proved as useful: when light intensity was
increased to only 0.5 lux, M and E components
started to free-run with short and long period,
respectively, before the flies became arrhythmic
(Rieger et al., 2006). All this is in accordance with
the dual oscillator model.
A next logical step is to investigate how flies
with only functional M or E cells respond to dim
light in the night or to constant dim light. This
has been investigated by Rieger et al. (2009). Let
us start with the constant dim light experiments
because these are largely in line with the LL exper-
iment conducted in flies with compromised CRY
signaling (see above). LL of 0.03 lux was already
too intense for the “LNv-M-oscillator-only flies”
(per only in the PDF-positive LNvs) (Rieger
et al., 2009). These became arrhythmic as did the
same flies with compromised CRY signaling under
LL of higher light intensity (Picot et al., 2007). In
contrast, the “E-oscillator-only flies” (per only in
the 5th s-LNv and three LNds) remained rhythmic;
and again this is in concert with the previously
mentioned results and fits to the model (Grima
et al., 2004; Murad et al., 2007; Picot et al., 2007;
Stoleru et al., 2007).
72
Nevertheless, not every observation under
moonlight was along the expectations. Instead of
exhibiting only an E activity bout under light–
moonlight (LM) cycles, the “E-oscillator-only flies”
showed an additional prominent activity peak in
the second half of the night and this activity peak
persisted under constant moonlight (Rieger et al.,
2009). As a consequence, the activity pattern of
the “E-oscillator-only flies” was clearly bimodal
and not unimodal as expected. Although the nature
of this activity peak is not completely clear —it
might be a very early M peak as discussed in
Helfrich-Förster et al. (2007b)— this result
indicates that the fly clock is highly plastic and
not the same under all conditions. At the tested
LM-cycles, immunohistochemical analysis revealed
that two LNds behaved rather as M cells and the
5th s-LNv and one LNd behaved likely as E cells
(Rieger et al., 2009). Thus, the composition in M
and E cells may be variable and dependent on
the environmental conditions. We will come to this
important aspect later.
Flies adapting to different photoperiods
Simulation of dawn and dusk
Let us come back to the behavior of wild-type flies.
So far this was only regarded under artificial LD-
cycles, in which the lights were switched on in the
morning and switched off in the evening. As the
fly clock is very light sensitive and strongly responds
to moonlight, it is very likely that dim light during
dawn and dusk will affect M and E oscillators,
respectively. Rieger et al. (2012) compared the syn-
chronization of three different wild-type strains
under conventional rectangular LD-cycles and
LD-cycles with simulated dawn and dusk at differ-
ent photoperiods. As mentioned in the introduc-
tion, M and E activity peaks of fruit flies largely
follow lights-on and lights-off, and as a conse-
quence, CM,E increases with increasing photoperiod.
If M and E oscillators are mostly set by dim light
(and not by bright light that stops the oscillations),
one would expect that long photoperiods with
simulated dawn and dusk would extend CM,E to a
larger extent than long photoperiods with rectangu-
lar LD-cycles. This was indeed the case for all three
tested wild-type strains; they extended CM,E by
20–60 min, depending on the genetic background
(Rieger et al., 2012).
Adaptation of the clock to different photoperiods
occurs via light input through the photoreceptor
organs and not via CRY
CRY was already mentioned as a circadian photo-
receptor that is expressed in several clock neurons
and that interacts directly with TIM leading to its
degradation upon light. Although CRY action on
the clock is a direct and extremely efficient way
to reset the clock by light, CRY is not involved in
the adaptation of M and E peaks to different
photoperiods: mutants without functional CRY
can perfectly adapt their activity to short and long
photoperiod, but eyeless mutants cannot (Rieger
et al., 2003). Eyeless mutants are also not able to
shift their activity into moonlit nights (Bachleitner
et al., 2007), suggesting that the dim light receptor
must reside in the eyes and probably in an extra-
retinal eyelet, called Hofbauer–Buchner (H–B)
eyelet, that derives from the larval photoreceptor,
Bolwig’s Organ (Helfrich-Förster et al., 2002;
Veleri et al., 2007). Bolwig’s Organ and the H–B
eyelet directly target the LNv cells (Helfrich-
Förster et al., 2002; Malpel et al., 2002). The H–B
eyelet and certain photoreceptor neurons in the
eye express Rhodopsin 6 that still shows sensitivity
under longer wavelengths and could be responsi-
ble for the red light effects on M and E activity
peaks (Hanai et al., 2008; Fig. 4).
The PDF-positive l-LNvs play a crucial role in
mediating light input from the eyes
The l-LNvs are among the cells that may be directly
contacted by the H–B eyelet and some photorecep-
tor cells of the compound eyes (Helfrich-Förster
et al., 2002). Several studies suggest that these

clock neurons are neither M nor E cells but play a
crucial role in transferring light signals to the clock.
The l-LNvs respond to light with increased sponta-
neous action potential firing (Sheeba et al., 2008a)
and they modulate the light arousal and sleep
of Drosophila (Chung et al., 2009; Parisky et al.,
2008; Shang et al., 2008; Sheeba et al., 2008c).
Hyperexcitation of the l-LNvs increases the noctur-
nal activity of the flies (Parisky et al., 2008) as does
dim light during the night (see above). The arousal
effects of the l-LNvs are clearly mediated by PDF
because Pdf 01 mutants do not increase nocturnal
activity, neither after hyperexcitation of the l-LNvs
(Sheeba et al., 2008b) nor after exposition to dim
light during the night (Helfrich-Förster, 2009).
PDF from the l-LNvs can also synchronize the
LNds and DNs to LD-cycles in the absence of
CRY (Cusumano et al., 2009). Most interestingly,
an increase of PDF in the dorsal brain does lead
to internal desynchronization of the free-running
activity rhythm into two components as does LL
(Helfrich-Förster et al., 2000; Wülbeck et al.,
2008), and the same neurons seem to free-run with
short and long period, respectively, as observed
under LL (Yoshii et al., 2009a). This suggests that
PDF is the factor that accelerates the speed of the
M cells and decelerates the speed of the E cells.
In accordance with this, neither did Pdf01 mutants
show internal desynchronization upon light, nor
are they able to adapt M and E peaks to different
photoperiods (Yoshii et al., 2009a; see also discus-
sion in Helfrich-Förster, 2006, 2009).
The effects of temperature on M and E oscillators
As mentioned in the introduction, the environ-
mental temperature is extremely important for
poikilothermic animals, as it directly influences
their activity, reproduction, and survival (Garrity
et al., 2010). Majercak et al. (1999) nicely demon-
strated that the overall temperature strongly
affects the phases of M and E peaks in Drosophila
in the same way as the photoperiod: The M peak
advances and the E peak delays with increasing
temperature (see Fig. 1). Thermosensitive splicing
73
of the per and tim genes seem to account for the
temperature effects on the phase of the E peak
(Boothroyd et al., 2007; Collins et al., 2004;
Majercak et al., 1999, 2004), but no molecular
mechanism is known that shifts the M peak in
the opposite direction of the E peak.
It is also unknown how the overall temperature
level will influence the activity of flies that possess
only M or E cells, but some data are available
for such flies under T-cycles. Artificial rectangular
T-cycles in the lab (temperature immediately
increases in the morning and decreases in the even-
ing in DD) can synchronize the activity rhythm of
flies, whereby these exhibit the usual M and E
activity peaks (Busza et al., 2007; Tomioka et al.,
1998; Wheeler et al., 1993; Yoshii et al., 2002).
Under the same conditions, per0 mutants do not
show M and E peaks but are just more active dur-
ing the higher temperature. All this resembles the
activity pattern observed under LD conditions.
However, different neurons seem to be responsible
for M and E activity peaks under T-cycles: When
per is rescued in the PDF-positive M cells, an activ-
ity peak occurs in the middle of the warm phase
that seems to be neither an M nor an E peak (Busza
et al., 2007). Even the per rescue in all CRY-
positive neurons (with cry-gal4) did not lead to
wild-type-like M and E peaks. This suggests that
the CRY positive are dispensable for the normal
control of M and E peaks under T-cycles and that
the CRY negative are more important. Indeed,
flies that lack the CRY-positive clock neurons still
synchronize to T-cycles (Busza et al., 2007; Yoshii
et al., 2010). Among the CRY-negative clock
neurons, the LPNs seem to be most important
(Busza et al., 2007; Yoshii et al., 2005): The PER/
TIM oscillations in the LPNs do only synchronize
to T-cycles but not to LD-cycles. The DN2 cells
also seem to be more sensitive to T-cycles than to
LD-cycles (Miyasako et al., 2007; Picot et al.,
2009; Yoshii et al., 2009b). In contrast, the PDF-
positive l-LNvs do only entrain to LD-cycles, but
not at all to T-cycles (Yoshii et al., 2009b). This
result strongly underlines the above-mentioned
role of the latter cells (and PDF) in the light-input
pathway.
74
Flies under natural-like temperature cycles
In nature, T-cycles are not rectangular, but tem-
perature slowly increases in the morning until it
reaches its maximum  2 h after midday, and then
it decreases again reaching the lowest point in the
morning at sunset (Bhutani, 2009; Yoshii et al.,
2009b). When such natural-like T-cycles are
simulated in the lab, the flies show their M peak
few hours after the temperature minimum and
the temperature maximum, respectively (Currie
et al., 2009; Yoshii et al., 2009b). Recently, we
subjected flies to simulated short and long days
of natural T-cycles of 26/16  C under DD
conditions (Bywalez et al., 2012). The two peaks
nicely changed their phase-angle to adapt to dif-
ferent day lengths as was observed under differ-
ent photoperiods: CM,E was shorter in shorter
days and broader in longer days. This suggests
that CM,E is similarly flexible under T-cycles as
it is under LD-cycles. Thus, the dual oscillator
model seems to be valid also for T-cycles,
although different clock neurons may be responsi-
ble for M and E activity under LD- and T-cycles.
Which CRY-negative clock neurons control M
activity and which control E activity under T-
cycles has to be determined in the future.
Interaction of light and temperature
When natural-like LD- and T-cycles are applied in
phase with each other, they work synergistically on
entrainment: the flies show narrow M and E peaks
and the molecular clock cycles with high amplitude
in all clock neurons (Yoshii et al., 2009b). Interest-
ing effects are observed when LD- and T-cycles
are applied out of phase with each other or when
the clock is restricted to certain clock neurons
(Busza et al., 2007; Yoshii et al., 2010; Zhang
et al., 2010a).
Under conflicting LD- and T-cycles, wild-type
flies entrain to the LD-cycles, but CRY-less flies to
the T-cycles (Yoshii et al., 2010). This shows that
(1) light is a stronger Zeitgeber than temperature
—a fact that was reported already a long time ago
(Pittendrigh et al., 1958)— and (2) it underlines
the above-mentioned importance of the CRY-
positive neurons for entrainment to light.
But what happens when the PDF-positive l-LNvs
that seem to be most important for light input are
ablated? Busza et al. (2007) performed this impor-
tant experiment and found that such flies entrain
faster to T-cycles than wild-type flies. Busza et al.
concluded that the PDF cells may even inhibit the
temperature entrainment of CRY-negative neurons
and that this inhibition is important for entrainment
in nature because it guarantees that the clock does
not overreact to erratic temperature changes caused
by weather changes.
More complex results are found when per ex-
pression is restricted to the CRY-positive DN1ps
that are designated as M cells under LD conditions
(Zhang et al., 2010a; see above). Such flies show a
prominent M peak under LD 12:12 (500 lux) and a
constant temperature of 25  C. However, when
light intensity was reduced to 50 lux or the temper-
ature decreased to 20  C, an additional E peak
appeared. The same happened when the flies were
subjected to T-cycles in DD. All these suggest that
the temperature interacts with light in a complex
way. The most important result of the study of
Zhang et al. (2010a) is that the CRY-positive
DN1ps are sufficient to generate M and E peaks
but that the expression of the two peaks depends
on the environment. A possible explanation is that
the E peak of the flies may be suppressed at higher
light intensities and temperatures, but this has to
be proven by future studies.
A completely different approach to dissect the
temperature-sensitive clock is to make the light-
sensitive clock arrhythmic by applying LL of rather
high light intensity (Tomioka et al., 1998; Yoshii
et al., 2002). Amazingly, T-cycles can override the
LL effect on the clock, although the amplitudes of
PER and TIM oscillations under such conditions
are quite low (Glaser and Stanewsky, 2005; Yoshii
et al., 2005). Nevertheless, the low-amplitude
molecular rhythms restored by T-cycles in LL are
sufficient for robust behavioral rhythms (Yoshii
 75

(a) (b)
DD T-cycles LL T-cycles
26 °C/16 °C 26 °C/16 °C

2
n=30 n=32
2

Activity
1
1

0 0
18 0 6 12 18 18 0 6 12 18
Zeitgeber time (hr) Zeitgeber time (hr)

(c) (d)
(n=27) (n=28)

5 5

6 hr
10 10 delay
Days

15 15

6 hr
20 20 advance

25 25

Fig. 5. Activity rhythms under natural-like temperature cycles (T-cycles) in DD (a, c) and LL (b, d). (a) The averaged activity
pattern in DD T-cycles indicates that one main activity peak occurs in the evening and another peak occurs in the morning
(arrowheads; Yoshii et al., 2009b). Those two peaks run in parallel when T-cycles were shifted by 6 h delay and advance (c). In
contrast, only one robust peak at evening is visible in LL T-cycles, and the phase of E peak is delayed compared with that in
DD T-cycles (b, d). (c, d) Average actograms from 27 flies for DD T-cycles and from 28 flies for LL T-cycles, respectively.

et al., 2002; 2007). Interestingly, the restored behav- The dual oscillator model appears too simple
ioral rhythms in LL do not display an M peak except
for a startle response at the sudden temperature Though the M and E oscillator model turned out
increase in the morning (these studies were per- to be very useful to explain the adaptation of flies
formed under rectangular T-cycles). More informa- to long and short days, it became clear that it is
tive are natural-like T-cycles that show clearly that too simple to understand all observations made
the M peak is absent in LL but is present in DD so far. Several studies show that there are more
conditions (Fig. 5). In contrast, the E peak is more than two clocks in the fly brain. These should
robust under T-cycles in LL than in DD. These be briefly summarized in the following.
findings fit to the idea mentioned in the beginning
that the M oscillator runs in DD and the E oscillator
in LL (Picot et al., 2007; Stoleru et al., 2007). Never- E cells alone can drive two or even more
theless, the relationship between light and tempera- activity components
ture is clearly more complicated. The phases of
the E peak are different in DD and LL (Fig. 5), Sheeba et al. (2010) ablated specifically the PDF-
and the above-mentioned results for flies with positive s-LNvs by expressing the human neurotoxic
restricted clock function (especially the ones with protein Q128-Huntingtin. One may expect that
the DN1p-only flies) clearly show that we do not such flies lack the M peak, but this was clearly not
yet completely understand how light and tempera- the case: M and E peaks remained present. In this
ture act on M and E oscillators. study, still the CRY-positive DN1ps may account
76
for the remaining M peak, but this cannot be the
explanation for the two activity peaks present in
per0 flies in which per was rescued only in the four
LNs (the 5th s-LNv and three LNds; Rieger et al.,
2009). Most convincing that these four putative E
cells can drive several activity components are per-
haps the results shown in Fig. 3b. Here, the same
flies were put additionally into the cryb background
and subjected to LL. Instead of the expected single
activity band, up to three free-running components
were present and these did not even originate from
the E peak in LD (Fig. 3b). Rieger et al. (2009)
suggested that not all of these neurons are E cells,
but alternatively, the nature of the neurons could
be influenced by the environment (here the light
intensity).
Under certain circumstances, M cells alone can
also drive two activity components
As outlined above, even the M cells alone —in this
case, the CRY-positive DN1ps— can drive M and
E peaks. This is the case at high light intensities
and higher temperatures (Zhang et al., 2010a).
Either these six or seven cells represent not exclu-
sively M oscillators as was suggested for the four
E cells above, or again the environment (here tem-
perature and light intensity) determines whether
they behave as M or E cells.
All these results suggest that we are dealing with a
flexible multi-oscillator system that may be com-
posed of interacting M and E cells (see also
Umezaki and Tomioka, 2008). Alternatively, the
activity pattern may be determined by a flexible net-
work of interacting clock neurons without the neces-
sity of the designated M and E cells. The latter view
is strongly favored by Sheeba et al. (2008b).
Welcome to complexity
Understanding nature and biological mechanisms
is hard. Seven years after the Stoleru and Grima
papers, we have to realize that it is very difficult
to define M and E cells in the clock network.
People may think that only 150 clock neurons of
Drosophila should be easy to study. Yes, perhaps
they are easier to study than the 10,000 neurons in
the mammalian clock, but still more complicated
than we wished. Nevertheless, we should not capit-
ulate. To reach the goal, we need much more
pieces in the puzzle. People are successfully
searching for further useful gal4 drivers and devel-
oping new efficient methods to manipulate subsets
of neurons (Choi et al., 2009; Gummadova et al.,
2009; Sheeba et al., 2010; Wu et al., 2008). The bac-
terial LexA-LexAop-system and the Q-system of
Neurospora are new binary expression methods
that can be combined with the gal4-uas/gal80 sys-
tem allowing to dissect the clock network further
(Lai and Lee, 2006; Potter and Luo, 2011; Shang
et al., 2008). Although we can only expect minor
effects on the flies’ activity rhythm of the flies after
manipulating smaller subsets of clock neurons, one
day these may represent extremely useful pieces
for the puzzle we need to solve in order to under-
stand the whole system. We would like to give a
recent example: We have just ablated the NPF-
positive clock cells, which are three LNd, the 5th
s-LNv, and few l-LNv, and found subtle but signifi-
cant opposing effects on the E activity peak and
the free-running period in DD (Hermann et al.,
2012) that have not been reported before (Lee
et al., 2006): the knock-out advanced the E peak
but prolonged the free-running period—an effect
that is hard to reconcile with the present M and E
model. Clearly, more pieces of the puzzle are
needed to understand what this means.
Tiny differences after defined manipulations
ask also for careful observations and analyzing
methods. Defining the presence of M and E
peaks by looking at anticipatory activity before
lights-on and lights-off is probably not enough.
An activity peak can be delayed or masked by
certain circumstances, but this does not mean
that the peak is absent (see Helfrich-Förster,
2009 for further discussion).
It is also important to choose the best experi-
mental conditions for studying M and E
oscillators. The dual oscillator system is important

to adapt the activity rhythm to different
photoperiods and temperatures, meaning that
investigations under different photoperiods and
temperatures are warranted (Bywalez et al.,
2012; Hermann et al., 2012; Majercak et al.,
1999; Rieger et al., 2003, 2012). Monitoring phase
response curves for each oscillator to light pulses
is also an important issue as was suggested by
Schwartz (2004) in his comments on the Stoleru
and Grima papers. Given the predictions by Daan
et al. (2001), the M clock should only show phase-
advances upon light pulses, whereas the E clock
should show only phase-delays. Such fundamental
investigations are still lacking.
Stoleru and Grima and the following papers
provided a good start how to find clock neurons
in the fly brain that control different aspects of
rhythmic behavior. Now we have to continue this
research—a task that is possible due to the unique
genetic methods available in the fruit fly. Under-
standing the fly’s dual or multiple oscillators and
their role in seasonal adaptation will not be easy,
but will certainly remain fascinating.
Acknowledgments
We thank Pamela Menegazzi, Francois Rouyer,
and Irina Stahl for commenting on and editing
the chapter and Francois Rouyer for providing
the fly strains used in the LL experiment shown
in Fig. 3. The work from our groups was finan-
cially supported by the European Community
(the 6th Framework Project EUCLOCK no.
018741) and that of the C.H.F. group additionally
by the DFG (Fo207/10).
Abbreviations
CLK clock
CRY cryptochrome
CYC cycle
DD constant dark
DN dorsal neuron
77
E evening
LD light–dark
LL constant light
LN lateral neuron
M morning
PDF pigment-dispersing factor
PER period
T-cycles temperature cycles
TIM timeless
CM,E the phase-angle between M and E
peak
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 5

Circadian system from conception till adulthood

Alena Sumova*, Martin Sladek, Lenka Polidarova, Marta Novakova and

Pavel Houdek

Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Abstract: In mammals, the circadian system is composed of the central clock in the hypothalamic
suprachiasmatic nuclei and of peripheral clocks that are located in other neural structures and in cells of
the peripheral tissues and organs. In adults, the system is hierarchically organized so that the central
clock provides the other clocks in the body with information about the time of day. This information is
needed for the adaptation of their functions to cyclically changing external conditions. During
ontogenesis, the system undergoes substantial development and its sensitivity to external signals
changes. Perinatally, maternal cues are responsible for setting the phase of the developing clock, while
later postnatally, the LD cycle is dominant. The central clock attains its functional properties during a
gradual and programmed process. Peripheral clocks begin to exhibit rhythmicity independent of each
other at various developmental stages. During the early developmental stages, the peripheral clocks are
set or driven by maternal feeding, but later the central clock becomes fully functional and begins to
entrain the periphery. During the perinatal period, the central and peripheral clocks seem to be
vulnerable to disturbances in external conditions. Further studies are needed to understand the
processes of how the circadian system develops and what degree of plasticity and resilience it possesses
during ontogenesis. These data may lead to an assessment of the contribution of disturbances of the
circadian system during early ontogenesis to the occurrence of circadian diseases in adulthood.

Keywords: ontogenesis; suprachiasmatic nucleus; peripheral circadian clocks; clock gene.

Introduction organism grows and matures. Increasing awareness

The mammalian internal timekeeping system,
which temporally controls many processes in our
body, undergoes developmental changes as an
*Corresponding author.
Tel.: þ420-24106-2528; Fax: þ420-24106-2488
E-mail: sumova@biomed.cas.cz
of the mechanisms of how the system operates at
molecular level facilitates studies of the develop-
ment of the system from its very early stages, that
is, far before the overt rhythms driven by the
system can be used as a marker of its functional
stage. These studies may uncover the mystery of
when the circadian clock begins to tick indepen-
dent of the maternal environment. It is possible

http://dx.doi.org/10.1016/B978-0-444-59427-3.00005-8 83
84
that the circadian clock is functional early prena-
tally when it resides in the maternal body and the
mother’s role is just to adjust its phase according
to the external light/dark (LD) cycle to ensure that
her offspring are born with fully entrained clocks.
Alternatively, it is also possible that the developing
circadian clock is first directly driven by maternal
signals as a slave oscillator in utero and develops
into a self-sustained clock that is sensitive to
the external LD cycle only gradually after birth.
Both possibilities would ensure that the newborn
leaves the maternal body with its clock properly
entrained with the external environment, but it
remains unclear which of these possibilities would
be evolutionally more advantageous. It is also pos-
sible that different species utilize various scenarios
of the development. This issue is important for
understanding the significance of mother-to-fetus
and mother-to-neonate communication for proper
development of the circadian system. Enormous
amounts of data have been accumulated to dem-
onstrate that a malfunction of the circadian system
leads to the distortion of temporal control of phys-
iology and results in the development of serious
diseases. Studies on the ontogenesis of the circa-
dian system may reveal whether its malfunction
in adulthood may result from an insult during early
developmental stages, which might arise from the
disruption of the endogenous timekeeping system
during pregnancy.
The circadian system is composed of hierar-
chically organized circadian clocks forming an
integral regulatory system that gives the organism
the daily temporal program and helps it adapt to
the external environment. Circadian clocks reside
in nearly every, if not all, mammalian cells (for a
review, see Schibler et al., 2003). These clock cells
are equipped with a set of so-called clock genes,
which are genes that are indispensable for circa-
dian function. In mammals, Per1, Per2, Cry1,
Cry2, Bmal1, Clock, Rev-erba, and Rora have
been recognized as clock genes (for a review,
see Takahashi et al., 2008). Cellular clocks are
regularly entrained by the master pacemaker
located in the suprachiasmatic nuclei (SCN) of
the ventral hypothalamus just above the optic chi-
asm (Klein et al., 1991). The SCN is a complex
structure, which is composed of a cluster of inde-
pendent cellular oscillators (Welsh et al., 1995)
that are mutually coupled by a web of synapses.
This intercellular communication is responsible
for synchrony among the cells (Liu et al., 2007).
Moreover, the SCN cells are organized into
morphologically and functionally distinct sub-
populations (Klein et al., 1991) which also
communicate among each other. The most prom-
inent subpopulations are located in the ventrolat-
eral (core) and dorsomedial (shell) regions of the
SCN (for a review, see Welsh et al., 2010), but
there are also other subdivisions, including a func-
tional specialization in populations of cells in the
rostro-caudal dimension of the SCN (Brown and
Piggins, 2009; Morin, 2007; Sosniyenko et al.,
2009). As a result of this intercellular communica-
tion, the SCN generates a synchronized output
signal to the rest of the body that entrains the
peripheral clocks with the external LD cycles.
This chapter provides a summary of our latest
knowledge and offers some hypotheses on the pro-
cesses by which the circadian system develops in
mammals from its origin through weaning and
adulthood. The summary includes mostly data from
rodent models. For a more extensive overview of
the subject, the readers are encouraged to refer to
previous reviews (e.g., Weinert, 2005; Sumova
et al., 2006a,b, 2008; Seron-Ferre et al., 2007).
Developing circadian system in utero
Prefetal development
The development of circadian clocks in mammals
is intrinsically programmed and does not depend
on presence of a functional maternal clock (Davis
and Gorski, 1988; Jud and Albrecht, 2006;
Reppert and Schwartz, 1986; Shibata and Moore,
1988). At the cellular level, the first appearance
of circadian rhythmicity has been detected in
differentiated, multipotent cells derived from

embryonic stem cells but not at earlier stages such
as pluripotent cells (Kowalska et al., 2010; Yagita
et al., 2010). Unfertilized mouse oocytes contain
transcripts of the clock genes (Johnson et al.,
2002), but their levels do not exhibit rhythms in
oocytes and preimplantation embryos (Amano
et al., 2009). The embryonic stem cells first differ-
entiate into neural precursor cells, which further
divide and may differentiate into neurons and
astrocytes (Gotz and Sommer, 2005). The differ-
entiated cells possess the circadian clock, which
is lost if they are forced to dedifferentiate back
into embryonic stem cells (Yagita et al., 2010).
Thus, the earliest detection of the circadian clock
is in studies employing differentiated neural
precursor cell lines. The presence of a clock in
these studies was detected by rhythmic clock
and clock-controlled gene transcription, using
Bmal1- and Dbp-promoter-driven luciferase
reporters (Kowalska et al., 2010; Yagita et al.,
2010) and RT-PCR (Kowalska et al., 2010). How-
ever, information about the levels of protein
products of the clock genes at these stages is
lacking. Also, these studies must rely on employing
cell lines and it is possible that the cells and the
process of their differentiation may not exactly
match the situation in vivo. Nevertheless, these
studies provide insight into the origin of the cellu-
lar circadian clock in mammalian development.
It is intriguing that the presence of transcripts
of the clock gene Bmal1 was confirmed already
in unfertilized eggs at 2-cell and 16-cell stages
(Ko et al., 2000). This finding may suggest that this
gene is expressed intermittently, skipping some
developmental stages. Moreover, this finding
may imply that Bmal1 may be involved in various
processes already during ontogenesis, such as the
timing of cell division in the mouse embryo (Ko
et al., 2000).
Development of the fetal SCN clock
At the structural level, the development of the
circadian clocks in the SCN or peripheral organs
85
Rat
E0 Conception
E15 SCN formation
E22/P0 Birth
P5
Photic entrainment
synaptogenesis completed
P10
P15 Eye opening
P21
Weaning
P28
Fig. 1. Schematic diagram of the main circadian milestones of the
rat ontogenesis. Vertical bar represents a timescale from
embryonic day (E) 0 till postnatal day (P) 28 (for details see text).
during the prenatal period depends on the differ-
entiation and maturation of the structure in ques-
tion. The main circadian milestones of the rat
ontogenesis are depicted in Fig. 1.
Morphologically, the rodent SCN develops
gradually (Moore, 1991). For example, in the
rat, neurogenesis of the SCN begins on embry-
onic day (E) 14 and continues through E17,
originating from a specialized zone of the ventral
diencephalic germinal epithelium as a component
of periventricular cell groups. Neurons of the ven-
trolateral SCN are generated at E15–E16 and
those of the dorsomedial SCN at E16–E17. Neu-
rogenesis is completed at E18, but the morpho-
logical maturation of the SCN neurons gradually
proceeds until postnatal day (P) 10. The genetic
86
program that is responsible for the formation of
the SCN seems to be dependent on the presence
of the Six3 transcriptional factor (VanDunk
et al., 2011).
Well before the circadian molecular clock
mechanism was deciphered, studies in mammals
using overt circadian rhythms as a marker of the
functional state of the circadian clock strongly
suggested the presence of a functional circadian
clock during the fetal stage, and this statement
has been widely accepted for decades (for a
review, see Weinert, 2005). The question of when
exactly during the fetal stage the molecular
machinery begins to operate was reinitiated by a
rather surprising finding that clock gene expres-
sion profiles in the rat SCN of 19-day-old fetuses,
that is, 3–4 days before delivery, do not exhibit
circadian rhythms (Sladek et al., 2004). Moreover,
levels of protein products of the clock genes
PER1, PER2, and CRY1, which are indispens-
able for clock function, could not be detected by
conventional immunocytochemistry in the SCN
of these fetuses (Sladek et al., 2004). This finding
suggests that the population of SCN cells might
not be able to generate synchronized rhythms at
this developmental stage and facilitated further
studies to identify the developmental stage at
which the circadian clock begins to tick.
After completing neurogenesis, the SCN fur-
ther develops gradually during ontogenesis
(Moore and Bernstein, 1989). In rodents, the
newly formed SCN neurons begin to spread their
processes across the nuclei to make contact with
other neurons and to create an intercellular web
during the late prenatal and mainly early postna-
tal period. In the rat SCN, very sparse synapses
may be observed at E19–E22, but not earlier, so
indicating that fetal SCN cells are virtually devoid
of synapses (Moore and Bernstein, 1989).
Similarly, astroglia were only sparse in the SCN
during the embryonic stage (Munekawa et al.,
2000). Therefore, during the prenatal period,
neurogenesis of the SCN is completed, but the
multilevel intercellular coupling may not yet be
functional due to the lack of reasonable number
of synapses. The presence of a functional network
in the SCN seems to be one of the basic
mechanisms of how the SCN generates circadian
rhythms. Interestingly, the network has been
recently attributed to maintenance of noise-
induced stochastic circadian rhythms that may
arise as its emergent property (Ko et al., 2010;
Mohawk and Takahashi, 2011). It was hypothe-
sized that due to this function, circadian rhythms
may exist even in the absence of cell-autonomous
circadian oscillators (Ko et al., 2010). Accord-
ingly, the population of clock cells with low
degree of intercellular communication, and there-
fore with a low level of the stochastic rhythmicity,
should generate rhythms with lower amplitude
than the population with a high degree of commu-
nication. This is in agreement with the finding
that, during fetal stage, the rodent SCN with an
undeveloped intercellular communication system
exhibited no or only low-amplitude rhythms in
clock gene expression within the population of
the cells (Ansari et al., 2009; Li and Davis, 2005;
Sladek et al., 2004). Moreover, the amplitudes of
the clock gene expression rhythms gradually
increased as a function of the web development
during the postnatal stage and achieved adult-like
levels only when the development was completed
(Kovacikova et al., 2006). These data suggest that
the stochastic rhythms likely develop later during
ontogenesis together with the formation of the
intercellular web as an additional mechanism to
fortify the cell-autonomous rhythms and the clock
rhythmicity. It remains to be deciphered whether
the stochastic oscillations are conditional for the
clock function.
Studies where gene expression was recorded in
real time from SCN slices in vitro have suggested
that the fetal SCN as a whole structure might have
a potential to oscillate. The SCN slices explanted
from 22-day-old fetuses of transgenic rat exhibit
rhythm in Per1:luciferase when cultured in vitro
(Ohta et al., 2008). Interestingly, at this time, only
faint rhythms were detected in the SCN in vivo
(Kovacikova et al., 2006). It is possible that these
luminescence recordings from the SCN slices are

more sensitive than the detection of gene expres-
sion by in situ hybridization. However, it may also
imply that a coherent signal synchronizing the indi-
vidual oscillators is missing in vivo, but when the
cells are explanted, the culture medium and/or
the dissection procedure might be sufficient to syn-
chronize them. This hypothesis was supported by a
recent observation that at E18, various mouse
peripheral tissues exhibit rhythmicity in clock gene
expression in culture but not in vivo (Dolatshad
et al., 2010). Similarly, the time when the
organotypic SCN slice is dissected can influence
the phase of the resulting in vitro oscillations
(Yoshikawa et al., 2005). Importantly, placing the
rat SCN tissue from arrhythmic animals into culture
can even induce a rhythm in vitro (Yoshikawa et al.,
2005). These data underline the importance of the
presence of a synchronizing cue for existence of cir-
cadian rhythmicity at early developmental stages.
Although detectable rhythms in clock gene
expression are absent in the fetal SCN cells
in vivo, the population of cells likely oscillates in
its metabolic activity (Reppert and Schwartz,
1983, 1984), in spite of the fact that an absence
of the rhythm in the fetal SCN was reported in
an earlier report (Fuchs and Moore, 1980). More-
over, the neurotransmitter arginine vasopressin
(Avp) (Reppert and Uhl, 1987) was also found
to be rhythmic in a population of SCN cells dur-
ing the prenatal stage. It means that these
functions must be driven rhythmically in a syn-
chronous manner even though the population of
SCN cells does not evince rhythmicity in clock
gene expression. It is difficult to understand how
this situation arises, especially because at least
some of these rhythms are known to be driven
by the molecular clock mechanism in adults (Jin
et al., 1999). There is a possibility that during
the fetal stage, these rhythms are directly driven
by rhythmic maternal signals independent of the
molecular clockwork. These signals may be medi-
ated by a common pathway, which likely involves
activation of cAMP and phosphorylation of
CREB, because these steps are necessary for
activation of transcription of c-fos, Avp, and
87
vasoactive intestinal peptide (Vip) (Ginty et al.,
1993; Hahm and Eiden, 1998; Iwasaki et al.,
1997; Obrietan et al., 1999). In this context, it is
interesting to point out that maternal melatonin,
whose role in the entrainment of the fetal SCN
is still being questioned, seems to be a likely can-
didate of such rhythmic cue; it acts through this
pathway by inhibiting production of cAMP (for
review, see Hastings, 1997; Vanecek, 1998) and
no direct immediate effect of melatonin on clock
gene expression has been detected thus far
(Poirel et al., 2003). Similarly, the cAMP pathway
is employed by dopamine, which is also a likely
maternal signal (Viswanathan and Davis, 1997;
Viswanathan et al., 1994). If this scenario is true,
it would mean that even though the SCN cells lack
mutually synchronized molecular clocks, they may
respond synchronously to melatonin or any other
mediator using a cAMP-signaling pathway. This
response would appear at any time and would be
independent of the phase of the desynchronized
individual cellular oscillators. In contrast, once
the SCN cells establish their communication web
later in development, the response becomes highly
dependent on the circadian phase (Ginty et al.,
1993; Shearman et al., 1997; Shigeyoshi et al.,
1997). Therefore, it is plausible to hypothesize that
the cAMP signaling might play a role in entraining
the phases of various rhythmical processes in the
individual cells even before their clocks are mutu-
ally synchronized. Interestingly, once functional
synapses are formed during postnatal stages, the
cAMP-signaling pathway is likely to be involved
in coupling and strengthening the oscillation of
molecular core clock mechanisms (O’Neill et al.,
2008). It remains unclear why the molecular clock
mechanism lacks this property during the syn-
apse-lacking, fetal stage.
The development of the SCN clock gene rhyth-
micity may be dependent on the species-specific
degree of fetal maturation at the time of delivery.
In the capuchin monkey, a rhythm in the expres-
sion of Per2 and Bmal1 was reported in the
fetal SCN at the time corresponding to 90% of
gestation (Torres-Farfan et al., 2006), although
88
these rhythms could only be fitted on the basis of
the levels at three time points when the animals
were sampled during the 24-h cycle. For obvious
reasons, using nonrodent models cannot provide
temporal resolution sufficient for reliable deter-
mination of the rhythmicity and phase of the fetal
clock in vivo.
Development of the fetal peripheral clocks
Only a few studies have examined the develop-
ment of peripheral clocks during the prenatal
stage and those have mostly focused on the liver.
In mice, genetic markers of liver cells are first
detected in tissue arising from the primitive gut
between E8.5 and E9 (Cascio and Zaret, 1991).
The early liver primordium originates from endo-
dermal tissue and contains mostly bipotential
liver progenitor cells capable of differentiating
into either hepatocytes or biliary epithelium
(Dabeva et al., 2000; Shiojiri and Mizuno, 1993).
The fetal liver is also the anatomic site of hemato-
poiesis. Hematopoietic progenitor cells, which are
distinct from the hepatoblast lineage, occur in the
liver beginning at E11 (Dzierzak and Medvinsky,
1995). The later phase of fetal development
is characterized by extensive growth of the
organ mass, differentiation of the bipotential
hepatoblasts into mature parenchymal cell types,
and cessation of hematopoiesis (Walker et al.,
2001). At E16, the rat liver is mainly composed
of relatively well-differentiated hepatoblasts. As
liver development progresses, the liver structure
starts to resemble that of the adult (Van Eyken
et al., 1988).
Studies on the development of rhythmicity of
clock gene expression in the peripheral tissue rev-
ealed results similar to those seen in the SCN;
the explanted fetal liver exhibited circadian rhyth-
micity in vitro (Dolatshad et al., 2010), but in vivo
expression of most clock genes detected by
RT-PCR did not exhibit significant rhythms
(Dolatshad et al., 2010; Sladek et al., 2007). The
most likely explanation is that the individual
hepatocytes were desynchronized during early
developmental stages because the undeveloped
SCN was not able to synchronize them. Indeed,
in adult animals, ablation of the signaling from
the SCN to the peripheral clocks disrupts the
synchrony among the individual cells, which leads
to gradual loss of the rhythm at the level of the
entire structure (Akhtar et al., 2002). However,
this simple explanation was seemingly opposed
by the finding that one of the clock genes, namely,
Rev-erba, exhibited high-amplitude rhythms in the
fetal rat liver (Sladek et al., 2007). Theoretically,
there might be two explanations: (i) the fetal
hepatocytes are already synchronized at the fetal
stage but their molecular clockwork is not yet fully
functional or (ii) the fetal hepatocytes are not syn-
chronized and Rev-erba expression is selectively
driven rhythmically and synchronously by putative
external signals derived from the mother; this pro-
cess would be analogous to the cAMP-signaling
cues in the fetal SCN (see above). The Rev-erba
gene is likely sensitive to various maternal sig-
naling molecules such as glucocorticoids (Torra
et al., 2000), which can pass from mother to fetuses
despite their partial metabolic degradation in the
placenta (Staud et al., 2006). The latter possibility
seems to be more plausible because, at the cellular
level, a functional clock mechanism has been
shown to exist in peripheral tissues at early prena-
tal stages (Pando et al., 2002; Yagita et al., 2001).
In the rat liver, the expression of Rev-erba peaked
in the subjective night at E20, which was opposite
in phase to the expression in the adult liver
(Sladek et al., 2007). Therefore, while the SCN
clock is born in proper phase with the external
LD cycle, the liver clock is born with a clock gene
cycling in the opposite phase (Fig. 2). This differ-
ence might be an adaptation of the peripheral
clock to the diurnal pattern of breast feeding in
nocturnal animals (see below). It would be inter-
esting to determine whether peripheral clocks also
develop at an opposite phase in diurnal animals
and to compare the development of the circadian
clock in the liver with a clock located in another
peripheral tissue that is not directly related with
feeding, such as the lung or heart. Currently
 89

SCN Liver
Per1 Bmal1 Per1 Rev-erba

Fetal

P2

P10

P30

AD

Fig. 2. Schematic drawings of the development of circadian clocks in the rat. Daily profiles of clock genes Per1 and Bmal1 mRNA
in the SCN and Per1 and Rev-erba in the liver are depicted in fetuses; pups at postnatal day 2 (P2), P10, and P30; and adult rats.
X-axis represents daytime with the shaded area defining night hours. Y-axis represents relative mRNA levels. Drawings are
based on results published previously (Kovacikova et al., 2006; Sladek et al., 2004, 2007) and are adapted according to Sumova
et al. (2008) (with permission).

available data on the development of the clock
gene expression rhythms also lead to speculation
that, in the liver, the rhythmic expression of
Rev-erba might trigger the newly appearing
rhythms in clock gene expression during the post-
natal stage because a constant phase relationship
between the rhythms in Rev-erba and other clock
genes is maintained during all the developmental
stages studied (Sladek et al., 2007).
In addition to the liver, circadian clock gene
expression has also been studied in the fetal heart
and kidney (Dolatshad et al., 2010). Similar to the
liver, both tissues exhibited a circadian rhythm in
Per2:luciferace in vitro, but no rhythm in Per2 and
Bmal1 expression was detected in vivo (Dolatshad
et al., 2010).
Entrainment of the clocks during the fetal stage
The above summarized data suggest that develop-
ment of the molecular clock mechanism during
the prenatal stage is still not fully understood.
Specifically, a possibility exists that detection of
a rhythm at the cell population level in the unde-
veloped central and peripheral clocks might not
a priori mean that the molecular oscillations in
the individual cells of the clocks are mutually syn-
chronized. A putative external rhythmical cue
derived from the maternal environment might
impinge on the desynchronized clock cells and
induce or reduce non-clock gene expression in a
rhythmical manner according to its appearance
without a change in the phase of the cycling cells
(Fig. 3). If this hypothesis is true, one might fur-
ther speculate that, in the SCN, this imposed
rhythmicity may help to set the phase to which
the whole population of cellular oscillators fixes
during development once the intercellular web is
formed. This scenario could also explain why the
clock may shift due to prenatally imposing cues
even though the individual cells are not synchro-
nized. Under such conditions, the maternal cues
likely affect the desynchronized cells at any time
90

Fig. 3. Hypothesis on development of the synchronized circadian rhythms in the SCN during ontogenesis. During the fetal stage, the
individual oscillators are not interconnected, and therefore, no circadian rhythm in clock gene expression can be detected in
population of the SCN cells. Putative external rhythmic cues derived from the maternal environment might impinge on the
desynchronized clock cells and induce or reduce a non-clock gene expression (e.g., Avp, c-fos) in a rhythmical manner according
to their appearance without a change in the phase of the cycling cells. Apparently, such effects may appear any time of the day.
During the late prenatal and early postnatal stage, the SCN oscillators become gradually interconnected via a web of synapses.
Due to the gradual synchronization, the clock gene expression begins to exhibit low-amplitude rhythms, which may fortify
expression of clock-controlled genes (e.g., Avp). The maternal or external cues may entrain the oscillators only during a specific
window of time according to the development of the gating mechanism. During the late postnatal period, both the intercellular
communication and the gating mechanism are fully developed and achieve the adult-like stage, resulting in high-amplitude
oscillations of the clock and clock-controlled gene expression (for details, see text).

of the day, while in the case of a highly synchroni-
zed population, the effect would be gated to a
specific time window, as happens in adults. This
might be the reason of a lack of evolutionary
pressure to develop intercellular communication
among the fetal SCN neurons. There are numer-
ous pieces of experimental evidence that the
phase of the developing SCN clock may already
be set prenatally. The evidence comes from ear-
lier studies in which the phase of the fetal clock
was extrapolated from the phase of behavioral
activity when it developed in pups postnatally
(Davis and Gorski, 1988; Reppert and Schwartz,
1984; Viswanathan and Davis, 1997; Weaver and
Reppert, 1987). More recently, a shift of the fetal
SCN by maternal cues has been demonstrated by
the detection of the phase of gene expression
rhythms in the SCN of rat pups sampled immedi-
ately after birth, which more closely reflects the
phase of the fetal clock (El-Hennamy et al.,
2008). This study explored the dynamics of fetal
entrainment and demonstrated that several days
elapse between the time when the maternal clock
sends its resetting signal and the time when the
fetal clock entrains accordingly. As mentioned
above, clock gene expression profiles exhibit
low-amplitude rhythms at this developmental
stage; therefore, rhythms in c-fos and Avp expres-
sion were used as reliable markers of the activity
of the fetal SCN cells.
The nature of the maternal cue(s) that entrain
the fetal clock has been studied for decades,
and melatonin and dopamine have been the ever-
green candidates as previously mentioned. Mela-
tonin is a plausible molecule for many reasons,
and studies that provide evidence supporting
or excluding its role have been previously
summarized elsewhere (Bellavia et al., 2006;
Davis, 1997; Seron-Ferre et al., 2007). Dopamine
likely has the opposite effect than melatonin on
signaling pathways in the fetal SCN (Weaver
et al., 1992). Thus, melatonin is thought to medi-
ate the subjective night and dopamine the subjec-
tive day, regardless of when they are present (for
a review, see Weinert, 2005). In fact, this putative
91
mechanism supports the hypothesis mentioned
above regarding imposed rhythmicity on the
desynchronized fetal SCN cells.
Although the participation of the melatonin/
dopamine couple in fetal entrainment seems
likely, it is not the only possible mechanism by
which the fetal clocks perceive temporal cues.
Other likely candidates are related to maternal
behavior and may involve temperature or feeding
cycles. Exposure of pregnant rats with surgically
removed SCN to a restricted feeding regime was
able to shift the phase of the SCN clocks in their
fetuses as measured by a shift in the phase of
the drinking activity of pups after weaning
(Weaver and Reppert, 1989). Recently, another
study demonstrated that the fetal clock is
entrained by a maternal-restricted feeding regime
by measuring the phase of gene expression in the
SCN of pups immediately after birth; however,
this entrainment only occurs when the maternal
SCN clock is disturbed due to prolonged expo-
sure to constant light (Novakova et al., 2010). In
contrast, when the pregnant rats were maintained
under a standard LD cycle, the restricted feeding
had no effect on the phase of gene expression
profiles in the SCN of newborn rats. This result
did not support the previous finding of Ohta
et al. (2008), who demonstrated the effect of
maternal-restricted feeding on the phase of the
rhythms in Per1:luciferase in the SCN slices from
fetuses of transgenic rats maintained under the
LD cycle. It remains unclear whether differences
in the experimental design of these two studies
accounted for the different results. Nevertheless,
as the restricted feeding entrained the in vivo
fetal clock only when the maternal SCN was
absent or nonfunctional, the outcome of these
studies questioned the relative entraining potency
of the signaling from the maternal SCN and
restricted feeding to the fetal SCN. Apparently,
the signaling from the feeding regime did not
override the signaling from the maternal SCN
and may thus be redundant. Moreover, the results
of Novakova et al. (2010) provided evidence that
the entrainment of the fetal SCN is possible even
92
without melatonin. Because the pregnant rats
exposed to a restricted feeding regime were
maintained under constant light during the entire
pregnancy, their endogenous melatonin was likely
suppressed, and melatonin could not serve as a
messenger. However, this redundant mechanism
of fetal SCN entrainment by food remains
unknown. The redundancy of the mechanism also
suggests that formal properties of the fetal SCN
clock more closely resemble the adult SCN than
the adult peripheral clocks. The only difference
is in the way how information about the LD cycle
is conveyed to the adult or fetal SCN. While pho-
tic cues directly delivered from the retina are
dominant for the entrainment of the adult SCN,
the information about the LD cycle is transduced
to the fetal SCN by the putative signaling from
the maternal SCN.
Development of the circadian system under
maternal care
SCN develops postnatally
With the postnatal progression of synaptogenesis
in the SCN (Moore, 1991), the coupling among
individual cellular oscillators is strengthened and
reaches an adult-like state at P10. At the same
time, the number of astrocytes in the SCN rapidly
increases (Munekawa et al., 2000). Moreover, the
intrinsically photosensitive retinal ganglion cells
that mediate photic information from the retina
begin to innervate the SCN only during this
period (McNeill et al., 2011). Although this inner-
vation occurs postnatally, the axons of those cells
are already present in the optic chiasm during the
fetal stage. The terminals of the intrinsically pho-
tosensitive retinal ganglion cells gradually spread
throughout the retinorecipient zone of the SCN,
and the dynamics of this process spatially and
temporally correlate with the pattern of c-fos pho-
toinduction in the mouse and rat SCN (Mateju
et al., 2009; Sekaran et al., 2005). Therefore, dur-
ing the early postnatal period, the rodent SCN
undergoes substantial reorganization and func-
tional specialization that results in formation of
various cell subpopulations. Because intercellular
coupling has been shown to have a significant role
in the clock mechanism (reviewed by Mohawk
and Takahashi, 2011), it would not be surprising
if the rhythms in clock gene expression as
measured in the population of the SCN cells grad-
ually gain their robustness in parallel with the for-
mation of the intercellular web. Indeed, the
amplitudes of the rhythms in clock gene expres-
sion in the rat SCN gradually increased up to
P10, when they attained an adult-like state
(Kovacikova et al., 2006; Sladek et al., 2004).
Interestingly also in vitro, the amplitudes of
Bmal1:luciferase rhythms in SCN slices from
mouse pups at P6 were much smaller than those
in SCN slices from adult mice (Nishide et al.,
2008). Although both of these processes are
remarkably correlated, there is no direct evidence
thus far that they are mutually conditional.
Maternal entrainment of the SCN clock
after birth
Entrainment of a pup’s SCN with the external LD
cycle already occurs during the prenatal period
through maternal cues (see above). The pups
are thus born with their SCN clocks already
set to the proper phase, and during the early post-
natal period, they only need to maintain the
phase until their own SCN clock becomes fully
entrainable by light. The postnatal maternal
entrainment gradually loses its significance
(Reppert and Schwartz, 1986; Shimoda et al.,
1986; Viswanathan, 1999) with the increasing
probability of incidental exposure of pups to
external light. In rats, this might happen after
about P10 when they begin to move around the
nest in the dark burrow even though their eyes
are opened only around P15 (see below). During
the period when the pups are in darkness in the
nest, they are synchronized exclusively by the
maternal circadian system. The mechanism of

how the mother entrains her pups is not yet
understood, although various candidate pathways
have been suggested.
During the period of lactation, the mother
keeps behaving like a nocturnal animal and
leaves the nest during most of the night to forage
(Viswanathan, 1999). During the daytime, she
stays in the nest and takes care of her pups by
making contact with them and keeping them
warm and satiated. Thus, all these mutually cou-
pled factors, such as social contact, temperature,
and food, are presented to the pups rhythmically
and may entrain their circadian system. Melato-
nin which is present in milk (Illnerova et al.,
1993) and may thus theoretically provide the
pup’s SCN with temporal information likely does
not play such a role (Rowe and Kennaway, 2002).
Only few studies have focused on the effect of
these maternal factors on the molecular clock in
the SCN. It has been demonstrated that if a foster
mother rears pups that are blind since birth under
the opposite LD cycle than the original mother,
this has only marginal effect on the rhythms of
Per1 and Per2 expression in the pups SCN at P6
(Ohta et al., 2002). This implies that the opposite
phase of maternal care since birth was not able to
reprogram the pup’s SCN rhythmicity. These
results suggest that the role of maternal entrain-
ment during the early postnatal period is only to
keep the phase of the clock that was already set
prenatally. However, it is not clear whether the
same result would be obtained under a natural sit-
uation where the mother needs to go a long dis-
tance from her pups to chase for food and
would therefore need to leave them alone for a
much longer time than in laboratory conditions
where they remain in the same cage and the food
is available ad libitum. Nevertheless, the maternal
entrainment, though likely of low potency, is
present during the early postnatal period because
the pups do not free-run but remain synchronized
with their mothers. The inability of the circadian
clock to reverse its phase due to maternal care
provided in the opposite phase, as shown in the
above-mentioned foster experiment, is likely
93
related to the fact that during the early postnatal
period, the SCN is under intensive structural
development when intercellular communication
is formed and the cells become mutually coupled.
With creation of this web, the SCN cells might
lose their round-the-clock responsiveness, and
the rhythmical maternal cues might become effec-
tive only during a specific window of time similar
to mature animals.
A more potent effect on the pup’s circadian
system results from the deprivation of maternal
care (Ohta et al., 2003). It has been shown in
blind pups that are subjected to a complete
absence of their mothers from their home cages
during the daytime from birth until P6. Due to
this maternal deprivation, circadian rhythms in
Per1 and Per2 expressions were completely
phase-reversed in the SCN of these pups. This
experimental arrangement appears to have been
a much stronger entraining cue to the neonatal
SCN than the previously described cross-
fostering. Under the cross-fostering experiment,
a mother still provided care but it was reversed
in phase when compared with the pup’s clock.
However, under the deprivation experiment, the
pups were more extensively deprived of their
mother’s care because the mother was removed
during the daytime when she would take care of
the pups and returned at the nighttime when she
would normally not take care of them. It can be
expected that the nighttime care of pups was not
appropriate. Therefore, this latter condition was
life-threatening for the pups which were thus
likely deprived from food, warmth, and social
contact. Thus, it is not surprising that such a situ-
ation activated stress-related gene expression in
the pups (Ohta et al., 2003). Whether the activa-
tion of this pathway was responsible for the phase
reversal of the pup’s clock remains to be deter-
mined, but data from studies in adults suggest
the feasibility of such a possibility (Reddy et al.,
2007; Yamamoto et al., 2005).
The effect of experimentally induced temporal
restriction of maternal care on the pup’s circadian
system (as described above) resembles the situation
94
that naturally occurs in rabbits. During early
development, the rabbit pups live in complete
darkness in subterranean burrows. In contrast to
rats, rabbit mothers visit their pups only once a
day to nurse them for a brief period of < 5 min
(Zarrow et al., 1965). It is vitally important for
the pups not to miss their chance to be fed, and
therefore, they present a behavioral arousal in
anticipation of the feeding. The time of feeding
is a strong entraining cue for the rabbit pups,
and it can entrain many physiological variables
such as core body temperature and hormonal,
metabolic, and neural functions independent of
the time of the day (Caba and Gonzalez-Mariscal,
2009). However, similar to the previously de-
scribed foster mother experiment in rats (Ohta
et al., 2002), only a small shift in the rhythm in
the SCN (as measured by PER1 immunoreactiv-
ity) was found in pups that were fed during
the day when compared with those that were fed
during the night (Caba et al., 2008). Therefore,
the feeding-induced arousal in rabbit pups was
entrained by a food-entrainable oscillator rather
than by the central clock in the SCN.
It seems that maternal care attains a significant
efficiency as an entraining cue for the pup’s circa-
dian system only under such conditions when it
becomes restricted and chancy. Therefore, it is
highly probable that more than one pathway is
involved in maternal entrainment and the mecha-
nism is likely very complex.
Maternal entrainment of the peripheral clocks
after birth
During the postnatal period, molecular clock
mechanism seems to mature at different rates in
various peripheral tissues. Evidence for this the-
ory comes from both in vivo and in vitro studies
that share some similar, but not identical, out-
comes. In studies using an in vivo approach, it
was shown that the rhythms in most clock
genes started gradually during postnatal develop-
ment. In the brain, ontogenesis of the mouse
striatal molecular clock machinery was studied
(Cai et al., 2009). The data demonstrated that at
P3, none of the measured clock genes (Bmal1,
Clock, Npas2, Cry1, Per1, and Rev-erba)
exhibited a circadian rhythm. At P14, only Rev-
erba and Npas2 expression was driven rhythmi-
cally, and the rhythms of all these clock genes
were at least borderline significant only at P60.
In peripheral organs, development of clock gene
expression profiles was studied in detail in both
the liver and the heart; information about other
tissues is sparse. In the rat heart and liver, the
rhythms of all clock genes studied were expressed
only around P10 and P20, respectively (Sakamoto
et al., 2002; Sladek et al., 2007). The only excep-
tion was the rhythm in Rev-erba expression which
was present in the liver since the embryonic stage
(Sladek et al., 2007). However, the adult-like
waveform and amplitude of the rhythms were
present in most of the clock genes in the liver only
around weaning (Sladek et al., 2007). Studies
employing transgenic rats and measuring the
Per1 expression in vitro revealed that circadian
rhythms in the expression of this gene were pres-
ent in pineal, liver, and thyroid tissues since birth.
In the adrenals, the rhythm was present but
increased its amplitude until P25, and in the lungs,
the rhythm was absent until P12 (Yamazaki et al.,
2009). Thus, the lung does not seem to be capable
of expressing an autonomous rhythm in Per1:
luciferase until P10, although the expression is
transiently inducible by addition of forskolin or
serum to the incubation medium, and all other
genes of the clockwork are present (Yamazaki
et al., 2009). However, the authors do not report
whether the other clock genes were expressed
rhythmically in the “arrhythmic” lungs. This pos-
sibility arises from in vivo data published so far
by several groups that clearly demonstrates that
the rhythmic expression of each individual clock
genes may not occur at the same time during
development (Sakamoto et al., 2002; Sladek
et al., 2007). These results are difficult to explain
in the context of the current view on the mecha-
nism of how the molecular clockwork operates
and could merely reflect differences in assay sen-
sitivity for various genes.

Both in vivo and in vitro studies confirmed that,
during development, the phase of the clock gene
expression rhythms in some tissues gradually
shifted until the adult stage was achieved. The
shifts in the phases of the rhythms were detected
in the heart (Sakamoto et al., 2002), liver (Sladek
et al., 2007; Yamazaki et al., 2009), and pineal
gland (Yamazaki et al., 2009). These data indicate
that the circadian clocks in these tissues are
entrained by maternal cues likely related to
maternal behavior and breast feeding. They likely
reflect the fact that during lactation, the newborn
pup of a nocturnal animal is fed as a diurnal ani-
mal (see above), and the “nocturnality” appears
with increasing independence from maternal milk
during gradual weaning. Indeed, the circadian
clock in the liver was completely reversed by
P30 when weaning was complete. Importantly,
the SCN clock remains in the same phase during
this developmental period (Fig. 2). Thus, the
selective sensitivity of peripheral tissues to food
that is observed in the adult (Damiola et al.,
2000; Stokkan et al., 2001) is already present
immediately after birth and likely represents the
adaptation of the organism to the developmen-
tally dependent timing of food availability.
In summary, the available data are in favor of
the view that the synchronized rhythms of clock
gene expression develop earlier in the central
SCN clock than in the peripheral oscillator.
A putative role of the developing SCN in the syn-
chronization of the peripheral clocks during the
early postnatal period remains to be determined.
Tissue-specific differences in the programming of
the development of molecular oscillations have
also been suggested. Such differences might help
to elucidate specific roles of the individual compo-
nents of the molecular core clockwork in the SCN
and in peripheral organs during development.
LD cycle entrains the developing
clock in the SCN
As previously mentioned, the mechanism of photic
entrainment of the SCN likely develops once the
95
pups begin to move independently in the burrow
and the probability of their light exposure in-
creases. In rats, photic entrainment was indi-
cated already at P6 (Duncan et al., 1986; Weaver
and Reppert, 1995). The development of this
mechanism is not completely understood. Mor-
phologically, the neuronal projection from the ret-
ina to the SCN develops gradually during the
postnatal period. At P1, the innervation termina-
tes close to the ventral border of the SCN;
later, the area gradually extends and achieves an
adult-like pattern at around P10 (Speh and Moore,
1993). Although it is undeveloped, this innervation
is already functional because both the retina and
the SCN are sensitive to light immediately after
birth (Hannibal and Fahrenkrug, 2004; Leard
et al., 1994; Mateju et al., 2009, 2010), despite the
fact that the eyes are closed until about P15 and
the light is thus perceived only through the eyelids.
The SCN is connected to the retina via ret-
inohypothalamic tract, which is formed by pro-
cesses of a small subset of retinal ganglion cells
containing the photopigment melanopsin (Hattar
et al., 2002; Provencio et al., 2002), and projects
to the retinorecipient zone of the SCN. These cells
are already present within the retina at prenatal
stages in mice and rats; thus, they may transmit
information about external lighting conditions dur-
ing the early postnatal period, that is, well before
the visual photopigments maturate (Fahrenkrug
et al., 2004; Hannibal and Fahrenkrug, 2004;
Sekaran et al., 2005). Recently, it has been shown
that while the image-forming brain targets are
innervated from the retinal ganglion cells prena-
tally, the innervation from the intrinsically photo-
sensitive subset of the cells to the SCN is delayed
and proceeds into postnatal period (McNeill et al.,
2011). The dynamics of the innervation remarkably
correlate with the spread of the retinorecipient area
within the rat SCN during the postnatal period
(Mateju et al., 2009) (Fig. 4). However, despite
the high sensitivity to light, the rodent SCN is likely
not able to be entrained by light during the first
days of life. The exact mechanism of how the func-
tionality develops is not yet known; although it
likely depends on the development of the SCN, a
96
P1
c-fos
Per1
Shell
Per2 Core
OC
Fig. 4. The development of photosensitivity in the rat SCN. In situ hybridization emulsion autoradiographs demonstrating spatio-
temporal distribution of c-fos, Per1, and Per2 expression in SCN sections of rat pups exposed to a 30-min light pulse during
subjective night at postnatal day (P)1, P3, P5, and P10 (for more data, see Mateju et al., 2009).
role for retinal development cannot be excluded.
The development of photic entrainment is difficult
to follow in vivo because the ability to read a phase
shift in response to light exposure requires using a
reliable marker that reflects the functional state of
the clock both at the time of the light exposure
and after the phase shift. However, most overt
rhythms are not yet present at these early develop-
mental stages. Also, dissection of the immediate
response from the developmental change of the
variable is experimentally difficult. An insight into
this process was provided in a study where develop-
ment of a mechanism gating the SCN photosensitiv-
ity to a time-specific window was used as a marker
of photic entrainment (Mateju et al., 2009). In
adults, light entrainment is accomplished so that
light induces signaling pathways that are responsi-
ble for induction of expression of the clock genes
that are involved in entrainment of the molecular
clockwork, that is, Per1 and Per2 (for a review,
see Meijer and Schwartz, 2003). Importantly, this
response is possible only during a restricted window
of time when the endogenous expression of these
genes is low, that is, during the subjective night
(Albrecht et al., 1997; Shearman et al., 1997;
Shigeyoshi et al., 1997). Mateju et al. (2009) demon-
strated that immediately after birth, the SCN
responds to light pulses delivered any time of the
P3 P5 P10
day by induction of Per1 gene expression; thus,
the response in the SCN is not yet gated by the
clock. The gating mechanism develops gradually
between P3 and P5 but was only fully completed
at P10 (Mateju et al., 2009). Interestingly, the devel-
opment of photoinducibility of Per2, its spatial dis-
tribution, and its gating mechanism differed from
Per1 (Mateju et al., 2009) and might thus be con-
trolled by a different mechanism from the begin-
ning. The development of the mechanism that
gates the photoinducibility of Per1 and Per2 to sub-
jective night depends on the development of the
retinal innervation of the SCN, but it also likely
depends on development of intercellular communi-
cation among the individual SCN oscillators (see
above). It appears that this gating mechanism
develops in parallel with the developmental stage
when population of SCN cells begins to express
Per1 and Per2 rhythmically, which likely occurred
because these cells attained synchrony (Mateju
et al., 2009). It seems that the development of the
time-specific response to light goes hand-in-hand
with the decline in spontaneous levels of expression
during the night in control animals. Again, these
data suggest that for the SCN to fulfill the role of
a central clock, postnatal maturation must extend
far beyond the development of the rhythmical con-
trol of activation of a clock gene promoter within

one cell. This view is even more obvious when we
consider the development of the mechanism by
which the SCN codes the day length, that is, photo-
period. Apparently, photoperiodic modulation of
the SCN is highly dependent on communication
among individual cells and among populations of
cells (Schaap et al., 2003; VanderLeest et al.,
2007). In the adult rat SCN, the photoperiod
modulates markedly the rhythm of photosensitivity
in the ventrolateral SCN (Sumova et al., 1995) and
rhythm of spontaneous neuronal activity in the
dorsomedial SCN (Sumova et al., 2000). The photo-
period also modulates the waveform of the clock
gene expression profiles as measured in a popula-
tion of SCN cells within the medial part of the
rostro-caudal axis (Sumova et al., 2003), and the
mutual phasing of these profiles measured in
the rostral and caudal parts of the SCN (Hazlerigg
et al., 2005; Sosniyenko et al., 2009, 2010). The
photoperiod begins to gradually modulate the
waveforms of the clock gene expression profiles in
the medial part of the SCN from P10 until weaning
(Kovacikova et al., 2005). The development of pho-
toperiodic modulation of the mutual phasing
among the rostral and caudal parts of the SCN has
not been studied.
Effect of disturbing the circadian system during
early ontogenesis may persist through adulthood
From data summarized above, it appears that the
circadian system is sensitive to disturbances in the
external environment during early developmental
stage; no matter whether they are related to
changes in LD cycles or to the feeding regime.
It is still unclear whether these changes may have
long-lasting effects on the organism and whether
they may persist till adulthood.
Some results indicate that light experience during
the early postnatal period may affect clock function
and clock output in adulthood. For example, expos-
ing pups to abnormal lighting conditions, such as
constant light during lactation, has both acute and
lasting effects on circadian organization that result
97
in an increased vulnerability to disruption by con-
stant light (Cambras et al., 1997; Canal-Corretger
et al., 2000; Ohta et al., 2006; Smith and Canal,
2009). However, the disrupted rhythms may be
restored by exposure to LD cycles (Ohta et al.,
2006). Similarly, the effect of pharmacological inter-
vention of the circadian clock during the fetal/early
neonatal stage was completely eliminated by
subsequent entrainment to a LD cycle (Lorelli
et al., 2011). These data suggest a remarkable resil-
ience of the central clock in the SCN to disturbances
in the external environment during early develop-
mental stages. In contrast, a recent report suggested
that although the presence of seasonal photoperiods
during perinatal period does not have a long-lasting
effect on the network-level timing of neuronal
rhythms in the SCN, it might have a persistent effect
on waveform and periods of individual SCN
neurons (Ciarleglio et al., 2007).
Recent data suggest that changes in perinatal
nutrition may induce long-lasting alterations of
the circadian control of food intake and metabo-
lism. The effect of nutrient restriction during this
period had a significant effect on the circadian
expression of genes regulating food intake in the
hypothalamus and on energy metabolism in
the liver, which persisted in rats after weaning
(Orozco-Solis et al., 2010). These data indicate that
the circadian system undergoes nutritional pro-
gramming during perinatal development and that
such programming may contribute to the develop-
ment of metabolic disorders in adulthood. Vice
versa, disruption of circadian rhythmicity during
gestation resulted in metabolic consequences for
adult offspring (Varcoe et al., 2011). Because these
data are likely translatable to human medicine,
they reinforce the importance of maternal nutri-
tion together with integrity of the circadian system
during the perinatal period.
Conclusion
Studies focused on unraveling the basic prin-
ciples of circadian clock development during
98
early ontogenesis have uncovered an enormous
plasticity of the whole system, which seems
dependent on the development of its individual
bricks of which it is built. These studies also point
out the necessity of a detailed understanding of
the processes by which the circadian system
develops. This importance is underscored by the
findings that intervention of the system by various
cues during development may permanently impact
the temporal control of physiological functions
in adulthood. Such interventions become more
and more frequent in the modern lifestyle that
has been adopted during the past decades. More-
over, pregnant women often are not excluded from
the socio-economic pressure that underlies some of
these interventions. It has been widely recognized
that the circadian system is involved in the tempo-
ral control of most physiological functions and
that distortion of the temporal regulation may
promote various diseases including cancer and psy-
chiatric, metabolic, and cardiovascular disorders.
Therefore, understanding how interventions dur-
ing the perinatal period impact on the circadian
system may help in the prevention and treatment
of many disorders.
Acknowledgments
The study was supported by grant nos. 305/09/
0321, P303/11/0668, P303/12/1108, NT11474-4/
2010, and LC554, and by Research Project
AV0Z 50110509.
Abbreviations
Avp arginine vasopressin
Bmal1 clock gene Brain and muscle Arnt-
like protein
Clock clock gene Circadian Locomotor
Output Cycles Kaput
CREB cyclic AMP response element-binding
protein
Cry1 clock gene Cryptochrome 1
Cry2 clock gene Cryptochrome 2
E embryonic day
LD light/dark
SCN suprachiasmatic nucleus
P postnatal day
Per1 clock gene Period 1
Per2 clock gene Period 2
Rev- clock gene Reverse erb A alpha
erba
Rora clock gene RAR-related orphan
receptor A
Vip vasoactive intestinal peptide
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 6

When does it start ticking? Ontogenetic

development of the mammalian circadian system

Elmar Christ{,*, Horst-Werner Korf{,{ and Charlotte von Gall{,{,1

{
Dr. Senckenbergische Anatomie II, Fachbereich Medizin, Goethe-Universität Frankfurt,
Frankfurt am Main, Germany
{
Dr. Senckenbergisches Chronomedizinisches Institut, Goethe-Universität Frankfurt,
Frankfurt am Main, Germany

Abstract: Circadian rhythms in physiology and behavior ensure that vital functions are temporally
synchronized with cyclic environmental changes. In mammals, the circadian system is conducted by a
central circadian rhythm generator that resides in the hypothalamic suprachiasmatic nucleus (SCN)
and controls multiple subsidiary circadian oscillators in the periphery. The molecular clockwork in
SCN and peripheral oscillators consists of autoregulatory transcriptional/translational feedback loops
of clock genes. The adult circadian system is synchronized to the astrophysical day by light whereas
the fetal and neonatal circadian system entrains to nonphotic rhythmic maternal signals. This chapter
reviews maturation and entrainment of the central circadian rhythm generator in the SCN and of
peripheral oscillators during ontogenetic development.

Keywords: circadian clock; ontogenesis; development; suprachiasmatic nucleus; pars tuberalis; adrenal
gland; peripheral clock.

The circadian system and its molecular clockwork

Based on the rotation of the Earth around its axis,

*Corresponding author.
Tel.: þ49-069-6301-83156; Fax: þ49-069-6301-6017
E-mail: E.Christ@med.uni-frankfurt.de
1
Current address: Zentrum für Anatomie und Hirnforschung,
Institut für Anatomie II, Universitätsklinikum Düsseldorf, Life
Science Center, Düsseldorf, Germany
the light conditions change rhythmically with a
period length of 24 h (day). During phylogeny,
circadian systems have evolved in nearly all living
organisms to anticipate these rhythmic changes in
environmental light conditions (Dunlap, 1999;
Dvornyk et al., 2003; Johnson and Golden,
1999) and to drive rhythms in physiology and

http://dx.doi.org/10.1016/B978-0-444-59427-3.00006-X 105
106
behavior. In mammals, the circadian system is
organized in a hierarchy of multiple oscillators.
The central circadian pacemaker is located in the
suprachiasmatic nucleus (SCN) of the anterior
hypothalamus. The SCN integrates light informa-
tion conveyed by the retinohypothalamic tract
(RHT) and coordinates peripheral oscillators
distributed throughout the body. In each SCN
neuron, a molecular clockwork consisting of
autoregulatory transcriptional/translational feed-
back loops of clock genes drives rhythmic cellular
properties and rhythmic output signals with a
circadian period length ( 24 h) (Okamura et al.,
2002; Reppert and Weaver, 2002). The transcrip-
tional activators in the core molecular clockwork,
CLOCK/NPAS2 and BMAL1 are characterized
by a basic helix–loop–helix DNA-binding domain
and two Per-Arnt-Sim protein interaction domains
that allow for the formation of heterodimers.
These heterodimers activate gene expression
through E-box (like) enhancer elements located
in the promoter region of the clock genes Per1,
Per2, Cry1, and Cry2 and of so-called clock-
controlled genes. The protein products of the
Per and Cry genes form repressor complexes
that comprise additional proteins such as casein
kinase 1 e and d. After translocation into the
nucleus, the repressor complex inhibits CLOCK/
NPAS2:BMAL1-mediated transcription and thus
suppresses its own transcription. A new cycle
starts after hyperphosphorylation, ubiquitination,
and proteasomal degradation of the repressor
complex. This core negative feedback loop is
modulated by accessory feedback loops that
involve the orphan nuclear receptors REV-ERBa
and RORa. By binding to ROR enhancer
elements REV-ERBa and RORa control expres-
sion of Bmal1. As CLOCK and BMAL1 are con-
stitutively present and bound to the E-box
element throughout the circadian cycle, the circa-
dian rhythm in gene expression depends mainly
on the presence of the repressor complex (von
Gall et al., 2003). The molecular clockwork genet-
ically determines the endogenous circadian period
length that varies with the species, for example, in
Syrian hamster it is 22.5 h (Ralph and Menaker,
1988), in laboratory mouse strains 23.6 h
(Schwartz and Zimmerman, 1990; von Gall et al.,
1998), and in humans 25 h (Aschoff, 1965). Period
and phase of the circadian oscillation is entrained
to the external rhythm by so-called zeitgebers
(see below). The molecular clockwork controls
the rhythmic expression of clock-controlled genes
such as arginine vasopressin (AVP) (Jin et al.,
1999), an important rhythmic SCN output signal
(see below). Importantly, molecular clocks are
not restricted to the SCN, but are present in
peripheral oscillators such as other brain regions
and peripheral organs (Balsalobre, 2002;
McNamara et al., 2001; Oishi et al., 1998; Sun
et al., 1997; Yoo et al., 2004; Zylka et al., 1998).
Robust sustained oscillation of clock gene expres-
sion in the SCN and in peripheral oscillators
depends on intercellular coupling (Liu et al.,
2007). Within the SCN paracrine signaling via
neuropeptides such as vasoactive intestinal peptide,
AVP, and gastrin-releasing peptide provides pow-
erful interneuronal communication (Maywood
et al., 2011).
Synchronization mechanisms within the adult
circadian system
In the adult circadian system, phase and period
length of the SCN molecular clockwork is
entrained to the environmental day/night cycle
by light. This entrainment is essential for normal
physiology because long-term internal temporal
desynchronization causes sleep disorders and
chronic illnesses, such as cardiovascular disease,
metabolic syndrome, and cancer (Hastings et al.,
2003). Light during early or late night is a strong
stimulus for delaying or advancing the phase of
the molecular clockwork, respectively (Reppert
and Weaver, 2002). Light information is transmit-
ted to the SCN by glutamate and the neuropep-
tide PACAP released from specialized retinal
ganglion cells projecting into the SCN (Reppert
and Weaver, 2002). The activation of glutamate

receptors during night activates the transcription
factor Ca2 þ/cAMP-responsive element-binding
protein (Gau et al., 2002), which activates the
expression of mPer1 and mPer2 in the SCN
(Albrecht et al., 1997; Gau et al., 2002; Shearman
et al., 1997). The photic induction of Per expres-
sion is crucial for the daily adjustment of the
endogenous rhythm of the molecular clockwork
because light during early night delays the rhythm
by activating Per in its declining phase, whereas
light during late night leads to a precocious
increase in Per expression, resulting in a phase
advance (Hastings et al., 2003).
The SCN transmits its phase and period to
peripheral oscillators via paracrine, neuronal, and
neuroendocrine output pathways. Behavioral
rhythms such as circadian locomotor activity
depend on diffusible signals from the SCN such as
transforming growth factor (Kramer et al., 2001),
cardiotrophin-like cytokine (Kraves and Weitz,
2006), and prokineticin 2 (Cheng et al., 2002;
LeSauter et al., 1996; Ralph et al., 1990; Silver
et al., 1996). In contrast, the circadian control of
neuroendocrine rhythms requires intact neuronal
projections from the SCN (Kalsbeek et al., 2000;
LeSauter et al., 1996; Perreau-Lenz et al., 2003).
Vasopressinergic SCN neurons provide an impor-
tant output pathway of the SCN: they project to
the paraventricular nucleus (PVN) of the hypothal-
amus. The PVN is a prime center for the control of
both the autonomic nervous system and the neuro-
endocrine system. The autonomic nervous system
that controls body homoeostasis originates from
the dorsal parvocellular portion of the PVN which
contains presympathetic and preparasympathetic
neurons (Kalsbeek et al., 2006; Vrang et al.,
1995). The presympathetic neurons send their
axons to the intermediolateral column of the upper
thoracic spinal cord, where they contact sympa-
thetic preganglionic neurons. Postganglionic nerve
fibers originating from the superior cervical ganglia
regulate the nocturnal secretion of melatonin via
the rhythmic release of norepinephrine (reviewed
by Klein et al., 1991). GABAergic neurons in the
SCN are involved in the inhibition of melatonin
107
release by light as well as in the control of the circa-
dian rhythm in melatonin synthesis (Kalsbeek
et al., 2006). Melatonin is an important rhythmic
endocrine signal within the circadian system
and can adjust the phase of the SCN rhythm gener-
ator (reviewed by von Gall et al., 2002b). Melato-
nin influences insulin production (reviewed by
Peschke, 2008), modulates pain (reviewed by
Ambriz-Tututi et al., 2009), synchronizes slave
oscillators such as the pars tuberalis (PT) of the
anterior pituitary (Jilg et al., 2005; Messager et al.,
2000; Unfried et al., 2009, 2010; von Gall et al.,
2001, 2005), and is believed to play a role in
maintaining sleep throughout the night (reviewed
by Cajochen et al., 2003). In addition, the SCN con-
trols sensitivity of peripheral glands to pituitary
hormones (Buijs et al., 2003) and the circadian
rhythm in plasma glucose levels (Kalsbeek et al.,
2004) via the autonomic nervous system.
Neurons in the medial parvocellular PVH syn-
thesize corticotropin-releasing hormone (CRH)
which is secreted into the portal vasculature
of the hypophysis (Kalsbeek et al., 2004) and
regulates the secretion of adrenal corticosteroids
via the hypothalamo-pituitary-adrenal (HPA)
gland axis (Buijs and Kalsbeek, 2001). Vaso-
pressinergic SCN output signals inhibit CRH
release from the PVN and thus control the diur-
nal glucocorticoid (GC) rhythm (Kalsbeek et al.,
1992; Tousson and Meissl, 2004). In mice with a
corrupted molecular clockwork, the regulation of
the HPA axis is defective (Oster et al., 2006b).
Light can affect GC release from the adrenal
by influencing either the HPA or the SCN-
sympathetic nervous system (Ishida et al., 2005).
Moreover, the molecular clockwork in the
adrenal gland gates GC production in response
to adrenocorticotropin (ACTH) and controls
rhythmic expression of a variety of genes involved
in corticosterone biosynthesis (Oster et al., 2006a,
b). GC levels show a strong daily oscillation in
both laboratory rodents and humans, and repre-
sent an important phase entrainment signal within
the circadian system (Balsalobre et al., 2000). In
addition to rhythmic cues from the SCN, external
108
rhythmic cues such as food intake also provide a
strong entrainment signal for peripheral
oscillators (Bur et al., 2010; Damiola et al., 2000;
Schibler et al., 2003; Shibata et al., 2010; Stephan,
2002; Yamazaki et al., 2009).
Development of the central endogenous
rhythm generator
A complex and dynamic process of development
prepares the embryo for the living conditions
which it will experience after birth. In mammals,
the embryos and fetuses receive important mater-
nal signals for maturation and synchronization of
the fetal circadian system via the placental sys-
tem. In the early postnatal period, the intense
social interaction with the mother as well as
hormones in the milk still provides rhythmic
maternal signals for the developing circadian sys-
tem. These maternal signals might directly drive
neonatal oscillators until the light input pathway
and the SCN endogenous rhythm generator have
fully developed.
The SCN is formed during the third wave of
hypothalamic neuron generation and develops as
a component of the periventricular cell group.
In rats (mean gestation period: 22 days), SCN
neurons start to develop and differentiate
between embryonic days 14–17 (E14–E17) and
the adult morphology of the SCN is reached
within postnatal day 10 (P10). The ventrolateral
subdivision of the SCN develops around
E14–E16, prior to the dorsomedial subdivision
which develops around E16–E17 (Moore, 1991;
Moore and Leak, 2001; Weinert, 2005). Circadian
rhythms in SCN activity in rodents can be
detected already in utero. The fetal rat SCN
shows circadian rhythms in metabolic activity
monitored by a 2-deoxyglucose uptake (Reppert
and Schwartz, 1984a,b), Avp mRNA expression
(Ansari et al., 2009; Reppert and Uhl, 1987),
and neuronal firing rate between E19 and E22
(Shibata and Moore, 1987; Weinert, 2005). In
melatonin-proficient C3H mice (mean gestation
period: 20 days), a circadian rhythm in Avp
mRNA expression is evident as early as E18
(Ansari et al., 2009). However, it is still a matter
of debate whether Avp expression is rhythmic in
the fetal SCN as heteronuclear Avp RNA, which
is supposedly a more reliable marker of transcrip-
tion rate than mRNA, is not rhythmic until P1 in
rat SCN (Kovacikova et al., 2006).
The expression of clock genes in the fetal SCN
is first detected at E13.5 in hamsters (mean gesta-
tion period: 16 days), at E17 in mice, at E19 in
rats, and at E142 in capuchin monkeys (gestation
period: 180 days). Thus, the molecular clockwork
in the SCN starts to develop at around 90% of
gestation in these species. In hamster SCN, circa-
dian rhythms in clock genes (Bmal1, Per1, and
Cry1) occur for the first time around P0 and clear
rhythms can be detected at P2 (Li and Davis, 2005).
In rat SCN, a circadian rhythm in Per1 occurs
already at E20, while circadian rhythms of Bmal1
and Per2 start at P1 and of Cry1 at P2 (Kovacikova
et al., 2006; Sumova et al., 2004, 2006). In acute
SCN slice preparations from genetically modified
rats bearing a Per1 promoter-driven luciferase
(luc) reporter gene, a circadian rhythm in Per1
expression was found to persist for at least 4 days
in vitro (Ohta et al., 2008). Clock gene proteins
PER1, PER2, and CRY1 are undetectable in rat
SCN at E19 (Sumova et al., 2004). In the SCN of
capuchin monkey, a circadian rhythm in Bmal1
and Per2 becomes apparent at E142 (Torres-
Farfan et al., 2006a). In mouse SCN, mPer1, and
mPer2 mRNAs are detectable at E17 (Shearman
et al., 1997), but only mPer1 mRNA levels show a
significant circadian rhythm during this fetal stage
(Shimomura et al., 2001). There is a small but sig-
nificant circadian variation in mPER1 and mPER2
immunoreaction in the SCN of C3H mice at E18,
coincident with a low-amplitude circadian rhythm
in Avp mRNA levels (Ansari et al., 2009). How-
ever, the rhythm of mCRY2 and mCRY1 levels
starts only after birth at P2 and P10, respectively
(Ansari et al., 2009; Shearman et al., 1997; Shim-
omura et al., 2001). This suggests that immature
molecular clockwork driving rhythmic mPer

expression might already be present in individual
fetal mouse SCN neurons. Taken together, these
results show that the endogenous circadian
rhythm generator in the SCN starts to develop
during late fetal stages and matures gradually dur-
ing the postnatal period. In rodents, a mature
molecular clockwork clock is established around
P10. Coincidently, synapse formation, which is
sparse at the time of birth, increases dramatically
between P4 and P10 (Moore and Bernstein,
1989). Thus, synapse formation and consequently,
synchronization between SCN neurons accounts
for robust and coherent circadian rhythmicity in
the developing SCN neuronal network (Moore,
1991). Circadian rhythms in AANAT activity
in the pineal gland can be observed from P10
(Reppert et al., 1984) accurately reflecting rhyth-
mic output from the developing SCN (Deguchi,
1982). Interestingly, the postnatal maturation of
the molecular clockwork seems to be independent
of rhythmic maternal cues as robust rhythms
in Per1 and Per2 expression can be observed in
organotypic SCN slice cultures derived from new-
born (P4–P7) genetically modified mice and rats
bearing luc reporter genes after 1 week up to sev-
eral months in vitro (Yamaguchi et al., 2001;
Yamazaki et al., 2000).
It is still not known whether circadian rhythms
in fetal SCN activity are driven by fetal SCN
molecular clockwork. Although few mouse SCN
cells show circadian rhythms in mPER1 and
mPER2 levels (Ansari et al., 2009), there is no
evidence that this is sufficient for driving rhythmic
Avp expression. Moreover, the rhythms in clock
gene expression in the fetal SCN might not be
self-sustained as in the adult endogenous circa-
dian rhythm generator but rather driven by rhyth-
mic maternal cues in a peripheral oscillator-like
manner. Importantly, an intact maternal SCN is
indispensible for the synchronization of the
embryonic/fetal circadian system. In newborn
Syrian hamsters, the synchrony in rest/activity
within the litter is lost after SCN lesion of the
mother between E7 and E14 (Davis and Gorski,
1988). In rats, a lesion of the maternal SCN at
109
E7 leads to the disruption of circadian rhythms
in SCN metabolic activity at E21 and pineal
AANAT activity at P10 within the litter (Reppert
and Schwartz, 1986). Synchronization within the
fetuses of SCN-lesioned dams can be restored by
rhythmic feeding cues (Weaver and Reppert,
1989). Thus, during fetal development, the circa-
dian system is primed to prevailing lighting
conditions by the maternal circadian system via
endocrine and/or metabolic substances crossing
the placenta (Reppert et al., 1984). Rearing of
pubs born to SCN-lesioned dams by intact dams
partially restores circadian rhythms in pineal
AANAT activity at P10 (Reppert and Schwartz,
1986). This suggests that rhythmic cues from the
mother are necessary and sufficient for entrain-
ment of the newborns. However, the nature of
these rhythmic maternal cues is still enigmatic;
they can be provided by the social maternal behav-
ior and/or the milk. As far as chemical messengers
are concerned, both dopamine and melatonin have
been shown to entrain the fetal circadian rhythms
in metabolic activity (Davis and Mannion, 1988;
Weaver et al., 1995). The melatonin receptor 1
(MT1) is expressed in the fetal SCN of different
species (Thomas et al., 2002; Torres-Farfan et al.,
2006a; Weaver and Reppert, 1996) and melatonin
can readily cross the placenta (McMillen et al.,
1990; Yellon and Longo, 1988). In capuchin
monkeys, suppression of maternal melatonin by
constant light leads to low expression levels of
Per2 and MT1 and increased expression levels of
Bmal1 in the fetal SCN. The effects on clock gene
expression levels by constant light condition could
be reversed with melatonin injections (Torres-Far-
fan et al., 2006a) indicating that maternal melato-
nin is capable to influence clock gene expression
in the fetal SCN. This assumption is further
supported by the observation that in the SCN of
melatonin-deficient C57BL mice, rhythmicity of
clock gene expression becomes evident only sev-
eral days later (at P5; Huang et al., 2010) than in
the SCN of melatonin-proficient C3H mice
(Ansari et al., 2009). The hypothesis that maternal
melatonin drives rhythmic clock gene expression in
110
the fetal mouse SCN needs to be corroborated by
comparative studies with melatonin (receptor)-
proficient and -deficient mice. Another rhythmic
maternal cue that entrains rodent fetuses is dopa-
mine acting on D1 receptors in the SCN (Vis-
wanathan et al., 1994) and this pathway may
provide the daytime signal. In addition to melato-
nin and dopamine, maternal behavior such as loco-
motor activity, body temperature, and periodic
food intake could entrain the fetal SCN molecular
clockwork. In Per1-luc transgenic rats, maternal
feeding has been shown to entrain the fetal SCN
independent of both the maternal SCN and the
light–dark cycle (Ohta et al., 2008).
Environmental lighting has no direct effect on
the phase of fetal SCN metabolic activity (Reppert
and Schwartz, 1983) and this is consistent with the
observations that the light input pathway into the
SCN develops postnatally (Mateju et al., 2009;
Munoz Llamosas et al., 2000; Sumova et al., 2003;
Weaver and Reppert, 1995). In rats, light stimuli
were shown to induce the immediate early gene
c-fos in the SCN as early as P0 (Leard et al., 1994;
Weaver and Reppert, 1995). Per1 and Per2 mRNA
expression in the SCN can be induced by light at P1
and P3, respectively (Mateju et al., 2009). At
variance, in hamster and mouse SCN, Fos/Fos-like
immunoreaction is inducible by light at P4
(Kaufman and Menaker, 1994; Reppert et al.,
1984). This species difference might be a con-
sequence of developmental differences in the
formation of the RHT. However, under natural
conditions, newborn rodents experience environ-
mental light only rarely as they are raised in dens.
Feeding and social interaction with the mother
might be the most important rhythmic signals for
the newborns. Rodents feed their pups during
the day while the mother takes food during the
night (Ohta et al., 2003). Most likely, melatonin
is absent in the milk during day (Illnerova et al.,
1993) and thus provides a reliable rhythmic
maternal signal for the newborns. After weaning,
photic input becomes the most important rhyth-
mic cue for entrainment of the circadian system
in the juvenile animals.
Ontogenesis of peripheral circadian oscillators
Peripheral oscillators have different capacities for
autonomous rhythmic expression of clock genes
in vitro (Yoo et al., 2004). Some peripheral
oscillators such as the liver show robust rhythmic-
ity over several weeks while others damp out rap-
idly in vitro demonstrating their dependency on
periodic rhythmic input for sustained rhythmic
clock gene expression.
An excellent model for a strongly input-
dependent oscillator is the PT of the anterior pitu-
itary. The PT is part of the anterior pituitary devel-
oping from a distinct antero-ventral area of
Rathke’s pouch (Stoeckel et al., 1979). The adult
PT is a major target of the hormone melatonin,
which conveys photoperiodic information to the
endocrine system (Hazlerigg, 2001; Lincoln, 2002;
Morgan et al., 1994; Roca et al., 1996). Melatonin
by acting on PT cells affects prolactin secretion
from the anterior pituitary (Lincoln, 2002; von
Gall et al., 2002a) presumably via PT-derived
paracrine factors (called tuberalins) that act
anterogradely on lactotrophs in the pars distalis
(Morgan and Williams, 1996; Yasuo and Korf,
2011; Yasuo et al., 2010a,b). In addition, melatonin
controls PT-derived thyrotropin which acts retro-
gradely and activates Dio2 expression in the
ependymal cell layer of the infundibular recess
(Yasuo et al., 2007, 2009, 2010b), thereby
regulating local thyroid hormone levels in the
mediobasal hypothalamus. The adult PT shows a
rhythmic expression of clock genes which strongly
depends on melatonin signaling via the MT1
receptor. In pinealectomized animals or in mice
with a targeted deletion of the MT1, no rhythms
in clock genes/proteins can be detected in the adult
PT (Jilg et al., 2005; Messager et al., 2001; von Gall
et al., 2002a, 2005). Importantly, circadian rhythms
in clock proteins are already present in the fetal
(E18) PT of melatonin-proficient mice. These
rhythms are in phase with and show the same
amplitude as those in the maternal PT (Ansari
et al., 2009). This suggests that the PT circadian
oscillator is already fully established in the

embryonic stage. As the PT circadian oscillator is
driven by the rhythmic melatonin signal and fetal
rodents are not capable to produce melatonin
rhythmically (Deguchi, 1975; Kennaway et al.,
1992; Nowak et al., 1990), the oscillations in clock
proteins in the fetal PT are presumably driven by
maternal melatonin.
The cortex of the adult adrenal gland controls
metabolic homeostasis and circadian rhythms in
energy balance via the hormone corticosterone.
This hormone also represents an important rhyth-
mic cue for other peripheral oscillators like the
liver (Balsalobre et al., 2000; Reddy et al., 2007).
Rhythmic corticosteroid production in the adult
adrenal cortex is driven by the SCN by influencing
both the hypothalamus-pituitary-adrenal (HPA)
neuroendocrine axis (Abe et al., 1979; Buijs and
Kalsbeek, 2001; Buijs et al., 2003) and the sympa-
thetic nervous system (Buijs et al., 2003; Ishida
et al., 2005; Lemos et al., 2006; Ulrich-Lai et al.,
2006). Melatonin inhibits the activation of cortisol
secretion induced by ACTH (Torres-Farfan et al.,
2003, 2004). The adult adrenal cortex possesses a
molecular clockwork (Bittman et al., 2003;
Fahrenkrug et al., 2008; Ishida et al., 2005; Lemos
et al., 2006; Oster et al., 2006b; Torres-Farfan
et al., 2006b; Valenzuela et al., 2008) which gates
rhythmic cortisol secretion in response to ACTH
(Oster et al., 2006b). Melatonin-proficient mouse
strains show circadian rhythms in clock gene
expression in the adrenal cortex in contrast to
melatonin-deficient mouse strains (Torres-Farfan
et al., 2006b) suggesting an important role of mel-
atonin for driving the adrenal cortex peripheral
oscillator. The fetal rat adrenal gland (E18) shows
circadian rhythms in clock gene expression and in
cortisol secretion which persist at least for 2 days
in vitro (Torres-Farfan et al., 2011). Melatonin
affects the phase of clock gene expression in
cultured adrenal glands (Torres-Farfan et al.,
2011), suggesting that maternal melatonin might
trigger the molecular clockwork in this develop-
ing peripheral oscillator. However, in the adrenal
gland of fetal capuchin monkeys, the circadian
rhythm in clock gene expression is not
111
synchronized by maternal melatonin (Torres-
Farfan et al., 2006a), demonstrating species-specific
differences in the control of fetal peripheral
oscillators.
The liver has a very strong capacity for autono-
mous rhythmic expression of clock genes in vitro
(Yoo et al., 2004). In the adult liver, a large num-
ber of genes (Akhtar et al., 2002; Panda et al.,
2002; Reddy et al., 2007) and proteins (Reddy
et al., 2006) involved in plasma protein synthesis,
glycogen metabolism, detoxification, and the core
molecular clockwork show circadian fluctuations.
About 60% of the circadian liver transcriptome
is driven by GCs (Reddy et al., 2007). In the fetal
rat liver (E20), detection of clock genes by in situ
hybridization did not reveal any circadian
rhythms except for a low amplitude in Cry1
(Sladek et al., 2007). This study has also shown
that the molecular clockwork in the liver matures
even more slowly during postnatal development
than the fetal SCN oscillator and rhythmic expres-
sion of all clock genes has been achieved only at
P30 (Sladek et al., 2007; Sumova et al., 2004). In
contrast, the fetal liver of Per1-luc transgenic rats
shows a circadian rhythm in Per1 expression for
at least 2 days in vitro that, similarly to the fetal
SCN and the adult liver, can be entrained by
maternal feeding (Ohta et al., 2008). Thus, the
fetal liver represents a damped circadian oscilla-
tor that gradually matures during postnatal life.
Summary
In fetal rodents, the SCN, adrenal gland, and liver
show a circadian rhythm of clock gene expression,
which persists for at least 2 days in vitro,
demonstrating the existence of circadian fetal
oscillators. These can be entrained by maternal
cues such as melatonin (impact on SCN and adre-
nal gland), dopamine (SCN), and maternal feed-
ing (SCN and liver). It is still a matter of debate
whether the fetal SCN and the fetal adrenal gland
produce rhythmic output signals, such as vaso-
pressin, and cortisol, respectively, which might
112

Mother

Rhythmic Melatonin
signals
(e.g.,
feeding)

Placenta

Fetus

Rhythmic Fetal peripheral

signals? oscillators
(e.g., liver,
adrenal, PT)

Fig. 1. Model depicting synchronization of fetal circadian oscillators by rhythmic maternal cues. The endogenous rhythm generator
in the maternal SCN is synchronized by light and generates circadian rhythms in melatonin and other hormones, body temperature,
and metabolites. Via the placenta these rhythmic maternal signals reach the fetus in which they synchronize the SCN, and self-
sustained peripheral oscillators, such as the liver and the adrenal gland, and drive input-dependent peripheral oscillators, such as
the pars tuberalis of the pituitary (PT). It is still a matter of debate whether vasopressin or other rhythmic signals generated by
the fetal SCN are capable of driving/synchronizing fetal peripheral oscillators. Modified after Reppert and Weaver (2002) and
Ansari et al. (2009).

1200
Relative optical density
Number of cells/SCN

1.0 Adult

900 P10
P2
600 0.5 E18

300

0 0.0
00 06 12 18 24 00 06 12 18 24
Circadian time Circadian time

Fig. 2. Analysis of mPER1-immunoreaction (Ir) in SCN (left) and PT (right) during mouse ontogenetic development (mean  SEM
of five animals per time point and ontogenetic stage. Data points at CT00/24 are double-plotted). Fetal SCN and PT show low- and
high-amplitude circadian oscillation of mPER1-Ir, respectively. Modified after Ansari et al. (2009).
 113

provide intrinsic entrainment signals to other the mouse liver transcriptome, as revealed by cDNA micro-
peripheral oscillators in the fetus (Fig. 1). Alter- array, is driven by the suprachiasmatic nucleus. Current
Biology, 12, 540–550.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 7

The circadian output signals from

the suprachiasmatic nuclei

Jia-Da Li{,*, Wang-Ping Hu{ and Qun-Yong Zhou}

{
The State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, PR China
{
Department of Pharmacology, Hubei University of Science and Technology, Xianning, Hubei, PR China
}
Department of Pharmacology, University of California Irvine, Irvine, CA, USA

Abstract: The suprachiasmatic nuclei (SCN) of the anterior hypothalamus comprise a self-sustained
biological clock generating an endogenous
24-h circadian rhythm, driving many overt daily rhythms in
the body. An important remaining question is how the SCN neurons communicate with their efferent
targets to control the daily oscillations in behavior and physiology. In this chapter, we summarize several
signaling factors that may serve as such SCN output factors. Whereas vasopressin may be involved in
the regulation of circadian hormone rhythms, SCN-derived prokineticin 2 (PK2), TGF-a, and
cardiotrophin-like cytokine (CLC) may serve as output factors for other circadian rhythms, including
locomotor activity, body temperature, and energy metabolism. The circadian rhythm in firing activity of
SCN neurons is also likely to be a critical output signaling mechanism. The likely involvement of these
output factors in the generation of the circadian rhythm in SCN neuronal firing activity is also discussed.

Keywords: circadian; suprachiasmatic nuclei; prokineticin 2; vasopressin; TGF-a; cardiotrophin-like

cytokine.

Introduction earth. In mammals, the endogenous pacemaker

Almost all organisms, from bacteria to mammals,
exhibit circadian (
24 h) rhythms in physiology
and behavior to adapt to the environmental
changes imposed by the daily revolutions of the
*Corresponding author.
Tel.: þ0086-731-84805339; Fax: þ0086-731-84805339
E-mail: lijiada@sklmg.edu.cn
that drives circadian rhythms resides in the
suprachiasmatic nuclei (SCN) of the anterior hypo-
thalamus (Lowrey and Takahashi, 2004; Reppert
and Weaver, 2002; Schibler, 2005). The SCN coor-
dinate daily cycles of behavior and physiology,
such as the daily rhythms of locomotor activity
and body temperature (Lowrey and Takahashi,
2004; Reppert and Weaver, 2002; Schibler, 2005).
In the past decades, the molecular mechanisms

http://dx.doi.org/10.1016/B978-0-444-59427-3.00028-9 119
120
of this endogenous clock in the SCN have been
elucidated as consisting of several autoregulatory
transcriptional/translational feedback loops
(Dibner et al., 2010; Lowrey and Takahashi, 2004;
Reppert and Weaver, 2002; Schibler, 2005). How-
ever, how the information in the SCN is mechanis-
tically transmitted out of them to regulate the overt
circadian rhythms is still largely unknown.
Dye tracing experiments have revealed that the
primary efferent target areas of the SCN are quite
limited and predominantly located in the hypothala-
mus and the midline thalamus. Primary SCN target
areas include lateral septum, bed nucleus of the stria
terminalis, subparaventricular zone, para-
ventricular hypothalamic nucleus, dorsomedial
hypothalamic nucleus, as well as paraventricular
thalamic nucleus (Buijs, 1996; Kalsbeek et al.,
1993; Leak and Moore, 2001; Morin and Allen,
2006; Watts and Swanson, 1987; Watts et al., 1987).
Lesion and transplant studies have indicated that
the SCN drive locomotor activity cycles by rhythmi-
cally releasing factors that act on receptors in the
hypothalamus near the wall of the third ventricle
(Lehman et al., 1987; Ralph et al., 1990). The dem-
onstration that transplanted SCN tissue encaged in
a semipermeable membrane can still restore a circa-
dian rhythm in locomotor activity proved that dif-
fusible factors emanate from the transplanted SCN
(Silver et al., 1996). However, transplantation of
embryonic SCN tissue does not restore the endo-
crine and other physiological rhythms, underscoring
the importance of axonal connections between the
SCN and their target sites for the regulation of many
other circadian processes (Meyer-Bernstein
et al., 1999). In this chapter, we summarize a few
candidate factors that may transmit the circadian
information from the SCN to other brain areas.
Circadian control of hormone rhythms by
arginine vasopressin
Arginine vasopressin (AVP) is one of the first
neurotransmitters identified in the SCN (Burlet
and Marchetti, 1975; Swaab et al., 1975; Vandesande
et al., 1974). The vasopressin-containing neurons
constitute an important subpopulation of the het-
erogeneous SCN neurons: between 10% and 30%
of the approximately 10,000 SCN neurons on each
side of the rat brain may contain AVP (Kalsbeek
et al., 2010). The concentration of AVP in the cere-
brospinal fluid varies in a circadian cycle, with
morning levels approximately five times higher
than those during night hours (Reppert et al.,
1981), corresponding to the circadian variation of
AVP content in the SCN (Jansen et al., 2007;
Sodersten et al., 1985; Tominaga et al., 1992; Uhl
and Reppert, 1986). Molecular studies have rev-
ealed that the transcription of the AVP gene in
the SCN is rhythmically regulated by the same pos-
itive and negative elements that control the core
molecular loops. The CLOCK/BMAL1 complex
positively activates the expression of the AVP
gene, which is suppressed by the period (Per) and
cryptochrome (Cry) proteins (Jin et al., 1999).
The role of AVP as an output factor was elegantly
demonstrated by Tousson and Meissl with electro-
physiological recordings (Tousson and Meissl,
2004). They demonstrated circadian rhythms in
spontaneous firing rate in the SCN and adjacent
hypothalamic regions, with the activity rhythms
recorded from structures outside the SCN in phase
with the rhythms in the SCN. Cyclic firing in the
hypothalamus was lost after ablation of the SCN
but could be restored by SCN grafts. Further, the
restoration of a circadian rhythm in the para-
ventricular nucleus cocultured with an SCN graft
was abolished by the AVP receptor antagonist.
Conversely, periodic application of AVP was able
to induce firing rhythmicity in the hypothalamus
(Tousson and Meissl, 2004).
However, central infusion of either vasopressin or
a vasopressin V1-antagonist failed to produce signif-
icant effects on the daily patterns of wheel-running
behavior (Albers et al., 1984; Stoynev and Nagai,
1996). Moreover, AVP-deficient Brattleboro rats
did not show dramatic changes in the locomotor
activity rhythms (Groblewski et al., 1981); thus, it
was supposed that AVP may not be critically
involved in the control of circadian locomotor

rhythm. Nevertheless, deficiency in AVP receptor
V1a, a dominant AVP receptor that is expressed in
the SCN, led to an attenuated circadian rhythmicity
of locomotor activity (Li et al., 2009). Interestingly,
the rhythmicity of prokineticin 2 (PK2), one of the
candidate output genes, was attenuated in the SCN
of V1a-deficient mice (Li et al., 2009), suggesting a
role of AVP/V1a signaling in SCN gene expression
and, subsequently, in the control of behavior and
physiology. By using bioluminescence recording,
Maywood et al. recently demonstrated that AVP
signaling within the SCN plays a role in the synchro-
nization of SCN cellular pacemaking (Maywood
et al., 2011). Recent studies in common voles
also indicate a possible role of SCN vasopressin in
circadian locomotor activity. Arrhythmic voles
exhibited constantly high populations of vasopres-
sin-immunoreactive neurons in the SCN (Jansen
et al., 2007). Further, vasopressin release from
cultured SCN neurons was attenuated in arrhythmic
voles (Jansen et al., 2000).
The role of AVP in the circadian regulation
of hormone release has been extensively studied.
Circulating plasma corticosterone levels display a
circadian rhythm, whereas SCN-lesioned animals
display constant, but elevated, levels of plasma
corticosterone. Kalsbeek and colleagues demon-
strated that AVP was able to suppress elevated
levels of corticosterone in SCN-lesioned animals
to basal daytime values. In agreement with this
suppressive effect of AVP signaling, infusion of
an AVP antagonist induced a sevenfold increase
in basal corticosterone levels in intact animals
(Kalsbeek et al., 1992, 1996a,b).
AVP release from the SCN is probably also
important for the control of the daily rhythm in
other hormonal axes, such as the hypothalamic–
pituitary–gonadal axis. SCN lesions lead to basal,
nonfluctuating luteinizing hormone (LH) levels,
but a 2-h administration of AVP in the medial
preoptic area is sufficient to reinstate a complete
LH surge comparable to the estrogen-induced
surges in SCN-intact animals, both in shape and
amplitude (Palm et al., 1999). Funabashi et al.
measured the release of gonadotropin-releasing
121
hormone (GnRH) and AVP in cocultures of the
preoptic area and the SCN (Funabashi et al.,
2000). The period of the GnRH circadian rhythm
was the same as that of the AVP circadian rhythm.
Administration of AVP significantly increased
GnRH release in single preoptic area cultures in
the presence of estrogen (Funabashi et al., 2000).
Further, Clock mutant mice lack the LH surge on
the afternoon of proestrus. Miller et al. showed that
intracerebroventricular (ICV) injection of AVP on
the afternoon of proestrus is sufficient to induce
LH secretion in Clock mutant mice. The effect of
AVP on the Clock mutant LH surge seems to be
mediated by V1a receptors, as coinfusion of AVP
and a V1a-specific antagonist prevents the AVP-
induced LH release (Miller et al., 2006).
The circadian role of PK2 as an output molecule
Prokineticins are a pair of cysteine-rich peptides
involved in a variety of biological functions, includ-
ing gastrointestinal smooth muscle contraction,
angiogenesis, neurogenesis, nociception, feeding,
and circadian and emotional regulation (Zhou
and Cheng, 2005). Prokineticins execute their
functions by activating two closely related
G-protein-coupled receptors, PKR1 and PKR2
(Lin et al., 2002; Masuda et al., 2002; Soga et al.,
2002). Activation of these receptors leads to mobi-
lization of calcium, phosphoinositide hydrolysis,
and activation of the mitogen-activated protein
kinase and protein kinase B pathways (Lin et al.,
2002; Masuda et al., 2002; Soga et al., 2002).
The possible link of PK2 with the circadian clock
was first indicated by the observation that PK2
mRNA levels in the SCN oscillate with a high
amplitude, with high levels during the day and
low or undetectable levels at night (Cheng et al.,
2002). Studies using a bacterial artificial chromo-
some (BAC) transgenic mouse line, in which the
enhanced green fluorescence protein (EGFP)
reporter gene expression was driven by the PK2
promoter, indicated a circadian oscillation of a
number of EGFP-positive neurons in the SCN
122
(Zhang et al., 2009). The data from this strain of
transgenic mice also revealed EGFP-expressing
neurons in the SCN projected to many known
SCN target areas, including the ventral lateral
septum, medial preoptic area, subparaventricular
zone, paraventricular nucleus, dorsomedial hypo-
thalamic nucleus, lateral hypothalamic area, and
paraventricular thalamic nucleus (Zhang et al.,
2009). In the same brain areas, the receptor for
PK2 (PKR2) was also found to be expressed, as
identified with in situ hybridization (Cheng
et al., 2002). In situ hybridization studies also rev-
ealed that PK2 mRNA-positive neurons were
scattered in both the dorsomedial and ventrolat-
eral SCN (Masumoto et al., 2006). Double label-
ing in situ hybridization indicated that more than
50% of the PK2 mRNA-containing neurons co-
expressed vasoactive intestinal peptide (VIP),
gastrin-releasing peptide (GRP), or AVP in the
SCN (Masumoto et al., 2006).
The rhythm of PK2 is directly regulated by
the transcriptional/translational feedback loop of
the central clock (Cheng et al., 2002). Both the
human and mouse PK2 promoters contain four
E-boxes within the first 2 kb upstream of the tran-
scription start site. In vitro transcription assays
indicate that PK2 transcription is tightly regulated
by clockwork gene products through activation of
the E-boxes residing in its proximal promoter.
These in vitro findings were validated in vivo, as
PK2 mRNA expression in the SCN is completely
absent or blunted in mutant mice lacking a func-
tional clockwork, including Clock mutant mice
and Cry1/Cry2/ mice (Cheng et al., 2002).
ICV delivery of PK2 into the lateral ventricle
during subjective night, when endogenous PK2
was low, inhibited the nocturnal wheel-running
activity of rats (Cheng et al., 2002). The impor-
tance of PK2/PKR2 signaling in the circadian
regulation was also demonstrated in mice defi-
cient in PK2 or PKR2, respectively. The PK2/
mice showed attenuated circadian rhythmicity in
a variety of behaviors and physiology, including
locomotor activity, sleep/wake, body tempera-
ture, hormone as well as peripheral clock gene
expression (Hu et al., 2007; Li et al., 2006). The
PKR2/ mice showed a similar attenuation in
their daily rhythms (Prosser et al., 2007). Further,
clock gene expression is not altered in the SCN of
either PK2/ or PKR2/ mice (Li et al., 2006;
Prosser et al., 2007), consistent with the supposed
role of PK2 as an output molecule. Moreover,
Morton et al. observed a correlation between
increased daytime activity and reduced expres-
sion of the PK2 molecular rhythm in the SCN of
transgenic mice expressing a mutant Huntington’s
gene (Morton et al., 2005). This finding suggests
that the reduced PK2 rhythm may contribute to
the sleep disturbances and abnormal circadian
behavior seen in this strain of transgenic mice.
In addition to expression in the primary SCN
targets, PKR2 is also intensively expressed in the
SCN neurons, implying a possible signaling role
of PK2/PKR2 signaling within the SCN. Recently,
Ren et al. investigated the effect of PK2 on sponta-
neous firing and miniature inhibitory postsynaptic
currents (mIPSCs) of SCN neurons using whole-
cell patch-clamp recordings (Ren et al., 2011).
PK2 dose-dependently increased spontaneous
firing rates in most neurons from the dorsal SCN.
Further, PK2 seems to act postsynaptically to
reduce g-aminobutyric acid (GABA)-ergic func-
tion within the SCN (Ren et al., 2011). This study
indicated that the local effect of PK2 on the cellular
activity of SCN neurons may also be linked to
its regulatory role in behavioral and physiological
circadian rhythms.
TGF-a and CLC are identified as inhibitory
factors by systematic screening
In their elegant temporal chimera experiments,
Vogelbaum and Menaker demonstrated that there
are both stimulatory and inhibitory output signals
from the SCN, at least for locomotor activity
(Vogelbaum and Menaker, 1992). Weitz and
coworkers utilized a systematic approach to identify
peptides that inhibit locomotor activity when infused
into the third ventricle (Kramer et al., 2005). As a

result of this approach, Weitz et al. reported two
secreted proteins that could be involved in the gener-
ation of circadian locomotor rhythmicity. In 2001,
they reported TGF-a as such a candidate output
molecule (Kramer et al., 2001). When TGF-a
was infused into the third ventricle, it reversibly
inhibited locomotor activity and disrupted circadian
sleep–wake cycles (Kramer et al., 2001). The mRNA
of TGF-a is expressed rhythmically in the SCN, with
peak and trough levels around subjective dawn and
dusk, respectively (Kramer et al., 2001). However,
in contrast to the circadian rhythms of TGF-a
mRNA levels in the hamster SCN, there is no signif-
icant circadian change in the levels of TGF-a protein
in the hamster SCN (Lindley et al., 2008). TGF-
a-positive cells were found throughout the SCN,
but more abundantly in the core than the shell
area, while AVP was predominantly found in the
shell. Fluorescent double labeling revealed a total
lack of coexpression for the two proteins in SCN
cells. Further, TGF-a was found to be colocalized
with glial fibrillary acidic protein (GFAP), but not
with the general neuronal marker NeuN, or
calbindin-D28K (Li et al., 2002; Lindley et al.,
2008). Thus, the role of TGF-a in the circadian regu-
latory system remains controversial.
In 2006, Weitz and coworkers identified car-
diotrophin-like cytokine (CLC) as another candi-
date circadian regulator of locomotor activity
(Kraves and Weitz, 2006). When CLC was
acutely infused into the third ventricle, it pro-
duced a transient blockade of locomotor activity
without affecting the core molecular oscillation
(Kraves and Weitz, 2006). In the mouse, CLC is
expressed in a subpopulation of SCN vasopressin
neurons with a circadian rhythm that peaks dur-
ing the late day, a daily period with locomotor
quiescence. The rhythm of CLC seems directly
regulated by the transcriptional/tranlational nega-
tive feedback loop of the central clock (Kraves
and Weitz, 2006).The CLC receptors flank the
third ventricle, and the hypothalamic infusion of
neutralizing antibodies to the CLC receptor pro-
duces extra daily locomotor activity at a time
when CLC is maximally expressed (Kraves and
123
Weitz, 2006). However, genetic studies to verify
the possible role of CLC in circadian rhythms
have not been reported.
Neuronal firing as an SCN output
The SCN neurons express a cell autonomous circa-
dian rhythm in spontaneous action potential fre-
quency (Kuhlman and McMahon, 2006). In vivo
recordings revealed circadian rhythms in the elec-
trical activity in the SCN, with spike activity being
high during the day and low at night (Inouye and
Kawamura, 1979). Rhythmic SCN electrical activ-
ity is likely a critical component of SCN output.
Several ion channels have been identified in the
SCN. One class of fast delayed rectifier (FDR)
channels, the Kv3.1/3.2 Kþ channels, is rhythmically
expressed in the SCN, with its members’ expression
peaking during the day phase. Blocking the FDR
potassium currents appeared to block the daily
rhythm in firing rate in SCN neurons (Itri et al.,
2005). Further, the iberiotoxin-sensitive BK
channels are rhythmically expressed in the SCN,
and the daily expression of BK channel in the
SCN is controlled by the intrinsic circadian clock
(Meredith et al., 2006). The mRNA level of the tran-
script encoding for BK channel KCNMA1 is high
during the night phase, so is the BK current. Both
pharmacological blockade of BK current and
genetic knockout of the channel gene (Kcnma1/)
blunt, but do not completely block, the circadian
rhythm in spike frequency (Meredith et al., 2006).
Specifically, Kcnma1/ mice have increased spon-
taneous firing rates in SCN neurons at night, imply-
ing that the nightly BK current might reduce SCN
spike frequency. Kcnma1/ mice also displayed
reduced circadian amplitudes in multiple behaviors
timed by the SCN, including locomotor activity
and core body temperature. Further, Kcnma1/
mice showed normal expression of clock genes such
as Bmal1, consistent with a role of BK channels
in SCN output, rather than in the intrinsic time-
keeping mechanism of the master pacemaker
(Meredith et al., 2006).
124
PK2
B C
AVP
E
CLC
Fig. 1. A diagram depicting the relationship between three putative circadian output molecules. The transcription of PK2, AVP,
and CLC is regulated by Bmal1/Clock heterodimer by binding to the E-box located at their promoters. However, the PK2,
AVP, and CLC mRNA levels peak at approximately CT 3, CT 6, and CT 9, respectively. In addition, they influence different
behavioral and physiological rhythms.
Perspectives
Several candidate signaling factors have been
reported as output signals connecting the SCN
and their target areas. Temporally, these factors
might exert overlapping functions, as knockout
mice for a single output gene or blockade of a
single pathway did not result in complete
arrhythmicity. PK2, AVP, and CLC mRNA peak
at approximately circadian time 3 (CT 3), CT 6,
and CT 9, respectively (Cheng et al., 2002; Jin
et al., 1999; Kraves and Weitz, 2006). Thus, the
dawn and dusk time may represent unique zones
for PK2 and CLC, respectively (Fig. 1). Defi-
ciency in PK2 resulted in increased locomotor
activity, wakefulness, body temperature, as well
as corticosterone levels during the early morning
period (Li et al., 2006), whereas blocking CLC
signaling by infusion of anti-gp130 caused an
increase in locomotor activity specifically during
CT 9–12 (Kraves and Weitz, 2006).
The circadian rhythms of neural, metabolic, neu-
rotransmitter, and clock gene expression in the
SCN neurons are similarly phased in nocturnal
and diurnal animals. It is thus challenging to iden-
tify the switch responsible for diurnality. Some
research has been performed to see if there is
any difference in the output genes in the diurnal
Thermal regulation
Sleep-wake
Hormone
Locomotor activity
rodents. However, as it seems that PK2, AVP,
and TGF-a expression is similar in diurnal and
nocturnal animals (Lambert et al., 2005; Mahoney
et al., 2009; Tournier et al., 2007), there is no evi-
dence indicating that a difference in the phase of
output gene expression accounts for diurnality.
Although the temporal chimera experiments
carried out by Vogelbaum and Menaker proposed
the existence of both stimulatory and inhibitory
signals in the SCN (Vogelbaum and Menaker,
1992), no experiments have thus far proposed
SCN factors that promote arousal or locomotor
activity. Further, most of the research appeared
to focus on locomotor activity. As the SCN also
regulate many other physiological and neuroendo-
crine rhythms, it might be worthwhile to investi-
gate the other putative output pathways.
Acknowledgments
The research in Jia-Da Li’s lab was partly supported
by the Projects in the Major State Basic Research
Development Program of China (973 Program)
(grant number 2012CB517904), National Natural
Science Foundation of China (grants 30970958
and 81070481), and the Lotus Scholar Professorship
Funds from Hunan Province Government, China.
 125

The research in Qun-Yong Zhou’s lab was Dibner, C., Schibler, U., & Albrecht, U. (2010). The mamma-
supported in part by grants from NIH (NIMH67753, lian circadian timing system: Organization and coordination
of central and peripheral clocks. Annual Review of Physiology,
HD15788) and the American Heart Association 72, 517–549.
(855156 F). Funabashi, T., Shinohara, K., Mitsushima, D., & Kimura, F.
(2000). Gonadotropin-releasing hormone exhibits circadian
rhythm in phase with arginine-vasopressin in co-cultures of
Abbreviations the female rat preoptic area and suprachiasmatic nucleus.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 8

Suprachiasmatic nucleus: Cellular

clocks and networks

Sato Honma{,{,*, Daisuke Ono{,}, Yohko Suzuki}, Natsuko Inagaki{,1,

Tomoko Yoshikawa}, Wataru Nakamura{,} and Ken-ichi Honma{

{
Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo, Japan
{
Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan
}
Advanced Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University
Graduate School of Medicine, Sapporo, Japan
}
Department of Chronodentistry, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

Abstract: The suprachiasmatic nucleus (SCN), the master circadian clock of mammals, is composed of
multiple circadian oscillator neurons. Most of them exhibit significant circadian rhythms in their clock
gene expression and spontaneous firing when cultured in dispersed cells, as well as in an organotypic
slice. The distribution of periods depends on the SCN tissue organization, suggesting that cell-to-cell
interaction is important for synchronization of the constituent oscillator cells. This cell-to-cell interaction
involves both synaptic interactions and humoral mediators. Cellular oscillators form at least three
separate but mutually coupled regional pacemakers, and two of them are involved in the photoperiodic
regulation of behavioral rhythms in mice. Coupling of cellular oscillators in the SCN tissue compensates
for the dysfunction due to clock gene mutations, on the one hand, and desynchronization within and
between the regional pacemakers that suppresses the coherent rhythm expression from the SCN, on the
other hand. The multioscillator pacemaker structure of the SCN is advantageous for responding to a
wide range of environmental challenges without losing coherent rhythm outputs.
Keywords: suprachiasmatic nucleus; neural network; clock genes; multi-electrode array; oscillatory
coupling; photoperiods; bioluminescence imaging.

*Corresponding author.
Tel.: þ81-11-706-4778; Fax: þ81-11-706-4737
E-mail: sathonma@med.hokudai.ac.jp
Introduction

1
Present address: Laboratory of Cell Growth and Differentia- The suprachiasmatic nucleus (SCN), the master
tion Institute of Molecular and Cellular Biosciences, The circadian clock of mammals, is composed of about
University of Tokyo, Tokyo, Japan. 20,000 neurons in rats and mice (Abrahamson

http://dx.doi.org/10.1016/B978-0-444-59427-3.00029-0 129
130
and Moore, 2001; van den Pol, 1980). Monitoring
spontaneous firing activity from SCN tissue
cultured on a multi-electrode array dish (MED)
showed that most of the neurons exhibited robust
circadian firing rhythms with periodicities slightly
different from each other (Herzog et al., 1997;
Honma et al., 1998; Welsh et al., 1995). These
results suggest that each SCN neuron contains a
cellular circadian oscillator. The current under-
standing of the molecular core of the cellular
oscillator involves a transcriptional–translational
feedback loop in which heterodimeric transcrip-
tion factors, CLOCK and BMAL1, activate tran-
scription of Period (Per) and Cryptochrome
(Cry) genes and the protein products, PERs and
CRYs, in turn, suppress their own transcription,
thus closing the auto-feedback loop, whose single
turn takes about 24 h (Reppert and Weaver,
2002). Robust circadian rhythms in the transcrip-
tional activity of most clock genes and in the pro-
tein levels are advantageous for monitoring the
tick of the circadian clock by bioluminescence
and fluorescent reporters (Kuhlman et al., 2000;
Yamazaki et al., 2000). This recent progress
in molecular neurobiology enabled long-term,
real-time monitoring of multiple functions from
hundreds of SCN neurons simultaneously. Com-
parison of circadian rhythms of different functions
from a single dispersed SCN cell makes it possible
to analyze intracellular circadian signal transduc-
tion. Further, long-term and simultaneous moni-
toring of different circadian rhythms from single
cells of an SCN explant revealed novel roles of
the oscillatory networks. Dependence of regions
in the SCN and developmental stages is also of
special interest. Here, we review cellular and net-
work properties of the SCN circadian rhythms
revealed by these new rhythm monitoring tools.
Cellular oscillators and coupling
Enzymatically isolated SCN neurons of rats and
mice exhibit circadian rhythms in spontaneous
firing and clock gene expression (Herzog et al.,
2004; Welsh et al., 1995). In the SCN neurons of
wild-type Wistar rats, circadian periods of sponta-
neous firing range from 20 to 30 h when cultured
as enzymatically dispersed ones, whereas they
range from 22 to 27 h when the SCN neurons
are cultured as an organotypic coronal slice
(Honma et al., 2004; Fig. 1). Periods from both
cultures followed a Gaussian distribution. The
mean periods were not statistically different, but
the distribution range was substantially different
between the two cultures. The circadian periods
are well synchronized within a cultured SCN slice,
where cell-to-cell interactions are much stronger
than those in the dispersed cell culture. In both
cultures, the average periods are close to the free-
running period of animals’ behavior, suggesting
that mutual synchronization among the constituent
cellular oscillators in the SCN determines the
rhythm output from the SCN for behavioral
rhythms (Fig. 1a; Honma et al., 2004). Considering
the narrow distribution of free-running periods in
the behavioral rhythms, intact neural networks and
close cell–cell apposition for efficient paracrine sig-
naling seem to effectively increase the coupling
intensity among oscillator cells in the SCN in vivo.
Further, input signals from extra SCN tissues may
also affect the stability of periodicity. Interestingly,
at a small percentage, anti-phasic firing rhythms
were observed in a cultured SCN slice, indicating
a large diversity of neuronal outputs from the pace-
maker (Nakamura et al., 2001).
We also analyzed circadian firing rhythms of
single SCN neurons in the slice and dispersed
cell cultures of C57BL/6 J mice (Fig. 1b). The
results were basically the same as those for rats
except for the periods, which were slightly
shorter than the respective period for rats. In
both rats and mice, mean circadian period of
behavioral rhythm was slightly but significantly
longer than the respective SCN neurons. The
reason for the difference is not known. Although
the circadian period is systematically changed
depending on the environmental light in con-
stant conditions, the periodicity in the behavioral
rhythms is not due to the environmental light,
 131

(a) rats (Wistar) (b) mice (C57BL/6J)

30 15
23.1±1.9 h

Number of neurons
dispersed

Number of neurons
25 disperse 24.1±1.5 h
72.4% (223/308) 98% (121/123)
20 10
15
10 5
5
0 0
16 18 20 22 24 26 28 30 16 18 20 22 24 26 28 30
circadian period (h) circadian period (h)
70 15
24.2±0.7 h slice
Number of neurons

slice

Number of neurons
60 23.3±0.4 h
82.6% (185 / 224)
50 100% (n=53)
10
40
30
20 5
10
0 0
16 18 20 22 24 26 28 30 16 18 20 22 24 26 28 30
circadian period (h) circadian period (h)
80 60
behavior behavior
Number of animals

Number of animals

24.4±0.2 h 50 23.8±0.1 h
60 (n=86)
(n=147) 40
40 30
20
20
10
0 0
16 18 20 22 24 26 28 30 16 18 20 22 24 26 28 30
circadian period (h) circadian period (h)

Fig. 1. Distribution of circadian periods of spontaneous firing rhythms in individual SCN neurons and behavior. Data show the
distribution of circadian periods of spontaneous firing rhythms in rats (a) and mice (b) together with that of behavioral rhythms.
Upper, middle, and lower panels demonstrate period distribution of SCN neurons in dispersed cell culture, that in organotypic
slice culture, and spontaneous behavior, respectively. Behavioral rhythms were monitored by an infrared thermal sensor from
blinded rats and mice kept in constant darkness. Numbers in each panel indicate average period (SD) and percentage of
neurons with significant circadian periodicity. Numbers in parentheses are those of SCN neurons with significant rhythms out of
the total number examined.

because behavioral rhythms were monitored Pacemaker composed of oscillator and

from blinded rats and mice kept in constant non-oscillator cells
darkness. Since the SCN had to be sliced for cul-
turing, we might have lost some components of The percentage of neurons exhibiting a significant
the SCN, which would have lengthened the peri- circadian rhythm in a culture depends on the cell
odicity during the culture preparation. Alterna- density and cellular architecture (Herzog et al.,
tively, some factors in the culture media might 2004; Nakamura et al., 2002), suggesting that not
affect the periodicity in vitro. every SCN neuron is a cell-autonomous clock cell.
132

The percentage of neurons exhibiting a circadian (a)

20
firing rhythm was significantly higher in slice dispersed 22.8±2.1 h
cultures than that in dispersed cell cultures 100% (n=168)
15

Number of cells
(Honma et al., 2004). Luciferase reporters and
bioluminescence imaging of clock gene expres-
10
sion made the analysis of single cell rhythms
much easier compared to that of firing rhythms.
In addition, cellular rhythms can now be analyzed 5
in adult animals that have been exposed to
different experimental conditions. Using an SCN 0
16 18 20 22 24 26 28 30
slice culture from transgenic mice carrying a circadian period (h)
mouse Per1 promoter-driven luciferase reporter (b)
50
gene (Per1-luc) (Inagaki et al., 2007), we com- newborn slice 24.0±0.3 h
pared circadian periodicities of single SCN cells 40 100% (n=109)

Number of cells
in the slice and those in dispersed cell cultures.
30
All bioluminescent cells exhibited significant cir-
cadian Per1-luc rhythms in dispersed cell cultures 20
as well as in organotypic slice cultures (Fig. 2). In
the slice culture, circadian periods of single SCN 10
cells of adult mice were distributed in a wider
0
range than those of newborns. The reason for this 16 18 20 22 24 26 28 30
difference is not known. Culture condition might circadian period (h)
have affected the period distribution, since new- (c) 50
adult slice 23.7±0.9 h
born SCN slices were precultured 1–2 weeks
40 100% (n=108)
before the start of recording, which might have
Number of cells

promoted the network formation within a slice. 30

On the other hand, neural networks might have
been dissected by the slice preparation and never 20
recovered in adult SCN.
10
The percentage of neurons with significant
circadian periods depends on the cell density in 0
dispersed cell cultures: the higher the density, 16 18 20 22 24 26 28 30
the higher the percentage of rhythmic neurons. circadian period (h)
Using dispersed SCN cells from mice carrying
Fig. 2. Distribution of circadian periods in Per1-luc rhythms
a PERIOD2::LUCIFERASE fusion protein as depends on culture system and age. Data show the
a real-time reporter (PER2::LUC, Yoo et al., distribution of circadian periods of Per1-luc rhythms in
2005), Webb et al. (2009) demonstrated that 71% individual mouse SCN cells. Distribution in dispersed cell
of dispersed SCN cells did not show PER2::LUC culture of newborns (a), slice culture of newborns (b), and
rhythms longer than 3 days when cultured in a adults (c) are shown. For the details, see the legend of Fig. 1.
very low density of 100 cells/mm2. Among the
remaining cells, 65% were rhythmic with intrinsic rhythm-positive and -negative cells, suggesting that
circadian periods. They also reported that the neither the AVP nor the VIP cells are solely the
marker neuropeptides of the SCN, vasopressin oscillator cells (Webb et al., 2009). Surprising is a
(AVP) and vasoactive intestinal polypeptide (VIP), relatively small number of rhythm-positive cells
were detected at almost the same percentage in (19%) in the culture with almost no physical contact.

Mathematical models predict a stable oscillation
by coupling among cells with self-sustained and
damped oscillators (Abraham et al., 2010; Ko
et al., 2010). Therefore, the oscillatory coupling in
the intact SCN seems to be a specific character of
the central clock rather than the oscillation within
single neurons.
Regional oscillators in the SCN for
different functions
An organotypic slice culture of the SCN enables
us to analyze networks and regional specificity
within the SCN. Previously, we reported desyn-
chronization between circadian rhythms in AVP
and VIP releases (Shinohara et al., 1995). We
measured the concentration of the two peptides
in the culture medium of an SCN slice cultured
in a roller tube every 2 h, and demonstrated
robust circadian rhythms in both peptides. They
were synchronized in the control cultures, but in
the cultures treated with anti-mitotics in the begin-
ning of culturing, the rhythms of the two peptides
free-ran with different periods and eventually
desynchronized with each other during the culture.
This result suggests that the SCN contains at least
two regional pacemakers, one composed of AVP-
releasing neurons and the other composed of
VIP-releasing neurons. The two pacemakers were
uncoupled by treatment with anti-mitotics; never-
theless, synchronization among cellular oscillators
of each peptidergic neuron was kept basically
intact. We still do not know the mechanism for
the desynchronization by anti-mitotics. Anti-
mitotic treatment prevents glial overgrowth; thus,
treated SCN slices tended to extend, whereas
untreated slice remained rather compact, which
might promote better physical contact. Alterna-
tively, the glia might exert some functions in
rhythm coupling among SCN neurons mechani-
cally or chemically. A similar desynchronization
between the dorsal and ventral SCN was also
observed in the rat SCN in vivo after an abrupt shift
in the light–dark (LD) cycle (Nagano et al., 2003).
133
Clock gene expression rhythms in the ventrolateral
SCN immediately shifted to the new LD cycle,
whereas those in the dorsomedial SCN gradually
shifted with several days of transients. These
data not only confirmed the uncoupling of two
regional oscillators seen previously in vitro but
also suggested a functional significance of these
oscillators.
Recently, we found at least three photoperiodi-
cally regulated oscillator cell groups in the SCN
by measuring Per1-expressing rhythms. Further,
two of them were phase-locked to the onset and
offset of behavioral activity separately (Inagaki
et al., 2007). Animals living in the middle and
high latitudes change their behavioral rhythms
depending on season, which is important for their
survival in different seasons. The long-lasting
hypothesis for photoperiodic regulation of behav-
ioral rhythms of nocturnal rodents consists of
two separate, but mutually coupled, oscillators:
evening (E) and morning (M) oscillators, which
regulate activity onset and offset, respectively
(Pittendrigh and Daan, 1976b). We exposed
Per1-luc mice, which were reared under a LD
cycle of 12:12 h, to either short day (LD 6:18) or
long day (LD 18:6) conditions for 3 weeks. Two
serial coronal SCN slices of 300-mm thickness
were made and their Per1-luc bioluminescence
was measured for more than 5 days (Fig. 3). In
mice kept under LD 12:12 and LD 6:18, the circa-
dian peak of the Per1-luc rhythm in the anterior
SCN always phase-lagged compared with that in
the posterior counterpart. The phase difference
was smaller in LD 6:18 than in LD 12:12. Surpris-
ingly, only in the anterior SCN of mice under LD
18:6, Per1-luc exhibited two peaks per day, which
tended to merge during culturing (Fig. 3). Biolumi-
nescence imaging revealed that this bimodal Per1-
luc pattern was due to two differentially peaking
cell groups with a period of about 24 h (Inagaki
et al., 2007). Since the morning peak in the anterior
SCN is expressed only in the long photoperiods,
the evening Per1-luc peak was selected for compar-
ison with those in other photoperiods. We found
that, irrespective of the photoperiods, the anterior
134

anterior SCN
posterior SCN Time of day (hr)
(a)
20 0 4 8 12 16 20 24
RLU (⫻102 counts/min)

Days in culture (day)

10 1

0 2

3
-10
4
-20
12 24 12 24 12 24 12
Time of day (hr)
(b) Time of day (hr)
20 0 4 8 12 16 20 24
RLU (⫻102 counts/min)

Days in culture (day)

10 1

2
0

3
-10
4
-20
12 24 12 24 12 24 12
Time of day (hr)

Fig. 3. Per1-luc rhythms in anterior and posterior SCN slices from mice exposed to long photoperiod. Data show Per1-luc rhythms
of cultured SCN slices from two representative mice (a and b) exposed to LD 18:6. Left panels indicate Per1-luc records of two
consecutive SCN slices, which were detrended by 12 h-moving average from the original data. Solid and broken lines indicate
Per1-luc records of the posterior and anterior slices from the same mouse, respectively. Horizontal dark and white bars for the
first day of culture indicate dark and light phases to which mice were exposed. Right panels indicate peak phases of Per1-luc
rhythms shown in the left panels for 5-day culture. (Reproduced from Fig. 3 of Honma et al., 2008, with permission.)

Per1-luc peak phase-locked with the onset of
the behavioral activity rhythm and the posterior
peak with the offset of behavioral rhythms (Fig. 3).
Neither anterior nor posterior Per1-luc peaks
phase-locked to light on or light off. The findings
suggest that the discrete regions of the SCN were
separately involved in the regulation of the phase
of behavioral rhythms: the anterior SCN being
involved in the regulation of the activity onset (thus
the site of E oscillator) and the posterior SCN, in
that of activity offset (M oscillator). Previously,
on the basis of the phase responses of various gene
knock-out mice, Daan et al. (2001) formulated
a hypothesis that proposed E and M oscillators
consisting of two molecular clocks; the E oscillator
tracks evening lights and is composed of Per2 and
Cry2, and the M oscillator tracks morning light
and is composed of Per1 and Cry1. Our data sug-
gest that Per1 is involved in both E and M clocks,
but that only the region in the SCN is different.
The role of the third oscillator exhibiting an
early morning peak in the anterior SCN is not
known. Per1-luc rhythms in horizontal SCN slices,
by covering entire rostro-caudal parts in a single
 135

SCN slice, demonstrated phase difference of cel-
lular rhythms (Fig. 4). Time of Per1-luc expres-
sion corresponded roughly to the subjective light
phase of an LD cycle to which mice were
exposed. Cells in the caudal SCN peaked earlier
than the rostral ones. In our experiments, we only
detected Per1-luc peaks in LD 18:6 very early in
the morning at around light on in the lateral part
of the anterior-to-middle SCN (indicated by an
arrow in Fig. 4). Newly developed spatiotemporal
maps based on Fast Fourier Transform (FFT)
analysis (Fricker et al., 2007) clearly demonstra-
ted the localization of these different cell groups
(Fig. 5, see the online colored version). Since
the late Per1 peak in the anterior SCN appeared
a few hours before the onset of subjective night
on the first day of culture, in total, the peak
phases were distributed over about 15 h in
the subjective day. The SCN cells dynamically
change their circadian phases depending on the
photoperiod. These results suggest that cell groups
in specific regions of the SCN separately entrain to
light on or off and regulate the offset and onset of
behavioral rhythms separately. On the other hand,
in the SCN slices from mice exposed to short days,
the Per1 peaks are distributed over only a few
hours. Irrespective of the photoperiods, the phases
of circadian Per1-luc rhythms in the anterior SCN
were distributed over a wider range than those in
the posterior SCN, indicating that not only phase
position but also phase distribution is different
depending on the region in the SCN.
Coupling of oscillator cells and that of
regional pacemakers
Although it still remains to be studied as to how
SCN cells communicate their rhythmic informa-
tion to express coherent circadian rhythms,
several candidates have been found. Among
them, VIP seems to be an important coupling

LD6:18
2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00
C

R
LD12:12
C

LD15:9
C

LD18:6
C

Fig. 4. Per1-luc bioluminescence images in horizontal SCN slice. Horizontal SCN slices were cultured from mice exposed to four
different photoperiods, LD 6:18 (L 9:00–15:00), LD 12:12 (L 6:00–18:00), LD 15:9 (L 4:30–19:30), and LD 18:6 (3:00–21:00).
Bioluminescence images were captured by a CCD camera (ORCA-II, Hamamatsu Photonics), cooled at 60  C, by exposing
cultured SCN for 59 min. Exposure was started at the local time indicated at the top of each image. Shown are the time series
images from 2:00 to 22:00 on the second day of culturing. Scale bars indicate 80 mm. Arrows in images at 2:00, 6:00 and 16:00 of
LD18:6 indicate regions where Per1-luc differentially peaked.
136

Amplitude high Phase

caudal
(a) (b)
VIII

rostral

OC

low

0 6 12 18 0
Local time (h)

Fig. 5. Amplitude and phase maps of Per1-luc expression rhythms in a horizontal SCN slice. Time series of bioluminescence images
were analyzed at pixel level by Fast Fourier Transform (FFT). Resultant amplitude maps (a) and phase maps (b) are demonstrated
in psuedocolor. Rhythm amplitude of Per1-luc was high in the posterior SCN. The relative scale of amplitude is shown at the right
side of the amplitude map. The phase map shows differentially phasing cell groups in specific regions in the SCN on the second day
of culturing. Arrows in the SCN from LD 18:6 indicate localization of three cell groups. Horizontal dark and yellow bar indicates
subjective night and day. Horizontal pseudocolor bar shows peak phase of Per1-luc rhythm in each pixel on the second day of
culture. OC, optic chiasm; VIII, the third ventricle; scale bar measures 50 mm.

cue within the SCN. VIP deficiency and VIP
receptor, VPAC2, knockout cause severe dis-
turbances in the behavioral rhythms, which
are basically due to the desynchronization of
SCN cellular oscillators (Maywood et al., 2006).
Furthermore, Maywood et al. (2011) recently
demonstrated, by co-culturing SCN slices from
mice with clock gene mutations with a wild-type
SCN slice, that paracrine signals are able to
restore circadian PER2::LUC rhythms. Diffusible
signals are important not only in synchronization
but also in sustaining the cellular oscillation.
Synaptic interactions are also involved in rhythm
synchronization. We have studied the role of synap-
tic interaction in rhythm coupling by measuring the
spontaneous firing of single SCN neurons before
and after treatment with tetrodotoxin (TTX), a
sodium channel blocker. Dispersed SCN neurons
showed synchronized circadian rhythms when they
had functional synapses (Shirakawa et al., 2000),
but the inter-spike intervals of the synchronized
neuron pairs were lengthened after TTX treatment
(Honma et al., 2000), suggesting a reduced efficacy
of the synaptic transmission. Prolonged suppression
of synaptic interaction by TTX may have reduced
the number of synapses.
TTX treatment also affected the synchroniza-
tion among neurons of cultured SCN slices. Webb
et al. (2009) treated the SCN slice with TTX
twice, with each treatment continuing for 6 days.
About 13% of cells remained rhythmic during
both treatments, suggesting that they are cell-
autonomous clock cells. But these cells were not
localized in specified regions of the SCN. In addi-
tion, they also observed switching of rhythmic
neurons during the repeated TTX challenges.
Some rhythm-positive cells became rhythm nega-
tive in the second trial, and vice versa. Thus, they
concluded that circadian rhythm generation is not
localized to a specific cell groups, but that instead
each SCN cell has about 25% of chance to exhibit
a circadian rhythm. In their experiment, however,
two SCN slices were treated with TTX for 6 days.
The duration of treatment might be long enough
to exert an irreversible change in synaptic trans-
mission. We suppressed synaptic transmission with
TTX for 48 h in the wild-type rat SCN slices
cultured on the MED probe with the method
 137

described elsewhere (Nakamura et al., 2001, 2002).
Following the baseline recording of spontaneous
firing for more than 5 days, SCN slices were treated
with TTX (final concentration 200 nM) for 48 h.
The medium was changed daily. After the treat-
ment, they were washed with the recording
medium twice and spontaneous discharges were
monitored for an additional 1–4 weeks. TTX treat-
ment completely abolished the spontaneous firing
of the SCN neurons. After the washout of TTX,
most neuronal rhythms started to free-run from
the phase predicted by the rhythm before the treat-
ment as shown in Fig. 6, as was observed in those of
dispersed SCN cell cultures (Honma et al., 2000;
Welsh et al., 1995). We analyzed circadian period
by a chi-square periodogram using a record of five
consecutive days at the significance levels of
p  0.01. In each slice, differences between the circa-
dian periods before and after the TTX treatment
were statistically analyzed using two-way ANOVA,
and difference in the period variance within a slice
was analyzed by F-test. The circadian periodicity in

(a) ch12 ch14 ch15 ch18

1
25.1 25.3 25.4 25.1

10 24.0 27.5
24.8 25.1
(days)

20
24.0 25.6 23.9 24.0

0 12 0 12 0 0 12 0 12 0 0 12 0 12 0 0 12 0 12 0
(b) ch1 ch6 ch8
1 ch5
25.0 24.7 25.0 25.2

10 26.1. 24.0 25.7 24.0

(days)

20
24.0 23.9
23.9 23.7

0 12 0 12 0 0 12 0 12 0 0 12 0 12 0 0 12 0 12 0
Time of day (h) Time of day (h) Time of day (h) Time of day (h)

Fig. 6. Effects of TTX treatment on the synchronization of spontaneous firing rhythms. SCN slices of newborn rats were cultured on
an MED probe, and the numbers of spontaneous discharges with signal-to-noise ratio >2 were collected every 20 s from eight
electrodes covered by an SCN slice. Four firing rhythms simultaneously monitored from a single SCN slice are double-plotted.
Firing rhythms from two SCN slices (a and b) are plotted in histogram of mean firing rates against the time of day at 15-min
bins. The number at the upper left margin of each panel indicates channel number of the MED probe. Shadowed areas are the
time of TTX treatment (200 nM, 48 h). Numbers and arrows at the right margin of each panel are the circadian periods of firing
rhythm at the indicated time analyzed by a chi-square periodogram.
138

each channel was designated as altered by TTX
treatment when it showed a larger change than that
observed in vehicle-treated controls (> the mean
period difference þ 2 SD ¼ 1.06 h). Before the treat-
ment, neuronal rhythms were basically synchroni-
zed within a slice. Out of 15 SCN slices examined,
only two SCNs showed no significant difference in
their circadian periods after the TTX treatment
(Fig. 7a). On the other hand, TTX treatment
desynchronized the circadian neuronal rhythms in
seven SCNs and significantly shortened the periods
in six SCNs (Fig. 7c). In the remaining SCN slice,
rhythms either appeared or disappeared, and we
could not compare the difference. Interestingly,
desynchronized rhythms were partially or fully
resynchronized in 1–2 weeks. In this experiment,
we recorded five to eight neuronal rhythms from a
single SCN slice. Out of 95 channels recorded, circa-
dian periods were shortened in 36 and lengthened in
10. Period change was smaller than 1.06 h (the mean
change þ 2 SD of controls) in 40 channels, was
abolished in five channels and appeared in four
channels. In five control SCN slices, the circadian
periods were not significantly changed, but the vari-
ation was changed in two slices: increasing in one
and decreasing in the other. Since circadian periods
were either desynchronized or shortened by TTX
treatment in most SCNs examined, it is apparent
that the suppression of synaptic transmission for
only 48 h modified coupling of cellular oscillators.
In addition, neuronal rhythms were abolished or
appeared in some channels, suggesting a loss or
acquisition of the interaction between neurons with
an autonomous oscillator. The present data suggest
that the ensemble of cellular rhythms is critical for
the expression of synchronized firing rhythms, which
is, at least partly, accomplished through Naþ-chan-
nel dependent mechanisms.
TTX-resistant circadian rhythms have also been
reported. Using the Ca2 þ-sensitive fluorescent pro-
tein Cameleon, Ikeda et al. (2003) reported that
TTX blocked multi-unit activity (MUA) but not
intracellular Ca2 þ rhythms in the SCN cells. On
the other hand, TTX application was reported to

(a) Unchanged (b) Desynchronized (c) Shortened

28 24.0±0.1 24.1±0.2 24.7±0.3 23.8±0.3

26

24
Circadian period (h)

25.4±0.3 25.0±1.3
22

28 24.5±0.2 24.5±0.2 23.7±0.1 23.5±1.0 24.9±0.8 23.8±0.3

26

24

22
Pre Post Pre Post Pre Post

Fig. 7. TTX effects on the synchronous circadian firing rhythm of rat SCN. Effects of TTX treatment (48 h) on the circadian
periodicity were examined in each SCN slice on a MED probe. The presence of significant circadian periodicity and its period
were evaluated by a chi-square periodogram using 5-day records immediately before (pre) and after (post) the treatment.
Results of representative SCN slices in which circadian periodicity of neuronal rhythms was unchanged (a), desynchronized (b),
and shortened (c) are shown. Numbers in each panel are mean circadian periods (SD) of individual firing rhythms.

dramatically suppress the amplitude and to
desynchronize the Per1-luc rhythms in SCN
neurons (Yamaguchi et al., 2003), and it also
suppressed AVP expression rhythms in the SCN
(Arima et al., 2002).
Clock gene functions in the SCN
cellular networks
A lack or mutation of clock genes results in a
genotype-specific rhythm phenotype. Clock is
the first clock gene cloned in mammals. Upon
exposure to DD, the D19 clock-mutant mice
exhibited circadian rhythms with extremely long
periods, which eventually became aperiodic
(Vitaterna et al., 1994). SCN neurons of
organotypic slice cultures from clock-mutant mice
exhibit robust and synchronized circadian firing
rhythms with a period comparable to that of the
behavioral rhythms; however, the percentage of
neurons with significant circadian rhythms was
much less than that of wild-type mice (Nakamura
et al., 2002). The Clock mutation similarly
affected the circadian periodicity of the SCN
neurons in the slice and dispersed cell cultures how-
ever, the mutation dramatically reduced the per-
centage of rhythm-positive neurons in the
dispersed cell cultures. Genotype difference (clock
mutant vs. wild type) and culture system differences
(organotypic slices vs. dispersed cells) suggest that
not all of the SCN neurons possess a cell-autono-
mous circadian clock, as described above, and that
the clock mutation affects rhythm synchronization,
especially that between neurons with and without
a cell-autonomous clock. The mutation may directly
reduce rhythm synchronization by reducing either
coupling signals or receptors. Our results suggest
that SCN tissue structures compensated the effects
of the clock mutation on single neurons (Nakamura
et al., 2002). Such compensation was also observed
in various other clock gene knockouts (Liu et al.,
2007). Therefore, an SCN tissue structure with close
cell-to-cell appositions may be essential to transmit
the rhythm information for synchronization, such
139
as paracrine signals (Maywood et al., 2006, 2011).
Furthermore, extreme circadian periodicities with
a large period distribution may have affected the
synchronization among cellular rhythms. Clock-
mutant SCN cells exhibit much longer periods with
larger variability than wild-type cells. Various clock
gene-deficient mice also exhibit either shorter or
longer periodicity compared with wild-type mice.
Even in wild-type rats, the synchronization within
an SCN slice becomes weaker as the periodicity of
individual neuronal rhythms deviates more from
24 h (Honma et al., 2004). Pittendrigh and Daan
(1976a) demonstrated in rodents that also the circa-
dian period of behavioral rhythms becomes more
variable as the species average deviates more from
24 h, which thus seems to be a general rule for circa-
dian rhythms.
In conclusion, in the SCN, multiple oscillator
neurons with relatively diverse circadian periodic-
ity are mutually synchronized to form a few
regional pacemakers, which further couple to
express coherent rhythm outputs. The multi-
oscillator neuronal pacemaker structure is advan-
tageous for adapting cyclic environments of wide
variability without losing stable periodicity and
phase adjustment. Desynchronization within and
between the regional pacemakers results in an
arrhythmic phenotype even if each cellular clock
can generate a circadian rhythm. Not all of the
SCN cells seem to possess a cell-autonomous clock;
thus, mammals have developed a central circadian
clock in the SCN via oscillatory coupling with neu-
ronal and paracrine signals.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 9

Dynamic neuronal network organization of the

circadian clock and possible deterioration in disease

Johanna H. Meijer{,*, Christopher S. Colwell{,{, Jos H. T. Rohling{, Thijs Houben{ and

Stephan Michel{

{
Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University Medical Center,
Leiden, The Netherlands
{
Laboratory of Circadian and Sleep Medicine, Department of Psychiatry and Biobehavioral Sciences, David Geffen
School of Medicine, University of California, Los Angeles, CA, USA

Abstract: In mammals, the suprachiasmatic nuclei (SCNs) function as a circadian pacemaker that drives
24-h rhythms in physiology and behavior. The SCN is a multicellular clock in which the constituent
oscillators show dynamics in their functional organization and phase coherence. Evidence has emerged
that plasticity in phase synchrony among SCN neurons determines (i) the amplitude of the rhythm, (ii)
the response to continuous light, (iii) the capacity to respond to seasonal changes, and (iv) the phase-
resetting capacity. A decrease in circadian amplitude and phase-resetting capacity is characteristic
during aging and can be a result of disease processes. Whether the decrease in amplitude is caused by
a loss of synchronization or by a loss of single-cell rhythmicity remains to be determined and is
important for the development of strategies to ameliorate circadian disorders.

Keywords: circadian; SCN; network; plasticity; synchronization; entrainment; photoperiod; jet lag; aging;
circadian disorders.

Introduction environment, brought about by the rotation of the

Most animals show clear 24-h rhythms in physiol-
ogy and behavior. These rhythms have developed
as an adaptation to the recurring changes in the
*Corresponding author.
Tel.: þ31 71 526 9760; Fax: þ31 71 526 8270
E-mail: j.h.meijer@lumc.nl
earth around its axis. In order to anticipate these
changes, innate clocks have evolved that allow
organisms to prepare for the predictable onset of
night and day. In mammals, the central clock is
located in the suprachiasmatic nucleus (SCN).
The SCN is a bilateral structure, located at the base
of the brain, with the ventral aspect immediately
above the optic chiasm. The SCN contains about

http://dx.doi.org/10.1016/B978-0-444-59427-3.00009-5 143
144
10,000 neurons on each side and maintains direct
and indirect connections with many parts of the
central nervous system (Kalsbeek et al., 2006;
Morin and Allen, 2006). Light is the major external
stimulus that synchronizes the endogenous clock
to the external 24-h cycle. It reaches the neurons
of the SCN via a monosynaptic pathway, formed
by melanopsin containing retinal ganglion cells
(Berson et al., 2002; Hattar et al., 2003), that proj-
ect with glutamate- and PACAP-containing fibers
to the SCN (Golombek and Rosenstein, 2010;
Hannibal et al., 2000). A distinction has been made
in the core and shell region of the SCN (Antle and
Silver, 2005; Gamble et al., 2007; Ibata et al., 1989;
Kiss et al., 2008; Moore and Silver, 1998). The core
contains gastrin-releasing peptide (GRP)- and
vasoactive intestinal polypeptide (VIP)-expressing
neurons and is retinorecipient (Abrahamson and
Moore, 2001; Antle et al., 2005). The shell contains
vasopressin and receives input from the core of
the SCN (Moore et al., 2002), while the core is
only sparsely innervated by the shell (Romijn
et al., 1997).
Generation of circadian rhythmicity occurs at
the single-cell level and is based on an intertwined
negative feedback loop between clock genes and
their protein products (Herzog, 2007; Reppert
and Weaver, 2002). The genetic basis for rhythm
generation can explain that isolated cells of the
SCN are capable of generating circadian rhythms
and do not require rhythmic input (Webb et al.,
2009; Welsh et al., 1995, 2010). The important
implication is that the SCN functions as a multi-
oscillator structure in which the different neurons
are mutually synchronized in phase in order to
function as a coherent pacemaker. In this chapter,
we review the role of synchronization within the
SCN for the functional adaptation of the SCN as
a clock and cover specific questions: How is the
multioscillator structure used for the adaptation
to shifts of the light–dark cycle? What is the
response of the SCN to constant light and to sea-
sonal changes? What are the consequences of
changes in neuronal synchrony for rhythm wave-
form, amplitude, and resetting capacity? Finally,
we raise the possibility that one mechanism by
which aging and disease can alter the function of
the circadian system is by reducing the strength
of the intracellular coupling within the SCN circuit.
SCN waveform is an ensemble property
Recordings of electrical impulse frequency have
been performed both in vivo in the intact animal
and in vitro in brain slices that contain the SCN.
Pioneers of SCN in vivo recordings were Inouye
and Kawamura (1979) who performed these
recordings to show that the SCN functions as an
endogenous oscillator. SCN in vitro recordings
confirmed that the SCN functions as an endoge-
nous oscillator and does not require rhythmic
input to sustain rhythmicity (Green and Gillette,
1982; Groos and Hendriks, 1982; Shibata et al.,
1982). A consistent finding of in vitro and in vivo
electrophysiological recordings is that during the
day, electrical activity is high, and during the night
it is low, both in nocturnal and in diurnal species.
In nocturnal animals, high activity of the SCN
corresponds with the resting phase and low activity
with the active phase of the animal (Brown and
Piggins, 2007; Colwell, 2011; Gillette, 1996).
In diurnal species, this is reversed and electrical
activity is in phase with the behavioral activity pat-
tern (Challet, 2007). A major difference between
nocturnal and diurnal animals is therefore the
phase relationship between the SCN and other
parts of the central nervous system.
The waveform of the SCN electrical activity
rhythm is almost sinusoidal. Simultaneous
recordings of electrical activity and behavioral
activity have allowed us to determine at what
level of electrical activity, behavior is initiated or
arrested (Houben et al., 2009). The onset of activ-
ity appears to occur at around the 50% level of
the rhythm amplitude. Within a certain range
around the 50% level, the chance for a transition
from rest to activity is maximal. Also for the off-
set of activity, a close correlation with the
50% level is observed. It is concluded that the

on- and offset regulation of behavioral activity
is rather predictable from the level of SCN elec-
trical activity and suggests a direct relationship
between SCN neural activity and the onset–offset
of behavioral patterns. These findings have
implications for situations in which the waveform
of the SCN changes, which will be discussed in
this chapter.
The waveform of the SCN molecular and elec-
trical activity reflects the combined activity of
many neurons and is thus a composite tissue-
level property (Evans et al., 2011; Maywood
et al., 2006; Quintero et al., 2003; Schaap et al.,
2003; Yamaguchi et al., 2003; Yamazaki et al.,
2000). Decomposition of the ensemble pattern
has revealed that small subpopulations and single
SCN neurons exhibit much shorter periods of
enhanced electrical activity. Single units in the
mouse and rat SCN appear to be active for
durations of about 5 h (Brown and Piggins, 2009;
Brown et al., 2006; Schaap et al., 2003;
VanderLeest et al., 2007). The time of maximal
activity of most neurons of the SCN is during the
day, but some neurons are active during the night
(Fig. 1). The distribution of individual neuronal
activity patterns can account for the ensemble pat-
tern of the SCN, as the highest density of active
neurons occurs during midday, while the lowest
density of active neurons occurs at midnight. This
phase distribution renders a sinusoidal pattern in
SCN multiunit activity at the ensemble level.
Constant light effects
Exposing organisms to continuous light causes
robust changes in the circadian rhythms including
a strong reduction in the amplitude of overt
rhythms, lengthening of the endogenous cycle
length (period) (Aschoff, 1979), and can even
lead to splitting of circadian locomotor behavior
and arrhythmicity (Depres-Brummer et al., 1995;
Eastman and Rechtschaffen, 1983; Pittendrigh
and Daan, 1976). Previous data suggest that the
impact of constant light is to desynchronize
145
(a)
800
MUA (Hz)
700
600
500
400
(b)
0 12 24 36 48 60
Time (h)
Fig. 1. Distribution of single neuron electrical activity pattern
explains the multiunit electrical activity pattern of the SCN.
(a) An in vivo electrical activity recording of 60 h, with the
gray background indicating night and the white background
indicating day. (b) One typical recording of an electrical
activity pattern of a mouse housed in a 12:12 light–dark cycle
was used and plotted repetitively according to a Gaussian
distribution, to simulate the phase distribution of neuronal
activity patterns in the SCN. The electrical activity pattern of
the population renders the characteristic multiunit electrical
activity pattern.
single-cell oscillations. For example, recordings
from individual SCN cells in vitro of rhythms
in Period1 promoter-driven GFP fluorescence
rhythms have revealed that individual neurons
from arrhythmic mice kept in constant light are
not compromised in their rhythm-generating abil-
ity (Ohta et al., 2005). In fact, the individual
neurons remained rhythmic, but the population
shows a severely distorted phase distribution.
The strong disruption of phase coherence by
exposure to constant light leads to a decline in cir-
cadian amplitude at the tissue level. Similarly,
in vivo recordings in the SCN of mice kept in
continuous light result in reduced overt rhythmic-
ity and confirm these findings for electrical activ-
ity of the SCN. These recordings revealed a
gradual decline in amplitude and a distortion of
electrical rhythmicity upon prolonged exposure
to continuous light (Fig. 2). The results show the
necessity of coupling mechanisms, in addition to
the presence of rhythm-generating units, and indi-
cate the vital importance of the network-level
146

(a)

700
MUA (Hz)

600

500

400

300
0 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816
Time (h)
(b)
100
Rhythm amplitude (Normalized)

75

50

25

0
DD 1 2 3 4 5 6 7
Circadian cycle

Fig. 2. Effect of constant light on SCN neuronal activity and behavior. (a) Long-term in vivo recording of SCN neuronal activity in a
freely moving mouse. SCN activity was recorded with implanted microelectrodes and is depicted in 10-min bins (black trace). Gray
background indicates lights off and white background lights on. During the first days in constant light, a decrease in amplitude is
observed. During prolonged exposure to constant light, the amplitude of the rhythm was variable as the SCN appeared to lose
and then regain coherence. This may reflect temporal synchronization and desynchronization of the constituent oscillators.
(b) Effect of constant light on the mean amplitude of the SCN rhythm in seven animals. The animals were exposed to LL for at
least 7 days following exposure to constant darkness. The rhythm was normalized for each animal and expressed relative to the
amplitude in constant darkness (gray bar, SEM). White bars depict the amplitude during the first 7 days in constant light.

organization of the SCN to obtain robust and high- factors determine the phase coherence within the
amplitude rhythms. SCN and hence influence the waveform of the
Synchronization among the neurons of the SCN ensemble rhythm. Coupled oscillator models have
is critically determined by a variety of coupling found that large groups of oscillators such as
mechanisms (for review, see Welsh et al., 2010). SCN neurons can organize themselves into many
Several coupling factors have been implicated such different configurations to meet environmental
as VIP, GABA, and even gap junctions. These challenges. These conformational changes are

rapidly driven by alterations in the strength of
neuronal coupling mechanisms (Golubitsky
et al., 1999; Strogatz, 2003; Strogatz and Stewart,
1993). The flexibility in coupling strength between
neuronal populations enables the system to cope
with different environmental conditions such as
changes in the light–dark cycle and responses to
constant light. In the next section, we will consider
how changes in day length affect the phase syn-
chrony among SCN neurons.
Seasonal changes in waveform by changes in
phase distribution
In response to the changes in day length that
occur over the course of the year, many animals
undergo strong alterations in their anatomy,
physiology, and behavior. Seasonal breeding
organisms show drastic changes in behavioral
activity, metabolism, and reproductive physiology
(Ebling and Barrett, 2008; Hazlerigg and Loudon,
2008). The duration of the melatonin signal is a
critical cue for some seasonal breeders, while
other seasonal adaptations do not appear to be
driven by melatonin. To provide an example,
C57 mice have very low melatonin and yet display
robust photoperiodic driven changes in behavioral
activity. When these mice are exposed to long or
short photoperiods, the duration of activity
(alpha) is systematically lengthened in short days
and shortened in long days. The changes in alpha
are maintained even when the mice are placed in
constant darkness. The behavioral activity pattern
that is then displayed reflects the previous photo-
period, indicating an endogenous memory, or
encoding mechanism, which is present for days
to weeks (Sumova et al., 2004).
The SCN plays an important role in the seasonal
encoding process. The sinusoidal-like waveform of
the SCN electrical activity profile is strongly
altered under influence of long and short days, as
shown by in vivo recordings of the SCN, or by in
situ analysis of clock gene expression. The electri-
cal activity profile becomes compressed (narrow
147
peak) in short days and decompressed (broad
peak) in long days (Houben et al., 2009; Mrugala
et al., 2000). The same modulation at the tissue—
or population—level is observable in a number
of clock genes (Carr et al., 2003; Johnston
et al., 2003, 2005; Nuesslein-Hildesheim et al.,
2000; Sumova et al., 2002, 2003; Tournier et al.,
2003), Fos expression (Jac et al., 2000b; Sumova
et al., 1995; Vuillez et al., 1996), and vasopressin
(Jac et al., 2000a) and prokineticin 2 (PK2)
mRNA levels (Cheng et al., 2005). The peak width
appears to serve as an internal representation of
the length of the day. Importantly, the photope-
riodic driven changes in the SCN waveform and
activity patterns are maintained for weeks even
when the animal is released back into constant
conditions. We view these changes as a type of
plasticity in SCN physiology.
While photoperiodic encoding of day length is
explainable by the change in the width of the elec-
trical activity peak, a valid question is whether also
changes in triggering level account for the modula-
tion in activity duration. Presumably, a higher trig-
gering level would result in an increase in the
activity period and a lowering of the triggering
level in an increase in the activity duration. This
mechanism was proposed by Wever (1960) as a
possible underlying mechanism for modulation of
long- and short-activity periods in finches. In order
to explore this possibility, we have recorded the
SCN in vivo under long and short photoperiods
and have simultaneously scored the onset and off-
set of behavioral activity (Houben et al., 2009).
However, we found no evidence for changes in
triggering level, and instead, we observed that
both in long and in short days, transitions between
activity to rest were most likely to occur at 50% of
the electrical activity rhythm. We conclude that
the change in peak width represents the major
mechanism by which photoperiod is internally
encoded by the SCN, and that no changes in
triggering level are involved.
The plasticity in the waveform of the SCN,
observable in behavioral activity patterns, and in
the SCN in vivo is preserved when the SCN is
148

recorded in vitro, in a brain slice preparation (a)

(Mrugala et al., 2000). This finding opens up
the important possibility to investigate the mecha-
nism underlying SCN waveform changes in vitro. 8
Two studies have independently proposed that

Activity (Hz)
SCN neurons show oscillations in their molecular 6
or electrical activity pattern that are out of phase
and that these phase differences could contribute 4
to the photoperiodically induced changes in
2
waveform (Quintero et al., 2003; Schaap et al.,
2003). These predictions were confirmed in 0
recordings of subpopulations of individual SCN 0 6 12 18
neurons from mice held in different photoperiods ZT (h)
(Brown and Piggins, 2009; Hazlerigg et al., 2005; (b)
Inagaki et al., 2007; Naito et al., 2008; Sosniyenko
et al., 2009; VanderLeest et al., 2007). These stud-
ies all found evidence that exposing mice to long 8
days causes a desynchrony in the electrical activ-
Activity (Hz)
ity and gene expression rhythms within the SCN 6
(Fig. 3).
Surprisingly, the neural activity patterns 4
recorded from individual SCN neurons show little
2
change in their activity pattern in long and short
days (Brown and Piggins, 2009; Naito et al., 2008; 0
VanderLeest et al., 2007). Photoperiod changes 18 0 6 12
primarily the phase distribution but not the dura- ZT (h)
tion of electrical activity within individual cells.
While Brown and Piggins (2009) observed longer Fig. 3. The distribution of single-unit electrical activity patterns is
duration activity of cells in the dorsal SCN under responsible for the difference in multiunit electrical activity
pattern in long and short photoperiod. The multiunit electrical
long days, they also showed that these changes activity pattern in short (a) and long (b) photoperiod is shown
render very little difference in the duration of as a thick black line. This pattern is derived from an ensemble
the population activity pattern in agreement with of single-unit activity patterns that are distributed over the 24-h
simulations and predictions from other studies cycle according to a Gaussian distribution. The single-unit
(Rohling et al., 2006a,b).There is consensus on activity patterns for short and long photoperiod were determined
from a number of recorded single-unit activity patterns (25 for
the conclusion that the increment in the active short photoperiod, 22 for long photoperiod). The half-maximum
duration of the population is caused by changes values of the single-unit patterns were determined and the time-
in phase distribution, rather than by changes in point that expressed the middle between both half-max
single-cell activity pattern. values was used to align all single-unit patterns. The mean of
all aligned single-unit patterns was then determined, and
As a consequence of this decreased synchrony in
these averaged single-unit patterns were distributed over the
long photoperiods, neural activity rhythms are 24-h cycle according to a narrow Gaussian distribution for
broader and exhibit a lower peak–trough ampli- short photoperiod (a) or broad Gaussian distribution for long
tude. In contrast, exposure to short days leads to photoperiod (b). The multiunit activity pattern was determined
enhanced synchrony among SCN neuronal by summating these distributed single-unit activity patterns.
populations with a resulting narrowing of the peak Above the figure, the light–dark schedule for each photoperiod
is shown.
and increased amplitude of the SCN rhythm.

Phase-shifting capacity as a function of neuronal
synchrony
The photoperiod to which animals have been
exposed determines the phase-shifting capacity of
the circadian system. The impact of photoperiod
on the amplitude of the phase response curve
(PRC) was first demonstrated by Pittendrigh and
coworkers (1984). Hamsters kept on short days
exhibited a larger amplitude PRC than animals
kept on long photoperiod. The findings (also see
Evans et al., 2004; Refinetti, 2002) raise questions
about the underlying mechanisms. One possibility
is that long light exposure desensitizes the retinal
ganglion cells to light, and that the smaller shifts
in long days reflect a difference in retinal light
response. In a recent study, we exposed mice to
short and long photoperiods but doubled the
hourly number of photons in short photoperiods
(VanderLeest et al., 2009). Accordingly, the
animals received the same amount of photons in
long and short photoperiod but distributed over a
different time span. No decline in the phase-
shifting response was observed in the short days,
and the response remained significantly larger than
in long days. Although other interpretations are
possible, these findings provided a first indication
that retinal information processing is not responsi-
ble for the photoperiod-driven changes in the mag-
nitude of light-induced phase shifts.
The other possibility is that the difference in
phase-shifting capacity is not determined at the
level of the retina but is determined at the level
of the SCN neuronal network. In this case, the
difference in phase-shifting capacity observed in
long and short days should be preserved in vitro.
To test this, we prepared brain slices from mice
held on long and short photoperiods and exam-
ined the phase-shifting effects of the application
of NMDA. A variety of evidence suggests that
NMDA receptors are critical for light-induced
phase shifts and we, and others, have found that
bath application of NMDA can produce phase
shifts in the neural activity rhythms (Ding et al.,
1994; VanderLeest et al., 2009). While the acute
149
response to NMDA was not different in the short-
and long-day preparation, the phase-shifting
effect was significantly larger in slices from short
days. The data indicate that reduction in the mag-
nitude of phase shifts observed in long days is
intrinsic to the SCN network organization.
In long days, the neuronal populations of the
SCN are desynchronized, yielding an ensemble
pattern with a low-amplitude rhythm. In short
days, the neurons are highly synchronized in
phase, leading to a high-amplitude rhythm of the
population. Our finding that large shifts are
obtained under conditions in which the neural
activity rhythm is exhibiting a high-amplitude
rhythms is in some ways surprising in that it does
not fit predictions from the limit cycle oscillator
theory that the magnitude of the phase shift is
inversely related to the amplitude of the rhythm
(Pittendrigh et al., 1991; Pulivarthy et al., 2007).
This assumption is based on the notion that a per-
turbation of similar strength changes the phase
angle of a low-amplitude rhythm more than the
phase angle of a high-amplitude rhythm because
the perturbation is a larger fraction of the radius
of the cycle. This prediction has been confirmed
in many biological (and nonbiological) systems
and is valid for single-cell rhythms (Gonyaulax:
Johnson and Kondo, 1992; Neurospora: Johnson,
1999; Lakin-Thomas et al., 1990). We believe that
the results of vanderLeest et al. (2009) show that
the limit cycle theory does not hold for the net-
work of the SCN in explaining differences in
phase-shifting capacity in long and short days.
Instead, we favor the view that under long-day
conditions, the SCN network is desynchronized
and the individual neurons receive the NMDA
pulse at different phases of their circadian cycle,
leading to diverse phase-shifting responses. When
the neurons are more synchronized, they would
respond more consistently, leading to a larger
net-shift of the ensemble. Using computer
simulations, we have found that the phase-shifting
response of the population can be simulated by
distributing neurons over the cycle, according to
the observed phase distribution in long and short
150
days (VanderLeest et al., 2009). We supposed
that the individual neurons showed limit cycle
oscillator behavior, and that they had the same
PRCs. Distribution of neurons, in the simulations,
was therefore similar to distribution of PRCs.
Because the PRCs of individual SCN neurons
are not known, we gave the neurons either type
1 or type 0 PRCs, to test the feasibility of our
explanation (but only one type per simulation).
When we distributed neurons according to the
distribution observed in long photoperiods, the
delaying and advancing parts of the PRC do not
fully overlap. As a result, a particular pulse will
lead to delays in some neurons, but to advances
in others. The net result of these divergent shifts
is a minor shift of the population as a whole.
When the PRCs are fully synchronized, a particu-
lar pulse will lead to a unidirectional shift in all
neurons, and the net result will be a much larger
phase response of the population (Fig. 4).
We are aware that the present simulations pres-
ent only one possible explanation for the observed
difference in phase response magnitude in long and
(a) (b)
Pulse Pulse
Single cells
6 12 18 24 6 12
Population
0 6 12 18 24 0 6 12
CT (h) CT (h)
Fig. 4. Distributed single-cell phase response curves (PRCs)
determine the ensemble PRC. If the single-cell PRCs are all
in the same phase of the cycle, the resulting ensemble PRC
will have a large amplitude as a single light pulse (arrow)
will trigger a similar phase-shifting response in all cells (a). If
the single-cell PRCs are out of phase, the resulting ensemble
PRC will have a lower amplitude as the cells will have
divergent responses (b).
short days which needs to be confirmed experi-
mentally. Notwithstanding, it has become clear
that in the neuronal network of SCN oscillators,
at least one prediction of the limit cycle oscillator
theory does not hold, that is, the larger amplitude
rhythms showed the larger instead of the smaller
phase-shifting responses. The experiments demon-
strate that properties emerge at the population
levels that are not present at the underlying
single-cell level.
Phase resetting is driven by a small population of
highly synchronized neurons
Shift work and jet lag cause a temporal disruption
of circadian rhythms that are evidenced by sleep
disturbances, intestinal problems, and fatigue
(Arble et al., 2010; Eastman and Burgess, 2009;
Waterhouse et al., 2007). Animals have been
exposed to an abrupt shift of the light–dark cycle
to investigate the mechanism underlying phase
adjustment of the SCN. Following such a shift, a
dissociation of the electrical profiles and the
molecular expression patterns of the ventral and
dorsal SCN becomes visible (Albus et al., 2005;
Davidson et al., 2009; Nagano et al., 2003;
Nakamura et al., 2005; Reddy et al., 2002; Yan
and Silver, 2004). Desynchrony between the
dorsal and ventral SCN areas can also be induced
by exposure of rats to short light–dark cycles
(de la Iglesia et al., 2004), but in the case of a jet
18 24 lag, the phase desynchrony is transient and cou-
pling is eventually restored. Following a shifted
light–dark cycle, the neurons in the ventral SCN
appear to shift rapidly followed by a much slower
18 24
shift of the dorsal SCN (Albus et al., 2005;
Nagano et al., 2003; Nakamura et al., 2005). The
difference in phase-resetting speed may relate to
the differential innervation of the ventral versus
the dorsal SCN area by the retina (Antle and
Silver, 2005, 2009; Card et al., 1981; Gamble
et al., 2007; van Esseveldt et al., 2000). The ventral
SCN receives most of the retinal input (Ibata et al.,
1989; Kiss et al., 2008; Morin and Allen, 2006)

and shows more robust light-induced changes in
electrical activity (Meijer et al., 1998; Shibata
et al., 1984) and gene expression (Dardente et al.,
2002; Guido et al., 1999; Karatsoreos et al., 2004;
Kuhlman et al., 2003; Schwartz et al., 2000; Yan
et al., 1999). The initial shift of the ventral SCN
may thus be explainable by the stronger influence
of environmental light input on this part of the
nucleus.
The rapid shift of the ventral SCN can be easily
observed in an SCN slice, prepared after expo-
sure to a 6-h delay of the light–dark cycle (Albus
et al., 2005). The differential shifts of ventral
and dorsal SCN become visible by two peaks in
the multiunit activity rhythm. The one peak is
delayed by nearly 6 h, while the other peak shows
no significant shift. A surgical cut, made between
the ventral and dorsal part, made it clear that the
shifted component originates from the ventral
part, and the unshifted component from the
dorsal part. The findings also show that, in the
intact slice, communication and transfer of electri-
cal activity occur within the SCN circuitry, as in
intact slices, both components are visible in the
dorsal and ventral SCN. The transmittance of
information may rely on GABAergic activity as
bicuculline blocked the information transfer, and
single peaks (i.e., a shifted peak in the ventral
SCN and an unshifted peak in the dorsal SCN)
were observable in intact slices in the presence
of bicuculline.
GABA plays a role in the transfer of information
between ventral and dorsal SCN. SCN neurons
receive a tonic GABAA receptor-mediated synap-
tic input that, at least partly, originates within the
SCN itself and peaks during the night (Itri et al.,
2004; Jiang et al., 1997; Kim and Dudek, 1992;
Strecker et al., 1997). Although GABA is normally
an inhibitory transmitter within the SCN, for a
certain percentage of cells within the SCN circuit,
GABA can play an excitatory role in communica-
tion (Choi et al., 2008; Irwin and Allen, 2009). This
GABA-mediated excitation is critically dependent
on the activity of the chloride pump NKCC1
(Belenky et al., 2010; Choi et al., 2008). Recent
151
work in other hypothalamic neurons demonstrates
that the activity of NKCC1 can be actively
regulated such that GABA can switch from being
inhibitory to excitatory as a function of physiologi-
cal demands (Kim et al., 2011). It is quite possible
that a similar dynamic regulation of GABA signal-
ing may be occurring in the SCN. Functionally, the
exogenous application of GABA can synchronize
the electrical activity of SCN neurons (Liu
and Reppert, 2000; Shirakawa et al., 2000), and
GABA may synchronize ventral and dorsal SCN
following a shifted light cycle (Albus et al., 2005);
however, GABAergic signaling does not appear
to be required for cultured SCN neurons to remain
synchronized (Aton et al., 2006).
It is evident that the shift of the ventral SCN
ultimately causes a phase shift of the whole SCN
network. When an SCN slice is prepared follow-
ing the shift of the light–dark cycle, and after
three days in constant darkness, the dissociation
within the SCN is still observable (Albus et al.,
2005; Nagano et al., 2003; Nakamura et al.,
2005). After day 6 in constant darkness, the
dorsal and ventral SCNs have resynchronized,
and the final peak time is near the time of the
shifted ventral SCN. These findings indicate a
strong effect of the ventral on the dorsal SCN,
while, in contrast, the dorsal SCN has a small
effect on the ventral SCN. The idea that light
information flows from ventral to dorsal is consis-
tent with anatomical studies (Moore et al., 2002;
Romijn et al., 1997).
An intriguing observation in our experiments
was that the shifted component of the multiunit
activity pattern was consistently narrower than
the unshifted component (Fig. 5). A curve fitting
analysis revealed that this observation was highly
consistent (Rohling et al., 2011). We performed
recordings of neuronal subpopulations following
exposure to the shifted light–dark cycle and
observed that only 20% of the neurons peaked
in the middle of the new light phase. This
indicates that only 20% of the neurons seemed
to have shifted on the first day after the shift in the
light cycle (Rohling et al., 2011). The narrowness
152
(a)
300
MUA (Hz)
200
100
0 responsiveness (Meijer et al., 1986) and with the
18 0 6
ZT (h)
(b) (c)
100 10
80 8 the light–dark cycle.
Variance (h)
Area (%)
60 6
40 4 have a bigger influence on less synchronized
20 2 2003; Winfree, 1967). Thus, when two groups of
0 0
Unshifted shifted
Fig. 5. After a 6-h delay of the LD cycle, one small population of
neurons shifts its activity immediately to the new LD regime,
while another, larger population of cells remains active in the
old phase. (a) Example of a typical bimodal SCN rhythm,
following a phase delay (black line). The timescale indicates the
new Zeitgeber time, that is, ZT 6 is the middle of the shifted
day. When Gaussian functions were fitted through the MUA
pattern, two different Gaussian functions were found in this
example. One Gaussian function represents an unshifted
population and one represents a shifted population. In this
example, the percentage of action potentials responsible for the
unshifted population is 83%, while the shifted component
contains 17% of the action potentials. (b) For all recordings,
subpopulation patterns were recorded and the variance
between the different phases of the subpopulations was
assessed. The subpopulations before the trough, in the
unshifted component, had a much larger variability than the
subpopulations after the trough, in the shifted component (7.9
vs. 0.7 h, respectively). (c) Our data indicate that the neurons in
the shifted component are much more synchronized than the
neurons in the unshifted component.
of the peak of the shifted component is not a triv-
ial finding when a low number of subpopulations
is recorded, as can be shown by Monte Carlo
simulations (Schaap et al., 2003). Instead, it
indicates that the shifted neurons are highly syn-
chronized in phase. Recordings of the phase dis-
tribution of the shifted neurons confirmed this
prediction (Rohling et al., 2011). The percentage
of neurons that shifts immediately corresponds
with the fraction that shows direct light
12 18 subtype of VIP cells that receive direct retinal
input (Kawamoto et al., 2003). These neurons
showed an absence of endogenous rhythmicity
(Kawamoto et al., 2003), which could make them
more susceptible to phase-shifting influence of
From the coupled oscillator theory, we can
explain that more tightly synchronized neurons
neurons (Mirollo and Strogatz, 1990; Strogatz,
neurons are out of phase, the more synchronized
Before After
group will exert a strong phase-shifting effect on
the less synchronized group. This can be under-
stood as, in a firmly synchronized population,
each neuron is exerting its maximal phase-shifting
effect at the same time of the cycle, rendering a
strong signal of the group at a particular phase
of the cycle. Previous studies reported that the
ventral neurons are dominant over the dorsal
neurons and determine to a large extent the final
phase (Albus et al., 2005; Nagano et al., 2003;
Nakamura et al., 2005). The synchronization
among the shifted neurons may contribute to
their dominance over the unshifted population.
Adjustment to phase advances is more complex
than adjustments to phase delays and requires
more days. This cannot be explained by the endog-
enous period of the rhythm, as in nocturnal
animals, the period is generally shorter than 24 h,
which would facilitate a faster resetting. The slow
adaptation to a phase advance results in part from
feedback of the central nervous system to the
SCN (Vansteensel et al., 2003). In the isolated
SCN, when the SCN is deafferented from this feed-
back input, the phase-advancing capacity is there-
fore strongly enhanced (Vansteensel et al., 2003).

Cav2.1 calcium channels play a role in the influence
of the central nervous system on the SCN (van
Oosterhout et al., 2008). The CACNA1A gene
encodes the ion-conducting, pore-forming a1A sub-
unit of voltage-gated Cav2.1 (P/Q type) calcium
channels (Ophoff et al., 1996). These channels are
predominantly localized at presynaptic nerve
terminals in several brain regions (Westenbroek
et al., 1995), including the SCN (Chen and van
den Pol, 1998; Cloues and Sather, 2003; Nahm
et al., 2005), and play a key role in mediating
neurotransmitter release (Mintz et al., 1995;
Wu et al., 1999). Animals with a gain of function
in these channels (used as a migraine model), show
enhanced phase-advancing capacity, while their
phase delays are unaltered (van Oosterhout et al.,
2008). However, the phase-resetting capacity of
the SCN in isolation is unaltered in these animals.
These findings suggest a role for Cav2.1 channels
in mediating influences of the central nervous sys-
tem on the SCN, and indeed, the synaptic input to
dorsal SCN neurons is enhanced in these animals
(van Oosterhout et al., 2008). The complexity of
phase advances is thus a consequence of an addi-
tional level of organization that attenuates the
phase-shifting capacity of the circadian system.
Analysis of the SCN phase synchrony following
an advance has shown that also in response to
phase advances, the ventral SCN shifts first and
is followed by a shift of the dorsal SCN, which
is similar to the response to delays (Albus
et al., 2005; Nagano et al., 2003; Nakamura
et al., 2005; Reddy et al., 2002; Yan and Silver,
2002, 2004). However, advances induce a larger
degree of desynchrony within the SCN as com-
pared to phase delays (Rohling et al., 2011). The
desynchrony may contribute to the inertia in
response to phase advances, and possibly, it is
related to the role of the central nervous system
in phase advances, as the activation of input pat-
hways from other areas may well contribute to
phase desynchrony within the SCN. However,
the role of extra SCN areas on the SCN is to date
largely unexplored and deserves attention in
future studies.
153
Collectively, the phase-resetting studies lead to
the following model for resynchronization:
In response to a shifted light cycle, only the
oscillators in the ventral SCN are initially shifted.
Other areas of the SCN become synchronized to
the shifted light cycle through interoscillator-
coupling mechanisms. GABA may be involved in
the transmission between ventral and dorsal
oscillators. The strong effect of the small group of
ventral neurons on the dorsal neurons may rely
on the excitatory effect of GABA on the dorsal
SCN as well as on the high degree of synchrony of
the shifted versus the unshifted cells.
The differences in phase synchronization among
neuronal populations as observed under light–dark
cycles, under constant light, under shifted light
cycles, and under changes in photoperiod have
made clear that the multioscillator structure of the
SCN is strongly involved in the adaptive function of
the SCN. The different environmental conditions
can lead to reconfiguration of the phases among
the SCN population and determine the waveform
of the SCN rhythm. These findings are in agree-
ment with the theory of coupled oscillators, which
proposes that the same system of coupled neurons
can account for many different states just by chang-
ing its configuration (Golubitsky et al., 1999;
Mirollo and Strogatz, 1990; Strogatz, 2003;
Strogatz and Stewart, 1993). These findings also
lead to the question of how the coupling among
SCN oscillators is affected by aging and diseases.
Effects of aging and disease
Disruptions in the circadian system, including
decreased amplitude of rhythmic behaviors and
fragmentation of the activity–rest episodes, are
commonly associated with aging in humans and
other mammals (Carrier and Bliwise, 2003; Van
Someren, 2000). While undoubtedly many factors
contribute to these changes, a variety of data
(Aujard et al., 2001; Biello, 2009; Satinoff
et al., 1993; Turek et al., 1995; Watanabe et al.,
1995) are emerging that is consistent with the
154
hypothesis that an age-related decline in the output
of the central circadian clock in the SCN may be
key. For example, in recent work, in vivo multiunit
recordings were carried out from the SCN and
a brain region that receives robust innervation
from the SCN (subparaventricular zone) in freely
moving animals. The amplitude of the day–night
difference in neural activity was substantially
reduced in both brain regions of middle-aged mice
(Nakamura et al., 2011). Another striking feature
was the increase in variation in the levels of the
spontaneous activity. In contrast, the molecular
clockwork in the SCN as measured by PERIOD2
levels was not disrupted in middle-aged mice.
These results suggest that the age-related disrup-
tion in the circadian output occurs before any
disruption of the molecular clockwork. The
mechanisms underlying the age-related decline in
SCN neural activity are unknown but are an impor-
tant area for future research. As both sleep states
(Deboer et al., 2003) and locomotor activity
(Meijer et al., 1997; Schaap and Meijer, 2001;
Yamazaki et al., 1998) can “feedback” to regulate
SCN neural activity, future studies will need to
address the issue of to what extent the age-related
decline in sleep and activity contributes to the
reduced SCN neural activity seen in vivo. Several
studies have shown that the electrophysiological
activity of aged SCN neurons in vitro is altered in
situations where these feedback mechanisms
would not be in operation (Aujard et al., 2001;
Biello, 2009; Nygard et al., 2005; Satinoff et al.,
1993; Watanabe et al., 1995). The firing rate
changes found in the SCN itself could be medi-
ated by age-related alterations in synaptic trans-
mission within the circuit as well as changes in
ion mechanisms and cellular metabolism intrinsic
to single SCN neurons.
Patients suffering from neurodegenerative
disorders including Alzheimer’s disease (AD),
Parkinson’s disease (PD), and Huntington’s dis-
ease (HD) commonly exhibit sleep disorders.
These patients have difficulty sleeping at night
and staying awake during the day. These symptoms
have a major impact on the quality of life of the
patients and on their caregivers. While the underly-
ing pathology has not yet been identified, several
studies have been carried out using mouse models
of these neurodegenerative diseases (Kudo et al.,
2011a,b; Morton et al., 2005; Oakeshott et al.,
2011; Sterniczuk et al., 2010). Most of these mouse
models exhibit circadian disruptions and there is at
least some evidence that treatments designed to
stabilize these rhythms can improve other, non-
motor symptoms of these mice (Maywood et al.,
2010; Pallier et al., 2007). In one of the mouse
models of HD (the BACHD line) in which the
mutated human Htt gene is expressed, the output
of the circadian system as measured by locomotor
activity, heart rate, and body temperature was pro-
foundly disrupted early in the life span (Kudo et al.,
2011b). The neural activity rhythms in the SCN,
but not rhythmic PER2 expression, were also
reduced in the BACHD mice. A very similar story
is emerging from a study of a line of alpha-
synuclein-overexpressing (ASO) mice (Kudo
et al., 2011a). Selective deficits were found in the
expression of circadian rhythms of locomotor
activity, including lower nighttime activity and
greater fragmentation in the wheel-running activ-
ity in this model of PD and other synucleinopathies.
The temporal distribution of sleep was also altered
in the ASO mice compared to littermate controls.
In the ASO mice, the peak–trough expression of
the clock gene PERIOD2 was normal in the SCN;
however, the daytime firing rate of SCN neurons
was reduced in the mutant mice. Together, these
data in mouse models of AD, HD, and PD in com-
bination with the clinical symptoms raise the possi-
bility that a weakening of circadian output is a core
feature of neurodegenerative diseases.
Could disease processes alter the synchrony of
the SCN circuit?
One mechanism by which aging and disease can
alter the function of the circadian system is by
reducing the strength of the intercellular coupling
within the SCN circuit. Recent work in a variety

of mouse models of neurodevelopmental and psy-
chiatric disorders suggests that alterations in the
balance between synaptic excitation and inhibi-
tion are at the heart of the pathophysiology in
these conditions (Dani et al., 2005; Gogolla
et al., 2009; Milnerwood and Raymond, 2010;
Nelson and Turrigiano, 2008; Shepherd and Katz,
2011). Within the SCN circuit, glutamate and
the peptide PACAP are the neurotransmitters
released from the RHT that drive the effects of
light on the retinorecipient SCN neurons
(Colwell, 2011; Morin and Allen, 2006). For
neurons within the circuit, the main transmitter
within the SCN is GABA (Moore and Speh,
1993; Okamura et al., 1989) with most neurons
receiving a constant flux of GABA signaling (Itri
et al., 2004; Jiang et al., 1997; Kim and Dudek,
1992; Strecker et al., 1997). Interestingly, GABA
can mediate both inhibitory and excitatory
postsynaptic effects within the SCN (Choi et al.,
2008; Gribkoff et al., 1999; Irwin and Allen,
2009; Wagner et al., 1997) with the activity of
the chloride pump NKCC1 being the critical
determinant (Belenky et al., 2010; Choi et al.,
2008; Irwin and Allen, 2009). So, one likely conse-
quence of a number of diseases of the central ner-
vous system is a change in the balance between
excitation and inhibition synaptic transmission.
While we have no specific evidence for a disease
that alters synaptic transmission or coupling within
the SCN circuit, this remains a very plausible
mechanism to explain the disruption in circadian
function. Recent work shows that the blockade
of synaptic transmission within the SCN disrupts
both circadian gene expression and neural activity
rhythms (Deery et al., 2009; Kim et al., 2009).
In addition, the SCN circuit highly expresses sev-
eral peptides including VIP, GRP, vasopressin,
and PK2. We do not know whether these peptides
function as neurotransmitters or more as cofactors
within this circuit. Among these peptides, perhaps
the best studied is VIP and there is overwhelming
evidence for this peptide playing a critical role in
coupling or synchronizing cellular oscillators within
this circuit (Freeman and Herzog, 2011; Maywood
155
et al., 2011; Vosko et al., 2007; Welsh et al., 2010).
A number of studies have found evidence that the
levels of VIP in the SCN are reduced with aging
and neurodegenerative diseases in humans and
rodents (e.g., Duncan et al., 2010; Fahrenkrug
et al., 2007; Mazurek et al., 1997; Pereira et al.,
2005; Zhou et al., 1995). These data add a key
support to our suggestion that an important con-
sequence of disease pathology will be to alter
intra-SCN coupling within the circadian circuit
and decrease the synchrony of the SCN popula-
tion. Based on the work described in this chapter,
the loss of coupling and decreased synchroniza-
tion would have the consequence of reducing
the phase-shifting effects of light and other
phase-shifting agents. The reduced synchrony
would also reduce SCN neural activity and, as a
consequence, reduce the amplitude of any SCN-
driven output. A reduction in output would be
expected to change the phase relationships
between the SCN and the peripheral oscillators.
Thus, many of the key circadian symptoms
exhibited with aging and diseases of the nervous
system could be explained by a reduction in the
synchrony of the SCN cell population.
These types of disruptions of circadian system
caused by altered coupling within the SCN circuit
are likely to have profound consequences on
patient health (Hastings et al., 2003; Reddy and
O’Neill, 2010; Takahashi et al., 2008). We believe
that robust circadian rhythms are essential
to good health. In recent years, a wide range of
studies have demonstrated that disruption of the
circadian system leads to a cluster of symptoms,
including metabolic deficits (Gale et al.,
2011; Marcheva et al., 2010; Turek et al., 2005),
cardiovascular problems (Bray et al., 2008; Scheer
et al., 2009), difficulty sleeping (Reid and Zee,
2009; Wulff et al., 2009), and cognitive deficits
(Gerstner et al., 2009; Loh et al., 2010; Wang
et al., 2009). Many of these same symptoms are
seen in aging and neurodegenerative diseases.
This suggests that we should put a greater empha-
sis on the development of pharmacological tools
and behavioral interventions that can boost
156
neural activity rhythms and the synchrony of the
SCN cell population in situations in which the
molecular clock may still be working.
Acknowledgment
This work was supported by NWO travel grant
(040.11.242) for collaboration between C. S. C.
and S. M.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 10

Interaction of central and peripheral clocks

in physiological regulation

Johanna L. Barclay{, Anthony H. Tsang{ and Henrik Oster{,{,*

{
Circadian Rhythms Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
{
Medical Department I, University of Lübeck, Lübeck, Germany

Abstract: In mammals, circadian rhythms of physiology and behavior are regulated by a complex
network of cellular molecular oscillators distributed throughout the brain and peripheral tissues.
A master clock in the hypothalamic suprachiasmatic nuclei (SCN) synchronizes internal time with the
external light–dark cycle, thus entraining the overall rhythmicity of the organism. Recent findings have
challenged the dominant role of the SCN in physiological regulation and it becomes increasingly
evident that close interaction between different central and peripheral clocks is necessary to maintain
robust circadian rhythms of physiology and metabolism. In this review, we summarize recent findings
regarding circadian organization in the SCN and in other central and peripheral tissues. We outline
the communication pathways between different tissue clocks and, exemplified by the regulation of
glucocorticoid release from the adrenal gland and glucose homeostasis in the blood, characterize the
interaction between different clocks in the regulation of physiological processes.

Keywords: SCN; adrenal; circadian clock; glucose; metabolism; glucocorticoids; liver; clock genes;
mammals; pancreas.

Introduction its axis. The biological timing system that organizes

Living organisms—from unicellular prokaryotes
to multicellular metazoans—have evolved diverse
strategies to internalize the diurnal environmental
changes brought about by Earth’s rotation around
*Corresponding author.
Tel.: þ49-551-201-2738; Fax: þ49-551-201-2705
E-mail: henrik.oster@mpibpc.mpg.de
such 24-h oscillations is known as the circadian
clock. The two major functions of the circadian clock
are to optimize the temporal manifestations of
different biological activities along the course
of a day through anticipating recurring environ-
mental fluctuation and to separate incompatible
biological processes such as feeding and sleeping.
Unicellular organisms can internalize external
diurnal rhythms by employing a single set of

http://dx.doi.org/10.1016/B978-0-444-59427-3.00030-7 163
164
molecular clockwork; multicellular organisms,
such as mammals, on the other hand, have devel-
oped a highly complex circadian timing system
that is intricately intertwined with different physi-
ological systems. Mammalian circadian rhythms
are a result of a close interaction between differ-
ent central (i.e., inside the central nervous system
or CNS) and peripheral oscillators. An ever-
growing body of evidence has demonstrated
that a misalignment of central and peripheral cir-
cadian clocks (internal desynchrony) predisposes
individuals to physiological complications and dis-
eases. This chapter describes the current state of
knowledge concerning the properties of clocks in
the CNS and the periphery. We then discuss gen-
eral mechanisms of central-to-peripheral clock
interaction in the regulation of physiology using
two extensively studied processes, glucose metab-
olism and glucocorticoid (GC) secretion.
Molecular clockworks
In the past decades, our knowledge of the molec-
ular make-up of the cellular circadian clock has
been significantly advanced. The current model
suggests that the central mechanism of the mam-
malian molecular clock is composed of a set of
clock genes intertwined with a delayed interlocked
transcriptional–translational feedback loop (TTL)
with several auxiliary mechanisms reinforcing
robustness and stability (Zhang and Kay, 2010).
The positive limb of this TTL comprises the basic
helix–loop–helix transcription factors—circadian
locomotor output cycles kaput (CLOCK) and brain
and muscle aryl hydrocarbon receptor nuclear
translocator like (BMAL1 or ARNTL). They form
heterodimers via their PER–ARNT–SIM (PAS)
domains and activate E-box element containing
genes (Hardin, 2004; Zhang and Kay, 2010) by rec-
ruiting several transcriptional co-activators, chro-
matin modifying proteins, and finally, RNA
polymerase II. In certain tissues such as forebrain
or the vasculature, CLOCK is functionally replaced
by its homologue neuronal PAS domain protein
2 (NPAS2; McNamara et al., 2001; Reick et al.,
2001). Period (PER1–3) and cryptochrome
(CRY1–2) constitute the negative limb of the core
clock. CLOCK–BMAL1 complexes activate the
expression of Per and Cry genes during the subjec-
tive day. PER and CRY interact to form complexes
and translocate into the nucleus. When PER/CRY
complexes accumulate to a critical concentration,
they interact with CLOCK and BMAL1 and
thereby inhibit their transactivator function, thus
shutting down Per and Cry transcription (Lee
et al., 2001). The progressive degradation of PER/
CRY complexes throughout the subjective night
releases the inhibition on CLOCK–BMAL1 tran-
scriptional activity and, thereby, completes the neg-
ative feedback loop of the circadian clock.
In addition to the core clock TTL described
above, additional auxiliary TTLs have also been
described. Though principally dispensable, they sta-
bilize the oscillation of the core clock TTL and help
to translate time-of-day information into physiolog-
ical signals via transcriptional control of clock target
genes (Zhang and Kay, 2010). Such loops include
the nuclear receptors REV-ERBa (NR1D1) and
RORa (NR1F1) that regulate Bmal1 expression
via retinoid orphan receptor-responsive elements
(ROREs) (Preitner et al., 2002; Ueda et al., 2002)
as well as the PAR basic leucine zipper (bZIP)
proteins, D-box albumin-binding protein (DBP)
and E4 promoter-binding protein (E4BP; NFIL3;
Cowell, 2002; Ripperger and Schibler, 2006), that
feedback on the expression of Per genes via D-box
promoter elements (Ripperger et al., 2000).
Recently, there are accumulating evidences
showing that the circadian clock is tightly inter-
twined with multiple metabolic pathways. While
the temporal manifestation of several metabolic
functions is one of the most important functional
outputs of the circadian clock (see below), the cel-
lular circadian clock, on the other hand, constantly
receives the feedbacks from the metabolic sig-
nalings of the cells. For example, the redox status
of the cells has been demonstrated to regulate
the molecular clock, in both direct and indirect
manners. The ratio of oxidized to reduced

nicotinamide adenine dinucleotide (phosphate)
(NAD(P)þ/NAD(P)H) reflects the cellular redox
and metabolic status. This ratio oscillates in a circa-
dian manner (Nakahata et al., 2009; Ramsey et al.,
2009). The binding of the CLOCK–BMAL1 and
NPAS2–BMAL1 heterodimeric complexes to the
E-box elements is inhibited by the oxidized NADþ
and NADP but stimulated by the reduced NADH
and NADPH (Rutter et al., 2002). NAD is an
important cofactor that involves in many cellular
enzymatic reactions, for example, sirtuins-mediated
protein deacetylation. SIRT1 catalyzes the removal
of acetyl group from acetylated lysine residues of
proteins thereby modulating their activities in the
expense of NAD(P)H. It has recently been shown
that the activity and level of SIRT1 oscillate in a cir-
cadian manner (Asher et al., 2008; Nakahata et al.,
2008). Importantly, SIRT1 physically interacts with
the CLOCK–BMAL1 complex and mediates
BAML1 and histone H3 deacetylation, which is
important for the transcriptional activating activity
of the CLOCK–BMAL1 complex (Nakahata et al.,
2008). In addition to acting on the positive limb
components, SIRT1 also deacetylates PER2, which
promotes its degradation (Asher et al., 2008). More
recently, another NAD-dependent poly (ADP-
ribose) polymerase-1 (PARP-1)-mediated protein
poly (ADP-ribosyl)ation also showed a circadian
oscillation pattern (Asher et al., 2010). PARP-1,
on the other hand, can also poly (ADP-ribosyl)ate
CLOCK and thereby inhibit the CLOCK–BMAL1
DNA-binding capacity (Asher et al., 2010). The
nutrient-sensing AMP-dependent protein kinase
(AMPK) represents another elegant example
integrating the metabolism to the circadian clock.
AMP/ATP ratio is another indicator of the meta-
bolic status of the cells. AMPK is the major sensor
of such ratio. The elevated AMP level stimulates
the AMPK activity via liver kinase B1 (LKB1;
Mihaylova and Shaw, 2011). The activation of
AMPK acts on the negative limb of the core clock
TTL in both direct and indirect manners. AMPK
directly phosphorylates CRY1 (Lamia et al., 2009)
and indirectly leads to the phosphorylation of
PER2 via casein kinase 1e (CK1e) (Um et al., 2007).
165
In both scenarios, AMPK activation promotes
their degradation and thereby influences the core
clock oscillation cycle.
These and other similar examples illustrate that
the molecular clockwork and several metabolic sig-
naling pathways are so tightly intertwined that
the clear distinction between them is becoming
somewhat blurrier. Here, we only reviewed a few
examples of such metabolic feedback pathways
on the circadian clock. Several elegant reviews dis-
cussing the orchestration of the metabolic homeo-
stasis and the circadian clock and the deleterious
consequences if this orchestration is disrupted have
recently been published (Asher and Schibler, 2011;
Huang et al., 2011; Reddy and O’Neill, 2010). Most
of these metabolic feedback mechanisms have
been implicated in the food entrainment of the
peripheral clock but have only a little if not no
effect on the suprachiasmatic nuclei (SCN) clock.
However, it would be interesting to know how
these pathways influence the extra-SCN clocks in
the brain, particularly for those regions involved
in organizing the feeding schedule.
The master circadian pacemaker of the SCN
In vertebrates, almost all cells express clock genes.
Without synchronization on a systemic level, these
autonomous clocks could not produce physiologi-
cally meaningful signals. In mammals, circadian
regulation is organized in a hierarchical fashion
with the hypothalamic SCN housing the master
circadian pacemaker. The SCN is a bilaterally
paired compact brain nucleus comprising about
20,000 neurons in mice, located directly adjacent
to the third ventricle and atop the optic chiasm
(Welsh et al., 2010). Electrical ablation of the
SCN renders animals behaviorally arrhythmic
(Moore and Eichler, 1972; Stephan and Zucker,
1972). Transplanting SCN tissue to SCN-lesioned
animals restores circadian rhythmicity (Ralph
et al., 1990). Importantly, the restored rhythm of
recipients is determined by the donor SCN period,
indicating that the SCN indeed generates the
166
timing information that synchronizes oscillators
throughout the body (Ralph et al., 1990). SCN
explants as well as dispersed neurons display
robust circadian oscillations in firing rate in vitro,
suggesting that the rhythmicity of the SCN is
autonomous in nature (Green and Gillette, 1982;
Groos and Hendriks, 1982; Shibata et al., 1982;
Welsh et al., 1995).
The endogenous circadian clock in mammals
possesses a rhythm with an approximate 24-h
free-running period. However, the daily fluctua-
tion of the external environment is not constant,
and variables such as photoperiod, temperature,
and food availability are subject to seasonal
changes. In order to synchronize the internal circa-
dian rhythm to the external diurnal fluctuation
patterns, circadian clocks are constantly reset by
external environmental cues, so-called zeitgebers,
every day in a process known as entrainment. The
major zeitgeber in mammals is light, with the SCN
acting as a relay between the external light–dark
cycle and the endogenous timing system (Hankins
et al., 2008). Other, nonphotic zeitgebers exist,
some of which act through the SCN, for example,
arousal (Welsh et al., 2010), while others, such as
food intake, are more directly affecting the periph-
eral circadian machinery (Huang et al., 2011).
The SCN makes use of the same TTL molecu-
lar timekeeping machinery as the peripheral
oscillators (see below). However, the particular
robustness and resilience of SCN circadian rhyth-
micity are achieved through the formation of a
tight interneuronal network (Welsh et al., 2010).
SCN slices cultured in vitro exhibit robust and
persistent circadian oscillations in electrophysio-
logical activity and clock gene expression for sev-
eral weeks, while rhythms in slice explants from
most other brain regions and peripheral tissues
dampen after a couple of days (Guilding and
Piggins, 2007; Guilding et al., 2009). SCN explant
rhythms are also more resistant to temperature
fluctuations or clock gene mutations (Abraham
et al., 2010; Buhr et al., 2010; Liu et al., 2007).
This rigidity and robustness has been attributed
to the intercellular coupling of individual SCN
neurons (Aton et al., 2005; Buhr et al., 2010; Liu
et al., 2007; Welsh et al., 1995).
The SCN innervates numerous brain nuclei,
thereby passing time information to other CNS
clocks. The paraventricular hypothalamic nucleus
(PVN) is one of the main regions transducing SCN
circadian function to the periphery (Saeb-Parsy
et al., 2000). The PVN is a relay hub for energy
homeostasis and projects predominantly to the pitu-
itary where it regulates the release of hormones
such as adrenocorticotrophin (ACTH; see below)
and thyroid-stimulating hormone. The PVN also
innervates the sympathetic limb of the autonomous
nervous system which allows the SCN to indirectly
control melatonin release from the pineal gland
(Buijs et al., 2003b). Further projections of the
SCN have been described to the dorsomedial hypo-
thalamic nucleus (DMH; Luiten et al., 1987), the
nucleus accumbens (NAc; Phillipson and Griffiths,
1985), and the paraventricular thalamic nucleus
(Watts and Swanson, 1987; Watts et al., 1987)
enabling the SCN to affect a plethora of physiologi-
cal processes such as the reward system, fee-
ding–fasting cycles, cognitive function, locomotor
activity, and body temperature (Dibner et al.,
2010). In addition, the SCN secretes diffusible
factors which can function as timing cues. This
notion has been substantiated by an elegant experi-
ment showing that membrane-encapsulated fetal
SCN tissue grafts, which only allowed for small mol-
ecule passage, could restore the periodicity of loco-
motor activity in SCN-lesioned hamsters in the
absence of axonal outgrowth (Silver et al., 1996).
Transforming growth factor (TGF) alpha (Kramer
et al., 2001; Li et al., 2002), prokineticin-2 (PK-2)
(Cheng et al., 2002), and cardiotrophin-like cyto-
kine (CLC) (Kraves and Weitz, 2006) have been
implicated as SCN-secreted peptides capable of
regulating behavioral rhythmicity.
Extra-SCN clocks in the brain
The classical view of the role of SCN as the exclu-
sive circadian pacemaker that controls all circadian
 167

aspects of behavior (Fig. 1a) has been changing
(Guilding and Piggins, 2007). The emerging the-
ory is that the SCN synchronizes and coordinates
numerous semiautonomous circadian clocks
residing in different brain regions and peripheral
tissues (Fig. 1b). Thanks to recent technical
advancements, long-lasting and self-sustained
circadian oscillations of clock genes have been
revealed in a number of brain nuclei in vitro
(Abe et al., 2002; Guilding and Piggins, 2007).
These data provide compelling evidence for the
existence of other circadian clocks in the brain.
Here, we discuss some examples of these extra-
SCN neural oscillators.
The retina was the first neuronal tissue outside
the SCN shown to possess a circadian oscillator.
Cultured hamster retinae show a circadian pat-
tern of melatonin synthesis in vitro (Tosini and
Menaker, 1996). Importantly, this rhythm persists
in constant darkness (self-sustainment) but can be
reset by a light–dark cycle (entrainment). More-
over, the period of the retina clock is resistant to

A B

SCN SCN

Peripheral clocks Systemic rhythms Peripheral clocks Behavioral rhythms

Leptin ng/mL

ZT

Peripheral transcriptional Peripheral transcriptional

rhythms rhythms

Physiological rhythms Physiological rhythms

Fig. 1. Communication routes of the mammalian circadian timing system. (a) The SCN pacemaker entrains peripheral clocks.
Via transcriptional regulation of clock controlled genes in target tissues, peripheral physiological functions are reset and
synchronized to the light–dark cycle. (b) Pathways of interaction between central and peripheral clocks. The SCN resets
physiological rhythms via the entrainment of peripheral tissue clocks, but at the same time regulates behavioral and systemic
(e.g., endocrine) functions in a more direct manner. Peripheral rhythms may, in turn, directly or indirectly feedback on the SCN.
This interlocked balance system creates plasticity in the entrainment of the circadian timing system and promotes adaptation to
complex changes in environmental parameters.
168
temperature changes (temperature compensation),
thereby fulfilling all the formal requirements of
a true autonomous circadian oscillator (Tosini
and Menaker, 1996). More recently, circadian
oscillations of clock gene expression have also
been detected in the retina, further supporting
the clock properties of this tissue (Kamphuis
et al., 2005). Genetic disruption of retinal clock
function results in the loss of circadian rhythm
of electrical responses to light (Storch et al.,
2007). The olfactory bulbs (OBs), similar to the
retina, have also been shown to comprise an
autonomous circadian clock (Granados-Fuentes
et al., 2004a,b). OB Per1 expression and electro-
physiological activity display rhythmic circadian
patterns. Importantly, these rhythms persist in
SCN-lesioned animals and under constant light
conditions—when behavioral rhythms are dis-
rupted—indicating that the OB’s circadian oscilla-
tion is autonomous and independent from the SCN
(Granados-Fuentes et al., 2004a). Circadian rhythms
of clock gene expression and of odor responses
have been described in the OB (Granados-Fuentes
et al., 2006), but molecular evidence for an autono-
mous OB clock is still missing.
The mediobasal hypothalamus (MBH), situated
posterior to the optic chiasm and overlying the
pituitary, is a collective anatomical structure com-
prising the DMH, ventromedial (VMH), PVN,
supraoptic, and arcuate (ARC) nuclei. The MBH
is deemed as an integrating center regulating a
diverse array of physiological processes such as
growth, feeding, maturation, and reproduction
(Luiten et al., 1987). Clock gene rhythms have
been demonstrated in the DMH (see below), the
PVN, and the ARC, as well as in the adjacent
median eminence and the pituitary (Abe et al.,
2002; Guilding et al., 2009). However, the physio-
logical relevance of potential MBH clocks remains
largely unknown. A recent study revealed that,
despite the fact that ARC slices in culture possess
a sustained (more than 1 week) molecular rhythm,
electrical firing rate rhythms dampen within 48 h,
suggesting that in the MBH molecular clocks may
not—or only weakly—couple to electrical activity
(Guilding et al., 2009). A subpopulation of dopami-
nergic (DA) ARC neurons displays robust circadian
oscillations of Per1 and Per2 transcripts (Sellix et al.,
2006). These neurons receive projections from
the SCN (Gerhold et al., 2001) and themselves proj-
ect to the anterior pituitary, where they regulate
prolactin secretion. The DA turnover in these
neurons exhibits a circadian rhythm which is light
entrainable and has an approximate 24-h free-
running period in constant conditions (Sellix and
Freeman, 2003). It has been reported that several
ARC neuropeptides display circadian/diurnal
expression patterns including Agouti-related protein
(AgRP; Lu et al., 2002), cocaine and amphetamine-
regulated transcript (CART; Vicentic et al., 2005),
neuropeptide Y (NPY), and pro-opiomelanocortin
(POMC; Kalra et al., 1999). Also, orexin, secreted
from the lateral hypothalamic area (LHA), shows
rhythmic expression (Willie et al., 2001). Impor-
tantly, many of these rhythms are altered in
diet-induced obese animals (Kohsaka et al., 2007),
suggesting a link between the hypothalamic clock
and metabolic regulation.
The reward system controls and regulates ani-
mal behavior by inducing pleasurable sensations
upon perceiving certain stimuli. Such stimuli can
be primary, that is, in response to food, sex, and
water, which are important for the survival of
individuals and propagation of species, or second-
ary, for example, in the context of money/profits
and power/reputation (Chen et al., 2010). The
major anatomical structure of reward is found in
the mesolimbic DA system. DA neurons from
the ventral tegmental area in the midbrain signal
to the NAc, the dorsal striatum, and the frontal
cortex (Chen et al., 2010). The links between
circadian rhythms and the reward system are
multifaceted (Dibner et al., 2010; Webb et al.,
2009). Patients that suffer from defective reward
function, for example, in bipolar disorder or major
depression, often show altered behavioral rhythms
and sleep patterns (Westrich and Sprouse, 2010).
Conversely, patients with genetic sleep disorders
are often predisposed to addiction (Shibley
et al., 2008). The most striking example, seasonal

affective disorder (SAD), provides a mechanistic
link between altered mood status and altered circa-
dian behavior. SAD patients suffer from depres-
sive episodes upon seasonal change, mostly in
winter, suggesting that alterations in circadian light
entrainment might trigger disease states (Levitan,
2007). Along the same line, psychostimulants
such as cocaine and methamphetamine that can
activate the mesolimbic reward system and induce
pleasurable effects are known to affect circadian
clock. Early studies revealed that chronic exposure
to methamphetamine can disrupt the circadian
rhythms of rats (Honma et al., 1986). On the other
hand, many aspects of addictive behavior show
a time-of-day-dependent pattern with a period
of approximately 24 h (Abarca et al., 2002).
Drug intake can induce clock gene expression in
several brain areas including the NAc and the stri-
atum (Uz et al., 2005; Yuferov et al., 2003). In
rodents, clock genes have been shown to modify
psychostimulant responses (Abarca et al., 2002;
Rosenwasser et al., 2005; Spanagel et al., 2005).
Importantly, ClockD19 mutant mice show increased
excitability of DA neurons and a higher rate of
DA synthesis, indicating a general excited, mania-
like state of the DA circuitry (McClung et al.,
2005; Roybal et al., 2007). Recently, Per2 has been
identified as a positive regulator of monoamine
oxidase A (MAOA) expression and activity, a
degrading enzyme of DA, and hence a negative
regulator or DA release (Hampp et al., 2008).
Taken together, these data illustrate an active
role of the circadian clock in modulating the
mesolimbic reward pathway. It remains to be
shown, however, whether the DA neurons them-
selves contain a functional circadian clock and
how such a system affects reward responses to dif-
ferent stimuli.
Two phenomena tightly linked to the reward
system suggest that circadian rhythms may also
emerge from structures that are potentially very
differently organized than the cellular TTL clocks
described above. The food-entrainable oscillator
or FEO is a putative timing system that has drawn
much attention in the past 30 years. It has been
169
shown that the lost rhythmic locomotor activity
of SCN-lesioned arrhythmic rats can be partly
restored by temporally restricted feeding (RF)
schedules (Stephan et al., 1979), a phenomenon
known as food anticipatory activity (FAA; Boulos
and Terman, 1980; Mistlberger, 1994). RF can
also restore the rhythmicity of pineal melatonin
release (Feillet et al., 2008), thermogenesis,
plasma rhythms of nutrient-related blood-borne
hormones, and drinking patterns (Boulos and
Terman, 1980; Mistlberger, 1994; Stephan, 2002).
Such feeding-related rhythms, once established,
can persist (or free-run) for several days with an
approximate 24-h period under fasting conditions.
Lesion studies have tried to determine the
anatomical locus of the FEO, but to date, no
study has unequivocally identified a structure
essential for the generation of feeding-related
rhythms (Davidson, 2009). Moreover, it remains
controversial whether the known clock genes
are involved in the regulation of FAA (Challet
et al., 2009; Feillet et al., 2006; Storch and Weitz,
2009). The current prevailing opinion is that the
FEO is a diffuse system emerging from the inter-
play of different circuits within the CNS, and
likely even within peripheral organs. Within the
CNS it may comprise multiple brain regions as
well as various signaling pathways.
The concept of the methamphetamine-sensitive
oscillator (MASCO) originated in the 1980s;
Honma and colleagues showed that chronic treat-
ment of SCN-lesioned rats with methamphetamine
could reinitiate circadian locomotor activity, core
body temperature, and plasma corticosterone
rhythms (Honma et al., 1987, 1988), which can per-
sist for up to 2 weeks after withdrawal from the
drug (Ruis et al., 1990). The emergence of meth-
amphetamine-induced rhythms is independent of
functional circadian clock machinery (Honma
et al., 2008; Masubuchi et al., 2001). Interestingly,
methamphetamine treatment can reset the rhythm
of clock gene expression in several brain areas such
as the caudate putamen, the striatum, and the pari-
etal cortex, but not the SCN (Masubuchi et al.,
2000). The anatomical structure, the endogenous
170
zeitgebers, and the output pathways of the
MASCO, however, remain largely unknown.
Peripheral clocks
Outside the brain, “canonical” circadian clocks
have been identified in several peripheral organs
and tissues, capable of generating oscillations with
a periodicity of approximately 24 h. The intrinsic
properties of such peripheral clocks have been
characterized using primary cell culture models
and tissue explants cultures (Yoo et al., 2004).
Immortalized rat fibroblast (Rat-1) cells display
robust oscillations of clock gene expression after
brief stimulation with high concentrations of serum
(serum shock; Balsalobre et al., 1998). Using single
cell imaging techniques, Nagoshi et al. showed that
individual fibroblasts possess sustained endoge-
nous circadian expression rhythms of clock genes,
although populations of cells quickly become
desynchronized from each other because of indi-
vidual differences in period (Nagoshi et al., 2004).
Serum shock (or stimulation with forskolin,
GCs, or phorbol esters) synchronizes the individ-
ual cells, yielding a transiently phase coherent pop-
ulation (Nagoshi et al., 2004). These results point
to the notion that the peripheral cellular clock is
actually self-sustained and autonomous in nature
but fails to maintain the coherence with neighbor-
ing cells, in contrast to the coupled nature of SCN
(see above). In other words, at the cellular level,
fibroblasts do not differ from SCN neurons in
terms of the molecular circadian machinery (Liu
et al., 2007). In line with this observation, tissue
explants from a wide array of peripheral organs
including heart, lung, kidney, liver, spleen, pan-
creas, stomach, cornea, thyroid gland, and adrenal
gland all show robust clock gene expression
rhythms (Yamazaki et al., 2000; Yoo et al., 2004).
It is still not fully understood how the SCN tra-
nsmits its timing signal to peripheral clocks. Endo-
crine signals such as GCs play a role (Balsalobre
et al., 2000; Kiessling et al., 2010). Some clocks
respond to neuronal cues (Ishida et al., 2005;
Kalsbeek et al., 2004; Oster et al., 2006a), while
others are affected by behavior-associated changes
in temperature (Brown et al., 2002; Dibner et al.,
2010). Circadian transcriptome profiling studies
suggest that local peripheral clocks, while being
reset by the SCN, independently control tissue
physiology via the regulation of output genes com-
prising 5–10% of the active transcriptome (Akhtar
et al., 2002; Hughes et al., 2009; Kornmann
et al., 2007a; McCarthy et al., 2007). In line with
this, peripheral clocks have been implicated in a
plethora of physiological functions such as cardiac
contraction (Bray and Young, 2009), renal excre-
tion (Firsov et al., 2011), adipogenesis and lipid
metabolism (Gimble et al., 2011), digestive pro-
cesses (Gimble and Floyd, 2011), and xenobiotic
metabolism (Claudel et al., 2007), to name but a
few. However, recent data suggest that the organi-
zation of circadian molecular and physiological
functions is more complex than originally thought
and involves tight interaction between different
central and peripheral clocks (Kornmann et al.,
2007b; Fig. 1b). In the following section, we will
discuss glucose metabolism and the regulation of
GC secretion from the adrenal cortex to exemplify
the intricate interplay between the central and
peripheral circadian clocks which is essential for
the maintenance of physiological homeostasis.
Glucocorticoid (GC) secretion
GCs are steroids produced in the adrenal gland, cor-
tisol in humans and corticosterone in rodents, which
are essentially involved in energy metabolism,
immune function, and stress responses. Disruption
of GC secretion is associated with severe patho-
physiology. Patients affected with Cushing’s Syn-
drome, characterized by excess GC levels, often
present with diabetes mellitus, osteoporosis,
hypertension, dyslipidemia, and sleep disorders
(Carroll and Findling, 2010). Conversely, GC
insufficiency results in Addison’s disease, which is
characterized by stress sensitivity, hypoglycemia,
hypotension, mood disturbances, and weight loss

(Anglin et al., 2006). In addition, chronic fatigue
syndrome is associated with perturbations in GC
regulation (Chung et al., 2011b). Clock-related life-
style factors can affect GC levels, such as shift
work, jet lag, and nighttime eating, and are
associated with diabetes and metabolic syndrome
and increased risk of heart attack and cancer
(Boivin et al., 2007). GC secretion shows a very
prominent diurnal/circadian rhythm peaking
around wake-up time (morning in humans, even-
ing in rodents). SCN and adrenal clocks are both
required for the circadian production of GCs. Fur-
ther, GC actions are circadian gated through the
rhythmic expression of its receptors. Finally, GC
feedback directly affects the phase of clock gene
transcription in other peripheral oscillators, com-
pleting an elegant cycle of integration.
The nycthemeral production of GCs occurs
through the hypothalamus/pituitary/adrenal (HPA)
axis. The role of the SCN was determined in
the 1970s and 1980s, with studies demonstrating
that the ACTH release from the pituitary is no lon-
ger rhythmic in SCN-lesioned animals, disrupting
adrenal GC secretion rhythms (Cascio et al., 1987;
Moore and Eichler, 1972; Szafarczyk et al., 1983).
The neuropeptide arginine vasopressin (AVP) is
released rhythmically from SCN neurons (Earnest
and Sladek, 1986; Gillette and Reppert, 1987)
projecting into the rostral PVN. There, it inhibits
the production of corticotrophin-releasing hormone
(CRH; Buijs et al., 1993; Gomez et al., 1997;
Kalsbeek et al., 1992), which is ultimately responsible
for ACTH release from hypophyseal adreno-
corticotrophs in the pituitary. In this manner, circa-
dian rhythms in the SCN result in the circadian
release of ACTH, peaking at the beginning of the
active phase. ACTH then regulates the production
and release of GC from the adrenal gland.
Anatomically, the adrenal gland is divided into
the cortex and the medulla, which are structurally
and functionally discrete. The medulla is responsi-
ble for the secretion of epinephrine and norepi-
nephrine, while the cortex produces various
steroid hormones. GCs are produced in the zona
fasciculata cells of the cortex, which express ACTH
171
receptors (Chung et al., 2011a). Rhythmic GC
secretion is regulated by an intrinsic circadian clock
located in the cortex of the adrenal gland (Oster
et al., 2006a,b; Son et al., 2008). Using a model of
adrenal transplantation between wild-type and
mutant mice lacking a functional clock, the role of
the adrenal clock was elucidated (Oster et al.,
2006b). In the absence of an SCN clock, GC
rhythms remain entrainable by light, but in the
absence of light, the rhythm is rapidly lost. Con-
versely, in the absence of a functional adrenal clock,
GC rhythms are dampened, suggesting that the
function of the adrenal clock is to gate the
responsiveness of the adrenal to ACTH through
the rhythmic expression of steriodogenic enzymes
or modulators. A Bmal1 knockdown study supports
these findings, suggesting that the adrenal clock
plays a dominant role in the regulation of local GC
production in the adrenal, though not of circulating
GC levels in the blood (Son et al., 2008).
In addition to HPA axis control, GC release is
regulated by neuronal signals. Virus tracing stud-
ies reveal multisynaptic autonomic connections
between the SCN and the adrenal gland (Buijs
et al., 1999). Jasper et al. showed that splanchnic
denervation results in dampening of diurnal GC
rhythms and increased sensitivity to ACTH stimu-
lation (Jasper and Engeland, 1997). A direct effect
of light on the adrenal gland was characterized by
Ishida et al., who showed that light exposure
induces Per gene expression in the adrenal gland
via the SCN sympathetic nervous system, resulting
in an upregulation of GC release (Ishida et al.,
2005), thus offering a mechanism for the observed
light entrainment of adrenal clock transplants in
otherwise arrhythmic animals mentioned above
(Oster et al., 2006b).
GC receptors are expressed throughout the
periphery and the brain, with the notable excep-
tion of the SCN (Rosenfeld et al., 1988). Activated
GC receptors act as transcription factors via activa-
tion or repression of GC target genes (Surjit et al.,
2011). Disruption of GC signaling, for example, by
adrenalectomy, affects gene transcription in the
periphery and the brain. In the central nucleus of
172
the amygdala, it causes a loss of PER2 rhythmic
expression, while in the liver, it alters the regulation
of numerous genes involved in metabolism (Lamont
et al., 2005; Oishi et al., 2005). Timed GC or GC ana-
log treatment in mice has a powerful resetting effect
on liver (clock) gene rhythms (Balsalobre et al.,
2000; Reddy et al., 2007; Segall et al., 2006; Son
et al., 2008). Additionally, GC receptors are rhyth-
mically transcribed in various tissues and subjected
to acetylation—and subsequent inactivation—by
CLOCK (Nader et al., 2009; Yao et al., 2006). In
conclusion, GC production, secretion, and signaling
are an example of the complex system of integration
afforded by the presence of multiple circadian
clocks organized in a hierarchical manner.
Glucose metabolism
The maintenance of glucose homeostasis is essential
for mammalian physiology. Plasma glucose levels
display diurnal rhythms in mammals, peaking
before the onset of activity while remaining constant
throughout the remainder of the day. This peak
does not coincide with food intake, clearly
illustrating the extent of endogenous regulation
dedicated to glucose circulation (La Fleur et al.,
1999). Circulating glucose is altered by absorption
from the gut following feeding, glucose uptake into
tissues, and glucose production. These latter pro-
cesses are tightly regulated in a temporal manner
to assure sufficient glucose availability, for example,
for the brain, while avoiding extended postprandial
hyperglycemia. The liver plays a pivotal role in this
process as a site of glucose uptake from the circula-
tion, as well as being the major source of de novo-
synthesized glucose in times of need (Kalsbeek
et al., 2010). The regulation of the diurnal glucose
rhythm has been shown to be maintained by
both neuroendocrine and neuronal pathways and
involve a large number of different central and
peripheral circadian clocks.
Early studies on the autonomic innervation of
the liver in regard with glucose homeostasis show
that sympathetic input predominantly increases
hepatic glucose output, while parasympathetic
input stimulates insulin-dependent glucose uptake
and storage in the form of glycogen (Puschel,
2004). The influence of the master clock on glu-
cose regulation was shown by Buijs and colleagues
using SCN-lesioned rats, which have no diurnal
glucose rhythm, compared to fasted and arrhyth-
mic-fed (six daily feeds) animals, which retain their
glucose rhythms (La Fleur et al., 1999). The pre-
cise nature of this regulation has been dissected
in a series of studies using euglycemic hyper-
insulinemic clamps in combination with selective
autonomic denervation of the liver. Initial retro-
grade tracing studies from the liver revealed
projections via both sympathetic and parasympa-
thetic systems to third-order neurons in the hypo-
thalamus, specifically the SCN (La Fleur et al.,
2000). Studies designed to distinguish between
sympathetic and parasympathetic output from the
SCN showed that exclusive populations of neurons
within the SCN are responsible for each of these
signals, projecting to preautonomic neurons in the
PVN (Buijs et al., 2003a). In 2004, it was shown
that the SCN-derived sympathetic inputs to the
PVN were inhibitory GABAergic inputs, and their
inhibition resulted in increased hepatic glucose
production (Kalsbeek et al., 2004). It was addition-
ally demonstrated that these GABAergic inputs
provide the circadian timing information for the
liver as well as for the insulin response of the pan-
creas (Kalsbeek et al., 2008). Interestingly, it was
shown that complete denervation of the liver in
conjunction with constant feeding does not abolish
diurnal glucose rhythms; however, this can be
achieved by inactivation of either the sympathetic
or the parasympathetic inputs (Cailotto et al.,
2008). Collectively, these studies indicate that the
autonomic modulation of glucose rhythms requires
a balance in both branches of the autonomic ner-
vous system by the SCN.
Further studies revealed that orexin, a hypo-
thalamic neuropeptide involved in wakefulness
and feeding behavior, is an important regulator
of glucose homeostasis and the main effector in
the preactive phase glucose peak (Yi et al., 2009).

Intracerebroventricular infusion of orexin results
in increased glucose production. Inhibition of
GABAergic inputs—originating from the SCN—
to the perifornical orexin area (PF-Oa) has a
similar effect, correlating with activation of
orexin-positive neurons. Given that GABAergic
inhibition of hyperglycemia, GABAergic inputs
to orexin neurons in the PF-Oa, and orexin
release all show clear diurnal rhythmicity (Alam
et al., 2005; Kalsbeek et al., 2008; Zhang et al.,
2004), it is feasible to propose that orexin-
containing PF-Oa neurons translate SCN-derived
GABAergic rhythms into glucose rhythms via
the sympathetic nervous system.
Interestingly, hepatic sympathetic denervation
of the liver results in the loss of diurnal glucose
rhythms, without affecting gene expression rhythms
of liver clock genes, indicating that the liver clock is
not essential in this process (Cailotto et al., 2005).
However, glucose production is generally thought
to be further regulated via clock target genes
involved in glucose metabolism in the liver. The
liver clock has been well described, and rhythmic
liver genes are highly enriched for metabolic func-
tion (Lamia et al., 2008; Oishi et al., 2003; Panda
et al., 2002; Storch et al., 2002). SCN ablation studies
show that liver clock gene and clock output gene
rhythmicity are abolished or severely dampened in
the absence of synchronization by the central clock
(Akhtar et al., 2002). The liver clock is highly
responsive to RF regimes, which change the phase
of core clock gene expression as well as the diurnal
rhythm of circulating glucose (Damiola et al., 2000;
Escobar et al., 1998). Interestingly, these RF-
induced changes to the liver clock are inhibited
by GCs (Le Minh et al., 2001). Conversely, regular
short-period feeding paradigms prevent dis-
turbances in clock gene expression in the liver and
leave circulating glucose rhythms intact (La Fleur
et al., 1999).
Many clock-deficient animal models show per-
turbations in glucose metabolism. Clock mutant
mice display impaired glycogen storage, which cor-
relates with dampened glycogen synthase 2 (Gys2)
expression rhythms in the liver (Doi et al., 2010).
173
Microarray analyses of the ClockD19 mutant liver
transcriptome reveal that metabolic gene rhyth-
micity is dampened (Oishi et al., 2003). ClockD19
mutant and Bmal1/ mice show impaired
gluconeogenic potential, correlating with dec-
reased phosphoenolpyruvate carboxykinase 1
(PEPCK) expression in the liver (Rudic et al.,
2004). More recently, studies have shown that
CRY1 negatively regulates gluconeogenesis
through the inhibition of G-protein-coupled recep-
tor-mediated cAMP accumulation (Zhang et al.,
2010). Perhaps most significantly, liver-specific
Bmal1/ (L-Baml1/) mice display per-
turbations in rhythmic expression of glucose regu-
latory genes and glucose metabolism, including
circulating blood glucose (Lamia et al., 2008).
These mice display hypoglycemia in the middle
and end of the inactive phase and increased glucose
uptake during this time, arguing for a significant
role of hepatic clocks in the maintenance of glucose
homeostasis.
Glucose uptake is dependent on insulin and can
be influenced by insulin release from the pancreas
or by insulin sensitivity in peripheral tissues. Insu-
lin levels display diurnal rhythms, and similarly to
glucose, time-RF results in changes to this rhythm
in rodents (Diaz-Munoz et al., 2000). In the case
of insulin, however, the diurnal rhythm is lost fol-
lowing fasting and appears to be primarily depen-
dent on feeding rhythms rather than being clock
driven (La Fleur et al., 1999). On the other hand,
feeding-stimulated insulin responses are rhythmic
(Kalsbeek and Strubbe, 1998). Indeed, changes in
glucose uptake over the course of the day correlate
to alterations in insulin sensitivity rather than insu-
lin release, and this rhythm is lost in SCN-lesioned
animals, indicating central clock involvement (La
Fleur et al., 2001). Conflictingly, SCN-lesioned
animals display increased glucose uptake as well
as decreased meal-induced insulin secretion. This
is hypothesized to be due to insulin-independent
glucose uptake, perhaps via autonomic inputs
to muscle tissues (La Fleur, 2003). Functional
clocks have been identified in the pancreatic b-cells
(Marcheva et al., 2010; Sadacca et al., 2011).
174
In ClockD19 mutant mice, a diabetic phenotype com-
prising increased blood glucose levels, loss of active
phase insulin peak, reduced glucose tolerance, and
retarded glucose-stimulated insulin release is seen
(Marcheva et al., 2010). Generation of a b-cell-
specific Bmal1/ mouse confirmed that these
phenotypes are due to the loss of a functioning clock
in islets of Langerhans and seem to be the result of
impaired glucose-stimulated insulin release rather
than insulin production (Sadacca et al., 2011).
As discussed above, euglycemia is maintained in
the fasting state predominantly through hepatic pro-
duction and release of glucose. Of note, GCs can
increase gluconeogenesis directly through the acti-
vation of a number of key enzymes involved in this
pathway (Jin et al., 2004; Sasaki et al., 1984; Vander
Kooi et al., 2005) as well as through the production
of suitable substrates from increased lipolysis
(Campbell et al., 2011), thus suggesting indirect
effects of adrenal—and possibly adipose—clocks
in glucose regulation. GCs further affect gluconeo-
genesis by decreasing the sensitivity of the liver to
insulin, the major inhibitor of gluconeogenesis. This
is achieved in two ways, first, by directly decreasing
the release of insulin from b-cells in the pancreas
and, second, by decreasing the insulin-mediated glu-
cose uptake in adipocytes and muscle (Delaunay
et al., 1997; Sakoda et al., 2000; Weinstein et al.,
1998), potentially involving further circadian clocks
active in the respective tissues. Under conditions of
HPA axis dysregulation where GC rhythms are
affected, abnormal glucose homeostasis is observed,
promoting the development of diabetes.
The hypothalamus contains distinct populations
of neurons, identifiable according to their signa-
ture expression profiles of receptors and neuro-
transmitters. These nuclei form a complex
network of excitatory and inhibitory signals that
regulate peripheral nutritional status and both
homeostatic and hedonic feeding. These feeding
nuclei receive inputs from the SCN, as discussed
above, and many have been suggested to contain
functional molecular clocks (Guilding et al.,
2009). Arguably the most important input in the
regulation of feeding is leptin, which shows a diur-
nal circulation rhythm, and is disrupted in mouse
models lacking a functional clock (Turek et al.,
2005). Leptin signals predominantly in the ARC,
which contains two distinct populations of neurons:
the first expressing NPY and AgRP and possessing
orexigenic function and the second expressing
POMC and CART and possessing anorexigenic
function. In addition to receiving leptin signals,
these cells may also be directly responsive to glu-
cose and function as sensors of peripheral glucose
fluctuations. Some studies have demonstrated that
NPY neurons are inhibited by glucose, whereas
POMC neurons are excited, although it should be
noted that other studies have failed to see these
effects (Claret et al., 2007; Fioramonti et al., 2007;
Ibrahim et al., 2003; Muroya et al., 1999). Despite
discrepancies, it seems plausible that ARC neurons
are capable of responding directly to changes in
circulating glucose levels. ARC neurons signal to
various regions of the brain such as the LHA, the
VMH, the mediodorsal nucleus of the thalamus,
the dentate gyrus, the piriform cortex, the ventral
basolateral amygdala, and the bed nucleus of stria
terminalis (DeFalco et al., 2001; Muroya et al.,
2004), and some of these neurons may also possess
glucose-sensing properties. For example, orexin/
hypocretin-containing neurons of the LHA are
activated by hypoglycemia (Cai et al., 2001) and
inhibited by glucose in mice (Guyon et al., 2009).
Melanin-concentrating hormone (MCH)-containing
neurons—also in the LHA—respond to glucose
stimulation (Burdakov et al., 2005). Given the
apparent opposing functions of these subsets of
neurons, with orexin/hypocretin neurons involved
in wakefulness and MCH-containing neurons
involved in sleep and decreased activity, circulating
glucose levels can feedback to the hypothalamus
to direct appropriate behaviors. How clock disrup-
tion in these neurons affects metabolic homeostasis
and glucose levels, however, remains to be shown.
Conclusion
While the circadian clock is traditionally seen as a
top-down-controlled system in which the SCN pace-
maker synchronizes peripheral clocks throughout

the body, which, in turn, regulate local physiolog-
ical rhythms via transcriptional programs (Fig. 1a),
recent data clearly suggest that a coordinated
interplay between different central and peripheral
clocks is necessary to maintain robust rhythms of
certain humoral factors (e.g., GCs) or restrict
fluctuations in others within physiologically toler-
able ranges (e.g., blood glucose; Fig. 1b). We are
only now developing the genetic tools to dissect
the role of organ-specific circadian clocks in these
processes in living animals, and new technologies
will be needed to specifically manipulate several
tissue clocks in a coordinated manner which would
allow the interactivity of the circadian oscillatory
network at the systemic level to be analyzed.
Acknowledgments
H. O. is an Emmy Noether Fellow of the German
Research Foundation (DFG) and a Lichtenberg
Fellow of the Volkswagen Foundation. A. H. T.
is supported by a GGNB Fellowship of the
University of Göttingen.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 11

Circadian rhythms in white adipose tissue

Rianne van der Spek{,*, Felix Kreier{, Eric Fliers{ and Andries Kalsbeek{,{

{
Department of Endocrinology and Metabolism, Academic Medical Center (AMC),
University of Amsterdam (UvA), Amsterdam, The Netherlands
{
Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience, an Institute
of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands

Abstract: Adipose tissue is an important endocrine organ. It is involved in the regulation of energy
metabolism by secreting factors (adipokines) that regulate appetite, food intake, glucose disposal,
and energy expenditure. Many of these adipokines display profound day/night rhythms, and
accumulating evidence links disruption of these rhythms to metabolic diseases such as obesity and type
2 diabetes. Here, we briefly present the circadian system, describe the development of white adipose
tissue (WAT) and its depot-specific characteristics and innervation, we discuss energy storage in WAT
and, lastly, review recent findings that link circadian rhythmicity to adipose tissue biology and obesity.

Keywords: white adipose tissue; circadian rhythm; SCN; suprachiasmatic nucleus; ANS; autonomic
nervous system; corticosterone; food intake; energy metabolism.

Introduction glucose and lipid metabolism. Once the amount

The prevalence of obesity is taking on enormous
proportions. Obesity is defined by the World
Health Organisation as the accumulation of exces-
sive fat tissue, to the point that it may impair
health (World Health Organisation, 2011). Fat,
or white adipose tissue (WAT), is a metabolically
active tissue and a key player in the regulation of
*Corresponding author.
Tel.: þ31 20 566 6071; Fax: þ31 20 691 7682
E-mail: r.d.vanderspek@amc.uva.nl
of fat has accumulated to such an extent that it
hinders tissue function, insulin resistance of
WAT may occur. Both human and rodent studies
point toward an important role for the circadian
timing system in energy metabolism. Disturbed
day/night rhythms are closely correlated with the
development of obesity and type 2 diabetes
mellitus (T2DM). Here, we briefly present the cir-
cadian system and describe the development of
WAT and its depot-specific characteristics and
innervation, we discuss energy storage in WAT
and review recent findings that link circadian
rhythmicity to adipose tissue biology and obesity.

http://dx.doi.org/10.1016/B978-0-444-59427-3.00011-3 183
184
Circadian rhythms
From an evolutionary perspective, circadian
(Latin; circa diem ¼ approximately a day) rhythms
may have ensured continuous energy availability
throughout the 24-h day for organisms relying
on photosynthesis (Hut and Beersma, 2011),
in addition to protecting the replicating DNA
from high ultraviolet radiation exposure during
daytime (Asher and Schibler, 2011). In mammals,
the circadian timing system allows the anticipation
of recurring events during the course of a day,
along with temporal segregation of incompatible
processes such as sleep and wakefulness (Asher
and Schibler, 2011). By definition, circadian
rhythms have a  24 h cycle, persist in the absence
of environmental cues (i.e., they are endogenous),
adjust to match local time (so-called entrainment)
and maintain intrinsic periodicity over a range
of physiological temperatures (i.e., temperature
compensation) (O’Neill and Reddy, 2011).
In mammals, the biological master clock resides
in the suprachiasmatic nuclei (SCN) in the anterior
hypothalamus. The endogenous rhythmicity of
mammals is thus generated within the SCN and
distributed to the rest of the brain and to the body.
To maintain synchrony with the outside world, the
SCN integrate entraining signals (Zeitgebers) of
which light, food intake, and locomotor activity
are the most important ones. At least three major
input systems feed information to the SCN. The
first consists of melanopsin-containing ganglion
cells within the retina projecting via the ret-
inohypothalamic tract directly to the SCN using
glutamate and pituitary adenylate cyclase-activating
polypeptide (PACAP) as neurotransmitters. The
second input system runs from the retina to the
intergeniculate leaflet (IGL) and from there via
the geniculohypothalamic tract to the SCN, using
neuropeptide Y (NPY), enkephalin, and GABA
as its main neurotransmitters. The third input sys-
tem projects from the medial and dorsal raphe
nuclei to the SCN and the IGL and uses serotonin
(5-HT) as its main transmitter (Dibner et al., 2010).
Circadian oscillators have virtually the same
molecular make-up in SCN neurons and non-SCN
cells. However, while within the SCN these
oscillators are synchronized via synaptic and para-
crine signals, they do not appear to communicate
as much between cells in other tissues (Asher and
Schibler, 2011). Oscillators in non-SCN cells,
known as peripheral clocks, are synchronized and
entrained by the SCN via direct (neural or humoral
factors) and indirect (e.g., control of sleep–wake
cycle, food intake, body temperature) cues.
Regulation of rhythms in peripheral tissues
Since the discovery of the SCN in 1972, numerous
neuroanatomical tracing studies have been per-
formed to reveal target areas for the circadian
information generated in the SCN. Generally,
these studies showed that the projection fibers
from the SCN are surprisingly limited and mainly
restricted to a few hypothalamic nuclei (Watts
and Swanson, 1987; Watts et al., 1987). In these
neuroanatomical tracing studies, the hypotha-
lamic paraventricular nucleus (PVN) showed up
as an important target area of the SCN. Although
the PVN is considered to be the neuroendocrine
“headquarters” of the hypothalamus, it also
contains a prominent population of neurons
projecting to the autonomic nervous system
(ANS), i.e., preautonomic neurons.
Within the preautonomic neurons in the PVN,
there is a clear separation of neurons projecting
to the sympathetic and the parasympathetic
branch of the ANS (Buijs et al., 2003; Kreier
et al., 2006). Via these ANS projections, the
SCN are able to convey rhythmic information to
various peripheral tissues. For example, the
rhythmic release of melatonin by the pineal gland
is controlled by SCN input to the preautonomic
neurons in the PVN that regulate the sympathetic
input to the pineal gland (Moore, 1996). Besides
this circadian control, the ANS may play a
role in resetting peripheral clocks after phase
shift-inducing light exposure (Cailotto et al.,
2009).
However, peripheral rhythmicity is not solely
entrained by the ANS. Although a complete

autonomic hepatic denervation (both the sympa-
thetic and parasympathetic branch) abolished
the daily rhythm in plasma glucose concentrations,
it had no overall effects on the molecular rhythms
in the liver (Cailotto et al., 2005). Catecholamines
like epinephrine and nor-epinephrine can modulate
clock function in vitro (Reilly et al., 2008), however
dopamine b-hydroxylase (Dbh, the enzyme that
converts dopamine into nor-epinephrine) knock-
out mice displayed no disrupted molecular rhythms
in peripheral tissues (Reilly et al., 2008). Moreover,
Dbh/ mice treated with a- and b-adrenergic
receptor antagonists showed undisturbed molecu-
lar rhythms in heart, aorta, liver, and brown
adipose tissue (BAT; Reilly et al., 2008), thus
excluding the possible compensatory effect of
dopamine.
Parabiosis experiments have shown that non-
neural signals suffice for the maintenance of circa-
dian rhythms of clock gene expression in liver and
kidney, but not in heart, spleen, or skeletal mus-
cle (Guo et al., 2005). Moreover, in the submaxil-
lary glands, sympathetic input modulated but did
not determine the phase of peripheral circadian
oscillators (Vujovic et al., 2008). Together, these
data indicate that peripheral oscillators may
receive multiple signals that contribute to their
phase of entrainment.
Another example of this multiple signaling princi-
ple is found in the hypothalamo–pituitary–adrenal
(HPA) axis, where the SCN use a two-stage mecha-
nism to control the daily rhythm in plasma cortico-
sterone concentrations. Plasma glucocorticoid
hormone levels exhibit robust daily oscillations,
and glucocorticoids are potent Zeitgebers, both
in vitro and in vivo (Balsalobre et al., 2000), by
binding to the glucocorticoids response element
in the promotor region of Period1 (Per1) and
possibly Period2 (Per2) (Balsalobre et al., 2000;
So et al., 2009; Yamamoto et al., 2005). On the
one hand, the SCN act on the corticotrophin-
releasing hormone (CRH)-containing neuroendo-
crine motorneurons in the PVN to bring about the
release of adrenocorticotrophic hormone (ACTH)
from the pituitary, while on the other hand, it
also acts—through the preautonomic neurons in
185
the PVN and the ANS—on the adrenals and affects
their sensitivity to ACTH (Kalsbeek et al.,
2012; Lilley et al., 2012). Adrenal denervation
abolishes the circadian corticosterone rhythm, the
light-induced corticosterone secretion, and the
daily change in ACTH sensitivity (Engeland
and Arnhold, 2005; Ishida et al., 2005; Kalsbeek
et al., 2012).
Next to innervation and hormonal control regu-
lation of the feeding–fasting cycle provides
several indirect pathways for the SCN to entrain
peripheral tissues. These pathways include daily
rhythms in hormones secreted upon feeding and
fasting (e.g., cholecystokinin, peptide YY, ghrelin,
leptin), changes in plasma concentrations of food
metabolites (glucose, cholesterol, fatty acids,
heme), postprandial temperature elevations, and
intracellular changes in redox state (see section
on nongenetic clock) (Dibner et al., 2010).
At present, it is unclear to what extent each of
these Zeitgebers contributes to the regulation
of circadian rhythms in peripheral clock systems,
in general, or to WAT in particular. Figure 1
summarizes the different pathways via which the
SCN could relay its timing information to WAT.
Cellular timekeeping
Transcriptional–translational feedback model
Cellular timekeeping is regulated on several
levels. On a genetic level, messenger RNA
(mRNA) is synthesized from DNA (transcrip-
tion). This mRNA may contain coding regions
(exons) and non-coding regions (introns). To
form a protein, introns are spliced out, so the
ribosome can translate the mRNA into an amino
acid sequence, thereby producing a polypeptide
(translation). Subsequent posttranslational modi-
fication attaches other biochemical functional
groups (e.g., phosphates) to the polypeptide, to
activate or inactivate the protein, while protein
folding gives the polypeptide a 3D structure,
enabling interaction with other proteins. Changes
186
P
SCN
Autonomic
Behavior innervation
Hormonal
Grip strength Adipokines Glucose
Fig. 1. Three pathways via which the SCN could relay timing
information to peripheral tissues.
in the transcriptome do thus not necessarily
reflect altered protein expression or function,
and vice versa, as has been shown for the liver
(Reddy et al., 2006).
In the transcriptional–translational feedback
model, the molecular clock is based on a core
loop formed by Clock:Bmal1 and Period 1–3
(Per1–3) and Cryptochrome 1 and 2 (Cry1–2).
The Clock:Bmal1 heterodimer stimulates the
transcription of Per1–3 and Cry1–2, and Pers
and Crys subsequently heterodimerize, translo-
cate to the nucleus, and inhibit Clock:Bmal1
activity. As a consequence, Clock:Bmal1 tran-
scriptional activity drops, which reduces the tran-
scription of Per and Cry genes, thereby activating
CRYs
PERs +
–
Lipolysis
Clock Bmal1 Lipogenesis
Glucose transport
–
RevErba +
RORs
Fig. 2. Simplified version of the molecular core clock
mechanism. The core loop is formed by Clock:Bmal1 and
Period 1-3 (Per1–3) and Cryptochrome 1 and 2 (Cry1-2). The
Clock:Bmal1 heterodimer stimulates the transcription of Per1–3
and Cry1–2. Subsequently, Pers and Crys heterodimerize,
translocate to the nucleus, and inhibit Clock:Bmal1 activity. As
a consequence, Clock:Bmal1 transcriptional activity drops,
which reduces the transcription of Per and Cry genes, thereby
activating Clock:Bmal1 again. Additional loops formed by
RevErbs and RORs enhance the robustness of the core loop.
Components of the core clock regulate energy metabolism, for
example, lipolysis, lipogenesis, and glucose transport.
Clock:Bmal1 again. Figure 2 shows a simplified
model of this molecular clock mechanism.
Additional loops enhance the robustness of the
core loop. The retinoic acid-related orphan
nuclear receptors, RevErbs and RORs, represent
additional regulatory loops by binding to retinoic
acid-related orphan receptor response elements
on the Bmal1 promotor (Ko and Takahashi,
2006).
Nongenetic clock
Cycles of transcription and translation feedback
generally operate on a timescale of up to a few
hours and maintaining an oscillatory rhythm on
a daily basis thus requires a significant delay
between activation and repression of transcrip-
tion. Posttranslational modifications such as phos-
phorylation, histone acetylation, methylation,
sumoylation, and ubiquitination affect the stabil-
ity and nuclear translocation of core clock

proteins, thereby delaying the cycle to  24 h
(Cardone et al., 2005; Gallego and Virshup,
2007; Ko and Takahashi, 2006; Reppert and
Weaver, 2001). A more extended review of the
genetic clock can be found in Ko and Takahashi
(2006) and Gallego and Virshup (2007).
Some observations cannot be explained suffi-
ciently by this transcriptional–translational feed-
back model. In the past few decades, several
nongenetic pathways were found to play a role
in cellular timekeeping.
Nicotinamide adenine dinucleotide (NAD) is
involved in reduction–oxidation (redox) reactions;
it can accept (becoming NADH), carry, and
donate electrons (returning to NAD). In vitro
experiments have shown that the redox state of
NAD can regulate DNA binding activity of the
Clock:Bmal1 heterodimer. Interestingly, in vivo
NAD levels are subjected to daily variations,
thereby giving rhythmic input to the genetic clock
(Asher and Schibler, 2011; Merrow and
Roenneberg, 2001; Rutter et al., 2001), but there
are also several indirect pathways via which
the redox state can be linked to the clock. The
enzymes silent information regulator protein
(SIRT) and poly (ADP-ribose) polymerase 1
(PARP-1) are both NAD-dependent enzymes.
SIRT is expressed rhythmically and interacts with
Clock:Bmal1 heterodimers, leading to rhythmic
deacetylation of Clock:Bmal1, histone H3, and
Per2 (Asher and Schibler, 2011). When subjected
to daytime feeding the liver of Parp-1/ mice
showed a significantly delayed phase inversion of
clock gene expression, when compared with wild
type mice, suggesting that PARP-1 activity is
indeed implicated in the phase entrainment of
peripheral oscillators. However, PARP-1 activity
is probably driven by feeding rhythms rather
than by local circadian clocks (Asher and
Schibler, 2011).
The energy status of a cell influences the redox
state, implying that via this pathway, food intake
might be able to entrain the circadian system
(Arble et al., 2010; Maury et al., 2010; Merrow
and Roenneberg, 2001).
187
Adenosine monophosphate-activated protein
kinase (AMPK) activation has an impact on clock
function through various mechanisms. AMPK
senses the increase in AMP/ATP ratio in
conditions that deplete energy, such as hypoxia,
ischemia, and glucose deprivation. During these
conditions, AMPK may activate pathways that
generate ATP and suppress ATP-consuming pro-
cesses. For instance, in the hypothalamus, AMPK
activity stimulates food intake, whereas in the
periphery AMPK activity stimulates uptake and
oxidation of fatty acids in addition to glucose
uptake (Um et al., 2011). AMPK is part of the
core clock mechanism by degrading Per2 (via
CKIe), phosphorylating Cry1 and thereby
decreasing its stability. In line with this concept,
studies in mouse embryonic stem cells lacking
both isoforms of the catalytic subunit (AMPKa1/
a2 /) and in mice lacking either AMPKa1 or
AMPKa2 show altered free-running periods,
indicating that AMPK plays a fundamental role
in the core clock (Um et al., 2011).
Cyclic adenosine monophosphate (cAMP) is a
further example of an acute signaling pathway
that is tightly intertwined with the core clock.
cAMP is a second messenger derived from ATP
and is used for signal transduction in the cell, for
example, transferring the postsynaptic effects of
hormones that cannot pass the cell membrane.
In a series of elegant experiments, O’Neill et al.
showed that cAMP is not solely an output of the
SCN, but an integral component of the SCN pace-
maker, regulating transcriptional cycles (O’Neill
et al., 2008).
Heme has various biological functions; its most
common function is as a component of hemoglo-
bin, the red pigment in blood that transports
oxygen and carbon dioxide. However, heme also
functions as a ligand for the—formerly orphan—
RevErba and -b receptors. Binding of heme to
the RevErbs enables the receptors to recruit
NCoR, their corepressor, and subsequently to
repress the transcription of target genes (e.g.,
Bmal1), thereby regulating genes that are essen-
tial to the core clock. Furthermore, Per2 contains
188
heme as a prosthetic group (Burris, 2008), and
Bmal1, Per1, and Per2 regulate the expression
of aminolevulinic acid synthase 1 (Alas1), a rate-
limiting enzyme in heme production. Moreover,
PGC1a (a transcription factor that mediates tran-
sition toward FA oxidation and gluconeogenesis
when glucose is low) directly regulates the expres-
sion of Alas1, identifying heme as another signal-
ing molecule via which nutritional status can
influence the core clock mechanism.
Intriguingly, circadian rhythmicity has recently
been found in red blood cells, which have no
nucleus or indeed any other organelles, and
therefore do not undergo transcription or trans-
lation. Peroxiredoxins (PRX) are highly con-
served antioxidant proteins that scavenge
cellular reactive oxygen species, and in red
blood cells, PRX oxidation shows a clear circa-
dian rhythm (O’Neill and Reddy, 2011). This
illustrates very clearly that, in the absence of
transcription, circadian rhythms remain observ-
able in basic biochemical reactions. However,
when PRX rhythms were measured in an
arrhythmic mouse model (Cry1/2 /), PRX
rhythms differed from controls, implying that,
although purely biochemical mechanisms are
able to sustain 24-h rhythms, they must normally
reciprocally interact with gene expression cycles
(Reddy and O’Neill, 2011).
Taken together, these observations indicate
nongenetic pathways that have a fundamental
role in the core clock mechanism and represent
molecular routes via which nutritional status
may entrain the clock.
White adipose tissue
Adipose tissue is one of the largest organs in the
body, making up percentages of bodyweight from
5% in lean men to over 50% in the morbidly
obese. In mammals, three functionally different
types of adipose tissue have been described:
BAT, WAT, and bone marrow adipose tissue
(BMAT). The role of BMAT is poorly
investigated. It appears to be related to the con-
trol of hematopoiesis and osteoblastogenesis by
acting as an energy store, but also via the release
of paracrine factors (Casteilla et al., 2008). BAT
and WAT share the ability to store lipids as
triglycerides, but use them for different purposes.
BAT produces heat and plays an important role
in nonshivering thermogenesis. WAT, besides
functioning as mechanical and thermal protection
of vital organs and as an important long-term
energy store, secretes several proteins that influence
processes as diverse as hemostasis, blood pressure,
immune function, angiogenesis, and energy balance
(Christodoulides et al., 2009). Besides adipocytes,
WAT comprises a stromavascular fraction including
macrophages, mesenchymal stem cells (MSCs),
preadipocytes, endothelial and epithelial cells.
WAT development
Adipocytes derive from MSCs cells residing in
the adipose tissue stroma. The differentiation of
MSCs into mature adipocytes (adipogenesis)
occurs in two phases. The first phase, known
as determination, involves the commitment of a
pluripotent stem cell to the adipocyte lineage.
Determination—an as yet poorly characterized
process—converts the stem cell into a
preadipocyte. The second phase is terminal dif-
ferentiation, in which the preadipocyte takes on
the characteristics of the mature adipocyte: it
acquires the machinery that is necessary for
lipid transport and synthesis, insulin sensitivity,
and the secretion of adipocyte specific proteins.
Terminal differentiation involves a cascade of
transcriptional events, mainly regulated by
CCAAT/enhancer-binding proteins and peroxi-
some proliferator-activated receptor gamma
(PPARg) (Christodoulides et al., 2009; Rosen
and MacDougald, 2006). Each fat depot (see
the section “WAT depots”) has a unique pattern
of developmental gene expression and these
differences are largely independent of the func-
tional state (Gesta et al., 2007).

WAT depots
WAT is abundantly present throughout the body.
The main depots in rodents can be divided in those
found underneath the skin (subcutaneous; cervical,
dorsal, lumbar, abdominal, mammary, and glu-
teofemoral) and those found in the thoracic
and abdominal cavities. These internal fat
depots may be further subdivided in visceral and
nonvisceral. Visceral depots are found intratho-
racic, intraperitoneal (omental, mesenteric, and
umbilical), and extraperitoneal (retroperitoneal,
perirenal, gonadal and urogenital). The non-
visceral depots are mainly found in muscle tissue
(intramuscular and perimuscular; a.o. orbital).
Among white adipocytes, cells from different
depots may have distinct molecular and physiologi-
cal properties (Poulos et al., 2010; Rosen and
MacDougald, 2006). A striking example of this is
illustrated by the fact that, in humans, accumulation
of excess fat in the visceral compartment carries
increased metabolic risk, even if subcutaneous fat
mass is normal (Yamamoto et al., 2010). Regional
differences result from variations in molecular cha-
racteristics, such as expression of developmental
genes, hormone receptor distribution, adipokine
expression pattern, and secretory profile, as well as
from depot-specific innervation and vascularization
(Fruhbeck, 2008; Turer et al., 2011; Yamamoto
et al., 2010). For instance, adipocytes in visceral
depots are sensitive to lipolytic stimuli, whereas
adipocytes from nonvisceral depots do not release
stored lipids easily (Rosen and MacDougald,
2006). However, the proteome of visceral and
subcutaneous WAT in humans displays more
differences in the stromavascular cells than in
isolated adipocytes, suggesting that besides intrinsic
molecular characteristics in adipocytes, also inner-
vation and vascularization, and cellular heterogene-
ity in WAT are important to explain physiological
differences between depots (Fain et al., 2004). Fur-
thermore, WAT distribution is influenced by age,
sex, and endocrine determinants such as growth
hormone, cortisol, and sex steroids, leading to an
apple-shaped android (visceral) fat distribution in
189
males and postmenopausal females, and a pear-
shaped gynoid (subcutaneous) fat distribution in
premenopausal females (Elbers et al., 1999).
WAT innervation
A role for the ANS in the regulation of fat metab-
olism was proposed as early as the 1920s, as
reviewed by Kreier and Swaab (Kreier and
Swaab, 2010). In 1921, the neurologist Klien pres-
ented a case of lipodystrophy (abnormal body fat
distribution) and suggested a role for the hypothal-
amus in the development of this syndrome. More-
over, he hypothesized that dedicated regions of
the hypothalamus would be involved in the accu-
mulation of fat tissue, while other regions would
be involved in the mobilization of energy stores
(Klien, 1921). It took nearly a century to provide
some neuroanatomical evidence for this concept.
The sympathetic branch of the ANS regulates
lipolysis (Bamshad et al., 1998). However, the pro-
posed role for the parasympathetic innervation of
WAT in the storage of lipids is still debated
(Giordano et al., 2006; Kreier et al., 2006).
Sympathetic nerve fibers entering WAT mainly
innervate blood vessels; only 2–3% of the
adipocytes receive parenchymal innervation
(Slavin and Ballard, 1978). Nevertheless, these
sparse nerve-endings might be able to affect many
surrounding cells, and thereby potentially account
for a functionally significant contribution to WAT
regulation (Bartness et al., 2010a). Neuronal
tracing methods have been a valuable tool in
unraveling neuronal connections between the
brain and WAT. The Bartness lab reported the
connection from brain to WAT via the sympathetic
nervous system (SNS) in hamsters using pseudo
rabies virus (PRV), which is a neuroinvasive retro-
grade viral tracer that can be transported between
functionally connected neurons (Bamshad et al.,
1998). Using two PRV strains with a different
reporter, Kreier et al. identified separate neuronal
pathways innervating different fat compartments
(Fig. 3) (Kreier et al., 2006). These separate path-
ways allow a differential ANS drive to internal
190

(a)
MPO d
Hypothalamus:
c
3
PVN
b
Spinal cord:
IML
SCN
a

CeA

Subcutaneous
Intraabdominal body compartment

(b)
a b

c d e

Fig. 3. Separate neuron sets in the hypothalamus, amygdala, and spinal cord project to different body compartments. (A) Five
microliters of PRV B80 and PRV GFP were injected into parasympathetically denervated intraabdominal adipose tissue and in
subcutaneous adipose tissue. The intermediolateral (IML) cell column of the spinal cord shows a separate control of the
compartments; therefore, the survival time of the animals was chosen so that either only second-order or third-order neurons
were labeled. (B) In an upstream direction, the PVN of the hypothalamus shows specialized sets of neurons projecting to only
one compartment. The same specialization can be seen in the MPO (c), the central biological clock of the hypothalamus (SCN,
d), and the amygdala (e). (Bar in SCN: IML/PVN ¼ 50 mm; MPO/CeA/SCN ¼ 100 mm) (Kreier et al., 2006).

and subcutaneous fat compartments. Thus, these
tracing data may provide a neuroanatomical basis
for depot-specific differences in lipolytic rate
among WAT depots, as seen in, for example,
Siberian hamsters exposed to short winter-like
days (Bartness, 1995), or in patients treated with
pharmacological doses of glucocorticoids (Peckett
et al., 2011).
Parasympathetic innervation of WAT was
reported after injecting a retrograde PRV tracer
in retroperitoneal WAT, in combination with sur-
gical sympathetic denervation of the same WAT
depot, inducing intense neuronal labeling in the
dorsal motor nucleus of the vagus in rats
(DMV) (Kreier et al., 2002). The DMV is the
main cranial motor nucleus for the vagal nerve,
which is the major parasympathetic nerve, and
thus neuronal labeling in this nucleus implies
parasympathetic innervation of retroperitoneal
WAT. However, an experiment using chemical
denervation in a different fat pad and in another
species did not replicate this finding (Giordano
et al., 2006), while immunohistochemical markers
of parasympathetic nerves were shown to be
absent in various fat pads of several animal
models (Giordano et al., 2006).
In order to confirm the existence of parasympa-
thetic innervation of WAT, evidence of the pres-
ence of parasympathetic ganglia within the
adipose tissue, the presence of biochemical
indicators of PNS (peripheral nervous system)
innervation or the presence of neurochemical
markers of PNS innervation are needed (Bartness
et al., 2010a). Such histological or biochemical
evidence is currently not available. However, sev-
eral physiological studies suggest a role for the
PNS in WAT metabolism. In cultured rat eWAT
adipocytes, acetylcholine decreased insulin-
stimulated glucose uptake in a dose-dependent
manner. This effect was blocked completely
by atropine (nonselective muscarinic receptor
agonist), and partially by 4-diphenylacetoxy-
N-methylpiperdine methiodide (4-DAMP), a M3
muscarinic receptor antagonist. This suggests a
role for the M3 receptor in insulin-mediated
191
glucose uptake (Liu et al., 2004; Yang et al.,
2009). Moreover, pharmacological observations
in human subcutaneous adipose tissue micro-
dialysis studies showed cholinergic effects on lipol-
ysis (Andersson and Arner, 2001). In addition,
functional studies provided evidence for the ana-
bolic function of parasympathetic innervation of
adipose tissue, as euglycemic hyperinsulinemic
clamp studies revealed a > 30% reduction in the
insulin-mediated uptake of glucose and FFAs in
rat adipose tissue after selective removal of its
parasympathetic input. Furthermore, the activity
of the catabolic enzyme hormone sensitive lipase
(HSL) increased by 51% in the denervated adi-
pose tissue (Kreier et al., 2002).
As well as receiving information from the CNS,
WAT also provides neural feedback to the brain
via sensory nerves. In 1987, Fishman and Dark
showed that application of the anterograde tract
tracer True Blue in rat inguinal WAT (iWAT)
or dorsosubcutaneous WAT stained neurons in
the dorsal root ganglia (Fishman and Dark,
1987). Substance P and calcitonin gene-related
peptide were identified as neurotransmitters in
these neurons (Bartness et al., 2010a), and by
using the H129 strain of herpes simplex virus-1,
a viral anterograde transneuronal tract tracer
injected into iWAT and eWAT of Siberian
hamsters, Song and colleagues identified the spi-
nal cord and brain sites that receive this sensory
input (Song et al., 2009). Earlier, labeling in the
gracile nucleus—the sensory nucleus in the brain
stem involved in the perception of information
from the lower body—had been found using chol-
era toxin B, which is a monosynaptic anterograde
neuronal tracer (Kreier et al., 2006). This obser-
vation was later confirmed with the H129 antero-
grade tracer (Song et al., 2009).
The function of these sensory nerves is not fully
understood; however, studies by Song et al. sug-
gest that sensory nerves could be informing the
brain about lipid reserves, which might be the
neural equivalent of the humoral information that
leptin provides to the brain (Bartness et al.,
2010b; Song et al., 2009).
192
Regulation of energy storage
Lipogenesis
The adipocyte is unique among cells in that one
organelle, the lipid droplet, can encompass over
95% of the entire cell body. This lipid droplet
serves as a storage vessel for triglycerides pro-
duced by lipogenesis and released through lipoly-
sis (Trujillo and Scherer, 2006). Lipogenesis is
the anabolic process by which simple sugars
together with glycerol are converted to fats. Sim-
ple sugars such as glucose are converted to pyru-
vate by glycolysis, and subsequently pyruvate
dehydrogenase converts pyruvate into acetyl-
CoA. Following the formation of acetyl-CoA, fatty
acid synthesis combines glycerol with three acetyl-
CoAs to form triglycerides, which are transported
from the liver as very low-density lipoproteins, to
be stored in the adipose tissues.
Bar the proposed role of the PNS, lipogenesis is
regulated by insulin. Insulin plasma levels increase
proportionally with rising blood glucose levels.
Thus, high insulin levels are associated with the
fed state and might therefore be expected to
increase the storage of energy. Insulin stimulates
lipogenesis in two main ways, namely, by (1) the
upregulation of pyruvate dehydrogenase and (2)
the dephosphorylation (activation) of acetyl-coA
carboxylase.
Much of what we know about lipogenesis
has been elucidated through the study of
thiazolidinediones (TZDs), a class of antidiabetic
drugs that improve insulin sensitivity. TZDs
increase the expression of PPARs and thereby
promote energy storage. Upregulation of PPARs
in the adipocyte results in upregulation of
lipoprotein lipase, fatty acid transporter protein,
adipocyte fatty acid binding protein, malic
enzyme, glucokinase, and the GLUT4 glucose
transporter (Trujillo and Scherer, 2006).
In obese patients, lipids may be stored ectopi-
cally in nonadipose tissues, including the pan-
creas, heart, liver, kidney, and blood vessel wall.
This process and its deleterious consequences,
termed lipotoxicity, has been implicated in b-cell
loss during the progression of type 2 diabetes,
and in the pathogenesis of diabetic complications
through loss of cardiomyocytes, hepatocytes,
renal parenchymal cells, and endothelial cells
(Brookheart et al., 2009).
Lipolysis
Lipolysis is the catabolic process leading to the
breakdown of triacylglycerols (TAGs) into FFAs
and glycerol. After release into the blood, FFAs
are transported and taken up by other tissues to be
utilized for b-oxidation and subsequent ATP gener-
ation. Some FFAs do not leave the fat cell and are
reesterified into intracellular TAG. During lipolysis,
intracellular TAG undergoes hydrolysis through
the action of three major lipases: adipose triglycer-
ide lipase (ATGL/desnutrin/phospholipase A2z),
HSL, and monoacylglycerol (MGL) lipase. ATGL
hydrolyses TAGs into diacylglycerol (DAG) and
one FA, followed by HSL converting DAG into
monoacylglycerol (MAG) plus one FA, MGL then
hydrolyses MAG to produce glycerol and a third
FA (Ahmadian et al., 2010; Lafontan and Langin,
2009).
Lipolysis is regulated by the ANS (Bartness
et al., 2010a) and by several humoral factors, such
as catecholamines (phosphorylation of HSL),
glucocorticoids (upregulation of ATGL), natri-
uretic peptides, and growth hormone (Ahmadian
et al., 2010; Lafontan and Langin, 2009). While
systemic regulation of lipolysis has been relatively
well characterized, much remains to be
investigated regarding the local regulation of
lipolysis in adipocytes by (autocrine/paracrine)
factors. Adipocytes secrete several factors able
to regulate lipolysis locally, such as TNFa, which
stimulates lipolysis, and adenosine, which inhibits
lipolysis (Ahmadian et al., 2010).
Circadian rhythms in WAT
Like most other tissues, WAT gene expression
shows circadian rhythmicity (Ando et al., 2005;

Ptitsyn et al., 2006). Although diurnal variations
in adipose tissue metabolism are undisputedly
regulated by neurohumoral factors, the circadian
clock within the adipocyte probably plays a signif-
icant role as well, by altering the sensitivity of the
adipocyte to specific stimuli throughout the day
(Bray and Young, 2007). Indeed, several rodent
models with alterations in whole body clock gene
expression display disturbed lipid metabolism
(Gimble et al., 2011; Grimaldi et al., 2010; Shimba
et al., 2005). For instance, serum leptin levels
increased during the light phase in ClockD19
mutant mice fed a regular diet, and this increase
was enhanced in mice fed a high-fat diet (Turek
et al., 2005). Moreover, both in vitro and in vivo,
lack of Bmal1 results in reduced differentiation of
adipocytes and reduced lipid storage in the
adipocytes (Bunger et al., 2005; Kondratov et al.,
2006; Shimba et al., 2005, 2011). ClockD19 mutant
mice on a C57BL/6J background have hypercho-
lesterolemia, hypertriglyceridemia, hyperglycemia,
and hypoinsulinemia. However, the severity of
the ClockD19 phenotype is dependent on the
genetic background of the mice (Kennaway et al.,
2012; Oishi et al., 2006). Furthermore, Per2-
deficient mice display altered lipid metabolism
with a drastic reduction of total TAG and nones-
terified fatty acids. Per2 exerts its inhibitory func-
tion by blocking PPARy recruitment to its target
promoters and thereby its transcriptional activa-
tion (Grimaldi et al., 2010). Yet RORa mutant
mice resist obesity when placed on a high-fat diet
(Lau et al., 2008). In sum, these observations dem-
onstrate a tight relationship between genes of the
core clock mechanism and lipid metabolism.
WAT plays a central role in the regulation of
energy metabolism, mainly via the secretion of
factors (adipokines) that regulate appetite, food
intake, glucose disposal, and energy expenditure
(Wang et al., 2008). Adipokines are secreted by
adipocytes and/or the stromavascular fraction of
WAT. Originally, the term adipokine described
cytokines secreted specifically from adipocytes;
however, as many cell types in adipose tissue
have been found to secrete proteins, and other
193
proteins besides cytokines are being produced,
the term adipokine now incorporates all proteins
secreted from adipose tissue (Stryjecki and
Mutch, 2011; Wang et al., 2008).
Extensive reviews on the metabolic functions of
adipokines can be found in Halberg et al. (2008),
Maury and Brichard (2010), Poulos et al. (2010),
and Trujillo and Scherer (2006).
Leptin
Leptin is a hormone secreted by adipose tissue in
proportion to body fat amount and relays fat stor-
age information to the brain. High levels of leptin
signal satiety and reduce food intake, whereas
low levels of leptin stimulate food intake (Schwartz
et al., 2000). The discovery that leptin could regu-
late body weight through effects on food intake
and energy expenditure represented a major
breakthrough in our understanding of the neuro-
anatomical and molecular components of the sys-
tems involved in energy homeostasis (Farooqi,
2011). The prevalence of mutations in this system
range from 0.5% to 1% in adult obesity up to
6% in subjects with severe obesity starting in child-
hood (Farooqi, 2007; Larsen et al., 2005). For the
discovery of this previously unknown endocrine
system, Coleman and Friedman received the
Albert Lasker Award for Basic Medical Research
in 2010 (Flier and Maratos-Flier, 2010).
Plasma levels of leptin are regulated by the
biological clock, leading to a clear day/night
rhythm. Furthermore, WAT mass and feeding reg-
ulate leptin plasma levels; long periods of fasting
eliminate the leptin rhythm (Elimam and Marcus,
2002). However, under constant and continuous
feeding conditions, a circadian rhythm in leptin
persists, indicating a role for the circadian clock
in regulating leptin levels during fed conditions
(Kalsbeek et al., 2001; Simon et al., 1998). In
healthy volunteers, misalignment between behav-
ior and endogenous circadian timing leads to
lower overall leptin levels (Scheer et al., 2009),
suggesting that leptin responds to the endogenous
194

circadian clock, independent of behavioral factors
such as feeding. Although SCN lesions eliminate
leptin circadian rhythmicity (Kalsbeek et al.,
2001), cultured adipocytes still show rhythmic lep-
tin mRNA expression, implying regulation by an
endogenous clock within the adipocytes (Otway
et al., 2009). Figure 4 shows plasma leptin levels
in rats under varying conditions.
In addition to being regulated by the clock, lep-
tin serves as an input factor for the biological
clock, as the leptin receptor is expressed in SCN
cells, and in vitro leptin can advance the SCN
(Prosser and Bergeron, 2003).
In sum, leptin is a pivotal factor in the interplay
between feeding cues, metabolic state, and circa-
dian timing.

(a) Other adipokines

4
Besides leptin several other adipokines exhibit sig-
3
nificant day/night rhythmicity. Adiponectin is an
adipokine that is involved in glucose and lipid
Leptin (ng/ml)

metabolism by increasing fatty acid oxidation and

2 potentiating insulin-mediated inhibition of hepatic
gluconeogenesis, thus promoting insulin sensitivity
(Barnea et al., 2010). Interestingly, although
1
adiponectin is produced by adipose tissue, its serum
levels and WAT gene expression decrease in obe-
sity and in animals fed a high-fat diet (Barnea
0 6 12 18 24 et al., 2010; Boucher et al., 2005; Turer et al., 2011).
(b) Both in vitro and in vivo, adiponectin has a sig-
8
nificant day/night rhythm (Barnea et al., 2010;
Garaulet et al., 2011; Gavrila et al., 2003; Otway
6 et al., 2009; Scheer et al., 2009, 2010), with a
trough at night for humans, and a trough during
Leptin (ng/ml)

daytime for rats (Oliver et al., 2006; Scheer

4 et al., 2010). In lean men, this rhythm is not
2
0 6 12 18 24
Zeitgeber time (h)
Fig. 4. Diurnal plasma leptin profiles in different experimental
conditions. The diurnal plasma leptin profile (mean  SEM) of
nonoperated control animals (n ¼ 7; ○) is compared with that
in SCN-lesioned (n ¼ 9; ; a) or ADXþCort (n ¼ 8; △) and
regular-fed (n ¼ 6; ▪) animals (b). Asterisks indicate that
plasma leptin values are significantly different (P < 0.01)
from trough values between ZT0 and ZT6. (b) The shaded
area in (b) indicates the mean  SEM for the nonoperated
control animals (Kalsbeek et al., 2001).
driven by the feeding/fasting cycle (Scheer et al.,
2010). ClockD19 mutant mice that retain melato-
nin rhythmicity (ClockD19þ MEL) show increased
eWAT adiponectin gene expression, which may
contribute to the improved insulin resistance
found in ClockD19þ MEL mice compared to
ClockD19 mice (Kennaway et al., 2012).
Resistin is a cytokine that is produced in WAT
(adipocytes in rodents, macrophages in human)
and is a potential mediator of T2DM and cardio-
vascular disease (Ando et al., 2005; Fain et al.,
2003; Oliver et al., 2006; Schwartz and Lazar,
2011), with higher expression rates in omental
versus subcutaneous WAT of obese female
subjects (Fain et al., 2003). Resistin mRNA

expression is rhythmic in several WAT com-
partments in rats, with a peak in the late dark/
early light phase (Oliver et al., 2006). Resistin is
downregulated by fasting and upregulated by
(re-)feeding (Oliver et al., 2006). However,
WAT gene expression levels of resistin are
decreased in obese and high-fat diet-fed mice
(Boucher et al., 2005). Rotating shift workers
have elevated plasma levels of resistin compared
to day work controls (Burgueno et al., 2010).
WAT circadian rhythms in obesity
Obesity and disturbed rhythmicity in WAT are
tightly correlated. For example, in humans,
chronic desynchrony of internal circadian time
with the external environment (e.g., during
shiftwork) correlates with increased incidence
of obesity and type 2 diabetes (Pan et al.,
2011). Furthermore, circadian misalignment in
healthy subjects decreases leptin levels (Scheer
et al., 2009). In visceral and subcutaneous
WAT (sWAT) biopsies of severely obese
men, the expression level of clock genes was
correlated with waist circumference (Gomez-
Abellan et al., 2008). However, when gene
expression was measured in human sWAT
explants across a 24-h cycle, no differences in
expression rhythm of clock genes were observed
between overweight study participants and type
2 diabetes participants or lean controls. The
authors suggest this might be due to the early
stage of the disease and the high level of
glycemic control in the group of participants.
However, in cultured WAT of severely obese
women, slight differences between visceral and sub-
cutaneous fat compartments in acrophase and
amplitude of the expression of several clock and
metabolic genes have been found (Garaulet et al.,
2011; Martinez-Agustin et al., 2010). Together,
these results indicate that circadian WAT rhythms
may be disturbed in obesity, and that results from
one WAT compartment cannot be downright
extrapolated to another WAT compartment.
195
Due to limited sampling possibilities in human
experiments, most of the research on the relation-
ship between obesity and WAT rhythms has been
done in animal models. Below, we describe 3 rodent
models with obesity due to altered leptin function.
ob/ob mice
ob/ob mice exhibit a mutation in the leptin gene,
rendering leptin unable to bind to its receptors.
As a result, these mice become severely obese, with
hyperphagia, hyperglycemia, hyperinsulinemia,
high levels of corticosteroids, hypothyroidism,
dyslipidemia, decreased body temperature, de-
fective thermogenesis, and infertility due to
hypogonadotropic hypogonadism. The broad and
severe behavioral, metabolic and neuroendocrine
changes in these animals represent the classic star-
vation response designed to protect viability and
can all be remedied by leptin administration (Arble
et al., 2010; Robinson et al., 2000).
The mutated leptin gene in ob/ob mice results
in disturbed sleep and attenuates diurnal and
overall locomotor activity. However, reports are
indecisive on the effect of the mutation on the
day/night pattern of food intake (Ando et al.,
2011; Laposky et al., 2006). Daily mRNA expres-
sion rhythms of clock genes in the SCN are unaf-
fected; yet the daily rhythms of these genes were
substantially damped down in the liver and
eWAT of ob/ob mice (Ando et al., 2011). Daily
leptin injections modestly but significantly
improved the mRNA expression rhythms of sub-
sets of clock genes in liver and eWAT as they
did the activity levels during the dark phase. At
present, it is unclear whether the increased noc-
turnal activity is a prerequisite for the improved
rhythms in liver and eWAT clock genes.
db/db mice
db/db mice have a mutation in the long form of
the leptin receptor, leading to ineffective leptin
196

signaling and thereby to a phenotype similar to Zucker rats, but not in the SCN, mesenteric
the ob/ob mutation. These mice are characterized WAT, and heart. The authors suggest that leptin
by impaired regulation of core components of the may play a role in the regulation of the clock in
clock mechanism in WAT, possibly due to the liver (Motosugi et al., 2011).
impaired AMPK and SIRT1 function (Caton
et al., 2011). Clock, Bmal1 and Per2 mRNA,
Conclusion
and/or protein levels are decreased; treatment
with the antidiabetic drug metformin reversed
The role of the circadian timing system in adi-
these decreases (Caton et al., 2011).
pose biology represents an exciting new field
of study that will give us a greater insight into
the pathogenesis of obesity and its health con-
Obese Zucker rat
sequences. The discovery of the ob/ob gene
and its product leptin, along with the other
A third rodent model manifesting altered leptin
adipokines, revealed an intriguing endocrine sys-
signaling is the Zucker rat. These rats are rela- tem that regulates feeding and metabolism via
tively insensitive to leptin, again due to a muta-
communication between adipose tissue and the
tion in the long form of the leptin receptor and
biological clock in the brain. The monogenetic
consequently have a similar phenotype to ob/ob
mutations in this system leading to obesity have
and db/db mice. As early as 1977, Becker and
taught us valuable lessons on the regulation of
Grinker observed that the normal pattern of pre-
energy metabolism. Moreover, there seems to
dominantly nocturnal feeding was absent in the
be a clear link between circadian misalignment
Zucker rat (Becker and Grinker, 1977). A more
and metabolic disorders, such as obesity and
detailed study 10 years later found no significant type 2 diabetes. In view of the recent development
differences between obese (fa/fa) and lean (Fa/–)
of a 24/7 h society, combined with globalization
Zucker rats in light and dark feeding, expressed
with its increased intertime zone travelling, it will
as a percentage of 24 h intake. The increased food
be increasingly important to expand our knowl-
intake of the Zucker was mainly due to an increase
edge on the health consequences of circadian
in meal size (Alingh et al., 1986; Fukagawa et al.,
misalignment.
1988). Nevertheless, some circadian disturbance
was noted: only in Zucker rats was a significant dif-
Acknowledgments
ference observed between meal sizes in the light
and dark phase, and Zucker rats ate fewer but
We thank Wilma Verweij for correction of the
larger meals during the first half of the dark phase.
manuscript. Rianne van der Spek was financially
Body temperature, activity, and feeding rhythms
supported by the Netherlands Organization for
were phase-advanced in obese Zucker rats, proba-
Scientific Research Foundation (ZonMw
bly because of altered SCN resetting in response
TOP91207036).
to light (Fukagawa et al., 1992; Mistlberger et al.,
1998). All in all, the conclusion after almost 20 years
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 12

Circadian modulation of sleep in rodents

Roman Yasenkov1 and Tom Deboer*

Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University Medical Center,
Leiden, The Netherlands

Abstract: Sleep is regulated by circadian and homeostatic processes. The sleep homeostat keeps track of
the duration of prior sleep and waking and determines the intensity of sleep. In mammals, the homeostatic
process is reflected by the slow waves in the non-rapid eye movement (NREM) sleep electroencephalogram
(EEG). The circadian process is controlled by a pacemaker located in the suprachiasmatic nucleus of the
hypothalamus and provides the sleep homeostat with a circadian framework.
This review summarizes the changes in sleep obtained after different chronobiological interventions
(changes in photoperiod, light availability, and running wheel availability), the influence of mutations
or lesions in clock genes on sleep, and research on the interaction between sleep homeostasis and the
circadian clock.
Research in humans shows that the period of consolidated waking during the day is a consequence of
the interaction between an increasing homeostatic sleep drive and a circadian signal, which promotes
waking during the day and sleep during the night. In the rat, it was shown that, under constant
homeostatic sleep pressure, with similar levels of slow waves in the NREM sleep EEG at all time
points of the circadian cycle, still a small circadian modulation of the duration of waking and NREM
sleep episodes was observed. Under similar conditions, humans show a clear circadian modulation in
REM sleep, whereas in the rat, a circadian modulation in REM sleep was not present. Therefore, in
the rat, the sleep homeostatic modulation in phase with the circadian clock seems to amplify the
relatively weak circadian changes in sleep induced by the circadian clock.
Knowledge about the interaction between sleep and the circadian clock and the circadian modulation of
sleep in other species than humans is important to better understand the underlying regulatory mechanisms.
Keywords: circadian; electroencephalogram; hamster; mouse; rat; rodent; photoperiod; REM sleep; SCN
lesion; sleep; sleep homeostasis.

*Corresponding author.
Tel.: þ31 71 526 9771; Fax: þ31 71 526 8270
E-mail: tom.de_boer@lumc.nl

1
Present address: Faculty of Health and Medical Sciences,
University of Surrey, Guildford, Surrey, United Kingdom.

http://dx.doi.org/10.1016/B978-0-444-59427-3.00012-5 203
204
Introduction
Sleep is regulated by homeostatic and circadian
processes (Achermann and Borbely, 2011).
In mammals, homeostatic sleep pressure is
reflected by electroencephalogram (EEG) slow-
wave activity (SWA, EEG power density
between  1 and 4 Hz) in undisturbed non-rapid
eye movement (NREM) sleep. In all mammalian
species investigated until now, SWA increases
after sleep deprivation, and in several species, a
dose–response relationship between waking dura-
tion and subsequent SWA was established
(Deboer and Tobler, 2003; Dijk et al., 1987;
Huber et al., 2000a; Lancel et al., 1991; Strijkstra
and Daan, 1998; Tobler and Borbely, 1986).
A study involving an afternoon nap showed that
also predictable decreases in sleep homeostasis
can be observed in the NREM sleep EEG SWA
(Werth et al., 1996b). Mathematical models,
simulating the homeostatic response, have been
applied successfully in humans (Achermann
et al., 1993), rats (Franken et al., 1991), and mice
(Franken et al., 2001; Huber et al., 2000a).
The circadian process is controlled by an endoge-
nous pacemaker, which is located in the
suprachiasmatic nucleus (SCN) of the hypothala-
mus (Meijer and Rietveld, 1989). This pacemaker
is thought to provide the sleep homeostat with a
circadian framework.
Normal sleep in rodents
Rodents show a large variety of sleep patterns.
In Table 1, all rodent species are listed in which
at least one full 24 h cycle of EEG and EMG
was recorded under a 12 h:12 h L:D cycle
(Table 1). We do not claim completeness and in
addition realize that, particularly in mice, a broad
range of strains and knockout models is available
that could also have been included. For most spe-
cies, only one publication is available, but for
some species (mouse, rat, and hamster), several
studies have been published and not all of them
are covered in this review. Particularly, the rat
and the (C57BL/6) mouse were recorded by sev-
eral more authors than listed here. In Table 1,
the “doubles” are included which are also
discussed in Tables 2 and 3.
Many rodent species recorded in the laboratory
are night active and therefore have their
main sleep phase during the light period (rat,
mouse, Syrian hamster, Djungarian hamster, chin-
chilla), others are day active (13-lined ground
squirrel, European ground squirrel, Siberian chip-
munk, golden-mantled ground squirrel), and a
few species show crepuscular or cathemeral
behavior, with no preference for activity in the
light or dark period. The latter is confirmed by
the ratio in total sleep time (TST) between light
and dark being close to 1 and the percentage of
TST in the rest period being close to 50 (naked
mole rat, guinea pig, degu, Mongolian gerbil).
These sleep patterns are relatively fixed when
the animals are left undisturbed in the laboratory
(but see Table 3 for some exceptions), whereas in
the wild, rest–activity patterns seem to be more
flexible and may be very dependent on environ-
mental influences (Daan et al., 2011; Gattermann
et al., 2008; Levy et al., 2007).
In contrast to humans, rodents show a strong
ultradian component in their sleep, and even in
species with a clear preference for activity in the
light or dark phase, no single period of con-
solidated waking is seen during the active period
or consolidated sleep during the rest period.
Despite these differences, sleep homeostatic the-
ory, as it predicts the depth of sleep, seems to
apply just as well to polyphasic sleep in rodents
as it does to monophasic sleep in humans. How-
ever, due to the polyphasic nature of their sleep,
rodents are more flexible in their response to
environmental influences on sleep. This may
explain the relatively large differences in 24-h
TST found in the same species between different
laboratories in the Sprague–Dawley rat, Syrian
hamster, and C57BL/6 mice (Table 1). The range
of TST in the C57BL/6 mouse is between 40.6%
and 48.6%, which is a difference of almost 2 h
over 24 h. Since these mice are supposed to be
genetically identical, this indicates that the
 205

Table 1. List of rodent species and their electroencephalographic confirmed total sleep time under 12 h:12 h light:dark conditions

% TST % TSTL % TSTD Ratio % TST in rest period References

Diurnal species
Spermophilus citellus
European ground squirrel 57.5 46.6 68.3 1.47 59.4 Strijkstra (1999)
Eutamias sibericus
Siberian chipmunk 52.8 29.1 74.7 2.57 70.7 Dijk and Daan (1989)
Citellus lateralis
Golden-mantled ground
squirrel 60.6 40.9 80.4 1.97 66.3 Haskell et al. (1979)
Citellus tridecemlineatus
Thirteen-lined ground squirrel 57.5 54.8 60.2 1.10 52.3 Van Twyver (1969)a
Nocturnal species
Mesocricetus auratus
Syrian hamster 64.7 78.2 51.2 1.53 60.5 Tobler and Jaggi (1987)
60.1 76.0 44.2 1.72 63.2 Van Twyver (1969)a
Mus musculus
Mouse CRI 54.8 64.5 45.1 1.43 58.9 Van Twyver (1969)a
Mouse C57BL/6 48.6 64.1 33.1 1.94 65.9 Huber et al. (2000a)
40.6 56.4 24.7 2.28 69.5 Franken et al. (1999)
46.6 63.4 29.8 2.13 68.0 Deboer et al. (2007b)
Rattus norvegicus
Sprague–Dawley rat 47.9 68.9 26.9 2.56 71.9 Franken et al. (1995)
49.2 68.0 30.4 2.24 69.1 Tobler et al. (1994)
57.1 78.1 36.0 2.17 68.4 Mistlberger et al. (1983)
Long Evans rat 55.2 65.7 44.7 1.47 59.5 Van Twyver (1969)a
Chinchilla langer
Chinchilla 52.2 57.6 46.8 1.23 55.2 Van Twyver (1969)a
No clear diurnal preference
Cavia porcellus
Guinea pig 32.2 35.7 28.9 1.24 55.4 Tobler and Franken (1993)
Octodon degus
Degus 37.6 36.7 38.5 1.05 48.8 Kas and Edgar (1998)
Meriones unguiculatus
Mongolian gerbil 54.5 57.1 51.8 1.10 52.4 Susic and Masirevic (1986)
Spalax ehrenbergi
Mole rat 51.8 48.8 54.7 1.12 52.8 Tobler and Deboer (2001)

TST, total sleep time over 24 h; TSTL, total sleep time during the light period; TSTD, total sleep time during the dark period; Ratio, ratio between
TSTL and TSTD, a measure for diurnality or nocturnality.
a
TSTL and TSTD values calculated from the LD ratio.

differences in TST are caused by (in most cases,
unknown) differences in environmental conditions
between the laboratories. The same variability
may also be responsible for the difference in the
ratio of TST between light (TSTL) and dark
(TSTD; an index for diurnality/nocturnality of
the sleep–wake rhythm) which ranges from 1.94
to 2.28 in the examples for C57BL/6 mice in
Table 1. However, this ratio also depends on the
total amount of sleep (low 24-h TST results in
higher ratios). Another index for the amplitude,
the percentage of 24-h TST spent in the main
rest period, is found in a much closer range
(65.9–69.5%), suggesting that this variable may
be less susceptible to differences in environmental
conditions.
206

Table 2. Comparison of mouse strains recorded in the same laboratory

% TST % TSTL % TSTD Ratio % TST in rest period

Huber et al. (2000a)

129/Ola 51.2 63.6 38.7 1.64 62.1
129/SvJ 58.6 66.9 50.3 1.33 57.1
C57BL/6J 48.6 64.1 33.1 1.94 65.9
Franken et al. (1999)
AKR/J 48.0 63.9 32.1 1.99 66.6
BALB/cByJ 43.9 52.2 35.6 1.47 59.4
C57BL/6J 40.6 56.4 24.7 2.28 69.5
C57BR/cdJ 39.9 59.1 20.2 2.93 74.1
DBA/2J 36.4 55.3 17.5 3.16 76.0
129/Ola 40.7 58.3 23.0 2.53 71.6

TST, total sleep time over 24 h; TSTL, total sleep time during the light period; TSTD, total sleep time during the dark period; Ratio, ratio between
TSTL and TSTD, a measure for diurnality or nocturnality.

Table 3. Effects of SCN lesion, photoperiod, and wheel availability

Condition % TST % TSTL % TSTD Ratio % TST in rest period References

LD/DD
Mouse C57BL/6 LD 46.6 63.4 29.8 2.13 68.0 Deboer et al. (2007b)
DD 47.8 65.0 30.0 2.17 78.7
Sprague–Dawley rat LD 49.2 68.0 30.4 2.24 69.1 Tobler et al. (1994)
DD 50.3 67.5 33.0 2.05 67.1
Wheel availability
Degu No wheel 37.6 36.7 38.5 1.05 51.2 Kas and Edgar (1998)
Wheel 38.2 39.2 36.2 1.08 51.3
Mouse C57BL/6 No wheel 43.8 63.3 24.2 2.62 72.7 Vyazovskiy et al. (2006)
Wheel 39.6 68.3 10.8 6.32 86.2
SCN lesion
Sprague–Dawley rat SCN 57.1 78.1 36.0 2.17 68.3 Mistlberger et al. (1983)
SCNx 60.7 60.8 60.7 1.00 50.1
Rata SCN 51.2 67.9 38.2 1.78 66.3 Coindet et al. (1975)
SCNx 52.5 59.2 45.8 1.29 56.4
Photoperiod
Siberian chipmunk LD 18/6 48.7 39.3 77.6 1.97 39.8 Dijk and Daan (1989)
LD 12/12 52.8 29.1 74.7 2.57 70.7
LD 6/18 53.0 17.2 65.7 3.82 82.6
Djungarian hamster LD 16/8 59.2 67.2 42.8 1.57 87.3 Deboer and Tobler (1996)
LD 8/16 61.8 71.7 56.7 1.26 38.7
Rat LD 16/8 52.7 64.4 28.3 2.28 94.0 Franken et al. (1995)
LD 12/12 47.9 68.9 26.9 2.56 71.9
LD 8/16 55.8 73.3 45.7 1.60 43.8

TST, total sleep time over 24 h; TSTL, total sleep time during the light period; TSTD, total sleep time during the dark period; Ratio, ratio between
TSTL and TSTD, a measure for diurnality or nocturnality.
a
Strain not specified.

Another aspect that becomes clear from the
data in Table 1 is that some species which, from
the behavioral point of view, are considered to
be clearly diurnal or nocturnal do not show a
large difference in TSTL and TSTD. The Euro-
pean and 13-lined ground squirrel are considered
to be day active, and the Long Evans rat and the
chinchilla are known as night active species. They
spent, however, less than 60% of their 24-h TST
in their main rest period. The guinea pig is, on
the basis of its day–night difference in sleep, not
different from these four species. Yet, when ana-
lyzing the hourly distribution of activity and
TST, it is considered to be a species with no pref-
erence for sleep in the light or dark period. It is
the detailed distribution of sleep and waking dur-
ing the active phase, and particularly the expres-
sion of a relatively long consolidated waking
period and the overt behavior during that active
period (i.e., increased activity), which determines
whether an animal is considered to have a clear
diurnal or nocturnal preference.
When comparing the variation between mouse
strains, it seems that at least part of the diurnal
modulation of sleep has a genetic basis. Different
mouse strains recorded under the same cir-
cumstances in the same laboratory have different
amounts of TST over 24 h (ranging from 40% to
50%,  2.5 h difference) and show also strong
differences in the light–dark ratios (Table 2).
Strains like 129/SV and BALBc have relatively
low ratios, whereas C57 strains, 129/Ola, and par-
ticularly DBA show large differences between
light and dark. However, quantitative trait loci
analysis did not result in a putative genetic locus
responsible for diurnal variation in TST (Tafti
et al., 1997).
Clocks, photoperiod, running wheels, and light
Chronobiologically relevant factors that may
influence sleep are light and darkness, the photo-
period (day length), the presence or absence of a
running wheel, and the presence or absence of an
207
endogenous circadian pacemaker. Changes in
these factors result in significant changes in overt
rest–activity behavior and sleep.
Mice and rats, acutely transferred from a
12 h:12 h L:D cycle into constant darkness (DD),
do not show large changes in their TST over
24 h (Table 3). In addition, no clear changes in
the preferences for sleep in the rest or active
phase were observed. In albino Sprague–Dawley
rats, the distribution of sleep and waking across
the circadian day did show a small increase in
REM sleep and a clear increase in SWA in
NREM sleep in the rest period (Tobler et al.,
1994). In contrast, in pigmented C57BL/6 mice,
no changes were observed from LD to DD
(Deboer et al., 2007b). These differences between
rats and mice may be related to the difference in
pigmentation between the animals but may also
be based on species-specific differences.
It may be obvious that removal of the central
pacemaker will influence the distribution of sleep.
After SCN lesions, the difference in TST ratio
between the light and the dark period decreases,
but sometimes a preference for sleep in the light
period remains (Coindet et al., 1975). The latter
may be a masking effect of light due to a suppres-
sion of activity (Deboer and Tobler, 2000) and
direct induction of sleep by light (Alfoldi et al.,
1991; Benca et al., 1998; Deboer et al., 2007b).
In most SCN lesion experiments in mice and rats,
performed under constant dark conditions, the
amount of sleep over 24 h did not change,
indicating that the endogenous clock does not
determine the amount of sleep (Ibuka et al.,
1980; Mistlberger et al., 1983; Tobler et al.,
1983), but also here exceptions have been
reported (Easton et al., 2004).
An increase of sleep in the rest phase was seen
in mice which were provided with a running
wheel (Vyazovskiy et al., 2006). Here, TST over
24 h hardly changed, but the preference for sleep
in the light period became stronger. Without a
running wheel, the Degu is a crepuscular species
with a slight preference for sleep in the dark
period. The availability of a running wheel
208
reversed this preference, making the animals
slightly nocturnal (Kas and Edgar, 1998). These
data suggest that the distribution of sleep, and
possibly also the amount of sleep, is subject to
time management “decisions,” just like other
behavioral activities observed in mice (de Visser
et al., 2005).
Under different photoperiods, TST over 24 h
does not change systematically in rodents
(Table 3); however, the ratio of the difference in
TST between light and dark changes. In the diur-
nal Siberian chipmunk, the amplitude increased
(Dijk and Daan, 1989), whereas in the nocturnal
rat and Djungarian hamster, the amplitude
decreased (Deboer and Tobler, 1996; Franken
et al., 1995) when photoperiod shortened. This
suggests that diurnal and nocturnal rodents may
show an opposite response to photoperiodic
changes. The distribution of sleep of the rodent
species confirms this (Fig. 1). In humans, the
effect of photoperiodic changes seems to differ
slightly from the changes in the Siberian chip-
munk as the percentage of TSTD decreased
(Wehr et al., 1993), reducing the daily amplitude
in the short photoperiod. What is also clear from
these data is that when the duration of the oppor-
tunity to sleep in the preferred phase of the day
(light or dark) is reduced, rodents decrease the
relative amount of TST in this period without
changing TST over 24 h. This is in contrast with
the available human data, where in general, no
sleep is observed during the light period. The lat-
ter may be because most experimental designs in
human sleep research do not allow sleep in the
light period, but it may also be because human
sleep is more disturbed by light. The flexibility
in the timing of sleep in rodents results, under
certain photoperiods, in the rather extreme con-
dition that the Siberian chipmunk and the
Djungarian hamster spent less than 40% of their
TST over 24 h in what is considered to be their
main “sleep” or rest period. The remaining sleep
is fulfilled in their main “waking” or active
period. This means that, although the animals
are more active compared to the main rest period,
they will spend a substantial amount of time dur-
ing this active period also in sleep. In general, the
data show that rodents are very flexible in their
choice of the time of day for their sleep.
Circadian–homeostatic interactions
As mentioned in the section “Introduction,” sleep
is regulated by homeostatic and circadian
mechanisms. This begs the question whether
these two mechanisms regulate sleep indepen-
dently or whether there is an interaction between
the two. Experiments in rodents have contributed
extensively to answer this question. From sleep
deprivation experiments in SCN-lesioned rats
(Mistlberger et al., 1983; Tobler et al., 1983;
Trachsel et al., 1992), it became clear that sleep
homeostatic mechanisms function without the
presence of an intact circadian clock. Phase-
shifting experiments in humans showed that sleep
homeostatic regulation does not follow the
circadian timing system (Dijk et al., 1989). There-
fore, it was assumed that sleep homeostasis
and circadian regulation of sleep function inde-
pendently. However, different types of continu-
ous regulatory interactions were also proposed
(Achermann and Borbely, 1994; Edgar et al.,
1993; Mistlberger, 2005).
The notion that changes in sleep pressure can
influence the functioning of the circadian clock
came from data in mice and hamsters, which
showed that sleep deprivation attenuates phase
shifts induced by light (Challet et al., 2001; Mis-
tlberger et al., 1997). It was also shown that sleep
deprivation can change circadian phase and FOS
expression in the SCN (Antle and Mistlberger,
2000). Recently, it was shown that partial sleep
deprivation reduces phase advances to light in
humans as well (Burgess, 2010).
When sleep–wake behavior and SCN neuronal
activity are recorded simultaneously, the influ-
ence of sleep on SCN neuronal activity becomes
very clear. At the onset of NREM sleep, SCN
neuronal activity decreases, whereas at the
 209

100 100
Human
75 75

50 50

25 25

0 0

100 100
Chipmunk
75 75

50 50

25 25
Sleep (%)

0 0

100 100
Rat
75 75

50 50

25 25

0 0

100 100
Djungarian hamster
75 75

50 50

25 25

0 0
0 6 12 18 24 0 6 12 18 24
Time since mid-active period (h)

Fig. 1. Mean 24-h profiles total sleep time in four different species in two different photoperiods. Mean 24-h profiles of EEG
confirmed sleep in humans, Siberian chipmunks, rats, and Djungarian hamsters in a short (left) and long (right) main sleep
period. Human data adapted from Wehr et al. (1993), Siberian chipmunk data adapted from Dijk and Daan (1989), rat data
adapted from Franken et al. (1995), and Djungarian hamster data adapted from Deboer and Tobler (1996).

transition from NREM sleep to REM sleep or in NREM sleep was found, suggesting that deep
waking, the activity increases again (Deboer sleep reduces SCN activity. Further experiments
et al., 2003). In addition, a significant negative cor- showed that SCN neuronal activity is attenuated
relation between SWA and SCN neuronal activity after sleep deprivation for approximately the same
210
duration as SWA in NREM sleep is increased
(Deboer et al., 2007a). Taken together, the data
show that information about vigilance state
changes and changes in sleep pressure reach the
SCN and influence its neuronal firing pattern.
Recent experiments applying functional magnetic
resonance imaging show similar effects in humans
(Schmidt et al., 2009). The data suggest that sleep
homeostatic feedback on the output of the endog-
enous clock may be continuous instead of being
restricted to distinct time points in the circadian
cycle. Recently, a model which integrates the
traditional two process model of sleep regulation
with these recent findings was proposed (Dijk
and Archer, 2010). In this conceptual model,
sleep homeostasis and the circadian clock
function independently, but sleep homeostatic
pressure feeds back on the circadian clock,
influencing its output.
Whether information on circadian time also
influences sleep homeostasis, other than intervening
with the timing of sleep, remains unclear. However,
some evidence that the circadian clock may influ-
ence sleep homeostatic mechanisms comes from
mathematical simulations of sleep homeostasis in
rodents. In these simulations, the time course of
the sleep homeostatic process (Process S) is pre-
dicted on the basis of prior sleep–wake history and
correlated with SWA in NREM sleep. In rodents,
the time course of Process S is determined itera-
tively on the basis of the vigilance states. During
waking and REM sleep, S increases, whereas during
NREM sleep, S decreases (Deboer, 2009; Franken
et al., 1991; Huber et al., 2000a; Vyazovskiy et al.,
2007). Both increase and decrease of Process S
occur with corresponding fixed time constant
(Deboer, 2009; Franken et al., 1991; Huber et al.,
2000a; Vyazovskiy et al., 2007). For mice, this
approach was sufficient to reach an optimal fit in
most strains (Franken et al., 2001; Huber et al.,
2000a). However, in the rat, an additional modula-
tion of the decreasing time constant (determining
the speed of decrease of Process S during NREM
sleep) was needed, as SWA was consistently higher
than S in the light period and lower than S in the
dark period (Franken et al., 1991). In an experiment
where the effect of a 6-h sleep deprivation was
tested in the light and in the dark period, SWA
was lower than S in the dark period (Vyazovskiy
et al., 2007). Even in DD conditions, a circadian
modulation of the decreasing time constant was nec-
essary to obtain an optimal fit, suggesting that there
is an endogenous circadian influence on the expres-
sion of SWA in NREM sleep (Deboer, 2009).
Detailed analysis of the behavior of SWA revealed
that the rate of increase of SWA at the onset of a
NREM sleep episode showed a circadian modula-
tion, which may explain part of the systematic circa-
dian deviations observed in the simulations of
Process S in the rat (Deboer, 2009).
Clock genes and sleep
The discovery of clock genes has brought new per-
spective to the relationship between the circadian
clock and sleep. The search for clock genes has
been highly successful, resulting in five to seven
central clock genes (Cry1, Cry2, BMAL1, Clock,
Per1, Per2, and Per3, reviewed by Albrecht,
2002). Knocking out one or more of these genes
results in animals with a changed or disabled circa-
dian clock in which sleep regulation can be
investigated, and in some cases, obstructing normal
clock functioning also seemed to change sleep
homeostasis.
For instance, Cry1 and Cry2 double-knockout
mice showed increased NREM sleep time and
consolidation, and increased EEG SWA in
NREM sleep (Wisor et al., 2002), which are all
signs of high homeostatic NREM sleep pressure.
The BMAL1 mutation exhibits more sleep and
an attenuation of the homeostatic response to
sleep deprivation (Laposky et al., 2005). Also
the Clock mutation seems to alter sleep homeo-
stasis, since the mutant mice sleep less than
wild-type control animals (Naylor et al., 2000).
All three clock gene mutations induce arrhyth-
mic behavior (Bunger et al., 2000; van der Horst
et al., 1999; Vitaterna et al., 1994), similar to

SCN-lesioned animals. However, the difference is
that SCN lesions in rodents generally (for
exceptions, see Easton et al., 2004) do not change
the time spent in sleep (Ibuka et al., 1980; Mis-
tlberger et al., 1983; Tobler et al., 1983; Trachsel
et al., 1992). NPAS2-deficient mice also show a
reduction in sleep time (Dudley et al., 2003) and
an attenuated response after sleep deprivation
(Franken et al., 2006). Modeling sleep homeostasis
in Cry1,2 double knockout mice showed that the
parameters describing the increase in sleep pres-
sure during waking are altered such that sleep pres-
sure builds up faster compared to wild type (Wisor
et al., 2002). The results indicate that Cry, BMAL1,
NPAS2, and Clock are not only involved in
generating circadian rhythms but may also play
a role in sleep homeostasis, probably in brain
areas outside the SCN. However, these genes are
knocked out in the entire organism from the begin-
ning of development. It is therefore unclear which
part of these results is caused by changes in circa-
dian regulation in the SCN, and which by loss
and/or compensation of gene function in other
regions of the brain. Until studies become available
where the mice underwent SCN lesions or the
clock genes are mutated/knocked out locally in
the SCN, the exact meaning of these results may
not be resolved.
The sleep data obtained in mPer1 and mPer2
mutant mice are better understood and suggest a
role for these genes in the circadian timing of sleep.
Particularly, the mPer2 mutant shows a clear phe-
notype with advanced sleep reflecting the phase
advance of motor activity onset in the mutant
(Kopp et al., 2002; Shiromani et al., 2004), which
corresponds with similar observations in humans,
with a mutation in the Per2 gene in a familial
form of advanced sleep phase syndrome (Toh
et al., 2001).
The correspondence between mPer3 mutant
mice and human polymorphism in the Per3 gene,
associated with delayed sleep phase syndrome,
is less clear. An explanation for this discrepancy
may be that in primates the Per3 gene contains
a number of tandem repeat polymorphisms
211
which are not present in other mammals (Jenkins
et al., 2005).
The homeostatic response after sleep depriva-
tion is intact in most mentioned clock gene
mutants, which again supports the notion that
sleep homeostasis can function independently
from the circadian clock.
Separating circadian and homeostatic processes
Experimental “separation” of circadian- and sleep-
dependent processes can help in understanding the
basic mechanisms in the functioning of these two
major regulating systems. This separation is com-
plicated by the synchronous progression of circa-
dian time and changes in sleep and wakefulness.
In humans, several protocols (ultrashort day,
forced desynchrony) have been established to
uncouple sleep homeostasis from the output of
the circadian pacemaker (Carskadon and Dement,
1975; Dijk and Czeisler, 1995; Lavie and Scherson,
1981). In animals, research in this direction was
done either after SCN lesions (Mistlberger et al.,
1983; Tobler et al., 1983; Trachsel et al., 1992) or
in spontaneously arrhythmic animals (Deboer
and Tobler, 2003; Larkin et al., 2004; Tobler
and Franken, 1993), mainly showing that sleep
homeostasis can function independently from the
circadian clock.
The basic problem is to differentiate daily
changes in observed variables, caused by the
central clock itself (endogenously), from environ-
mental influences of light, temperature (exoge-
nous), and/or masking effect of behavior, such as
sleep, rest, and activity, or food and water intake,
which are only in part or indirect clock driven.
As early as 1938, Nathanial Kleitman per-
formed in a pilot study a forced desynchrony pro-
tocol where the subjects were forced to sleep on a
daily schedule outside of the reach of the endoge-
nous circadian pacemaker. Unfortunately, only
one of the two participants in this experiment
was able to follow the forced schedule of a 21-
or a 28-h day (Kleitman, 1963). Other attempts
212
were made, but it took until the 1990s before the
protocol was applied systematically in humans
(Dijk and Czeisler, 1995). This research brought
many new insights into the interaction between
sleep homeostasis and the circadian clock in
humans. It showed that in humans, waking is
enforced very strongly by the endogenous
clock, particularly at the end of the day (around
10 p.m.), and that also REM sleep has a strong
circadian component, peaking in the early morn-
ing. In rodents, a forced desynchrony protocol with
repeated sleep deprivations and rest according to
a 20 h:20 h schedule was applied only once, but
unfortunately no sleep was recorded (Strijkstra
et al., 1999).
In another type of protocol in rats, a 22-h dim
light–dark cycle is applied which results in a split
of the rest–activity rhythm into two components.
One component follows the 22-h light–dark cycle,
whereas the other component ( 25 h) follows the
endogenous circadian pacemaker (de la Iglesia
et al., 2004). After releasing into DD, the pattern
immediately disappears and only a single circa-
dian period remains. With this protocol, it was
possible to induce a 22-h rhythm in NREM sleep
and waking, but not REM sleep, next to the nor-
mal endogenous 25-h circadian rhythm in sleep
and waking (Cambras et al., 2007). Unfortu-
nately, the authors did not separate sleep homeo-
stasis from the 25-h rhythm induced by the
circadian clock, as was done in the forced
desynchrony experiment in humans (Dijk and
Czeisler, 1995). This limits the interpretation of
the data concerning the relative role of the circa-
dian clock and the sleep homeostat in sleep regu-
lation in the rat. As a result, the clock-driven
circadian component of sleep regulation is proba-
bly overestimated in this protocol.
Several experiments were carried out in
humans using the short “90-min day” protocol
(Carskadon and Dement, 1975, 1980), in which
30 min of rest and 60 min of enforced wakefulness
are alternated for several days. Variations on
this protocol were the “ultrashort sleep–wake
schedule,” with 15 min of waking–5 min sleep
alternations (Lavie and Scherson, 1981), and the
10/20 min ultrashort sleep–wake schedule (Liu
et al., 2000; Tagaya et al., 2002). Also here a
strong waking peak was found around 10 p.m.
and a peak in REM sleep in the early morning.
Particularly, the inability to sleep properly around
10 p.m. made that subjects lost 30% of TST over
24 h compared to normal baseline conditions
(Carskadon and Dement, 1975, 1980).
A similar approach, to specifically separate the
circadian- and sleep-dependent components of
sleep regulation, was performed recently in freely
behaving rats. Here the animals were exposed to
a 2 h/2 h short-sleep deprivation protocol in
constant darkness (Yasenkov and Deboer, 2010,
2011). The protocol aims to stabilize the homeo-
static sleep pressure over the circadian day,
without reducing the opportunity to sleep. In the
case of the rat, this means that the animals should
be able to sleep approximately 50% of 24 h. For
that reason, the sleep deprivation paradigm con-
sisted of 2-h sleep deprivation periods alternated
with 2-h periods of rest (2 h/2 h protocol). This
schedule was maintained for at least 48 h to
ensure an equilibrium can be reached. Another
crucial part of the experimental design was the
constant DD condition which guaranteed that
any physiological changes in the circadian range
observed during the experimental procedures
are due to intrinsic properties of the endogenous
circadian clock, and not caused by the changes
of the light regime.
The protocol was successful in stabilizing
homeostatic sleep pressure as SWA in NREM
sleep was equally distributed across the circadian
day (Yasenkov and Deboer, 2010, 2011). Despite
constant DD conditions and constant sleep pres-
sure, the animals still showed a circadian rhythm
in NREM sleep, but with a lower amplitude,
whereas REM sleep was equally distributed
across the circadian day (Fig. 2, top panels). The
remaining circadian rhythm in NREM sleep was
caused by a significant modulation of NREM sleep
episode duration (Fig. 2). NREM sleep episode fre-
quency, and REM sleep episode frequency and
 213

NREM sleep REM sleep

100 20

75 15
Amount
of sleep
(%)
50 10

25 5

0 0
8
of sleep eposides

6
(episodes/h)

7
Frequency

6 4

5 2
4
0
9
of sleep episodes

8
7 1.5
Duration

(min)

6
1.0
5
4 0.5
3
2 0
0 12 24/0 12 24/0 0 12 24/0 12 24/0
Circadian time (h)

2 h/2 h Baseline

Fig. 2. Circadian profiles of NREM and REM sleep (amount, frequency, and duration) in baseline and during constant sleep
pressure in the rat. Top panels—amount of NREM sleep and REM sleep plotted as percentage of recording time. Middle
panels—sleep episode frequencies (events per hour). Bottom panels—sleep episode duration (min) for corresponding vigilance
states. All data computed for the undisturbed 2-h periods of the 2 h/2 h protocol and corresponding baseline intervals. Each data
point represents 2-h mean values (n ¼ 8) for 2 h/2 h protocol (dots) and corresponding baseline intervals (circles). Data are
double plotted to visualize the circadian modulation. Top horizontal bars indicate the periods of subjective rest (gray) and
activity (black). *p < 0.05 and **p < 0.01: two-tailed paired t-test after significant ANOVA or rANOVA, factor “day.” Data
adapted from Yasenkov and Deboer (2010).

duration did not show a circadian rhythm during
the 2 h/2 h protocol. These findings are of interest
because this is much harder, if not impossible, to
see such changes in species in which sleep episodes
are much longer, like humans.
During the 2 h/2 h protocol, the animals lost
only 7% of their TST over 24 h, which was caused
by a decrease in NREM sleep, as the amount of
REM sleep did not change (Yasenkov and
Deboer, 2010). This is in strong contrast with
humans, where under similar conditions sleep
dropped to less than 5 h per 24 h (Carskadon
and Dement, 1975; Dantz et al., 1994). This differ-
ence between humans and rodents is probably
caused by the polyphasic nature of sleep in
rodents. From these experiments, it can be con-
cluded that, in contrast to humans, the daily mod-
ulation of REM sleep in rats is less pronounced
and possibly more sensitive to changes in sleep
homeostatic pressure. In contrast, the amount of
214
NREM sleep and waking still shows a clear influ-
ence of the circadian clock in the rat. In addition,
the experimental conditions resulted in a clear
separation of circadian- and sleep-dependent
components between the baseline day and the
2 h/2 h protocol, and explicit sleep- and circadian-
dependent EEG frequencies in both NREM and
REM sleep were identified (Yasenkov and
Deboer, 2011). The data indicate that in the rat
the circadian changes in homeostatic sleep pres-
sure, which accompany the circadian changes in
waking, strongly amplify the circadian pattern in
NREM and REM sleep. Therefore, despite the
larger flexibility in sleep timing in rodents, still a
clear circadian modulation of sleep can be
observed when the two are in phase under undis-
turbed conditions.
Future perspectives
There are still several open questions related to
sleep regulation. The EEG SWA data obtained
during NREM sleep suggest the presence of some
kind of homeostatic process. However there is no
clear evidence where the homeostat is located in
the brain. This question has become increasingly
pressing after the discovery of cortical differences
in local sleep homeostasis, where time constants
of homeostatic increase and decrease of EEG
SWA (and therefore of Process S) differed
between cortical areas (Huber et al., 2000b; Werth
et al., 1996a). Also the discovery of a cortical sleep
homeostat in rats (Vyazovskiy et al., 2009) or the
finding that learned tasks influence the level of
EEG SWA in subsequent NREM sleep in the cor-
tical area related to this task (Huber et al., 2004;
Vyazovskiy et al., 2004) adds to the complexity. It
is also unknown whether there is a neuroanatomi-
cal substrate for the interaction between sleep
homeostasis and the circadian clock. It seems that
the interaction may take place in the SCN, as
changes in sleep pressure influence SCN neuronal
firing (Deboer et al., 2003, 2007a) and can influ-
ence circadian phase resetting (Antle and
Mistlberger, 2000), but it can also be that changes
in sleep homeostatic pressure merely change the
output of the SCN.
Protocols and techniques
An application of new protocols and techniques
may elucidate some of the questions. For the
understanding of the interaction between the cir-
cadian and sleep homeostatic component in
rodents, the 2 h/2 h protocol may be an attractive
tool. We have shown that in the protocol, any
changes obtained in the course of the circadian
day are clearly not induced by the sleep
homeostat, but probably by the circadian clock.
The protocol can be applied on several different
experimental levels from gene expression to
behavior and cognition.
However, the constant sleep pressure in the
protocol can also be a disadvantage. A true
forced desynchrony protocol in a rodent, where
sleep homeostatic changes occur out of phase
with the circadian clock, will probably elucidate
more complex interactions. These type of exper-
iments are time consuming, as they are in
humans, and difficult to perform in rodents, as
rodents are not as good as humans in following
instructions from the experimenter.
Modeling
The modeling of sleep homeostasis has seen many
new developments since its implementation in the
two process model of sleep regulation, and the
time course of SWA in the course of a sleep epi-
sode can be predicted very precise (Achermann
et al., 1993). Unfortunately, simulation of sleep
homeostasis in rodents did not reach this level
of detail. In both humans and rodents, the circa-
dian process is not incorporated routinely in
present-day modeling. There are, however, some
indications that the circadian clock may influence
sleep homeostatic time constants and that an

effort should be made to incorporate circadian
time as a variable in the modeling of sleep
homeostasis. For instance, when modeling sleep
homeostasis in the rat, time-of-day-dependent
deviations are observed in the fit between model
and data, even under constant dark conditions
(Deboer, 2009; Franken et al., 1991). In addition,
it was also shown that, in animals lacking the Cry1
and Cry2 genes, the buildup rate of the homeo-
static process was significantly shortened (Wisor
et al., 2002). Discrepancies were observed
between the data and the simulation when the
homeostatic sleep process was simulated for sleep
scheduled outside the normal sleep phase, like an
afternoon nap (Werth et al., 1996b), sleep in the
early evening, or extended sleep (Achermann
et al., 1993). From the data presented in this
review, it is clear that the circadian clock does
probably more than mark the onset and end of
the rest and activity phase, and this should be
implemented into the models. In addition, more
data of different species (and not only rodents)
are needed to be able to eventually come up with
a model that can be applied to general mamma-
lian sleep.
Acknowledgment
This research was supported by the Netherlands
Organization for Scientific Research (NWO,
Grant 818.02.016 to T. D.).
Abbreviations
2 h/2 h the repeated short-sleep depriva-
tion protocol
CT circadian time
DD constant darkness
EEG electroencephalogram
EMG electromyogram
LD light/dark cycle
NREM non-rapid eye movement sleep
sleep
215
REM rapid eye movement sleep
sleep
SCN suprachiasmatic nucleus
SWA slow-wave activity
TST total sleep time
TSTD total sleep time in the dark
TSTL total sleep time in the light
Glossary of sleep-related terms
Electroencephalogram Electrical activity in the
brain recorded from scalp electrodes (in
humans) or epidural electrodes (in animals).
Non-rapid eye movement sleep One of the two
basic sleep states (see also rapid eye movement
sleep) characterized in animals by slow waves
(< 5 Hz) in the electroencephalogram and low
muscle activity (see also slow waves).
Rapid eye movement sleep One of the two basic
sleep states (see also non-rapid eye movement
sleep) characterized by rapid eye movements,
muscle atonia, muscle twitches, and low-amplitude
fast-frequency electroencephalogram.
Sleep intensity Depth of sleep.
Sleep homeostasis A balancing process that
maintains the amount and intensity of sleep
within certain boundaries. Slow waves in undis-
turbed non-rapid eye movement sleep are
thought to be a reflection of the level of the
sleep homeostatic process.
Slow waves Oscillations in the sleep electroen-
cephalogram below 5 Hz which are a character-
istic of non-rapid eye movement sleep.
Slow-wave activity spectral power in the slow-
wave frequency range of the electroencephalo-
gram, which is a measure of non-rapid eye
movement sleep intensity.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 13

Local aspects of sleep: Observations from

intracerebral recordings in humans

Lino Nobili{,#,*, Luigi De Gennaro{, Paola Proserpio{, Fabio Moroni{,},

Simone Sarasso||, Andrea Pigorini||, Fabrizio De Carli# and Michele Ferrara**

{
Centre of Epilepsy Surgery “C. Munari,” Center of Sleep Medicine, Niguarda Hospital, Milan, Italy
{
Department of Psychology, University of Rome “Sapienza,” Roma, Italy
}
Department of Psychology, University of Bologna, Bologna, Italy
||
Department of Clinical Sciences L. Sacco Università degli Studi di Milano, Milan, Italy
#
Institute of Bioimaging and Molecular Physiology, Genoa Unit, National Research Council, Genoa, Italy
** Department of Health Sciences, University of L’Aquila, L’Aquila, Italy

Abstract: Human sleep is considered a global phenomenon, orchestrated by central specialized neuronal
networks modulating the whole-brain activity. However, recent studies point to a local regulation of sleep.
Sleep disorders, such as sleepwalking, suggest that electroencephalographic (EEG) features of sleep and
wakefulness might be simultaneously present in different cerebral regions. Recently, intracranial EEG
recording techniques, mainly applied for the presurgical evaluation of drug-resistant epileptic patients,
have provided new and interesting information on the activity of different cortical and subcortical
structures during sleep in humans. In particular, it has been observed that the thalamus, during the
transition between wake and sleep undergoes a deactivation process that precedes the one occurring
within the cortex, with extensive cortical territories maintaining an activated pattern for several minutes
after the thalamic deactivation. Very recent intracerebral EEG studies have also shown that human
NREM sleep can be characterized by the coexistence of wake-like and sleep-like EEG patterns in
different cortical areas. Moreover, unit-firing recordings in multiple brain regions of neurosurgical
patients evidenced that most sleep slow waves and the underlying active and inactive neuronal states do
occur locally. These findings add a new dimension to the concept of local sleep regulation and opens new
perspectives in the interpretation of the substrates underlying behavioral states of vigilance. The
implications for sleep medicine are also discussed.

Keywords: local sleep; arousal; cortical activations; hippocampus; parasomnias; dissociated state; sleep
onset.

*Corresponding author.
Tel.: þ 39-0264442917-2918; Fax: þ 39-0264442868
E-mail: lino.nobili@ospedaleniguarda.it

http://dx.doi.org/10.1016/B978-0-444-59427-3.00013-7 219
220
Introduction
Human sleep is traditionally considered a global
phenomenon orchestrated by central specialized
neuronal networks modulating whole-brain activ-
ity. Specifically, the reduced reticular formation
tone is interpreted as the main determinant of
the electroencephalographic (EEG) synchroniza-
tion characterizing the sleep state (Fuller et al.,
2011; Moruzzi and Magoun, 1949).
However, it has been recently proposed that
sleep is local in nature, being a fundamental prop-
erty of small neuronal groups (Krueger and Obál,
1993; Krueger et al., 2008). According to this view,
global sleep (behaviorally and electroencephalo-
graphically defined) emerges when the altered
input–output state, that characterizes the sleep-
like state at the local network level, involves a
large and widespread number of cortical regions
(Krueger et al., 2008).
Moreover, experimental studies have shown
that sleep and wakefulness can be restricted to
small groups of neurons (Pigarev et al., 1997), to
individual cortical columns, (Rector et al., 2005)
or to larger brain regions. For instance, some birds
and marine mammals can simultaneously exhibit
sleep in one cerebral hemisphere and wakefulness
in the other one in order to continue flying or swim-
ming (Lyamin et al., 2008; Mukhametov, 1984).
To a broader extent, clinical evidence deriving
from patients affected by sleep disorders, such as
sleepwalking, suggests that sleep and wakefulness
might be simultaneously present in different
cerebral regions indicating that the boundaries
between these different behavioral states are not
strictly defined (Mahowald and Schenck, 2005).
According to these observations, brain-sleep
state may be spatially non-uniform, and along
the same line, sleep and wakefulness may not be
temporally discrete behavioral states. As an exam-
ple, scalp EEG studies have shown that large
regional frequency-specific EEG differences char-
acterize NREM sleep, with a frontal predominance
of delta activity (1–4.5 Hz), the main index of sleep
intensity (Ferrara et al., 2002; Finelli et al., 2001;
Werth et al., 1996). Moreover, it has been demon-
strated that sleep EEG delta power can be locally
increased or reduced as a function of previous
waking activity (Cantero et al., 2002; Huber et al.,
2004, 2006).
Recently, the spread of intracranial EEG record-
ing techniques, mainly exploited for the presurgical
evaluation of drug-resistant epileptic patients,
allowed to overcome the intrinsic spatial limitations
of scalp EEG techniques, providing detailed infor-
mation on the activity of different cortical and sub-
cortical structures, both during wake and sleep
(Moroni et al., 2008, 2012).
The aim of the present work is to review the avail-
able published data regarding local aspects of sleep
supported by novel observations from our and
other research groups, obtained using scalp high
density and intracranial EEG techniques. Finally,
capitalizing on these observations, we will propose
novel insights for sleep medicine.
From wake to sleep—A behavioral transition
through local brain changes
A complex and widely distributed neural system,
with functionally distinct but integrated
components, orchestrates sleep (Rosenwasser,
2009). Schematically, there are three main sleep
regulatory processes: (1) the circadian process
(Process C) that couples the timing for sleep and
wakefulness with the light–dark cycle; (2) the
homeostatic process (Process S), modulating sleep
intensity and strongly dependent on wakefulness
duration and activity; and (3) the ultradian process,
which regulates the intra-sleep NREM–REM
alternation (Borbély, 1982; Borbély and
Achermann, 1999; Ferrillo et al., 2007). In normal
conditions, the interaction of these processes
allows an unambiguous separation between the
state of wakefulness and sleep, from both a behav-
ioral and a neurophysiological perspective. Indeed,
during the aroused state, when we are fully respon-
sive and alert, cortical and thalamocortical neurons
display fast and complex patterns of activation;

when we fall asleep and become progressively less
responsive, this activated state is in turn replaced
by pervasive and synchronized low-frequency
oscillations of neural activity (Steriade et al.,
1993). This phenomenon is reflected in the interrup-
tion of fast rhythms (alpha and beta activity) and the
appearance of slow waves on the EEG. These dra-
matic changes in behavioral and electrophysiologi-
cal states, which in humans may take from few
seconds to several minutes, require specific
switching mechanisms in the brain, enabling rela-
tively rapid and complete state transitions (Fuller
et al., 2006).
However, recent experimental evidence in
animals and humans demonstrated that this transi-
tion mechanism might work with different timing
for different brain areas, partially redefining the
classical distinction between wake and sleep as
separate, incompatible states.
Sleeping locally during wakefulness
Under extreme conditions, as is the case of pro-
longed wakefulness, brain activity undergoes
dramatic changes. Classically, the EEG traces
gradually show a progressive increase in the
amount of slow signal components (delta–theta
activity) as a function of time spent awake,
(Cajochen et al., 2002; De Gennaro et al., 2007;
Finelli et al., 2000; Franken et al., 1991; Strijkstra
et al., 2003) reflective of increased homeostatic
sleep pressure (Process S). Experimental studies
suggest that the progressive slowing of waking
EEG may be associated with the appearance of
local sleep states during wakefulness. Indeed, it
has been shown that in sleep-deprived monkeys
performing a visual discrimination task, some
neurons in the extrastriate visual cortex display
the characteristic sleep firing pattern (bursting
mode), while neurons in the striate cortex show
the waking pattern of activation (tonic firing)
(Pigarev et al., 1997). These episodes of “local
sleep” were much more frequent in the animal that
apparently experienced stronger sleep pressure
221
during recording sessions. These results indicated
that, taking into account the neuronal firing pat-
tern, sleep onset (SO) may occur in a strictly local
manner; the animal can be behaviorally awake,
but local cortical “islands of sleep” can appear.
In a very recent experimental study, Vyazovskiy
et al. (2011) focused their analysis on the presence
of slow EEG activity during wakefulness. Slow
EEG rhythms (< 4 Hz) represent a typical feature
of NREM sleep reflected, at the cellular level, by
a bistable behavior of cortical neurons, alternating
between two distinct states: one characterized by
depolarized membrane potential with burst of
action potentials (upstate) and the other associated
with hyperpolarized membrane potential and neu-
ronal silence (downstate) (Contreras and Steriade,
1995; Crunelli and Hughes, 2010). Exploiting
multiunit activity recordings, Vyazovskiy et al.
(2011) showed that, in sleep-deprived but awake
rats, relatively small populations of neurons in
different cortical areas may suddenly manifest
“downstates”—resembling those typical of NREM
sleep—associated with low-frequency ( 2 Hz)
EEG activity and deficit in performance at a pellet
reaching task. Interestingly, these events occurred
in a strictly local fashion, involving only a small
group of neurons at a time. In other words, subsets
of neurons may enter an “off” period in one corti-
cal area but not in another and, even within
the same cortical area, some neurons may be
“off” while others remain “on.” Finally, such local
“off periods” increased with the duration of wake-
fulness, suggesting a relationship with a local
increase of sleep pressure.
The authors interpret these findings as possibly
representing a form of neuronal tiredness due to
use-dependent factors, such as synaptic overload
(Tononi and Cirelli, 2006). This view assumes
that local, use (and possibly learning)-dependent
increase of synaptic strength is followed by a
corresponding local increase in subsequent sleep
pressure, reflected in increased EEG slow-wave
activity (SWA). Along the same line, after
showing that a homeostatic regulation of SWA
is present also in subcortical structures such as
222
the hippocampus (Moroni et al., 2007), we
recently performed intracerebral EEG recordings
of the hippocampal formation investigating the
effects of both procedural and declarative
learning on the qualitative and quantitative
measures of hippocampal sleep EEG (Moroni
et al., 2008). We reasoned that, if the hippocampus
is directly involved in the memory consolidation
process, in agreement with the local use-dependent
theory, significant changes in the hippocampal
EEG slow rhythms should be expected. We indeed
showed that an intensive training on a sequential
finger-tapping task is followed by an increase in
the amount of scalp recorded SWS, as well as
in the hippocampal EEG power in the very low-
frequency range (0.5–1.0 Hz) during the first
postlearning NREM period. The magnitude of
procedural performance improvement was signifi-
cantly correlated with the subsequent EEG low-
frequency power increase. This supports the
existence of a direct link between procedural
memory consolidation and very low hippocampal
EEG rhythms. To our best knowledge, these are
the first reported subcortical (local) effects of
learning on sleep EEG recorded from both scalp
and intracerebral derivations.
Falling asleep locally
So far, very few studies in humans have analyzed
the EEG changes during the wake–sleep transi-
tion, and most of these were based on scalp
recordings. They clearly differentiate topographi-
cal EEG modifications showing, after SO, a
fronto-central predominance of delta/theta activ-
ity, a centro-parietal maxima of the sigma activity,
and an anteriorization of the alpha activity (De
Gennaro et al., 2001; Werth et al., 1996). The
whole pattern indicates that the anterior areas
are the first to show a synchronized EEG activity,
while the occipital areas are the last ones (De
Gennaro et al., 2001). Moreover, SO seems to be
characterized by a posterior-to-anterior functional
cortical coupling replacing the anterior-to-
posterior one observed during the presleep phase
(De Gennaro et al., 2004, 2005). The local nature
of EEG and corticographic changes during the
wake-sleep transition and the existence of an
antero-posterior gradient are coherent with the
spatiotemporal dynamics of the < 1 Hz slow oscil-
lation during full-fledged NREM sleep. This
EEG activity originates more frequently at the
anterior cortical regions and propagates in an
antero-posterior direction, with a highly reproduc-
ible origin, speed, and direction (Massimini et al.,
2004).
Electrocorticographic recordings of 13 epileptic
patients undergoing presurgical assessment (Bódizs
et al., 2005) have confirmed these scalp observations.
During the wake–sleep transition, cortical delta
activity showed an antero-posterior gradient with
the frontal power peaking earlier than the temporal
and the temporal earlier than the occipital. The
time course of parahippocampal changes in delta
activity during the transition paralleled those of the
frontal cortex (Hangya et al., 2011).
Although an asynchronous SO between differ-
ent cortical areas has been repeatedly demonstra-
ted; only very recently it has been shown in
humans that the thalamus and the cortical mantle
are not strictly coupled during the wake–sleep
transition, as firmly believed in the past (Magnin
et al., 2010). Indeed, Magnin et al. (2010), using
intracerebral recordings in drug-resistant epileptic
patients, showed that the thalamus, during physi-
ological SO, undergoes a deactivation process
that precedes the one occurring within the cortex.
In particular, the authors observed that extensive
cortical territories maintained an activated pat-
tern for several minutes after the thalamic deacti-
vation. Moreover, the study confirmed that the
cortical deactivation showed marked topographi-
cal differences at SO both within and between
patients.
Based on these findings, in order to explore the
possibility of transient decoupling between limbic
and neocortical structures during wake–sleep
transition, we investigated simultaneous intrace-
rebral hippocampal and neocortical recordings in

five patients with refractory epilepsy. Our prelim-
inary results (Sarasso et al., in preparation) show
that several minutes before SO (detected on scalp
EEG by the emergence of the first spindle or
K-complex), spindles appeared in the hippocam-
pus with the typical 4 s periodicity. Moreover, in
one of our patients, we had the possibility to analyze
also the thalamic EEG activity. We observed that
during wakefulness the thalamus (in particular, the
anterior thalamic nucleus) is characterized by a fast
rhythm, with a peak around 25 Hz (Fig. 1, upper
panel). Few minutes before SO, periodic slow
oscillations in the low-delta range (< 2 Hz) appeared
in the thalamic EEG recording, almost simulta-
neously with the emergence of hippocampal spindle
activity (Fig. 1, middle panel). Quantitative EEG
analysis by fast fourier transform confirmed the
observations derived from visual analysis. Figure 2
shows the time course of normalized absolute values
of spectral power in the slow-delta (0.5–2.0 Hz)
and sigma (12.0 –16.0 Hz) frequency range. Several
minutes before SO, a progressive raise in the
thalamic delta power is evident; when such an
increase becomes steeper, also the hippocampal
delta power progressively increases. Interestingly,
the appearance of spindles precedes the delta power
raise in the hippocampal EEG recording.
Magnin et al. (2010) hypothesized that the
delay between thalamic and cortical deactivation
at SO could reflect a thalamus-driven process or
a higher sensitivity of the thalamus to firing
patterns of brainstem and hypothalamic afferents.
This hypothesis could explain also our findings
regarding a limbic structure, such as the hippo-
campus. In the future, it would be interesting to
evaluate whether these features are common to
other archi-cortical regions. This would suggest
that the phylogenetic development of a cortical
structure influences its sensitivity to the brainstem
and hypothalamic SO switch (Fuller et al., 2006).
On the other hand, we cannot exclude that the
early hippocampal SO reflects an increased pres-
sure of the homeostatic process in this structure.
According to this alternative interpretation, one
should find a higher relative low-frequency
223
activity as a function of time spent awake in the
hippocampus compared to other neocortical
areas.
Local cortical awakenings within the
sleeping brain
Since local “islands of sleep” can appear during
wakefulness, could also “local wakefulness” appear
during sleep?
In order to probe the coexistence of dissociated
(wake-like and sleep-like) electrophysiological
behaviors within the sleeping brain, we recently
analyzed intracerebral EEG activity drawn
from sleep recordings of five patients with
pharmacoresistant focal epilepsy without sleep
disturbances, who underwent presurgical intrace-
rebral EEG investigation (Nobili et al., 2011).
We observed that sleep can be characterized by
the coexistence of wake-like and sleep-like EEG
patterns in different cortical areas, as indicated
by a high number of local activations (abrupt
increases in EEG frequency, including alpha
and/or beta rhythm) within the motor cortex
(Mc), occurring synchronously with deep-sleep
EEG patterns in the dorsolateral prefrontal cor-
tex (dlPFc) and scalp (Nobili et al., 2011). In par-
ticular, we found that the physiological and
progressive decay of SWA across NREM sleep
cycles was comparable between the derivations
(dlPFc, Mc, scalp) investigated. However, in spite
of this similarity, the Mc showed a higher rate of
local activations during NREM sleep compared
to the dlPFc and scalp. Moreover, the local
activations in the Mc increased across sleep cycles
and within each cycle toward the end of each
NREM period, being possibly associated, respec-
tively, with the decrease of SWA and the
approach of REM sleep. Interestingly, recent
observations derived from an intracerebral study
conducted in epileptic patients showed that the
spatial dynamics of cortical slow oscillations,
the hallmark of full-fledge NREM sleep, change
throughout the night, involving the entire cortical
 Th

Hc

P cx

Scalp

EOG

Chin

EKG

Th

Hc

P cx

Scalp

EOG

Chin

EKG

Th

Hc

P cx

Scalp

EOG

Chin

EKG

1s

Fig. 1. Three 30-s epochs of intracerebral and scalp EEG recordings. Upper panel: patient behaviorally awake; all the EEG traces
show a typical waking pattern of activity. Middle panel: patient behaviorally awake, 10 min before sleep onset; appearance of
periodic slow oscillations in the low-delta range (<2 Hz) within the thalamic EEG trace and of sleep spindles in the
hippocampus. The parietal cortex and scalp EEG maintain a waking pattern of activity. Lower panel: stage 2 of NREM sleep; all
the EEG traces show a NREM sleep pattern of activity. Abbreviations: Th, thalamus; Hc, hippocampus; P cx, parietal cortex;
Scalp, scalp EEG (Fz-Cz); EOG, electroculogram; Chin, submental electromyogram; EKG, electrocardiogram.
 225

250
Scalp EEG Thalamus Hippocampus Parietal cortex

12.0–16.0 Hz

Spectral power (normalized values)

200

150

100

50

0
200

180
0.5–2.0 Hz
Spectral power (normalized values)

160

140

120

100

80

60

40

20 N2
N1
W
0
0 5 10 15 20 25 30
Time (min)

Fig. 2. Time course of normalized absolute values of spectral power in the sigma (12.0–16.0 Hz, upper panel) and slow-delta range
(0.5–2.0 Hz, lower panel), in a patient with both intracerebral and scalp EEG derivations. Several minutes before the sleep onset (as
indicated by the start of N2 sleep), a progressive raise in the thalamic delta power is evident; when such an increase becomes
steeper, also the hippocampal delta power progressively increases. Abbreviations: W, wakefulness; N1, stage 1 of NREM sleep;
N2, stage 2 of NREM sleep.

mantle in early sleep and only limited cortical sometimes last up to 120 s and more than 50% of
areas toward the end of sleep (Nir et al., 2011). them were not accompanied by EMG activation.
In general, the Mc local activations (LAs) had an They could occur as isolated or cyclical events.
about  10 s duration (Fig. 3), but they could When LAs occur in a cyclical fashion, they strongly
226

(a) 250 mV
Mc
250 mV
dIPFc
100 mV
Fz-Cz

100 mV

Eog
100 mV

Chin

(b)
250 mV (c)
Mc
250 mV

dIPFc
100 mV

Fz-Cz
100 mV

Eog
100 mV

Chin

10 s

Fig. 3. Sample patterns of intracerebral and scalp EEG recordings. (a) NREM sleep: slow waves prevail in all the three EEG traces;
(b) and (c): NREM sleep: local activations appearing in the motor cortex accompanied by bursts of slow waves in the other two
EEG derivations. Abbreviations: Mc, motor cortex; dlPFc, dorsolateral prefrontal cortex; Fz-Cz, bipolar scalp EEG; EOG,
electroculogram; Chin, submental electromyogram.

resemble the scalp EEG pattern corresponding to
the definition of the cyclic alternating pattern
(CAP; Parrino et al., 2012; Fig. 4).
Around most LAs, the time–frequency analysis
of the EEG showed an increase of high-frequency
EEG activity (mainly in the 8–12 Hz frequency
range) in the Mc, paralleled by an increase of low-
frequency EEG activities (peaking at 0.5–2 Hz) in
the dlPFc and scalp sites (Fig. 5).
In the transition from sleep to the aroused
state, the ascending activating systems abolish
the slow rhythmic burst firing mode in thalamo-
cortical circuits and induce the appearance of a
high-frequency tonic firing pattern (Moruzzi and
Magoun, 1949; Steriade et al., 1993). This allows
the brain to recover full responsiveness enhancing
the sensory-motor processing (McCormick and
Feeser, 1990). Our data suggest that this state-
switching can be confined to restricted thalamo-
cortical pathways.
On the basis of our results, we hypothesized
that a lower arousal threshold and a higher level
of activation in the Mc during NREM sleep may
have been evolutionary selected, because they
increase the probability of survival by facilitating
motor behaviors in case of sudden awakenings
(Nobili et al., 2011). Accordingly, the significant
enhancement of slow frequencies in the dlPFc
immediately before and during the Mc LAs
could be interpreted as a mechanism preventing
a full awakening and allowing sleep as a global
process to continue uninterrupted, even when a
 227

GC
dlPfcx
sF gyrus
mF gyrus
SMA
Mcx
Mcx
Pcx
Supr. gyrus
PC
PC
Intr. P sulcus
Is GC
Sup P lob
Precuneus
Scalp EEG
EOG
Chin
EKG

GC
dlPfcx
sF gyrus
mF gyrus
SMA
Mcx
Mcx
Pcx
Supr. gyrus
PC
PC
Intr. P sulcus
Is GC
Sup P lob
Precuneus
Scalp EEG
EOG
Chin
EKG

5s

Fig. 4. Sample patterns of intracerebral and scalp EEG recordings. Cyclical appearance of local activations in the supplementary
motor area, motor cortex, and parietal cortex. Abbreviations: GC, gyrus cinguli; dlPfcx, dorsolateral prefrontal cortex; sF gyrus,
superior frontal gyrus; mF gyrus, middle frontal gyrus; SMA, supplementary motor area; Mcx, motor cortex; Pcx, parietal cortex;
Supr. gyrus, supramarginal gyrus; PC, parietal cingulus; Intr. P sulcus, intraparietal sulcus; Is GC, isthmus of cingulate gyrus; Sup
P lob, superior parietal lobe; EOG, electroculogram; Chin, submental elecromyogram; EKG, electrocardiogram.

local activation appears. Our data are in accor-
dance with results obtained from PET studies,
which showed that the cortical deactivation
during NREM sleep is not a homogeneous phe-
nomenon, with the associative cortices of the fron-
tal and parietal lobes being the most deactivated
areas, while the primary cortices are the least
deactivated (Braun et al., 1997; Dang-Vu et al.,
2005). Finally, our findings are also coherent
with the observation of similar cortical excitability
of human primary Mc, as assessed by transcranial
magnetic stimulation, in wake and NREM sleep
(De Gennaro et al., 2007).
Local aspects of sleep—Implications for sleep
medicine
Considering sleep not only as a global phenome-
non but also as a locally regulated process might
provide novel understanding also in the field of
sleep medicine.
It is well known that sleep deprivation can
affect various aspects of performance (Bocca and
Denise, 2006) reducing attention and vigilance,
decision-making ability and memory functions
(Curcio et al., 2006; Harrison and Horne, 2000;
Killgore et al., 2006).
228

16–32
1
8–12
Hz

2–4 0.5
0.5–1
0

16–32
1
8–12
Hz

2–4 0.5
0.5–1
0

16–32
1
8–12
Hz

2–4 0.5
0.5–1
0
-20 -16 -12 -8 -4 0 4 8 12 16 20
sec

Fig. 5. Time–frequency distribution of EEG patterns recorded in Motor cortex (upper part), dorsolateral prefrontal cortex (middle
part), and scalp (bottom part) in association with motor cortex local activations (LAs) in a single subject. A 40-s time window was
set around the start (zero time) of each Mc LA and signal power was calculated by wavelet transform for each time unit within a
time–frequency grid (0.125 s time resolution and frequency-dependent bandwidth). Time–frequency distribution was averaged
within each recording. The distribution of averaged signal amplitude (square root of the power) is presented for each brain
region by a color scale. The abrupt shift toward higher frequencies (8–12 Hz; 16–32 Hz) in the motor cortex is accompanied by
an increase of EEG activity in the low frequencies (0.5–2.0 Hz) in the other regions, in association with the onset of the Mc local
activation.

We might hypothesize that cognitive impairment
and performance deficits induced by sleep depriva-
tion could be due to the occurrence of cortical and
subcortical local “islands of sleep” in behaviorally
fully awake subjects. In the future, the application
of high-density EEG in sleep-deprived subjects
may allow the detection of subtle signs of “neuronal
tiredness” expressed by local slow waves. Consider-
ing the fundamental involvement of the hippocam-
pus in memory processing, the observation that this
subcortical structure displays typical sleep features
(i.e., spindles) many minutes before the occurrence
of sleep on scalp EEG might explain specific mem-
ory alteration or amnesia for events preceding the
unequivocal occurrence of behavioral SO (Wyatt
et al., 1994, 1997).
The different temporal dynamics of state-
synchronization in different cortical areas (as
observed in our data and by Magnin et al., 2010)
could explain the frequent dissociations between
EEG and behavioral or subjective measures of
SO (Ogilvie and Wilkinson, 1988). Indeed, since
sleep does not simultaneously begin in all cortical
and subcortical areas, it is not surprising that the

subjective definition of SO does not fit with scalp
EEG electrophysiological observations. In the
near future, coregistering patients with intracere-
bral electrodes and high-density EEG, together
with the assessment of behavioral and vegetative
variables (EKG, heart rate variability, respiration,
etc.), could allow extracting noninvasive parameters
able to sensibly detect early signs of sleepiness even
in confined regions. This could be extremely useful
for the prevention of accidents related to excessive
sleepiness in the general population.
In the clinical practice, an impairment in the
process of state-synchronization among different
cortical regions, with cortical territories that
remain activated for long time after the thalamic
and hippocampus deactivation, could explain
both long sleep latency in insomniac patients and
the mismatch between subjective and objective
measures frequently observed in subjects with par-
adoxical insomnia (Manconi et al., 2010; Marzano
et al., 2008; Parrino et al., 2009). In particular, in
these patients, it has been observed that the per-
ception of a nonrestorative sleep can be correlated
with a disproportionate increase of sleep instabil-
ity, expressed by a high CAP rate also in the pres-
ence of normal sleep duration compared to healthy
controls (Parrino et al., 2012; Stone et al., 2008;
Terzano et al., 2003). We suggest that an increase
in sleep instability, expressed by a disproportional
number of K-complexes and delta bursts, could
be related to the presence of unrecognized local
cortical activations characterized by higher EEG
frequencies (Fig. 4). A high number of local corti-
cal activations could alter the normal evolution of
sleep playing an important role in the perception
of a nonrestorative sleep or in the genesis of mis-
perception insomnia.
The appearance of localized simultaneous sleep-
like and wake-like activity during NREM sleep
may also explain paradoxical sleep phenomena
such as sleepwalking and confusional arousal,
which have been attributed to a breakdown of
the boundaries between wakefulness and NREM
sleep (Mahowald and Schenck, 2005). Indeed, dur-
ing these phenomena the subjects lack of conscious
229
awareness though they can show automatic or
more organized motor behaviors. During these
events, scalp EEG generally shows high-amplitude
slow waves, mainly expressed over the frontal
regions, together with alpha and beta activity
(Schenck et al., 1989; Zadra et al., 2004). Con-
firming our prediction, we recently captured a
confusional arousal in a subject affected by drug-
resistant epilepsy who was undergoing an intrace-
rebral EEG investigation during presurgical
assessment. Intracerebral EEG showed the pres-
ence of local activations of the motor and cingulate
cortices associated with increased delta activity in
the frontoparietal associative cortices (Terzaghi
et al., 2009).
Currently, we have demonstrated that the
appearance of local dissociated states is an intrin-
sic feature of NREM sleep (Nobili et al., 2011),
supporting the hypothesis that paradoxical phe-
nomena could be the result of a coexistence of
different local states: an aroused state (very simi-
lar to wakefulness) and sleep. Genetic factors or
external triggers such as sleep deprivation (Zadra
et al., 2008), modifying the arousal threshold, may
favor the persistence of local dissociated activity
allowing the appearance of motor phenomena as
those observed in NREM parasomnias.
Finally, if the aroused state can appear locally
during sleep, we cannot exclude that such a dis-
sociation may also occur during awakenings (in
this case with cortical areas showing local sleep
features). This could explain other phenomena
such as sleep inertia (Ferrara et al., 2006;
Marzano et al., 2011), the subjective feeling of
grogginess accompanied by decreased levels of
cognitive and behavioral performance which typ-
ically follows the awakening.
In conclusion, intracerebral EEG recordings
offer a unique opportunity to investigate simulta-
neously several cortical and subcortical structures
during the entire vigilance spectrum, going from
active wakefulness to deep sleep. Due to the inva-
sive nature of the technique, it cannot be applied
to study sleep physiology or sleep disorders in the
general population.
230
In the future, by coupling intracerebral and
scalp hd-EEG recordings, we can obtain novel
and more precise interpretation of the nature
of scalp recorded events, therefore overcoming
the intrinsic spatial limitation of surface EEG
recordings.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 14

The circadian clock component PERIOD2: From

molecular to cerebral functions

Jürgen A. Ripperger* and Urs Albrecht

Department of Biology, Unit of Biochemistry, University of Fribourg, Fribourg, Switzerland

Abstract: The circadian clock is based on a molecular oscillator, which simulates the external day within
nearly all of a body’s cells. This “internalized” day then defines activity and rest phases for the cells and the
organism by generating precise rhythms in the metabolism, physiology, and behavior. In its perfect state,
this timing system allows for the synchronization of an organism to its environment and this may optimize
energy handling and responses to daily recurring challenges. However, nowadays, we believe that
desynchronization of an organism due to its lifestyle or problems with its circadian clock not only causes
discomfort but also may aggravate conditions such as depression, metabolic syndrome, addiction, or cancer.
In this review, we focus on one simple cogwheel of the mammalian circadian clock, the PERIOD2
(PER2) protein. Originally identified as an integral part of the molecular mechanism that yields overt
rhythms of about 24 h, more recently multiple other functions have been identified. In essence, the
PER proteins, in addition to their important function within the molecular oscillator, can be seen not
only as integrators on the input side of the circadian clock but also as mediators of clock output. This
diversity in their function is possible, because the PER proteins can interact with a multitude of other
proteins transferring oscillator timing information to the latter. In this fashion, the circadian clock
synchronizes many rhythmic processes.

Keywords: metabolism; mood; synchronization; Per2.

Introduction (Dibner et al., 2010; Ko and Takahashi, 2006; Liu

The circadian clock generates a projection of the
external day into the individual cells of the body
*Corresponding author.
Tel.: þ41-26-300-8674
E-mail: juergenalexandereduard.ripperger@unifr.ch
et al., 2007). However, under normal conditions
we may not become aware of the existence of such
a timing device in us. The reason for this is that the
circadian clock is responsive to external timing
cues, the so-called Zeitgebers (german for “time-
giver”), such as the light/dark cycle. Hence, the
effects of the circadian clock are generally masked

http://dx.doi.org/10.1016/B978-0-444-59427-3.00014-9 233
234
by external factors. To observe the characteristic
properties of a circadian clock, it is necessary to place
an organism into conditions without external
Zeitgebers (e.g., constant darkness or constant light
of constant light intensity). In these free-running
conditions, solely the circadian clock governs the
rhythmic metabolism, physiology, and behavior of
an organism. The advantage of such a process is
obvious. It provides a temporal network allowing
synchronization of many metabolic pathways that
otherwise would run each on their own pace, just
driven by substrate availability, for example. Daily
separation of metabolic pathways yielding or con-
suming energy is already observed in simple
organisms such as cyanobacteria (Dong et al., 2010;
Johnson et al., 2011). Experiments performed with
cyanobacteria also provide compelling evidence for
the biological advantage of possessing such timing
systems: the growth and survival rate of strains
with mutations in their circadian clock are generally
not affected unless these mutants are kept in
coculture with another strain that is in better reso-
nance with the environmental light/dark cycle
(Ouyang et al., 1998). Under the latter conditions,
the better adjusted one completely wipes out the less
adjusted strain. Hence, there is a substantial
biological advantage to possess a well functioning
timing system, and it is therefore not surprising that
circadian clocks are found throughout the bacteria,
plant, and animal kingdoms (Hut and Beersma,
2011; Yerushalmi and Green, 2009). However, a
recent field study with wild type and circadian
rhythm-affected Period2 (Per2) knockout mice did
not reveal a strong selective disadvantage of having
an impaired function of the circadian clock in
mammals (Daan et al., 2011). Here, we would like
to elaborate the basic mechanisms of the mammalian
circadian clock and then focus on the functions of
the circadian clock component PER2 in liver metab-
olism and the brain.
General mechanism of circadian clocks
Circadian clocks arose probably independently
during evolution in different taxa such as cyano-
bacteria, plants, and animals (Rosbash, 2009).
However, interesting similarities and common con-
cepts can be found over the borders of the taxa, such
as the occurrence of posttranslational circadian
oscillations of the redox state of peroxiredoxins,
which are observed in human red blood cells
(O’Neill and Reddy, 2011) and also the green algae
Ostreococcus tauri (O’Neill et al., 2011). The mam-
malian circadian oscillator is related to the one
found in the fruit fly Drosophila melanogaster
(Dunlap, 1999). The basic concept is quite simple
and is found in essentially all cells of an organism.
At the center of the clock is a negative transcrip-
tional feedback loop. A pair of transcriptional
activators increases the transcription of a set of
repressors. The corresponding repressor proteins
accumulate until their concentration is sufficient
to shut down the transcription of their own genes.
Consequently, transcription and translation of the
repressors cease and the activity of the repressor
proteins declines. As soon as the transcription block
is relieved, a new cycle of about 24 h restarts, yield-
ing robust oscillations with a periodicity of about
a day. Obviously, such a simple mechanism would
not be sufficient to produce all of the robust rhythms
observed in the organisms. There would be the
risk that the oscillations generated by the feedback
loop dampen rapidly, nearly reaching equilibrium
conditions (Ripperger and Brown, 2010). Hence,
there exist regulatory posttranslational mechanisms
and an associated network of other feedback loops
that stabilizes and reinforces the circadian rhythms.
This network also provides input information to the
core circadian oscillator to allow synchronization of
the self-sustaining loop to the environment. On the
other side, most of the primary output generated
by the circadian oscillator is mediated primarily by
rhythmic transcriptional regulation of target genes.
The history of Period
In the early 1970s, Konopka and Benzer performed
a mutagenesis screen in D. melanogaster to identify
mutations in its circadian timing system (Konopka
and Benzer, 1971). Surprisingly, the identified
mutations provoking shortening or lengthening of
 235

the free-running period length or complete
arrhythmicity in constant darkness mapped all to
the same gene, which was baptized Period (Per)
(Konopka and Benzer, 1971; Reddy et al., 1984;
Zehring et al., 1984). Nearly 20 years after the iden-
tification of the Per mutants, it was realized that Per
regulated the accumulation of its own mRNA and
the concept of the feedback loop generating overt
rhythms of about 24 h was born (Hardin et al.,
1990). However, it was not until about 10 years later
that the mammalian Per genes were identified and
cloned (Albrecht et al., 1997; Shearman et al.,
1997; Sun et al., 1997; Tei et al., 1997), and that
the function of these Per genes for the mammalian
circadian oscillator (at least of Per1 and Per2)
was verified genetically by analyzing the corre-
sponding knockout mouse strains (Bae et al., 2001;
Cermakian et al., 2001; Zheng et al., 1999, 2001).
At about the same time the mammalian transcrip-
tional activators, CLOCK (Gekakis et al., 1998;
King et al., 1997) and BMAL1/MOP3 (Bunger
et al., 2000; Hogenesch et al., 1998), and a pair of
additional repressors, the CRYPTOCHROME
(CRY) 1 and 2 proteins were also found (Griffin
et al., 1999; Kume et al., 1999; van der Horst et al.,
1999). These proteins cooperate and make up the
core loop of the molecular oscillator (Fig. 1). As
mentioned above, all of the mammalian genes have
their counterparts in Drosophila, maybe with the
exceptions that the mammalian CRY proteins

Bmal1 Rev-Erbα

Clock

Per2

Fig. 1. The network of the mammalian molecular oscillator. The core loop (red shaded circle), responsible for generating rhythms
of about 24 h, is composed of activation of the Per genes by BMAL1 (red) and CLOCK (blue) and the increasing repression of Per
gene expression by the accumulation of its own gene product. The feedback inhibition by the PER proteins is delayed by
posttranslational modifications and interaction with the CRY proteins (not shown). In the stabilizing loop (blue circle), the Rev-
Erba gene is also activated by BMAL1 and CLOCK (not shown) and later on repressed by the PER proteins but immediately
REV-ERBa starts to inhibit transcription of the Bmal1 and Clock genes and of its own gene. PER2 can interact with REV-
ERBa (or PPARa, not shown) to regulate the Bmal1 gene. The overall organization of the network allows for a tight
synchronization of the core and stabilizing loops. Extended and adapted from Ko and Takahashi (2006).
236
are no (or less) important light-sensitive compo-
nents of the circadian oscillator (Griffin et al.,
1999; Okamura et al., 1999), and the function of
the Drosophila Timeless protein (which functions
similar as the mammalian CRY proteins) in the
mammalian circadian clock is less pronounced
(Gotter et al., 2000).
CRYs or PERs?
Ever since the discovery of the main repressor
molecules of the mammalian circadian oscillator,
the question was posed, which of both kinds of
molecules were the more important ones. The
genetics were not really helpful to unravel the
precise functions of these supposed repressor
molecules. Mutations that affect the free-running
period of the circadian oscillator are thought to
be integral parts of the clock mechanism or are
affecting its integral parts. Single knockout mice
deficient for Per1 (Bae et al., 2001; Cermakian
et al., 2001; Zheng et al., 2001), Per2 (Bae et al.,
2001; Zheng et al., 1999), or Cry1 (van der Horst
et al., 1999; Vitaterna et al., 1999) had shorter
free-running circadian oscillators and mice defi-
cient for Cry2 (van der Horst et al., 1999;
Vitaterna et al., 1999) had a longer free-running
circadian oscillator, while the knockout for Per3
did not affect circadian rhythmicity (Bae et al.,
2001). Interestingly, depending on the strain,
Per2 single knockout mice had a shorter free-
running period length but became arrhythmic
after 1–2 weeks in constant darkness, suggesting
that PER2 has a more prominent function
for the circadian clock than PER1. Hence, most
of these individual components have an impact
on the clock mechanism. The combination of
knockout mice for Per1 and Per2 (Zheng et al.,
2001) and the combination of knockout mice for
Cry1 and Cry2 (van der Horst et al., 1999) had
nearly the same phenotype, yielding arrhythmic
behavior immediately after placing the mice in
constant conditions. Consequently, both classes
of proteins appear to be equally important to
maintain circadian oscillator function. Interestingly,
the combination of any Per knockout with any Cry
knockout yielded a whole plethora of phenotypes
ranging from a rescue of the Per2’s arrhythmic phe-
notype in Per2:Cry2 double knockout mice to
completely arrhythmic Per2:Cry1 double knockout
mice (Oster et al., 2002, 2003a,b). These genetic
interactions suggest that the PER and CRY proteins
form multiple different complexes with each other
to fulfill their function(s). Interestingly, all of
the mammalian clock components are redundant
(i.e., there exist Bmal1 and Bmal2, Clock and
Npas2, Per1 and Per2, Cry1 and Cry2) (Ripperger
et al., 2011). A reason for this is unknown yet. Are
there two oscillators running in parallel in the same
cell or do the possible combinations mediate, for
example, tissue-specific gene regulation? At least
for CLOCK and Npas2, it was established that
CLOCK may be more important in the periphery,
and Npas2 may be more important in the brain
(DeBruyne et al., 2007a,b).
Function of PER proteins on the core loop of
the circadian oscillator
Considering the role of Per within the Drosophila
circadian oscillator (Hardin et al., 1990), it was
tempting to speculate that the mammalian PER
proteins fulfilled a similar function to create a tran-
scriptional feedback loop. However, cotransfection
experiments in vitro revealed that, by contrast to
the CRY proteins (Kume et al., 1999), the impact
of the PER proteins on BMAL1 and CLOCK-
mediated transcription was quite subtle (Kume
et al., 1999). This was surprising because the bio-
chemical analysis clearly indicated that PER and
CRY proteins can be coimmunoprecipitated and
consequently they are in the same (repressor?)
complex (Brown et al., 2005; Duong et al., 2011;
Lee et al., 2001). Indeed, the regions within both
classes of proteins mediating the interaction were
biochemically mapped (Fig. 2) (Langmesser et al.,
2008). Nevertheless, nonrhythmic overexpression
of PER2 but not CRY1 strongly interfered with
 237

PAS A
PAS B
PAC Pro-rich Coiled-
coil
HLH

H3N COOH
1 1257
CLD NLS
NES1 NES 3 NES 2
CoRNR
LXXLL LXXLL

GSK3β β−TrCP CKI Cry, E4BP4

Fig. 2. Protein–protein interaction regions of PER2. The protein belongs to the class of PAS domain-containing factors. The PAS A
and B domains and the associated PAC domain mediate the interaction with other PER proteins and the BMAL1 and CLOCK
heterodimer. The region is flanked by binding sites for the regulatory kinase GSK3b on the left and the ubiquitin ligase b-TrCP
on the right. Both enzymatic activities affect the half-life of PER2. The basic helix-turn-helix (HLH) motif resembles a DNA-
binding motif but is probably not functional. The protein has three nuclear-export sites (NES), one nuclear-localization site
(NLS) and a cytoplasm-localization domain (CLD), which influence the nuclear localization of the protein. Interaction with the
CRY proteins (and E4BP4) is mediated by the C-terminus. The protein contains a sequence of casein kinase 1 (CKI)
phosphorylation sites organized as a relay, from which the priming site is affected in FASPS. PER2 also harbors some potential
motifs, which resemble binding sites for nuclear receptors (LXXLL and CoRNR), but only for the most N-terminal LXXLL
motif an interaction with nuclear receptors was identified. Finally, the protein contains potential interaction motifs resembling
proline-rich (Pro-rich) motifs and coiled-coil interaction motifs. Interactions with further kinds of proteins have been described,
such as with WDR5 (an adaptor for the histone methyl transferase Set1), NONO and SFPQ1, and histone demethylases of the
Jarid1 family; however, these were mainly identified in complexes containing also PER1 and a direct interaction has not been
demonstrated yet. Adapted from Albrecht et al. (2007).

normal circadian oscillator function, which is a
strong indicator for a prominent function of the
PER2 protein for the circadian oscillator (Chen
et al., 2009). The more detailed analysis suggested
that the PER proteins form a kind of scaffold for
the CRY proteins to bind to their target sequences
in the genome (Chen et al., 2009). Interestingly,
both kinds of proteins can interact with other
proteins that repress transcription via the modifica-
tion of the chromatin structure. For CRYs, these
include histone deacetlyases (HDAC) (Naruse
et al., 2004), which attenuate local transcription
by removing specific acetyl groups from 50 -termini
of histones, and for PER deacetylases (Duong
et al., 2011), but also proteins that remove methyl
groups from (DiTacchio et al., 2011) or add methyl
groups to 50 -termini of histones (Brown et al.,
2005; Etchegaray et al., 2006). However, it is
necessary to decipher the precise biochemical
function(s) and their impact on the molecular
oscillator of both classes of repressor molecules in
more detail. Taken together, the available litera-
ture on the function of PER proteins in vivo and
in vitro suggests that it is not the protein itself that
acts as a regulator within the circadian oscillator
mechanism, but it is the interaction of PER
proteins with other proteins, which mediates the
effect(s).
238
Role of PER2 in the stabilizing loop
Initially, the characterization of the available cir-
cadian oscillator mutant and knockout mice
uncovered a surprise (Shearman et al., 2000).
The PER2 protein, but not PER1, was suggested
as activator of circadian Bmal1 transcription. This
puzzle resolved itself about 2 years later, when an
additional feedback loop was discovered based on
the transcriptional repressor REV-ERBa (Pre-
itner et al., 2002; Ueda et al., 2002). Hence, the
model of the circadian transcriptional feedback
loop was extended by the stabilizing loop (Fig. 1).
In the core loop, BMAL1 and CLOCK activate
transcription of the Per genes until the PER
proteins (together with the CRY proteins) feed
back onto their own synthesis. To achieve this,
there is a typical delay in the action of the repres-
sor molecules as a prerequisite for the near 24 h
oscillations. In parallel, BMAL1 and CLOCK
also activate transcription of Rev-Erba. The
accumulating REV-ERBa subsequently represses
transcription of the Bmal1 and also the Clock
genes by replacing the transcriptional activator
RORa (Akashi and Takumi, 2005; Sato et al.,
2004) or PPARa (Canaple et al., 2006), depen-
dent on the cell type, and also by recruiting
HDAC3 activity to the target genes (Feng et al.,
2011). Because the action of REV-ERBa does
not involve a delay mechanism typical for the
action of the PER proteins, the phases of tran-
scription of the Per and Rev-Erba genes on
one hand and of the Bmal1 and Clock genes on
the other hand are separated by about 12 h. As
conclusion, loss of repression of Bmal1 transcrip-
tion as observed in Per2 knockout mice was prob-
ably due to the reduction of REV-ERBa and a
concomitant increase in Bmal1 expression. The
story became even more complicated about
2 years ago. It was found that PER2 rather than
PER1 directly interacted with both REV-ERBa
and PPARa, contributing to about 10% of the
repression by REV-ERBa and 20–30% of activa-
tion by PPARa of the Bmal1 gene in the liver
(Schmutz et al., 2010). This interaction was due
to a specific amino acid motif in PER2, which res-
embles a typical nuclear receptor/coregulator
binding site (Fig. 2). However, the functional
extension of PER2 due to its interaction with
nuclear receptors appears not to be restricted to
the core circadian oscillator alone (see below).
The rhythmic metabolism of the liver
As revealed by DNA-microanalysis experiments,
many metabolic processes in the liver occur
rhythmically (Miller et al., 2007; Panda et al.,
2002; Storch et al., 2002; Vollmers et al., 2009).
At the center of this regulation is glucose homeo-
stasis to keep the blood glucose concentration at a
constant level (Lamia et al., 2008). Hence, over
the day, the liver has to switch steadily from glu-
cose uptake from the blood and the storage of
glucose as glycogen to the mobilization of glucose
from its glycogen stores and its secretion into
the blood stream. About 15% of the transcripts
found in the liver accumulate in rhythmic fashion
(Vollmers et al., 2009); however, only about 10%
of those rhythmic transcripts can be assigned to
the circadian oscillator. This means that the bulk
of rhythmic transcripts is induced solely by the
presence of their substrates. What is the advantage
of either way of regulation by induction through
substrates or by the circadian clock? Induction
through substrates necessitates the presence of
substrates, but the response is normally fairly pro-
portional to the substrate concentration. Hence, it
is a quite economical way of gene regulation
according to the needs. On the other hand, the cir-
cadian clock allows the expression of genes before
the substrates become available, obviously with
the drawback that the gene is expressed even
if the substrates never come. However, the antici-
pation of daily recurring events may be the main
biological advantage of possessing a circadian
clock as evidenced by the increase of fitness of cya-
nobacteria that are in better resonance with the
environment. The circadian oscillator provides sta-
ble rhythms of about 24 h, but over the course of a

year the photoperiod (i.e., the day/night ratio)
changes with an impact on the time when the ani-
mal gets active and starts eating (Schultz and Kay,
2003). Consequently, there exist mechanisms in
the liver that uncouple anticipation mechanisms
from the circadian oscillator to keep the anticipa-
tion potential intact (Stratmann et al., 2010). As
example, some detoxification enzymes of the
P450 family are rhythmically regulated by the cir-
cadian transcription factor CAR, which itself is
regulated by the circadian transcription factors of
the PAR-Zip family (Gachon et al., 2006). The
reason may be that those detoxification enzymes,
in the absence of their substrates, produce radical
oxygen species, which may cause damage to the
cell. Hence, basal expression of these enzymes
is restricted to a phase when the occurrence of
substrates is probable to provide some basic pro-
tection. In addition, the genes of these enzymes
can be super-induced by the nuclear receptor
CAR according to the presence of their substrates.
Consequently, the risk of damage to the cell is
optimally reduced. The expression of CAR is
adjusted to the photoperiod by the PAR-Zip
factors, demonstrating that the precise temporal
action of these regulators and their target genes
relative to the activity phase of the animals is
important (Stratmann et al., 2010). In conclusion,
temporal organization of metabolic processes in
the liver optimizes not only energy expenditure
but also protects the organ from excessive damage.
The circadian clock represents an elegant means
to fulfill such a coordinating function. Interest-
ingly, the analysis of Per knockout mice revealed
a function of PER proteins in the concerted
action of DNA damage repair enzymes and
proto-oncogenes, rendering the Per knockout
animals more prone to tumor formation under
certain circumstances (Fu et al., 2002).
Role of PER2 in the liver metabolism
Many of the metabolic processes in the liver are
regulated by members of the nuclear receptor
239
superfamily (Yang et al., 2006). There are 49
members of this family, of which about one half
is regulated in a circadian fashion. In essence,
rhythmic expression of the transcriptional regu-
lators of the nuclear receptor family suffices to reg-
ulate their target genes in a rhythmic fashion. In
addition, many of these nuclear receptors require
specific ligands for their activity. Some of these lig-
ands are produced and secreted in a rhythmic fash-
ion. Under normal conditions, this system could
stably regulate rhythmic processes but appears to
be circuitous and inflexible to respond rapidly to
changes of the circadian clock. The extended
capacity of PER2 to directly interact with these
nuclear receptors provides an elegant way to trans-
mit circadian clock information to the nuclear
receptor regulated target genes (Fig. 3) (Schmutz
et al., 2012). Nuclear receptors involved in the reg-
ulation of metabolic pathways and able to interact
with PER2 in vitro include REV-ERBa, PPARa
and PPARg, and HNF4a and affect the lipid
and glucose metabolism, respectively. Interest-
ingly, PER2 acts as nuclear receptor coregulator,
context-dependent as coactivator or corepressor.
However, further experiments are required to
fully understand these differences in its action.
Nevertheless, PER2 transmits circadian oscillator
information directly to nuclear receptor-mediated
metabolic pathways.
Function of PER2 in the brain
Per2 expression was initially observed in the
suprachiasmatic nuclei in the ventral part of the
hypothalamus and various other brain regions as
well as in peripheral tissues (Albrecht et al.,
1997). Because of its inducibility by light, food
restriction, and temperature pulses, Per2 was
recognized soon as a link between signals from
the environment (the input) and the clock mecha-
nism. Per2 acts as a responder to environmental
signals and is a component of the core clock mech-
anism, but it can also affect physiological pathways
downstream of the clock (the output, Fig. 3).
240

Input Clock Output

NR homo/hetero Target gene

dimers promoters

Nurr1 Dopaminergic system,

Light inflammation
PER2
Hnf4a Glucose metabolism
Food

TRa Temperature

PPARa Fatty acid metabolism

Rev-Erba Circadian timing

? Others

Fig. 3. Transmission of circadian clock information to nuclear receptor-target genes by PER2. PER2 mediates primary output from
the molecular oscillator. By direct interaction with the nuclear receptor (NR) homo- or heterodimers, it can affect the corresponding
target gene promoters and metabolic or physiological processes. The activity of PER2 may be modulated by the input (e.g., light or
food) to the circadian oscillator. Adapted from Schmutz et al. (2010).

Hence, it appeared that Per2 is an important
player in determining the transcription status of
the genome in a specific environmental context.
Therefore, it was not astonishing to find that
Per2 plays a role in many brain related functions.
The reward system regulating addiction appeared
to be at least partially affected when the Per2
gene was mutated (Abarca et al., 2002; Hampp
et al., 2008; Spanagel et al., 2005). In particular,
it appeared that the rate-limiting step in dopa-
mine degradation involving the monoamine
oxidase A was under the control of the circadian
clock mechanism including the Per2 gene
(Hampp et al., 2008). In line with this view is
the observation that PER2 variation in humans
is associated with depression vulnerability
(Lavebratt et al., 2010; Partonen et al., 2007).
Recent findings indicate that the reward system
and addiction processes share neurobiological
mechanisms with overeating and obesity (Simerly,
2006). Interestingly, a mutation in the Per2 gene
of mice resulted in the loss of food anticipatory
activity, when animals were kept under a timed
feeding schedule (Feillet et al., 2006). Taken
together, it appears that the Per2 gene is at the
crossroads of the neurobiological circuitry that is
common to feeding signals and drugs of abuse.
In support of this view is the fact that Per2 is
expressed in regions of the midbrain such as the
arcuate nucleus and the ventral tegmental area,
which regulate food uptake and reward pro-
cessing, respectively. Many of the metabolic pro-
cesses in the brain involve also nuclear receptors
such as Nurr1, which can physically interact with
PER2 (Ripperger et al., 2010; Schmutz et al.,
2010), and hence, brain function is at least par-
tially regulated by this avenue.
Function of PER2 in sleep
Per2 appears to play a role in various parameters
of sleep. Loss of Per2 affected proper sleep timing
(Kopp et al., 2002), which is also evidenced in

advanced sleep-phase syndrome (Jones et al.,
1999). Per2 may affect parameters of sleep
homeostasis as well (Franken et al., 2007), espe-
cially sleep deprivation and recovery sleep appear
to involve PER2. However, whether this function
of PER2 is only attributable to its role in the
brain is not known, because metabolic demands
in the liver involving this protein may also impact
on sleep mechanisms (Albrecht, 2011). Sleep
appears to be regulated by remodeling of neuro-
nal connectivity either strengthening or weaken-
ing synaptic connections. The finding that PER2
is involved in gating the light/dark information
to vesicular glutamate transporter 1 (vGLUT1)
content on synaptic vesicles (Yelamanchili et al.,
2006) was of great interest, because the number
of vGLUT1 molecules on the synaptic vesicle
correlates with the glutamate filling state of this
vesicle and hence affects glutamate release poten-
tial, which may impinge on synaptic strength.
Hence, light may impact via PER2 on brain
function and therefore, alterations in lighting
schedule as experienced in jet-lag and shift work
may affect behavior.
Posttranslational modifications of PER2
How is the interaction potential of PER2 with
other proteins regulated? One possibility is the
accumulation of the PER2 protein over the circa-
dian cycle (Lee et al., 2001). The peaks of tran-
scription and translation of the Per genes are
characteristically separated by a couple of hours.
In addition, the accumulation of the PER proteins
in the nucleus occurs with high amplitude every
day and consequently, the interaction of PER
proteins with other proteins would follow their
affinity of interaction (i.e., first the high affinity
binders and then the low affinity binders).
Exchange of interaction partners would occur by
simple competition mechanisms. This simple affin-
ity model may be extended by posttranslational
modifications (Vanselow and Kramer, 2010). In
the extended model, the dynamic combination of
241
phosphorylation of the PER2 protein reflects
directly oscillator time and each phosphorylation
site may represent a docking site for a specific
protein. Specific phosphorylation of a particular
site is achieved by equilibrium of the action of
kinases on one side and of phosphatases on the
other side. For PER2, about 21 phosphorylation
sites have been identified in fibroblasts although
the kinetics of phosphorylation of the individual
sites remains to be established (Maier et al., 2009).
Some sites when phosphorylated by casein kinase
1 (CK1) d or e serve as binding sites for b-TrCP1/2,
which add ubiquitination marks to the PER2 pro-
tein, rendering it unstable and finally provoke deg-
radation by the proteasome (Reischl et al., 2007;
Shirogane et al., 2005). An interesting case is the
familial advanced sleep-phase syndrome (FASPS)
(Jones et al., 1999). Individuals with specific
mutations in either CK1d (Xu et al., 2005) or the
CK1d binding site on PER2 display sleep rhythms
advanced by several hours (Toh et al., 2001) (a sim-
ilar mutation in CK1e is provoking the familial
delayed sleep-phase syndrome, (Takano et al.,
2004)). Detailed analysis revealed that the ratio of
kinase to substrate was important for the free-
running period length of the molecular oscillator
(Xu et al., 2007). On the phosphatase side, regu-
latory subunits of the protein phosphatase 1 were
shown to regulate the nuclear-localization PER2
and the free-running period length (Lee et al.,
2011; Schmutz et al., 2011). Are the interactions of
nuclear receptors affected by reversible phosphory-
lation? At the moment, there is no direct evidence
in favor of this hypothesis. The interaction motif
appears not to be adjacent to a known phosphoryla-
tion side. However, it could be that phosphor-
ylation at a distant side affects the accessibility of
the interaction motif. As speculation, the region
between the two PAS domains is flexible to
dynamically shift from a conformation facilitating
interaction with BMAL1 and CLOCK to another,
which confers interaction with nuclear receptors.
Phosphorylation mediating interaction with PER2
could also be present on the nuclear receptors, pos-
sibly regulated by the presence of specific ligands.
242
Conclusions
Synchronization of the individual branches of the
circadian oscillator and the different routes of
input and output might necessitate more effort
than simple transcriptional feedback loops can
provide. Temporally controlled protein–protein
interactions enable the coupling of the high-
precision timer of the circadian clock to, for
example, nuclear receptor-target genes. The high
flexibility of these interactions allows PER2 to
act as coactivator or corepressor depending on
the regulatory context and hence to fine-tune
the activity of these nuclear receptors. To fully
understand the interaction potential of PER2 and
its regulation by posttranslational modifications,
however, requires further experiments. These
interactions may form the base of the many addi-
tional functions of PER2 in the metabolism and
the brain. Other members of the core circadian
oscillator can interact with a variety of other
proteins and regulators, and it is tempting to spec-
ulate that the overall function(s) of these
interactions are similar to those of PER2. How-
ever, it is also tempting to speculate that PER2
can directly interact with many more classes of
transcriptional regulators.
Acknowledgments
We thank for the support of our laboratory by the
State of Fribourg and the Swiss National Science
Foundation.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 15

Generation of mouse mutants as tools in

dissecting the molecular clock

Sneha N. Anand, Jessica K. Edwards and Patrick M. Nolan*

Neurobehavioural Genetics, MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire,
United Kingdom

Abstract: Elucidation of the molecular basis of mammalian circadian rhythms has progressed
dramatically in recent years through the characterization of mouse mutants. With the implementation
of numerous mouse genetics programs, comprehensive sets of mutations in genes affecting circadian
output measures have been generated. Although incomplete, existing arrays of mutants have been
instrumental in our understanding of how the internal SCN clock interacts with the environment and
how it conveys its rhythm to remote oscillators. The use of ENU mutagenesis has proven to be a
significant contributor, generating mutations leading to subtle and distinct alterations in circadian
protein function. In parallel, progress with mouse gene targeting allows one to study gene function in
depth by ablating it entirely, in specific tissues at specific times, or by targeting specific functional
domains. This has culminated in worldwide efforts to target every gene in the mouse genome allowing
researchers to study multiple gene targeting effects systematically.

Keywords: ENU; mutagenesis; mouse; transgenics; gene-driven screen.

Introduction might play a role in the progression of complex

A major challenge for scientific research in the
twenty-first century will be to assign functions
to ca. 25,000 genes of the mammalian genome.
With innovations made in high-throughput geno-
mics techniques, a large number of genes that
*Corresponding author.
Tel.: þ44-01235-841091; Fax: þ44-01235-842000
E-mail: p.nolan@har.mrc.ac.uk
diseases and behaviors have now been identified.
Questions such as how these genes act and
what pathways they are involved in can be
answered by determining the consequences of
gene disruption using mutants (Picciotto and
Wickman, 1998). The systematic generation of
mouse mutants through the use of two comple-
mentary approaches, forward and reverse genet-
ics, has been key in advancing our knowledge
of how genes function in biological systems.

http://dx.doi.org/10.1016/B978-0-444-59427-3.00015-0 247
248
Forward genetics begins with the investigation of
a given phenotype and makes use of genetics
and genomics tools to identify the causative gene.
Conversely, reverse genetics uses our knowledge
of a candidate gene or gene sequence, its expres-
sion patterns and presumed functions, and then
introduces mutations in the gene using available
technologies to study phenotype.
These two complementary approaches have been
equally invaluable in contributing to our under-
standing of how the molecular clock ticks. In this
chapter, we provide a brief overview of the
approaches and technologies currently used to gen-
erate mouse clock mutants and review the progress
in behavioral and molecular analyses using these
tools. Finally, we consider new developments and
directions in this area that promise to provide signif-
icant mouse mutant resources for the future.
Forward genetics
The benefit of forward genetics approaches as a
complement to gene targeting strategies is that it
is unbiased, requiring no a priori assumptions
about gene function. Importantly, it is hypothesis
generating, meaning that novel genes may be
attributed to known pathologies and behaviors.
Most behaviors are complex traits, relying upon
genetic and environmental interactions, with most
genetic components currently unknown. This is
true for the circadian system. Insights into the
molecular basis of circadian function have been
possible primarily through the use of forward
genetics screens and the analysis of mutant
phenotypes in model organisms such as Drosoph-
ila (Konopka and Benzer, 1971) and Arabidopsis
(Ahmad and Cashmore, 1993). Mammalian circa-
dian function has depended on the analysis of
mutations in orthologous genes in Drosophila
and Arabidopsis, the period and cryptochrome
genes (Bae et al., 2001; Cermakian et al., 2001;
Shearman et al., 2000; van der Horst et al., 1999;
Zheng et al., 1999, 2001). Nevertheless, several
important insights into circadian function have
been established from the analysis of mutant
phenotypes in mice and other rodents. This strat-
egy continues to provide functional insight into
the molecular basis of circadian rhythm genera-
tion and maintenance.
The mouse as a model organism
The house mouse, Mus musculus, has long been
used as a vital tool for molecular genetics. There
are currently more than 450 inbred mouse strains
available (Beck et al., 2000), the accessibility of
which has allowed for finely detailed genetic
maps to be generated. The mouse is an ideal
model, given its high homology with the human
genome. The structure of the mouse genome is
similar to humans, with approximately the same
number of genes that display extensive synteny
(Boguski, 2002). The mouse also displays measur-
able endophenotypes of emotional states, such as
anxiety, depression, and fear, which allow for
complex behavioral conditions and traits to be
analyzed. The genetic composition and behav-
ioral characteristics of the mouse have ensured
that it is at the frontier of mammalian molecular
genetics research across a range of disciplines.
Random mutagenesis in mice
Prior to the era of gene targeting, our knowledge of
mouse genes and genomes was established through
the analysis of spontaneous or induced mutations.
Spontaneous mutations occur at a low rate of
approximately 5  10 6 per locus (Silver, 1995).
Nevertheless, many spontaneous mutants with vis-
ible or easily detectable phenotypes were used to
establish genetic maps in the mouse and these, in
turn, were used as a backbone to establish the
order of numerous molecular markers on each
chromosome, progress that eventually led to the
sequencing of the entire mouse genome. Given
the rarity of their occurrence, it is unusual for spon-
taneous mutants to be identified solely on the basis
of circadian phenotypes. Exceptions to this can, of

course, be found in other mammalian species.
Cloning of the spontaneous hamster mutant, tau
(Ralph and Menaker, 1988), has been crucial for
the investigation of posttranslational mechanisms
in clock function (Lowrey et al., 2000). Spontane-
ous mutants affecting other components of circa-
dian regulatory pathway have been identified in
mice through phenotypes associated with other
gene functions, for example, the staggerer mutant
carries a mutation in the orphan nuclear receptor
Rora (Sidman et al., 1962). Despite the historical
role of spontaneous mutations, their utility in the
future of functional genetic analysis is limited due
to the large colonies required.
Several mutagenic agents have been used in
mouse genetics to increase the rate of identifying
functional mutations, including those that affect
behavior. X-ray mutagenesis was one of the first
mutagens used, increasing mutation frequency by
20–100 times (Cordes, 2005). The disadvantage
of X-ray mutagenesis is that it commonly induces
chromosomal rearrangements which inevitably
affect multiple genes or may result in positional
effects. Chemical mutagenesis emerged as an effi-
cient alternative although many chemicals, includ-
ing chlorambucil, induced similarly confounding
contiguous gene effects (Rinchik et al., 1990). Of
all the chemical mutagens available for mamma-
lian studies, ENU (N-ethyl-N-nitrosourea) is
unique in that it almost exclusively induces point
mutations at an extremely high efficiency. It acts
through direct alkylation of nucleic acids, showing
a slight bias toward alkylating A-T bases, at a rate
of 1 mutation every 1–1.5 Mb (Quwailid et al.,
2004). ENU has a high affinity for spermatogonial
stem cells allowing for mutations to be inherited
through the germline. Crucially, it does not cause
complete sterility in males; rather, after adminis-
tration, it results in a period of temporary sterility
as spermatogonia are depleted from, and then
repopulate, the testis with individual mature
sperm carrying unique catalogs of mutations in
their genome (Nolan et al., 2000b).
The most common point mutations are trans-
versions (A-T! T-A) or transitions (A-T ! G-C)
249
(Cordes, 2005). The resultant mutations are
diverse, including missense or nonsense, loss or
gain of STOP codons, or those which affect splic-
ing or transcriptional activity. In this way, ENU
can create an allelic series to finely dissect gene
function. ENU may therefore be considered a
complementary approach to transgenic strategies,
since issues such as redundancy or pleiotropy,
may be avoided if the induced mutation still
enables gene function.
ENU mutagenesis screening strategies
ENU screens are generally used to identify domi-
nant or recessive traits. Screens for dominant
traits are easiest to carry out as progeny of
ENU-mutagenized males (G0) crossed to wild-
type females are directly phenotyped (Fig. 1).
Each product of this cross (G1 founder) can
potentially carry mutations at up to 100 different
loci. Each G1 individual can be phenotyped for
dominant mutations either using a high-through-
put strategy or for specific phenotypes using a
specialized phenotyping pipeline (Nolan et al.,
2000a). G1 founders carrying abnormal traits are
then bred to assess inheritance of the trait. This
approach is quick, since only one generation of
breeding is required before phenodeviants are
identified. Dominant mutations, however, are
recovered on average 10 times less frequently
than recessive mutations. A further disadvantage
to this screening approach lies in the fact that only
mutations of moderate to large effect size are lia-
ble to be detected. This can be especially con-
founding if a particular trait is subject to high
variability. An alternative method, though much
less high throughput, is to screen for recessive
mutations. The recessive screen requires two
more generations of breeding to generate a
screening population. Here, the G1 progeny are
again mated to a wild-type animal to produce
G2 offspring. The ENU mutations will still be in
a heterozygous state, so these offspring are inter-
crossed to produce a G3 pedigree, with 25% now
250

ENU

X
G0
+/+ m/+

G3
Backcross recessive
screen

G1
X
Dominant screen +/+ m/+
+/+

X
m/+ m/m
X
G2

m/+ +/+ +/+ m/+

G3
Intercross
recessive screen m/m m/+ +/+

Fig. 1. ENU mutagenesis dominant and recessive breeding scheme. Dominant mutations may be observed in G1 progeny, whereas
recessive mutations may be observed in G3 progeny using either a backcross or intercross strategy.

homozygous for any of the ENU-induced
mutations. Alternatively, G2 female progeny
may be backcrossed to the male founder (G1)
and again, G3 pedigrees containing homozygous
animals will be produced (Balling, 2001). Gener-
ally, 20–40 animals per pedigree are screened
ensuring that 5–10 animals will carry the homozy-
gous mutant phenotype.
Several modifications of either approach can be
used to target specific phenotypic domains or
molecular pathways. One modification is to screen
for dominant mutations in a two-generation
screen. Using this strategy, mutations with smaller
effect size can be readily screened (Kumar et al.,
2011), for example, in screens such as open field
activity that are subject to high variability.
Another approach is to target a specific molecular
pathway by screening for mutations that alter
reporter gene expression on a specific sensitized
background (Stottmann et al., 2011).
Screening ENU phenodeviants for circadian
phenotypes
Circadian phenodeviants can be identified in a
screen based upon wheel-running activity under
an automated lighting regime (Bacon et al.,
2004). Each animal, from either G1 or G3 litters,
is singly housed in a cage fitted with a running

wheel. Each revolution of the wheel is recorded
and plotted as a measure of the animal’s activity
throughout the entire screen to produce a
double-plotted actogram. A typical primary
screen consists of an initial 1-week period under
a 12:12 light–dark schedule to entrain the
animals. The animals are then exposed to a
period of constant darkness for up to 5 weeks.
The screen may then continue with a period of
constant light, under which a wild-type animal
will normally lengthen its period. Other lighting
schedules may include light pulses to assess mas-
king or phase shifting, or returning to a 12:12
light–dark cycle to assess reentrainment after
exposure to constant light conditions. As well
as period length, levels of activity, phase angle,
and responses to light intensity may also be ana-
lyzed to further dissect the circadian phenotype
on a behavioral level. Typical examples of the
diversity of mutants identified in wheel-running
screens are shown in Fig. 2.
Mapping and positional cloning of
ENU mutations
Following identification of phenodeviants,
mutations are tested for inheritance and pene-
trance. Phenodeviants are mated to wild-type
animals and the progeny are tested for the
observed phenotype. Those mutations which
are inherited and maintain high levels of pene-
trance are then selected for mapping and posi-
tional cloning of the mutation. The principles of
genetic mapping and positional cloning have been
followed in identifying the after-hours (Afh) circa-
dian mutant (Godinho et al., 2007). This mutant
was identified in an ENU mutagenesis dominant
screen and was subsequently intercrossed to pro-
duce homozygous animals displaying a period
length of approximately 27 h in constant darkness.
The first step in identifying a causative muta-
tion is to generate a high resolution map of the
mutant locus and a region of linkage identified.
Typically, 10–20 phenodeviants are selected for
251
mapping. In the case of Afh, 13 of the most severe
phenodeviants for period length in constant dark-
ness were selected. This region of linkage is
known as a “candidate region” and sequencing
of the region is required to identify the functional
mutation. Technological advances now make it
possible for the whole genome to be sequenced
following a genome-wide scan, such that all
sequences within a critical region can be aligned
and the functional mutation identified. This is
becoming the most cost-effective and time-efficient
method by which to identify mutations.
Once a mutation has been identified, it is
important to prove that it is causative of the phe-
notype, through techniques such as bacterial arti-
ficial chromosome (BAC) rescue (Antoch et al.,
1997; Justice et al., 1999). Further phenotypic
analyses may be required to fully characterize
the mutant, and often further crosses to other
mutants or inbred lines may be useful in deter-
mining whether there are genetic interactions
with other known and characterized genes. For
circadian research, forward genetic screens and
the utilization of techniques such as ENU muta-
genesis has so far been essential in expanding
our knowledge of the molecular basis of the circa-
dian oscillator and the vital role of the circadian
clock on gross behavior and physiology.
Classes of mutants identified in mouse
ENU screens
Mutations that uncover new clock elements
The use of similar forward genetics screens in Dro-
sophila and Arabidopsis has informed our knowl-
edge of how the mammalian circadian oscillator
functions, uncovering core elements such as
period, timeless, and cryptochrome that play
important roles in multiple species. Although this
approach is certainly more laborious in mammals,
it has led to key discoveries that have, in turn,
informed our knowledge of circadian function in
other species. The identification of the Clock gene
252

(a) (b)

(c) (d)

(e) (f)

(g) (h)
 253

in a mouse ENU mutagenesis program was the cat-
alyst for the elucidation of the molecular basis of
mammalian circadian function and the establish-
ment of functional conservation across multiple
species (Antoch et al., 1997; King et al., 1997b;
Vitaterna et al., 1994). The identification of this
core element through the forward genetics
approach was particularly relevant as the mutation
was shown to be antimorphic (King et al., 1997a).
The Clock mutation, an A ! T transversion, was
identified in the 50 splice donor site of intron 19
resulting in skipping of exon 19 and a 51-amino
acid deletion in the mutant protein. Subsequently,
generation of a Clock/ mutant produced
surprising results (Debruyne et al., 2006). The pre-
diction was that the circadian oscillator in Clock/
mice would be severely compromised and circa-
dian rhythmicity would be lost. Unexpectedly,
circadian rhythmicity was maintained in the homo-
zygous null mutants. Only a mild phenotype was
observed—a shortening of the period length in
constant darkness by approximately 20 min and
an advanced onset of activity of 2 h. In retrospect,
it would appear that the significance of Clock gene
function might not have been identified if one were
to use reverse genetics alone, and this early exam-
ple highlights the complementarity of both
approaches in understanding gene function. It
would now appear that the paralog Npas2 may
compensate for the loss of Clock gene function.
More recently, the discovery of multiple muta-
tions within the Fbxl3 locus in mice (Godinho
et al., 2007; Siepka et al., 2007) has highlighted
the importance of the proteasomal system in the
timed degradation of core mammalian circadian
proteins. Both mutations are missense mutations
resulting in amino acid substitutions at the
C-terminal end of the protein. Although neither
mutation affects Fbxl3 stability or the ability of
Fbxl3 to associate with the ubiquitin ligase com-
plex, both affect the ability of Fbxl3 to associate
with mammalian cryptochromes, reducing their
efficiency in targeting them for degradation. The
ability of any cell to rapidly clear protein levels
over circadian time or even in response to changes
in environmental condition appears to be highly
functionally conserved across a number of species
and would suggest that multiple additional com-
ponents in this regulatory pathway remain to be
identified and characterized.
Additional mutations in known clock genes
The idea that one mutant is sufficient to study gene
function is redundant. Even in Drosophila, where
there is a single period gene, multiple allelic
mutations with varying effects on phenotype have
been established resulting in period lengthening,
period shortening, or complete arrhythmicity.
Flybase (http://flybase.org/) lists over 10 Per
alleles identified in chemical mutagenesis screens
that result in defective circadian rhythms. Each
of these mutants can add to our knowledge of
how a particular protein functions by mapping
functional domains onto each protein sequence
(protein interaction, DNA binding, enzyme re-
cognition, etc.). The same strategy applies to

Fig. 2. Selected mutants identified in an ENU mutagenesis screen. Representative images illustrating wheel-running activity rhythm
records for mutant lines identified in dominant and recessive ENU screens. Each image is a double-plotted actogram of wheel-
running activity under light/dark conditions (LD), followed by constant dark conditions (DD) and, in some cases, constant light
conditions (LL). Occasionally, animals also received light pulses during the LD phase of the screen. Shaded blocks represent
times at which the lights are on and short vertical bars represent periods of wheel-running activity. Records of littermate activity
patterns for (a) a wild-type mouse; (b) a mutant line with a long period in DD and arrhythmicity in LL; (c) a mutant line free-
running in LD with an extremely long period in DD and activity suppression in LL; (d) a mutant line with reduced entrainment,
low amplitude short period rhythms in DD, and low amplitude rhythms in LL; (e) a mutant line with reduced period lengthening
in LL; (f) a mutant line with extreme period lengthening in LL; (g) a wild-type mouse; and (h) mutant line with extreme short
period in DD.
254
mammalian ENU mutagenesis screens although,
with exceptions, the identification of multiple alle-
lic mutations is far more infrequent. In one
instance, a hypomorphic mutation in Cry1, named
part-time (prtm), has recently been described
(Siepka et al., 2007) indicating that ENU screens
in mice have the potential to identify other func-
tionally relevant mutations in clock genes apart
from nulls.
Mutations in genes that may affect clock circuitry
By measuring locomotor output rhythms, ENU
mutagenesis screens also have the capacity to
uncover mutations that affect clock circuitry.
The earlybird mutation was identified in a modest
screen of 500 animals (Kapfhamer et al., 2002).
Although the dominant screen identified a mild
short period phenotype, homozygotes display a
circadian period as short as 22 h as well as sleep pat-
tern disturbances. Sequence analysis identified a
mutation in the synaptic vesicle trafficking protein,
Rab3a. The nature of the mutation would suggest
that a deficit in synaptic transmission could be
responsible for the short period phenotype. In
another dominant screen, a seizure susceptibility
mutant was identified, where heterozygotes showed
additional secondary phenotypes including length-
ened free-running period with associated sleep dis-
turbances (Kirshenbaum et al., 2011). The mouse
carries a missense mutation in the Naþ,Kþ-ATPase
a3 isoform, resulting in a I810N substitution.
Investigating redundancy and epistasis in
ENU mutants
The robustness of the circadian system is evident
in that many of the components involved in the
regulation of the system exist as paralog pairs,
often serving partially overlapping functions.
One reason for this could be to ensure that
deficits in any single gene need not dramatically
affect the ability of an organism to drive or adjust
rhythms in response to changing environmental
cues. Although this leads to an efficient biological
process, it has consequences when trying to inves-
tigate the function of any single gene by knocking
it out. Such effects on output rhythms are evident
when comparing the Csnk1etau/tau mutant in mice
to the Csnk1e/ mutant; whereas the null allele
shows only a slight deficit in period, introduction
of the tau point mutation results in a large and sig-
nificant shortening of period (Meng et al., 2008).
Due to the complexity of the circadian system,
the presence of multimolecular interactions for
proper functioning of the clock is essential. It is
evident that analyzing a specific locus indepen-
dently does not provide enough knowledge to dis-
sect the function and interaction of particular
genes in such a complex system. Hence, it is neces-
sary to consider the epistatic interactions that
contribute to the variability of the circadian system.
For example, Maywood et al. showed that
Csnk1etau and Fbxl3Afh showed additive and inde-
pendent effects on regulation of the circadian feed-
back loop (Maywood et al., 2011). As illustrated,
the use of ENU mutagenesis has proved and will
continue to prove its power to identify new alleles
which will allow us to better understand the
mechanisms of the circadian clock.
Reverse genetics
Gene knockout through gene targeting
The most common way to study gene function
in vivo is to ablate its function (gene knockout).
This approach makes use of homologous recombi-
nation in embryonic stem (ES) cells to introduce a
nonfunctional gene into the host genome. Initially,
mammalian genes were targeted on the basis of
phenotypes identified in other organisms such as
Drosophila. However, as more genomic data
became available, genes were knocked out on the
basis of their oscillating expression patterns, gene
or protein interactions, or in vitro gene expression
or knockdown data. Undoubtedly, this approach

has led to many important advances in our under-
standing of the molecular basis of rhythm genera-
tion and maintenance. Nevertheless, limitations of
this approach in the characterization of circadian
mutants can often be attributed to functional
redundancy as discussed earlier. The implication
here is that no one gene is necessary to maintain
circadian rhythms in mammals. In fact, the only
single gene knockout to result in arrhythmicity,
Bmal1 (Bunger et al., 2000), does so by virtue of
the fact that Bmal1 regulates the expression of its
paralog, Bmal2 (Shi et al., 2010).
Interactions between paralogous genes
This approach identifies where functional red-
undancies in paralogs or gene families might be
present. A systematic investigation would be
highly informative, as it has been in species
such as Drosophila melanogaster, Caenorhabditis
elegans, and Arabidopsis thaliana; nevertheless,
the time, cost, and space involved in carrying out
these studies in mice means that only limited
focused work has been carried out. As all three
mammalian homologues of the Drosophila Per
gene were cloned and knocked out, an initial focus
was to determine whether they had distinct or
overlapping functions in period regulation. All
three individual Per mutants display short free-
running period phenotypes, Per2/ exhibiting
the strongest phenotype and Per1/ and Per3/
progressively weaker (Cermakian et al., 2001;
Shearman et al., 2000; Zheng et al., 1999, 2001).
The generation of double mutants led to the con-
clusion that Per1 and Per2 had overlapping roles
as the mice became completely arrhythmic when
released into constant darkness. Per3, on the other
hand, seemed to have a distinct and less vital role
in rhythm generation as double mutants with it
and either Per1 or Per2 showed no additional phe-
notype (Bae et al., 2001). Similar conclusions were
drawn following the generation of double Cry1/;
Cry2/ mutants. With a shorter circadian period
observed in Cry1/ mice and a longer circadian
255
phenotype in Cry2/ mice, their functions could
be interpreted as being nonredundant. However,
Cry1/; Cry2/ double knockout mice are
arrhythmic in constant darkness suggesting that
the two paralogs may have partial overlapping
functions (van der Horst et al., 1999). The double
knockout mutant phenotype of paralogous positive
regulators Clock and Npas2 was also shown to
be arrhythmic in constant darkness, while each sin-
gle mutant had negligible effects on clock function
(DeBruyne et al., 2007). Although the basic helix-
loop-helix genes Dec1 and Dec2 (Bhlhe40 and
Bhlhe41) were proposed as negative regulators
of the clock, neither null mutant had significant
effects on clock function. Again, double mutants
exhibited a significant lengthening of the circadian
period (Rossner et al., 2008). Additional molecular
analysis of these mutants provided further evi-
dence that may well act as transcriptional
coactivators as well as repressors depending on
how they interact with the Clock/Bmal complex.
Mutant interaction studies may also help in
defining roles for paralogous genes outside their
circadian function. All three PAR bZIP transcrip-
tion factors (D site albumin promoter binding
protein, DBP; hepatic leukemia factor, HLF;
and thyrotroph embryonic factor, TEF) oscillate
in brain and liver. Although Dbp/ animals were
shown to have a small but significant shortening of
free-running period (Lopez-Molina et al., 1997),
no further circadian phenotype was observed when
all three genes were knocked out (Gachon et al.,
2004). However, their role in other aspects of
CNS function was identified as triple mutants
showed an increased susceptibility to spontaneous
and audiogenic seizures.
Interactions between nonparalogous genes
Investigation of compound mutant phenotypes can
confirm how two unrelated genes may be inter-
acting in a circadian pathway. These investigations
can produce surprising results that question our
prior understanding of how molecular rhythms
256
are orchestrated. Oster et al. refined the roles of
period and cryptochrome by comparing double
mutant phenotypes (Oster et al., 2002, 2003).
Unexpectedly, they found that Per1/; Cry1/
mice had free-running periods similar to wild-type,
while single gene mutants had short free-running
periods. A similar paradoxical result showed that
Per1/ further lengthened Cry2/ period. Con-
versely, Per2/ mice appeared to maintain their
phenotype in a Cry/ background. Per2/;
Cry1/ double mutants lose rhythmicity immedi-
ately upon release into free-running conditions,
whereas Per2/; Cry2/ mutants maintain a
rhythm in constant darkness that is slightly shorter
than wild-type. This study would support the belief
that Per2 and Cry1 are the strongest negative reg-
ulators of clock function. Evidence for molecular
interactions between Per2 and Rev-erba has also
been identified. Support for this was elucidated
through generation of double mutants, where the
double mutant phenotype is significantly shorter
than either single mutant (Schmutz et al., 2010).
As well as assigning a genetic interaction for these
two proteins, the authors provide evidence that
these two proteins can act as transcriptional
corepressors.
Inducible and tissue-specific knockouts
It is becoming increasingly evident that many
components involved in regulation of clock func-
tion can serve multiple purposes from develop-
ment and differentiation through to metabolic
homeostasis and cell cycle regulation in adults.
Although gene knockouts have provided essential
insights into clock gene function, they can also
result in confounding pleiotropic phenotypes. To
overcome these effects, the spatiotemporal
expression of mutant alleles can be controlled
through the generation of inducible and tissue-
specific knockouts. By this method, the use of
two engineered mutants is required, one where
loxP sites flank a critical target exon in the gene
of interest and a second where Cre recombinase
is driven by a promoter that will express the
recombinase in a specific spatiotemporal pattern.
As technologies are advancing, conditional circa-
dian mutants have become available for use, includ-
ing those for Bmal1 (Marcheva et al., 2010), Clock
(Debruyne et al., 2006), Csnk1d and Csnk1e
(Etchegaray et al., 2009), and Sirt1 (Nakahata
et al., 2008). As we discuss later, the numbers and
availability of conditional or “conditional-ready”
resources and mutants should start to increase and
become readily available through worldwide efforts
such as EUCOMM (the European conditional
mouse mutagenesis program) and KOMP (the
knockout mouse project).
The power of the conditional mutagenesis
approach will develop with the emergence of
new Cre driver lines. Reports in the literature to
date have predominantly been on conditional
ablation of gene expression in liver or pancreas
for two reasons. First, the consequences of clock
gene dysfunction in these organs can help in dis-
secting the role of these genes in metabolic
homeostasis. The Sirt1/ and Csnk1d/ mutants
are both homozygous lethal, whereas conditional
ablation in the liver shows that they are both
effective in disrupting molecular oscillations. On
the other hand, Bmal1/ is not lethal but condi-
tional ablation in pancreatic islets results in defec-
tive beta-cell function. The second reason for
these lines of study is that mouse lines that
express Cre in these tissues or cells are more read-
ily available. Up to now no mouse lines driving
Cre expression exclusively in the suprachiasmatic
nucleus (SCN) have been available. One of the lat-
est mouse lines that may be useful has been
reported recently (Husse et al., 2011). Although
not exclusively expressed in SCN, this
Synaptotagmin-10 Cre line is highly expressed in
this region with limited expression in other brain
regions and no expression in peripheral organs
apart from testis. Using this line, conditional dele-
tion of Bmal1 results in arrhythmicity.
New Cre driver lines currently being generated
may be archived in public resources (Table 1).
One such resource is the GENSAT transgenic
 257

Table 1. Information on and access to useful mouse resources

Resource Acronym Web details Description

International Mouse Phenotyping IMPC http://www. Information on and portal to worldwide mouse
Consortium mousephenotype. genetics programs and infrastructures
org/
European Mouse Mutant EMMA http://www.emmanet. Searchable database of mouse mutant strains;
Archive org/ available as cryopreserved material or live mice
Mutant Mouse Regional MMRRC http://www.mmrrc. Database of mouse mutant strains, KOMP lines,
Resource Centers Archive org/ Cre driver lines, ES cell lines, etc.
Europhenome Mouse Europhenome http://www. Searchable database of mouse strains and mutant
Phenotyping Resource europhenome.org/ lines phenotyped in primary phenotyping pipelines;
databrowser/viewer. searchable by gene or phenotype
jsp
International Knockout Mouse IKMC http://www. Searchable database of mouse mutant strains;
Consortium knockoutmouse.org/ status on mutant generation for any gene provided
European Mouse Phenotyping EMPReSS http://empress.har. Database of standard operating procedures for
Resource of Standardized mrc.ac.uk/ screens in primary phenotyping pipelines
Screens
Gene Expression Nervous GENSAT http://www.gensat. Database of neuroscience-relevant mouse
System Atlas org/index.html transgenic BAC-EGFP and BAC-Cre recombinase
driver lines
Coordination of Resources for CREATE http://creline.org/ Unified portal to access worldwide Cre line
Conditional Expression of home resources
Mutated Mouse Alleles
RIKEN ENU-Based Gene- RGDMS http://www.brc.riken. Archive of ENU mutants identified in reverse
Driven Mutagenesis System jp/lab/mutants/ genetics screens; details of how to request
genedriven.htm additional screens
MouseBook MouseBook http://www. Archive of mouse mutants archived at Harwell
mousebook.org/ including ENU lines; searchable by phenotype or
index.php gene

BAC-Cre recombinase driver line resource Transgenics

(Gong et al., 2007), providing access to and in-
formation on over 100 Cre driver lines expressed
in region-specific patterns in mouse brain (see
http://www.gensat.org/cre.jsp). This includes
many driver lines of interest to the circadian com-
munity including Avp, Vip, Vipr2, Prokr2, and
Dbp lines. Other resources and searchable
databases include CREATE, a coordination of
resources for conditional expression of mutated
mouse alleles (http://creline.org/home). This site
acts as a portal to access Cre line resources and
searchable databases worldwide. The diverse
expression patterns of these lines will no doubt
enable biologists to dissect circadian function in
a systematic way.
Expression of transgenes is quite commonly used in
circadian research to help identify specific functions
of genes. One way in which to do this is by pronu-
clear injection of a BAC expressing the gene of
interest and containing all or most of the elements
required for the expression of the native protein.
This approach was used to generate a Bmal1 trans-
genic as one of a series of mouse genetic tools to
distinguish the effects of Bmal1 dysfunction in brain
and muscle (McDearmon et al., 2006). Transgenic
constructs can also be generated and genetically
modified to constitutively overexpress a gene and/
or to introduce specific mutations or tags. These
modifications have been used to complement
258
mouse knockout research and can often identify
functionally relevant domains or uncover novel
roles for clock proteins. In two cases, transgenic
mice have been generated to model the human
inherited condition, familial advanced sleep phase
syndrome (FASPS). Transgenic mice expressing
the Ser662Gly mutation in Per2 had an advanced
phase of activity in a light–dark schedule and a
short free-running period of activity (Xu et al.,
2007). A second missense mutation in Csnk1d is
also associated with FASPS (Xu et al., 2005), the
mutation resulting in reduced enzymatic activity
in vitro. Again, the phenotype in a mouse trans-
genic line mimicked that of the human subjects.
Consequences of Cry1 overexpression were
investigated using a Cry1 cDNA construct driven
by the chick b-actin promoter and a CMV
enhancer (Okano et al., 2009). Although a 5- to
10-fold ubiquitous overexpression of Cry1 had
no evident circadian phenotype, the introduction
of a Cys414Ala substitution resulted in a circa-
dian period of ca. 28 h, indicating that this domain
of Cry1 has a crucial rhythm-related function. In
defining the role of the Bmal1 paralog, Bmal2,
in biological rhythms, a Bmal2 transgenic con-
struct constitutively driven by the human EF-1a
promoter was generated (Shi et al., 2010). By
crossing these mice to Bmal1/ mice, behavioral
wheel-running and metabolic rhythmicity could
be restored. This finding was crucial in defining
an otherwise ambiguous role for Bmal2 in circa-
dian function and suggested that Bmal1 and
Bmal2 may function as a paralogous pair.
Implementing temporal control over the expres-
sion of transgenes can further dissect gene function
(Picciotto and Wickman, 1998). Tetracycline-
controlled transcriptional activation is widely used
for this purpose and makes use of a specific mouse
mutant line where the tetracycline transactivator
(tTA) is controlled by a promoter directing tissue-
specific expression and the expression of a target
gene is under the control of the tetracycline
operator. This system has been used in a number
of circadian studies, for example, examining
the phenotypes associated with tissue-specific
transgenic rescue of the Bmal1/ phenotype
(McDearmon et al., 2006). In this study, Bmal1
was constitutively expressed in either brain using
the secretogranin-2 promoter or in muscle using
the human a-actin-1 promoter in Bmal1/ mice.
While the brain-rescued mice displayed circadian
rhythmicity, the muscle-rescued mice displayed no
circadian rescue but activity and body weight
phenotypes were alleviated. Using the same tTA
mouse line, transgenic overexpression of the wild-
type Clock gene in brain shortened circadian
period, whereas it was lengthened by over-
expressing the dominant negative transgene. Both
effects could be reversed by treating animals with
doxycycline (Hong et al., 2007). In a similar study,
Kornmann et al. used a hepatocyte-specific pro-
moter driving tTA to constitutively express a Rev-
erba transgene in the liver, thus effectively turning
off the liver clock while retaining a functional
SCN clock (Kornmann et al., 2007). They con-
cluded that some molecular oscillations in the liver
could still be driven by systemic factors coming
from the SCN clock.
Targeted gene knock-ins can be useful in deter-
mining whether mutations or polymorphisms in
humans or in other species are functional and
can specifically be used to investigate the
functions of particular protein domains or amino
acids. Rather than using pronuclear injection to
achieve random integration of the DNA of inter-
est, this technique uses an approach similar to the
knockout approach where DNA is integrated spe-
cifically at the target locus via homologous recom-
bination. In this way, a mutant allele can replace
the wild-type allele. This approach circumvents
positional effects (site of integration effects) and
gene dosage effects. The technique has been used
rarely in the circadian field but has been used to
introduce the hamster tau mutation (Arg178Cys)
into mice (Meng et al., 2008).
Reverse genetics using ENU
Using ENU mutagenesis in a reverse genetics
strategy is a powerful tool, complementary to for-
ward genetic and transgenic approaches. The high
efficiency mutation induction rate of ENU enables

the potential detection of multiple mutations
within a single gene (Quwailid et al., 2004). As
ENU-induced mutations are predominantly mis-
sense mutations, diverse mutations can be uncov-
ered at a single locus that might differentially
affect protein–protein interactions, protein–DNA
interactions, and amino acids that are likely to be
posttranslationally modified (e.g., phosphorylation,
acetylation, and ubiquitiation).
A number of research centers using ENU
mutagenesis have been developing archives of
frozen material from progeny of mutagenized
animals or from mutagenized ES cells. In the
Harwell ENU archive (http://www.har.mrc.ac.uk/
services/enu-dna-archive), parallel DNA and
sperm samples have been archived from over
10,000 animals, each animal potentially carrying
one nonsynonymous or nonsense mutation
per  2 Mb of DNA. Mutation detection within
the DNA archive can be carried out rapidly using
a number of methods including high-throughput
sequencing (Fig. 3). Many technologies have been
developed for high-throughput detection of
mutations including heteroduplex analysis and
denaturing high-performance liquid chromatogra-
phy (Quwailid et al., 2004). To date, users of the
Harwell ENU archive have screened coding
regions of interest and more than 250 mutations
have been found in over 80 genes, with a proven
multiple hit rate in all genes tested. Mouse lines
can subsequently be resurrected from the parallel
frozen sperm archive and mutant phenotypes
characterized. Many of the mutations identified
in the archive have been rederived in this way,
resulting in  70 new mouse mutant lines. To
date, specific genes have been screened using
this approach. With the advent of next-generation
sequencing, it is becoming increasingly realistic to
carry out whole exome sequencing on the entire
ENU archive uncovering thousands of new and
unique mouse mutations (Gondo et al., 2010).
Identification of ENU mutations in this
way posits the challenge of determining whether
the mutation identified will be functional. Conse-
quently, it is advisable to conduct in silico analysis
before deriving a particular line. For example,
259
phylogenetic analysis can be carried out to
determine whether the mutation lies in an evolu-
tionarily conserved motif. Moreover, the ability
to perform functional in vitro assays prior to
generating mutants is desirable. This type of
in vitro prescreen is ideally suited to investigating
mutations that might affect the circadian system.
In vitro assays in well-established cell lines are
widely used to determine phenotypes and pri-
mary effects of mutations in circadian biology.
Cell lines such as U2OS, NIH3T3 (Akashi and
Nishida, 2000), and Rat-1 fibroblasts (Balsalobre
et al., 1998; Izumo et al., 2003) are known to
express clock genes. The protein products of
these genes are also accumulated in a circadian
fashion and model events in vivo.
Real-time monitoring assays, making use of
clock gene promoters such as Per2, Bmal1, Rev-
erba, driving the expression of luciferase
reporters have been of great value in determining
the effects of a gene on circadian function (Baggs
et al., 2009). This approach can be modified to
focus on particular target molecules. For example,
Maier et al. carried out a large-scale RNAi screen
by knocking down all the known and predicted
kinases, phosphatases, and F-box proteins indi-
vidually and studied the effect on molecular
oscillations (Maier et al., 2009). This same
approach can be used to determine whether
overexpression of mutant proteins identified in
ENU screens can affect oscillator function.
The utility of the ENU gene-driven approach is
evident in the mouse behavioral field as more
novel mutant phenotypes are being identified,
including those where mouse knockout phenotypic
analysis has failed to reveal convincing data.
Mutants in Disrupted-in-Schizophrenia-1 (Disc1)
and Serine racemase (Srr) are recently identified
examples known to be associated with behavioral
and psychiatric disorders in humans. Q31L and
L100P are two missense mutations identified in
Disc1, the human homolog of which is a genetic
risk factor for schizophrenia (Clapcote et al.,
2007). Q31L displayed a recessive major depres-
sion phenotype, while the L100P proved to be a
dominant schizophrenia mouse model. Srr is also
260

Candidate gene selection

Exon specific amplification

by PCR

Wild-type female ENU treated male

(G0) (G0)

Heteroduplex analysis for

mutation detection

Wild-type Mutant Heteroduplexes Homoduplexes

Male G1 founder heat

cool
+ A C G T
A T G C A T G C

Sperm DNA
archive archive Mutation confirmation by DNA
sequencing

ENU mutant rederivation from

archived frozen sperm

Fig. 3. ENU gene-driven screen. Parallel DNA and sperm samples are archived from G1 founder males. Any gene can be targeted
using this approach. The DNA archive is amplified using primers flanking exons of candidate genes. The light scanner is one of a
number of high-throughput apparatus used to identify heteroduplexes in pools of amplified DNA. The presence of a mutation is
confirmed in individual DNA samples and by sequencing. After in silico analysis to confirm that the mutation is likely to be
functional, the mutant line is rederived from the parallel frozen sperm sample.

considered as a genetic risk factor for schizophre-
nia. ENU archive screening lead to the identifica-
tion of a nonsense mutation in Srr. Mice carrying
this mutation were found to display a knockout-
like phenotype with altered Srr mRNA levels
(50% reduction) and no SRR protein (Labrie
et al., 2009). An independent study that generated
Srr/ mice using the gene targeting approach also
reported similar phenotypes (Basu et al., 2009).
Large-scale mouse mutant production and
phenotyping pipelines
Although forward and reverse genetic screens car-
ried out by individual institutes and researchers
are proving extremely fruitful in terms of identifi-
cation and annotation of novel genes, much work
remains in annotating the whole genome. It is clear
that manipulation of the genome, through muta-
genesis, gene targeting, transgenics, or gene silenc-
ing in vitro, has become an effective strategy for
determining gene function, but now efforts are
focused on scaling these approaches up through
sharing resources and working systematically on
functional characterization of the genome, gene
by gene. Over the past few years, collaborative
efforts have been initiated that aim to broadly
annotate each gene in the mouse genome, through
generating and phenotyping mouse gene
knockouts, and share this information with the

wider research community. This is being achieved
through projects such as EUCOMM, KOMP,
EUMODIC, the European Mouse Disease Clinic;
and IMPC, the International Mouse Phenotyping
Consortium (Table 1).
EUCOMM, KOMP, and IKMC
These programs were set up to generate large
numbers of mutations by gene targeting in mouse
ES cells (Friedel et al., 2007; Ringwald et al.,
2011; Skarnes et al., 2011). Currently, many of
these mutations are being generated such that
they can be rendered into conditional alleles,
allowing Cre recombinase-mediated disruption
of gene function in a time- and tissue-specific
manner. The cell lines and mutants are available
as “knockout first” and “conditional-ready” mice.
The programs are now organized in such a way
that ES and mutant line generation is been shared
among the worldwide mouse genetics community.
EUMODIC
EUMODIC was originally established to com-
plement the EUCOMM program. The purpose
of the program was to generate mutant lines
from ES cells and phenotype  500 of these. In
phenotyping, the program uses a selection of care-
fully selected high-throughput tests (EMPReSS,
http://empress.har.mrc.ac.uk/) that act as a primer
for further characterization by interested inves-
tigators. Age-matched cohorts of 10 males and 10
females are subjected to phenotyping pipelines
that consist of morphological, metabolic, cardio-
vascular, and bone assessment as well as neuro-
behavioral, sensory, hematological, and immune
system characterization. Phenodeviants may be
selected by individual researchers for more specific
phenotypic assessment. For example, animals dis-
playing particular neurobehavioral phenotypes
may then be selected for circadian and sleep
phenotyping through wheel-running screens and
261
home cage analysis. Data for each of the several
hundred lines produced under this program is
now collated in the Europhenome database
(http://www.europhenome.org/databrowser/
viewer.jsp) (Mallon et al., 2008; Morgan et al.,
2010) and is searchable by phenotype or by gene
name. The database currently includes information
on circadian-relevant genes such as Nr1d1 (REV-
ERBa), Nr1d2 (REV-ERBb), and Hdac3. Nr1d2
mice, for example, show significant deficits in
respiratory, blood biochemistry, and immune
function parameters.
IMPC
As an extension of EUMODIC (in terms of scale
and number of partners involved), the IMPC was
formed with the aim of characterizing mouse
mutants produced by the International Mouse
Knock-Out Consortium, IMKC. The consortium
consists of large mouse genetics centers, including
institutes based in Europe, Australia, Japan,
USA, and Canada, which have the capacity
for large-scale, high-throughput phenotyping as
well the ability to generate “conditional-ready”
knockout mutants from targeted ES cells. This
approach will allow researchers to complement
their own reverse or forward genetic research by
having access to general multisystem phenotypic
data for individual gene knockouts. Mouse
mutants themselves will be available through a
number of searchable mutant archives including
the European Mouse Mutant Archive (EMMA,
http://www.emmanet.org/) and the Mutant Mouse
Regional Resource Centers Archive (MMRRC,
http://www.mmrrc.org/).
IMPC, like forward genetics, uses an unbiased
approach to understanding gene function where
no assumptions or hypotheses are made
concerning phenotypes expected. In this way, it
is likely that many new associations between gene
and biological function will be identified. As pre-
viously discussed, relying on knockout studies
may not always reveal all true functions of a gene
262
because of confounds such as redundancy and
homozygous lethality. This approach will, how-
ever, provide researchers with complementary
information regarding candidate genes and may
also highlight critical function of novel genes in
a particular biological domain. These programs
will also allow the researcher to consider spatio-
temporal aspects of gene function by conditional
ablation. Among important considerations for the
future in the identification and characterization of
mutants will be the continued development of high
throughput, automated data-capture technologies
to phenotype even complex behaviors, such as cir-
cadian rhythm disturbances.
The aim and subsequent success of the past
decade in genetic research has been the comple-
tion of genome sequencing of a number of differ-
ent model organisms and the development of
efficient methods for generating mutants. Future
aims lie in the domain of functional genomic
research, to annotate and assign function to each
gene through the phenotypic analysis of mouse
mutants. Large-scale projects such as IMPC will
propel this research forward, with the aim that
this should be completed within the next decade.
Future challenges will lie in developing an under-
standing of molecular pathways and interactions
of individual components within systems. It will
be important to consider strategies to model
multigenic traits, complex conditions, epistasis,
quantitative trait loci, and gene–environment
interactions. This remains a challenge for devel-
oping our understanding of circadian systems as
circadian phenotypes are frequently associated
with other disorders and systems such as metabo-
lism and diabetes (Froy, 2011), the cell cycle and
cancers (Rosbash and Takahashi, 2002), and
behavior and psychiatric disease (Wulff et al.,
2010). In order to rise to this challenge, it will be
necessary to combine forward and reverse genetic
approaches together with in vitro approaches.
Our ability to target multiple components of a par-
ticular system will pave the way to understanding
how genes interact to direct mammalian biological
processes.
Acknowledgments
This work has been supported by the MRC and
by the 6th Framework Project EUCLOCK
(No. 018741).
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 16

In search of a temporal niche: Social interactions

Alexandra Castillo-Ruiz{,*, Matthew J. Paul{ and William J. Schwartz{

{
Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA
{
Department of Psychology, University of Massachusetts Amherst, Amherst, MA, USA

Abstract: Circadian rhythms can be entrained to periodic cues in the environment including the solar
day, food resources, and temperature. Work on a variety of organisms has suggested that social
interactions within and between species may also influence circadian rhythmicity, but conceptual and
technical difficulties relating to animal models, housing environments, rhythm assays, and experimental
design have complicated mechanistic investigations in the laboratory. We review these issues and
introduce the gregarious Nile grass rat, Arvicanthis niloticus, as a suitable model for research on this
problem. Understanding social influences on temporal organization at this supra-organismal,
community level is of considerable translational value, as its implications range from conservation
biology to human health.

Keywords: circadian clock; circadian rhythm; entrainment; masking; behavioral ecology.

Introduction environment. These temporal programs are not

Circadian clocks are pervasive across all
kingdoms of life, from unicellular to multicellular
organisms. Their ubiquity has been taken as evi-
dence for their adaptive value. It is believed that
circadian rhythms evolved not only to provide
an internal temporal organization for physiologi-
cal and behavioral processes but also to help
organisms anticipate challenges and opportunities
that occur predictably and periodically in the
*Corresponding author.
Tel.: þ1-508-856-1128; Fax: þ1-508-856-6778
E-mail: alexandra.castillo-ruiz@umassmed.edu
rigid, however, and respond to changes in photo-
period, food resources, temperature, as well as
to other organisms. The influence of abiotic
factors on circadian rhythmicity is reviewed by
Hut and collaborators in this issue (Hut et al.
2012), while here we focus on inter-organismal
effects on circadian clock function, a topic which
we term socio-chronobiology.
Unlike the typically uneventful life in the labo-
ratory setting, life in the natural world is a con-
stant struggle to survive, procure resources, and
reproduce. Survival hinges on finding favorable
spatial and temporal niches, and conspecific or
heterospecific individuals can impact the niche

http://dx.doi.org/10.1016/B978-0-444-59427-3.00016-2 267
268
through cooperation, competition, or predation,
among other interactions. Previous critical reviews
have discussed the effects of social cues on circadian
rhythmicity (Davidson and Menaker, 2003; Favreau
et al., 2009; Mistlberger and Skene, 2004; Regal and
Connolly, 1980), but they have focused mainly on
whether social cues can entrain organisms, sp., on
their ability to act as zeitgebers to synchronize the
behavioral rhythms of conspecifics. Here, we pro-
vide some examples in different taxa of the wide
range of social interactions that might be considered
to be socio-chronobiological, outline the search for
underlying mechanisms, and highlight the concep-
tual and technical difficulties that have complicated
research progress.
Interspecific relationships and circadian rhythms
Mutualism
Cooperation between individuals of different
species is known as mutualism. Theoretically,
the coordination of timing of behavioral and
physiological events between allies in a beneficial
relationship ought to be extremely important for
conservation of the partnership. We are aware
of only a handful of studies that have investigated
circadian rhythms in mutualistic species. As an
example, Flint et al. (2005) found that bacterial
symbionts (Epulopiscium spp.) living in the intes-
tinal tract of the surgeonfish, Naso lituratus, show
a prominent daily cycle, remaining dormant dur-
ing the night (when the fish is mostly inactive)
and proliferating during the day. The authors
argue that this temporal pattern may be driven by
the lower availability of host nutrients during the
night. Wier et al. (2010) analyzed the symbiotic
relationship between the Hawaiian squid,
Euprymna scolopes, and its symbiont, the lumi-
nous bacteria Vibrio fischerii. Rhythmic bacterial
bioluminescence, highest during the night, is used
by the host for its nocturnal behaviors; and rhyth-
mic bacterial expulsion from the squid’s light-
emitting organ, highest at dawn, insures stability
of symbiont population size. Study of gene expres-
sion patterns revealed corresponding rhythms of
transcription that time the changes in squid epithe-
lial (light organ) anatomy with switches in bacterial
metabolic pathways. Although the cues that couple
such interlocked rhythms are not known, or
whether they are circadian clock-driven, these
studies of mutualism suggest that temporal coordi-
nation may be key for the evolution and conserva-
tion of interspecific alliances.
Antagonistic interactions
Among species, sharing the same time preference
for activity can have detrimental consequences
for one of the parties, such as infestation by a par-
asite, death from starvation, or being eaten by a
predator.
Parasitism
Parasites depend on their hosts for survival and
reproduction. Presumably, they have been sub-
jected to a strong selective pressure to evolve
traits that increase their chances of interactions
with hosts, and one of these traits likely involves
temporal coordination of behavior. For instance,
schistosomiasis is a parasitic disease caused by
trematodes of the genus Schistosoma. The worms
have complex life cycles, in which eggs released
into fresh water hatch into miracidia, infect an
intermediate (snail) host to develop as sporocysts,
and then emerge as cercariae to infect and produce
eggs in their definitive mammalian host. Cercarial
emergence is rhythmic, appears to be genetically
determined (Theron and Combes, 1988), and
may exhibit a phase that matches the activity
rhythm of the intended host (for Schistosoma
japonicum, late afternoon for a nocturnal rodent
reservoir, early morning for a diurnal cattle reser-
voir; Lu et al., 2009). The possible significance of
such coordination is exemplified by a recent study
(O’Donnell et al., 2011) demonstrating that fitness

in malaria parasites (Plasmodium chabaudi) is
increased when both the parasites and their rodent
hosts are synchronized to the same light–dark
(LD) cycle as compared to when they are under
a mismatch condition. Such data hint that circadian
rhythms may be a force driving evolution.
As with mutualism, the mechanisms underlying
such interspecific coordination are not known.
Toledo et al. (1999) examined the rhythmic pat-
tern of larval emergence of the intestinal parasite
Euparyphium albuferensis; this rhythm does not
appear to be under the control of an endogenous
clock because it cannot be identified under con-
stant conditions. Future studies on the chronobi-
ology of parasitism will need to assess the
rhythmicity of hosts as well as of parasites, given
that parasites may affect the rhythms of their
hosts (Lima-Camara et al., 2011).
Competition
Competition in the face of limited resources can
be mitigated by the separation of species in space
and by diet but also in time (Schoener, 1986). For
example, a redistribution of nighttime activity
patterns has been demonstrated in Gerbillus
allenbyi and G. pyramidum, two species of sand
dune gerbils, studied separately or together in
100 m2 seminatural grids. When separated,
both species show nocturnal activity mostly con-
fined to the first part of the night; but when
together, the activity of the subordinate species,
G. allenbyi, is displaced to the second half of the
night (Ziv et al., 1993). Temporal partitioning
also occurs between the cotton rat (Sigmodon
hispidus) and prairie vole (Microtus ochrogaster)
studied in 4 m2 arenas, with voles becoming most
active at phases when rats are least active. Spatial
partitioning arises additionally only in the pres-
ence of reproductive rats, which show higher
levels of interspecific aggression than nonrepro-
ductive rats (Glass and Slade, 1980).
Even more dramatic are reports of interspecific
interactions that lead to an inversion of daily
269
activity patterns. Two species of closely related
spiny mice (Acomys cahirinus and A. russatus)
cohabit the same rocky terrain of the Dead Sea
Valley, with overlap in microhabitat use, home
ranges, food habits, and reproductive period (for
review, see Kronfeld-Schor and Dayan, 2008).
Notably, A. cahirinus is nocturnal and A. russatus
is diurnal. Shkolnik (1971) attributed this pattern
to interspecific competition; upon removal of
A. cahirinus from the field site, A. rusattus expands
its activity profile to the night, suggesting that the
presence of A. cahirinus prevents A. russatus from
being active at its preferred phase of the daily
cycle. When A. cahirinus is released back into the
field site, both species reestablish their temporal
segregation. The nature of the limiting resource
(s) is unknown. More recently, laboratory studies
of Acomys have been informative and are
reviewed in a later section of this review.
A second example relates to the American
mink (Neovison vison), which was introduced to
Great Britain in the early 1900s and became
established in the absence of its natural com-
petitors, the Eurasian otter (Lutra lutra) and the
European polecat (Mustela putoris). Under these
conditions, Harrington et al. (2009) observed in
the 1990s that the mink was predominantly active
during the night. Ten years later, however, after
the native competitor populations had recovered,
the mink appeared to have switched to a day-
active pattern.
To our knowledge, only one laboratory study
has explored whether time-dependent exposure
to an interspecific competitor can induce acute
phase shifts of activity rhythms. Scheibler and
Wollnik (2009) introduced a Mongolian gerbil
(Meriones unguiculatus) housed in a small cage
into the home cage of a desert hamster (Pho-
dopus roborovskii) for 2 h at eight time points
spanning the 24-h circadian cycle in constant
darkness; in this pairing, gerbils are believed to
be dominant and hamsters subordinate. The
authors reported that hamsters exhibited phase
advances of their circadian wheel-running rhythm
during the subjective day and mid-subjective
270
night. Shortening of hamster free-running period
with increased wheel running was also observed
after gerbils were introduced during the subjective
day, perhaps related to increased arousal.
Predator–prey
Throughout the day/night cycle there are times at
which activity outside the nesting site or burrow
can be extremely dangerous due to the presence
of predators. Thus, most predator avoidance
strategies involve the avoidance of increased pre-
dation risk times, and it has been postulated that
these strategies have enabled the coexistence of
predator and prey (for review, see Kronfeld-
Schor and Dayan, 2003). The sudden arrival of a
novel predator to a species niche could poten-
tially induce a redistribution of activity to less
dangerous times, and examples of such behavioral
plasticity have been reported. Naturally, noctur-
nal wild rats (Rattus norvegicus) become diurnal
when a predator, the red fox (Vulpes vulpes), is
in the vicinity. When exposure to the fox is exper-
imentally prevented, the rats revert to their noc-
turnal behavior (Fenn and Macdonald, 1995).
A similar phenomenon was observed for Santa
Cruz foxes (Urocyon littoralis; Swarts et al.,
2009). In the 1990s, before predatory golden eagles
(Aquila chrysaetos) colonized Santa Cruz Island,
foxes were active at all times of day and night.
After the eagles arrived, foxes showed a prefer-
ence for nocturnal activity. The authors argued
that this change was related to predator-induced
selection for a nocturnal trait but a more recent
report demonstrates that eagle removal efforts
have been associated with a rapid vulpine rever-
sion to diurnality (Hudgens and Garcelon, 2011).
The neurobiological mechanisms responsible for
adaptive temporal switches in response to interspe-
cific antagonistic interactions are unknown. Specif-
ically, might they represent altered phase angles of
entrainment to the day–night cycle secondary to
changes in the circadian clock’s free-running
period, perhaps related to arousal? Or are they
caused by changes in the strength and/or sign of
coupling mechanisms that link the clock to subor-
dinate downstream oscillators responsible for
rest-activity rhythms? Or do social interactions
modulate or modify rhythm phase and waveform
directly, by circumventing or overriding the clock
(a mechanism referred to as masking), rather than
by resetting (entraining) the clock’s oscillation?
Conspecific relationships and circadian rhythms
Eusocial insects
Of insects that live in colonies with highly devel-
oped social structures—including cooperative
care of the young and individual division of
labor—the most extensively studied from a socio-
chronobiological view is the honeybee (Apis
mellifera; for reviews, see Bloch, 2010; Moore,
2001). Worker honeybees include younger nurses,
which care for the brood continuously and are active
during both the day and night; and older foragers,
which periodically leave the hive and express robust
circadian activity rhythms. Social mediation of this
temporal organization becomes apparent when
the colony experiences a shortage of nurses. Some
foragers return to nursing and adopt an arrhythmic,
persistently active state (Bloch and Robinson,
2001). Nursing arrhythmicity represents a context-
dependent behavioral plasticity, since robust circa-
dian rest-activity rhythms can be expressed by
nurse-age bees if they are restricted to broodless
combs or transferred from the cyclically illuminated
hive to constantly dark laboratory conditions
(Shemesh et al., 2010). These authors have also
provided indirect evidence to suggest that
arrhythmicity induced by brood contact is not due
to an arrest of the central circadian pacemaker; it
seems instead that at least some clock cells must be
keeping track of the passage of time. The lack of
overt behavioral rhythmicity could be the result
of desynchronized or low amplitude cellular
oscillators, and/or masking or uncoupling of oscilla-
tor outputs from downstream effectors.

Maternal entrainment
In mammals, the circadian clock in the fetus
appears to be oscillating in utero and entrained by
the mother’s rhythm; in altricial rodent species
(sp., hamsters, mice, and rats), there is also evi-
dence that mothers entrain the rhythms of their
immature offspring during the postnatal period,
before the pups become responsive to the external
LD cycle (for review, see Davis and Reppert,
2001). The influence of maternal care has been
examined by cross-fostering and maternal depriva-
tion studies; in the former, prenatal natural
mothers are replaced by postnatal foster mothers,
and in the latter, nursing mothers are cyclically
present or absent from their pups. Maternal rhyth-
micity is required for pup synchronization (Davis
and Gorski, 1988; Jud and Albrecht, 2006; Reppert
and Schwartz, 1986), and in its absence pups within
a litter are unable to entrain each other’s rhythms
(Hiroshige et al., 1982; Reppert et al., 1984). Ohta
et al. (2003) studied the entrainment of two groups
of neonatal rats (Rattus norvegicus) blinded by
bilateral orbital enucleation at birth, and subse-
quently, nursed by foster mothers entrained to a
LD cycle; in one of the two groups, mothers were
removed during the light phase (when they nor-
mally would be nursing) and returned during the
dark phase each day for 6 days, after which they
were present continually until the pups were
weaned at postnatal day 20. This maternal depriva-
tion schedule resulted in the phase reversal of pup
rhythms of “clock” gene expression in the
suprachiasmatic nucleus (SCN) on postnatal day 6
and of locomotor activity rhythms after weaning.
While the data are consistent with maternal
entrainment of the molecular circadian clock in
the neonatal SCN, the authors also noted the possi-
bility that entrainment may not have been via
maternal time cues but instead due to the stress
that was associated with her periodic absence.
The strength and persistence of postnatal
maternal entrainment differs among species. In a
precocious rodent species, the common spiny
mouse (A. cahirinus), the eyes open shortly after
271
birth and postnatal maternal influences on pup
rhythmicity appear to be minimal (Weaver and
Reppert, 1987). In a marsupial species, the gray
short-tailed opossum (Monodelphis domestica),
the offspring are born extremely immature but
are directly exposed to the environmental LD
cycle while attached to their pouchless mothers;
in this species also, the dam wields no influence
on the timing of pup rhythmicity (Rivkees and
Reppert, 1990). At the other extreme is the Euro-
pean rabbit (Oryctolagus cuniculus): rabbit
mothers are absent from the nest except to nurse
their pups for a 3- to 4- min interval once daily
(for review, see Jilge and Hudson, 2001). An
endogenous anticipatory rhythm in the pups is
entrained to this maternal rhythm, including a
rhythm of “clock” gene expression in their SCN
(Caldelas et al., 2009).
Social synchronization
Social synchronization of the temporal behaviors
of adult conspecifics should allow for the realiza-
tion of common goals, for example, defending
against predators, hunting in packs, or huddling
as a means of energy conservation. While there
have been tantalizing hints of social entrainment
in some (but not all) studies, it has been difficult
to find robust, reliable, and experimentally tracta-
ble animal models for further analysis.
Field and laboratory
In the field, temporal synchronization among
animals has been inferred from studies of Canadian
beavers (Castor canadensis) living together as a
family unit, as they exhibit a single coherent free-
running rhythm in daily noise while they overwinter
in constant darkness under thick ice (Bovet and
Oertli, 1974). In the laboratory, several studies have
demonstrated that co- or group-housed animals—
including killifish (Fundulus heteroclitus; Kavaliers,
1980b), deer mice (Peromyscus maniculatus;
272
Crowley and Bovet, 1980), palm squirrels
(Funambulus pennanti; Rajaratnam and Redman,
1999), honey bees (Apis mellifera; Frisch and
Koeniger, 1994), and fruit flies (Drosophila
melanogaster; Levine et al., 2002)—may display
coherent or converging activity rhythms in constant
lighting conditions but their reliance on group activ-
ity data has generally prohibited much further
mechanistic analyses. The expression of a single
free-running rhythm in group-housed individuals
could be due to social entrainment (mutual or unidi-
rectional), but it could also reflect similar phases and
periods before cohabitation or a masking effect of
one, possibly socially dominant, individual.
Researchers have used different strategies to dis-
tinguish individual activity under social conditions.
Some have housed animals in close proximity to,
but not in direct contact with, each other, or
together but separated by a barrier (wire mesh,
clear perforated plastic, or a restraining cage);
others have restricted the interval of social contact
to a few hours per day. Under such housing
conditions, social effects on the circadian system
have been inconsistent (e.g., in golden hamsters,
Mesocricetus auratus; Davis et al., 1987; Gattermann
and Weinandy, 1997; Mistlberger et al., 2003;
Mrosovsky, 1988; Refinetti et al., 1992) or relatively
modest, with observed alterations in activity levels,
reentrainment rates to a photically induced phase
shift, or free-running periods in constant darkness
(e.g., in degus, Octodon degus; Goel and Lee,
1995, 1996, 1997). One study has demonstrated syn-
chronous activity due to social cues in individuals
separated by a barrier. Schneider’s roundleaf bats
(Hipposideros speoris) individually caged in a cave
without access to light synchronize their activity
rhythms to local time, in phase with their free-living
conspecifics that leave the nest daily around sunset;
a control bat caged in a cave devoid of other
bats instead exhibited a free-running rhythm
(Marimuthu et al., 1981).
Refinetti et al. (1992) co-housed two hamsters
in direct physical contact with each other but with
markedly different free-running periods: one was
entrained to a short 23.3 h LD cycle while the
other was blinded and free ran with a period
> 24 h. There was no synchronization or relative
coordination of the activity of the blind hamster
to that of the entrained one. The conclusion from
all these studies has been that social factors can
provide only weak inputs to the circadian clock.
Mixed sex interactions
Strategies similar to those described above have
been employed to study the possible interactions
of paired male and female hamsters or degus.
Male and female hamsters either closely or co-
housed without physical contact, either continu-
ally for 6 weeks (Davis et al., 1987) or for 3 h
per day for intervals ranging from about 2 to 6
weeks (Aschoff and von Goetz, 1988), show no
change in the free-running periods of their activ-
ity rhythms, although the males show masking of
their rhythms by the females. Honrado and
Mrosovsky (1989) exposed males for 3 h to
estrous females in restraining cages—or for 1 h
to actually mate—and reported more rapid
reentrainment to a phase-advanced LD cycle for
the exposed but not for the mated males. When
these authors entrained males to an LD cycle
and then exposed them to females in restraining
cages for 1 h daily at a phase 4 h before lights-
off, the males exhibited advances in locomotor
activity onsets that habituated after repeated
exposure (Honrado and Mrosovsky, 1991). In
degus housed together (separated by a wire mesh
barrier), females reentrain faster to a phase-
advanced LD cycle when co-housed with another
female but not with a male (Goel and Lee, 1995).
Challenges in bringing social interactions into
the laboratory
While experimentation in the laboratory has obvi-
ous advantages, especially for elucidation of pos-
sible molecular and neurobiological mechanisms,
it is an unnatural setting. Researchers intent on

analyzing social interactions under these con-
ditions need to be aware of special considerations
with regard to their choice of animal subjects,
caged environment, selected assays, and experi-
mental design.
Animals
One difficulty with domesticated animals is that
their physiology and behavior may be quite dif-
ferent than that of their counterparts in the
wild (for review, see Calisi and Bentley, 2009).
It is now well known that some prototypically
“nocturnal” laboratory rodents are not necessar-
ily nocturnal in their native habitat (e.g., emer-
gence from the burrow in the golden hamster;
Gattermann et al., 2008); laboratory-bred and
wild-derived hamsters also differ in other physio-
logical and behavioral traits (Krause and
Schuler, 2010). Golden spiny mice (A. russatus)
and cururos (Spalacopus cyanus), while nocturnal
in captivity (Begall et al., 2002; Cohen and
Kronfeld-Schor, 2006), are evidently diurnal
when free-living (Levy et al., 2007; Urrejola
et al., 2005). Obviously, these kinds of differences
between laboratory and field could also impact
the effects, and even the presence, of social
influences on rhythms.
Not often considered in socio-chronobiological
experiments is whether the subjects are familiar
with each other. The significance of this variable
is exemplified by work with common marmosets
(Callithrix j. jacchus), which are social primates;
paired animals may synchronize their free-
running circadian periods if they are in full social
contact and know each other prior to the experi-
ment (Erkert and Schardt, 1991). Awareness of
sex differences in activity patterns in the wild
(e.g., as dramatically expressed by some mustelid
species; Marcelli et al., 2003; Zschille et al., 2010)
is also important for interpreting observed
patterns in the lab. Lastly, dominant/subordinate
relationships must be considered. There is a
report of laboratory group housing of long-tailed
273
field mice (Apodemus sylvaticus), in which three
subordinate mice were observed to avoid the time
of peak out-of-burrow activity of a dominant
mouse (Bovet, 1972). In rats, a single episode of
social defeat influences the amplitude of locomo-
tor, heart rate, and temperature rhythms for sev-
eral cycles, apparently by a masking mechanism
(Meerlo et al., 1999). And Hansen and Closs
(2005), investigating how food depletion can alter
the daily activity pattern in the giant kokopu
(Galaxias argenteus), demonstrated that domi-
nant fish are the ones that switch from nocturnal
to diurnal activity when food is limited.
Environment
In the field, species must make complex decisions,
“eat or be eaten,” so the relative safety of the lab
undoubtedly eliminates most of the abiotic and
biotic factors that drive the phenotypic plasticity
of animals in their natural temporal niche. In a
wild population with underlying genetic variation,
traits that are induced in response to such envi-
ronmental factors may then become genetically
determined, by a process of directional selection
on extreme phenotypes (Ghalambor et al.,
2007). In this way, chronotypic plasticity that
was originally induced by a social interaction
might become selectively assimilated over time
as an evolved circadian activity pattern, that is,
as a “ghost of selection past” (Ghalambor et al.,
2007) that outlasts the initial social stimulus. It is
now appreciated that such adaptive evolution
can occur on ecological time scales (Carroll
et al., 2007).
Consider the two species of spiny mice
(A. cahirinus and A. russatus) described in an ear-
lier section of this review; A. cahirinus is noctur-
nal while A. russatus is diurnal, and observations
in the 1970s suggested that the presence of
A. cahirinus competitively displaces A. russatus
from the nocturnal niche (Shkolnik, 1971).
Indeed, individually caged A. russatus maintained
in the laboratory in a 12 h:12 h LD cycle primarily
274
exhibits nocturnal activity and temperature
rhythms (Cohen and Kronfeld-Schor, 2006).
However, A. russatus exposed to A. cahirinus in
the room, or in the same cage, does not become
diurnal (Cohen, 2004); and, on the other hand,
A. russatus held in 1000 m2 field enclosures in
the Judean desert, without A. cahirinus present,
is diurnal, becoming immediately nocturnal when
introduced into the laboratory (Levy et al., 2007).
Thus, diurnality in A. russatus does not appear to
be fully explained by behavioral plasticity in
response to interference or resource competition
with A. cahirinus. Perhaps such competition in
the past, and/or other nonsocial factors, produced
selective forces to which A. russatus became
highly adapted.
Assays
If the experimental question is whether a social
cue is capable of acting as an independent zeitge-
ber for entraining the master circadian clock in
the SCN, then longitudinal assessment of a single,
precise, circadian marker rhythm that exhibits
unambiguous phase and period (e.g., wheel-
running activity in rodents) makes sense. But if
social forces act on alternative substrates—on
rhythm amplitude or waveform, phase of entrain-
ment to a geophysical cycle, or mechanisms
downstream or separate from those that generate
the locomotor rhythm—inspection of circadian
wheel-running records will be an insensitive
and/or irrelevant assay. In root voles (Microtus
oeconomus) under winter snow and lacking daily
photo-entrainment, social interactions synchronize
the activities of individuals at the ultradian rather
than the circadian level (Korslund, 2006). The right
assays are also required for understanding the
effects of social cues on the timing of bird song. Male
chipping sparrows (Spizella passerina) shorten their
dawn chorus in response to the removal of neigh-
boring males but not to the removal of their female
mates; they do increase their daytime singing
after removal of their mates but not neighboring
males (Liu, 2004). In black-capped chickadees
(Poecile atricapillus) it is the phase (onset) of the
dawn chorus that is modified (advanced) by the
appearance of a simulated conspecific competitor
(Foote et al., 2011).
Design
Assuming that “social coupling” is relatively weak
(at least as compared to light), and that individual
animals represent nonidentical oscillators
governed by their own intrinsic properties, then
one would predict that strong interindividual
effects might be precipitated only under particu-
lar circumstances, sp., when oscillators are in
direct physical contact for a relatively long period
of time and their respective free-running periods
are relatively close to one another (Winfree,
2001). Thus, actual prolonged cohabitation might
lead to robust effects on the rhythmicity of co-
housed individuals, effects not easily reproduced
by indirect or pulsatile social contacts. As
described previously in this review, relatively few
studies have recorded the rhythms of individuals
during long-term cohabitation.
Theoretically, network strength under such
conditions should increase with the number of
coupled oscillators (Kuramoto, 1984). Indeed,
groups of fruit flies (Levine et al., 2002), colonies
of honey bees (Frisch and Koeniger, 1994), and
shoals of white suckers (Catostomus commersoni
Kavaliers, 1980a) do exhibit more precise and/or
stable circadian periods than do the individual
flies, bees, or fish. At a different level of biological
organization, similar effects have been reported
for SCN neuronal oscillators (Herzog et al., 2004;
Liu et al., 2007; Webb et al., 2009).
Advances have been made in identifying candi-
date cues that might mediate social coupling,
especially chemical (in spiny mice: Fluxman and
Haim, 1993; Haim and Rozenfeld, 1993; in degus:
Governale and Lee, 2001; Jechura et al., 2006; in
fruit flies: Krupp et al., 2008), and gonadal hor-
monal (in degus: Jechura and Lee, 2004; Jechura

et al., 2003). But it is probable that multiple inter-
acting signals play physiological roles, as is likely
the case for melatonin and food intake in mater-
nal-fetal entrainment (for review, see Davis and
Reppert, 2001). Arousal may be a critical variable.
Some bird species are reported to entrain to
daily playbacks of conspecific birdsong (Gwinner,
1966; Menaker and Eskin, 1966), but house
sparrows (Passer domesticus) can also entrain to
periodic noise (Reebs, 1989), highlighting a possi-
bly permissive role for nonspecific arousal.
For the future
We have recently begun a research program
in mammals (initially in hamsters; Paul and
Schwartz, 2007) that seeks to address some of
these socio-chronobiological challenges.
The unstriped Nile grass rat, Arvicanthis
niloticus (Fig. 1), native to sub-Saharan Africa,
was introduced to circadian rhythms research in
the early 1990s (Katona and Smale, 1997) and
should be an interesting animal subject for future
research on social interactions and rhythms. It
has already served as a useful model for studies
on the neural substrates of diurnality, as it
exhibits robust diurnal activity patterns in the
field and in the lab (Blanchong and Smale, 2000;
McElhinny et al., 1997). Notably, some individuals
Fig. 1. Grass rat family in captivity (photo courtesy of A. C. R.).
275
show behavioral plasticity in the lab, with a noc-
turnal activity pattern upon exposure to a running
wheel, returning to a diurnal pattern after wheel
removal (Blanchong et al., 1999). Another nota-
ble trait of the grass rat is its social behavior. Wild
colonies consist of several males, females, and
their offspring; adult males migrate to other col-
onies while adult females do not disperse (Packer,
1983; Senzota, 1990). Additionally, although little
is known about the male’s role in parental care in
the wild, observations in captivity suggest that he
participates in the rearing of offspring; in the lab,
the male not only nests with the female and her
offspring but also grooms the pups even as
weanlings. Importantly, due to continuous efforts
to prevent inbreeding in captivity, grass rats are
more likely than the nocturnal rodents commonly
used in circadian research to resemble their wild
counterparts.
In order to longitudinally measure rhythmicity in
individual, co-housed grass rats, we are implanting
miniature temperature sensors (ibuttons; Dallas
Semiconductor DS1922L-F5, Maxim Integrated
Products, Inc., Sunnyvale, CA), which are com-
mercially available for this purpose. Temperature
can be recorded at an accuracy of 0.5  C from
 10  C to þ 65  C and a resolution of 0.0625  C
by a digital thermometer at a sampling rate
from 1 s to 273 h. Each ibutton contains 8 kB of
datalog memory for storage of up to 4096 16-bit
temperature readings. Data collection can begin
immediately after implantation or programmed
to begin after a user-defined delay. The device is
encased in stainless steel, resulting in a height,
radius, and weight of 5.89 mm, 17.35 mm, and
2.95 g, respectively.
Of note, we believe that a crucial part of our
program will be the application of a rigorous
quantitative method—based on wavelet analysis—
that will allow us to analyze rhythm phase, ampli-
tude, and period at multiple scales. In fact, other
circadian researchers have begun to use wavelet
transforms, for their rigor, utility, and minimal
assumptions for analyzing nonstationary datasets
(Leise and Harrington, 2011). Wavelet transforms
276
do not have a single set of basis functions (like
the Fourier transform, which utilizes only sines
and cosines) but an infinite set of possible basis
functions.
Coda
Understanding how circadian rhythmicity is
endogenously generated and entrained to the
LD cycle has been the focus of considerable
research—from molecular to organismal levels—
for the past several decades. Relatively, less
attention has been directed to investigating the
generation and entrainment of rhythmicity at
the community level. We know relatively little
about the importance and mechanisms that regu-
late the timing of social interactions, whether syn-
chronizing or segregating; or the role of circadian
timekeeping at the supra-organismal level; or how
the interaction of social and circadian systems
ensures adaptation and economy in the temporal
domain. New ideas, technologies, and paradigms
are now emerging to build on the pioneering
work reviewed here. Chronobiologists have
already benefited from their collaborations with
molecular biologists, photobiologists, and applied
mathematicians, and we are looking forward to
expanded interactions with behavioral ecologists,
evolutionary biologists, and social neuro-
scientists. And in so doing, we need to attend
to ultimate-level (why) questions in addition to
proximate-level (how) ones.
Progress is also being catalyzed by new attitudes.
While past research has concentrated on entrain-
ment of the clock as a mechanism for social effects,
masking of rhythms has been viewed—at least in
some chronobiological circles—almost pejora-
tively. For social interactions that ultimately confer
survival and reproductive benefits to individuals,
there is beauty in elucidating diverse mechanisms
and convergent evolution in multiple species, with
differential roles for entrainment, masking, and
other mechanisms not fully understood (Thornton
et al., 2010). Inbred lines of model organisms may
not be the most informative animals to investigate
these questions (Smale et al., 2005), in part because
countless generations have been selected for par-
ticular traits (like tameness).
Socio-chronobiology has enormous transla-
tional value. Its implications range from pest con-
trol (e.g., to plan strategies to control population
growth), animal welfare (e.g., to identify the best
housing situation for laboratory animals), conser-
vation biology (e.g., to understand human effects
on animal populations, like coyotes, Canis latrans;
Kitchen et al., 2000), and clinical medicine (e.g.,
to develop strategies to prevent and control para-
sitic infection while enhancing the activities of
beneficial gut flora). Many blind people somehow
entrain to the 24-h day (Klerman et al., 1998),
presumably through nonphotic cues, and a better
understanding of the role of social influences may
lead to new strategies for healthy adjustment to
life on a rotating world.
Acknowledgments
This work is supported in part by R01 GM094109
(W. J. S.). The contents of this review are solely
the responsibility of the authors and do not neces-
sarily represent the official views of the National
Institutes of Health.
Abbreviations
LD light–dark
SCN suprachiasmatic nucleus
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 17

In search of a temporal niche: Environmental

factors

Roelof A. Hut{,*, Noga Kronfeld-Schor{, Vincent van der Vinne{ and

Horacio De la Iglesia}

{
Chronobiology unit, Center for Behaviour and Neurosciences, University of Groningen, Groningen, The Netherlands
{
Department of Zoology, Tel Aviv University, Tel Aviv, Israel
}
Department of Biology and Program of Neurobiology and Behavior, University of Washington, Seattle, USA

Abstract: Time as an ecological niche variable or “temporal niche” can be defined in the context of the
most prominent environmental cycles, including the tidal cycle, the lunar day and month, the solar day,
and the earth year. For the current review, we focus on the 24-h domain generated through the earth’s
rotation around its axis (solar day). The daily environmental cycles of light and temperature are a
dominant ecological factor generating a variety of adaptations among animals. In this review, we
describe these adaptations with a special focus on the visual system and on the adaptive plasticity of
activity patterns. Our goals are: (1) Underscore the importance of the 24-h time axis as critical
variable in the ecological niche. (2) Highlight cases of temporal niche switches at the evolutionary
timescale (phylogenetic level). (3) Review temporal niche switching within an individual’s lifespan. (4)
Evaluate possible underlying mechanisms for temporal niche switching. (5) Describe a new hypothesis
of circadian thermoenergetics which may explain several cases of temporal niche switching in
mammals. With this, we hope to inspire experiments under natural conditions or more complex
laboratory environments, aimed to reveal environmental factors and mechanisms underlying specific
temporal programs.

Keywords: temporal niche switching; circadian thermoenergetics hypothesis; nocturnality; diurnality; eye
properties; visual system; environment; evolution.

Introduction

*Corresponding author. More than half a century after G. Evelyn

Tel.: þ31503632060; Fax: þ 31503632148 Hutchinson’s crisp definition of ecological niche
E-mail: r.a.hut@rug.nl as a multidimensional space delineated by the

http://dx.doi.org/10.1016/B978-0-444-59427-3.00017-4 281
282
range of resources in which a species survives
and reproduces (Hutchinson, 1957), “time” still
struggles to make it into the list of critical niche
orthogonal axes (Kronfeld-Schor and Dayan,
2003, 2008). Those of us studying how time
shapes physiology and behavior take the inclusion
of time in this multidimensional space for granted;
after all, it is hard to imagine the survival of Arctic
birds hatching just before the winter solstice or
honeybees foraging at midnight. Yet, we feel that
studies that have addressed which environmental
factors and mechanisms shape this temporal niche
deserve a closer look.
The daily temporal niche of a species is usually
defined as the time of day at which its individuals
display locomotor activity. Thus, diurnality and
nocturnality are defined as temporal patterns with
activity, respectively, during daytime and night
time. Similarly, crepuscularity is characterized by
activity around dawn and dusk and cathemerality
by activity both during the daytime and nighttime.
These definitions have been biased to a great
extent by the methodology to measure locomotor
activity. For instance, measuring running-wheel
activity may portray a different locomotor-activity
pattern than patterns emerging from infrared-beam
interruptions. The presence of a running-wheel per
se can even cause a shift in temporal distribution of
locomotor activity as it is the case in Nile rats
(Blanchong et al., 1999), degus (Kas and Edgar,
1999), Mongolian gerbils (Weinert et al., 2007),
and golden spiny mice (Table 1; Cohen et al.,
2009). Importantly, locomotor activity displayed by
an animal under laboratory conditions may—
regardless of the measurement method—represent
the expression of different behavioral programs
under natural conditions (Fig. 1). In other words,
wheel running may reflect foraging behavior,
predator avoidance, mate searching, or even escape
behavior. Thus, it is important to define the con-
ditions under which locomotor activity is measured
and the methodology.
Circadian studies under narrow laboratory
conditions have led to a dichotomous view of
temporal niche in which animals display either
nocturnal or diurnal phenotypes. However, the
temporal programs under natural conditions,
where environmental factors are more variable
and the behavioral repertoire of an animal has a
wider range, are likely more complex and more
plastic. A classic example of this complexity is
the locomotor-activity patterns migratory birds
display in the laboratory. Most birds are diurnal
and display behaviors such as foraging during
the daytime; under laboratory conditions, this is
usually manifested by increased locomotor activ-
ity during the light phase. During the migratory
season, birds migrate during the night; under the
proper photoperiod, this migratory activity is
manifested in the laboratory as nocturnal restless-
ness (Bartell and Gwinner, 2005; Coverdill et al.,
2008; Gwinner, 1996). This nocturnal-locomotor
activity can be distinguished from the activity
birds display during the light phase by an experi-
enced observer. However, an automatic locomo-
tor-activity detection system would only portray
a bimodal pattern of activity but would provide
no information about the respective behaviors
each activity about represents.
The temporal distribution of behavior can also
shift under different environmental conditions.
In fact, more and more examples of “temporal niche
switches,” in which individuals of a species can
switch from one temporal niche (e.g., nocturnality)
to another (e.g., diurnality) are described both in
nature and under laboratory conditions (Table 1;
Mrosovsky and Hattar, 2005). Similarly, temporal
niche switches occurred across phylogenetic bra-
nches of closely related groups. Moreover, within
many taxa, these switches occur independently in
different phylogenetic branches (e.g., Roll et al.,
2006), indicating considerable changes in selective
pressure on temporal niche phenotypes and conse-
quently considerable adaptive plasticity in this trait.
Temporal niche switching across phylogeny
Evolutionary plasticity is limited, to a certain
extent, by phylogenetic constraints. However,
 283

Table 1. Examples of temporal niche switching in different species

Species TNS Cause Con References

House mouse Mus musculus N>D Natural conditions F Daan et al. (2011)
House mouse Mus musculus N>D Reduced food intake L Hut et al. (2011)
House mouse Mus musculus N>D Photoreceptor ablation L Mrosovsky and Hattar (2005),
Doyle et al. (2008)
House mouse Mus musculus N>D Subthreshold light L Doyle et al. (2008)
House mouse Mus domesticus N>D Metabolic demand L Perrigo (1987, 1990)
Golden spiny Acomys russatus N>D Interspecies competition F Shkolnik (1971)
mouse
Golden spiny Acomys russatus D>N Lab conditions F/L Levy et al. (2007)
mouse
Common vole Microtus arvalis N > Da Winter F Erkinaro (1969)
Common vole Microtus arvalis D > Ca Spring F Lehmann and Sommersberg
(1980)
Field vole Microtus agrestis N > Da Winter F Erkinaro (1969), Bäumler
(1975)
Field vole Microtus agrestis N>A Cage size reduction L Lehmann (1976)
Montane vole Microtus montanus N>D Short photoperiod L Rowsemitt et al. (1982)
Tundra vole Microtus N > Da Winter F Erkinaro (1969)
oeconomus
Syrian hamster Mesocricetus N > C, D Natural conditions F/L Gattermann et al. (2008)
auratus
Desert hamster Phodopus N > Nb Interspecies competition F Scheibler and Wollnik (2011)
roborovskii
Mongolian gerbil Meriones D>N Running wheel L Weinert et al. (2007)
unguiculatus
Kangaroo rat Dipodomys N>D Drought condition F Lockard (1978)
spectabilis
Ord’s kangaroo Dipodomys ordii N>D Drought condition F Boal and Giovanni (2007)
rat
Rat Rattus norvegicus N > N, Dc Lactation L Strubbe and Gorissen (1980)
Rat Rattus norvegicus N>D Natural conditions F/L Fenn and Macdonald (1995)
Rat Rattus norvegicus N>D Predation F Fenn and Macdonald (1995)
Rat Rattus norvegicus N>D Hypothalamic lesion L Richter (1978)
Rat Rattus norvegicus; A>N Development L Honma and Hiroshige (1977)
pup
N. American Castor canadensis N > FR Winter F Potvin and Bovet (1975),
beaver Bovet and Oertli (1992)
Rabbit Oryctolagus N>D Predation F Bakker et al. (2005)
cuniculus
Degu Octodon degus D, C > N, C Lab conditions, running F/L Fulk (1976), Bacigalupe et al.
wheel (2003), Garcia-Allegue et al.
(1999), Kas and Edgar (1999)
Degu Octodon degus D>N High temperature L Vivanco et al. (2010a),
Hagenauer and Lee (2008),
Refinetti (2006, 2008)
Coruro Spalacopus cyanus D>N Lab conditions F/L Ocampo-Garcès et al. (2006),
Begall et al. (2002)
Tuco tuco Ctenomys cf. D>N Lab conditions F/L Tomotani et al. (2011)
knighti

(Continued)
284

Table 1. Examples of temporal niche switching in different species (Continued)

Species TNS Cause Con References

Blind mole rat Spalax ehrenbergi D>N High temperature, F Kushnirov et al. (1998), Oster
summer et al. (2002)
Reindeer Rangifer taradus D>U Loss of LD cycle F Van Oort et al. (2007)
Roe deer Capreolus capreolus C, N > C, D Winter F Bubenik (1960) (in Aschoff,
1962; Tscherkowitsch, 1953)
Coyote Canis latrans N, D > Nd Hunting F Kitchen et al. (2000)
Bat-eared fox Otocyon megalotis D>N High temperature F Lourens and Nel (1990)
Geoffroy’s cats Leopardusgeoffroyi N>D Food shortage F Pereira (2010)
Owl monkey Aotus azarai C>N Moonlight F Fernandez-Duque et al. (2010)
Rhesus macaque Macaca mulatta A>D Development L Tscherkowicz (1953)
(in Aschoff, 1962)
Human Homo sapiens; A>D Development F Kleitman and Engelmann
infant (1953)
Whiskered bat Myotis mystacinus N>D Spring, autumn F Nyholm (1965)
Vampire bat Desmodus rotundus LN <> EN Moonlight avoidance F Turner (1975)
Leaf nosed bat Artibeus lituratus LN <> EN Moonlight avoidance F&L Erkert (1974), Häussler and
Erkert (1978)
Fat-tailed dunnart Sminthopsis N>D Winter F/L Warnecke et al. (2008),
crassicaudata Morton (1995), M.P. Gerkema
(personal communication)
Green toad Bufo viridis N>D Rainfall F R.A. Hut (personal
observation)
Cane toad, Bufo marinus D, N > De Cannibalism L Pizzatto et al. (2008)
metamorphs
Atlantic salmon Salmo salar A>N Low temperature F&L Fraser et al. (1995), Eriksson
(1973)
Atlantic salmon Salmo salar N>D Reduced food L Metcalfe et al. (1999)
Atlantic salmon Salmo salar N>D Growth demand L Metcalfe et al. (1998)
Atlantic salmon Salmo salar D>C>N Winter > summer L Eriksson (1973)
Brown trout Salmo trutta trutta D>C>N Winter > summer L Eriksson (1973)
Golden-lined Siganus lineatus D>N Shore > reef F Fox and Bellwood (2011)
rabbit fish
Mediterranean Nephrops N>D Deep water (>200 m) F Chiesa et al. (2010)
lobster norvegicus
Fruitfly Drosophila C>D Short photoperiod, low L Dubruille and Emery (2008)
melanogaster temperature
Fruitfly Drosophila C, D > C, N Moonlight L Kempinger et al. (2009)
melanogaster
Midge Chironomus N>D Low temperature F&L Kureck (1979)
thummi
Seed-harvesting Pheidole spp. D > N, C High temperature F Whitford et al. (1981)
ants

TNS, temporal niche switch; Con, condition; F, field study; L, lab study; F/L, comparison between field and lab study; F&L, effect observed in field
and lab. Switch in the dominant temporal niche indicated as N, nocturnal; D, diurnal; C, crepuscular; U, ultradian rhythms with no diurnal/nocturnal
preference; A, arrhythmic; FR, free running circadian rhythm; LN, late night active; EN, early night active.
a
Strong ultradian organization of activity always in either temporal niche preference.
b
Earlier-nocturnal activity.
c
Increased-diurnal feeding.
d
Reduced-diurnal activity.
e
Reduced-nocturnal activity.

Master
oscillator
LD
Temperature
Food
Fig. 1. Complexity of temporal distribution of behaviors. A complex scenario of behavioral programs is likely to be present in
natural habitats. Multiple environmental cycles can act as Zeitgebers to entrain a master circadian pacemaker that in turn
synchronizes peripheral oscillators. Specific Zeitgebers could also directly entrain peripheral oscillators and bypass the control by
the master circadian pacemaker (dotted lines). Peripheral oscillators regulate specific physiological and behavioral outputs that
could each have a different temporal niche.
studies focusing on phylogeny as an evolutionary
constraint limiting temporal niche switches are
scarce. Here, we focus on mammalian taxa for
which a phylogenetic framework has been used to
map temporal niches.
The mammalian nocturnal bottleneck
The nocturnal bottleneck hypothesis (Crompton
et al., 1978; Foster and Menaker, 1993; Heesy
and Hall, 2010; Young, 1962) proposes that all
mammals share a common nocturnal ancestor.
Endothermic mammals evolved from ectothermic
reptiles during the Mesozoicum between 205 and
65.5 mya. The first mammal-like species are
thought to have evolved endothermia in order to
occupy the nocturnal temporal niche to avoid
ectothermic predators and competitors that were
tied to the diurnal temporal niche because of their
need for behavioral body temperature regulation
through ambient temperature (Crompton et al.,
1978). The term “bottleneck” indicates that all
mammalian taxa decent from species that faced a
common selective pressure favoring nocturnality.
285
Peripheral
oscillators
Nocturnal care
of offspring
Diurnal
foraging
Crepuscular
mating
X Arrhythmic
predator
avoidance
The main arguments in favor of the nocturnal
bottleneck hypothesis are threefold: (1) most cur-
rent mammalian species seem to be nocturnal in
their behavior (Roll et al., 2006), (2) mammals
have developed endothermia in order to occupy
the nocturnal niche, and (3) most mammals have
a rod dominated visual system (Table 2) and sev-
eral cone types (except S and M cones) were lost
in mammals, indicating specialization toward a
nocturnal life style.
The mass extinction of dinosaurs at the end of
the Mesozoicum (65.5 mya; Cretaceous–Paleogene
extinction event or K/Pg boundary, also called the
Cretaceous–Tertiary extinction event or K–T
boundary) opened the way for mammalian species
to diversify their temporal niches. Diurnality and
other forms of temporal niche exploitation
emerged through evolutionary radiation and all
known temporal niche patterns in mammals were
probably present by the end of the Eocene (37
mya) when most modern mammalian taxa had
evolved.
Several arguments against the nocturnal bottle-
neck hypothesis can be raised. First, ectothermia
is not exclusively linked to diurnal behavior.
286

Table 2. Dominant temporal niche and cone photoreceptor percentages

Species DTN Cone (%) References

European ground squirrel Spermophilus citellus D 97 Szél and Röhlich (1988)

Mexican ground squirrel Ictidomys mexicanus D 95–96 West and Dowling (1975)
13-lined ground squirrel Ictidomys D 95–96 West and Dowling (1975)
tridecemlineatus
Tupaia Tupaia belangeri D 95 Müller and Peichl (1989)
Black-tailed prairie dog Cynomys ludovicianus D 90 West and Dowling (1975)
California ground squirrel Otospermophilus D 85 Kryger et al. (1998)
beecheyi
Eastern gray squirrel Sciurus carolinensis D 60 West and Dowling (1975)
Nile grass rat Arvicanthis niloticus D, C 35–40 Gaillard et al. (2008)
Degu Octodon degus D, N 32 Jacobs et al. (2003)
Plains pocket gopher Geomys bursarius N, D 25 Feldman and Phillips (1984), Benedix (1994)
Talas tuco tuco Ctenomys talarum D, N 14–31 Schleich et al.– 2010
Magellanic tuco tuco Ctenomys magellanicus D, N 10–31 Schleich et al. (2010)
Agouti Dasyprocta aguti D, N 10–20 Rocha et al. (2009)
Pig Sus scrofa domestica D, N, C 10–20 Hendrickson and Hicks (2002)
Guinea pig Cavia porcellus C, A 8–17 Peichl and González-Soriano (1994)
Mongolian gerbil Meriones unguiculatus N, D 13 Govardovskii et al. (1992)
European mole Talpa europaea A, U 10–12 Glösmann et al. (2008)
Ansell’s mole rat Cryptomys anselli N, D, A 10 Peichl et al. (2003)
Giant mole rat Cryptomys mechowi N, D, A 10 Peichl et al. (2003)
Coruro Spalacopus cyanus N, D 10 Peichl et al. (2005)
Common shrew Sorex araneus A, U 6–13 Peichl et al. (2000), Crowford (1953)
Greater white-toothed Crocidura russula N 5–6 Peichl et al. (2000), Crowford (1953)
shrew
Human Homo sapiens D 4.7 Curcio et al. (1990)
Fraser’s musk shrew Crocidura poensis N 4–5 Peichl et al. (2000)
Rabbit Oryctolagus cuniculus N, D, C 4 Peichl (1997)
Mouse Mus musculus N 3 Carter-Dawson and LaVail (1979)
Lesser hedgehog, tenrec Echinops telfairi N 1.5–2.3 Peichl et al. (2000)
Rat Rattus norvegicus N 1 LaVail (1976), Szél and Röhlich (1992)
Eastern woodrat Neotoma floridana N 1 Feldman and Phillips (1984)
Mouse opossum Thylamys elegans N 0.4–1.2 Palacios et al. (2010)

Cones are functional at higher light intensities than rods and therefore thought to be indicative of diurnal vision. Consequently, cone densities, as per-
centage of the total number of photoreceptors in the whole retina, may indicate the dominant temporal niche of a species. DTN, dominant temporal
niche; D, diurnal; N, nocturnal; C, crepuscular; A, arrhythmic; U, ultradian.

Geckos, for instance, are small ectothermic reptiles
that hunt for prey during the night. Second, it is
debatable whether all dinosaurs were ectotherms.
The arguments indicating that dinosaurs during
the Mesozoicum may have evolved forms of endo-
thermic capacity include the discovery of insulating
feather structures, fossils of breeding dinosaurs in
arctic regions suggesting heat transfer from adults
to offspring, raised body posture, fibrolamellar
skeletal bone structure, and the presence of nasal
turbinate structures (Nespolo et al., 2011). Even
without endothermia, large dinosaurs may have
been able to retain enough body heat to support
activity during the night through “inertial hom-
eothermy” or “mass homeothermy.”. Third, intra-
ocular and orbital bone sizes indicate that several
dinosaur species—including relatively small, feath-
ered predatory Theropoda like Velociraptor—
seemed to have had eye shapes that facilitate
nocturnal vision (Schmitz and Motani, 2011).

Taken together, it seems probable that the
nocturnal bottleneck indeed has occurred in early
mammals. Recent evidence, however, seems to
indicate that potential predators of these early
mammals (like some Therapoda species) may have
responded to this novel invention of mammalian
nocturnal activity by developing endothermic cap-
acities, enabling these dinosaurs to also exploit the
nocturnal niche (as indicated by changes in eye
morphology). This leads to the interesting conclu-
sion that, already during the Mesozoicum (i.e.,
before the great extinction of the dinosaurs), the
mammalian nocturnal bottleneck triggered an evo-
lutionary arms race between predator and prey for
temporal niche occupation. This in turn may have
led to considerable adaptive plasticity in temporal
niche usage among the earliest mammals.
Temporal niche switching in mammals
Rodents
A literature survey of activity patterns of 700
rodent species found that activity patterns at
the family level are significantly different from
the expected random patterns, emphasizing the
strong relationship between intrafamily taxo-
nomic affiliation and daily-activity patterns. Large
families (Muridae and Sciuridae) analyzed by
subfamilies and tribes showed a similar but stron-
ger pattern than that of the family level. The
researchers concluded that phylogeny constrains
the evolution of activity patterns in rodents and
may limit their ability to use the time niche axis
for ecological separation (Roll et al., 2006). Nev-
ertheless, there are some striking examples of
temporal niche dichotomy within relatively small
taxa. For instance, ground squirrels form a species
group which is typically exclusively diurnal. Euro-
pean ground squirrels (Spermophilus citellus),
unlike diurnal birds, are not even active around
dawn and dusk, but appear above ground  3 h after
sunrise and retreat below ground  3 h before sun-
set (Hut et al., 1999). In stark contrast to this
287
extreme diurnal phenotype are flying squirrels, an
example of strictly nocturnal Sciuridae (DeCoursey,
1960, 1986).
Primates
The temporal niche of primates ranges from
diurnality to nocturnality with many species showing
“cathemeral” patterns of activity. Although the
term cathemeral has been widely accepted in the
primatology literature, a careful analysis of so-called
cathemeral patterns of activity indicates that these
patterns are crepuscular, with varying degrees of
nocturnality and diurnality, which is in many cases
determined by environmental factors (see Lumi-
nance and Temporal niche switching). Strepsirhine
primates (lemurs and lorisiforms) are predomi-
nantly nocturnal. Haplorhine primates (tarsiers,
monkeys, and apes), in contrast, are predominantly
diurnal, with two genera representing the nocturnal
exception: Tarsius (tarsiers) and Aotus (owl
monkeys). Thus, owl monkeys represent a remark-
able exception among anthropoid primates (Old
World monkeys, New World monkeys, apes, and
humans), as the only nocturnal genus. Because
strepsirhine primates and tarsiers are typically noc-
turnal and both groups show more primitive
features than anthropoid primates, it is traditionally
believed that the ancestral Eocene primate was
probably nocturnal (Martin and Ross, 2005). This
notion, however, has been challenged by molecular
evolution studies of the S-opsin, which usually
shows relaxed selection in nocturnal species, of
several tarsiers and lemurs (Tan et al., 2005).
The authors show great variability in the degree
of relaxed selection among nocturnal species,
suggesting that nocturnality has emerged at differ-
ent times and multiple times among nocturnal
primates, and that the ancestral primate may have
been diurnal or cathemeral. Regardless of the tem-
poral niche of the ancestral primate, it is likely that
nocturnality–diurnality switches, and diurnality–
nocturnality switches have emerged indepen-
dently and multiple times throughout primate
288

evolution (Ankel-Simons and Rasmussen, 2008), nocturnal emergence from their burrows and
and this is consistent with the multiple mechanisms foraging during the night when they live in the
that could potentially lead to niche switches. shallow sea shelf (10–50 m deep), they have a cre-
puscular phenotype on the lower shelf (50–200 m
deep), and they are diurnal in the deeper slope
Temporal niche switching within species
(200–400 m deep; Table 1). Whereas fishermen
trolling for this commercially important species
Changes in the daily temporal niche have been
had long recognized this depth-dependent temporal
reported in a wide range of animals. These changes
niche switch (reviewed in Aguzzi and Sarda, 2008),
can be associated with environmental cycles such as
a recent study has shown that light–dark (LD) cycles
seasons and lunar month, with development and
that, respectively, mimic the brighter blue light of
aging, and with changing ecological factors such as
the shallow waters and the dimmer blue light
the presence of competitors, prey, or predators. The
of the deeper slope are sufficient to determine the
golden spiny mouse (Acomys russatus) seems to be
nocturnal and diurnal phenotypes (Chiesa et al.,
forced to the diurnal niche under natural conditions,
2010). Although similar experiments have not been
possibly through resource competition with the com-
done in other species with wide ocean-depth ranges,
mon spiny mouse (Acomys cahirinus). When golden
it is likely that the dramatic changes in light intensity
spiny mice are placed under laboratory conditions,
and wavelength across water depths represent a key
they immediately switch back to the nocturnal niche
determinant of temporal niche in other species that
(Fig. 2; Levy et al., 2007). Many such cases of tempo-
inhabit the continental margin.
ral niche switching are briefly summarized in Table 1.
Nocturnal species depending on vision may use
Here, we limit our discussion to environmental
moonlight to increase their foraging success.
determinants of niche switches, both in laboratory
In fact, most nocturnal mammals rely on moon-
and in field settings. Social determinants of niche
light, and nocturnal dim light stimulates their loco-
switches, such as inter- and intraspecific interactions
motor activity. In these species, nocturnal-activity
are reviewed by Castillo-Ruiz et al. (2012).
patterns can often be synchronized with moon
phases (Erkert, 2008). In the southernmost owl
Luminance and temporal niche switching monkey (Aotus azarai), this moonlight depen-
dence leads to predictable temporal niche switches
The Mediterranean lobster (Nephrops norvegicus) with striking nocturnal activity during moonlit
represents a unique case of diurnal–nocturnal nights and crepuscular activity during new moon
temporal niche switching. These animals show nights (Table 1, Fernandez-Duque et al., 2010).
Field
Field

Lab (DD)
Lab (DD)

5:00 18:30 5:00 18:30 5:00 18:30 5:00 18:30

Fig. 2. Temporal niche switching in golden spiny mice: field versus laboratory. Two examples of temporal niche switching in golden
spiny mice. When the animals were taken from the field to the laboratory under continuous dim light to assess the phase of their
activity rhythm, they show a clear switch from diurnal activity in the field to nocturnal activity in the lab.

In contrast to the stimulatory effects of moonlight
on locomotor activity in nocturnal primates and
rodents, bats, which rely more on echolocation
than on vision, seem to avoid the moonlit part
of the night (Erkert, 1974; Häussler and Erkert,
1978; Turner, 1975). This behavior could in-
crease their foraging success by timing hunting
when their prey exhibits minimal avoidance
behavior and also by reducing the risk of being
predated upon.
Ambient temperature and temporal niche
switching
In general, temperature differences in the terres-
trial environment are much higher than in the
buffered aquatic environment. Many ectothermic
species may need the high daytime temperatures
to augment their body temperature in order to
facilitate locomotor activity. In doing so, they face
the potential problem of increased evaporative
water loss. These effects of temperature can
explain why amphibians (with high evaporative
water loss through their skin) in general choose
the nocturnal niche for terrestrial locomotion,
while reptiles (with reduced evaporative water
loss through their scaled skin) choose the diurnal
niche and even start their active phase with thor-
ough sun basking in the morning. The terrestrial
green toad (Bufo viridis) escapes daytime high
temperatures on the Hungarian puszta plains by
hiding in abandoned ground squirrel burrows
(Hut and Scharff, 1998). Rainfall allows this
animal to emerge above ground in daytime,
sometimes in extremely high numbers (Table 1,
R.A. Hut, personal observation). A more subtle
response to temperature was described in the
midge (Chironomus thummi), which emerges
from the water at dusk to swarm and reproduce
at night. Colder water temperatures, however,
trigger dawn emergence and daytime swarming.
The low water temperature is apparently used by
the insects as an indicator for night temperatures
being too low to swarm and successfully repro-
duce (Table 1, Kureck, 1979). The bat-eared fox
289
(Otocyon megalotis) of the South African deserts
forages during the night in the summer when mid-
day soil temperatures reach 70 C and during the
day in the winter when night air temperatures
drop to 10 C (Lourens and Nel, 1990). Desert
seed-harvesting ants forage during the day in
winter but avoid the heat of summer by foraging
crepuscularly, nocturnally, or on cloudy days
(Whitford et al., 1981). Several rodent species
switch activity patterns between seasons, appar-
ently for thermoregulatory reasons. For example,
montane voles (Microtus montanus) become
more diurnal during the winter (Rowsemitt
et al., 1982). Similarly, the subterranean mole rat
(Spalax ehrenbergi) is predominantly diurnal dur-
ing winter but predominantly nocturnal during
summer (Kushnirov et al., 1998; Oster et al.,
2002), a behavioral switch which seems to be
regulated at the molecular light input pathway
of the core clock mechanism (Oster et al., 2002).
Also, degus that are maintained at cooler
temperatures (e.g., 18  C) with food ad libitum,
demonstrate diurnal rhythms in temperature and
locomotor activity, whereas degus that are
maintained under the same conditions but at
more standard animal room temperatures
(21–23  C) shift to more crepuscular or noc-
turnal patterns (Hagenauer and Lee, 2008).
Interestingly, in the field, there is no evidence of
degus ever leaving their shelters at night. These
data suggest that apart from thermoregulatory
costs associated with ambient temperature,
animals also integrate other variables like risk
of predation, poor nocturnal eyesight, or other
variables that may shape activity patterns
(Jesseau et al., 2009).
Factors limiting temporal niche switches
Factors limiting temporal niche switches may be
divided into internal (the organism anatomy and
physiology, such as its sensory and thermoregula-
tory capabilities) and external (environmental
conditions, both biotic and abiotic). As in the case
of the factors leading to temporal niche switches,
290
factors limiting temporal niche switches can be
associated with environmental factors, and with
inter- and intraspecies interactions. The latter
are reviewed by Castillo-Ruiz et al. (2012).
Day and night differ in many environmental
factors other than light levels (e.g., temperature,
humidity), and therefore expose animals to differ-
ent challenges that require different anatomical,
physiological, and behavioral adaptations. Diurnal
animals usually use vision for predatory avoid-
ance and foraging, while nocturnal animals use
tactile probing, smell, and hearing. Communica-
tion is usually through sound and olfaction in
nocturnal animals (and also, although rarely,
through bioluminescence). Nocturnal animals
may use camouflage (e.g., moths or night jars)
and burrowing for concealment from their diurnal
predators during the day, whereas diurnal animals
on the other hand use visual signals or even con-
spicuous aposematic coloration (Daan, 1981).
All these complex adaptations to the specific
temporal niche may limit temporal niche switches.
Here, we focus on some properties of the visual
system, the sensory system mostly studied in the
context of adaptation to the nocturnal or diurnal
temporal niche.
Visual adaptations to specific temporal niches
Night- and day-adapted vision
Because the activity pattern within an individual
may show considerable plasticity, it may not be
the best parameter to determine the dominant
temporal niche phenotype for longer time scale
evaluations. Physiological and anatomical deter-
minants for temporal niche usage likely integrate
over a much longer time frames (generations)
than individual activity patterns do. The best can-
didate for such a determinant is likely to be found
in the eye, since this organ is specifically adapted
to light levels that range—in up to five orders of
magnitude—between the darkness of the night
and the bright light during the day. These visual
adaptations can take place at the anatomical,
physiological, or molecular level to define a gen-
erally nocturnal or diurnal life style for a specific
species.
The adaptive value of a temporal niche will
likely present trade-offs due to selective forces
that act upon specific physiological and anatomical
systems. For instance, in a species forced to
become diurnal to escape nocturnal predators
new physiological and behavioral strategies may
need to be selected to cope with activity at the
warmer phase of the day. The most prominent
and predictable environmental variable that may
exert selective pressure in association with spe-
cific temporal niches is the dramatic change in
luminance across the 24-h day. Several studies
have focused on adaptations of the visual system
as key features of evolutionary adaptation to
specific temporal niches.
The large majority of reef fish families are pri-
marily diurnal. Nocturnality has been observed in
at least 13 families across teleost reef fishes
(reviewed in Schmitz and Wainwright, 2011). An
analysis of morphological traits of the eyes in 265
species of teleost reef fish in 43 different families
(Schmitz and Wainwright, 2011) revealed that the
eye morphology of nocturnal reef teleosts is
characterized by better light sensitivity, resulting
from large relative eye size, high optical ratio, and
large, rounded pupils. Improved dim-light image
formation comes at the cost of reduced depth of
focus and reduction of potential accommodative
lens movement. Diurnal teleost reef fish have
much higher morphological and optical diversity
than nocturnal species, with large ranges of optical
ratio, depth of focus, and lens accommodation.
The authors suggest that the trade-off between
improved dim-light vision and reduced optical
diversity may be a key factor in explaining the
lower trophic diversity of nocturnal reef teleosts.
Thus, it is likely that the trade-off associated with
nocturnal vision may represent a limiting factor
for evolutionary radiation in nocturnal taxa.
Several studies on different taxonomic groups
tested the effect of switching from diurnal; vision

based foraging, to nocturnal foraging, showing
that low light levels can constrain visual abilities
and reduce foraging success. For example, the
common murre (Uria aalge), a diurnal, diving
predator seabird, extends its activity to the night
during the breeding season. Diving depths
decreased with decreasing nocturnal light levels,
probably decreasing foraging efficiency (Regular
et al., 2011). Juvenile salmonids are visual for-
agers; they switch from being predominantly diur-
nal for most of the year to being nocturnal in
winter. Their foraging efficiency is high at light
intensities down to those equivalent to dawn or
dusk but drops markedly at lower levels of illumi-
nation; even under the best night condition (full
moon and clear sky), the feeding efficiency is only
35% of their diurnal efficiency (Fraser and
Metcalfe, 1997).
In the nocturnal house mouse, the retina is
dominated by rods but also contains 3% cones
(Table 2). This suggests that the mouse retina
retained diurnal visual function. Indeed, at the
functional level, Cameron et al. (2008) described
increased cone functionality in the subjective
day and increased rod functionality during the
subjective night. This indicates that an endoge-
nous circadian clock prepares the mouse retina
for visual function during daytime. The existence
of this daytime visual response indicates that phe-
notypic plasticity for temporal niche switching on
the behavioral level might have generated enough
selective advantage to retain some form of diur-
nal vision in this predominantly nocturnal species.
Eye properties: Rod-cone densities, visual acuity,
and sensitivity
The extreme diurnal life style in ground squirrels
is associated with two properties of their eyes:
the rod/cone ratio and the spectral filtering pro-
perties of the lens. The European ground squirrel
retina contains virtually only cone photoreceptors
(97%; Szél and Röhlich, 1988). Such extreme rod/
cone ratios are only found in diurnal mammals,
291
but the reverse conclusion that lower cone per-
centages are always associated with nocturnality,
cannot be drawn. Much lower retinal cone
percentages can be found in other species which
also display a diurnal preference, and indeed, they
may approach, or even overlap with, cone
percentages found in nocturnal species ( 10%,
Table 2). Some of these diurnal species with rela-
tive low cone percentages have a clear overrepre-
sentation of cones in the central retina, either in
an elongated region called the visual streak or
in a central area called the macula. These areas
are tuned to high acuity cone vision and can be
found in mammals, birds, amphibians, and reptiles
(Rodieck, 1973). Within the macula, some species
have a small indent or pit, packed with cone photo-
receptors to further increase the acuity in a small
central area of the visual field. This structure
is called the fovea and can be found in primates
(including humans) and birds, which may
have two or even three foveae. High density cone
areas like the macula and the fovea can be seen as
an adaptation to the diurnal niche. In fact,
most nocturnal haplorhines (Tarsius and Aotus),
which likely share a diurnal ancestor with
diurnal haplorhines, have a fovea; although in
Aotus individuals, it may be absent (Martin and
Ross, 2005).
A study comparing the retinal structure and
composition of golden spiny mice (A. russatus),
which are diurnal at their natural habitat when
this is shared with the nocturnal common spiny
mice (A. cahirinus), found that photoreceptor
cells of both species were uniform in shape and
tightly packed, and they conformed mainly to
the morphology of rods (Kronfeld-Schor et al.,
2001). This could indicate that no obvious
anatomical adaptation to the diurnal life style
has been found in the retina of this species so
far. Accordingly, golden spiny mice prefer to for-
age under boulders, where light intensities are
low (22–47 lux; Kronfeld-Schor et al., 2001).
These results suggest that the response of the
golden spiny mice to become diurnal is either
due to the phenotypic plasticity of this species or
292
that the diurnal life style is a relatively recent evo-
lutionary adaptation.
The increased visual acuity conveyed by
structures like the macula and fovea would obvi-
ously be advantageous for nocturnal species as
well. However, these structures require a reduced
pupil diameter to restrict the incoming images from
the paraxial regions, and a reduced pupil diameter
is not something nocturnal animals can afford as it
would reduce the light-gathering ability of the eye
(Heesy and Hall, 2010). The way in which noctur-
nal animals increase visual acuity is by increasing
the visual field overlap between eyes (reviewed in
Martin and Ross, 2005; Heesy and Hall, 2010). This
not only conveys higher acuity but also increases
luminance detection because each point in the
binocular visual field will gather twice as many
photons as a point in the monocular field.
The presence of a tapetum lucidum is seen as
an important adaptation to nocturnal life. The
tapetum is a membrane located behind the retina
that reflects back the light that has gone through
the retinal cell layers, increasing light detection
by photoreceptor cells. A tapetum is typically
present in nocturnal species, such as most strep-
sirhine primates. Interestingly, there is a wide var-
iation in the anatomy and histology of the
tapetum, indicating that it is an analogous organ
that has evolved multiple times from widely dif-
ferent cell types in the eye (Tan et al., 2005).
Eye properties: Spectral filtering and UV
protection
Eyes of diurnal species are under permanent pho-
tooxidative stress from UV radiation (Ringvold
et al., 2000a). It is thought that various levels of
yellowish coloration of the mammalian eye lens
shields the retina from the (DNA) damaging
effects of short-wavelength light (ultraviolet and
blue-violet; below  450 nm). Lenses of different
species may exhibit different amounts of colora-
tion resulting in different spectral filtering pro-
perties. The coloration occurs because of the
interaction of crystallines with a UV filter com-
pound, 3-hydroxykynurenine glucoside. Hence,
the mammalian eye lens could be viewed as a
low-pass filter with a species specific cutoff value
that relates to the species specific life style in the
nocturnal–diurnal gradient.
Ground squirrel eyes typically have lenses that
appear deep yellow to the human eye, indicating
stronger filtering properties in the blue region
of human visible light spectrum. Indeed, short-
wavelength blue light that is not absorbed by the
human lens (cutoff 445 nm) is shown to be
absorbed by the ground squirrel lens (cutoff
493 nm; Hut et al., 2000). In contrast, nocturnal
animals like the Syrian hamster have a cutoff
value  340 nm, allowing UV-A light penetration
to the retina (Table 3). The lens filtering property
in the 300–500 nm spectral band seems to reflect
the long-term dominant temporal niche usage of
a species (Fig. 3). This lens filtering property is
relatively easy to measure; it has a low within
species variance and is easier to interpret than
complex retinal properties like photoreceptor dis-
tributions (Table 3; Fig. 3). As such, it can be a
useful tool for the evaluation of temporal niche
switching within individuals.
Eye properties: Ascorbic acid concentration
Another possible mechanism for protecting the
eye from UV radiation damages is high con-
centrations of ascorbic acid in the aqueous humor
(Ringvold, 1980). High concentrations of ascorbic
acid in the aqueous humor of the eye—20–40
times higher than in the plasma—was reported
in many diurnal species including humans, ante-
lopes, tree shrews, golden spiny mice, and rhesus
monkeys. Conversely nocturnal mammals, includ-
ing the slow loris, fruit bats, cats, common spiny
mice, and owl monkeys have a concentration sim-
ilar to that in plasma (Koskela et al., 1989; Reiss
et al., 1986; Ringvold, 1980). Interestingly, such
a trend was absent in nocturnal and diurnal birds,
which apparently use a different mechanism for
 293

Table 3. Filtering properties of the eye lens relates to the species specific temporal niche

Lens cutoff
Species DTN (nm) References

European ground squirrel Spermophilus citellus D 493 Hut et al. (2000)

Black-tailed prairie dog Cynomys ludovicanus D 482 Yolton et al. (1974)
Mexican ground squirrel Ictidomys mexicanus D 478 Jacobs and Yolton (1971), Yolton et al.
(1974)
Golden mantled ground Callospermophilus D 471 Yolton et al. (1974)
squirrel lateralis
13-lined ground squirrel Ictidomys D 470 Chou and Cullen (1984), Yolton et al.
tridecemlineatus (1974)
California ground squirrel Spermophilus beecheyi D 464 Jacobs et al. (2003)
Agouti Dasyprocta aguti D, N 460 Peichl (1997)
Western gray squirrel Sciurus griseus D 447 Yolton et al. (1974)
Human (32years) Homo sapiens D 445 Stockman et al. (1999)
Guinea pig Cavia porcellus C, A 410 Zigman (1983)
Degu Octodon degus D, N 378 Jacobs et al. (2003)
Talas tuco tuco Ctenomys talarum D, N 370 Schleich et al. (2010)
Rat Rattus norvegicus N 343 Jacobs et al. (2003)
Syrian hamster Mesocricetus auratus N 341 Hut et al. (2000), Brainard et al. (1994)
Coruro Spalacopus cyanus N, D 330 Peichl et al. (2005)
Magellanic tuco tuco Ctenomys magellanicus D, N 325 Schleich et al. (2010)
European mole Talpa europaea A, U 310 Glösmann et al. (2008)

The eye lens absorbs short-wavelength light below a certain wavelength in the UV/blue band to protect the retina from tissue damaging properties of
high-energy short-wavelength light present during daytime. The wavelength at which transmission is reduced to 50% of the maximal transmittance is
presented as lens cutoff value (in nm). DTN, dominant temporal niche; D, diurnal; N, nocturnal; C, crepuscular; A, arrhythmic; U, ultradian.

UV protection. The end product of nitrogen
metabolism in birds is uric acid, which similarly
to ascorbic acid, is a powerful UV radiation
absorber. Mammals, and especially diurnal spe-
cies, have high concentration of ascorbic acid
and low concentration of uric acid in their aque-
ous, while birds have low concentration of
ascorbic acid and high concentration of uric acid
in their aqueous (Ringvold et al., 2000b). This
suggests that uric acid in birds may have the same
UV protection function as ascorbic acid in
mammals, and it will be interesting to compare
uric acid concentrations in the aqueous humor
of diurnal and nocturnal bird species.
Mechanisms underlying temporal niche switching
The timing of specific behaviors throughout the
day is the result of the interaction between the
output of circadian clock(s) that are entrained
to specific environmental cycles and masking,
namely, the direct inhibition or stimulation of
behavior by environmental factors. For example,
a circadian clock that sustains locomotor activity
during the night combined with an inhibitory effect
of light on activity will result in a robust nocturnal
niche. Three basic mechanisms could result in
switching of temporal niche usage, both across
phylogeny or within a species or individual (Fig. 4).
First, changes in circadian oscillator properties
could lead to changes in its phase angle of entrain-
ment, and therefore in the phase of the rhythms, it
regulates (see below). Second, a circadian oscilla-
tor with essentially the same properties could lead
to opposite temporal niches if a switch down-
stream from the oscillator translates the oscillator
output into divergent timing of activity. This seems
to be case for the switch between nocturnality
and diurnality in rodents. Both, diurnal rats
294

Fig. 3. Cone percentages and lens filtering property relate to dominant temporal niche. Percentage of cones of total number of
photoreceptors in the total retina plotted against lens filtering cutoff wavelength (in nm) correlate with each other such that
diurnal animals are positioned in the top right corner (with the exception of humans) and nocturnal animals in the bottom left
corner. Data taken from Tables 2 and 3, symbol fills correspond to dominant species specific temporal niche. EGS, European
ground squirrel; PD, prairie dog; MGS, Mexican ground squirrel; TLGS, 13-lined ground squirrel; CGS, California ground
squirrel; A, agouti; GS, gray squirrel (Western and Eastern); H, human; GP, guinea pig; D, degu; TTT, Talas tuco tuco; R, rat;
C, cururo; MTT, Magellanic tuco tuco.

(Arvicanthis sp.) and nocturnal laboratory rats
seem to carry a similarly phased master circadian
pacemaker within the suprachiasmatic nucleus
(SCN) of the hypothalamus, but the rhythms of
locomotor activity and corticosteroid release that
this oscillator regulates are oppositely phased
(Kalsbeek et al., 2008; Smale et al., 2003).
In accord, the anatomy and the physiology of the
SCN in the diurnal and nocturnal golden and com-
mon spiny mice show no differences (Cohen et al.,
2010a,b), and both, as well as the degu circadian
system (Hagenauer and Lee, 2008), are largely
consistent with that of nocturnal species. Third,
expression of a behavioral program during the
daytime or nighttime could be solely determined
by masking, regardless of the phase of the oscilla-
tor. Even if masking does not represent the sole
temporal niche determinant, it usually plays a crit-
ical role in shaping behavioral outputs of biological
timing systems. In the owl monkey A. azarai, noc-
turnal activity regulated by a circadian oscillator is
inhibited by darkness during new moon nights,
leading to a prominent nocturnal niche only
during moonlit nights. The negative masking by
darkness—or positive making by moonlight—was
evident after measuring activity during total moon
eclipses during full-moon nights, which leads
to dramatic decreases in nocturnal activity
(Fernandez-Duque et al., 2010). Moreover, in
order to switch to diurnal activity, animals have
to overcome the negative masking effect of light.
A study comparing masking response to LD pulses
in diurnal golden spiny mice and nocturnal com-
mon spiny mice found that whereas the nocturnal
common spiny mouse display the expected
responses, decreasing activity levels in response
to a light pulse and increasing activity level
in response to dark pulse, golden spiny mice
increased their activity in response to a dark pulse
(as expected from a nocturnal species), and show
insignificant and highly variable response to a light
pulse, indicating that in this diurnally active species
the negative masking effect of light was indeed
removed (Cohen et al., 2010b). Interestingly,
 295

Master
oscillator
Output
Input
pathways
pathways
Nocturnal
LD cycle Photoreceptors

Diurnal

Downstream
switch
Nocturnal

Diurnal

Nocturnal

Masking
(+)/(-)

Diurnal

Photic Nonphotic
input input

Fig. 4. Mechanisms underlying temporal niche switches in behavioral outputs. Simplified scenario portraying a single behavioral
output driven by a circadian oscillator. Top: The properties of the circadian oscillator could change, so that its phase relationship
to the LD cycle is different by almost 180 , leading to rhythms of activity that are oppositely phased. Middle: The properties of
the oscillator could be unaltered, but a switch in the output pathways regulating locomotor activity could alternate between
nocturnal and diurnal patterns of activity. Bottom: The properties of the oscillator could be unaltered, but positive and/or
negative masking could determine the rhythmic pattern of activity regardless of the oscillator-phase relationship to the LD cycle.
Modified from Chiesa et al. (2010).

diurnal and nocturnal degus present a stable mas-
king by light, each according to its respective
chronotype. Thus, whereas diurnal degus increase
their activity with light, in nocturnal degus light
induce a sharp drop in wheel-running activity
(Vivanco et al., 2010b). The resistance to inhibi-
tory effects of light on activity is likely to be essen-
tial for a switch into a diurnal niche and possibly
occur very early in every evolutionary transition
from nocturnality to diurnality.
The top panel of Fig. 4 portraits clocks with dif-
ferent properties leading to differently phased
rhythms. Subtle changes in the circadian period
of a circadian oscillator can lead to robust changes
in the phase angle of entrainment of the oscillator
(Daan and Aschoff, 1997; Johnson et al., 2003). In
the fruit fly Drosophila melanogaster, the tempo-
ral organization of locomotor activity is the result
of changes in period and phase of their circadian
oscillator. The circadian clock that regulates
296
locomotor activity is constituted by a neuronal
network of approximately 150 neurons that
express clock genes. The products of these genes
constitute the molecular clockwork of biological
clocks. D. melanogaster flies usually show crepus-
cular activity. Recent studies have shown that the
generation of the morning (M) and evening (E)
peaks of locomotor activity are associated with
the activity of anatomically identifiable subgroups
of cells within the clock neuronal network that
oscillate with different phase and period (Grima
et al., 2004; Stoleru et al., 2004, 2007; Yoshii
et al., 2012). Interestingly, the relative phase
between M and E activity is rather plastic, and
it is modulated by both room temperature and
photoperiod. Shorter photoperiods or lower
temperatures will bring the E and M peaks closer
together and lead to more consolidated diurnal
activity (reviewed in Dubruille and Emery,
2008). The combined effect of photoperiod and
ambient temperature likely provides adaptive
value; it will push activity to the warmer hours
during the winter but to the cooler dawn and dusk
during the summer. Remarkably, the shift in the
fly’s temporal niche relies to some extent on the
transcriptional machinery of the molecular clock-
work. The clock gene period (per) has two
mRNA isoforms. Under lower temperatures or
short photoperiods intron 8 of the gene is more
efficiently spliced, and this leads to an earlier
appearance of the PER protein, and of the E
locomotor-activity peak (Majercak et al., 1999).
This effect is further enhanced by the fact that
splicing of the per gene has a peak during the
day. This splicing occurs earlier under short
photoperiods, but it is inhibited—and pushed to
later times of the day—if temperature is high
(Majercak et al., 2004). The effect of photoperiod
on per mRNA maturation and timing of locomo-
tor activity is synergized by the photodegradation
of TIM, the product of the clock gene timeless
(tim). TIM dimerizes with PER and by doing so
protects PER from degradation. Under longer
photoperiods, because of the photic degradation
of TIM, it is hypothesized that it will take longer
for PER to accumulate and for activity to take
place. Other mechanisms contribute to the effects
of photoperiod and ambient temperature on the
timing of expression of molecular clock compo-
nents. Finally, the fact that the E and M locomo-
tor-activity components rely on different clock
neurons offers further plasticity in the fly’s
clock to independently regulate dawn- and dusk-
associated behaviors and their timing (Dubruille
and Emery, 2008).
The above scenarios only take into account a
master circadian oscillator and a single environ-
mental factor entraining it. The layout will obvi-
ously be more complex in natural environments
where multiple Zeitgebers will be present (Fig. 1).
Further, the presence of multiple peripheral
oscillators has been shown in all the species stud-
ied so far. The phase of these “slave” oscillators is
typically set by a master circadian oscillator, but
they can escape the master control. In mammals,
specific peripheral oscillators can be entrained
by environmental cycles other than light and in
an independent manner from the photic entrain-
ment of the master circadian oscillator within
SCN (Stokkan et al., 2001). Thus, the physiologi-
cal and behavioral temporal niche of a species in
nature may represent a complex array of rhythms
that emerge from a network of interacting circa-
dian oscillators—within and outside the central
nervous system—and their respective interactions
with several Zeitgebers with different abilities to
entrain them.
Adaptive value of temporal niche switching:
Unification through the hypothesis of circadian
thermoenergetics?
Temporal niche switching examples have been
described in many species and under various
circumstances (Table 1). The original descriptions
are often accompanied by explanations involving
the environmental cause which triggers the switch
(proximate mechanism) or the adaptive value
of the switching response (ultimate mechanism).

The proximate mechanisms may involve light, tem-
perature, or social interactions. Such mechanisms
are discussed in this chapter, in the accompanying
paper (social interactions; Castillo-Ruiz et al.,
2012) and in the original papers (Table 1). Studies
evaluating the ultimate mechanisms underlying
temporal niche switching rely more on parsimoni-
ous reasoning than on solid experimental evidence.
Here, we would like to propose a new hypothesis
of circadian thermoenergetics (CTE), which
may unify the adaptive value of several cases of
temporal niche switching.
The ultimate mechanism driving temporal
niche switching should involve the optimization
process in which energy balance, survival, and
reproduction are weighed against each other to
maximize fitness. In the temperate zone, noctur-
nal activity is expected to be more costly because
ambient temperatures are high during daytime
and low during the night. At nighttime, when
ambient temperatures are generally below the
thermal neutral zone of most mammals, endother-
mic animals will energetically profit from reduced
heat loss through insulation. This insulation is
often found during the rest phase when animals
retreat into burrows or nests, huddle together,
or adopt a typically sleep-associated thermal insu-
lation posture. Moreover, many endotherms
neglect homeothermy and use torpor during the
rest phase, making it even more energetically
profitable to rest during the night. From an ener-
getics point of view, all endothermic animals in
the temperate zone would profit from a diurnal
life style. However, other selective forces such as
daytime predation pressure or competition may
have forced endothermic animals to become or
remain nocturnal. This nocturnal phenotype can
only be sustained when enough food is available
to allow for the energetically costly nocturnal life
style. If food becomes scarce, nocturnal animals
may return to a diurnal life style to save energetic
costs of thermoregulation. Indeed, Daan et al.
(2011) observed robust periods of diurnal activity
in populations of normally nocturnal house mice
kept for 2 years in outdoor enclosures. Roughly,
297
the same amount of food was delivered in these
enclosures on a daily basis, but the mice were
allowed to reproduce freely. This led to the
hypothesis that food shortage might have trig-
gered the observed temporal niche switch at times
of high population density. This hypothesis was
tested in the lab by letting lab mice work for
their food to simulate natural food shortage.
This “work-for-food” protocol indeed induced
diurnality in house mice, showing that temporal
niche switching can be induced by reduced food
intake (Fig. 5, Hut et al., 2011). This phenomenon
has been reproduced in several strains of lab mice
(CBA/CaJ, C57bl6, CD1; R.A. Hut, personal
communication), as well as in rats (S. Daan, per-
sonal communication). The results confirm earlier
findings by Perrigo who showed that pregnant
female house mice and white footed deer mice
changed from nocturnal to a diurnal life style
when these mice had to combine working for
food with the high metabolic demand of lactation
(Perrigo, 1987, 1990). Together, these findings
indicate that, rather than reduced food intake
per se, the negative energy balance triggered the
switch from nocturnal activity to diurnal activity.
The CTE hypothesis can explain these
findings by assuming a preserved mechanism in
mammals, which associates a diurnal-activity
pattern with lower energy requirements than a
nocturnal-activity pattern. A negative energy bal-
ance (through reduced food intake, lactation,
etc.) would induce a switch from nocturnal to
diurnal-activity patterns, in order to save energy
expenditure through reduced heat loss through
increased insulation during the rest phase at night
when ambient temperatures are low. In a similar
vein, the CTE hypothesis would also predict that
heat loss reduction through daily torpor would
be optimal when it occurs at night time. This
indeed has been found in the house mice in which
food intake was reduced by letting these mice
work for their food. At the end of the protocol,
after the mice robustly occupied the diurnal tem-
poral niche and placed their rest phase at (end
of) the night, the mice showed various degrees
298

Fig. 5. Experimentally induced temporal niche switching in mice that work for food. Two representative examples (a, b) of mice in
which food intake (black line in side graphs) was successfully reduced through decreasing food reward for running-wheel
revolutions (gray bars in side graphs). As a result, activity patterns (black bars in actogram) shifted into the light phase of the day
(white bars and white lines in actogram). Peak body temperatures (color coded in actograms) also shifted into the day, but body
temperatures also gradually lowered over the course of the experiment eventually resulting in clear occurrences of torpor during
the rest phase at (end of) the night.

of daily torpor in the dark phase (Fig. 5) (Hut et al.,
2011). At this time, environmental temperatures
are expected to be the lowest and the energy
savings through torpor will be maximized. This
nocturnal timing of torpor has also been confirmed
in four species of elephant shrew [Elephantulus
rozeti, E. myurus (Lovegrove et al., 2001a,b); E.
rupestris, E. edwardi (Boyles et al., 2012)], in the
fat-tailed dunnart (Sminthopsis crassicaudata,
Warnecke et al., 2008) and in the golden spiny
mouse (A. russatus, Levy et al., 2011a,b). It may
be that the well studied Djungarian hamster (Pho-
dopus sungorus), with daily torpor timed at day-
time (Ruby and Zucker, 1992), is an energetically
suboptimal exception to this rule.
The fact that the work for food experiments
(Hut et al., 2011) and the effect of ambient tem-
perature on circadian rhythms in the degus
(Hagenauer and Lee, 2008) mentioned above
were carried out under a LD cycle under constant

temperature indicates that the underlying mecha-
nism can use the LD cycle and not temperature
cycle per se to predict the energetically optimal
timing of the activity phase.
The observation that ad libitum food supply
in the “work-for-food” model (Hut et al., 2011)
immediately restores the nocturnal phase under
continuous dim light indicates that the circadian
pacemaker did not change its phase angle relative
to the LD cycle. This leads to the interpretation
that the mechanism triggering behavioral temporal
niche switching is downstream from the circadian
pacemaker (SCN). This situation is schematized
in the middle panel of Fig. 4 and is indeed identical
to the temporal niche switch in diurnal mammals
compared to nocturnal mammals, where activity
patterns are in antiphase, but the SCN entrains
with a similar phase angle relative to the LD cycle
(reviewed in Challet, 2007).
Conclusions
Although temporal niche switching is a complex
phenomenon with a manifold of physiological
and neurobiological mechanisms and evolution-
ary causalities, we may be able to draw a number
of commonalities. This can be based upon the
observation that the main environmental differ-
ences between night and day, which are of direct
ecological importance for animals living in the
wild, relate to temperature and/or light.
Adaptations to the daytime as well as to the
nighttime niche are commonly apparent in the
eye. Mammals with robust nocturnality like
microchiroptera bats have strongly reduced vision
reflected in their tiny eye size, whereas exclusively
diurnal ground squirrels have well developed vision
reflected in their large eyes and cone dominated
retinas (Table 2; Fig. 3). In other species, long-term
adaptations to a specific temporal niche are also
present at the level of the retina (e.g., rod and cone
densities, Table 2), but sometimes difficult to inter-
pret due to variation in local retinal density (i.e.,
fovea, visual streak, photoreceptor gradients).
299
Protection of the retina against high energy short-
wavelength light present during daytime is strongly
reflected in the filtering properties of the lens
(Table 3). For most mammalian species, the lens fil-
tering property together with cone densities (and
possibly distribution; Fig. 3) offers a useful measure
for assessment of a species’ long-term adaptation to
the day- or nighttime niche.
Nocturnal animals need low light levels at night
to accommodate vision. The interaction with the
lunar cycle and the light properties of the envi-
ronment can explain several cases of temporal
niche switching. Such cases have been described
above for nocturnal primates and lobsters,
but similar responses may be wide spread among
nocturnal animals.
Temperature can exhibit important and pre-
dictable changes between night and day. When
temperatures are too high or too low, animals
may modify their activity patterns to reach their
optimal body temperature (in both ectotherms
and endotherms) or to minimize the energetic
needs to regulate their body temperature (in
endotherms only). As such, temperature patterns
can explain several cases of modification of activ-
ity patterns and temporal niche switching. In most
regions on earth, the environmental temperature
stays well below the lower critical temperatures
of most endotherms for a large part of the day.
This means that mostly energy is required to
maintain euthermic body temperatures. Energy-
saving strategies during the rest phase will cause
nocturnal activity to be energetically more costly
than diurnal-activity patterns. This feature has
led us to propose the CTE hypothesis which is a
useful tool to understand cases of temporal niche
switching induced by temperature, season, or
food abundance (Table 1).
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 18

Feedback actions of locomotor activity to the

circadian clock

Alun T. L. Hughes* and Hugh D. Piggins

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

Abstract: The phase of the mammalian circadian system can be entrained to a range of environmental
stimuli, or zeitgebers, including food availability and light. Further, locomotor activity can act as an
entraining signal and represents a mechanism for an endogenous behavior to feedback and influence
subsequent circadian function. This process involves a number of nuclei distributed across the brain
stem, thalamus, and hypothalamus and ultimately alters SCN electrical and molecular function to
induce phase shifts in the master circadian pacemaker. Locomotor activity feedback to the circadian
system is effective across both nocturnal and diurnal species, including humans, and has recently been
shown to improve circadian function in a mouse model with a weakened circadian system. This raises
the possibility that exercise may be useful as a noninvasive treatment in cases of human circadian
dysfunction including aging, shift work, transmeridian travel, and the blind.

Keywords: circadian; suprachiasmatic; locomotor activity; wheel-running; exercise; clock gene; VIP;
aging; arousal; NPY; serotonin.

Introduction physiological functions. A feature of these daily, or

The rotation of the Earth about its axis imposes
upon the vast majority of living organisms predict-
able daily cycles with a period of
24 h. In order
to perform optimally in an appropriate temporal
niche, organisms have developed corresponding
cyclic rhythms in their own behavioral and
*Corresponding author.
Tel.: þ44-161-306-0502; Fax: þ44-161-275-3938
E-mail: alun.t.hughes@manchester.ac.uk
circadian, rhythms (derived from the Latin circa,
meaning about, and dies, a day) is that they are gen-
erated by endogenous circadian clocks expressed
within the cells and tissues of an organism and
continue to oscillate with a period of close to 24 h
even when the organism is isolated from all
external timing influences. Endogenous circadian
oscillators are thought to confer a selective advan-
tage on organisms through predictive homeostasis;
organisms are able to anticipate predictable envi-
ronmental events, such as dusk and dawn or times

http://dx.doi.org/10.1016/B978-0-444-59427-3.00018-6 305
306
of high predator/prey activity, before they occur and
can therefore prepare appropriately (Piggins and
Guilding, 2011). Indeed, the lack of a circadian clock
can influence survival in a natural environment
(DeCoursey et al., 2000), and possession of a clock
that runs with an inappropriate cycle length is disad-
vantageous in laboratory-based competition studies
(Woelfle et al., 2004).
The mammalian circadian system consists of
input pathways (Fig. 1), a central “master” pace-
maker located in the suprachiasmatic nuclei
(SCN) of the hypothalamus, numerous extra-SCN
oscillators distributed throughout the brain and
body, and output pathways to effect changes in
the temporal organization of physiology and
behavior (Golombek and Rosenstein, 2010;
Kalsbeek et al., 2006). Anatomically, the SCN can
be loosely divided into two subregions, one
characterized by expression of the neuropeptide
IGL
Serotonin
Orexin
HT
G
AB Y
NP
LHA
G
Eye Glu
Orexin
RH
T
SCN
PA
Se
CA
P
Fig. 1. Schematic “wiring diagram” showing nuclei and
neurotransmitters of the mammalian central circadian system
involved with signaling photic and nonphotic entrainment
information. Photic signals are transmitted directly to the
SCN via the RHT utilizing glutamate and PACAP.
Nonphotic information, however, reaches the SCN via a
network of connections involving the DR and MR, the IGL, and
the LHA. These pathways use serotonin, NPY and GABA, and
orexin, respectively as their main transmitters. DR, dorsal raphe;
GABA, g-amino butyric acid; GHT, geniculohypothalamic
tract; Glu, glutamate; IGL, intergeniculate leaflet; LHA, lateral
hypothalamic area; MR, median raphe; PACAP, pituitary
adenylate-cyclase-activating peptide; RHT, retinohypothalamic
tract; SCN, suprachiasmatic nucleus.
vasoactive intestinal polypeptide (VIP) that
receives heavy external input from the retina and
other regions of the brain, and the remainder of
the SCN that receives somewhat lighter extra-
SCN innervation (Abrahamson and Moore,
2001; Morin, 2007). The SCN generates rhythms
of high- and low-neuronal discharge (Brown and
Piggins, 2007) driven by an interlocking network
of core clock genes and other rhythmic processes
(Glossop, 2011; Hastings et al., 2008; Ko and Taka-
hashi, 2006), though recent evidence indicates
that the electrical output of the SCN is more com-
plex than this simple model suggests (Belle et al.,
2009). Under normal environmental and physio-
logical conditions, the SCN dominates the circadian
system of mammals, coordinating the phase of
rhythms in the various tissue-specific extra-SCN
oscillators (see Dibner et al., 2010; Guilding and
Piggins, 2007).
To benefit an organism, endogenous circadian
rhythms must be maintained in phase with envi-
ronmental time cues. In addition to specific chem-
ical agents such as melatonin (van Geijlswijk
DR
et al., 2010), certain stimuli including light and a
variety of light-independent, or nonphotic, cues
are capable of phase-dependently phase resetting
the circadian system. Repeated exposure to these
O
re
xi
n
signals at appropriate times can synchronize, or
entrain, the circadian system to the environment
MR (Golombek and Rosenstein, 2010). The purpose
nin
ro
to of this review is to collate and summarize the
phase resetting and entraining actions of locomo-
tor activity feedback to the mammalian circadian
system.
Photic entrainment
The most ubiquitous entraining stimulus, or zeitge-
ber, encountered in daily life for the majority of
organisms is that of the light–dark (LD) cycle of
the solar day; therefore, to fully appreciate non-
photic influences on the circadian system, we must
first briefly consider photic entrainment. As the
endogenous SCN-driven period of circadian
 307

rhythms for most mammals differs slightly from
24 h, rhythms must be periodically shifted back
toward external environmental time in order to
maintain an appropriate phase relationship with
the environment (Challet, 2007). Light encoun-
tered during the early part of the night phase of
an animal’s endogenous rhythm, for instance, if a
nocturnal rodent were to become active before
dusk, delays the clock. This shifts activity to a
slightly later time on subsequent cycles, realigning
it with the external environment. Conversely, light
encountered during the late part of an animal’s
endogenous night phase, if a nocturnal rodent were
to remain active after dawn, advances the phase of
the clock. This shifts activity to a slightly earlier
time on subsequent days, again realigning behavior
and physiology with the external world. These
responses to light encountered during an animal’s
“subjective” night (i.e., night according to endoge-
nous circadian phase) are similar in both nocturnal
and diurnal species (e.g., Slotten et al., 2005) and
can be mimicked in a controlled laboratory envi-
ronment. Brief (5–10 min) pulses of light delivered
to animals otherwise housed in constant darkness
(DD) produce phase-dependent shifts in the timing
of the rest-activity cycle defining a phase response
curve (PRC; see Fig. 2) to photic stimulation (Daan
and Pittendrigh, 1976; Schwartz and Zimmerman,
1990). Such procedures, in addition to confirming
the advance and delay portions of the photic
PRC, also reveal a photically nonresponsive zone
during the subjective day, a time at which light
would be normal, so phase shifts would be inappro-
priate (Fig. 2).

(a) (b)
Nonphotic PRC
0 +3.0
Phase shift (h)

18
–3.0
Subjective day Subjective night
Days

(c) Photic PRC

+3.0

36
Phase shift (h)

–3.0
Subjective day Subjective night
54

Fig. 2. Stylized schematic representation of phase shift data for a laboratory rodent in constant darkness. (a) Bouts of induced
locomotor activity (open ellipses), stimulated by mechanisms such as novel wheel confinement or triazolam injection, produce
marked phase advances during the mid-to-late subjective day and modest phase delays during the late subjective night. Brief
light pulses (open circles) produce differential phase-dependent phase shifts in behavior when presented during the early and
late subjective night and fail to shift behavior during the subjective day. Each row represents 24 h and black bars represent
voluntary home-cage locomotor activity. (b) Phase response curve (PRC) summarizing the phase-dependent phase responses of
nocturnal rodents to a generalized “nonphotic” zeitgeber such as 3 h of induced locomotor activity. (c) PRC summarizing the
phase-shift responses of nocturnal rodents to brief (
5–60 min) light pulses. Reflecting the requirements of different circadian
systems, in species where tau is shorter than 24 h (as shown), the phase delay portion of the PRC is usually larger than the phase
advance portion. In species where tau is longer than 24 h, the advance portion is usually larger.
308
Phase shifts such as those described above in
response to light are typically assessed by measure-
ment of the daily onset or offset of locomotor or
consumptive activities of an animal, however,
these shifts in behavior reflect changes to the phase
of the underlying SCN oscillator. Light signals are
delivered to the central SCN oscillator via a gluta-
mate and pituitary adenylate-cyclase-activating
peptide-expressing projection (Fig. 1) from a
subset of intrinsically photosensitive melanopsin-
expressing retinal ganglion cells (Hannibal and
Fahrenkrug, 2006; Morin and Allen, 2006). Gluta-
mate agonists and antagonists infused or injected
into the third ventricle in vivo or applied to SCN
brain slices in vitro, mimic and block the effects of
light and optic nerve stimulation, respectively
(Abe et al., 1991; Ebling et al., 1991; Mintz and
Albers, 1997; Shibata et al., 1994; Vindlacheruvu
et al., 1992), confirming the role of glutamate in
signaling photic information to the SCN.
Negative masking by light
In addition to entrainment of a functional oscillator,
light can directly affect the expression of locomotor
activity in nocturnal species through an alternative,
more direct route: masking. Negative masking is
the direct suppression of locomotor activity by light
in nocturnal species (Doyle and Menaker, 2007) and
is mediated by the same melanopsin-expressing
retinal ganglion cells mentioned above, however,
the neural and neurochemical processes that medi-
ate masking in the brain are currently poorly under-
stood (Goz et al., 2008; Hattar et al., 2003; Li et al.,
2005; Panda et al., 2003).
Nonphotic entrainment
The concept that circadian rhythms could be
influenced by mechanisms other than light came
to prominence in the mid-1960s through a series
of experiments demonstrating that the rhythms of
birds could be entrained to the daily playback of
birdsong (Gwinner, 1966; Menaker and Eskin,
1966). These experiments, along with early reports
of social entrainment in humans (Aschoff, 1979;
Wever, 1979) elegantly revealed that circadian
rhythms could be altered using so-called nonphotic
mechanisms; that is, any phase shifting or
entraining stimulus independent of light itself or
the specific neural pathways activated by light. A
wide variety of different stimuli that fall into this
category are capable of signaling environmental
information to the circadian system and these
nonphotic stimuli can be broadly divided into
two major subclasses with very different functions
and behavioral responses: those associated with
feeding and those that induce locomotor activity
and/or arousal (Mistlberger and Antle, 2011).
Entrainment to restricted feeding and the
food-entrainable oscillator
The importance of regular food availability and
dire consequences of failure to acquire food
places special import on the ability of an animal
to track predictable feeding opportunities. As
such, animals have evolved a food-entrainable
oscillator (FEO) which functions independently
of the SCN and can become decoupled from it
(see Davidson, 2006 and Mistlberger and Antle,
2011 for review). FEO-driven food anticipatory
behavior can be driven out of phase from the
SCN-controlled main active phase by the restric-
tion of food presentation to a time during an
animal’s normal inactive phase (Boulos et al.,
1980; Honma et al., 1983). The anatomical and bio-
chemical bases of this FEO are not yet known,
though have been the focus of much debate and
controversy in recent years (e.g., Fuller et al.,
2009; Mistlberger et al., 2009). We do know, how-
ever, that this oscillator persists in the absence of
the SCN (Stephan et al., 1979a,b), in the absence
of a critical core clock gene (bmal1; Storch and
Weitz, 2012) and that daytime restricted feeding
not only induces food anticipatory activity prior
to food presentation, but also potently entrains

the phase of liver and other peripheral oscillators
associated with metabolism, while the SCN
remains entrained to the LD cycle (Damiola
et al., 2000; Stokkan et al., 2001).
Nonphotic stimuli based on locomotor activity
and/or arousal
The second major subclass of nonphotic stimuli is
composed of those associated with locomotor
activity and/or arousal. A great variety of different
stimuli of this nature can influence circadian func-
tion, including the introduction of novelty to the
environment, for instance, the presentation of a
new running wheel (Mrosovsky and Salmon,
1987); social interactions with conspecifics, and
in particular, proximity of inaccessible reproduc-
tively active females to male test animals (Honrado
and Mrosovsky, 1989); forced or voluntary loco-
motor activity (Edgar and Dement, 1991;
Marchant and Mistlberger, 1996); daytime pulses
of darkness to nocturnal animals (Boulos and
Rusak, 1982); and gentle handling (Antle and Mis-
tlberger, 2000). This wide range of stimuli involves
a common element, however: the induction of vig-
orous locomotor activity and/or the induction of
arousal. Moreover, injection of chemicals that
induce locomotor exercise, for instance, certain
benzodiazepines (see below) or morphine
(Marchant and Mistlberger, 1995), can act as non-
photic zeitgebers. This group of general locomotor
activity/arousal-associated stimuli differs funda-
mentally to feeding-related time cues and the
FEO in that they function through mechanisms
that, similarly to photic zeitgebers, converge and
act on the central SCN oscillator. Locomotor
activity/arousal-associated events induce a PRC
completely different to that of light, characterized
by large phase advances during the mid-to-late
subjective day and relatively small phase delays in
the late subjective night (Mrosovsky et al., 1989;
Fig. 2). It is this second group of nonphotic stimuli,
and, in particular, locomotor activity/exercise that
will form the main focus of this review.
309
Locomotor activity as a nonphotic stimulus
A number of techniques can be used to induce
locomotor activity in laboratory rodents including
confinement to a novel-running wheel, injections
of some benzodiazepines, and forced treadmill
running (e.g., Mistlberger, 1991a,b; Mrosovsky
and Salmon, 1987; Turek and Losee-Olson, 1986).
Each of these, along with scheduled voluntary
wheel access (Edgar and Dement, 1991), has been
used extensively to investigate the effects of overt
locomotor activity on circadian function.
The involvement of induced locomotor activity
in benzodiazepine-induced circadian effects was
originally the subject of some controversy. Initial
studies using triazolam, a short-acting benzodiaze-
pine, were designed to investigate the role of
GABA signaling in circadian biology (e.g., Turek
and Losee-Olson, 1986, 1987a) and the realization
that triazolam-mediated effects were reliant on
induced locomotor activity came later: hamsters
denied wheel access following injections show no
circadian effects, and crucially, the magnitude of
triazolam-mediated effects correlates with the
amount of time spent wheel-running during the
first 6 h after an injection (Mrosovsky and Salmon,
1990; Van Reeth and Turek, 1989a,b). It should be
noted however that, while locomotor activity
undoubtedly feeds back to the circadian system,
injections of chlordiazepoxide, a benzodiazepine
that suppresses locomotor activity, also cause
nonphotic-like phase shifts, demonstrating that
benzodiazepines in their own right can directly
confer some nonphotic information, presumably
through their effect on GABA signaling (Hastings
et al., 1998; Meyer et al., 1993). Indeed, injections
of muscimol, a specific GABAA agonist, produce
a nonphotic PRC (Smith et al., 1989).
Circadian responses to discrete “pulses” of
locomotor activity
Single, discrete, locomotor activity pulses induced
during the mid-to-late part of the subjective day
310
phase advance free-running behavioral rhythms in
hamsters. Both triazolam injections and confine-
ment to a novel-running wheel produce this effect,
in both DD and constant light (LL; Reebs and
Mrosovsky, 1989a; Reebs et al., 1989; Turek and
Losee-Olson, 1986, 1987a). Triazolam injections,
in addition to shifting the phase of locomotor activ-
ity rhythms, have also been shown to phase shift
the rhythm of LH release in female hamsters
(Turek and Losee-Olson, 1988). The efficacy of
single locomotor activity pulses to shift rhythms in
other species is less clear, however. For instance,
“pulses” of novelty-induced activity and 3-h forced
treadmill running produce only modest and incon-
sistent phase shifts in mice (Challet et al., 2000;
Marchant and Mistlberger, 1996).
PRCs to both triazolam-induced (Turek and
Losee-Olson, 1986) and novel wheel-induced
locomotor activity pulses (Reebs and Mrosovsky,
1989a) similarly define a classical nonphotic pro-
file, with maximal phase advances, of
2–3 h,
when activity is induced during the mid-to-late
subjective day and somewhat smaller phase
delays seen during the late subjective night
(Reebs and Mrosovsky, 1989a,b; Wickland and
Turek, 1991; Fig. 2). Following initial contradic-
tory reports, a careful assessment of the literature
including consideration of confounding factors in
individual experiments supports the notion that
the amount of locomotor activity performed does,
in the majority of cases, correlate with shift mag-
nitude (elegantly reviewed in Mrosovsky, 1996).
Shift magnitude in response to novelty-induced
locomotor activity correlates with the amount of
locomotor activity performed (Bobrzynska and
Mrosovsky, 1998; Janik and Mrosovsky, 1993;
Weisgerber et al., 1997; Wickland and Turek,
1991) and a minimum of
3 h of running is neces-
sary to induce maximal responses (Reebs and
Mrosovsky, 1989b; Wickland and Turek, 1991).
Individuals that neglect to run vigorously tend to
show minimal phase shifts. Further, the induction
of extremely large phase shifts, in the order of

12 h, is possible with the combined stimulus of
transfer to DD and presentation of a novel wheel
which is then permanently left in place (Gannon
and Rea, 1995).
The relatively long exposures to exercise
required for large shifts indicate that the circadian
system of nocturnal rodents has a relatively high
threshold for exercise to alter circadian function,
at least compared to light, which produces measur-
able phase shifts after as little as a few minutes of
exposure (Sharma and Chandrashekaran, 1997;
Takahashi et al., 1984). This relatively low sensitiv-
ity to locomotor activity may represent a buffering
system to prevent inappropriate phase shifting
to small amounts of activity which may be normal
outside the main active phase.
Interactions between nonphotic and photic
entraining stimuli
Previously in this review, we have considered
the well-characterized effects of both light and
nonphotic influences on the phase of behavioral
circadian rhythms. While in the laboratory we aim
to control variables in order to determine
responses to specific stimuli, in nature, it would
be more common for different zeitgeber modalities
to be encountered in combination. Responses of
the mammalian circadian system to photic and
nonphotic stimuli are both mediated by effects on
the central SCN oscillator (Challet, 2007). As such,
it is important to consider the convergent, yet dis-
tinct, responses of animals to photic and nonphotic
signals and to examine the interactions between
them when presented either together or in series.
Dark pulses
Behavioral responses to pulses of darkness pres-
ented to nocturnal rodents housed otherwise in
LL (Boulos and Rusak, 1982; Ellis et al., 1982)
produce a PRC that is somewhat similar to those
described for typical locomotor activity/arousal-
associated nonphotic stimuli (Canal and Piggins,
2006; Marston et al., 2008). Originally considered

a mirror image of the photic PRC (Subbaraj and
Chandrashekaran, 1978), these responses are
now thought to arise from a combination of both
nonphotic and photic components (Coogan and
Piggins, 2005; Rosenwasser and Dwyer, 2001),
though the contribution of locomotor activity to
dark pulse phase shifts has been debated. Total
wheel revolutions during a pulse do not correlate
significantly with shift magnitude, but a trend
toward more revolutions resulting in larger shifts
is evident (Coogan and Piggins, 2005; Marston
et al., 2008). However, specific assessment of
locomotor activity induced within an individual
by subjective day dark pulses, accounting for
baseline activity on previous days, does correlate
well with shift magnitude (Canal and Piggins,
2006). Further, the magnitude of phase shifts is
attenuated by restriction of wheel-running activity
during the pulse (Reebs et al., 1989). These data
suggest that locomotor activity does indeed form
the nonphotic component of a dark pulse, but
that a change in the level of activity within
an individual is important, rather than a pan-
individual threshold of activity. A photic compo-
nent arising from the resumption of LL at the
end of a pulse is postulated to give rise to the
differences between typical nonphotic PRCs and
the dark pulse PRC (Rosenwasser and Dwyer,
2001) and may be mediated at the molecular level
by interactions between photic and nonphotic
resetting as they converge at the SCN (Maywood
and Mrosovsky, 2001).
Reentrainment to an LD cycle shift and photic/
nonphotic conflict studies
Sudden shifts in the phase of the lighting regime
under LD simulate the jetlag encountered by
humans following transmeridian air travel. An
8-h advance of the LD cycle approximates the
temporal disruption experienced following a flight
from California to the United Kingdom and
requires a series of stepwise phase-advancing
shifts to resynchronize endogenous oscillators
311
and overt behavior with the environment. Under
normal conditions, reentrainment to an 8-h
advance such as this would require
8 days.
However, this latency to reentrainment can be
dramatically reduced in laboratory rodent models
by locomotor activity induced at the beginning of
the dark phase on the first day in the shifted LD
cycle, by either 3 h of novel-running wheel con-
finement or triazolam injection (Mrosovsky and
Salmon, 1987, 1990; Van Reeth and Turek, 1987,
1989a,b). These locomotor activity pulses result
in a rapid advance in the onset of subsequent
home-cage activity, with a shift of
7 h clearly
visible by the second cycle in the new lighting
schedule. In addition to accelerating behavioral
reentrainment, locomotor activity pulses such as
this also accelerate the reentrainment of some
peripheral oscillators (Yamanaka et al., 2008).
Rapid, locomotor activity-induced, phase
reentrainment to an LD shift and the previously
described PRCs to novel wheel/triazolam-induced
locomotor activity in DD provide an interesting
opportunity to examine the interaction of loco-
motor exercise and photic cues. The large
7h
phase advance seen in response to induced day-
time exercise shortly after an LD shift cannot be
explained by the peak of the advance portion of
the PRC to exercise alone, creating an apparent
discrepancy between the large phase shifts neces-
sary for rapid reentrainment and the 3-h peak
shift for discrete pulses of locomotor activity seen
in PRCs. This can be reconciled, however, by view-
ing the phase response of a hamster to a combina-
tion of an LD shift and exercise as synergistic of
both the nonphotic cue of induced activity and
the photic cue of light falling at inappropriate
phases of the circadian cycle (as a result of the
LD shift; Mrosovsky 1991; Reebs and Mrosovsky,
1989b). Indeed, presenting light pulses at different
phases relative to a phase-advancing exercise pulse
produces variable phase shifts depending on the
temporal relationship between locomotor activity
and light (Joy and Turek, 1992; Mrosovsky, 1991;
Reebs and Mrosovsky, 1989b). For instance, a light
pulse following within 3 h of a late subjective
312
daytime activity pulse would fall in the delay portion
of the photic PRC and all but cancels the phase-
advancing effect of locomotor activity, while a light
pulse presented 8 h after subjective daytime activity
enhances the resultant phase shift as a summation of
the two phase-advancing stimuli (Mrosovsky, 1991;
Reebs and Mrosovsky, 1989b).
Similarly, inducing locomotor activity during
subjective nighttime light pulses dramatically
attenuates phase shifts compared to those in
response to light alone (Ralph and Mrosovsky,
1992) and parallel treatment of SCN-containing
brain slices in vitro with neurochemicals that
mediate photic and nonphotic resetting recipro-
cally blocks the phase-resetting effects of the
other (Biello et al., 1997). This spectrum of
responses to the combined effects of photic and
nonphotic zeitgebers defines a complex relation-
ship between them, reflective of the complexity
of temporal signals organisms are exposed to in
the real world and the underlying complexity of
the extended circadian system itself.
Entrainment to exercise: Longer-term effects of
locomotor activity
We have, up to now, considered the short-term
effects of acute “pulses” of locomotor activity on
free-running circadian rhythms in constant
conditions and the ability of such pulses to modify
the reentrainment of behavior following LD
shifts. In addition to these acute effects, ad libitum
access to a running wheel, and hence daily,
though unscheduled, vigorous locomotor exercise
also feeds back to regulate central circadian func-
tion. The presence of a home-cage running wheel
shortens tau in rats and mice (Benus et al., 1988;
Edgar et al., 1991; Mistlberger and Holmes,
2000; Mistlberger et al., 1998; Yamada et al.,
1986, 1988) and has been suggested, though not
proven, to do so in hamsters (Pratt and Goldman,
1986; though see also Aschoff et al., 1973). In rats,
the magnitude of tau shortening by wheel-running
correlates with the number of wheel revolutions
performed (Shioiri et al., 1991; Yamada et al.,
1990), an effect also seen, though somewhat
inconsistently, in hamsters (Mrosovsky, 1999;
Weisgerber et al., 1997).
Entrainment to scheduled locomotor activity
To be of functional use for entrainment, however,
feedback information from locomotor activity must
demonstrate the ability to synchronize free-running
rhythms through repeated phase adjustments when
the opportunity to indulge in locomotor activity
is either offered, induced, or forced repeatedly on
sequential cycles.
Induced and scheduled locomotor activity can
alter the phase angle of entrainment to an LD cycle
(Mistlberger, 1991b; Mistlberger and Holmes,
2000; Salgado-Delgado et al., 2008; Turek and
Losee-Olson, 1987b). The direction of phase angle
changes in these studies is predictable based on
the PRCs to induced locomotor activity. Activity
induced near the light-to-dark transition, proximal
to the advance portion of the locomotor PRC,
results in a more positive (advanced) phase angle
of entrainment and activity late in the dark
phase results in a delayed phase angle. Other
reports, however, have suggested that under
some circumstances a more complex relationship
between photic and nonphotic zeitgebers may exist
(Sinclair and Mistlberger, 1997).
Stable entrainment of rodent behavioral rhythms
in the absence of an LD cycle has also been
shown. Serial daily injections of triazolam to
enucleated hamsters and daily novel wheel
confinement of hamsters in DD both entrain
behavioral rhythms (Reebs and Mrosovsky,
1989a; Van Reeth and Turek, 1989a,b, 1990).
Similarly, mice entrain to both voluntary and
forced scheduled locomotor exercise of between
2 and 12 h each day (Dallmann et al., 2007; Edgar
and Dement, 1991; Edgar et al., 1997; Laemle and
Ottenweller, 1999; Marchant and Mistlberger,
1996; Marchant et al., 1997; Power et al., 2010;
Figs. 3 and 4a) and while apparently a weak
 313

stimulus in rats, forced treadmill running entrains
free-running rhythms in this species too
(Mistlberger, 1991a). “Aftereffects” are com-
monly observed such that following scheduled
exercise, free-running tau is closer to the period
of the entraining signal than in initial free
run (Dallmann et al., 2007; Laemle and
Ottenweller, 1999; Marchant and Mistlberger,
1996; Power et al., 2010; Fig. 4a; see also Reebs
and St-Coeur, 1994). Radio frequency lesions in
mice have shown the SCN to be essential for
entrainment to scheduled locomotor activity;
mice rendered behaviorally arrhythmic by com-
plete SCN lesions do run on the treadmill, but
scheduled forced treadmill activity fails to either
induce or sustain behavioral circadian rhythms
(Marchant et al., 1997). Further, SCN efferent
connections appear to be essential for activity-
induced phase modulations; SCN-lesioned hamsters
with fetal SCN transplants, that lack extensive
neural connections with the host, fail to phase
advance in response to induced subjective day activ-
ity (Canbeyli et al., 1991).
Phase angle of entrainment to scheduled
locomotor activity and the nonphotic PRC
Importantly, in all successful locomotor activity
entrainment studies to date, the phase angle of
entrainment of behavioral rhythms to repeated
locomotor activity, be it voluntary, induced, or
forced, is dependent on the relative periods of the
entraining stimulus and endogenous free-running
rhythms of subject animals (e.g., Dallmann et al.,
2007). When entrainment by phase delay is
required, that is, endogenous period, tau, is
shorter than the entraining period, T, rhythms

(a) (b)
0 0

18 18
Days

Days

36 36

54 54
Entrainment by delay Entrainment by advance

Fig. 3. Stylized schematic representation of entrainment to scheduled locomotor activity. The scheduled opportunity for vigorous
locomotor activity, be it voluntary wheel-running or forced treadmill running, entrains behavioral rhythms in most species
examined. The black outline boxes in (a) and (b) indicate timing of scheduled activity and black bars represent behavior
recorded using a nonrunning wheel mechanism such as drinking or general locomotor activity. Each row represents 24 h. (a)
When an animal’s endogenous tau is shorter than the zeitgeber period (T) entrainment is achieved through daily phase delays.
Synchronizing the opportunity to run with the delay portion of the nonphotic phase response curve results in a positive phase
angle of entrainment. (b) When an animal’s endogenous tau is longer than T, entrainment is achieved through daily
phase advances, resulting in a neutral or small negative phase angle of entrainment. These characteristics are consistent with the
phase response curve shown in Fig. 2b and have been described across a range of both nocturnal and diurnal species.
314

(a) WT (b) Vipr2–/–

Wheel-running Drinking Wheel-running Drinking
LD
DD1
locomotor activity
Scheduled
DD2

0 24 48 0 24 48 0 24 48 0 24 48
Time (h)

Fig. 4. Scheduled locomotor activity acts as a potent zeitgeber to entrain behavior in a mouse model with a weakened SCN
pacemaker and induces lasting improvements in circadian function. (a) Wheel-running and drinking behavior for a wild-type
(C57BL/6J) mouse under LD, free-running in DD then subjected to 50 days of scheduled voluntary locomotor activity before a
second DD free run. (b) Wheel-running and drinking behavior for a mouse lacking functional expression of the VPAC2 receptor
(Vipr2/) under the same experimental protocol as (a). Black outline boxes indicate timing of scheduled activity, data are
double plotted such that each row shows 48 h. Wild-type mice gradually entrain to 6 h of daily scheduled voluntary locomotor
activity, visible on the drinking panel, and subsequently free-run with a modestly lengthened tau. Vipr2/ mice rapidly entrain
to scheduled activity and display a profound and persistent change in subsequent free-running tau of
24 h. Note that behavior
in (a) entrains according to the rules described in Fig. 3 and consistent with the PRC in Fig. 2b. Postscheduled activity behavior
in (b) free runs in phase with the previous opportunity to exercise. See Power et al. (2010) for further examples and interpretation.

entrain with a phase angle that places activity at
the end of the subjective night (Fig. 3a), the delay
portion of the nonphotic PRC (Fig. 2b). When tau
is longer than T, entrainment by advance is
required resulting in a phase angle that places
activity at the end of the subjective day/beginning
of the subjective night (Fig. 3b); the advance por-
tion of the PRC to locomotor activity (Fig. 2b).
The modulation of phase angle of entrainment
to LD cycles by scheduled locomotor activity
described above (Mistlberger, 1991b; Turek and
Losee-Olson, 1987b) is also consistent with this
model, as is the acceleration of reentrainment to
an LD cycle advance by activity induced during
what was previously the light phase, the subjec-
tive day phase advance portion of the nonphotic
PRC (e.g., Mrosovsky and Salmon, 1987; van
Reeth and Turek, 1987; Fig. 2b).
The roles of arousal and physiological correlates
of locomotor activity in entrainment
The role of exercise/locomotor activity in entrain-
ment is intimately linked with the arousal state of
an animal (Mrosovsky, 1996), and to date, even
25 years after the first reports of locomotor activity
feeding back to the central oscillator, the precise

nature of this signal has yet to be resolved. It
is clear that locomotor activity feedback can alter
circadian phase, both acutely and as stable entrain-
ment, however, it is unknown which aspect or com-
bination of aspects of locomotor activity are crucial
for this effect. Locomotor activity represents a
complex amalgam of behavioral, physiological,
and emotional factors. These include an underlying
arousal state related to both the motivation to
exercise and associated rewarding feedback,
physiological adaptations to demanding physical
activity, increases in body temperature during vig-
orous exercise, physical exhaustion, and increases
in consumptive behaviors inevitably associated
with increases in energy expenditure. A number
of studies have attempted to identify the key caus-
ative elements of locomotor activity-induced phase
shifts though no consensus has yet been reached.
Arousal and motivation
In general, the number of wheel revolutions
performed correlates with shift magnitude at a par-
ticular circadian phase (e.g., Biello et al., 1994;
Mrosovsky and Salmon, 1990; see Mrosovsky,
1996 for a detailed interpretation of different
methods of inducing activity). It has, however,
been suggested that locomotor activity per se may
not be sufficient for phase shifts to occur. One
alternative method to those discussed previously
that can induce wheel-running is lowering ambient
housing temperature, though increasing wheel-
running during a novelty-induced activity bout by
lowering ambient temperature does not necessarily
result in increased shift magnitude (Janik and
Mrosovsky, 1993; Mrosovsky and Biello, 1994).
This has been interpreted as a lack of some neces-
sary element of motivation or arousal associated
with cold-induced wheel-running increases; run-
ning in the cold may be necessary for thermoregu-
lation whereas running at normal temperatures
may simply be rewarding (Janik and Mrosovsky,
1993; Mrosovsky and Biello, 1994). This methodol-
ogy has, however, been questioned: Mistlberger
315
et al. (1996) report that with an appropriate exper-
imental design, cold-induced running can be as
effective at producing phase shifts as novelty-
induced running. Indeed, scheduled forced tread-
mill running, which potentially lacks the a priori
motivation to run, is as effective at inducing
entrainment in mice as the scheduled opportunity
for voluntary wheel-running (Marchant and
Mistlberger, 1996; Marchant et al., 1997).
Physiological correlates of locomotor activity
Few studies have examined the roles of physio-
logical correlates of locomotor activity in entrain-
ment, though a number of reports have been
published. Inadvertent scheduling of water intake
is an inevitable consequence of ad libitum water
availability during scheduled activity studies (e.g.,
see Edgar and Dement, 1991; Marchant and
Mistlberger, 1996; Power et al., 2010), but is not suf-
ficient for entrainment: Marchant and Mistlberger
(1996) report that scheduled drinking without
scheduled exercise fails to entrain behavior.
The possible role of inadvertent scheduled feed-
ing during scheduled exercise has yet to be specifi-
cally examined. While daytime restricted feeding
under an LD cycle is effective in entraining a com-
ponent of behavioral rhythms and can alter SCN
function (Verwey and Amir, 2009), it does not
entrain the SCN (Damiola et al., 2000). However,
daytime restricted feeding does entrain peripheral
oscillators (Damiola et al., 2000; Stokkan et al.,
2001) and scheduled feeding in DD in the absence
of other timing cues does entrain the SCN and
major SCN-controlled circadian systems, at least
in some mouse strains (Abe et al., 1989, 2007;
Castillo et al., 2004). The timing of feeding during
scheduled locomotor activity entrainment has yet
to be recorded though is likely to be high around
the times of increased locomotor activity. Indeed,
forced activity during the light phase of an LD
cycle is reported to increase feeding during this
time (Salgado-Delgado et al., 2008) and temporally
targeted feeding hastens reentrainment to an LD
316
shift (Angeles-Castellanos et al., 2011). As such, a
role for the inadvertent scheduling of feeding in
entrainment to exercise is a possibility that cannot
be discounted. A role for body temperature
rhythms, which will be higher during vigorous run-
ning also remains a possibility though has yet to be
systematically investigated.
Molecular, neural, and peripheral bases of
nonphotic phase-shifting and entrainment
Molecular basis of phase shifts
At the molecular level, circadian rhythms are gen-
erated within cell autonomous pacemakers
through the rhythmic expression of a network of
core clock genes and their proteins, including,
among others, the period (per) genes, per1 and
per2, the cryptochrome 1 and 2 genes and bmal1.
Our current understanding of the components
and workings of this core molecular oscillator has
been extensively reviewed elsewhere (Glossop,
2011; Ko and Takahashi, 2006), so this discussion
shall be limited to the role of per/PER in the con-
text of nonphotic phase shifting and entrainment.
The per genes are the main targets within the
core molecular oscillator for stimuli that phase shift
the clock (though photic influences on other clock
genes have been described, see Challet et al.,
2003 for review). Locomotor activity induced by
both novel wheel confinement and brotizolam
(a benzodiazepine with similar phase-shifting
actions to triazolam) suppresses the expression of
per1 and per2 mRNA in the SCN at times that
would result in a behavioral phase shift (Maywood
et al., 1999; Yokota et al., 2000). Further, and cru-
cially, injection of per1 antisense oligonucleotides
in the mid-subjective day, the approximate time
of maximal nonphotic phase shifts and maximal
per1 expression, suppresses per1 levels and induces
nonphotic-like phase advances (Hamada et al.,
2004). Interestingly, forced activity during the light
phase of an LD cycle, therefore in conflict with the
photic zeitgeber, appears to suppress levels of
PER1 expression in the SCN throughout the circa-
dian cycle, but fails to alter the phase of this rhythm
(Salgado-Delgado et al., 2010).
While the mechanisms of signal transduction
that mediate this suppressive action of locomotor
activity on per gene expression are poorly under-
stood, a number of reports indicate the involve-
ment of the extracellular signal-regulated kinase
(ERK) pathway in response to this and other
nonphotic stimuli. Dark pulses during the subjec-
tive day, that induce vigorous wheel-running in
nocturnal rodents, suppress ERK phosphoryla-
tion in the SCN (Coogan and Piggins, 2005),
while sleep deprivation, a (nonexercise related)
nonphotic arousal stimulus, suppresses ERK
phosphorylation in most areas of the SCN. In par-
allel to this general suppression, this stimulus
increases phospho-ERK levels in a distinct subre-
gion that corresponds with the area that receives
both retinal and geniculothalamic input (see
Section “Neuropeptide Y and the IGL” below;
Antle et al., 2008).
Further clues as to the mechanisms of nonphotic
control of per expression can be gleaned from the
somewhat better described mechanisms of signal
transduction for photic information. Representing
a point of convergence between the broadly oppo-
site effects of photic and nonphotic stimuli on the
circadian system, per1 and per2, and their proteins,
are induced by phase-shifting light pulses delivered
at appropriate nighttime phases (Albrecht et al.,
1997; Shigeyoshi et al., 1997; Yan and Silver,
2002). These photic signals induce per expression
through activating a number of second messenger
cascades, including ERK, protein kinase A, and
Ca2 þ/calmodulin-dependent protein kinase II sig-
naling and ultimately lead to transcriptional activa-
tion of the per genes via activation of cAMP
response elements in their promoters (Golombek
and Ralph, 1995; Tischkau et al., 2000; von Gall
et al., 1998). Given the synergy between per sup-
pression and induction by nonphotic and photic
stimuli, respectively, and the demonstration of
ERK pathway involvement in responses to both,
it is likely that at least some of the photic signaling

pathways mentioned above, and specifically
their suppression, may also function in mediating
nonphotic signals including those arising due to
locomotor activity.
Locomotor activity suppresses electrical output
of the SCN
The mechanistic connections between the core
molecular oscillator and membrane-level electri-
cal activity have yet to be fully elucidated
(Colwell, 2011). We cannot, therefore, describe
how the above effects of locomotor activity on
the core molecular oscillator are transduced into
long-term changes to the main output function
of the SCN, the phase of its electrical activity, or
indeed comment on information exchange in the
opposite direction. However, daily changes of
electrical activity in the SCN correlate with the
rest-activity cycle (Inouye and Kawamura, 1979)
and an acute effect of locomotor activity on
SCN electrical firing in vivo has been reported.
In vivo recordings of extracellular firing in the
SCN of freely moving rats and hamsters show that
bouts of increased locomotor activity are followed
by acute reductions in SCN firing rate that
recover after an activity bout ceases (Meijer
et al., 1997; Schaap and Meijer, 2001; Yamazaki
et al., 1998). These suppressions are not induced
by less vigorous activities such as feeding and
grooming (Schaap and Meijer, 2001) and the
magnitude of suppressions correlates with the
intensity of wheel-running (Yamazaki et al.,
1998). While further investigation will be required
to establish the causal nature of this interaction,
that is, whether locomotor activity suppresses
electrical activity or lower electrical activity
induces locomotor activity, reports that sup-
pressions of electrical activity follow increased
locomotor activity indicate the former (Meijer
et al., 1997; Schaap and Meijer, 2001). This raises
the possibility that locomotor activity suppression
of SCN electrical activity may, at least in part,
contribute to its phase-shifting effects.
317
Neuropeptide Y and the IGL
The intergeniculate leaflet (IGL) is a narrow
band of the visual thalamus that lies between
the dorsal and ventral parts of the lateral genicu-
late nucleus and abundantly expresses neuropep-
tide Y (NPY) and GABA, both of which are
also found in the major IGL efferent projection
to the SCN, the geniculohypothalamic tract
(GHT; Harrington, 1997; Fig. 1).
Evidence supporting a role for the IGL and NPY
in mediating nonphotic signals to the central circa-
dian system is extensive and includes convincing
data demonstrating a role specifically in the feed-
back effects of locomotor activity. Neurotoxic and
electrolytic IGL lesions attenuate locomotor activ-
ity-induced behavioral phase shifts in both
hamsters and mice (Janik and Mrosovsky, 1994;
Johnson et al., 1988; Koletar et al., 2011; Wickland
and Turek, 1994). A potential confound for the
interpretation of these studies, however, is that
IGL lesions also reduce wheel-running, though this
concern has been addressed: radio frequency
lesions of the IGL completely block entrainment
of mice to scheduled forced treadmill running with
no associated reduction in activity (Marchant et al.,
1997). In vivo electrical stimulation of the IGL
(Rusak et al., 1989) and NPY infusion into the third
ventricle (Albers and Ferris, 1984; Biello and
Mrosovsky, 1996) both result in behavioral phase
shifts that resemble the traditional nonphotic
PRC seen in response to induced locomotor activ-
ity. Finally, mice lacking Dexras-1, an intracellular
signaling component proposed to inhibit responses
to NPY receptor activation in vivo, are reported to
exhibit larger phase shifts to novel wheel-induced
exercise than WT mice (Cheng et al., 2004; Koletar
et al., 2011), though others have failed to replicate
this finding (Dallmann and Mrosovsky, 2007).
Further evidence also exists to place the IGL and
NPY specifically downstream of locomotor activity
in phase-shifting processes. Confining hamsters to
a nest box, and hence preventing wheel-running,
fails to block phase advances in response to NPY
infusion to the third ventricle during the day and
318
NPY antiserum injections severely attenuate novel
wheel-running-induced phase advances without
reducing wheel revolutions during the novelty
pulse (Biello et al., 1994). Moreover, novelty-
induced wheel-running at times when phase shifts
would occur activates NPY neurons in the IGL,
demonstrated by cFOS induction (Janik and
Mrosovsky, 1992; Janik et al., 1995; Webb et al.,
2008; though see Zhang et al., 1993). Interestingly,
though the IGL also receives photic input due to its
location in the visual thalamus and is activated by
phase-shifting light pulses, photically induced
cFOS in the IGL is not found in NPY-positive
neurons (Janik et al., 1995). Further, light
attenuates phase shifts due to both exercise and
NPY injection during the day (Biello and
Mrosovsky, 1995) and, in addition to effects
directly in the SCN, this integration of photic and
nonphotic stimuli is likely to be mediated, at least
in part, in the IGL as behavioral phase shifts
induced by activation of the IGL are blocked by
light exposure (Challet et al., 1998).
Increased locomotor activity results in increased
NPY levels in the SCN (Glass et al., 2010) and NPY
alters circadian function through changing clock
gene expression and electrical activity in the cen-
tral oscillator. Infusion of NPY into the region of
the SCN in vivo and directly onto SCN slices
in vitro suppresses per1 and per2 expression
(Fukuhara et al., 2001; Maywood et al., 2002); an
effect mediated by NPY Y1/Y5 and Y2 receptors.
NPY application to cultured SCN slices in vitro also
induces nonphotic-like phase shifts in electrical
activity as well as acute suppression of SCN firing
(Cutler et al., 1998; Gribkoff et al., 1998; Medanic
and Gillette, 1993; Shibata and Moore, 1993; van
den Pol et al., 1996). The GABAergic nature of
IGL neurons, including those that give rise to the
GHT, further supports a role for the IGL/GHT in
nonphotic signaling, given, as mentioned earlier,
that the GABAA agonist muscimol also produces
nonphotic phase-shifting responses when infused
centrally to hamsters (Smith et al., 1989).
Collectively, the above studies indicate a cen-
tral role for the IGL, utilizing NPY and GABA,
in mediating locomotor activity-induced phase-
shifting effects on the clock, with the lesion study
of Marchant et al. (1997) concluding that the IGL
may even be essential for locomotor activity-
mediated entrainment.
Serotonin and the raphe nuclei
The monoamine serotonin (5-HT; 5-hydroxy-tryp-
tophan) has long been reported to function in the
control of circadian responses to nonphotic stimuli
though specific details of the role played by seroto-
nin in mediating nonphotic-like and, in particular,
locomotor activity-induced phase shifts are, despite
22 years of research, still unclear (Mistlberger and
Antle, 2011). This perhaps surprising lack of clarity
arises due to a number of factors including the
complexity of serotonin receptor pharmacology,
differences in lesioning methods, and lesion extent
between studies, species differences, and potential
differences in the role of serotonin in mediating
phase shifts resulting from locomotor activity
induced in different ways (e.g., triazolam injection
vs. novel wheel). That serotonin is involved in
these processes in some capacity, however, is
beyond doubt.
Serotonin is released in the hypothalamus in
response to locomotor activity (Dudley et al.,
1998) and serotonergic projections from the
median raphe (MR) of the brainstem innervate
the SCN, while the IGL is innervated by seroto-
nergic projections from the dorsal raphe (DR;
Abrahamson and Moore, 200l; Fig. 1). Further,
circadian variation in serotonin content in the
SCN correlates with wheel revolutions performed
at different times of the day (Shioiri et al., 1991);
a number of markers of serotonin function in the
raphe nuclei are regulated by locomotor activity
(Greenwood et al., 2005; Malek et al., 2007) and
electrical stimulation of the raphe nuclei results
in serotonin release in the SCN, suppression of
SCN cFOS expression, and behavioral phase
shifts (Dudley et al., 1999; Meyer-Bernstein and
Morin, 1999).

Serotonin itself and a number of serotonin
agonists, effective variously against different receptor
subtypes (e.g., 8-hydroxy-2-di-n-propylaminotetralin
(8-OH-DPAT), 5-carboxamidotryptamine, qui-
pazine), administered to SCN slices in vitro and
whole animals in vivo broadly produce typical
nonphotic style PRCs, with large phase advances
during the day and somewhat smaller phase
delays at night, though not all studies report
the latter (Bobrzynska et al, 1996a; Challet
et al., 1998; Edgar et al., 1993; Ehlen et al.,
2001; Horikawa and Shibata, 2004; Lovenberg
et al., 1993; Medanic and Gillette, 1992; Prosser
et al., 1990, 1993; Shibata et al., 1992; Tominaga
et al., 1992; though see Antle et al., 2003). More-
over, suppressing endogenous serotonergic tone
with short-term constant light exposure, which
creates a state of serotonin-hypersensitivity,
strikingly potentiates phase shifts in response to
a locomotor activity pulse (Knoch et al., 2004,
2006; Landry and Mistlberger, 2005; Mistlberger
et al., 2002). Both 5-HT1a and 5-HT7 receptors,
potentially acting in consort, are likely involved
as transgenic mice lacking either subtype fail to
respond to 8-OH-DPAT injection (Gardani and
Biello, 2008; Smith et al., 2008).
Depletion of serotonin in the mouse SCN blocks
the tau-shortening effect of a home-cage running
wheel (Mistlberger et al., 1998), and general hypo-
thalamic serotonin depletion in hamster severely
attenuates locomotor activity-mediated phase
shifts induced by triazolam injection, without a
reduction in induced locomotor activity (Penev
et al., 1995a). Further, and similar to data for
NPY, preventing hamsters from exercising after
injections of 8-OH-DPAT does not affect the
amplitude of phase shifts (Bobrzynska et al.,
1996a). Perhaps most importantly, disruption of
serotonin signaling impacts on stable entrainment
to locomotor activity feedback; lesioning serotonin
terminals in and around the SCN blocks entrain-
ment of mice to scheduled voluntary exercise with-
out reducing running (Edgar et al., 1997) and
severely disrupts the ability of mice to entrain to
forced treadmill running (Marchant et al., 1997).
319
Taken together, these studies show that serotonin
acts downstream of locomotor activity in mediating
nonphotic phase shifts. Finally, serotonin is a likely
candidate for involvement in nonphotic modula-
tion of photic circadian control (Sanggaard et al.,
2003; reviewed in Yannielli and Harrington, 2004).
Serotonin signaling also alters core clock gene
expression and electrical activity in the SCN, fur-
ther confirming that serotonin is functionally
involved in mediating nonphotic and locomotor-
mediated phase shifts and entrainment. 8-OH-
DPAT injection during the subjective day, the
time at which this treatment produces phase
advances, markedly suppresses per1 and per2
mRNA levels (Duncan et al., 2005; Horikawa
et al., 2000) and cotreatment with both 8-OH-
DPAT and brotizolam potentiates both behav-
ioral phase shifts and the suppression of per
(Yokota et al., 2000). Moreover, in addition to
phase-shifting SCN firing rate rhythms in vitro
(see above), serotonin also acutely suppresses
SCN electrical activity, both in vitro (Medanic
and Gillette, 1992) and in vivo (Mason, 1986;
Nishino and Koizumi, 1977).
Inconsistencies, however, do exist. While
wheel-running does induce modest increases in
cFOS expression in the DR, this is not in seroto-
nergic neurons (Webb et al., 2010). Neurotoxic
lesions of serotonin neurons in the MR, which
result in complete depletion of serotonin fibers
in the SCN, have been reported to block
triazolam-induced but not novel wheel-induced
shifts (Meyer-Bernstein and Morin, 1998). More-
over, other studies have found serotonin deple-
tion in the SCN and serotonin antagonist
injections to neither attenuate phase shifts
resulting from novelty-induced wheel-running
(Antle et al., 1998; Bobrzynska et al., 1996b) nor
alter the acceleration of reentrainment mediated
by wheel-running (Smale et al., 1990). However,
Bobrzynska et al. (1996b) report only 88–95%
depletion of serotonin in and around the SCN
and the serotonergic DR projection to the IGL,
and IGL projection to the SCN, would have been
intact in this study and may account for the failure
320
to block shifts. Indeed, serotonin agonist injection
into the IGL during subjective day does induce
phase advances (Challet et al., 1998).
Methodological, pharmacological, and species
differences notwithstanding, serotonin signaling
from the raphe nuclei, acting either directly or
via the IGL appears, at least in part, to mediate
nonphotic-like phase shifts and entrainment to
scheduled exercise by suppressing clock gene
expression in the SCN.
Orexin and the lateral hypothalamic area
The lateral hypothalamic area (LHA) contains
orexin-expressing neurons that are critical for
appropriate control of arousal (Sakurai, 2007)
and innervate a number of the previously discussed
nuclei implicated in locomotor activity-mediated
entrainment (IGL, MR, and the periphery of the
SCN; Cutler et al., 1999; Peyron et al., 1998;
McGranaghan and Piggins, 2001; Fig. 1). Orexin
neurons themselves receive indirect afferent
projections arising from the SCN (Abrahamson
and Moore, 2001; Deurveilher and Semba, 2005),
suggesting reciprocal modulation between circa-
dian and arousal-promoting circuits and raising
the possibility that orexin neurons in the LHA
may play a role in mediating the actions of locomo-
tor activity on central oscillator mechanisms.
Indeed, orexin acutely alters both SCN and IGL
neuronal activity in vitro (Brown et al., 2008;
Farkas et al., 2002; Klisch et al., 2009; Pekala
et al., 2011), activation of orexin neurons in the
LHA precedes the onset of the main activity bout
(Marston et al., 2008), and electrical activity of
orexin neurons correlates with locomotor activity
(Mileykovskiy et al., 2005). Further, running in a
novel wheel and locomotor activity-promoting
dark pulses, which result in subjective day phase
advances, induce cFOS expression in LHA orexin
neurons while suppressing cFOS in the SCN
(Marston et al., 2008; Webb et al., 2008). It is not
currently clear, however, whether this activation of
LHA orexin neurons is a consequence of nonphotic
phase shifts or part of the mechanism controlling
them. However, demonstration of increased num-
bers of orexin-positive neurons in the LHA during
the “activity phase” of a forced daytime activity par-
adigm suggests that these neurons and the actions of
orexin function downstream of locomotor activity,
potentially to mediate influences on the circadian
system (Salgado-Delgado et al., 2010).
Locomotor activity and glucocorticoid signaling
The hypothalamic–pituitary–adrenal (HPA) axis
and sympathico-adrenomedullary system are the
main neuroendocrine pathways that are activated
in response to stressful stimuli and physical
challenges. Both are potently stimulated by loco-
motor activity, resulting in extensive physiological
adaptations that are now recognized not as stress
responses, but as normal responses to support
demanding behaviors (Beerling et al., 2011;
Koolhaas et al., 2011). The contributions of these
locomotor activity-induced physiological responses
to phase shifting by, and entrainment to, exercise
have yet to be thoroughly investigated. A key HPA
response to locomotor activity is increased cir-
culating glucocorticoid levels (Stranahan et al.,
2008) and glucocorticoids have been postulated to
play a role in mediating some activity-independent
nonphotic effects on the circadian system (Mis-
tlberger et al., 2003; Sumova et al., 1994).
A specific role in entrainment to locomotor activity
has not yet been determined however, but will be
required for a full understanding of the
mechanisms of entrainment to locomotor activity
feedback. Indeed, glucocorticoids play a key role
in maintaining internal synchrony between the var-
ious central and peripheral oscillators of the circa-
dian system (Dibner et al., 2010). It is likely that
glucocorticoid signaling will function downstream
of SCN entrainment to locomotor activity to syn-
chronize and entrain other oscillators; alternatively,
it remains possible that, given the direct induction
of glucocorticoids by locomotor activity and
their action to synchronize peripheral oscillators,

glucocorticoids may function as part of a mechanism
for organizing peripheral oscillators to allow a
coordinated feedback from peripheral clocks to the
central circadian system (Dibner et al., 2010;
Stranahan et al., 2008).
Nonphotic entrainment in diurnal species
Increasing numbers of studies demonstrating tem-
poral niche switching are blurring the distinction
between species traditionally considered in
the laboratory to be “nocturnal” or “diurnal”
(reviewed in Kronfeld-Schor and Dayan, 2008).
Nevertheless, the vast majority of nonphotic
entrainment research to date, and indeed circa-
dian research in general, has been carried out on
rodents that exhibit nocturnal activity patterns
under normal laboratory conditions. Studies of
species that exhibit diurnal activity under normal
laboratory conditions, though relatively few, have
however been performed.
Regardless of the temporal niche occupied by an
animal’s main active phase, central SCN pace-
maker phase is similar in all species studied; the
SCN exhibits high transcriptional, electrical, and
metabolic activity during the subjective day and
low at subjective night (e.g., Sato and Kawamura,
1984; reviewed in Smale et al., 2003). Entrainment
to scheduled locomotor activity has been reported
in diurnal European ground squirrels and common
marmosets (Glass et al., 2001; Hut et al., 1999) as
well as weak locomotor activity feedback effects
in diurnal/crepuscular degus (Octodon degus; Kas
and Edgar, 2001). The characteristics of nonphotic
entrainment in these “diurnal” mammals suggest
that the diurnal nonphotic PRC is similar in form
to that of nocturnal mammals; phase angle of
entrainment in all cases agrees (as described in Sec-
tion “Phase angle of entrainment to scheduled
locomotor activity and the nonphotic PRC”) with
a mid-to-late day phase advance portion and a
late-night delay portion. Nonphotic entrainment is
clearly determined by pacemaker phase and not
by activity phase.
321
Intriguingly, the degus and Nile grass rat
(Arvicanthus ansorgei) exhibit diurnal activity
when housed without a running wheel but a sub-
set switch to nocturnal behavior when a wheel is
present (Kas and Edgar, 1999; Smale et al.,
2001). In the grass rat, this locomotor activity-
induced switch is associated with changes in the
time of increased NPY neuron activation such
that cFOS levels are always high in NPY neurons
in the IGL during an animal’s active phase
(Smale et al., 2001), regardless of whether this
occupies a light or dark niche.
Feedback of exercise to the human circadian
system
Regular physical exercise is held as the cornerstone
for good physical and mental health and given the
well-documented entraining effects of locomotor
activity in animal models, exercise is attractive as
a cheap and noninvasive intervention in circadian
misalignment disorders and maladies. However,
due to the difficulty of maintaining humans in a
controlled environment for long periods and in
some studies a failure to adequately control lighting
during exercise pulses at times when light levels
should be low, reports of the phase responses of
humans to exercise have been variable (reviewed
in Atkinson et al., 2007). These difficulties notwith-
standing, a PRC to exercise for humans has been
postulated and generally resembles an archetypal
“nonphotic” profile similar to that of both noctur-
nal and other diurnal species; delay shifts occur
during the late night and advances are reported
during the late day and into the early evening
(Buxton et al., 1997a,b, 2003; Mistlberger and
Skene, 2005; Van Reeth et al., 1994).
Complementary to these studies, “real-world”
attempts have been made to assess the impact
of exercise on reentrainment following tra-
nsmeridian travel (Klein and Wegmann, 1974;
Shiota et al., 1996). Both studies report accelerated
reentrainment with exercise, though with the
caveat that neither study controlled for light
322
exposure or exercise intensity. Night shift studies
have reported more variable responses but similar
concerns over the control of a number of variables
exist (e.g., Baehr et al., 1999; Eastman et al., 1995).
Nevertheless, reports of beneficial outcomes in a
real-world setting are encouraging and a role for
exercise in humans as a phase modulator under
an LD cycle would well suit the use of exercise to
assist in reentrainment following transmeridian
travel. Studies of blind subjects, under controlled
conditions, performing defined duration and inten-
sity exercise protocols will be useful to determine
more precisely the parameters required for optimal
phase modulation by exercise in humans. Following
additional research, this may also lead to the use of
exercise as a component of a combined nonphotic
treatment, along with sleep scheduling, social
interactions, and scheduled meal times to improve
synchronization with society in either blind or
circadian-compromised individuals.
Locomotor activity feedback to the circadian
system in animals with altered circadian clocks
Aging
Age-related changes in circadian function in
animals are well documented. Phase angle of
entrainment to LD becomes more positive,
variability in onsets increases, and behavioral
rhythms fragment and lose amplitude (Weinert,
2000). SCN function also appears to weaken, with
lower amplitude oscillations in electrical function
(Biello, 2009; Nakamura et al., 2011; Watanabe
et al., 1995) and reduced responsiveness to photic
stimuli (Kolker et al., 2003). Older animals per-
form fewer spontaneous revolutions in a home-
cage running wheel (e.g., Penev et al., 1995b).
Changes in the circadian responses of aged
animals to locomotor activity and neurochemicals
involved in signaling these responses have also
been reported.
Aging hamsters fail to show behavioral phase
shifts following single injections of triazolam,
despite no reduction in induced wheel-running
(Van Reeth et al., 1992) and repeated injections
of triazolam fail to stably entrain behavioral
rhythms in most old hamsters (Van Reeth et al.,
1993). Dietary treatment of old hamsters with
the chronobiotic melatonin, however, improves
responses to triazolam-induced locomotor activity
(Kolker et al., 2002). Further, novel wheel confine-
ment also fails to induce phase shifts in old hamsters,
though it also induces less running in the wheel
than in young hamsters (Mrosovsky and Biello,
1994). In this study, increasing activity during novel
wheel confinement to normal induced levels with
either triazolam injection or positive motivation
(the presence of an estrus female) recovered
phase shifts. Together, these data indicate a reduced
responsiveness of the aging circadian system to
stimuli of this kind (though dark pulse-induced
phase shifts are reported to be unaltered with age;
Duncan and Deveraux, 2000). This may arise due
to reduction of one or more of the neurochemical
elements involved in mediating locomotor activity-
induced circadian feedback effects. Indeed, seroto-
nin levels in the brain have been reported to reduce
with age (Jagota and Kalyani, 2008) and both in vivo
and in vitro responses to 8-OH-DPAT are reduced
as age increases (Biello, 2009; Penev et al., 1995b),
suggesting some age-related deficiency in serotonin
receptor function may also be a factor. Consistent
with this hypothesis, many of the effects of ageing
on activity-induced entrainment events can be
simulated by monoamine depletion (as discussed
previously and reviewed in Turek et al., 1995).
Core clock gene mutations
Of the many core clock gene mutant strains that
exist, only two have been assessed for their
responses to locomotor feedback to the circadian
system: the tau mutant hamster (Ralph and
Menaker, 1988) and the clock mutant mouse
(Vitaterna et al., 1994).
Both strains exhibit altered responses to locomo-
tor activity feedback or associated neurochemical

treatments. Novel wheel confinement-induced loco-
motor activity produces a shifted and exaggerated
PRC in tau mutant hamsters (Mrosovsky et al.,
1992) with behavioral responses to NPY and
8-OH-DPAT infusion showing similar changes
(Biello and Mrosovsky, 1996; Colecchia et al.,
1996). The enhanced responses of tau mutant
hamsters to a nonphotic stimulus are consistent with
the responses of this strain to photic zeitgebers,
where “type 0” phase resetting is seen (Shimomura
and Menaker, 1994); clearly the circadian system of
tau mutants is more readily manipulated by both
external stimuli and behavioral feedback than WT
counterparts. In clock heterozygous mice, novel
wheel confinement results in phase delays during
the subjective day in addition to lengthening circa-
dian period in subsequent free run (Challet et al.,
2000), describing altered responses to locomotor
activity for this mutant strain also.
Entrainment to scheduled locomotor activity in
signaling mutants
To date, no rhythmic core clock gene mutant
strain has been examined for entrainment to the
feedback signals associated with scheduled loco-
motor activity. While WT mice and hamsters,
and to a lesser extent WT rats, readily entrain to
such stimuli (as discussed previously), mice ren-
dered arrhythmic through SCN lesion fail to
respond (Marchant et al., 1997). However, this
lesion model lacks a core fundamental component
of a functioning circadian system, the SCN itself.
As such, if the responses of the circadian system
to scheduled locomotor activity are assumed to
be mediated by the SCN and employ the core
molecular oscillator, then its lack of effectiveness
in this model is perhaps not surprising. One alter-
native model of disrupted clock function is mice
lacking either VIP (Colwell et al., 2003) or its cog-
nate receptor in the SCN, VPAC2 (Harmar et al.,
2002). VIP–VPAC2 signaling is critical for inter-
cellular communication within the SCN and as
such, mice lacking either VIP (Vip/) or VPAC2
323
(Vipr2/) suffer severe behavioral circadian
deficits ranging from arrhythmicity to weak, inco-
herent, short period oscillations (Aton et al.,
2005; Ciarleglio et al., 2009; Hughes and Piggins,
2008). These behavioral deficiencies are a mani-
festation of reduced numbers of rhythmic neurons
in the SCN, reduced amplitude and coherence of
the remaining rhythmic neurons, and a lack of
synchrony between their oscillations (Brown
et al., 2005, 2007; Ciarleglio et al., 2009; Cutler
et al., 2003; Hughes et al., 2008; Maywood et al.,
2006). While both Vip/ and Vipr2/ mice dis-
play altered responses to photic zeitgebers
(Dragich et al., 2010; Hughes and Piggins, 2008;
Hughes et al., 2004), Vipr2/ mice were recently
shown to manifest intact food anticipatory activity
in response to the nonphotic stimuli of restricted
daytime feeding (Sheward et al., 2007). The
responses of these strains to regularly scheduled
voluntary exercise were recently investigated
(Power et al., 2010).
While the entrainment of WT mice to sched-
uled voluntary exercise is relatively slow, taking

20–40 cycles to achieve stable entrainment,
depending on the relative phases of endogenous
activity and the opportunity to run (Edgar and
Dement, 1991; Marchant and Mistlberger, 1996;
Power et al., 2010), both Vip/ and Vipr2/
mice synchronize within just 2–4 days (Fig. 4). In
stark contrast to WT mice, which tend to show
small aftereffects on tau, following scheduled
activity most Vipr2/ mice show a marked
improvement in rhythms with a proportion of pre-
viously arrhythmic mice subsequently sustaining
behavioral rhythms (Power et al., 2010). Post-
scheduled exercise Vipr2/ rhythms free-run in
phase with the previous opportunity to run,
demonstrating entrainment and not simply posi-
tive masking, and tend to do so with a near 24-h
period which can be sustained for at least a month
(Fig. 4). A number of unknowns, however, do
remain. Few Vip/ mice show comparable
improvements in rhythmicity, indicating either a
more profound rhythm dysfunction in this strain
than in Vipr2/ mice, or that VIP acting through
324
non-VPAC2 receptors is involved in the main-
tenance of improved rhythms (Power et al.,
2010). Further, the relative potency of locomotor
activity-associated feedback to act on the SCN
and/or peripheral clocks in these mice (and
indeed in WTs) has yet to be examined. Indeed,
the possibility of potentially enhanced peripheral
feedback to the SCN in Vipr2/ mice to contrib-
ute to the more potent effects of scheduled activ-
ity on the Vipr2/ circadian system is thus far
unexplored. Changes in the activity of the NPY/
IGL and serotonin/raphe systems will also need
to be assessed in relation to the enhanced actions
of locomotor activity feedback in Vipr2/ mice.
Future directions
A number of key issues surrounding the feedback
influences of locomotor activity on the circadian
system have yet to be addressed. Species general-
ization continues to be an issue; the bulk of early
work was carried out on hamsters, with rats
and mice very much underrepresented. This has
been redressed to some extent but, as with most
research in the postgenomic age where mice have
become the species of choice, there is now a risk
of overspecializing and becoming limited to this
alternative model. Further, the majority of early
studies were performed before the discovery of
core clock genes and without a detailed knowl-
edge of the multioscillator nature of mammalian
circadian systems. As such, many are largely
restricted to behavioral investigations and few
have considered either the potential contributions
of extra-SCN oscillators to entrainment by loco-
motor activity or the effects of locomotor activity
specifically on these extra-SCN oscillators. We
do not yet know the relative sensitivity of SCN
and extra-SCN oscillators to locomotor/arousal-
associated nonphotic signals; this may be of par-
ticular interest for oscillators in tissues directly
involved in locomotion and metabolism including
cardiac muscle and adipose depots. Moreover, the
well-known positive effects of locomotor activity
on cognition and adult neurogenesis in the hippo-
campus (e.g., Kempermann et al., 2010; van
Praag, 2008), coupled with the demonstration that
circadian phase regulates the effects of exercise
on neurogenesis (Holmes et al., 2004), raises the
possibility that locomotor activity may be impor-
tant for the resetting of hippocampal clocks.
Again with consideration of the multioscillator
nature of circadian biology, investigating a role
for locomotor activity in ameliorating the effects
of internal desynchrony arising from circadian
disorders, shift work and transmeridian travel will
greatly enhance our current understanding of
nonphotic influences on the integrated circadian
system. Indeed, as internal desynchrony gains
increasing prominence as a contributory factor
in circadian disruption, further investigation of
the effects of locomotor activity on peripheral
and central oscillators offers an opportunity to
both develop our understanding of basic circadian
biology and may also lead to the development of
a unique noninvasive, nonpharmacological, tool
for the reorganization of the distributed circadian
system.
Related to the above points, a number of pow-
erful modern techniques are underrepresented in
research into locomotor activity feedback to the
circadian system and could be effectively
employed to improve our understanding of this
process. For example, lesion studies crucial to
our current understanding carry important
caveats. Nonspecific damage may be caused along
the path taken into the brain when electrolytic
lesions are performed and neurochemical lesions,
that destroy neurons rather than simply silencing
a gene of interest, may result in unwanted collat-
eral damage or pleiotropic effects related to the
neurons in question. These concerns could be
addressed, should appropriate targeting agents
become available, with the use of tissue-specific
conditional knockouts of key neurochemical
elements, for instance, NPY specifically in the
IGL or serotonin in either or both of the raphe
nuclei. Similarly, the plethora of reporter mice
now available (e.g., Cheng et al., 2009; Kuhlman

et al., 2000; Wilsbacher et al., 2002; Yoo et al.,
2004) will be crucial in addressing issues related
to entrainment at the cellular and molecular
levels, in particular in elucidating the effects of
locomotor activity on synchrony between oscilla-
tor cells within rhythmic tissues and identifying
the effects of locomotor activity feedback on neu-
rochemically defined neuronal populations.
Another key issue yet to be resolved is which
internal physiological correlates of locomotor
activity are critical for entrainment. In particular,
the inadvertent temporal organization of food
intake associated with scheduled activity and acti-
vation of the HPA axis by locomotion have yet to
be systematically assessed for their relative con-
tributions to the entraining effects of physical
exercise. These, along with consideration of the
importance of ultradian rhythmicity in glucocorti-
coid signaling (Lightman et al., 2008) may provide
fruitful avenues for future investigation.
Our own recent data (Power et al., 2010) dem-
onstrate that locomotor activity feedback is
clearly a potent entraining stimulus in a mouse
model with a “weakened” SCN pacemaker;
Vipr2/ mice. However, a key question remains
unanswered: are the entraining properties of
exercise similarly boosted in other models of a
weakened circadian system? Aged animals and
circadian gene mutants such as afterhours, a
mutation which alters tau and appears to weaken
circadian function (Godinho et al., 2007), would
be intriguing models with which to further probe
the effects of scheduled activity on a weakened
circadian system. Indeed, given the similarities in
circadian deficit between Vipr2/ mice and aged
individuals (i.e., weakened SCN function (as
discussed); aging-like alterations in circadian
function induced by VIP suppression in young
subjects (Gerhold et al., 2005)) and that VIP
levels in the SCN are reduced with age (Duncan
et al., 2001), Vipr2/ mice may prove to be a use-
ful model of aging in the circadian system. The
demonstration that aging laboratory animals, when
appropriately motivated, can be induced to gener-
ate locomotor activity-mediated phase shifts of
325
equal magnitude to younger animals, coupled with
the exaggerated responses of Vipr2/ mice to
scheduled activity make it possible that aged
animals will respond well to scheduled locomotor
activity, raising the possibility of a potential new
noninvasive treatment for age-related circadian
dysfunction.
Zeitgeber versus zeitnehmer?
Traditionally, chronobiologists have used the term
zeitgeber, literally meaning “time giver,” to describe
any external stimulus that is rhythmically encoun-
tered and acts as an entraining signal to the circa-
dian system. This term, and its implication of a
one-way transfer of information from the environ-
ment to the circadian system, appropriately
describes independent external stimuli such as the
LD cycle. However, the consideration of an inter-
nally controlled function, such as locomotor activity,
as an entraining stimulus raises questions over the
appropriateness of the term “zeitgeber” in a case
such as this. To address this issue, the introduction
of an alternative subclassification, “zeitnehmer” (lit-
erally meaning “time taker”—in analogy with zeit-
geber), has been suggested for endogenous process
that are themselves under circadian control but that
also feedback to actively participate in the entrain-
ment process (Lakin-Thomas, 2000; Merrow et al.,
2003; Roenneberg and Merrow, 1999; Roenneberg
et al., 1998). To date, this “zeitnehmer” term has
mostly been adopted by researchers investigating
plant circadian biology (e.g., Hicks et al., 2001;
McWatters et al., 2000; Thines and Harmon, 2010),
though need not be restricted in this way and offers
a more appropriate alternative to “zeitgeber” for
the description of locomotor activity feedback in
the context of entrainment. The adoption of
“zeitnehmer” in this case allows a clear distinction
to be drawn between independent geophysical
stimuli such as the LD cycle and endogenous, feed-
back stimuli including locomotor activity. If one is
to adopt the term “zeitnehmer,” then as in nature,
when a range of external zeitgebers and internal
326
zeitnehmers will always be encountered in combina-
tion, the use of both terms will always be necessary
when considering “real-world” entrainment.
Summary and conclusions
Once thought of as simply an output, locomotor
activity has for some time been known to also pro-
vide a feedback signal to the circadian system. Dis-
crete pulses of locomotor activity can shift
circadian phase or modify reentrainment to a shifted
LD cycle, while scheduled locomotor activity can
entrain circadian behavior and even induce lasting
improvements in pacemaker function in circadian
mutants. The precise nature of the feedback signal
is unknown but is likely to be complex, involving a
variety of physiological systems. Neurochemical pat-
hways in the brain that transmit this feedback infor-
mation to the SCN are somewhat better understood,
however. Arousal-related signals from the DR are
transmitted, via a serotonergic projection, to the
IGL which in turn innervates the SCN via the
GHT using GABA and NPY. Together with seroto-
nergic pathways from the MR and orexin signaling
from the LHA, these locomotor/arousal-associated
pathways act to suppress SCN electrical activity
and per gene expression, resulting in phase-depen-
dent phase shifts of the master circadian pacemaker
and hence the wider circadian system in general.
Though a number of key issues relating to the timing
and doses of exercise required for effective manipu-
lation of the human circadian system remain to be
addressed, feedback of locomotor activity to the cir-
cadian system has the potential to form part of novel
noninvasive interventions to a variety of circadian
dysfunctions, including those associated with aging,
shift work, transmeridian travel, and the blind.
Acknowledgments
The authors would like to thank Prof. Ralph
Mistlberger for critical comments on an earlier draft
of this review and acknowledge the BBRSC and
Wellcome Trust for financial support of their work.
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 19

The impact of the circadian timing system on

cardiovascular and metabolic function

Christopher J. Morris{,{,*, Jessica N. Yang{ and Frank A. J. L. Scheer{,{

{
Division of Sleep Medicine, Brigham and Women’s Hospital, Boston, MA, USA
{
Division of Sleep Medicine, Harvard Medical School, Boston, MA, USA

Abstract: Epidemiological studies show that adverse cardiovascular events peak in the morning (i.e.,
between 6 AM and noon) and that shift work is associated with cardiovascular disease, obesity, and
diabetes. The endogenous circadian timing system modulates certain cardiovascular risk markers to be
highest (e.g., cortisol, nonlinear dynamic heart rate control, and platelet activation) or to respond most
unfavorably to stressors such as exercise (e.g., epinephrine, norepinephrine, and vagal cardiac modulation)
at an internal body time corresponding to the time of day when adverse cardiovascular events most likely
occur. This indicates that the circadian timing system and its interaction with external cardiovascular
stressors (e.g., physical activity) could contribute to the morning peak in adverse cardiovascular events.
Moreover, circadian misalignment and simulated night work have adverse effects on cardiovascular and
metabolic function. This suggests that misalignment between the behavioral cycle and the circadian timing
system in shift workers contributes to that population’s increased risk for cardiometabolic disease.

Keywords: biological clock; circadian misalignment; glucose metabolism; heart; night work; shift work;
suprachiasmatic nucleus.

Introduction diabetes (Centers for Disease Control and Preven-

Western societies are rife with cardiovascular dis-
ease, diabetes, and obesity. In the United States, it
is estimated that 83 million adults have cardiovascu-
lar disease (Roger et al., 2011), 26 million have
*Corresponding author.
Tel.: þ1-617-525-9086; Fax: þ1-617-732-7337
E-mail: cjmorris@rics.bwh.harvard.edu
tion, 2011), and 80 million are obese (Flegal et al.,
2010). The influence of behaviors (e.g., poor diet
and physical inactivity) as underlying causes for these
diseases has been researched for decades. However,
more recently, evidence has been accumulating for
a contributing role of the endogenous circadian
timing system and its disruption in cardiovascular
and metabolic disorders. In this review, we dis-
cuss—with a focus on mammals and particularly

http://dx.doi.org/10.1016/B978-0-444-59427-3.00019-8 337
338
humans—the impact of the circadian timing system,
its interaction with behaviors (e.g., exercise), and its
disturbance on cardiovascular and metabolic
function.
The circadian timing system
Most life on earth—ranging from single cellular
organisms, plants, flies, rats, to humans—contains
an endogenous timing system that optimally
synchronizes physiology and behavior (e.g., rest/
activity and fasting/feedings cycles) with the solar
day. The system is known as the circadian (“circa,”
around; “dies,” day) timing system and has two
core characteristics: (1) endogenous rhythmicity
that cycles approximately every 24 h, even in the
absence of cyclic changes in external factors such
as light and temperature and (2) the capability to
adjust its timing in response to external factors
such as light and/or food intake. The circadian
timing system engineers a “biological day” and
“biological night” that transition in a cyclic man-
ner. Here, we define the biological night as the
endogenous circadian time window corresponding
to the habitual dark episode, that is, the time nor-
mally characterized by behavioral inactivity in
diurnal (day-active) species and behavioral activ-
ity in nocturnal (night-active) species. The oppo-
site holds true for the term biological day. In
mammals, the biological night is also the time
when melatonin plasma concentrations are high.
This is true both for diurnal and nocturnal
mammals. The transition between the biological
day and night is associated with relatively large
changes in many physiological variables, such as
circulating levels of melatonin and cortisol, and
core body temperature (Dijk et al., 1999; Gooley
et al., 2011; Scheer et al., 2009; Van Cauter
et al., 1994; Wehr et al., 2001).
In mammals, the circadian timing system is
composed of the suprachiasmatic nucleus (SCN)
and circadian oscillators in most peripheral
tissues. The SCN is situated in the anterior hypo-
thalamus on top of the optic chiasm and next to
the third ventricle. The SCN is a bilateral struc-
ture that contains approximately 50,000 neurons
in humans and 20,000 neurons in rats (Güldner,
1983; Hofman et al., 1988; Swaab et al., 1985;
van den Pol, 1980). Various levels of evidence
show that the SCN regulates circadian rhythms:
(1) physiological and behavioral rhythms are
abolished in SCN-lesioned animals (Moore and
Eichler, 1972; Stephan and Zucker, 1972; van den
Pol and Powley, 1979), (2) SCN-lesioned animals
regain circadian rhythms in locomotor activity fol-
lowing receipt of a donor-SCN (Lehman et al.,
1987), (3) SCN-lesioned animals that receive an
SCN transplantation exhibit the same period
length of the donor animal (Ralph et al., 1990),
and (4) neural firing rate of the SCN exhibits a cir-
cadian rhythm in vivo and in vitro (Green and
Gillette, 1982; Groos and Hendriks, 1982; Meijer
et al., 1997). Circadian oscillators—cells that
generate circadian rhythms autonomously from
others—are also located in the periphery (e.g.,
the heart, liver, and pancreas). In addition to the
SCN, they express circadian rhythms in gene
expression, which ultimately can produce endoge-
nous cyclic rhythms in biology independent of
input from the central pacemaker (Balsalobre
et al., 1998; Brown et al., 2005; Ko and Takahashi,
2006; Mühlbauer et al., 2004; Storch et al., 2002;
Yamazaki et al., 2000; Yoo et al., 2004). However,
the SCN is considered to be the master pacemaker
because synchrony between peripheral clocks
within organs is typically lost without input from
the SCN (Guo et al., 2006).
Circadian rhythms are generated and regulated
by the concerted expressions of core clock genes,
which compose the primary mammalian oscillatory
mechanism (Ko and Takahashi, 2006; Lowrey and
Takahashi, 2004). A complex molecular network of
positive and negative feedback loops drive circadian
rhythms in core clock genes, such as Clock (circa-
dian locomoter output cycles kaput), Bmal1 (brain
and muscle arnt-like protein-1), Cryptochrome
(Cry1, Cry2), and Period (Per1, Per2, and Per3;
Lowrey and Takahashi, 2004; Reppert and Weaver,
2001; Shearman et al., 2000b). Participating in the

primary feedback loop are the transcription factors,
Clock and Bmal1, which dimerize and activate the
transcription of target genes, such as Pers and Crys
(Gekakis et al., 1998; King et al., 1997; Reppert
and Weaver, 2002). Subsequently, Clock-Bmal1-
mediated transcription is negatively regulated by
the Per–Cry complex (Jin et al., 1999; Kume et al.,
1999; Reppert and Weaver, 2001; Zylka et al., 1998).
The importance of Bmal1 in generating circadian
rhythms is demonstrated by homozygous Bmal1-
deficient mice (Bmal1/), which cannot entrain
to light/dark cycles and are arrhythmic (Bunger
et al., 2000). Mice possessing the antimorphic (or
dominant-negative) mutation (ClockD19/D19) dis-
play a longer period ( 27.3 h) and arrhythmicity
in constant darkness (Vitaterna et al., 1994). These
studies suggest that the Clock:Bmal1 complex is a
crucial driving force in circadian clock function.
However, this theory is challenged by researchers
who recently demonstrated that Clock-deficient
mice (Clock/) maintain robust circadian rhyth-
micity in locomotor activity in constant darkness,
albeit with a shorter period ( 23.2 h; DeBruyne
et al., 2006). Disruptions of negative feedback
elements (e.g., Per1, Per2, Per3, Cry1, and Cry2)
also modify circadian rhythms (Cermakian et al.,
2001; Shearman et al., 2000a; Thresher et al., 1998;
Van Der Horst et al., 1999; Vitaterna et al., 1999;
Zheng et al., 1999, 2001).
Because the cycle length of the circadian timing
system is not exactly 24 h (Czeisler et al., 1999;
Duffy et al., 2011), external photic input (i.e.,
the light/dark cycle) and/or nonphotic input
(e.g., food intake) is needed to entrain it
with the environment. The strongest Zeitgeber
(German: “time giver”) to the central pacemaker
is light. For brevity, we only discuss entrainment
by light in this review. Intrinsically photosensitive
retinal ganglion cells containing the photopigment
melanopsin, together with rods and cones, ini-
tially detect light (Gooley et al., 2001; Hattar
et al., 2002). This signal is then passed along the
retinohypothalamic tract to the SCN (Moore
et al., 1995). The influence of light on the circa-
dian timing system is dependent on the circadian
339
time of exposure. In both diurnal and nocturnal
animals, light exposure during the biological
evening/early night phase delays circadian time
relative to clock time; the opposite occurs (i.e., a
phase advance) when light exposure takes place
during the biological morning (Khalsa et al.,
2003; Rosenberg et al., 1991).
The internal clock and the timing of adverse
cardiovascular events
Epidemiological data demonstrate a 24-h rhythm
in the frequency of adverse cardiovascular events
such as angina, myocardial infarction, stroke,
arrhythmias, cardiac arrest, and sudden cardiac
death (Fig. 1), with the highest incidence occur-
ring between approximately 6 AM and noon
(Cannon et al., 1997; Cohen et al., 1997; D’Avila
et al., 1995; Elliott, 1998; Goldberg et al., 1990;
Levine et al., 1992; Marler et al., 1989; Muller
et al., 1985, 1987; Twidale et al., 1989; Willich
et al., 1987, 1992). The above studies may
under-report the occurrence of adverse cardiovas-
cular events during nighttime due to people
normally sleeping at this time. However, this pos-
sible reporting bias does not apply to the morning
peak in arrhythmias because the precise timing
of the events was recorded by electrocardio-
graphic recordings (e.g., see Twidale et al., 1989).
Furthermore, data obtained from implanted
cardioverter-defibrillators—which also record the
precise timing of the events—show that the defi-
brillation threshold (i.e., the amount of energy
required) needed for successful defibrillation is
greatest in the morning (Venditti et al., 1996).
The use of plasma creatine phosphokinase MB
activity—which is elevated approximately 4 h after
the onset of a myocardial infarction—has con-
firmed that the incidence of myocardial infarction
peaks between 6 AM and noon (Muller et al.,
1985; Roberts et al., 1975; Willich et al., 1989).
In patients with sleep apnea, however, the peak in
adverse cardiovascular events occurs at night, pos-
sibly due to the acute hemodynamic, autonomic,
340

0.12

0.10

Relative frequency per 1-h interval 0.08

0.06

0.04

0.02

0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Clock time (h)

Fig. 1. Relative frequency histogram for the time of onset of sustained ventricular tachycardia across the day. The figure illustrates a
broad peak in the onset of sustained ventricular tachycardia between 8 and 11 AM. Redrawn and reproduced with permission from
Twidale et al. (1989).

and oxidative stress associated with apneas (Gami 2003; Sly et al., 1999). Moreover, the above men-
et al., 2005). This further suggests that, since the tioned organs themselves rhythmically express
peak for adverse cardiovascular events for the clock genes, suggesting that the cardiovascular
population as a whole (including those with sleep system could be influenced by the circadian timing
apnea) occurs in the morning, the morning peak system at the local level in addition to input from
in those people without sleep apnea may be even the central pacemaker (Valenzuela et al., 2008;
more pronounced. Yamamoto et al., 2004). In light of the above
In order to understand the underlying mech- findings and the 24-h rhythm in adverse cardiovas-
anisms for the morning peak in adverse cardio- cular events, researchers have investigated if car-
vascular events, studies have mainly focused on diovascular variables such as heart rate, blood
the impact of behavioral changes typical of the pressure, autonomic nervous system activity, and
morning, such as the change in posture from supine platelet function exhibit circadian rhythmicity.
to upright and the start of behavioral activity at From epidemiological studies, it is not possible to
awakening. However, the circadian timing system deduct the relative role of the circadian timing sys-
may also play a role in the morning peak in adverse tem versus behavioral and environmental effects
cardiovascular events. That the SCN may modulate because influences of behaviors and the internal
cardiovascular functioning is suggested by the pres- circadian timing system occur in synchrony. Thus,
ence of multisynaptic projections from the SCN to this calls for experimental designs that can isolate
the heart and organs that regulate cardiovascular these factors.
function through hormones and blood volume Circadian rhythms in humans can be assessed
regulation (e.g., adrenal cortex, adrenal medulla, using either a constant routine protocol or a
and kidneys; Buijs et al., 1993; Scheer et al., 2001, forced desynchrony protocol. The constant routine

protocol, developed by Mills et al. (1978), enables
the assessment of circadian rhythms because the
influences of behavioral and environmental factors
are minimized and equally distributed across the cir-
cadian cycle by maintaining constant wakefulness,
constant semi-recumbent posture; limiting physical
activity, consistent dim light conditions; and evenly
distributing isocaloric snacks or continuously infus-
ing nutrients. In the forced desynchrony protocol,
developed by Kleitman (1963), subjects are sched-
uled to live on a fixed sleep/wake cycle that is
adequately different from 24 h and in dim light
conditions, such that the sleep/wake cycle is outside
the range of entrainment of the master oscillator,
causing the internal clock to “free run” or drift
according to its own internal period. The forced
desynchrony protocol allows the separate assess-
ment of circadian and behavioral influence by evenly
spreading behavioral (e.g., sleep and wakefulness)
factors across the circadian cycle.
A circadian rhythm in resting heart rate has
been reported by different research groups in
healthy humans, with a broad peak occurring dur-
ing the middle of the biological day and a trough
during the biological night (Figs. 2 and 3; Burgess
et al., 1997; Kräuchi and Wirz-Justice, 1994; Scheer
et al., 2010; Shea et al., 2011). On the other hand,
the reactivity of heart rate to standardized exercise
and postural changes is not influenced by the circa-
dian timing system (Fig. 3; Hu et al., 2011; Scheer
et al., 2010). Considering that the circadian peak
in heart rate does not occur during the biological
morning and that heart rate reactivity to behav-
ioral stressors is not under circadian control, it is
unlikely that the circadian timing system’s influ-
ence on heart rate contributes to the morning peak
in adverse cardiovascular events. In rats, there is
also a circadian rhythm in resting heart rate under
constant dark conditions, thus even independent of
the circadian rhythm in behavioral activity (Scheer
et al., 2001). After lesioning the SCN, the circadian
rhythm is abolished and the level of resting
heart rate is intermediate between that during
the biological day and night in intact animals.
This suggests that the SCN has an inhibitory
341
and excitatory influence on resting heart rate
during the biological day and night, respectively.
This is reminiscent of the regulation of melatonin
and corticosteroids for which the SCN uses both
inhibitory (e.g., GABA) and excitatory neuro-
transmitters (e.g., glutamate; Kalsbeek et al.,
1996; Perreau-Lenz et al., 2003).
Despite the fact that blood pressure is often men-
tioned as a key example of a physiological variable
under circadian control, two studies using constant
routine protocols found no evidence for an endoge-
nous circadian rhythm in blood pressure (Kerkhof
et al., 1998; Van Dongen et al., 2001). However,
recently, we demonstrated a robust endogenous cir-
cadian rhythm in blood pressure in healthy humans,
with very similar timing and amplitude across three
different protocols (a constant routine and two
forced desynchrony protocols) that factor out envi-
ronmental (e.g., light) and behavioral (e.g., sleep/
wake cycle and physical activity) influences
(Fig. 2; Shea et al., 2011). The circadian peak in
blood pressure occurred during the biological even-
ing, corresponding to approximately 9 PM. The
lack of a circadian rhythm in blood pressure in pre-
vious studies was likely related to methodological
limitations, including lighting control, posture, and
circadian phase assessment (see Shea et al., 2011).
The reactivity of blood pressure to exercise, but
not postural change, is also controlled by the circa-
dian timing system, again with the greatest response
occurring during the biological evening (Fig. 3; Hu
et al., 2011; Scheer et al., 2010). Considering that
the circadian peak in blood pressure at rest and in
response to exercise does not occur during the
biological morning, it is unlikely that the circadian
timing system’s influence on blood pressure con-
tributes to the morning peak in adverse cardiovas-
cular events. However, future studies are required
to determine whether the timing or amplitude of
the circadian blood pressure rhythm is changed in
individuals at risk for or with underlying cardio-
vascular disease.
Platelets are a key component in the develop-
ment of thromboses that cause myocardial in-
farctions and ischemic strokes. Recent work has
342

28-h forced desynchrony protocol 20-h forced desynchrony protocol

Corresponding clock time (h) Corresponding clock time (h)
5 9 13 17 21 1 5 9 13 17 21 1
120
P = 0.015 P < 0.0001
102 114 102

SBP (% mean)
118
SBP (mm Hg)

116 100 112 100

114 98 110 98

112

66 104 104
P = 0.018 64 P = 0.0002

DBP (% mean)
DBP (mm Hg)

102 102
64
100 62 100

98 98
62
60
96 96

74 68
P < 0.0001 104 P < 0.0001 104
72 66

HR (% mean)
HR (bpm)

70 100 100
64
68
96 62 96
66
60
92 92
0 60 120 180 240 300 0 60 120 180 240 300
Circadian phase (°) Circadian phase (°)

Fig. 2. Circadian rhythm in resting systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) observed in
two (28- and 20-h) forced desynchrony protocols. Data are reported as mean  standard error of the mean and are expressed in
absolute values (left axes) and as percentages of individual participant’s averages (right axes). Data are plotted according to
circadian phase, that is, separated into six 60 -bins which all equate to 4 h. Gray bars represent the participant’s average
normal clock time for sleep under ambulatory conditions in the 2 weeks prior to admission to the laboratory. Solid lines
represent the cosinor model fits. Dashed vertical lines indicate the circadian phase at which SBP peaked in both forced
desynchrony protocols. Probability data indicate the likelihood of a circadian rhythm in blood pressure and heart rate.
Reproduced with permission from Shea et al. (2011).

demonstrated in healthy humans that platelet aggregation and adhesion to subendothelial tissue
size, count, aggregability, and platelet surface ex- (Merten et al., 2000; Phillips et al., 1988; Sadler,
pression of activated GPIIb-IIIa, P-selectin, and 1998)—are influenced by the circadian timing sys-
GP1b—factors involved in the pathway of platelet tem (Scheer et al., 2011). For platelet aggregability
 343

Corresponding clock time (hh:mm) Corresponding clock time (hh:mm)

4:30 12:30 20:30 4:30 12:30 20:30 4:30 12:30 20:30 4:30 12:30 20:30
300
80 160

Epinephrine (% mean)
140

Epinephrine (pg/mL)

SBP (% mean)

SBP (mmHg)
60
200 140
120
40 Rest: P < 0.0001
Reactivity: P = 0.03
100 120
20
Rest
100
Rest: P < 0.0001
Reactivity: P = 0.04(f2) Exercise
0 0

120
Norepinephrine (% mean)

Norepinephrine (pg/mL)
500 80
200

DBP (% mean)

DBP (mmHg)
400
110 75
300 Rest: P = 0.0005
100 Reactivity: n . s .
200
70

Rest: P = 0.0009 100 100

Reactivity: P = 0.01(f2)
0 0 65

200 140
15
150
Cortisol (% mean)

Cortisol (mg/dL)

120
HR (% mean)

HR (bpm)
100 10
150 100
Rest: P < 0.0001
Reactivity: n.s.
50 5 80
100
Rest: P < 0.0001 60
0 0

150
100
Aggregabilitty (% mean)

Aggregability (W ⫻ min)
1500 120
HF (% mean)

100 90
110
HF (s2)

1000

100
50 80
500
Rest: P = 0.003
Reactivity: P = 0.002 90 Rest: P = 0.04(f2)
Reactivity: n.s.
0 0 70

150
Platelet count ( ⫻ 10 /mL)
Platelet count (% mean)

40 120
pNN50 (% mean)

2.2
pNN50 (%)

100 30 110
2.0
20 100
50 1.8
Rest: P = 0.001 10 90
Reactivity: P = 0.002 Rest: n.s.
Reactivity: n.s. 1.6
0 0 80
0 120 240 0 120 240 0 120 240 0 120 240
Cricadian phase (°) Cricadian phase (°)

Fig. 3. Continued
344

and platelet count, a subset of the data regarding the
effect of exercise showed no significant circadian
rhythms, but a 12-h rhythm in aggregability (see
Fig. 3 for platelet aggregability and count data;
Scheer et al., 2010). A more comprehensive dataset
(more than four times larger) and analysis on the
effect of the circadian timing system, mental stress,
passive head-up tilt, and exercise demonstrated sig-
nificant circadian rhythms, not only in platelet
aggregability and platelet count, but also in platelet
size and platelet surface expression of activated
GPIIb-IIIa, P-selectin, and GP1b (Scheer et al.,
2011). Platelet count and aggregability peaked at a
circadian phase equivalent to 3–8 PM and thus, the
circadian timing system’s influence on platelet count
and aggregability is unlikely to contribute to the
morning peak in myocardial infarctions and ische-
mic strokes, unless the timing of its influence is dis-
turbed in people at risk for thrombotic events. On
the other hand, platelet size and surface expressions
of activated GPIIb-IIIa, P-selectin, and GP1b were
all greatest during the biological morning,
corresponding to 6 AM to noon (Scheer et al.,
2011), suggesting that the circadian timing system
may have a role in the greater incidence of adverse
cardiovascular events in the morning through these
thrombotic factors. The reactivity of the above
platelet measures to mental, postural, and physical
stress was not significantly influenced by the circa-
dian timing system, and thus, the influence of the cir-
cadian timing system and that of behavioral
stressors appear to be additive (Scheer et al., 2011).
In humans, resting plasma epinephrine and
norepinephrine levels—markers of sympathetic
nervous activity—exhibit endogenous circadian
rhythmicity with a broad peak during the middle
of the biological day (Fig. 3; Hu et al., 2011;
Scheer et al., 2009, 2010, 2011; Shea et al.,
2011). Therefore, if the timing would be similar
in vulnerable populations, the circadian peak
of catecholamines unlikely contributes to the
morning peak in adverse cardiovascular events.
Whether the doubling in epinephrine con-
centrations during the biological morning, possibly
coinciding with sensitized adrenergic receptors and
the circadian peak in cortisol, is of relevance to
the morning peak in adverse cardiovascular
events requires further study (Scheer et al.,
2010). Another cardiovascular biomarker that is
under circadian control is a measure of scale-
invariant, or fractal, behavior of heart beat
variability, which shows a peak at a circadian
phase equivalent to approximately 9 AM (Hu
et al., 2004). This fractal pattern breaks down
with disease and its change is predictive of mor-
tality (Hu et al., 2009; Mäkikallio et al., 2004;
Peng et al., 1995). Furthermore, lesioning of the
SCN abolishes such fractal patterns in heart beat
variability at time scales between 4 and 24 h, ren-
dering the pattern similar to random noise and
demonstrating the SCN as a critical node in
the neural feedback network underlying scale-
invariant patterns in physiology (Hu et al., 2008).
The reactivity of epinephrine and norepineph-
rine in healthy humans to exercise is dependent
on circadian phase, with two peaks: one in the
biological morning and the other in the biological
evening (Fig. 3; Scheer et al., 2010). These

Fig. 3. Influence of the endogenous circadian timing system on cardiovascular variables at rest and in response to standardized
bicycle exercise. Participants undertook an 11-day (including 12 times 20-h “days”) forced desynchrony protocol in which they
undertook 15 min of exercise (60% of maximum heart rate) at the same time into each wake period. Data are expressed relative to
each participant’s resting value averaged across the whole forced desynchrony protocol (left axes) and in absolute values (right
axes) and are plotted according to circadian phase that is separated into six 60 -bins that each equate to  4 h. Black lines and
closed circles indicate resting values, whereas gray lines with open circles represent data obtained during standardized exercise; error
bars represent the standard error of the mean. Gray bars indicate the group average habitual clock time for sleep in the 2 weeks
prior to admission to the laboratory. The vertical dotted line represents the timing of the group average core body temperature
minimum and curly brackets signify the most vulnerable period for adverse cardiovascular events according to epidemiological
research (6 AM to noon). Probability data were obtained via cosinor analysis. A statistically significant (P < 0.05) second harmonic
of a circadian rhythm is indicated by f2 appearing after the probability value. The arrow in the upper right plot is an example of the
reactivity of systolic blood pressure to exercise at particular circadian phase. Reproduced with permission from Scheer et al. (2010).

morning peaks coincide with the circadian peak in
cortisol (Scheer et al., 2009, 2010; Wehr et al.,
2001), which may further increase cardiac vulner-
ability since cortisol can potentiate the effects of
the sympathetic nervous system (Davies and
Lefkowitz, 1984). Also, the epinephrine response
to a passive head-up tilt in healthy humans is con-
trolled by the circadian timing system, with a
broad peak occurring during biological evening
(Hu et al., 2011). Taken all together, the circadian
timing system’s influence on the reactivity of epi-
nephrine and norepinephrine to exercise, but not
passive head-up tilt, may contribute to the morn-
ing peak in adverse cardiovascular events.
Markers of parasympathetic nervous activity dis-
play a circadian rhythm at rest in healthy humans,
with peaks in the biological night and morning,
and thus may be “protective” in the morning, at
least in healthy subjects at rest (Fig. 3; Burgess et al.,
1997; Hu et al., 2011; Scheer et al., 2010; Shea et al.,
2011). The circadian timing system also influences
the reactivity of the sympathetic nervous system to
behavioral stressors, including exercise and passive
head-up tilt, with the largest reduction in para-
sympathetic markers occurring in the biological
morning, corresponding to approximately 9 AM
(see Fig. 3 for responses to exercise; Hu et al.,
2011; Scheer et al., 2010). If confirmed in vulnerable
populations, the impact of the internal circadian
timing system on reactivity of parasympathetic
modulation of the heart in response to behavioral
stressors such as exercise could thus contribute to
the morning peak in adverse cardiovascular events
(Vanoli et al., 1991).
A recent study in mice demonstrated that
45 min of experimenter-induced ischemia of the
coronary artery at the beginning of the biological
night (start of the active phase, thus equivalent to
the human morning) as compared to the start of
the biological day resulted in greater infarct vol-
ume, fibrosis, adverse remodeling, and depression
of contractile function after 1 month of reperfu-
sion (Durgan et al., 2010). Moreover, deletion of
the cardiomyocyte circadian clock gene
attenuated the above mentioned outcomes. This
indicates that the cardiomyocyte circadian clock
345
gene contributes to the time of day variation in
responses to coronary artery ischemia and
subsequent reperfusion.
Circadian disruption and cardiovascular function
In the previous section, we provided recent
evidence for the impact of the circadian timing
system on cardiovascular risk markers that—if
confirmed in vulnerable populations—may con-
tribute to the morning peak in adverse cardiovas-
cular events. However, in healthy individuals,
the circadian timing system has been proposed
to optimally regulate many physiological pro-
cesses to prepare for the varying demands across
the sleep/wake cycle. If this would be the case, it
would be expected that circadian disruption
would have adverse health effects. Circadian dis-
ruption can be the result of, for example, circa-
dian misalignment (in which circadian function is
intact, but not properly aligned with either the
external environment or among different compo-
nents of the circadian timing system), neuroana-
tomical damage or changes to the SCN, and
genetic mutations and variance (for more details,
see Rüger and Scheer, 2009). Circadian misalign-
ment occurs when the circadian timing system
as a whole is desynchronized from the behavioral
and/or environmental cycles, and this can be
caused by external factors (e.g., rapidly shifting
the light/dark and sleep/wake cycle as seen in shift
workers and with jet lag) and internal factors
(e.g., blindness preventing entrainment by light
of the circadian timing system to the 24-h light/
dark cycle). Circadian misalignment can also
occur among the different circadian oscillators in
the body, for example, between the SCN and
peripheral oscillators in the liver, as a result of
“internal desynchrony.” Circadian disruption with
underlying neuroanatomical changes in the SCN
has been demonstrated in Alzheimer’s disease
and depression (Wu et al., 2006; Zhou et al.,
2001). Animal experimental SCN lesions and
human case studies of tumors in proximity to the
SCN have also been shown to lead to circadian
346
disruption (Moore and Eichler, 1972; Schwartz
et al., 1986). Examples of circadian clock gene
mutations which impact circadian function
include familial advanced sleep phase syndrome
in humans and the many clock gene mutations
in experimental animals (Toh et al., 2001; Turek
et al., 2005). These categories are not mutually
exclusive, for example, familial advanced sleep
phase syndrome can lead to circadian misalign-
ment. In this section, we will provide some
examples of the detrimental cardiovascular health
consequences of circadian disruption. Adverse
metabolic consequences of circadian disruption
will be discussed in the next section.
Epidemiological studies indicate that shift work is
associated with cardiovascular disease (Kawachi
et al., 1995; Knutsson et al., 1986, 1999; Suwazono
et al., 2008b). Because differences in socioeconomic
status and life style cannot fully explain these
observations, researchers have begun investigating
whether circadian misalignment itself may be one
of the underlying mechanisms of the above
associations. Human studies investigating the effect
of circadian misalignment or simulated night work
on cardiovascular function in controlled laboratory
conditions are scarce. Scheer et al. (2009) demonstra-
ted that circadian misalignment (i.e., desynchrony
between the circadian timing system and behaviors)
increases wake time blood pressure. Recent research
has demonstrated that an acute bout of evening exer-
cise significantly lowers blood pressure during
subsequent simulated night work, suggesting that
the adverse effect of circadian misalignment on
blood pressure could be negated by evening exercise
(Fullick et al., 2009). Clearly, more experimental
human research on the effects of circadian disruption
on cardiovascular function is needed.
There is also evidence of adverse cardiovascular
effects of desynchrony between the circadian
timing system and the imposed light/dark cycle
from animal experimental work. Cardiomyopathic
hamsters that are subjected to a 12-h shift in the
light/dark cycle every week have a shorter life
span than cardiomyopathic hamsters that maintain
a fixed 24-h light/dark cycle (Penev et al., 1998).
Furthermore, mice with surgically induced heart
disease (cardiac hypertrophy through transverse
aortic constriction) that live on an abnormal
light/dark (10:10 h) cycle following surgery show
impaired compensatory cardiac and vascular remo-
deling, reduced ventricular contractile strength,
and increased blood pressure as compared to
animals that live on a normal light/dark (12:12 h)
cycle (Martino et al., 2007). Phenotypic rescue,
including reversal/attenuation of abnormal pathol-
ogy and genes, only occurs when the external
rhythm is allowed to correspond with the animals’
innate 24-h internal rhythm.
Further evidence for a critical role of proper
synchrony between the circadian timing system
and the external environment comes from
follow-up studies in Tau mutant hamsters. Tau
mutant hamsters have a shorter period ( 22 h)
than their wild-type ( 24 h) counterparts (Ralph
and Menaker, 1988). Tau mutant hamsters have
a shorter life span and have cardiomyopathy,
fibrosis, and impaired heart contractility when
they live on a 24-h light/dark cycle which is
beyond their circadian timing system’s range of
entrainment and thus causes the animal’s behav-
ioral cycle and biological timing system to be out
of phase (i.e., desynchronized; Martino et al.,
2008). Remarkably, when Tau mutant hamsters
are placed in 22-h light/dark cycles, similar to their
endogenous circadian period, these pathological
changes are largely reversed. Taken together, the
above mentioned findings suggest that circadian
disruption, such as with shift work, may worsen
cardiovascular function in individuals with
preexisting heart disease.
It has been hypothesized that dysfunction of
the SCN is associated with hypertension. Levels
of three key neurotransmitters (vasopressin, vaso-
active intestinal peptide, and neurotensin) in the
human SCN, required for proper circadian func-
tioning, are lower in postmortem material of
patients with essential hypertension than in that
of control subjects (Goncharuk et al., 2001). At
the same time, more corticotropin-releasing hor-
mone (CRH) neurons and greater CRH mRNA

are present in the paraventricular nucleus (PVN)
of the hypothalamus of people with essential
hypertension than controls (Goncharuk et al.,
2002). Because CRH raises blood pressure when
administered intraventricularly, it has been pro-
posed that the elevated CRH levels in hyperten-
sive patients may contribute to their hypertension
(Kalin et al., 1983). The vasopressinergic neurons
from the SCN have an inhibitory effect on CRH
neurons in the PVN (Buijs et al., 1993; Hermes
et al., 1996). Thus, the reduced levels of vasopres-
sin in the SCN could result in disinhibition of
CRH production by the PVN and, in turn, contrib-
ute to the abnormal 24-h variation in blood
pressure observed in these patients (Goncharuk
et al., 2002). Alternatively or additionally, it
has been hypothesized that the reverse may be
true and that the observed more intense CRH
projection to the SCN in hypertensive patients
contributes to the decrease in the number of
vasopressin, vasoactive intestinal peptide, and
neurotensin neurons in the SCN (Goncharuk
et al., 2007). Based on the information above, the
evidence that the SCN is involved in cardiovascular
control (Scheer et al., 2003) and the observation
that melatonin administration can amplify or syn-
chronize circadian rhythms (Bothorel et al., 2002;
Koster-van Hoffen et al., 1993; Zaidan et al.,
1994), Scheer et al. (2004) investigated if 3 weeks
of nighttime melatonin administration can improve
blood pressure regulation in patients with essential
hypertension. Repeated nighttime melatonin
administration enhanced the day/night rhythm
amplitude of blood pressure and reduced blood
pressure during the patient’s scheduled sleep
period. Of interest, acute nighttime melatonin
intake did not influence blood pressure indicating
that the improvement in blood pressure after 3
weeks of melatonin intake was not due to the
drugs acute vasodilatory effect but possibly a result
of its influence on the circadian timing system. The
blood pressure lowering effect of melatonin after
repeated administration in hypertensive patients
has been replicated by others (Cagnacci et al.,
2005; Grossman et al., 2006).
347
Other examples of an intricate link between
the circadian timing system and cardiovascular
control come from recent data on the influence
of clock genes in hypertension and cardiac func-
tioning. Mice without core clock components
Cry1 and Cry2 develop hypertension when their
diet contains salt, whereas wild-type mice do
not (Doi et al., 2010). This finding may have been
due to Cry-null mice overexcreting the hormone
aldosterone which increases blood pressure
through stimulation of reabsorption of sodium
ions and water. Moreover, mice lacking the
Clock gene specifically within cardiomyocytes
have brachycardia, greater myocardial oxygen
consumption, and decreased cardiac efficiency
(Bray et al., 2008).
Circadian disruption and metabolic function
Human endogenous circadian rhythms have been
observed in many factors related to metabolism.
For example, glucose, insulin, cortisol, epineph-
rine, norepinephrine, and leptin display endoge-
nous circadian variation (Morgan et al., 1998;
Scheer et al., 2009, 2010; Shea et al., 2005; Van
Cauter et al., 1994; Wehr et al., 2001). Recent
research demonstrates that amino acid plasma
concentrations are under endogenous circadian
control, through Krüppel-like factor 15-control
of the rhythmic expression of multiple enzymes
involved in mammalian nitrogen homeostasis
(Jeyaraj et al., 2012). SCN-intact rats, that have
a fixed 6-meals-per-day feeding schedule (to uni-
formly distribute food intake across the circadian
cycle), also have a circadian rhythm in glucose
levels, showing that this is not dependent on the
feeding/fasting cycle, and this rhythm is abolished
in SCN-lesioned rats. Multisynaptic projections
from the SCN to the liver, pancreas, adrenal
cortex, and adipocytes have been discovered,
and these provide neuroanatomical pathways by
which the SCN can influence metabolic-related
factors (Buijs et al., 1999, 2001; Kreier et al., 2006;
la Fleur et al., 2000). The SCN can also influence
348
metabolic-related factors (e.g., cortisol) via hor-
monal pathways (Buijs et al., 1993, 1999) and
peripheral clocks per se can influence metabolism
(Marcheva et al., 2010). Taken together, the circa-
dian timing system is an important entity that con-
tributes to the control of many metabolic-related
processes, and thus it is conceivable that circadian
disruption results in impaired metabolic function.
In this section, we will provide some examples
of the adverse metabolic-related consequences of
circadian disruption.
Numerous epidemiological studies have demon-
strated that shift work is a risk factor for metabolic
disorders such as diabetes and obesity (Morikawa
et al., 2005; Niedhammer et al., 1996; Suwazono
et al., 2006, 2008a). As a consequence, researchers
have employed experimental models to investigate
the possible underlying mechanisms. A 9-h phase
advance of the circadian timing system—a simula-
tion of shift work—increased the glucose and insu-
lin response to a test meal given at the same clock
time when the test meal was preceded by a high-
fat but not low-fat meal (Hampton et al., 1996;
Ribeiro et al., 1998). Moreover, a 9-h phase
advance only had a detrimental effect on the aver-
age triglyceride response to a test meal when the
pretest meal is a low- rather than high-fat meal
(Hampton et al., 1996; Ribeiro et al., 1998). The
influence of macronutrient content of a meal on
responses to subsequent meals during night
work requires further research. Al-Naimi et al.
(2004) extended the work of the above researchers
by measuring glucose, insulin, and triglyceride
responses to multiple meals across simulated day
and night work shifts. The researchers reported
that integrated glucose (statistical trend) and tri-
glyceride levels were higher across the night shift
than day shift. Integrated insulin levels were not
different between the night shift and day shifts.
Furthermore, postprandial glucose, insulin, and tri-
glyceride levels are higher during night work than
day work under field conditions (Lund et al.,
2001). The glucose and insulin, but not triglyceride,
postprandial responses normalize 2 days after
ceasing night work, indicating that some—but not
all—of the adverse metabolic and hormonal effects
of night work quickly subside after short-term
exposure (Lund et al., 2001; Ribeiro et al., 1998).
How long it takes for adverse metabolic changes
to normalize after chronic exposure, such as fol-
lowing chronic shift work, requires further study.
The simulated shift work studies discussed
above did not replicate the duration of wakeful-
ness and behavioral activity before tests meals in
both experimental conditions. Thus, it is unclear
from those studies what the contribution is of cir-
cadian misalignment per se on metabolic and hor-
monal factors. Scheer et al. (2009), utilizing a
forced desynchrony protocol, demonstrated that
short-term exposure (after 3 days) to circadian
misalignment per se increases the postprandial
responses of glucose and insulin, demonstrating
reduced glucose tolerance and suggesting reduced
insulin sensitivity under these conditions (Fig. 4).
It has also been demonstrated, in rats prone to
the development of type 2 diabetes, that phase
advancing the light/dark cycle by 6 h every 3 days
for 10 weeks accelerates the onset of type 2 diabetes
by causing a hastened loss of beta-cell function and
mass (Gale et al., 2011). Two studies have assessed
the effect of more prolonged circadian misalignment
on human metabolism under controlled laboratory
conditions. Morgan et al. (1998) found no significant
changes in the concentrations of glucose, insulin, and
triglycerides as measured in two constant routine
protocols with small hourly liquid meals, before
and after a 19-day forced desynchrony protocol.
Recently, it was demonstrated in humans that 3
weeks of sleep restriction (5.6 h of sleep opportunity
per 24 h) concurrent with circadian disruption
increases postprandial glucose levels, possibly due
to a reduction in post-meal insulin concentrations
(Buxton et al., 2012). These adverse alterations were
abolished following 9 days of recovery sleep and cir-
cadian re-entrainment to a normal sleep/wake cycle.
Short-term circadian misalignment while
subjects consume isocaloric meals also decreases
circulating levels of leptin (Fig. 4; Scheer et al.,
2009). Such an effect, if persisted chronically,
could help explain the increased risk for the
 349

Aligned development of obesity through increased food

40 Misaligned intake. In addition, rats subjected to simulated
Leptin (% from mean)

12 night work (forced activity during the normal rest

20 phase) gain significantly more body mass than

Leptin (ng/ml)
10 rats undertaking simulated day work (forced
0 activity during the normal active phase; Salgado-
8 Delgado et al., 2008). Total daily food intake did
-20 P < 0.0001
not differ between the two groups, but the “night
6 workers” were less active and this may explain
-40
B L D S the above finding. Moreover, feeding mice for 6
0 4 8 12 16 20 24 28 weeks only during their biological day (normal rest
phase) rather than biological night (normal active
P < 0.0001
phase) increases their body fat percentage (statisti-
Glucose (% from mean)

60
160 cal trend) and body mass (Arble et al., 2009).

Glucose (mg/ml)
40 These increases may have been due to lower energy
140
expended via physical activity and greater food
20 120
intake in the mice fed during the biological day,
although neither of these observations reached
0 100 statistical significance. Exposing mice to light (150
lux of continues light or 16:8 150/5 lux cycle) during
-20 80 the habitual dark phase significantly increases
B L D S
body and fat mass and impairs glucose tolerance
0 4 8 12 16 20 24 28
(Fonken et al., 2010). These results were not
300 associated with any change in total 24-h food con-
P < 0.006
sumption or locomotor activity, but the mice in the
Insulin (% from mean)

60
200 16:8 light/dim light cycle altered their feeding behav-
Insulin (mIU/ml)

ior, with more food being consumed during the

100
0
-100 B L D S
0 4 8 12 16 20
Time since wake (h)
Fig. 4. The response of circulating levels of leptin, glucose,
and insulin to circadian misalignment. Data are reported
relative to each participant’s average values during circadian
alignment on the left axes and in absolute values on the right
axes. Error bars represent standard error of the mean. Gray
area indicates the participant’s sleep opportunity. The white
strips within the scheduled sleep opportunity represent when
participants were briefly awoken to perform pulmonary
function tests. B, breakfast; L, lunch; D, dinner; S, snack.
Reproduced with permission from Scheer et al. (2009).
40
habitual light phase.
Weekly 6-h phase advances and delays—
20 experimental models applicable to shift work—
significantly hasten mortality in mice (Davidson
et al., 2006). In an attempt to explore the mechanisms
0 underlying these findings, Castanon-Cervantes et al.
24 28 (2010) investigated the effect of four consecutive
weekly 6-h phase advances of the light/dark schedule
on immune responses in mice. Twenty-four hours
after a lipopolysaccharide (LPS) challenge, systemic
concentrations of the proinflammatory cytokines
interleukine-1b (IL-1b), granulocyte macrophage
colony-stimulating factor, IL-12, and IL-13 were sig-
nificantly higher in the shifted than control mice. The
circadian phase angle, as assessed by body tempera-
ture; locomotor activity; and Per2 expression in four
tissues (SCN, liver, thymus, and spleen), at the time
350
of the LPS challenge was similar between the shifted
and control animals, and therefore the findings were
not due to the stimulus being given at a different cir-
cadian time between the two groups. The increased
IL-6 release in the shifted mice was also observed
during in vitro LPS stimulation of macrophages. Fur-
thermore, the risk of death following the in vivo LPS
challenge was significantly greater in the shifted than
control mice. The above findings clearly indicate that
repeatedly shifting a mouse’s light/dark cycle has an
adverse effect on their immune response. Whether
similar adverse changes in immune function are also
observed in humans requires further study.
It is unclear how simulated night work and circa-
dian misalignment cause adverse effects such as
impaired glucose metabolism. Both night work
and circadian misalignment decrease total sleep
time which also decreases leptin levels and impairs
glucose tolerance and insulin sensitivity
(Åkerstedt, 1998; Buxton et al., 2010; Ohayon
et al., 2002; Spiegel et al., 1999, 2004). Thus, the
question rises to what extent the adverse effects
of night work and circadian misalignment are
mediated via a disturbance of sleep. A covariance
analysis performed by Scheer et al. (2009) suggests
that the effect of circadian misalignment on leptin
is likely to be at least in part independent of con-
current decreases in sleep efficiency. Moreover,
slow wave sleep suppression per se has adverse
effects on glucose metabolism (Tasali et al.,
2008). However, during circadian misalignment,
while sleeping during the biological day, there
was no decrease in the amount of slow wave sleep
(Morris et al., 2012), such that also changes in slow
wave sleep could not explain these results. Thus,
these data suggest that the adverse effects of night
work and circadian misalignment on endocrine
factors are not merely mediated by a reduction in
sleep quality or quantity. Another possible mecha-
nism underlying some adverse endocrine effects of
night work and circadian misalignment relates to
internal desynchrony caused by feeding schedules.
Feeding rodents only during the rest phase—as
similarly occurs during night work when they eat
during their night shift—can phase shift peripheral
oscillators in the liver, pancreas, kidney, and heart
while it has no effect on the phase of the SCN
(Damiola et al., 2000; Stokkan et al., 2001). Conse-
quently, such a feeding regime can uncouple
peripheral oscillators from the SCN. What the
physiological effects of internal desynchrony are
on metabolism and whether such internal
desynchrony occurs also in humans in a shift work
setting is currently unknown.
In addition to their participation in the generation
of circadian rhythms, molecular clock genes have
also been extensively reported to play a role in met-
abolic function. Homozygous ClockD19 mutant
(C57BL/6J) mice have a significantly attenuated
diurnal feeding rhythm and are hyperphagic and
obese compared to wild-type mice (Turek et al.,
2005). In addition, they exhibit adipocyte hypertro-
phy, lipid enlargement of hepatocytes with
pronounced glycogen build-up, hypercholesterol-
emia, hypertriglyceridemia, hypoinsulinemia, and
hyperglycemia. Marcheva et al. (2010) reported that
whole-body Clock mutation caused impaired glu-
cose tolerance in mice. The researchers also demon-
strated that glucose-stimulated insulin secretion is
reduced in isolated pancreatic islets from Clock
mutants, which may explain the observed impaired
glucose tolerance in these animals. Knockout of
Bmal1 alone in C57BL/6J mice showed that isolated
mouse embryonic fibroblast (MEF) cells could not
successfully differentiate into adipocytes (Shimba
et al., 2005). Further, restoration of Bmal1 function
by adenovirus gene transfer into the initial Bmal1-
deficient mice restored adipocyte differentiation
and lipid accumulation in the MEF cells. The pres-
ence of high Bmal1 mRNA levels in adipocytes
increased lipogenesis activity, further suggesting that
Bmal1 is involved in regulating lipid metabolism.
The hypoglycemic response following insulin
administration is exacerbated in Clock mutant and
Bmal1/ mice as compared in wild-type mice, pos-
sibly due to an impaired counterregulatory gluco-
neogenesis response (Rudic et al., 2004). In parallel
to the ClockD19 mutant mice (Turek et al., 2005),
mPer2/ mice also had a defective diurnal feeding
rhythm compared to their wild-type littermates.

These mice were also hyperphagic under a high-fat
diet and this may explain why these animals became
obese. In mice, genetic elimination of Cry increases
the expression of genes responsible for hepatic
glucogenesis and also raises fasting blood glucose
level (Zhang et al., 2010). The above studies indicate
that core clock genes participate in the regulation of
metabolic processes and many of the phenotypes
induced by dysfunctional/lost clock genes are
hallmarks of metabolic syndrome.
Experimenters have also investigated the effect of
organ-specific clocks on whole-body metabolism to
differentiate the effects from behavioral changes.
Some abnormal phenotypes displayed by global
Bmal1/ mice are observed in liver-specific knock-
outs. The loss of Bmal1 in the liver of mice leads to
hypoglycemia and to a loss of rhythmic expression
of hepatic genes that regulate glucose homeostasis
(Lamia et al., 2008). In recent work, young pan-
creas-specific Bmal1 mutant mice showed increased
ad libitum glucose levels, impaired glucose tolerance,
lowered insulin secretion, and decreased insulin
responsiveness to glucose (Marcheva et al., 2010).
These mice had normal body weight, activity, and
feeding rhythms, suggesting that the above men-
tioned phenotypes are attributable to pancreatic
clock disruption per se rather than to secondary
changes in behavior. These findings clearly reveal
that the abscission/disruption of the pancreatic clock
can lead to the onset of detrimental diseases such as
diabetes. However, more evidence is still needed
to determine which organ-specific clocks contribute
to global metabolic functions and what the underly-
ing mechanisms may be.
Countermeasures that prevent or attenuate the
adverse endocrine effects associated with night
work are needed. Researchers have demonstrated
in humans that appropriately timed light exposure
and/or melatonin intake can entrain the central
pacemaker to an inverted (relative to the solar
day) sleep/wake cycle (Boivin and James, 2002;
Crowley et al., 2003; Czeisler et al., 1990; Smith
et al., 2009). One would think that such a strategy
would attenuate the adverse effects of night work
on metabolic and hormonal factors; however, little
351
research has been undertaken in this area. Fur-
thermore, it is not known if the above strategy
would entrain, in addition to the central pace-
maker, pertinent peripheral oscillators, for exam-
ple, in liver and pancreas. Recent work has
demonstrated that restricting food intake in
rodents to the biological night (typical active
period) undertaking simulated night work prevents
the gain in body mass and the accumulation
of abdominal fat deposits induced by such a
work schedule; perhaps by preventing internal
desynchrony (Salgado-Delgado et al., 2010). This
appears to be a promising countermeasure, and
investigations are needed to determine if such a
strategy is helpful in humans. Morning exercise
can suppress daytime hunger and levels of acylated
ghrelin (Broom et al., 2007). Morris et al. (2010)
investigated if evening exercise suppresses hunger,
acylated ghrelin levels, and increases leptin con-
centrations during simulated night work. Evening
exercise increased levels of both acylated ghrelin
and leptin but had no influence on mean hunger
ratings. Participant’s activity—as measured by
a wrist accelerometer—was higher during the
simulated night shift that was preceded by exercise
which could have beneficial implications for their
energy balance. Clearly, more research is needed
to determine the optimal countermeasures for the
adverse endocrine effects of night work.
Summary
The circadian timing system orchestrates cyclic
variations in numerous cardiovascular and meta-
bolic functions independent of external influences
such as darkness/light, sleep/wakefulness, rest/
activity, and fasting/eating. At rest, the circadian
timing system causes some factors to peak during
the biological morning (e.g., cortisol, platelet
expression of activated GPIIb-IIIa, P-selectin,
and GP1b), which could potentiate the onset
of adverse cardiovascular events at that time.
This suggests that the morning peak in adverse
cardiovascular events may not be only due to
352
the acute transition from a resting (sleeping, inac-
tive, supine, and fasting) to an active (awake,
active, standing, and eating) state. Moreover, the
circadian timing system causes the greatest
increase in epinephrine level and the largest
decrease in parasympathetic nervous activity in
response to exercise to occur in the biological
morning. This further suggests that the circadian
timing system contributes to the morning peak
in adverse cardiovascular events. Previous work
in the above area has been conducted using
healthy people; thus, further research needs to
be undertaken in individuals who are at risk of
having an adverse cardiovascular event to deter-
mine whether there are changes in timing and
amplitude of the endogenous circadian rhythms
in cardiovascular risk markers. Research has also
demonstrated that circadian disruption has a pro-
found effect of cardiovascular and metabolic
function. More research is needed to fully under-
stand the underlying mechanisms. Such research
may help in the development of novel therapies
in the treatment of circadian related disorders.
Acknowledgments
C. J. M. was supported by the National Space
Biomedical Research Institute through NASA
NCC 9-58. F. A. J. L. S. was supported by National
Institute of Health Grants P30-HL101299 and R01
HL094806.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 20

Nutrition and the circadian timing system

Dirk Jan Stenvers{,*, Cora F. Jonkers{, Eric Fliers{, Peter H. L. T. Bisschop{ and
Andries Kalsbeek{,}

{
Department of Endocrinology and Metabolism, Academic Medical Center (AMC), University of Amsterdam,
Amsterdam, The Netherlands
{
Department of Nutrition, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands
}
Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience (NIN), An Institute of the
Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands

Abstract: Life on earth has evolved under the daily rhythm of light and dark. Consequently, most creatures
experience a daily rhythm in food availability. In this review, we first introduce the mammalian circadian timing
system, consisting of a central clock in the suprachiasmatic nucleus (SCN) and peripheral clocks in various
metabolic tissues including liver, pancreas, and intestine. We describe how peripheral clocks are
synchronized by the SCN and metabolic signals. Second, we review the influence of the circadian timing
system on food intake behavior, activity of the gastrointestinal system, and several aspects of glucose and
lipid metabolism. Third, the circadian control of digestion and metabolism may have important implications
for several aspects of food intake in humans. Therefore, we review the human literature on health aspects of
meal timing, meal frequency, and breakfast consumption, and we describe the potential implications of the
clock system for the timing of enteral tube feeding and parenteral nutrition. Finally, we explore the
connection between type 2 diabetes and the circadian timing system. Although the past decade has provided
exciting knowledge about the reciprocal relation between biological clocks and feeding/energy metabolism,
future research is necessary to further elucidate this fascinating relationship in order to improve human health.

Keywords: circadian clocks; circadian rhythm; biological clocks; suprachiasmatic nucleus; glucose; glucose
tolerance test; lipids; nutritional sciences; eating; appetite regulation; digestive system; gastrointestinal
system; parenteral nutrition; enteral nutrition; diabetes mellitus type 2.

Introduction

Ever since evolution started, life on earth is sub-

*Corresponding author.
Tel.: þ31 20 5663507; Fax: þ31 20 6917682 ject to the daily rhythm of light and dark. As a
E-mail: d.j.stenvers@amc.uva.nl consequence, many organisms experience a daily

http://dx.doi.org/10.1016/B978-0-444-59427-3.00020-4 359
360
rhythm in food availability. Most creatures, rang-
ing from bacteria to humans, developed a circa-
dian timing system to prepare for the alternating
daily periods of food intake and fasting. Recent
scientific work has generated a lot of knowledge
about the circadian control of digestion and
metabolism. There are several indications that
disturbances in daily rhythms may lead to obesity
and diabetes. Consequently, at present, scientists
aim to use the increasing knowledge about the
interaction between daily food intake rhythms
and the circadian timing system to improve
health.
The circadian timing system
Central clock
In mammals, including humans, the central bio-
logical clock resides in the bilateral hypothalamic
suprachiasmatic nucleus (SCN). The SCN
generates an autonomic rhythm of electrical activ-
ity with a period of approximately 24 h. This
rhythm continues to oscillate even when SCN cells
are removed from a living organism and brought
into culture (Bos and Mirmiran, 1990; Green and
Gillette, 1982; Groos and Hendriks, 1982; Welsh
et al., 1995). The SCN is located superior to the
optic chiasm and receives direct light information
from the retina via the retinohypothalamic tract.
Through this light input, the endogenous approxi-
mate 24-h rhythm of the SCN is synchronized with
the environmental rhythm with its period duration
of exactly 24 h (Dibner et al., 2010; Kohsaka and
Bass, 2007). The central clock signals to several
hypothalamic (e.g., the paraventricular nucleus
(PVN) and the subparaventricular zone (sSPZ)
and thalamic (e.g., the intergeniculate leaflet and
paraventricular nucleus of the thalamus) regions.
The central clock is of utmost importance for the
regulation of daily sleep/wake rhythms, daily
rhythms in the secretion of hormones, and daily
rhythms in feeding behavior (reviewed in Morin
and Allen, 2006; Saper et al., 2005).
Approximately 20 years ago, it was discovered
that the endogenous SCN rhythm is based on a
molecular clock mechanism. The core clock genes
(Clock, BMAL1, Period, and Cryptochrome) are
expressed in a transcriptional–translational feed-
back loop with a duration of approximately 24 h
(reviewed in Bass and Takahashi, 2010). In turn,
the clock genes regulate the expression of a pleth-
ora of other genes in SCN neurons and thereby
force SCN electrical activity into a 24-h rhythm
(Herzog et al., 1998; Nakamura et al., 2002).
Peripheral clocks
Soon after the discovery of the clock genes, it
became clear that virtually every mammalian cell
contains a molecular clock (Balsalobre et al., 1998;
Zylka et al., 1998). Animal studies have shown
peripheral clock gene oscillations in digestive
organs including liver (Balsalobre et al., 2000;
Damiola et al., 2000; Oishi et al., 2000; Sladek
et al., 2007; Stokkan et al., 2001; Zylka et al.,
1998), pancreas (Damiola et al., 2000; Muhlbauer
et al., 2004; Sadacca et al., 2011), stomach
(Bostwick et al., 2010; Hoogerwerf et al., 2007;
LeSauter et al., 2009), and intestine (Bostwick
et al., 2010; Hoogerwerf et al., 2007; Sladek et al.,
2007). In human adipose tissue, approximately
25% of the genes show a circadian rhythm of
expression (Loboda et al., 2009).
Elegant experiments targeting the molecular
clock revealed important aspects of the physiologi-
cal function of peripheral clocks. For instance, the
molecular liver clock regulates the expression of
important metabolic genes such as phosphoenol-
pyruvate carboxykinase (Kornmann et al., 2007;
Lamia et al., 2008; Panda et al., 2002; Roesler
et al., 1992; Zhang et al., 2010) and glucose-6-
phosphatase (Kornmann et al., 2007; Lamia et al.,
2008; Panda et al., 2002; Zhang et al., 2010), two
key enzymes in gluconeogenesis. Furthermore, the
liver clock controls the expression of major
enzymes in oxidative phosphorylation and lipid
metabolism (Kornmann et al., 2007; Panda et al.,

2002). On the other hand, in the pancreatic beta
cells, clock genes play an important role in insulin
secretion (Allaman-Pillet et al., 2004; Marcheva
et al., 2010; Sadacca et al., 2011). To date, the phys-
iological function of the molecular clocks in stom-
ach and intestine remains to be determined. The
function of the adipose tissue clock is reviewed else-
where in this volume (van der Spek et al., 2012).
Peripheral clocks also show an autonomic cycle
of approximately (but not exactly) 24 h (Brown
et al., 2008; Yamazaki et al., 2000) that needs syn-
chronization with the external light–dark cycle.
Since most peripheral clocks do not receive light
information, the SCN communicates its rhythm
to peripheral clocks. As indicated, the SCN signal
is passed on via neural projections to several
thalamic and hypothalamic areas. Subsequently,
the SCN signal is forwarded to peripheral clocks
via hormonal signals and the autonomic nervous
system. For example, the SCN controls the circa-
dian rhythm in the release of glucocorticoids
by the adrenal gland (Moore and Eichler, 1972;
Reppert et al., 1981) as well as the circadian
rhythm in the release of melatonin by the pineal
gland (Reppert et al., 1981). Glucocorticoids are
potent synchronizers of peripheral clock rhythms
(Balsalobre et al., 2000; Le Minh et al., 2001),
and melatonin may have a role in synchronizing
peripheral clocks as well (Torres-Farfan et al.,
2006; Valenzuela et al., 2008). In addition to
these hormonal pathways, the SCN has neural
connections with a variety of tissues including
liver (Buijs et al., 2003; Kalsbeek et al., 2004; La
Fleur et al., 2000; Niijima et al., 1992), pancreas
(Buijs et al., 2001; Niijima et al., 1992), and adi-
pose tissue (Kreier et al., 2006) via polysynaptic
autonomic projections. Evidence from in vivo
experiments supports an important functional
role in transmitting the circadian signal to periph-
eral clocks via these autonomic projections as
well (Cailotto et al., 2009; Guo et al., 2005;
Terazono et al., 2003; Vujovic et al., 2008).
In addition to the direct hormonal and neuro-
nal signals described above, peripheral clocks
are synchronized by several indirect signals. First,
361
peripheral clocks are strongly influenced by
energy metabolism. Animal studies show that clock
rhythms in liver and pancreas can be inverted by
feeding animals when they normally fast (Damiola
et al., 2000; Stokkan et al., 2001). Presumably, this
is due to the responsiveness of the molecular clock
mechanism to metabolites such as NADH and
AMP (Lamia et al., 2009; Ramsey et al., 2009;
Rutter et al., 2001) (see Fig. 1). Second, mammalian
peripheral clocks are synchronized by the daily
rhythm of body temperature (Brown et al., 2002;
Buhr et al., 2010). Third, peripheral clocks are
also influenced by hormones that are released upon
feeding, such as insulin (Kuriyama et al., 2004;
Tahara et al., 2011; Zhang et al., 2009).
Taken together, accumulating evidence indicates
that a major function of peripheral clocks is to pre-
pare the human body for the alternating periods of
food intake (day) and fasting (night) (Bass and Taka-
hashi, 2010; Green et al., 2008; Lamia et al., 2008).
Circadian aspects of digestion and metabolism
Food intake
During the day, most people grow hungry at reg-
ular intervals. At night, however, most people
sleep without the arousing effect of appetite
despite the much longer period of fasting. Does
the SCN modulate appetite and/or the timing of
food intake?
Since the discovery of leptin, knowledge about
the hypothalamic control of food intake has grown
exponentially, and the key role of the hypothalamic
arcuate nucleus has been revealed by numerous ani-
mal experiments. First, the arcuate nucleus contains
receptors for various homeostatic messages from
the body and is therefore considered the “window
of the brain.” These messages include ghrelin pro-
duction by the empty stomach, pancreatic insulin
production upon feeding, and adipose tissue leptin
production. Second, the arcuate nucleus contains
two sets of neurons that have a major role in the
control of food intake. One subset of neurons
362

Fig. 1. The central biological clock resides in the suprachiasmatic nucleus (SCN). The SCN communicates with peripheral clocks in
liver, pancreas, adipose tissue, and intestine via both indirect signals (sleep–wake rhythm, appetite) and direct signals (autonomic
nervous system, hormones). Figure adapted from Dibner et al. (2010) (with permission).

produces Agouti-related peptide (Ollmann et al.,
1997; Rossi et al., 1998) and Neuropeptide-Y (Clark
et al., 1984; Stanley and Leibowitz, 1985) that
serve as orexigenic signals. Another subset of
neurons in the arcuate nucleus produces a-melano-
cyte-stimulating hormone (Fan et al., 1997) that
serves as an anorexigenic signal. With their output
signals, both subsets of neurons regulate the activity
of so-called second order neurons in (1) the
PVN that releases neuropeptides including cortico-
trophin-releasing hormone (Arase et al., 1988) and
thyrotropin-releasing hormone (Ahima et al., 1996;
Legradi and Lechan, 1999; Vella et al., 2011) which
modulate food intake and (2) the lateral hypothala-
mus that releases orexin (Edwards et al., 1999;
Sakurai et al., 1998; Sweet et al., 1999) and thus

increases food intake (for an extensive review on the
hypothalamic control of food intake, see Woods and
D’Alessio, 2008).
The involvement of the SCN in the timing of
food intake was demonstrated using a number of
approaches. Lesioning the SCN or isolating it
from its surrounding tissue results in a loss of
diurnal rhythms in food intake (Nagai et al.,
1978; Nishio et al., 1979), and changes of the light-
dark regimen strongly affect the timing of food
intake in rodents (Plata-Salaman and Oomura,
1987; Zucker, 1971). Furthermore, several genetic
knockout models show that whole-body disruptions
of the molecular clock also abolish diurnal rhythms
of food intake (e.g., Turek et al., 2005; Yang et al.,
2009). However, most knowledge on the hypotha-
lamic regulation of food intake is derived from
rodent research, and food intake in rodents is tightly
coupled to wakefulness. Thus, the question whether
the SCN directly regulates food intake behavior and
appetite itself (apart from the SCN effects on wake-
fulness and activity) remains to be elucidated. Nev-
ertheless, a direct effect of the SCN on appetite or
food intake behavior is suggested by the SCN output
projections to both the CRF-producing neurons in
the PVN (Vrang et al., 1995) and orexin-producing
neurons in the lateral hypothalamus (Abrahamson
et al., 2001).
Gastrointestinal system
Since most people eat during the day, increased
daytime activity of the digestive system would
be convenient. Indeed, clear daily rhythms exist
in gastrointestinal motility, exocrine secretion,
and the activity of digestive enzymes.
First, human stomach emptying after identical
meals occurs faster in the morning than in the
evening (Goo et al., 1987). Furthermore, small
bowel propagation velocity of the migrating
motor complex (Kumar et al., 1986; Sanders and
Moore, 1992) as well as colonic motor activity
(Narducci et al., 1987; Rao et al., 2001) is higher
during daytime than during nighttime. Here, we
363
limit ourselves to data concerning the motor
activity of the human gastrointestinal system.
An extensive review covering animal experiments
on circadian gastrointestinal motor activity can be
found elsewhere (Hoogerwerf, 2010).
Second, the exocrine secretion of several digestive
fluids is influenced by the time of day. For example,
the production of saliva increases over the day in
humans (Dawes, 1975). During fasting, human gas-
tric acid secretion shows a daily rhythm with
increased production in the evening. Presumably,
this rhythm in gastric acid secretion is regulated by
the parasympathetic branch of the autonomic ner-
vous system as vagotomy (performed as a treatment
for gastric ulceration) abolishes this rhythm (Moore,
1991). Furthermore, animal research shows that the
exocrine production of pancreatic juice is increased
during the period of habitual food intake (Maouyo
et al., 1993; Thaela et al., 1998).
Third, diurnal rhythms exist in the activity of
digestive enzymes in the brush border of the small
intestine. Oligosaccharidases including sucrase
show a circadian rhythm of increased activity dur-
ing the period of food intake, and this rhythm
proceeds during several days of fasting (Saito
et al., 1976, 1980). Furthermore, transporters that
absorb glucose and protein from the intestinal
lumen are more active during the period of food
intake in rats fed ad libitum, but this rhythm does
not seem to persist under constant conditions
(Pan et al., 2002, 2004).
Carbohydrates
Considering the daily rhythms in many gastrointes-
tinal processes, it makes sense that metabolic meal
responses show daily rhythms as well. Indeed, in
healthy humans, plasma glucose excursions after
identical meals are higher at night than in the morn-
ing (Biston et al., 1996; Van Cauter et al., 1992).
However, intestinal processes are not the only
determinant of plasma glucose values. Plasma glu-
cose levels depend on glucose influx from intestinal
absorption and (during fasting) hepatic glucose
364
production, and glucose efflux to muscle, brain, adi-
pose tissue, and (after feeding) the liver.
Daily rhythms in human glucose metabolism
have been clearly demonstrated by studies in
which glucose is administered intravenously.
A single intravenous glucose bolus results in
lower plasma glucose levels in the morning than
in the evening (Verrillo et al., 1989; Whichelow
et al., 1974) (for an excellent review on human
circadian glucose metabolism, see Van Cauter
et al., 1997). This phenomenon is probably caused
by daily rhythms in the separate glucose fluxes.
Human fasting studies suggest the presence of a
day–night rhythm in hepatic glucose production
(Clore et al., 1989). Animal studies from our
department have shown a circadian rhythm in
hepatic glucose production as well, with increased
glucose production at the onset of the active
period, which is the night in nocturnal animals.
The SCN determines this rhythm in hepatic glu-
cose production (La Fleur et al., 2001) via a diur-
nal rhythm of GABAergic inhibitory output to
preautonomic neurons in the PVN (Kalsbeek
et al., 2004, 2008). From the PVN, the SCN signal
is transferred to the liver via a balance between
sympathetic and parasympathetic activity (Cailotto
et al., 2008; Kalsbeek et al., 2004). Interestingly,
one of our studies not only shows that hepatic
clock gene expression is independent from these
autonomic signals to the liver, but moreover that
rhythmic hepatic clock gene expression is insuffi-
cient to maintain the circadian rhythm in hepatic
glucose production after complete hepatic dener-
vation (Cailotto et al., 2005). This observation is
in contrast with the proposed role of the liver clock
in the regulation of gluconeogenesis (Kornmann
et al., 2007; Lamia et al., 2008; Panda et al., 2002;
Roesler et al., 1992; Zhang et al., 2010), and most
likely for an optimal glucose homeostasis, both
components are necessary.
In addition to glucose production, glucose
uptake by various tissues shows daily rhythmicity
as well. At rest, human skeletal muscle consumes
more glucose in the morning than in the evening
(Verrillo et al., 1989). This rhythm is probably
the net result of rhythms in insulin production,
insulin sensitivity, and insulin-independent glu-
cose uptake.
In healthy humans, a daily rhythm in pancreatic
insulin production is observed in response to the
administration of tolbutamide, with increased
insulin production in the morning compared to
the evening (Baker and Jarrett, 1972; Carroll
and Nestel, 1973; Sensi et al., 1973). The same
rhythm has been described both in response to
intravenous glucose (Carroll and Nestel, 1973;
Lee et al., 1992; Melani et al., 1976) and intrave-
nous glucagon (Melani et al., 1976). Cultured rat
pancreatic islets show an autonomous circadian
rhythm of insulin production with enhanced
nighttime insulin production, which persists for 7
days in culture (Peschke and Peschke, 1998).
In contrast, rats show a tendency toward increased
daytime insulin production after intravenous glu-
cose administration (La Fleur et al., 2001). This
paradox may be explained by the observation
that in vivo insulin-independent mechanisms
also determine the circadian rhythm in glucose
tolerance (see above and below), and insulin
production is in itself dependent on plasma glucose
levels.
Furthermore, mammals show a daily rhythm in
insulin sensitivity. After an intravenous insulin
bolus, blood glucose levels show a more rapid
decline at the onset of the activity period (day-
time in humans, nighttime in rats), both in
humans (Gibson and Jarrett, 1972; Gibson et al.,
1975) and in rats (Kalsbeek et al., 2003; La Fleur
et al., 2001). Finally, some studies indicate a cir-
cadian rhythm in insulin-independent glucose
uptake: After an intravenous glucose tolerance
test, the calculated evening insulin-independent
glucose uptake is lower than the calculated morn-
ing insulin-independent uptake (Lee et al., 1992),
and cultured adipocytes show a rhythm in glucose
uptake in the absence of insulin (Feneberg and
Lemmer, 2004).
Taken together, it is obvious that the circadian
timing system strongly influences virtually all aspects
of glucose metabolism. A likely physiological

explanation for the daily rhythms in glucose metab-
olism is that an organism needs to be prepared for
the active period, that is, the period of food intake
and increased metabolic needs (La Fleur, 2003;
Van Cauter et al., 1997).
Lipids
In addition to carbohydrate metabolism, lipid
metabolism seems adjusted to the daily rhythm
of fasting and feeding as well. In humans who
eat during the day, plasma free fatty acids (FFAs)
show a strong increase at night due to increased
lipolysis during the nighttime fast (Schlierf and
Dorow, 1973). Evidence for an underlying circa-
dian regulation of lipid metabolism is provided
by studies that compare morning and evening
responses to identical stimuli. Thus, in the
morning, fasting FFA levels are lower than in
the evening (Carroll and Nestel, 1973; Gibson
et al., 1975; Zimmet et al., 1974). Moreover,
plasma FFAs show a smaller decrease in the
morning than in the evening, not only after an
oral glucose tolerance test (Zimmet et al., 1974)
but also after identical meals (Pontiroli
et al., 1992; Romon et al., 1997) and after an
intravenous insulin bolus (Carroll and Nestel,
1973; Gibson et al., 1975; Morgan et al., 1999).
Finally, plasma levels of triglycerides and FFAs
show a slight increase during the night in humans
under continuous conditions of 3-h sucrose inges-
tion (Barter et al., 1971). The nighttime increase
of lipolysis is possibly regulated by the nightly
surge of growth hormone (GH) secretion, since
patients with GH deficiency do not show the
nightly increase of FFA levels, and GH replace-
ment restores this normal nocturnal increase of
lipolysis, albeit in a supraphysiological dosage
(Boyle et al., 1992). However, the autonomic ner-
vous system may have an additional role in
regulating the circadian rhythm of lipid metabo-
lism, considering the strong effects of the auto-
nomic nervous system on the metabolic activity
of adipose tissue (Fliers et al., 2003).
365
Furthermore, hepatic cholesterol production
shows a clear day–night rhythm. In humans,
cholesterol synthesis shows a strong increase
during the night (Cella et al., 1995). Interestingly,
in rats, hepatic cholesterol synthesis also shows
an increase during the night, due to increased
nighttime synthesis of the rate-limiting enzyme
3-hydroxy-3-methylglutaryl-coenzyme A reduc-
tase (Edwards et al., 1972). Thus, unlike many
other physiological processes, the day–night pat-
tern of cholesterol synthesis is not inverted in
nocturnal animals.
The circadian rhythm in hepatic cholesterol
synthesis has clinical implications. Several clinical
trials show that the cholesterol-lowering effect of
statins is increased when patients take the statin
in the evening compared to the morning (Lund
et al., 2002; Saito et al., 1991; Wallace et al.,
2003). Therefore, it is now common practice to
instruct patients to take the statin in the evening.
However, it may be interesting to note that these
trials all show an effect on morning (fasting) cho-
lesterol levels, which does not necessarily impli-
cate an effect on average (24 h) cholesterol levels.
Meal patterns and human health
The circadian control of digestion and metabo-
lism may have important implications for the con-
cept of healthy food intake in humans. Next, we
review the literature on the effects of meal timing,
meal frequency, and breakfast consumption on
human health. Furthermore, we discuss potential
implications of the circadian timing system for
the timing of enteral tube feeding and parenteral
nutrition.
Day and night
The biological clock has evolved under a pro-
nounced daily alteration of light and dark, cou-
pled to clear alterations of activity and sleep,
and feeding and fasting. In humans, activity and
366
feeding generally occur during the light period
(Roenneberg et al., 2012). However, in the pres-
ent 24-h society, the synchrony between these
rhythms is easily disturbed. Due to artificial light,
television and internet, average sleep duration
has decreased over the years (Spiegel et al.,
2009). Furthermore, many people become less
exposed to bright sunlight since they are living
and working inside. Finally, people perform less
physical exercise during the daytime (Brownson
et al., 2005). Several lines of evidence suggest that
mismatches between the daily rhythms of activity
and food intake (day), and rest and fasting (night)
lead to health complaints and disease in humans
(see Fig. 2).
First, shift workers are more prone to gastroin-
testinal problems such as constipation, diarrhea,
or gastric ulcers. In addition, they are at increased
risk of obesity, diabetes (Pan et al., 2011), cardio-
vascular disease, and cancer (reviewed in Wang
et al., 2011).
Second, not only shift workers but also short
sleepers are at increased risk of obesity and dia-
betes (reviewed in Spiegel et al., 2009). One sin-
gle night of sleep disruption already strongly
reduces glucose tolerance (Donga et al., 2010)
while two nights of short sleep (4 h) causes a
24% increase of appetite and an increased intake
of high calorie food in association with increased
Light
Wake
Food intake
Energy storage
Fig. 2. In the physiological situation, wakefulness, food intake, and anabolic metabolic processes coincide with the light period.
Sleep, fasting, and catabolic metabolic processes are coupled to the dark period. Disturbance of this circadian synchrony may
lead to obesity and diabetes.
ghrelin levels and reduced leptin levels (Spiegel
et al., 2004).
Third, people with night eating syndrome (NES)
often show obesity and an inability to lose weight.
NES is characterized by (1) excessive food intake
late at night, (2) difficulties falling asleep, and (3)
frequent nightly awakenings. NES occurs in
1–5% of the general population and in 6–14%
of patients with obesity (Sim et al., 2010). Fourth,
in an interesting experiment, humans showed
decreased glucose tolerance when they ingested
food at times that conflicted with their internal
clock rhythm (Scheer et al., 2009).
Thus, mismatches between day–night rhythms
have severe pathophysiological implications. Only
recently, clinical trials investigating the modula-
tion of day–night rhythms to prevent or treat dis-
eases have started. For example, one clinical trial
is investigating the treatment of obesity by
increasing sleep duration with 1.5 h per night
(Cizza et al., 2010).
Meal frequency
It is generally believed that a healthy meal pat-
tern consists of three meals per day, with a limited
number of snacks. The Netherlands Nutrition
Centre Foundation also advises to follow this
Dark
Sleep
Fasting
Energy use

meal pattern (http://www.voedingscentrum.nl/nl/
schijf-van-vijf/eet-gevarieerd/regelmatig-eten.
aspx, 2012). However, evidence supporting this
advice is scarce.
A comprehensive review on the relation
between meal frequency and energy balance
shows obvious discrepancies between a number
of epidemiological studies investigating the corre-
lation between meal frequency and obesity
(Bellisle et al., 1997). Furthermore, these epide-
miological studies are difficult to interpret, since
obese people tend to record food intake
unreliably (Bellisle et al., 1997; Lichtman et al.,
1992; Salle et al., 2006).
Thus far, intervention studies have not been
able to detect significant effects of meal frequency
on body weight, body composition, or 24-h energy
expenditure in people on a weight loss diet
(Bellisle et al., 1997; Palmer et al., 2009). Several
reviews into the effects of meal frequency inter-
ventions on cardiovascular risk markers found a
trend toward decreasing plasma total cholesterol
and low-density lipoprotein (LDL) levels with
higher meal frequencies. However, plasma high-
density lipoprotein levels may also decrease with
increasing meal frequencies, which make the
effect on the overall cardiovascular risk profile
uncertain (Bhutani and Varady, 2009; Mann,
1997; Palmer et al., 2009). The most firmly
established causal relation is that a high meal fre-
quency leads to an increase of dental caries
(Kandelman, 1997). In summary, a meal fre-
quency of three meals per day mainly protects
our teeth, but the evidence for other beneficial
health effects is very limited.
Another frequent advice is to eat regularly.
Considering the strong integration between the
circadian timing system and metabolism, this
advice seems to make sense. However, surpris-
ingly, little research has been performed into the
effects of regular food intake on health. One
interesting paper shows that in obese women, a
2-week regular meal pattern leads to reduced
energy intake, lower fasting cholesterol levels,
and increased insulin sensitivity, compared to an
367
irregular meal pattern (Farshchi et al., 2005a).
Thus, although the advice to eat regular meals
makes sense from a conceptual point of view,
sound scientific support still needs to be gathered.
Breakfast
Much literature exists on the pros and cons of a
daily breakfast, mostly based on epidemiological
studies. People who report to consume a regular
breakfast also report a higher intake of car-
bohydrates, fat, and protein than people who
report to omit breakfast. In addition, people who
report a regular breakfast are more prone to ingest
sufficient micronutrients. Many studies indicate
that children who consume a regular breakfast
have a lower body mass index (BMI), but there
are also studies showing no relation between
breakfast consumption and BMI (for systematic
reviews, see Rampersaud et al., 2005; Ruxton and
Kirk, 1997; Szajewska and Ruszczynski, 2010).
Only few intervention studies into the effects
of breakfast consumption have been performed.
One study suggested a beneficial effect of break-
fast consumption on insulin sensitivity and
plasma levels of total cholesterol and LDL
(Farshchi et al., 2005b). Furthermore, in devel-
oping countries, school performance improves
in children who consume breakfast regularly
(Jacoby et al., 1998; Powell et al., 1998). Thus,
several clues indicate positive effects of break-
fast consumption, but there is a need for more
controlled intervention trials.
Clinical nutrition
In the preceding paragraphs, the influence of the
clock system on physiological food intake was
discussed. However, many patients with gastroin-
testinal disease need nutritional supplements via
the enteral or parenteral route of administration.
Presently, the time schedule of administration
is mainly determined by practical arguments.
368
At home, both parenteral and enteral tube feed-
ing are usually administered during the night. In
contrast, patients who are submitted to the hospi-
tal usually receive enteral nutrition during the day
and parenteral nutrition continuously over 24 h.
Below, studies investigating different time
schedules for (par)enteral nutrition will be
discussed. Only few studies have addressed the
question whether patients receiving (par)enteral
nutrition therapy benefit from a schedule that res-
embles the physiological situation of feeding dur-
ing the day and fasting during the night. Most
studies investigated the differences between con-
tinuous and cyclic administration. Importantly,
long-term intervention trials investigating differ-
ent time schedules are still lacking.
Enteral tube feeding
Two studies showed no difference in appetite sup-
pression between daytime and continuous admin-
istration of enteral nutrition (reviewed in Stratton
and Elia, 1999). Healthy volunteers provided with
enteral tube feedings for 3 days (in addition to
regular food intake) showed no difference in
appetite, satiation, or resting energy expenditure
between different time schedules of administra-
tion (daytime, nighttime, or continuously over
24 h) (Stratton et al., 2003). Cyclic nighttime
administration of enteral nutrition caused lower
oxygen consumption, lower nitrogen balance,
and lower 24-h catecholamine excretion com-
pared to continuous administration in 18 patients
who had undergone surgery for malignancy in the
buccopharynx or larynx (Campbell et al., 1990).
Patients in a vegetative state showed a shift of
the daily peak of body temperature from 8 PM
to 4 AM after nighttime compared to daytime
administration (Nishimura et al., 1992) and a sim-
ilar shift in their daily cortisol peak (Saito et al.,
1989). In the intensive care unit setting, the
head-of-bed elevation influences microaspiration
risk, with the lowest risk of aspiration with a
head-of-bed elevation of 30–45 (Kattelmann
et al., 2006; Metheny, 2006; Metheny et al.,
2006). Since most people outside the intensive
care maintain an upright position during the day,
the time schedule of enteral tube feeding may
influence risk of aspiration outside the intensive
care, but this question remains to be assessed in
clinical trials.
Thus, several clues indicate metabolic effects of
the time schedule of enteral tube feeding on
nitrogen balance, body temperature, and auto-
nomic activity. Theoretically, the time schedule
may affect aspiration risk or general well-being,
but evidence is too limited to make an evidence-
based choice for a certain schedule. For the time
being, the practical advantage of being free of
enteral tube feeding during the day will probably
guide the choice.
Parenteral nutrition
In a recent systematic review, the metabolic
effects of cyclic parenteral nutrition were com-
pared to those of continuous parenteral nutrition
(Stout and Cober, 2010). No differences were
detected in nitrogen balance, total energy expen-
diture, or the rates of carbohydrate and lipid oxi-
dation, although cyclic administration did cause
rhythms in energy expenditure and substrate oxi-
dation (increased energy expenditure and carbo-
hydrate oxidation during administration). Cyclic
administration also reduced liver enzyme elevation
compared to continuous administration, especially
in patients with low baseline bilirubin levels (Stout
and Cober, 2010). In support, animal experiments
show that cyclic administration leads to less
hepatic steatosis than continuous administration
(Lanza-Jacoby, 1986). Another, less favorable,
effect of nightly cyclic parenteral nutrition is
increased urine production at night (Boncompain-
Gerard et al., 2000).
Thus, cyclic administration of parenteral nutri-
tion mainly serves to prevent hepatic steatosis
and plasma liver enzyme elevations. In humans,
no studies have compared daytime administration

with nighttime administration. Again, practical
arguments will probably influence the choice for
a schedule of administration. Many patients pre-
fer administration at night because of the large
infusion system involved, although the increased
nighttime urine production is a disadvantage.
Disturbed rhythms and type 2 diabetes
Type 2 diabetes is considered a multifactorial dis-
ease caused by genetic factors and obesity due to
excess caloric intake and reduced physical activ-
ity. However, several lines of evidence suggest a
role of the circadian system in the pathophysiol-
ogy of type 2 diabetes. We previously discussed
how disturbed day–night rhythms can lead to obe-
sity and type 2 diabetes. Interestingly, a number
of alterations in the 24-h rhythms of patients with
type 2 diabetes have been observed.
First, the daily rhythm in glucose metabolism
seems to be different in patients with type 2 diabe-
tes. Whereas healthy individuals show a decrease
of insulin sensitivity toward the night (Jarrett
et al., 1972; Whichelow et al., 1974), insulin sensi-
tivity (as measured with a 72-h hyperglycemic
clamp) increases toward the night in patients with
type 2 diabetes (Boden et al., 1996). Also, when
patients with type 2 diabetes are fed three identi-
cal meals, morning glucose excursions are higher
than evening glucose excursions (Peter et al.,
2010), again pointing to an increased insulin sen-
sitivity (or at least increased glucose tolerance)
toward the evening. On the other hand, daily
rhythms in glucose tolerance have never been
compared between patients with type 2 diabetes
and controls in a single design.
Second, a 24-h rhythm exists in the occurrence
of myocardial infarctions in the population at
large, with an increased incidence of myocardial
infarctions in the early morning. Patients with
type 2 diabetes do not show this rhythm, which
could be due to altered rhythms in the activity
of the autonomic nervous system, circulating
hemostatic factors, and/or platelet aggregation
369
(Rana et al., 2003). The first two factors are most
likely to cause the altered rhythm in the occur-
rence of myocardial infarctions, as one study in
patients with type 2 diabetes did find a diurnal
rhythm of platelet activation with an early morn-
ing peak (Spano et al., 1993), and the same
rhythm has been found in healthy individuals
(Dalby et al., 2000; Feuring et al., 2009).
Third, rhythms of peripheral clock gene expres-
sion may be affected in type 2 diabetes as patients
with type 2 diabetes show an altered daily rhythm
of clock gene expression in circulating leukocytes
(Ando et al., 2009a). Altered adipose clock gene
expression rhythms were detected in a murine
model of type 2 diabetes (Ando et al., 2005, 2011),
but not in two different rat models (Ando et al.,
2009b; Motosugi et al., 2011). In the only human
study thus far, no differences in the rhythms of
white adipose tissue clock gene expression were
detected between lean, obese, and diabetic
individuals (Otway et al., 2011).
In summary, mismatches in day–night rhythms
can lead to obesity and type 2 diabetes, while altered
circadian rhythms are observed in type 2 diabetes.
Thus, the circadian system is likely to be involved
in the pathophysiology of type 2 diabetes. But if
so, what could be the pathophysiological mecha-
nism? Does the molecular clock get damaged in
SCN, liver, pancreas, or adipose tissue, or do pro-
blems occur in the communication between the dif-
ferent clocks? Is the circadian system a potential
target in the treatment of type 2 diabetes? Future
research will have to answer these questions.
Conclusion
In order to adjust to the daily light–dark rhythm,
humans possess a central brain clock in the
SCN. Furthermore, virtually all cells in the body
contain molecular clocks. These peripheral clocks
are synchronized both by the SCN and through
metabolic signals. Daily rhythms are present in
appetite regulation, digestion and absorption,
and carbohydrate and lipid metabolism. Thus,
370
meal patterns may strongly influence human
health. Strong clues indicate that it is better to
maintain a rhythm of daytime food intake and
nighttime sleep than to eat at night. A limited
meal frequency helps to prevent caries, but evi-
dence for additional health effects remains scarce.
Eating regular meals including a daily breakfast
appears to benefit health, but more intervention
trials are needed.
In our modern society, the synchrony between
clock gene rhythms, metabolic rhythms, and
behavioral rhythms is easily disturbed due to the
around-the-clock availability of artificial light- and
high-caloric food. This may contribute to the
increasing incidence of obesity and type 2 diabetes.
The question whether in patients with gastrointes-
tinal disease, the desynchrony due to nighttime
administration of (par)enteral nutrition disturbs
metabolism needs further investigation. Undoubt-
edly, future research will shed more light on the
fascinating interaction between the circadian
timing system, metabolism, and human health.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 21

Managing neurobehavioral capability when social

expediency trumps biological imperatives

Andrea M. Spaeth{, Namni Goel{ and David F. Dinges{,*

{
Department of Psychology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA
{
Division of Sleep and Chronobiology, Department of Psychiatry, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA

Abstract: Sleep, which is evolutionarily conserved across species, is a biological imperative that cannot
be ignored or replaced. However, the percentage of habitually sleep-restricted adults has increased in
recent decades. Extended work hours and commutes, shift work schedules, and television viewing are
particularly potent social factors that influence sleep duration. Chronic partial sleep restriction, a
product of these social expediencies, leads to the accumulation of sleep debt over time and
consequently increases sleep propensity, decreases alertness, and impairs critical aspects of cognitive
functioning. Significant interindividual variability in the neurobehavioral responses to sleep restriction
exists—this variability is stable and phenotypic—suggesting a genetic basis. Identifying vulnerability to
sleep loss is essential as many adults cannot accurately judge their level of impairment in response
to sleep restriction. Indeed, the consequences of impaired performance and the lack of insight due to
sleep loss can be catastrophic. In order to cope with the effects of social expediencies on biological
imperatives, identification of biological (including genetic) and behavioral markers of sleep loss
vulnerability as well as development of technological approaches for fatigue management are critical.

Keywords: sleep deprivation; sleep duration; neurobehavioral functions; fatigue management; individual
differences; genetics; biomarkers.

Introduction

“Sleep persists in predators and prey, carnivores

and vegetarians, on the land and in the water, in
*Corresponding author. most mammals as they lie down relaxed, in rum-
Tel.: þ1-215-898-9665; Fax: þ1-215-573-6410 inants while they stand, in birds while they perch,
E-mail: dinges@mail.med.upenn.edu

http://dx.doi.org/10.1016/B978-0-444-59427-3.00021-6 377
378
and in dolphins which constantly swim . . . in
the smartest and in the dumbest of all mammalian
species” (Rechtschaffen, 1998). Although an organ-
ism cannot procreate, gather food, or protect itself
or offspring during sleep, sleep has been evolution-
arily conserved from the sloth to the human.
Indeed, some evidence suggests that sleep is vital
to life. A number of studies from 1894 to 1962
reported lethal outcomes from total sleep depriva-
tion in dogs, rabbits, and rats (Kleitman, 1963).
However, these studies lacked proper controls for
the procedures implemented to keep the animals
awake. This methodological failure was corrected
in 1983 when Rechtschaffen and colleagues devel-
oped a novel disk-over-water methodology with
yoked control procedures (Rechtschaffen et al.,
1983). Using this paradigm, mortality was more
likely in the sleep-deprived rats (death occurred
after 5–33 days) compared with yoked control rats
that experienced the same forced ambulation but
were allowed to rest. Research using Drosophila
also showed that prolonged sleep deprivation could
be deadly (Shaw et al., 2002). The results obtained
from these more recent studies as well as earlier
studies strongly suggest prolonged sleep depriva-
tion can be lethal and can cause death even more
rapidly than food deprivation (Everson et al., 1989).
Sleeping, like eating, is a biological imperative
that consumes approximately one-third of human
life. Just as missing a day’s worth of meals pro-
duces strong feelings of hunger, being deprived of
a night’s sleep leads to overwhelming feelings of
fatigue. Sleep resists being deprived; if exhausted
enough, humans can fall asleep under even dan-
gerous circumstances (Nansen, 1999). When sleep
is denied, there is an increase in the frequency
and duration of sleep episodes and an elevation
in the intensity of sleep (Banks et al., 2010;
Borbély and Tobler, 1996; Brunner et al., 1993;
Goel et al., 2009a, 2010; Van Dongen et al.,
2003). These properties of sleep and its ubiquitous
manifestation across species indicate that sleep
must serve an adaptive function. Despite our under-
standing that sleep plays a necessary and adaptive
evolutionary purpose, the universal function of
sleep, if one exists, remains elusive (Rechtschaffen,
1998; Siegel, 2005; Tobler, 1995). Our lack of knowl-
edge regarding sleep’s fundamental function has
led a select few to assume that humans need
only a small “core” amount of sleep to function nor-
mally (Horne, 1985, 1988); however, a large body of
research indicates that most humans who undergo
chronic partial sleep deprivation exhibit profound
neurobiological and physiological changes that can
impede performance and negatively impact health
(Banks et al., 2010; Dement and Greenber, 1966;
Dinges, 2004; Goel et al., 2009a; Leproult and Van
Cauter, 2010).
Healthy human sleep
Normal human sleep comprises two states—rapid
eye movement (REM) and non-rapid eye move-
ment (NREM)—that alternate cyclically during
a sleep episode. Using electroencephalography
(EEG), characteristics of these sleep states have
been well defined (Rechtschaffen and Kales, 1968;
Silber et al., 2007). NREM sleep shows synchro-
nous cortical EEG (sleep spindles, K-complexes,
and slow waves) and is associated with low muscle
tone and minimal psychological activity whereas
REM sleep shows desynchronized EEG and is
associated with muscle atonia and dreaming
(Kryger et al., 2011). In healthy humans, the timing,
intensity, and duration of sleep are primarily
regulated by two processes: homeostatic regulation
and circadian timing (Borbély, 1982, 1998).
Sleep homeostasis
Sleep homeostasis describes the drive for sleep
that increases progressively during wakefulness
and decreases progressively during NREM sleep
(Borbély, 1994). Organisms strive to maintain
sleep at a constant level; therefore, the propensity
for sleep is increased after prolonged wakefulness
and decreased after prolonged sleep. Similarly,
sleep restriction leads to an increase in the

intensity and duration of subsequent sleep,
whereas excess sleep leads to decreased sleep inten-
sity and duration (Borbély, 1982). Researchers have
found that slow waves, low-frequency EEG, likely
represent a measure of sleep intensity—extended
waking leads to increases in slow wave energy
(SWE) during the subsequent recovery night and
the extent of this SWE increase is a function of prior
wake duration (Åkerstedt et al., 2009; Brunner
et al., 1990). In addition to SWE, other biological
markers of sleep homeostasis have been identified
including extracellular adenosine, central nitrous
oxide levels, and salivary amylase; levels of these
markers increase with prolonged sleep–wakefulness
and thus may reflect an increased sleep drive
(Kalinchuk et al., 2006; Porkka-Heiskanen and
Kalinchuk, 2011; Scharf et al., 2008; Seugnet et al.,
2006). The homeostatic process of sleep–wake regu-
lation interacts with but is independent from circa-
dian control (Dijk et al., 1989).
Sleep as a circadian process
The circadian process that controls sleep is
described as the 24-h oscillatory variation in the
propensity for sleep. This 24-h period is the time
it takes the Earth to rotate about its own axis
which generates daily environmental cycles of
ambient temperature and illumination. The alter-
nation of light and darkness directly entrains an
organism’s circadian rhythms and thus influences
its life patterns, creating species that are active
primarily during the light (diurnal), the dark (noc-
turnal), twilight periods (crepuscular), or during
the light and the dark (cathemeral) (Pittendrigh,
1981). Environmental light is transmitted from
the retina to the suprachiasmatic nuclei in the ante-
rior hypothalamus which then transmit this infor-
mation to, among others, the pineal gland via a
multisynaptic pathway; the pineal gland secretes
melatonin, a hormone that regulates various
biological functions (Czeisler, 1995; Lucas et al.,
1999; Ralph et al., 1990; Sadun et al., 1984; Watts,
1991). In humans, melatonin increases during the
379
dark cycle which coincides with a period of inactiv-
ity and sleepiness and decreases during the light
cycle which coincides with a period of activity
and wakefulness (Aschoff and Wever, 1981;
Shanahan and Czeisler, 1991). This circadian pro-
cess interacts with but is independent from sleep
homeostasis (Åkerstedt and Froberg, 1978).
Scientific evidence shows that human sleep is
naturally regulated by these two processes, with
sunrise and sunset providing the photic signals
necessary to entrain the sleep–wake cycle (Wehr
et al., 2001). However, with the introduction of
artificial light and other technologies, television
and alarm clocks have replaced these natural
signals and thereby may not allow optimal sleep
duration. Additionally, industries that operate
24 h a day create light and noise that can interfere
with sleep (Basner et al., 2011) and require
employees to work during hours usually devoted
to sleep. Finally, with the increase in international
business and air travel, many adults are fre-
quently traveling across time zones which affects
the circadian timing system. Thus, many humans
are challenging basic biological pressures in order
to accommodate social norms and obligations.
Human sleep duration
For most healthy adults, physiological sleep dura-
tion appears to range between 7.0 and 8.5 h; how-
ever, habitual sleep duration among adults is
determined by a variety of factors and shows con-
siderable variance within and between individuals
(Van Dongen et al., 2005). Data from the
2005–2008 National Health and Nutrition Exami-
nation Survey suggest that self-reported sleep
duration is distributed approximately normally
and 2004–2007 National Health Interview
Survey-Sample Adult data reveal that 7.8% of
adults report sleeping less than 5 h per night,
20.5% report sleeping 6 h per night, 30.8% report
sleeping 7 h per night, 32.5% report sleeping 8 h
per night, and 8.5% of adults report sleeping
more than 9 h per night (Krueger and Friedman,
380
2009). One limitation of large epidemiological
studies is that sleep duration is examined using
self-report which can be subject to inherent biases
and thereby inaccurate.
Indeed, when comparing self-reported sleep
duration to objectively measured sleep duration
using actigraphy (an unobtrusive measure of gross
motor activity which is analyzed to objectively iden-
tify sleep periods), subjectively reported sleep aver-
aged 0.80 h longer than objectively measured sleep
(Bradshaw et al., 2007; Lauderdale et al., 2008).
Although wrist actigraphy provides an objective
measure of inactivity, it still overestimates poly-
somnography (PSG)-measured sleep duration,
which is the gold standard for determining physio-
logical sleep. One study showed this overestimation
was approximately 18 min on average (Blackwell
et al., 2008). Therefore, although epidemiological
studies are valuable in that they include data based
on a large number of subjects, the actual amount of
physiological sleep that adults receive is typically
less than what is self-reported in these studies.
There is considerable debate as to whether or
not sleep duration has been decreasing among
adults in recent decades and if so, whether this
reduction is resulting in higher rates of chronic
sleep restriction or sleep debt (Dinges, 2004;
Horne, 2004). However, according to the Center
for Disease Control Morbidity and Mortality
Weekly Report (2008), the percentage of adults
who reported an average of less than or equal to
6 h of sleep within a 24-h period significantly
increased from 1985 to 2004 (in both females
and males and among all age groups 18–75).
Among employed American adults, two epidemi-
ological studies found that the prevalence of
being a short sleeper (either  6 h/day (Knutson
et al., 2010) or < 6 h/day (Luckhaupt et al.,
2010)) has increased significantly in the past few
decades. Studies have suggested that habitual
short sleepers do not require less sleep than other
adults; rather, these individuals gradually accrue
sleep debt over time (Aeschbach et al., 1996;
Bradshaw et al., 2007; Klerman and Dijk, 2005).
Evidence from the American Time Use Survey
indicates that adult sleep duration is significantly
shorter during weekdays compared to weekends,
suggesting that adults attempt to recover sleep
debt by extending sleep when it is presumably
more convenient and when schedules are likely
more flexible (see Fig. 1a; Basner et al., 2007).
Causes of chronic partial sleep restriction
Paid work
Compensated work time may be the most potent
determinant of sleep duration (see Fig. 1b and c;
Basner et al., 2007). There is a higher prevalence
of short sleep duration among full-time employed
adults when compared to part-time workers,
students, retired individuals, homemakers, or un-
employed adults (Knutson et al., 2010; Luckhaupt
et al., 2010), and longer work hours are associated
with shorter sleep duration (Hale, 2005;
Nakashima et al., 2010; Virtanen et al., 2009).
Adults working more than 8 h/day have the same
bedtime compared to adults who do not work
more than 8 h/day, but they wake up much earlier
(Basner and Dinges, 2009). Using a prospective
study design, Virtanen et al. (2009) found that
working more than 55 h/week is a risk factor for
the development of shortened sleep and for diffi-
culty falling asleep. Although the average number
of hours spent working has remained relatively
stable during the past few decades, the prevalence
of individuals who work greater than 48 h/week
has increased (Rones et al., 1997). The duration
of required working hours varies by occupation,
with managerial, professional, manufacturing,
and transportation industries typically involving
longer work hours; interestingly, the prevalence
of short sleep duration is also highest in these
occupations (Luckhaupt et al., 2010). By contrast,
the lowest prevalence of short sleep duration is
observed in the “agriculture, forestry, fishing,
and hunting industry” (Luckhaupt et al., 2010).
Although workers within this category (such as
farmers) often work long hours and begin work
 381

(a) early in the morning, their work schedules are

11.0 Saturday more in sync with the natural light–dark cycle
Sunday
10.5 Monday
compared to other occupations, and thus their
Tuesday work hours presumably interfere less with sleep
10.0 Wednesday quality and duration.
Thursday
Sleep time (h)

Friday In contrast to the schedules of agriculture, for-

9.5
estry, fishing, or hunting industries, shift work
9.0 schedules often require work to occur during the
night when the circadian system is promoting
8.5 sleep and require sleep to occur during the day
when the circadian system is promoting wakeful-
8.0
ness. This work schedule creates a conflict between
7.5 the worker’s internal circadian time and his/her
15–24 25–34 35–44 45–54 55–64 65–74 75+ required sleep–wake schedule, leading to impaired
Age range (years) wakefulness and disturbed sleep (Åkerstedt, 2005;
(b) Kolla and Auger, 2011). According to the Bureau
150 of Labor Statistics (2005), approximately 15% of
Adjusted change in activity time (min)

Mon – Fri full-time wage and salary earners work shifts that
100
are not during the daytime. Night workers sleep
2–4 h less per day than day workers (Åkerstedt,
50
2003) and workers either permanently on a night
0
shift or on a rotation including the night shift are
significantly more likely to sleep less than 6 h and
–50 experience excessive sleepiness during situations
requiring a high degree of attention (Ohayon
–100 et al., 2010). Although some countermeasures,
such as strategic light exposure (Burgess et al.,
–150 2002), or the use of melatonin, stimulants, and other
<4.5 <5.5 <6.5 <7.5 <8.5 <9.5 <10.5 <11.5 ³11.5

(c)
150
Adjusted change in activity time (min)

Sat + Sun on Sunday followed by Saturday and was markedly shorter

100
during the week, gradually decreasing from Monday to
Friday for most age groups. (b and c) Average change in
50
weekday (b) (Mon–Fri) and weekend (c) (SatþSun) waking
activity time depending on sleep time category (N ¼ 23,325)
0 based on multiple linear regression models adjusting for age,
Work
gender, ethnicity, educational attainment, income, presence
–50 Work (>0 min) of partner, and presence of children. The 7.5 h to <8.5 h
Socializing, relaxing, leisure w/o TV
Travel sleep time category served as a reference. For work time,
–100 Personal care w/o sleep separate models were run for the whole group (Work) and
TV
Household activities those only who worked on the interview day (Work > 0 min).
–150 Education The largest reciprocal relationship to sleep was found for
<4.5 <5.5 <6.5 <7.5 <8.5 <9.5 <10.5 <11.5 ³11.5 work time, followed by commute/travel time. Short sleepers
Sleep time (h) spent more time socializing, relaxing, and engaging in
leisure activities, while both short and long sleepers watched
Fig. 1. (a) Average sleep duration with respect to age and day more television than the average sleeper. (Reprinted with
of the week. Average sleep time across age groups was longest permission from Basner et al., 2007.)
382
pharmacological agents related to sleep–wake reg-
ulation can promote wakefulness or improve sleep
among shift workers (Kolla and Auger, 2011), cur-
rently there is no treatment to completely normal-
ize a shift worker’s sleep duration that can be
widely used in real-world situations (Åkerstedt
and Wright, 2009).
Commute/travel
According to the Census American Community
Survey Report (2011), American workers spend
nearly an hour commuting to and from work each
day and 33.5% of workers have commutes that
are greater than 30 min each way. Travel time
(composed of traveling to work [commute],
stores, schools, and social events) is negatively
associated with sleep time (see Fig. 1b and c;
Basner et al., 2007). A majority of Americans
(55.4%) leave their homes between 06:00 and
08:30 in order to arrive at work on time (Census
American Community Survey Report, 2011),
and they do not arrive home until late in the
evening in part due to traffic; the result is an
increase in the length of the workday and a
decrease in time for other activities such as soci-
alizing, relaxing, and sleeping.
Leisure
Socializing, relaxing, and engaging in other leisure
activities are also negatively related to sleep dura-
tion (see Fig. 1b and c; Basner et al., 2007). For
many adults, this time usually involves sitting in
front of some type of screen, such as a television,
computer, or smartphone. For example, watching
television was the most common activity during
the 2-h period before bedtime, suggesting that
nighttime television viewing and sleep onset are
tightly correlated (Basner and Dinges, 2009; Basner
et al., 2007). Adults are now able to access e-mail,
engage in social networking and online gaming,
and use the web 24 h a day; devices have become
so portable that they can easily be brought into
bed and used right before going to sleep. Among
adults, computer and mobile phone use in the bed-
room is associated with greater variability in
sleep–wake schedules and poorer sleep habits
(Brunborg et al., 2011). In addition to the time used
engaging in these activities, exposure to light emit-
ted from these screens may also impede sleep. Blue
(short wavelength) light represents the most potent
portion of the light spectrum for suppressing mela-
tonin and thus promoting wakefulness (Brainard
et al., 2001; Lockley et al., 2003); moreover, blue
light from light-emitting diodes elicits a dose-
dependent suppression of melatonin (West et al.,
2011). Exposure to blue light immediately prior to
bedtime (via flat screen televisions, smartphones,
and tablet computers) may cause circadian phase
delays and disrupt sleep (Cajochen et al., 2006,
2011).
Chronotype
Although humans are diurnal, some individuals
prefer activity in the morning (larks) whereas
others prefer activity in the evening (owls)—this
preference influences the timing of sleep–wake
cycles. Morning-type and evening-type individuals
differ endogenously in the circadian phase of their
biological clocks (Baehr et al., 2000; Kerkhof and
VanDongen, 1996), which is partially determined
by genetic polymorphisms (Goel, 2011). In addi-
tion to genetic factors, age, and gender also
influence morningness–eveningness (Roenneberg
et al., 2007). The Earth’s light/dark cycle and the
work schedules of industrial societies complement
individuals who function best in the morning
rather than in the evening. Owls experience
heightened alertness in the late evening and typi-
cally stay awake longer and have a delayed bed-
time compared to larks; however, due to typical
work schedules owls often use an alarm clock to
wake up early in the morning, which produces
chronic sleep restriction, extended wakefulness,
and accumulated sleep debt during the work week

(Korczak et al., 2008; Roenneberg et al., 2003;
Roepke and Duffy, 2010; Taillard et al., 2003).
Social jetlag, a term coined by Roenneberg and
colleagues (Wittmann et al., 2006), describes the
misalignment of biological and social time caused
by social activities such as work, commuting, and
television viewing. For many adults, social jetlag
and/or a preference for eveningness can lead to
the accumulation of sleep debt during the work
week due to repeated episodes of shortened
sleep. This repeated and chronic partial sleep
deprivation has been shown to have serious neu-
robehavioral and physiological consequences
unlikely to be reversed by increasing sleep time
on weekends and days off (Banks et al., 2010;
Goel et al., 2009a; Van Dongen et al., 2003).
Consequences of chronic partial sleep restriction
According to the National Transportation Safety
Board (1989, 1995), 30–40% of all U.S. truck
accidents are fatigue related and several
catastrophes including the Exxon Valdez accident,
Chernobyl and Three Mile Island nuclear plant
meltdowns, and Space Shuttle Challenger tragedy
were partially due to human error resulting from
sleepiness and fatigue (Mitler et al., 1988). Sleepi-
ness due to long work hours has also been
implicated in medical errors: interns, surgeons,
physicians, and residents make significantly more
errors, including those that are injurious or fatal,
after working for an extended period of time
and/or after chronic and acute sleep loss (Czeisler,
2009; Lockley et al., 2004). As such, gaining a
greater understanding of the relationship between
sleep and waking performance is an important
focus of ongoing research.
Regulation of human performance
Similar to the regulation of sleep duration and inten-
sity, distinct sleep/wake-related physiological pro-
cesses regulate alertness and neurobehavioral
383
performance. The homeostatic sleep-dependent
process (process S) balances sleep propensity by
keeping track of recent sleep history (Daan et al.,
1984). As hours of wakefulness increase, homeo-
static drive increases the propensity for sleep. The
endogenous circadian process (process C) tracks
changes in light exposure (as well as other zeitgebers
or synchronizers) and entrains sleep propensity to
the light–dark cycle; when sleep propensity is
increased (during the night) waking performance is
often degraded (Czeisler et al., 1999). Sleep inertia,
a transient process that occurs immediately after
awakening, causes deficits in waking performance;
its effects depend on prior sleep history, circadian
phase, and the depth of sleep at the time of awaken-
ing (Dinges, 1990). Finally, a second homeostatic
process (process U) influences waking performance
by monitoring sleep–wake on a longer time scale
(nightly sleep duration) and interacting with the
other processes (McCauley et al., 2009). Process U
builds up over several days of prolonged wakeful-
ness, when sleep debt is accruing and the need for
sleep is increasing, and dissipates during sleep. The
status of process U, similar to a sleep reservoir being
full or empty, codetermines the rate by which the
original homeostatic process (process S) increases
sleep propensity and thus decreases waking perfor-
mance (McCauley et al., 2009).
All of these processes interact to form a
dynamic relationship between sleep propensity
and waking performance (Raslear et al., 2011;
Van Dongen and Dinges, 2005). During a 24-h
period, the interaction of these processes leads
to a period in the early morning when waking
performance is particularly vulnerable to sleep
restriction; neurobehavioral deficits are largest at
08:00 and become progressively smaller during
the day, especially between 16:00 and 20:00
(Mollicone et al., 2010). The incidence of single-
vehicle automobile accidents, human error, and
accidents related to work performance and sev-
eral industrial/engineering disasters also reveal
early morning hours as particularly vulnerable to
sleep restriction (Mitler et al., 1988). However,
when an individual lacks a sufficient amount of
384
sleep, either as a result of acute total sleep depri-
vation or chronic partial sleep restriction, homeo-
static pressure can increase to the point whereby
waking cognitive functions will be degraded even
during periods of time when the circadian drive
for wakefulness peaks (Doran et al., 2001).
Sleep propensity
Individuals who report sleeping less than 7.5 h/
day on average are more likely to exhibit a
stronger sleep propensity (the tendency to fall
asleep)—they report unintentionally falling
asleep during the day and nodding off or falling
asleep while driving (McKnight-Eily et al., 2011;
Punjabi et al., 2003). In the laboratory, sleep
propensity—operationalized as the speed of falling
asleep in both sleep-conducive and non-conducive
conditions—is among the most well-validated
measures of sleepiness (Roehrs et al., 2005). The
effects of chronic sleep restriction on daytime
physiological sleep propensity have been evaluated
using the Multiple Sleep Latency Test (MSLT)
(Carskadon and Dement, 1981) and the Mainte-
nance of Wakefulness Test (MWT) (Mitler et al.,
1982). During the MSLT, the subject is instructed
to close his/her eyes and try to fall asleep while
lying in a supine position, whereas during the
MWT, the subject is instructed to look straight
ahead and try to stay awake while seated upright.
In both tests, PSG recordings are made (including
EEG, electrooculogram and electromyogram) and
the time taken to fall asleep is a measure of sleep
propensity.
Whether attempting to fall asleep or resist
sleep, the latency from waking to sleeping is
reduced by chronic partial sleep restriction (see
Fig. 2a; Banks et al., 2010; Belenky et al., 2003;
Carskadon and Dement, 1981; Dinges et al.,
1997; Guilleminault et al., 2003; Rupp et al.,
2009). As the number of days of sleep restriction
increases or the amount of sleep available during
each night of sleep restriction decreases, the
latency to fall asleep decreases (Belenky et al.,
2003; Carskadon and Dement, 1981; Devoto
et al., 1999; Guilleminault et al., 2003; Rupp
et al., 2009). Spending 10 h in bed daily for 1
week prior to experiencing a week of sleep
restriction may attenuate the effects of sleep
restriction on sleep propensity as measured by
MWT (Rupp et al., 2009). By contrast, after
experiencing partial sleep restriction, one recov-
ery night of up to 10 h time in bed (TIB) is insuf-
ficient to return sleep propensity back to presleep
restriction levels (see Fig. 2b; Banks et al., 2010).
MWT sleep latencies appear normalized after two
8 h TIB nights of recovery sleep (Rupp et al.,
2009); however, due to the small sample size used
in this study, replication of this finding is needed.
Behavioral alertness: Microsleeps and wake-state
instability
The increased propensity for sleep due to
restricted sleep time can lead to the occurrence
of “microsleeps,” very brief sleep episodes that
intrude into wakefulness despite an individual’s
best effort to stay awake (Åkerstedt et al., 1987;
Bjerner, 1949; Torsvall and Åkerstedt, 1987).
Wake-state instability refers to the moment-to-
moment shifts in the neurobiological systems
mediating the motivated desire to sustain waking
alertness and those mediating the involuntary
homeostatic drive to fall asleep (Doran et al.,
2001; Saper et al., 2005). This type of interaction
between drives results in unpredictable behavior,
including increased variability in cognitive perfor-
mance and lapsing (i.e., brief periods of half a sec-
ond to many seconds of no response) (Doran
et al., 2001). Although individuals may not realize
they are experiencing microsleeps and perfor-
mance decrements, over time this instability can
progress into full blown sleep attacks—when
individuals will not spontaneously wake without
additional stimulation (Dinges and Kribbs, 1991;
Harrison and Horne, 1996; Kleitman, 1963).
Sleep restriction can increase the incidence of
microsleeps and produce decreased behavioral
 385

(a) (b)
0 0

5
MWT (min)

10

MWT (min)
10

20 15

20
30
25

(c) (d)
12 25

10 20
8

PVT lapses
PVT lapses

15
6
10
4

2 5

0 0
B2 SR1 SR2 SR3 SR4 SR5 0h 2h 4h 6h 8h 10 h
Protocol night Recovery night TIB

Fig. 2. (a and c) Daily means (SEM) of (a) MWT sleep onset latency and (c) PVT performance in the sleep restriction group
(n ¼ 142, 4 h TIB for five nights [SR1–SR5], solid line), and the control group (n ¼ 17, 10 h TIB on all nights, dashed line). All
subjects had 10 h TIB (22:00–08:00) on baseline day 2 (B2). Sleep restriction on SR1 to SR5 was from 04:00 to 08:00. Data are
plotted to show deficits in neurobehavioral functions increasing (upward) on the ordinate. Relative to the control condition,
sleep restriction degraded neurobehavioral function over days (decreased MWT sleep onset latency and increased PVT lapses
[>500 ms reaction times]). (b and d) Daily means (SEM) of (b) MWT sleep onset latency and (d) PVT performance following
the acute recovery (REC) night as a function of increasing TIB dose (0 –10 h, solid line), controlling for covariates (i.e., baseline,
cumulative deficits during sleep restriction, age, and sex). Sleep-restricted subjects (n ¼ 142) were randomized to either 0 h TIB
(n ¼ 13), 2 h TIB (n ¼ 27), 4 h TIB (n ¼ 29), 6 h TIB (n ¼ 25), 8 h TIB (n ¼ 21), or 10 h TIB (n ¼ 27). For comparison, horizontal
dotted lines show baseline night (B2, 10 h TIB) values, and horizontal dashed lines show control group (n ¼ 17) means on day
8 (10 h TIB), which is the study day equivalent to REC. These neurobehavioral outcomes showed improvement as recovery sleep
doses increased (MWT sleep onset latencies increased and PVT lapses decreased); however, even at the highest sleep recovery
dose, functioning did not return to baseline or control condition levels. (Reprinted with permission from Banks et al., 2010.)

alertness, even during goal-directed behaviors
(e.g., motor vehicle operation). The rapid intrusion
of sleep into wakefulness and consequent wake-
state instability can have profound adverse con-
sequences, especially if experienced by those in
safety-sensitive occupations, such as police officers,
firefighters, health care providers, and motor vehi-
cle operators (Barger et al., 2009; Lombardi et al.,
2010; Trew et al., 2011). Unfortunately, workers in
these professions are often subjected to chronic
sleep restriction due to shift work schedules and
long work hours and are thus particularly vulnera-
ble to microsleeps and wake-state instability.
Behavioral alertness, measured using sustained
attention tasks in the laboratory, has been shown
to be sensitive to sleep restriction. According to
386
the state instability hypothesis, sleep drive
escalates instability in attention which creates
increasingly variable neurobehavioral perfor-
mance (periods of accurate responding are inter-
rupted by errors of omission [lapses] comingled
with errors of commission) (Doran et al., 2001).
Studies support this hypothesis; cognitive perfor-
mance variability is influenced by prior wakeful-
ness, circadian phase, and the amount of time
spent on the task (Doran et al., 2001; Graw
et al., 2004; Mollicone et al., 2010; Zhou et al.,
2011b). When sleep is chronically restricted, sleep
debt accumulates across each night leading to
greater impairment over time (Banks et al.,
2010; Belenky et al., 2003; Dinges et al., 1997;
Goel et al., 2009a; Rupp et al., 2009; Van Dongen
et al., 2003; Wu et al., 2010). Thus, as the need for
sleep increases, the brain’s ability to maintain
alertness becomes progressively more over-
whelmed by the activation of sleep processes
which leads to microsleeps and consequent neuro-
behavioral instability (Doran et al., 2001). Even
when highly motivated, an individual’s attempt
to compensate for excessive sleepiness by
engaging in various behaviors ultimately fails to
prevent intrusions of microsleeps and impaired
neurobehavioral performance (Horne and Pettitt,
1985; Hsieh et al., 2010).
Driving performance
Driving a vehicle requires sustained attention and
is sensitive to the reduced alertness associated
with sleepiness. Every year, thousands of automo-
bile crashes, injuries, and fatalities are due to
drivers falling asleep (Strohl et al., 1997). Adults
who are employed, work more than 60 h/week,
work irregular hours, or work at night, as well as
those who are sleep deprived or chronically sleep
restricted are more likely to have a car accident
(Abe et al., 2010; Åkerstedt et al., 2005; Brown,
1994; Gander et al., 2005; Horne and Reyner,
1999; Philip et al., 2003, 2005; Scott et al., 2007;
Stutts et al., 2003). In the laboratory, studies have
primarily focused on short-term sleep restriction
and have found that driving performance on
simulators decreases (resulting in more crashes)
after short sleep duration (4–6 h TIB) (De Valck
and Cluydts, 2003; Lenne et al., 2004; Macchi
et al., 2002; Otmani et al., 2005; Vakulin et al.,
2007). Similarly, the less sleep obtained during
chronic partial sleep restriction, the more driving
performance (increase in number of driving simu-
lator accidents) is impaired (Russo et al., 2003).
Cognitive performance
In addition to its negative effects on alertness, sleep
deprivation degrades aspects of cognitive perfor-
mance (Harrison and Horne, 2000; Kleitman,
1963; McCoy and Strecker, 2011). There are
hundreds of published studies on the effects of total
sleep deprivation on cognitive performance (Alhola
and Polo-Kantola, 2007; Goel et al., 2009b; Lim
and Dinges, 2010), but far fewer studies on the
cognitive-impairing effects of chronic partial sleep
restriction.
Sleep deprivation induces a wide range of
effects on cognitive functions (Killgore, 2010;
Poe et al., 2010); however, cognitive tasks vary
considerably in their sensitivity to sleep loss.
In general, regardless of the task, cognitive per-
formance becomes progressively worse when time
on task is extended; this is the classic “fatigue”
effect that is exacerbated by sleep loss (Bjerner,
1949; Wilkinson, 1969). However, performance
on brief cognitive tasks that measure speed of
cognitive “throughput,” working memory, and
other aspects of attention are also sensitive to sleep
deprivation (Dinges, 1992). Two confounding
factors that can obscure the effects of sleep loss on
many cognitive tasks are intersubject variability
and intrasubject variability (Dorrian et al., 2005).
The intersubject confound describes differences in
aptitude: one individual’s poorest performance
during sleep deprivation may be superior to the
best performance of a non-sleep-deprived individ-
ual (Goel et al., 2009b). The intrasubject confound

describes a learning effect: a person may be cogni-
tively diminished by sleep loss, but continue to
improve on a repeated task due to the effects of
learning (Goel et al., 2009b). The nature of the
dependent variables selected for examining the
cognitive effects of sleep deprivation can also be
problematic. Sleep deprivation increases variability
within subjects (i.e., state instability) and between
subjects (i.e., differential vulnerability to the effects
of sleep deprivation); therefore, the effects of sleep
loss on cognitive measures may be missed due to
insensitive metrics or data analyses (Olofsen et al.,
2004; Van Dongen et al., 2004a; Whitney and
Hinson, 2010). To provide an accurate and useful
measure of performance during sleep loss as well
as the dynamic expression of waking neuro-
behavioral integrity as it changes over time, cogni-
tive assessments must be valid and reliable
reflections of fundamental waking functions altered
by sleep deprivation (Goel et al., 2009b). As such,
measures of attention, vigilance, and declarative
memory are often used, with reaction time as the
dependent variable.
The Psychomotor Vigilance Test (PVT), a mea-
sure of sustained attention, is free of aptitude and
learning effects and is widely used in sleep studies
due to its sensitivity to sleep loss, sleep pathology,
and functioning at an adverse circadian phase
(Balkin et al., 2004; Dorrian et al., 2005; Lim
and Dinges, 2008). The primary outcome measures
of the PVT are reaction time and errors, includ-
ing omissions (lapses) and commissions (false
responses). In 2003, two large-scale experimental
studies found that when sleep is reduced to 3, 4,
5, or 6 h TIB for several nights, sustained attention
performance (PVT) as well as working memory
and cognitive throughput (Serial Addition/Sub-
traction Task, Digit Symbol Substitution Task)
decreases in a dose-dependent manner (see Fig. 2c;
Belenky et al., 2003; Van Dongen et al., 2003).
As sleep debt accumulates across days, perfor-
mance becomes progressively worse over time;
14 days of chronic sleep restriction (4 or 6 h TIB
each night) produced comparable cognitive deficits
to those produced by 24–48 h of total sleep
387
deprivation (Van Dongen et al., 2003). This
finding has been replicated several times (Axelsson
et al., 2008; Banks et al., 2010; Bliese et al., 2006;
Cote et al., 2008; Dinges et al., 1997; Fafrowicz
et al., 2010; Goel et al., 2009a; Rupp et al., 2009;
Wu et al., 2010).
Research has also shown that the recovery rate
from chronic sleep restriction may be slower than
from acute total sleep loss (Banks et al., 2010;
Rupp et al., 2009). In a large-scale experimental
study examining dose–response effects of one
night of recovery sleep after five nights of sleep
restriction on PVT performance, recovery to
either a subject’s own baseline values or values
recorded for the sleep-satiated control group
was not achieved at any of the doses examined,
including the highest dose of 10 h TIB (see Fig. 2d;
Banks et al., 2010). Due to circadian limitations
on sleep duration, it is unlikely that recovery from
5 days of chronic sleep restriction can occur in
one night of more than 10 h TIB; therefore, resid-
ual attentional deficits still present after one night
of recovery may potentiate the effects of a
subsequent sleep restriction period (Banks et al.,
2010). Thus, attempting to recover lost sleep from
a work week by extending sleep on a weekend
night is likely insufficient for recuperating impaired
alertness and sustained attention. Other studies
examining recovery after chronic sleep restriction
have found that PVT performance remains sub-
standard even after 3, 5, or 7 recovery nights of
8 h TIB (Axelsson et al., 2008; Belenky et al.,
2003; Rupp et al., 2009). However, similar to results
for sleep propensity, prophylactic napping prior to
sleep deprivation significantly improved reaction
time performance and spending 10 h TIB daily for
1 week prior to experiencing chronic partial sleep
restriction attenuated the decrement in PVT per-
formance during sleep loss and facilitated improve-
ment of PVT performance during recovery (Dinges
et al., 1987; Rupp et al., 2009). Thus, chronic sleep
restriction induces slow changes in neural processes
mediating alertness and attention that cause perfor-
mance to become progressively worse over time,
producing the accumulation of severe performance
388
decrements. Similarly, the slow recovery rate from
chronic sleep restriction suggests that this type of
sleep loss may induce long-term neuromodulatory
changes in brain physiology (Banks et al., 2010;
Rupp et al., 2009).
Researchers have proposed that attention and
working memory, considered basic cognitive
actions, are essential to virtually all other cogni-
tive processes; therefore, sufficient attention and
memory performance are necessary for optimal
functioning of neural circuits that mediate higher
cognitive functions, such as executive function
(Balkin et al., 2008). Interestingly, there is a posi-
tive relationship between the level of sustained
attention required to perform a task and the
degree to which task performance is impaired by
sleep loss (Jennings et al., 2003). Although there
is evidence that sleep deprivation adversely
affects prefrontal cortex-related executive atten-
tion and working memory abilities, these cogni-
tive effects are often not as prominent or easy to
measure as those involving basic processes such
as cognitive and psychomotor speed (Goel et al.,
2009b). Therefore, more complex cognitive tasks
involving higher cognitive functions have been
regarded as insensitive to sleep deprivation by
some researchers (Harrison and Horne, 2000).
Sleep restriction affects mental flexibility,
attention shifting, and the inhibition of automatic
responses, all reflective of executive function
(McCoy and Strecker, 2011). Executive function
can be defined as “the ability to plan and coordi-
nate a willful action in the face of alternatives, to
monitor and update action as necessary and sup-
press distracting material by focusing attention on
the task at hand” (Jones and Harrison, 2001).
Many tasks believed to engage different aspects
of executive function have been used in sleep dep-
rivation studies. Examples of such tasks include
the Brixton Spatial Anticipation Test, Controlled
Oral Word Association Test (COWAT), Hayling
Sentence Completion Task, Torrance Tests of Cre-
ative Thinking, Tower of London, Thurstone’s
Verbal Learning Task, and Wisconsin Card
Sorting Task (Harrison and Horne, 2000; Jones
and Harrison, 2001). In experimental studies,
individuals who are sleep restricted exhibit deficits
on the Wisconsin Card Sorting Task (Herscovitch
et al., 1980) and Stroop test (Stenuit and Kerkhofs,
2008) but do not exhibit deficits on the Hayling,
Brixton, COWAT, or Tower of London Tasks
(Goel et al., 2009a). Although subjects are able
to accurately perform many cognitive assessments,
compensatory mechanisms become engaged in
response to chronic sleep restriction which
impedes the ability to think flexibly and increases
the use of automatic processing, impulsivity, and
rigid rule adherence to complete tasks (Stenuit
and Kerkhofs, 2008; Swann et al., 2006).
Plessow and colleagues studied new parents, a
population of healthy adults who are often sub-
jected to chronic sleep restriction for several months
due to nightly feedings and duties related to caring
for a new infant. Parents were administered an
explicit-cueing version of the task-switching para-
digm to assess their ability to flexibly adapt to
changing environmental demands. Sleep-deprived
new parents exhibited significantly slower reaction
times during task switches compared to new parents
reporting adequate sleep; correlational analyses
showed that greater self-reported sleep debt was
associated with greater impairment on the task
(Plessow et al., 2010). These results indicate that
chronic sleep restriction can impair an individual’s
ability to implement task goals in order to switch
tasks in a fast-changing environment—factors
important for doctors, pilots, and individuals in the
armed forces, who constantly rely on fast and flexi-
ble goal shifting and are likely to experience chronic
sleep restriction.
Interindividual differences in response to sleep
restriction
As stated previously, average sleep duration is
normally distributed and widely differs depending
on the individual (Van Dongen et al., 2005).
In addition, although humans are diurnal
mammals, there are individual differences in the
 389

timing of their behaviors; some prefer to be active
in the morning whereas others prefer to be active
in the late evening (Kleitman, 1963; Roenneberg
et al., 2003). Similarly, studies of total sleep depri-
vation as well as chronic sleep restriction have
reported substantial interindividual differences in
the magnitude of sensitivity/resilience to the
effects of sleep loss on neurobehavioral functions
(see Fig. 3a; Goel et al., 2009a, 2010; Van Dongen
et al., 2004b). Prior sleep history and endogenous
circadian rhythms likely play an important role in
an individual’s response to sleep loss; however, lab-
oratory studies that carefully control for these
factors still find marked individual differences in
neurobehavioral responses to sleep loss (Leproult
et al., 2003; Van Dongen et al., 2004b). In the stud-
ies described earlier, when sleep duration was
limited to less than 7 h per night for several consec-
utive nights, performance on sustained attention,
working memory, and cognitive throughput was
significantly impaired; however, not all subjects
were affected to the same degree (Axelsson et al.,
2008; Belenky et al., 2003; Bliese et al., 2006; Goel
et al., 2009a, 2010; Van Dongen et al., 2003).
Individuals are highly vulnerable, somewhat vul-
nerable, or highly resistant to the neurobehavioral
effects of sleep restriction (Goel and Dinges,
2011). It remains unknown whether the same
individuals are vulnerable to the adverse effects of
both acute total sleep deprivation and chronic sleep
restriction.
A few studies have found that these interindivid-
ual differences may be task dependent; one
individual’s sustained attention may be most
affected by sleep loss whereas another individual’s
working memory may be most affected (Frey
et al., 2004; Van Dongen et al., 2004b). In addition,
interindividual sensitivity to sleep loss is trait like;
when neurobehavioral performance was assessed
repeatedly (during separate laboratory visits), the
same individuals remained particularly vulnerable
or resistant to the effects of sleep loss even when
previously sleep satiated or sleep restricted (Van
Dongen et al., 2004b). Finally, these stable interin-
dividual differences account for a substantial
proportion of variance in cognitive performance
decrements induced by sleep loss. Demographic
factors (age, sex, IQ), baseline functioning, various

(a) (b)
13 7
Mean KSS sleepiness score

11 6
Mean PVT lapses

9
5
7
4
5

3 3

1 2
B SR1 SR2 SR3 SR4 SR5 B SR1 SR2 SR3 SR4 SR5
Day Day

Fig. 3. Neurobehavioral performance at baseline and during chronic partial sleep deprivation for the PER3 genotype groups. Mean
(SEM) (a) PVT lapses (> 500 ms reaction times) per trial and (b) Karolinska Sleepiness Scale (KSS) scores at baseline (b) for
each partial sleep restriction night (SR1–SR5) for PER34/4 (open circles), PER34/5 (gray triangles), and PER35/5 (closed circles)
subjects. All genotypes demonstrated large but equivalent cumulative decreases in cognitive performance (PVT) and increases in
sleepiness (KSS) across sleep restriction nights. As illustrated by increasing standard error bars in (a), interindividual differences
in PVT lapses increased across sleep restriction nights in the entire study group (from Goel et al., 2009a).
390
aspects of habitual sleep timing, and circadian
chronotype have not accounted for the robust and
stable differences in neurobehavioral responses
to sleep restriction (Doran et al., 2001; Goel and
Dinges, 2011; Van Dongen and Dinges, 2003; Van
Dongen et al., 2004b).
Due to the trait like, or phenotypic, nature of these
interindividual differences, research has focused on
understanding which genetic polymorphisms may
underlie vulnerability to sleep loss. Several genetic
mechanisms have been examined in relation to the
effects of chronic sleep restriction including the
PERIOD3 (PER3) variable number tandem repeat
(VNTR) polymorphism and the DQB1*0602 allele.
Although the VNTR polymorphism of the circadian
gene PER3 (PER34/4, PER34/5, and PER35/5) has
been shown to influence executive function deficits
in response to total sleep loss (Groeger et al., 2008;
Vandewalle et al., 2009; Viola et al., 2007), it does
not appear to influence neurobehavioral effects of
chronic sleep restriction. A large-scale experimental
study examining individuals with different PER3
genotypes found that PER34/4, PER34/5, and
PER35/5 genotypes exhibited similar baseline per-
formance on neurobehavioral assessments and
demonstrated large but equivalent cumulative
increases in sleepiness and sleep propensity and
cumulative decreases in cognitive performance and
physiologic alertness across five nights of sleep
restricted to 4 h per night (see Fig. 3; Goel et al.,
2009a). Daily intersubject variability also increased
across sleep restriction days similarly in all genotypes
(Goel et al., 2009a). Although PER3 genotypes did
not differ at baseline in habitual sleep, physiological
sleep structure, or sleepiness, during sleep restriction,
PER35/5 subjects had elevated sleep homeostatic
pressure, measured physiologically by EEG SWE
during NREM compared with PER34/4 subjects
(Goel et al., 2009a). Goel and colleagues also exam-
ined the human leukocyte antigen DQB1*0602 allele
which is closely associated with narcolepsy. Subjects
who were either positive or negative for the
DQB1*0602 allele differed in levels of sleep homeo-
static pressure, baseline sleepiness, and sleep physi-
ology but showed comparable cumulative decreases
in cognitive performance and increases in sleepiness
in response to chronic sleep restriction (Goel et al.,
2010). Additional investigations are underway to
identify other genetic polymorphisms that may medi-
ate an individual’s neurobehavioral vulnerability to
sleep restriction and acute total sleep deprivation
(Goel, 2011).
Identifying correlates of interindividual differ-
ences in neurobehavioral vulnerability to chronic
sleep restriction, such as biomarkers (including
genetic polymorphisms as well as adenosine and
salivary amylase levels) and behavioral predictors,
will provide a viable means to determine those
individuals in the general population who need lon-
ger habitual sleep durations and/or who require
effective interventions and countermeasures for
unavoidable sleep loss (Goel and Dinges, 2011). This
is a particularly important area of research since
adults are not adept at accurately judging how
affected they are by chronic sleep restriction (see
Figs. 3 and 4; Banks and Dinges, 2007; Van Dongen
et al., 2003; Zhou et al., 2011b). Subjective sleepiness
is measured using two widely used and well-
validated questionnaires: the Stanford Sleepiness
Scale (SSS) which requires subjects to rate how
sleepy they feel on a scale of 1–7 (Hoddes et al.,
1973) and the Karolinska Sleepiness Scale (KSS)
which requires subjects to rate how sleepy they feel
on a scale of 1–9 (Åkerstedt and Gillberg, 1990).
Although subjects are able to detect the rapid and
severe changes in levels of alertness and sleepiness
in response to total sleep deprivation, subjects are
much less sensitive in detecting the smaller changes
in levels of alertness and sleepiness that accumulate
during each day with sleep restriction (see Figs. 3
and 4; Banks and Dinges, 2007; Goel et al., 2009a;
Van Dongen et al., 2003; Zhou et al., 2011a). After
a week (or two) of sleep restriction, subjects show
marked cognitive impairment and severe decreases
in alertness but rate themselves as only moderately
sleepy (Belenky et al., 2003; Van Dongen et al.,
2003). Consequently, a subject’s rating of subjective
sleepiness does not accurately parallel the
continuing accumulation of cognitive performance
deficits associated with sleep loss. This finding
 391

Objective Subjective

PVT performance lapses SSS rating

15.0 4h TIB 3.0

10.0 2.0
4h TIB
6h TIB
6h TIB
5.0 1.0

8h TIB
0.0 0.0 8h TIB

BL 2 4 6 8 10 12 14 BL 2 4 6 8 10 12 14
Days of sleep restriction Days of sleep restriction

Fig. 4. Restriction of nocturnal sleep in healthy adults resulted in near-linear increases in PVT lapses (>500 ms reaction times) of
attention across 14 days (coefficients of change near 1.0), but subjective ratings of sleepiness, measured using the Stanford
Sleepiness Scale (SSS), showed a nonlinear coefficient below 0.5 for change over days. As objective performance continued to
decline near-linearly, there were only minor further increases in the subjective ratings of sleepiness. By the end of the 14 days of
sleep restriction, when PVT performance was at its worst levels, subjects in the 4 and 6-h sleep period conditions reported
feeling only slightly sleepy. Therefore, unlike performance measures, sleepiness ratings appeared to show adaptation to sleep
restriction (N ¼ 35 subjects; 8 h condition n ¼ 9, 6 h condition n ¼ 13, and 4 h condition n ¼ 13). (Reprinted with permission from
Van Dongen et al., 2003.)

suggests that people who are chronically sleep
restricted underestimate the impact of sleep restric-
tion and may overestimate their levels of alertness
and ability to perform various cognitive tasks
(Banks and Dinges, 2007; Czeisler, 2009; Van
Dongen et al., 2003; Zhou et al., 2011a).
Management of sleep restriction-induced
neurobehavioral impairment
Due to the high prevalence of chronic partial sleep
restriction in industrialized societies, the severe
consequences of sleep loss on alertness and cogni-
tive function, and our inability to accurately moni-
tor our own sensitivity to chronic sleep loss,
objective actions need to be employed to manage
sleep debt. Currently, fatigue management within
transportation, health professions, and other occu-
pational settings are primarily based on policy; a
regulatory organization imposes limits to daily/
weekly shift duration as well as the number of
consecutive shifts or rest hours (Czeisler, 2009;
Gander et al., 2011). Regulatory organizations
may also screen potential employers for sleep dis-
orders when occupational duties pose safety risks
(Czeisler, 2009; Gander et al., 2011). Although
these policies can improve workplace safety and
productivity, many regulations are not strictly
enforced or adhered to and it is nearly impossible
for an employer to ensure that workers are actu-
ally obtaining an appropriate amount of sleep each
night (Czeisler, 2009; Gander et al., 2011).
Balkin et al. (2011) summarized various tech-
nological approaches to fatigue management that
accurately predict and/or objectively monitor
sleepiness and fatigue effects in real time and that
allow for optimally timed administration of inter-
ventions or countermeasures (such as the use of
properly dosed pharmaceuticals or napping).
392

When discussing the ideal monitoring system, the sleep loss. These effects can have serious adverse
authors state that technological approaches consequences including fatal medical errors, cata-
should be valid, reliable, sensitive, specific, and gen- strophic oil spills, and vehicle crashes. Finding
eralizable. In order to adhere to these criteria, the biological and behavioral markers of sleep loss
approach would have (a) the ability to predict vulnerability and developing technological
fatigue, based on the factors that produce it (sleep approaches to fatigue management are two
history, hours of wakefulness, circadian rhythms, domains of research essential for offsetting the
time of day, and biomarkers or behavioral correlates negative effects of societal factors on the
of sensitivity to sleep loss); (b) the ability to measure biological need for sleep.
and monitor fatigue/performance online in the oper-
ational environment in order to detect downward
trends in alertness or performance before such Acknowledgments
trends reach the threshold at which operational
performance is actually impacted; and (c) the ability Supported by NIH NR004281; the National Space
to effectively intervene when potential deficits Biomedical Research Institute through NASA
are identified or anticipated, with interventions NCC 9-58; and the Department of the Navy, Office
calibrated so as to restore and sustain alertness/per- of Naval Research (Award No. N00014-11-1-0361).
formance as long as needed (e.g., until the operator
returns home after the work shift and can safely
obtain adequate, recuperative sleep) (Balkin et al., Abbreviations
2011). Examples of instruments currently being
examined for such use include EEG measurement COWAT Controlled Oral Word Association
of brain wave activity, ocular measures, video-based Test
monitoring systems and portable assessments of EEG electroencephalography
attention, and reaction time. Fatigue detection and KSS Karolinska Sleepiness Scale
prediction instruments, implemented in addition to MSLT Multiple Sleep Latency Test
fatigue-related regulations, will help ensure work- MWT Maintenance of Wakefulness Test
place and road safety and adequate levels of alert- NREM non-rapid eye movement
ness in workers while on duty (Balkin et al., 2011). PSG polysomnography
PVT Psychomotor Vigilance Test
Conclusion REM rapid eye movement
SSS Stanford Sleepiness Scale
Despite being treated as a commodity that can be SWE slow wave energy
traded for other activities, sleep is essential for TIB time in bed
optimal neurobehavioral functioning. The 24/7
schedule of many occupations in current indus-
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ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 22

Noisy and individual, but doable: Shift-work

research in humans

Thomas Kantermann{,1, Sophie M. T. Wehrens{, Melissa A. Ulhôa{, Claudia Moreno{

and Debra J. Skene{,*

{
Centre for Chronobiology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey,
United Kingdom
{
University of São Paulo, School of Public Health, São Paulo, Brazil

Abstract: Working around the clock is common for many occupations, as diverse as nurses, truck
drivers, physicians, steel workers, and pilots. Each shift-work profession is individual in more aspects
than just work hours and individual work scenarios, each posing a different impact on the health of
workers. Related health problems in shift workers, therefore, are also diverse and encompass sleep
problems, metabolic and cardiovascular system disturbances, as well as cancer. Little is known about
how all these individual factors influence a shift worker’s health status, partly because many shift-work
studies show inconsistent results. In addition, these individual factors create many methodological
difficulties for researchers who investigate such work scenarios. This chapter presents examples from
our laboratory and field studies of shift workers, which emphasize the importance of taking individual
circumstances into account. Both study approaches, laboratory and field based, are needed to fully
account for the difficulties that shift-work studies pose on both workers and researchers. Finally,
understanding the mechanisms that underpin interindividual differences in response to shift work will
advance our understanding of how to design better and healthier shift-work schedules in the future.

Keywords: chronotype; social jetlag; stress; sleep; health; shift work; cardiovascular system.

*Corresponding author. Introduction

Tel.: þ44 1483 689706; Fax: þ44 1483 686401
E-mail: d.skene@surrey.ac.uk
1
Current address: University of Groningen, Chronobiology
Research Unit, Life Science building, Nijenborgh 7, 9747AG
Groningen, The Netherlands
Studying shift workers means studying individuals
at a variety of workplaces employed in different
and often flexible working time arrangements.
Studying the effects of shift work on health is thus
complex and poses challenges to researchers to

http://dx.doi.org/10.1016/B978-0-444-59427-3.00022-8 399
400
control the many individual factors—economic,
social, and biological—that characterize modern
shift-work settings. Each of these individual factors
by themselves and in combination may negatively
affect health (Kantermann et al., 2010; Knutsson,
2004; Saksvik et al., 2011). Therefore, the effects of
shift work on health are very diverse (Costa,
2003b; Kantermann, 2008; Sookoian et al., 2007;
Wang et al., 2011), and studies on shift work and
health are often inconsistent in their findings.
Related health problems in shift workers broadly
fall into four categories: (i) sleep problems
(Åkerstedt and Wright, 2009; Åkerstedt et al.,
2007; Folkard et al., 2005; Lemos et al., 2009;
Moreno et al., 2004), (ii) metabolic problems
(Esquirol et al., 2009; Sookoian et al., 2007; Wang
et al., 2011), (iii) cardiovascular system disturbances
(Boggild and Knutsson, 1999; Hublin et al., 2010;
Puttonen et al., 2010), and (iv) cancer (Erren et al.,
2008; Stevens, 2009). Notably, none of the reported
health problems constitute an occupational disease
unique to shift workers. By contrast, all of these
health problems belong to a group of “normal” epi-
demiological and age-related health problems.
Interestingly, many of these epidemiological health
problems occur more frequently and at an earlier
age in shift workers than in the normal non-shift-
working population. To date, however, no study
has tested the hypothesis that exposure to shift work
is associated with advanced aging. Obviously, such a
broad range of health problems affects a worker’s
quality-of-life and life expectancy, which makes
shift-work studies highly relevant not only for the
health care system but also for society as a whole.
Little is known about how these individual factors
interact at an economic, social, and biological level
and how these interacting factors then affect health
(Kantermann and Roenneberg, 2009; Kantermann
et al., 2010; Knutsson, 2004; Wang et al., 2011).
Therefore, there is a need to understand the
mechanisms underlying shift work associated
health deteriorations to design better shift-work
conditions.
The complexity of the issue extends to under-
standing disruptions to the internal circadian
timing system by shift work. Human daily behav-
ior and physiology, including our mental and
physical performance, shows a clear time-of-day
variation, which is governed by an endogenous
circadian timing system. In temporal isolation,
such internal clocks produce internal days, which
in humans are usually longer than 24 h (Aschoff,
1965; Brown et al., 2008). Under normal con-
ditions, circadian clocks are synchronized to the
24-h day by zeitgebers (Roenneberg et al., 2003).
The light–dark cycle is the most important zeitge-
ber for the human circadian timing system
(Freedman et al., 1999; Kantermann et al., 2007;
Panda et al., 2002; Roenneberg et al., 2007b).
Synchronization of circadian clocks to a zeitgeber
cycle, called entrainment, is an active process that
works via response characteristics which, in turn,
depend on internal time of day (Roenneberg
et al., 2003, 2010): during the late internal night or
early morning, light shortens the internal day, and
lengthens it during internal afternoon or evening,
whereas there is little or no response to light
around internal midday. These principles of
entrainment are important for understanding the
disruption of the body clock in rotating shift
workers when they are exposed to light at unusual
and continually changing times. The relationship
between external and internal time is called phase
of entrainment, which shows large interindividual
differences, often referred to as chronotype
(Roenneberg et al., 2007a). In non-shift-work
populations, for example, late chronotypes have
been shown to be at higher risk of suffering from
bipolar disorders (Wood et al., 2009), headaches
(Bruni et al., 2008), seasonal depression (Natale
et al., 2005), depression (Levandovski et al.,
2011), and fibromyalgia syndrome (Kantermann
et al., 2012).
To date, only a few shift-work studies have
implemented the concept of internal time into
their analyzes. Complete adjustment of the circa-
dian clock to shift work is rare, even in permanent
night workers in conventional shift-work settings
(Folkard, 2008). Circadian adjustment supporting
work environments have been identified only in

highly specialized settings such as offshore oilrigs
(Barnes et al., 1998; Gibbs et al., 2002) or work-
stations in Antarctica (Lund et al., 2001; Midwin-
ter and Arendt, 1991; Ross et al., 1995). As an
alternative, recent shift-work simulation studies
have demonstrated that establishing a compro-
mise circadian phase position facilitated adjust-
ment in people switching between night shifts
and day work (Lee et al., 2006; Smith and
Eastman, 2008; Smith et al., 2008, 2009). These
observations may be of interest in the future
design of shift schedules. In addition, the few
studies that have implemented the concept of
internal time into their study design show evi-
dence of chronotype-specific differences in shift-
work adjustment: later chronotypes exhibited
higher shift-work tolerance and less rigid sleep-
wake behavior compared to earlier chronotypes
(Duffy et al., 1999; Härmä, 1993; Östberg, 1973;
Vidacek et al., 1988). Further, later chronotypes
appeared to accumulate less sleep deficit during
a shift-work cycle (Folkard and Monk, 1981).
These findings were also supported by reports of
better sleep quality in later chronotypes trying
to sleep during the daytime (Khaleque, 1999).
Later chronotypes are therefore assumed to suf-
fer less from night shifts (Burgess et al., 2002;
Folkard and Monk, 1981; Hildebrandt and
Stratmann, 1979). By contrast, later chronotypes
appear to be more challenged by early morning
shifts (Åkerstedt and Torsvall, 1981; Griefahn
et al., 2002). This latter result has also been con-
firmed in our study discussed in more detail
below (Kantermann et al., 2012a). Another
important factor in this context is the effect of
age, since younger people have later chronotypes
and chronotype, in turn, advances with age
(Roenneberg et al., 2007a). These findings have
been used to explain why older, earlier
chronotypes, on average, withdraw sooner from
shift work than younger, later chronotypes (Bohle
and Tilley, 1989; Costa et al., 1989; Hauke et al.,
1979). In addition, a study (Moreno et al., 2012)
involving 514 nursing professionals, found among
the day workers (DW) that the higher the
401
morningness, the more satisfied the workers were
with their job. By contrast, among night workers,
job satisfaction was associated with sleep quality
and hospital seniority rather than diurnal prefer-
ence. Although this study has been conducted
with night workers, these findings reinforce the
idea that individual preferences for evening
activities cannot be seen as the sole factor leading
to shift-work adaptation. Currently, however,
there are no conclusive data showing that later
chronotypes would develop less (chronic) health
problems than earlier chronotypes when working
in alternating shifts.
There are a number of comprehensive review
articles on shift work and health (Costa, 2003b;
Kantermann et al., 2010; Knutsson, 2004; Wang
et al., 2011). Aiming to add to this existing pool
of knowledge, in this chapter, we present results
from both our field and laboratory studies on shift
workers. These studies emphasize the importance
of taking individual factors—especially internal
time—into account. Clearly, the insights gained
when we study people under controlled labora-
tory conditions (e.g., by strictly controlling circa-
dian phase, posture, diet, light exposure, etc.)
helps to design better field studies and interpret
findings gathered in noisy and less controllable
real-life settings. Therefore, we have structured
this chapter into two sections: (1) studies per-
formed in the field, and (2) studies performed
under controlled conditions in the laboratory.
Studying shift workers in real-life settings
Stress and adverse effects on health in shift
workers has been related to adverse working
conditions, poor adaptation of work and social
life, sleep deprivation and interference in the syn-
chronization of the internal circadian clock by the
light–dark cycle (Costa, 2003a; Kantermann et al.,
2010; Knutsson, 2003; Thompson, 2009; Wang
et al., 2011). Therefore, we have performed a
number of shift-work field studies, to investigate
acute stress (measured via levels of morning and
402
evening cortisol), as well as chronic stress
(measured via arterial stiffness as a surrogate for
atherosclerotic risk), and consequences of shift-
work demand as estimated by the incidence rate
at work.
Cortisol awakening response and stress in shift
working truck drivers
In a cross-sectional study (Ulhôa et al., 2011), we
investigated the individual cortisol awakening
response (CAR) (Dahlgren et al., 2009) in truck
drivers working day shifts (n ¼ 21) or “irregular
shifts” including night work (n ¼ 21). In addition,
we investigated subjective and objective sleep
and cardiovascular blood parameters in these
workers. All participants worked for the same
transportation company in Brazil. Objective sleep
was obtained from actigraphy measurements. Sal-
ivary cortisol samples for the CAR analysis were
obtained at two time points: at waking time and
at 30 min after waking time. In addition, cortisol
at bedtime was also measured. These three corti-
sol samples were collected both during a workday
and on a day off work. The “irregular” shift
workers showed a significantly higher waist-hip
1.25
Salivary cortisol level (µg/dl)
1.00
0.75
0.50
0.25
0.00
Upon waking
Time of sample collection
Fig. 1. Salivary cortisol levels (mean  SEM) according to the time of sample collection in irregular shift workers. Samples were
collected upon awaking, 30 min after waking and at bedtime during a work day (■) and during a day off (□).
ratio (WHR) and elevated VLDL-cholesterol.
In addition, the “irregular” shift workers reported
more tiredness after work, more disturbances due
to truck vibration, and less job demand compared
to DW. In addition, the “irregular” shift workers
were employed as truck drivers for longer than
the DW. Cortisol collected both in the morning
and at bedtime in the “irregular” shift workers
was positively correlated to short sleep duration,
low job satisfaction, total cholesterol, HDL,
LDL, VLDL, and triglycerides. DW showed
higher cortisol at 30 min after waking and a
higher CAR on their workdays compared to their
days off. The “irregular” shift workers had higher
cortisol levels on their days off compared to the
DW. These results suggest that the “irregular”
shift workers might have had a prolonged stress
response on their days off, since they had higher
cortisol levels on these days compared to their
workdays (Fig. 1). An earlier study of 470 truck
drivers reported extended working hours as a
stressor at work, and this was associated with
minor psychiatric disorders (Ulhôa et al., 2010).
Regulation of working hours and avoidance of
extended work hours may thus be an important
way to reduce stress in truck drivers. Future stud-
ies of shift workers with irregular work hours are
30 min after waking At bedtime

warranted for a more detailed investigation of
stress responses.
Individual “shift-work load” in rotating shift
workers
Regarding the effects of shift work on the individ-
ual, the different shift types have become subject
to investigation. For example, clockwise (CW)-
rotating schedules (e.g., rotating from the morning
shift to the late shift to the night shift) compared to
counter-clockwise (CC) rotation (e.g., rotating
from the night shift to the late shift to the morning
shift) is considered the shift direction of choice as it
is assumed that CW rotation causes less sleep and
circadian rhythm disruption (Costa, 2003a; Härmä,
1993; Knauth, 1993). Conclusive evidence, how-
ever, in particular on chronic health parameters,
is limited. For the first time in a field study of rotat-
ing shift workers, we have measured pulse wave
velocity (PWV) in fast CW and slow CC shift
workers compared to DW (Kantermann et al.,
2012a). The aim of this study was to assess cardio-
vascular risk using arterial stiffness in workers
undertaking different shift rotations. Male workers
(n ¼ 77, mean ( SD) age 42  7.6 yrs) in a Belgian
steel factory with at least 5 years experience in their
current work schedule participated. All par-
ticipants completed questionnaires covering
demographics, details about their shift work sched-
ule, health, and stimulant consumption. All shift
and day workers also completed the Munich
Chronotype Questionnaire for shift workers
(MCTQshift) (Juda, 2010). Data obtained from the
MCTQshift were used to calculate each worker’s
chronotype (time of mid-sleep on free days
(MSFsc), corrected for the sleep deficit on
workdays) and the amount of their social jetlag
(SJL) (difference between the time of mid-sleep
on workdays (MSW) and MSF) (Wittmann et al.,
2006) in each work shift (early-, late-, night-, and
day shift). Shift workers also self-rated how sleep,
social, and work life were affected due to working
shifts. In 63 workers, we measured PWV, in
403
addition to blood pressure (BP) and heart rate
(HR) on one morning shift between 08:00 and
12:30 h (no caffeine/smoking/exercise). There
were no differences in chronotype, age, body mass
index (BMI), WHR, BP, HR, smoking, and coffee
consumption between shift workers and DW. By
contrast to DW, however, shift workers (CW and
CC combined) reported more stomach and diges-
tion problems, and more weight fluctuations. In
addition, as expected, shift workers had more
SJL. In previous investigations, SJL has been
found to vary with chronotype and work shift
(Juda, 2010; Vetter, 2010). This finding was also
corroborated in our study. The highest level of
SJL was found for early chronotypes while doing
night shifts, with only a small amount of SJL
observed for early chronotypes during morning
and late shifts. Late chronotypes, however, showed
the highest amount of SJL on the morning shift,
although this was less than observed in the early
chronotypes on night shifts. The two shift-work
groups (CW, CC) did not differ in the ratings of
how shift work affected their sleep, social, and
work life. In all workers combined (CW, CC, and
DW), HR and average SJL were significantly posi-
tively associated (r ¼ 0.309, p ¼ 0.021 adjusted for
age). This association also was significant for DW
alone (r ¼ 0.679, p ¼ 0.015 adjusted for age). There
was no statistically significant difference in PWV
between fast CW, slow CC, and DW.
In a subsequent step, and to introduce a new
and simple concept to shift-work research, we cal-
culated the “individual shift-work load” (ISL), to
measure the impact of a worker’s schedule, using
the following two formulae:
(i) For shift workers:
ISLshift ¼ (SJL/#WD)*#SWyears
(ii) For DW: ISLday ¼ (SJL/#WD)*#Wyears
The ISL is composed of a worker’s individual
SJL (difference between time of MSW and
MSF; Wittmann et al., 2006) divided by the
total number of days per shift cycle (#WD;
representing speed of rotation) and multiplied
404
by the individual’s total number of years of expo-
sure to shift work (#SWyears). As the DW were
not exposed to shift work, the respective number
of years of employment at the current company
(#Wyears) was used instead. There was a signifi-
cant positive correlation (r ¼ 0.493, p ¼ 0.005)
between ISL and PWV (PWVaBP, which is PWV
adjusted for age and BP). This finding provides
first evidence that arterial stiffness assessed by
PWV and the chronic strain of shift work
(estimated from measures of SJL) may be interre-
lated. The group differences in both speed and
direction of shift rotation, the small number of
subjects, and its cross-sectional design are
limitations of this study. We hope, however, that
the findings from this pilot study will initiate
future studies of this kind. Indeed, more studies
in workers employed in shift schedules with dif-
ferent speeds and directions of rotation but work-
ing in the same work setting are needed.
Effect of shift type on incidence rate in rotating
shift workers
Previous studies have shown that the night shift
in rotating shift schedules is characterized by the
lowest levels of alertness and vigilance, and the
highest amount of sleep deprivation and hence has
been suggested to lead to an elevated incidence risk
in night workers (Costa, 2003b; Folkard, 1997;
Mitler et al., 1988). Studies have shown influences
of, inter alia, work duration (length of time of being
at work), start time of a work shift, number of con-
secutive work shifts, type of occupation, and prior
sleep duration (Folkard et al., 2005), even when
the work tasks were comparable across shifts
(Folkard, 1997). Such findings are supported by
controlled circadian laboratory studies on alertness,
vigilance, sleepiness, and fatigue, which show a
clear 24-h variation in these parameters (Åkerstedt,
2007; Aschoff, 1965; Cajochen et al., 1999; Dijk
et al., 1992; Graw et al., 2004) and deterioration in
these parameters after sleep deprivation (Axelsson
et al., 2008; Dinges et al., 1997; Doran et al., 2001;
Franzen et al., 2008; Sallinen et al., 2008; Van
Dongen et al., 2003). We investigated the incidence
rate in rotating shift workers (undertaking
morning, late, and night shifts) employed in two
different shift-work rotations (Kantermann et al.,
2012b). A retrospective analysis of the incidence
data from 730 male shift workers employed in
either a CW (e.g., rotating from the morning shift
to the late shift to the night shift) or CC rotation
(e.g., rotating from the night shift to the late shift
to the morning shift) with comparable work
conditions at the same steel factory over a 5-year
period has been performed. Morning shifts
exhibited a significantly higher incidence rate com-
pared to night shifts, independent of shift-work
rotation. The incidence rate across the 24-h day
did not differ between CW and CC rotation. The
elevated incidence rate in the morning shift at this
steel factory could be related to the morning shift
being the most labor-intensive one of the three
work shifts in both the CW and CC rotation. In
addition, there could be an effect of sleep depriva-
tion due to the early start of the morning shift at
06:00 h, which means an even earlier get up time
for the workers. These findings suggest that, in
addition to, or even irrespective of, work shift and
direction of shift rotation, the impact of work time
and work demand may have a strong modulating
effect on the incidence rate.
Studying shift workers in controlled laboratory
settings
Advantages of laboratory studies
What is the benefit of performing studies in shift
workers under strictly controlled laboratory
conditions? To answer that question, one needs
to consider all the confounding factors in real life,
which are very difficult to control in field studies.
Such confounders encompass, inter alia interindi-
vidual and intraindividual differences in shift-work
history (e.g., work schedule, work tasks, number of
work hours, etc.), the lighting environment both at

and away from work, stress both mental and physi-
cal (which is often dependent upon job type and
work load), diet, sleep duration and sleep depriva-
tion prior to and after a shift, and individual inter-
nal time (chronotype). Epidemiological studies,
for example, often fail to take account of the exact
time (especially relative to internal time) samples
of biomarkers have been collected in relation to a
shift worker’s actual work hours (Boggild and
Knutsson, 1999; Sookoian et al., 2007), whereas
other studies have carried out such a control
(Esquirol et al., 2009). The lack of control of con-
founders is not merely a matter of the number of
study subjects, since there are also smaller field
studies with fewer subjects that have failed to
address one or more of these important points
mentioned above (Adams et al., 1998; Amir et al.,
2004; Rauchenzauner et al., 2009). Such con-
founding differences must be taken into account
else substantial information that could explain
group differences may be missed, for example,
when studying groups employed in different shift-
work rotations or when comparing shift workers
with non-shift-workers. To disentangle the differ-
ent causes that may contribute to the effects of shift
work on health and wellbeing, laboratory studies
are able to control many factors and hence are able
to manipulate single, specific variables at pre-
defined time points. Shift-work simulation studies
(Hampton et al., 1996; Ribeiro et al., 1998), for
example, were able to demonstrate that glucose,
insulin, and lipid levels following food intake were
dependent on a person’s internal time. In addition,
so-called forced desynchrony experiments showed
that circadian misalignment negatively affects
metabolism (Scheer et al., 2009). To our knowl-
edge, there is only one study that has investigated
real shift workers under laboratory conditions
(Simon et al., 2000), more commonly laboratory
studies investigate shift work naïve subjects that
are mostly healthy and young.
Therefore, we have compared experienced shift
workers (n ¼ 11, with at least 5 years of shift-work
experience) to matched (for age, BMI, and cho-
lesterol) non-shift-workers (n ¼ 14) in their
405
response to one night of total sleep deprivation
(TSD) (as a proxy for the first night of shift work)
and recovery sleep under controlled laboratory
conditions (Wehrens et al., 2010, 2012a, 2012b).
The effect of sleep deprivation on the participants’
metabolic and cardiovascular function as well as
alertness, mood, and performance was assessed.
Both groups followed a 7-day regular sleep-wake
cycle prior to the laboratory session. This was done
to ensure that shift-workers were either on day
shifts or days off in order to avoid sleep depriva-
tion and circadian misalignment in the study
subjects. The in-laboratory study protocol con-
sisted of one night of adaptation sleep, followed
by one night of baseline sleep, and one night of
TSD (30.5-h wakefulness). A subsequent daytime
nap opportunity of 4 h and a full recovery sleep
period were provided. All measurements were
performed relative to wake up time and controlled
for body posture, diet, and light exposure. Post hoc
measurement of dim light salivary melatonin onset
prior to the baseline night confirmed that there
were no significant differences in circadian phase
between shift-workers and non-shift-workers.
Compared to the non-shift-workers, however, shift
workers showed a lower HR variability (HRV)
variance and higher sympathetic activity, as well
as a trend towards lower endothelial function
(assessed by flow-mediated dilatation of the bra-
chial artery) throughout the study. In addition,
after the recovery sleep period, the postprandial
insulin response was significantly increased and
the non-esterified fatty acid (NEFA) response
reduced compared to after sleep deprivation
and/or baseline sleep in the non-shift workers
only. In this study, the shift workers also felt more
alert, more cheerful, more elated and were
calmer throughout the protocol compared to
the non-shift workers (Wehrens et al., 2012a).
In addition, shift workers showed a faster
median reaction time (RT) compared to non-shift
workers although four other psychovigilance
test parameters did not differ significantly bet-
ween the two groups. These findings suggest
that experienced shift workers cope better with
406
laboratory sleep deprivation than non-shift
workers. These group differences could be
explained inter alia by a selection bias into and
out of shift work, the absence of actual shift work
that was inherent to the laboratory study and
regular scheduled sleep prior to the laboratory
study. The data on HRV and endothelial function
(Wehrens et al., 2012b), however, suggest
increased cardiovascular risk in the shift workers,
although this was not significantly affected by
sleep deprivation. The observed effects of recov-
ery sleep on metabolism in the non-shift workers
suggest a tendency towards a state of insulin resis-
tance in non-shift workers compared to the shift
workers. This may either be a direct effect of
recovery sleep or a delayed response to sleep
deprivation. Such responses after recovery sleep
are similar to the findings of the delayed stress
response on days off in our study on truck drivers
in Brazil (Ulhôa et al., 2011). The results from
both these laboratory and field studies emphasize
that shift work schedules should be designed to
allow for optimal physiological recovery. From a
neurobehavioral point of view, shift workers
may underestimate how much recovery time they
actually need. A similar mismatch between the
subjective perception of increased sleep depriva-
tion and objective measures in performance has
been shown previously (Franzen et al., 2008;
Galliaud et al., 2008; Van Dongen et al., 2003,
2004) and remains an intriguing aspect for future
shift-work studies.
Disadvantages of laboratory studies
Despite all the advantages listed in the previous
section, there is one major limitation to laboratory
shift-work studies; only a small number of subjects
can be studied under strictly controlled conditions.
Often due to costs for research staff, consumables,
and laboratory space, time constraints or recruit-
ment issues (i.e., the more strict the inclusion
criteria, the more difficult recruitment becomes),
these studies can be statistically underpowered.
This latter aspect prevents detecting small
differences in study parameters, especially as many
biological parameters show large intra- and interin-
dividual variation. In addition, as laboratory stud-
ies are obviously not performed under “real-life”
conditions, information on many aspects of shift
work is not recorded, for example, the social and
economic reasons for choosing a shift-work job.
In addition, shift-work simulation studies often
use shift-work naïve subjects instead of experi-
enced shift workers. Although simulation studies
on non-shift workers can show the acute effects of
a shift-work schedule, they cannot assess the
chronic effects shift work poses on employees. By
contrast, field studies can achieve this by studying
the same subjects repeatedly and prospectively at
regular intervals (presuming no worker leaves the
company). However, from controlled laboratory
investigations, we can determine which parameters
to control when performing field studies and vice
versa—clearly a win-win collaboration.
Conclusion
With this overview of our laboratory and field
studies on shift workers and non-shift workers
we aimed to illustrate the importance of the fol-
lowing aspects:
 Studying shift workers and non-shift workers in
the same work environment is imperative to
best control for interindividual variation and
worksite-related effects (e.g., as we have done
in a Belgian steel factory study and in the study
of Brazilian truck drivers).
 Strictly controlled laboratory studies (as we
have presented) on real shift-workers and non-
shift workers are fundamental to understand
the immediate and acute effects that shift-work
and sleep deprivation pose on health, as these
studies allow for adequate matching of subjects.
 Findings from strictly controlled laboratory stud-
ies must be translated into field investigations,
first to validate these findings and second to
 407

The shift-clock-work

Real-life Controlled
field laboratory
studies studies
Healthier
Individual shift
predictors schedules

Data Computer
mining simulations

Fig. 2. The “shift-clock-work” illustrates the interconnectivity between field studies performed in real-life and controlled laboratory
investigations helping to cross-validate data, which in turn drives databases for data mining and computer simulations. The outcome
is “individual predictors” helping to design better and healthier shift-work schedules. Figure modified from Kantermann et al.
(2010).

discover new aspects, which in turn can be
translated back into laboratory studies—a clear
win-win circulation of scientific knowledge.
 The introduction of new, noninvasive (bio-)
markers into shift-work research has the poten-
tial to elucidate the underlying mechanisms of
the pathophysiological pathways involved in
shift work. These new (bio-) markers, for exam-
ple, are: chronotype (phase of entrainment),
SJL, ISL, PWV, HR, HRV, and flow-mediated
dilation.
Only by combining laboratory and field studies
and by comparing these data, will it be possible to
identify the individual predictors that constitute
important set points in the interaction between shift
work and health status. Further, although not
explicitly discussed in this chapter, these data can
then be subjected to shift-work models. Such
models will help, not only to rapidly generate new
hypotheses for future studies (both in the laboratory
and in the field), but also to develop better and
healthier shift-work schedules that take individual
factors into account (Fig. 2). Although there remain
some profound gaps in our knowledge, for example,
how shift work puts one individual but not another
at risk, the existence of good research tools and
our current knowledge makes this noisy and individ-
ual shift-work research doable.
Acknowledgments
T. K. is supported by the DFG (German Research
Foundation), S. M. T. W. is supported by the
BBSRC (BB/I008470/1), M. U. and C. M. are
supported by Santander, USP, FAPESP, and
Surrey (2011/50169-6), and D. J. S. is a Royal Soci-
ety Wolfson Research Merit Award holder. The
research was supported by an EU Marie Curie
Research Training Network grant (CT-2004-
512362), 6th Framework project EUCLOCK
(018741) and Stockgrand Ltd., University of
Surrey, Guildford, UK.
We especially thank our participants at
ArcelorMittal Industeel Belgium in Charleroi,
408
the truck drivers in São Paulo, Brazil, and the
participants in our laboratory shift-work study.
We also thank Shelagh Hampton, Françoise
Duboutay, Myriam Kerkhofs, Damien Haubruge,
and Arno Schmidt-Trucksäss for their valuable
help with the studies. Without their contribution,
these studies would not have been made possible.
Conflict of interest: None to declare.
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Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 23

The evolutionary physiology of

photoperiodism in vertebrates

David Hazlerigg*

Institute of Biological & Environmental Sciences, University of Aberdeen, Aberdeen, Scotland, United Kingdom

Abstract: The capacity to measure day length (photoperiod) is a trait subject to intense evolutionary
pressure, and the circadian system has become an important part of the photoperiodic machinery. With
the exception of mammals, vertebrates possess multiple sites of photosensitivity within the central
nervous system through which light responses may be coordinated. Of these, deep brain photoreceptors
play a special role in photoperiodism in nonmammalian vertebrates, independent of either retinal or
pineal pathways. In mammals, the pineal hormone, melatonin has assumed a function orthologous to
that of deep brain photoreceptors in other vertebrates. Contrasting with this dichotomy in photoperiodic
input pathways, downstream signal processing to produce switch-like seasonal responses is largely
conserved and appears to center on photoperiodic control of thyroid hormone bioavailability within the
hypothalamus. Recent studies implicate the clock-controlled gene eyes absent 3 in linking the circadian
clock to photoperiodic responses, dictating the seasonal level of expression of thyroid-stimulating
hormone by a specialized population of photoperiodically sensitive cells in the pituitary stalk which in
turn govern thyroid hormone metabolism in the basal hypothalamus. This ancestral control pathway
appears to have originated prior to the divergence of the mammalian and avian vertebrate lineages.

Keywords: photoperiodism; melatonin; thyroid-stimulating hormone; TSH; deep brain photoreceptor;

circadian; pars tuberalis; deiodinase; eyes absent.

Energy budgets, life history strategies,

and seasonality
The laws of natural selection dictate that species sur-
vival depends on individual success in reproduction.
*Corresponding author.
Tel.: (44) 1224 272871; Fax: (44) 1224 272396
E-mail: d.hazlerigg@abdn.ac.uk
Achieving breeding success depends on securing suf-
ficient energetic resources, through nutrient intake,
to reach reproductive maturity. This summarizes
the essential focus on bioenergetics as a fundamental
issue in evolutionary biology.
In managing the energy equation, a key prob-
lem is the variable nature of the environment,
with periods that are energetically favorable

http://dx.doi.org/10.1016/B978-0-444-59427-3.00023-X 413
414
interspersed with unfavorable episodes (drought,
famine, winter). Furthermore, evolution leads spe-
cies down avenues in which adaptive preparations
for breeding success (e.g., growth, gametogenesis,
development of reproductive ornaments, and other
secondary sexual characteristics) demand periods
of energetic favorability preceding the reproduc-
tive act itself. Offspring survival also depends crit-
ically on the environment, providing a further
temporal constraint on the timing of reproduction.
Hence, we can see that the phasing of reproductive
activity to windows of energetic favorability is a
basic evolutionary challenge for life on Earth.
Environmental variability is to a large degree of
predictable geophysical origin, derived from the
Earth’s periodic motion, both in rotation about
its polar axis, giving rise to day and night, and in
orbit around the Sun, giving rise to the seasons.
It is this latter annual–seasonal cycle that has a
major bearing on the energetic considerations per-
taining to the reproductive success for many spe-
cies, and as a result, mechanisms to synchronize
life history phases to the annual–seasonal cycle
have evolved. Except at the Equator, the orbit of
the Earth around the Sun leads to an annual cycle
of changing day length and total daily solar radia-
tion exposure, and together, these provide the ulti-
mate cause of the Earthly seasons and proximate
cues that organisms use to determine the time of
year and hence to predict forthcoming energetic
conditions. The use of changing day length to syn-
chronize life history cycles is a prevalent feature of
life, and this suggests that evolution of photope-
riodic time measurement is an ancestral feature
of life of comparable importance to the evolution
of circadian time keeping.
In the circadian realm, there is abundant evidence
for evolutionary conservation of circadian rhythm
generators among eukaryotes both in terms of
organization of the core transcription–translation
feedback loop and in terms of the molecular
elements involved in photic synchronization (e.g.,
the cryptochrome and period genes) (Bell-Pedersen
et al., 2005; Dardente and Cermakian, 2007). And,
it is therefore reasonable to ask whether similar
fundamental conservation can be found in elements
involved in photoperiodic time measurement. This
review considers this question from the perspective
of seasonal timing in vertebrates focusing on recent
developments in our understanding of neuroendo-
crine control of seasonal reproduction.
Thyroid hormone signaling as an ancestral
pathway controlling reproductive activation
It has been known since the 1930s (Benoit, 1936;
Woitkevitsch, 1940) that thyroidectomy interferes
with the seasonal cycle of reproductive activation
in birds, a finding that received renewed attention
with the work of Follett and colleagues in the Jap-
anese quail (Coturnix japonica) in the 1980s and
1990s (reviewed in Dawson et al., 2001), and
which was extended also to consider the role of
thyroid signaling in seasonal reproductive control
in sheep (e.g., Dahl et al., 1994; Follett and Potts,
1990). The current picture, reviewed thoroughly
elsewhere (Hazlerigg and Loudon, 2008; Nakao
et al., 2008b), places light-dependent regulation of
type 2 and type 3 thyroid hormone deiodinases
(Dio2 and Dio3, respectively) in the basal hypothal-
amus at the core of the seasonal photoperiodic
response in birds and mammals (Yoshimura et al.,
2003). These enzymes activate and inactivate
thyroxine, respectively, thereby controlling thyroid
hormone-dependent biology in a tissue-specific
manner (Lechan and Fekete, 2005; Fig. 1). The path-
way linking light to deiodinase regulation in birds
and mammals shows a high degree of conservation
and centers on light-dependent regulation of thyro-
tropin (thyroid-stimulating hormone, TSH) expres-
sion in cells in the pars tuberalis (PT) region of the
pituitary stalk (Hanon et al., 2008; Nakao et al.,
2008). TSH-expressing PT cells appear to constitute
a distinctive population of thyrotrophic cells, which,
in contrast to classical thyrotrophs found in the pars
distalis (PD), are insensitive to hypothalamic thyro-
tropin-releasing hormone and to peripheral thyroid
hormone feedback (Bockmann et al., 1997). This
distinctive regulatory profile is probably crucial to
 415

(a) Thyroid (b)

gland

T4 SP LP

Dio3 Dio3
OH- Outer Inner Ala

OH- Ala OH- Ala

Dio2 Dio2

T3 rT3

OH- Ala

T2

Fig. 1. Thyroid hormone metabolism in the hypothalamus. (a) Reaction scheme showing the enzymatic pathways leading to the
conversion of thyroxine (T4) produced by the thyroid gland into either triiodothyronine (T3) by outer ring deiodination
catalyzed by type 2 deiodinase (Dio2) or reverse T3 (rT3) by inner ring deiondination catalyzed by type 3 deiodinase (Dio3).
The former is an activating process leading to increased thyroid hormone actions through nuclear thyroid hormone receptors; the
latter is an inactivating process. Further metabolism of T3 or rT3 is possible, leading to diiodothyronine (T2) production, which
has no known signaling activity. (b) Reciprocal switching between states of high Dio2/low Dio3 expression and vice versa,
through photoperiodic control. Images are autoradiograms of radioactive in situ hybridization histochemistry for Dio2 or Dio3 in
coronal hypothalamic sections from sheep that were held on either SP (8 h light/24 h) or LP (16 h light/24 h) for 4 weeks prior to
sacrifice. Note strong labeling in the mediobasal hypothalamic area under LP for Dio2, but under SP for Dio3; note also that
Dio2 labeling extends into surrounding hypothalamic tissue, while Dio3 labeling is confined to the ependymal zone immediately
surrounding the third ventricle.

their role in photoperiodic time measurement.
Additionally, the current model states that PT-
derived TSH acts locally on Dio2/Dio3-expressing
cells in the hypothalamus or within the PT itself
(Hanon et al., 2008; Nakao et al., 2008; Ono et al.,
2008). Hence, recent work defines a fundamentally
novel role for TSH as seasonal photoperiodic signal
in neuroendocrine system for birds and mammals.
There are good reasons to consider this novel reg-
ulatory circuit as a reflection of ancestral function of
thyroid signaling in vertebrates. According to this
narrative, thyroid hormone signaling evolved from
an ancient nutrient-sensing pathway, reliant on
plant metabolites as indicators of environmental
favorability to cue the transition to reproductive
maturity (Heyland et al., 2005). Experiments to
manipulate levels of thyroid hormone in modern
day echinoderms (sand dollars) demonstrate that
increased TH promotes reproductive activation,
while reduced TH favors continued somatic
growth (Heyland and Hodin, 2004). It has been
proposed that there was a transition to endogenous
thyroid hormone production in ancestral chordates
with the establishment of a secretory structure
known as the endostyle, which can be seen in
modern day sea squirts (urochordates) and in
amphioxus (cephalochordates). The endostyle
appears to correspond to the thyroid gland of mod-
ern vertebrates, based upon orthologous develop-
mental gene expression. Comparative genomics
416

approaches have been used to explore how the
additional ingredients seen in the thyroid axis of
vertebrates appeared during the evolutionary
pathway originating with ancestral protochordates
(Paris et al., 2008). According to this scheme,
although a link between TH and reproduction
stems from the most primitive ancestors of the
vertebrates, the establishment of a neuroendocrine
pathway controlling TH production occurred
with the agnathans (lampreys and hagfish), with
further refinements appearing in subsequent bra-
nches leading to the current classes of modern
vertebrates (Fig. 2).
It remains unclear whether hypothalamic actions
of TSH, modulated by local deiodinase expression
are also at the center of the photoperiodic
response in modern vertebrate groups other than
birds and mammals. The bird/mammal story points
to subfunctionalization of TSH signaling (light-
dependent PT thyrotroph local actions and PD
thyrotroph actions on the thyroid) developing prior
to the divergence of the birds from the mammals.
On anatomical grounds, we might expect a similar
subfunctionalization to have occurred in other ver-
tebrate groups in which a distinct pituitary stalk
PT region is present—which would include the
amphibians and reptiles. Contrastingly, modern
teleosts appear to lack a distinctive pituitary stalk
or portal vasculature, relying instead on penetration
of neurosecretory fibers projecting from the hypo-
thalamus into the PD itself, which is directly
apposed to the basal hypothalamus without an
intervening stalk region. Clearly, experiments to
explore TSH and deiodinase gene expression in
the basal hypothalamus and pituitary of species
from a wider range of vertebrate groups would
be desirable to clarify this issue and give a proper
evolutionary perspective on this pathway.
The photoperiodic axis in mammals and its
coupling to brain thyroid signaling
The described effects of photoperiod on hypotha-
lamic deiodinase expression may be considered
as a (possibly the) key downstream target of the

Amphibians

TR duplication Mammals/sauropsids

Chondrichthyians
Neuroendocrine control
of TH production
Agnathans
TH-synthesizing
organ Cephalochordates/urochordates
Endogenous TH
production Echinoderms/hemichordates

Ancestral TR
Protostomes

Fig. 2. Evolutionary appearance of different elements of the thyroid signaling pathway during the evolution of modern chordates.
Sensitivity to TH-like molecules produced in the environment is thought to have occurred early in the deuterostomal lineage, prior
to the divergence of the protochordates. Endogenous TH synthesis may have preceded the cephalochordate and urochordate
lineages, but these see the first appearance of a TH-synthesizing organ, the endostyle. Neuroendocrine control of TH is a feature
of all vertebrates including the agnathans, with further refinements appearing in the jawed vertebrates. See Paris et al. (2008) for
further details.
 417

so-called photoperiodic axis in birds and mammals.
This comprises the set of interconnected neural
and endocrine elements that collectively govern
photoperiodic responses. In mammals, this axis is
seemingly linear from light reception in the retina,
through a core circadian pacemaker in the hypo-
thalamus and its effects on pineal melatonin
production and culminating with melatonin recep-
tor-expressing cells, notably in TSH-expressing
cells in the PT (Fig. 2, left). Comprehensive
reviews on photoperiodic influences at different
levels of the axis as a whole can be found else-
where (Iuvone et al., 2005; Morgan et al., 1994;
Schwartz et al., 2001; Simonneaux and Ribelayga,
2003). From the perspective of seasonal photope-
riodism in mammals, the key property of this axis
is that it produces a light-modulated circadian
rhythm of melatonin release. Melatonin secretion
is restricted to the night, continuing from after
dark onset, through to dawn—and changes in
the duration of this nocturnal melatonin signal
are both necessary and sufficient to drive seasonal
endocrine responses in mammals (Simonneaux
and Ribelayga, 2003).
Recent studies of the molecular-transduction
pathway linking melatonin to the seasonal photo-
periodic response have focussed on melatonin’s
effects on rhythmical gene expression in the PT,
and the way in which these might lead to a
photoperiod-dependent change in levels of TSH
release (Dardente et al., 2010; Masumoto et al.,
2010). Current thinking on this suggests that rhyth-
mical influences of melatonin on the expression of
the circadian clock-controlled gene thyrotroph
embryonic factor (Tef) and on the transcriptional
coactivator eyes absent 3 (Eya3) are critical to pro-
cessing of the melatonin signal in the PT (Fig. 3).
Of particular importance for this model is the con-
cept that melatonin has two influences on Eya3
expression, a phase-resetting influence such that
the phase of peak Eya3 expression follows the
evening rise in melatonin by a fixed interval of
approximately 12 h and a direct inhibitory influ-
ence of melatonin such that the amplitude of peak
Eya3 expression under conditions when melatonin
is still at elevated nighttime levels is markedly
lower than if the peak occurs when melatonin
is at low daytime levels. Together, these two

SP LP induction

Photoperiod

E-box
Melatonin
suppression Circadian signal

Ψ ≈ 12 h Ψ ≈ 12 h Transduction
Eya3 mechanism

EYA3 EYA3
TEF TEF
TSHβ Seasonal response
D D

Fig. 3. Photoperiodic switching in mammals through Eya3-dependent transcription of TSHb in the PT. Eya3 is a circadian clock-
controlled gene whose rhythmic expression is set by the phase of evening melatonin onset, and peak Eya3 expression
consistently occurs some 12 h after dark/melatonin onset independent of photoperiod. This means that under SP peak Eya3
expression occurs during the night, while the melatonin level is high and exerting a suppressive effect, and so the peak is small.
Contrastingly, under LP, the Eya3 peak occurs the following morning when the melatonin level is minimal, and so the peak is
large. This classic “external coincidence timer” mechanism, in which a circadian oscillation interacts with a light-dependent
stimulus, limits EYA3/TEF synergism to LP, dictating the onset of a summer phenotype.
418
influences appear to form what is known as a
“coincidence timing” mechanism to determine the
photoperiodic response (Hazlerigg and Wagner,
2006). It is probably no accident that the phase
lag between peak Eya3 expression in the PT, and
the evening rise in melatonin is approximately
12 h, leading to triggering of long day responses
around the spring equinox, and approximating to
the “critical day length” needed to trigger a sum-
mer phenotype in mammals (Elliott, 1980).
Deep brain photoreceptors and photoperiodic
signaling in birds
Eya3 is also prominent in the photoperiodic
response of quail (Nakao et al., 2008), and there
is clear conservation in the proximal promoter
organization of the TSH beta subunit (TSHb)
gene between birds and mammals—including the
proximal D-element (RTTAYGTAAY) which is
key to the TEF/EYA3 control of mammalian
TSHb (Dardente et al., 2010). Given that Eya3,
with TSHb is among the first genes to show
increased expression following a transfer to LP
in the quail (Nakao et al., 2008), it is most likely
that the TEF/EYA3-inductive mechanism for
control of TSHb in the PT is essentially conserved
between birds and mammals; nonetheless, a
direct demonstration of this in quail is lacking.
Where birds diverge intriguingly from the
mammalian scheme is upstream of Eya3 and
TSH control. The prevailing view is that con-
trasting with mammals, melatonin is not required
for the seasonal reproductive response of birds,
and that instead deep encephalic photoreceptors
within the mediobasal hypothalamus relay the
effects of light to the photoperiodic response sys-
tem. The evidence for this position derives from a
complex series of experiments, in bird species
including ducks and song sparrows as well as the
Japanese quail, and involving blinding, pinealec-
tomy, hypothalamic lesioning, and use of light
guides to directly deliver light to the basal hypo-
thalamus (reviewed in Dawson et al., 2001; Sharp,
2005). Deep brain photoreceptors are thought to
lie in the walls of the third cerebral ventricles,
and three recent studies have argued for specific
opsin-like photoreceptors (VA opsin, opn4x/
melanopsin, and opn5) as mediators of the photo-
periodic response in birds (Halford et al., 2009;
Kang et al., 2010; Nakane et al., 2010). In each
case, evidence is largely based on the demonstration
of immunoreactivity in the basal hypothalamus, and
projections from immunoreactive cells into the PT,
through which it is presumed that TSHb expression
is controlled. It is difficult to place these different
candidates in priority order in terms of likely contri-
bution to the photoperiodic response, but spectral
analysis of the quail photoperiodic response is
consistent with joint contributions from multiple
opsin-like photoreceptors in this region (Foster
et al., 1985; Nakane et al., 2010). Intriguingly, we
perhaps should not discount the possibility that mel-
atonin synthesis within the avian hypothalamus is
involved in the seasonal photoperiodic response of
birds, with evidence from turkeys for the expression
of two key enzymes in the melatonin synthesis
pathway (tryptophan hydroxylase 1 and arylamine
N-acetyl transferase) in dopaminergic neurons in
the premammillary hypothalamus of turkeys (Kang
et al., 2007). Whether this colocalization story
reflects an underlying link between melatonin and
photoperiodic timing in birds or a more general link
between melatonin synthesis and opsin-like photo-
receptor expression, as seen in the retina and pineal
gland (Iuvone et al., 2005), remains to be clarified.
I am unaware of any data demonstrating melatonin
receptor expression in the avian PT, but this may be
an avenue that should be reexplored.
The evolutionary origins of divergent melatonin
actions in different vertebrate lineages
Extra-retinal photoreception appears to be the
norm among vertebrates, with the mammals
forming an exception. The reasons for a shift to a
purely retinal route of photoreception in mammals
have been speculated upon, and a popular

hypothesis is that this is a further manifestation of
the “nocturnal bottleneck,” thought to have led
to reduced color vision among the mammals
(Menaker et al., 1997).
Sites of extra-retinal photoreception in the
brains of nonmammals include the pineal gland
as well as periventricular deep brain sites. Evi-
dence from birds and reptiles makes clear that
pineal production of melatonin plays a major role
in circadian coordination in nonmammalian
vertebrates (Underwood, 1990) which appears to
have become greatly attenuated in mammals (cir-
cadian effects of melatonin can be observed in
mammals, particularly in the fetal and neonatal
period (Davis, 1997), but in adults are largely
redundant, and loss of pineal melatonin secretion
does not lead to gross deficits in circadian func-
tion). Instead, the restriction of photoreception
to the retina in mammals has led to the
suprachiasmatic nucleus (SCN) assuming a domi-
nant role in coordinating circadian biology, and a
subordination of pineal gland as one output path-
way from the retinally entrained SCN pacemaker.
This framework predicts a decline in the extent of
direct control of circadian physiology through
melatonin responsiveness, and this is borne out
by our current understanding of melatonin recep-
tor biology in different vertebrate groups.
In the brains of birds (e.g., Siuciak et al., 1991),
fishes (e.g., Gaildrat and Falcón, 2000), and reptiles
(e.g., Larson-Prior et al., 1996), analysis of melato-
nin receptor distribution by in vitro autoradiogra-
phy, using the radioligand [125I]-2-iodo-melatonin
(IMEL) reveals very widespread expression of
binding, whereas in mammals, IMEL binding in a
given species shows a highly restricted distribution,
and in adult individuals, high levels of binding are
only observed consistently in the PT region of the
pituitary. Analysis of melatonin receptor RNA
expression by in situ hybridization serves to further
emphasize this point (e.g., compare Reppert et al.,
1995 with Reppert et al., 1994). Hence, in mammals,
the reorganization of light sensing in the brain has
led to a restriction of melatonin action to the point
where the only biology for which melatonin is
419
essential appears to be the seasonal photoperiodic
response. Why this is so remains unclear, but I am
tempted to suggest that it reflects an evolutionary/
developmental constraint within the photoperiodic
response circuit: it may be noteworthy that the
cell types producing TSH and Dio2/Dio3 are non-
neural in nature and possibly addressable only via
a humoral signal as opposed to a neural efferent
pathway.
Divergent evolution of melatonin receptors
The loss of extra-retinal photoreception appears
also to have been associated with a major change
in the structural evolution of melatonin receptor
subtypes. Three melatonin receptor subtypes
have been characterized and designated MT1
(previously Mel1a), MT2 (previously Mel1b),
and Mel1c. Of these, MT1 and MT2 are present
in all vertebrate groups, while Mel1c is found only
in nonmammalian vertebrates (Reppert et al.,
1996a). Contrastingly, peculiar to mammals is
the orphan melatonin-related receptor (gpr50),
which does not bind melatonin in in vitro expres-
sion assays and remains a puzzle from a func-
tional perspective (Reppert et al., 1996b).
Recently by synteny mapping, it was shown that
gpr50 is the mammalian orthologue of Mel1c
(Dufourny et al., 2008). Rapid evolutionary diver-
gence from the ancestral Mel1c appears to have
occurred with the branching of the mammalian
lineage, and in the duck-billed platypus, a Mel1c-
like Mel1c/gpr50 is found. The underlying causes
for this divergence are unclear but are most simply
interpreted as a consequence of a major chromo-
somal rearrangement, leading to gpr50 residing
near the end of the long arm of the X-chromosome
in eutherian mammals. X-chromosomal genes are
typically associated with sequence instability, and
the syntenic neighbors of gpr50 show a similar
rapid evolution (Dufourny et al., 2008). Despite
this, gpr50 shows highly localized expression in
the mammalian brain, consistent with maintained
function, and evolutionary sequence analysis
420

suggests that gpr50 has been subject to directional appear to link grp50 to metabolic regulation,
selection in mammals, rather than just neutral drift rather than directly to the photoperiodic
on an evolutionary pathway to a loss of function response, and this insight may help with the
(Dufourny et al., 2008). continuing search for ligands for gpr50.

A link between gpr50 and energy homeostasis Concluding remarks

These reflections get us little closer to establishing Photoperiodic seasonal responsiveness is an
a function for gpr50 in mammals or for identifying ancestral attribute of vertebrates, and its mecha-
possible ligands through which it may exert a nistic origins appear to lie in a thyroid signaling
signaling function. One school of thought, pro- pathway reflecting seasonal feast or famine,
pounded by Levoye et al. (2006), is that gpr50 acts coupled to a light-sensing pathway involving
as a dimerization partner for mammalian melato- retinal and extra-retinal photoreceptors, the cir-
nin receptors, thereby modulating melatonin signal cadian clock and melatonin. We are getting
transduction in a ligand-independent manner. A close to an integrated framework of understand-
difficulty with this hypothesis is the rather limited ing how these elements fit together, and how
overlap between melatonin receptor and gpr50 variant arrangements have evolved among the
expression, the former being concentrated in the vertebrates. Proper understanding of the role
PT, the latter showing the strongest expression in played by gpr50 in mammals will be necessary
brain sites in which melatonin receptor expression to complete this framework.
is weak or absent (Drew et al., 2001; but see also
Sidibe et al., 2010).
In the hypothalamus, gpr50 is expressed in
sites that have been linked to energy homeosta- Abbreviations
sis (e.g., dorsomedial hypothalamus and para-
ventricular hypothalamus) (Drew et al., 2001). Dio2, Dio3 type 2, type 3 deiodinase
Intriguingly, in rodents, gpr50 is also expressed Eya3 eyes absent 3
in the ependymal cell layer lining the wall of gpr50 orphan G-protein-coupled recep-
the third ventricle, overlapping the expression tor 50/melatonin-related receptor
distribution for Dio2 and Dio3 RNA. In Siberian IMEL 2-iodo-melatonin
hamsters (Phodopus sungorus), exposure to Mel1c type Mel1c melatonin receptor
short photoperiods leads to a loss of body fat as MT1 type 1 melatonin receptor
well as a reproductive switch off. At the same MT2 type 2 melatonin receptor
time, levels of grp50 are reduced in the medi- opn4x xenopus-type melanopsin
obasal hypothalamus (Barrett et al., 2006), while opn5 opsin 5
gpr50 null mice show a lean phenotype, with an PD pars distalis
unusual tendency to express torpor in response PT pars tuberalis
to food restriction (Ivanova et al., 2008). Inter- SCN suprachiasmatic nucleus
estingly, in sheep, a photoperiodic species in T4 thyroxine
which dramatic changes in adiposity are not a Tef thyrotroph-embryonic factor
feature, we have not observed photoperiodic TRH thyrotropin-releasing hormone
control of hypothalamic gpr50 expression TSH thyroid-stimulating hormone
(E. Krol, C.A. Wyse and D.G. Hazlerigg unpub- TSHb TSH beta subunit
lished observations). Collectively, these findings VA opsin vertebrate ancient opsin

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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 24

A kiss for daily and seasonal reproduction

Valérie Simonneaux*, Isabelle Bur, Caroline Ancel, Laura Ansel and Paul Klosen

Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Integratives, Strasbourg, France

Abstract: Reproduction is a fundamental biological function ensuring individual descendant survival

and species perpetuity. It is an energy-consuming process, and therefore, all underlying mechanisms
have to work in synchrony to ensure reproductive success. Synchronization of reproductive activity
with the best time of the day or the year is part of such adaptive processes. Recently, a neuropeptide
named kisspeptin, synthesized in two discrete hypothalamic nuclei, the anteroventral periventricular
nucleus and the arcuate nucleus, has been demonstrated to be a potent stimulator operating upstream
of the gonadotropic axis. In this review, we show how kisspeptinergic neurons integrate daily and
seasonal time cues to synchronize reproductive activity with the cycling environment.

Keywords: circadian clock; season; melatonin; reproduction; kisspeptin; RFRP; thyroid hormone;
hamster.

Hypothalamic kisspeptinergic neurons act drives the secretion of gonadotropins, luteinizing

upstream of the gonadotropic axis
In mammals, early studies on the regulation of the
reproductive axis have emphasized the pivotal role
of the pulsatile release of gonadotropin-releasing
hormone (GnRH), a decapeptide synthesized by
a few neurons (about 1000 in mice) located in the
rostral hypothalamus, mainly the preoptic area,
and released principally into the pericapillary
space of the pituitary portal system at the median
eminence (Hahn and Coen, 2006). GnRH further
*Corresponding author.
Tel.: þ33-3-88-45-66-71; Fax: þ33-3-88-45-66-54
E-mail: simonneaux@inci-cnrs.unistra.fr
hormone (LH) and folliculo-stimulating hormone
(FSH), by the pituitary, which in turn controls
gametogenesis and the production of sex steroids
by the gonads. Finally, sex steroids feed back on
the gonadotropic axis to modulate reproductive
activity (Witkin et al., 1982).
Recently, it has been discovered that GnRH syn-
thesis and release require the stimulatory action of
the hypothalamic peptide kisspeptin (Kp). Indeed
loss-of-function mutations of the Kp receptor
(KiSS1R/GPR54) in humans and rodents (de Roux
et al., 2003; Seminara et al., 2003) or the Kiss1 gene
coding for Kp (d’Anglemont de Tassigny et al.,
2007) prevent pubertal development and cause
infertility. Interestingly, genetic ablation of Kiss1

http://dx.doi.org/10.1016/B978-0-444-59427-3.00024-1 423
424
neurons or Kp-responsive neurons does not pre-
vent puberty onset or reproductive ability,
indicating that Kp signaling is only required when
this neuronal circuitry is present (Mayer and
Boehm, 2011). In mammals, the Kiss1 gene is
translated into a 145-amino acid (aa) precursor fur-
ther processed into Kp fragments of smaller sizes
(from 54 aa in humans—also named metastin—or
52 aa in rodents down to 10 aa). All fragments
share the same 10 C-terminus aa sequence with a
final RF-amidated motif, a hallmark of the
RFamide super family of peptides (Kotani et al.,
2001). The common 10 C-terminus aa sequence is
sufficient to induce a full activation of the KiSS1R
(Mikkelsen and Simonneaux, 2009); however, it is
not yet known what is(are) the naturally occurring
endogenous form(s) of the peptide.
The Kiss1 gene is expressed mainly in two
hypothalamic nuclei, the arcuate (ARC) and the
anteroventro-periventricular (AVPV) nuclei
(Fig. 1), the latter displaying a marked sexual
dimorphism, being larger in females compared
to males (Ansel et al., 2010; Clarkson and
Herbison, 2006; Gottsch et al., 2004; Smith et al.,
2005a,b). Kp neurons project to a few brain areas,
specifically to the preoptic area onto the GnRH
neurons (Clarkson and Herbison, 2006; Clarkson
et al., 2009; Desroziers et al., 2010; Kinoshita
et al., 2005; Mikkelsen and Simonneaux, 2009).
Low doses (from 1 fM) of Kp can potently induce
GnRH (Messager et al., 2005) and thus LH/FSH
(Gottsch et al., 2004) release. Indeed 80–90% of
GnRH neurons express c-FOS and are
depolarized after Kp administration (Han et al.,
2005; Irwig et al., 2004). The stimulatory effect
of Kp on GnRH release occurs via its binding to
KiSS1R which is highly expressed in these
neurons. Some experiments suggest that Kp may
also act at the level of GnRH nerve terminals
(Ansel et al., 2011; d’Anglemont de Tassigny
et al., 2008; Han et al., 2005); however, the cellu-
lar localization of the KiSS1R awaits the produc-
tion of a specific antibody.
Strikingly, Kp neurons are the main target for
the positive (in AVPV) and negative (in ARC)
AVPV ARC
Kiss1 Kiss1
neuron neuron
+ -
GnRH
neuron
Sex steroids
Sex steroids
Median
eminence
Gonadotrope
cell
Gonads
Fig. 1. Schematic representation of the mammalian
gonadotropic axis as currently accepted. Kiss1-expressing
neurons, located in two hypothalamic nuclei, AVPV
(anteroventro-paraventricular) and ARC (arcuate), project to
the GnRH neurons to induce the release of GnRH which
in turn stimulates the production of gonadotropins from
the pituitary. Sex steroids exert a positive (AVPV) or
negative (ARC) feedback on Kiss1-expressing neurons.
central feedback effects of sex steroids underlying
the tightly controlled positive/negative feedback
loops of the reproductive axis (Ansel et al.,
2010; Revel et al., 2006a; Smith et al., 2005a,b).
In addition, recent studies have reported that Kp
neurons integrate other factors, especially those
known to impact on reproductive activity, like
metabolic factors (Castellano et al., 2010).
Recently, we and others have reported that Kp
neurons integrate time of the day and time of
the year information to adjust reproduction to
the cycling environment (Clarke et al., 2009;
Khan and Kauffman, 2012; Revel et al., 2007;
Simonneaux et al., 2009).
Living on earth indeed imposes daily and annual
changes in light, temperature, and hygrometry,
which in turn cause cyclic changes in food quantity

and quality. Therefore, reproductive activity
should anticipate and adapt to these predictable
changes such that pups are born when food
resources are optimal for their survival. Mammals
have a dedicated photoneuroendocrine pathway
converting the physical light information into
nervous and endocrine signals which synchronize
biological functions with the environment
(Simonneaux and Ribelayga, 2003). Briefly, this
pathway involves a master biological clock located
in the suprachiasmatic nuclei (SCN). Circadian
clock activity is synchronized to the 24 h light/
dark cycle by the retino-hypothalamic pathway
originating in a specific set of melanopsin-
expressing ganglion cells of the retina. Impor-
tantly, the circadian clock also integrates annual
changes in photoperiod since the clock’s daily
activity is modulated according to the light/dark
ratio. Timing information built in the hypothalamic
biological clock is mainly forwarded to other hypo-
thalamic nuclei, which in turn set various biological
activities in phase with the time of the day and year
(Buijs and Kalsbeek, 2001; Kalsbeek et al., 2006).
As far as reproduction is concerned, two clock-
driven pathways have been reported to impact on
gonadotropic activity: a direct SCN nervous output
involving vasopressin (VP)- and vasoactive intesti-
nal peptide (VIP)-containing neurons and an indi-
rect SCN-driven endocrine pathway involving the
rhythmic release of the pineal hormone melatonin.
Recent reports have demonstrated that Kp
neurons are set at the interface of these clock out-
puts and the GnRH neurons to synchronize repro-
ductive activity with the time of the day and year.
The kisspeptinergic neurons of the AVPV
integrate circadian information to time the
preovulatory LH surge
In female rodents, the estrous cycle comprises
four stages of activity: metestrus, diestrus, proes-
trus, and estrus (the stage when ovulation occurs),
and lasts 4–5 days. Proestrus is characterized by a
marked and transient increase in LH, which
425
further stimulates ovulation at the estrus stage.
During the first part of the estrous cycle, cir-
culating levels of estrogen are low and exert a
negative feedback effect (Shupnik, 1996). On the
day of proestrus, the circulating levels of estrogen
increase and the sex hormone exerts a positive
feedback. This stage is a prerequisite to trigger
ovulation. Strikingly, the preovulatory LH surge
occurs only at a specific time window on the day
of proestrus, typically at the end of the afternoon.
It is generally acepted that the occurrence of the
LH surge on the late afternoon of the proestrus
requires the combination of two signals, a daily
signal arising from the hypothalamic clock and a
sufficient circulating level of estrogen. Recent
studies suggest that the Kp neurons of the AVPV
are the cellular site integrating these two signals
for the GnRH-induced LH surge (Fig. 2).
The estrogen signal
Estrogen binds specific receptors acting via genomic
(ERa and ERb) or nongenomic (GPR30) pat-
hways, ERa mediating the positive retroactive
effect of estrogen in the brain (Wintermantel et al.,
2006). ERa, however, are not located on GnRH
neurons but rather on the Kp neurons of the AVPV
(Smith et al., 2006), and various experiments of
gonadectomy or hormone administration have
demonstrated that Kiss1 expression in female
AVPV is strongly upregulated by estrogen (Ansel
et al., 2010; Smith et al., 2005a). In addition, central
administration of Kp antibody or KiSS1R antago-
nist prevents the preovulatory LH surge (Kinoshita
et al., 2005; Pineda et al., 2010a). Altogether these
observations demonstrate that estrogen exerts its
positive feedback via the Kiss1 neurons of the
AVPV to trigger the LH surge (Fig. 2).
The circadian signal
There is strong evidence that the circadian clock
governs the estrous cycle and more specifically
426

VP
neuron AVPV E2
Kiss1
VP neuron

SCN

Kp
POA
VIP
VIP GnRH
neuron neuron

GnRH

Gonadotrope E2
cell
LH secretion

LH surge

Metestrus Diestrus Proestrus Estrus

Ovarian E2 secretion

Fig. 2. Schematic representation of the circadian and estrogen signals controlling the LH surge in female rodents. Vasopressin (VP)
and vasoactive intestinal peptide (VIP) neurons located in the suprachiasmatic nuclei (SCN) signal the circadian time to the Kiss1
neurons of the anteroventro-periventricular nuclei (AVPV) and the GnRH neurons of the preoptic area (POA), respectively. The
coincidence of high circulating levels of estrogen (E2) and the circadian signal induces a surge of LH in the afternoon of the
proestrus.

the timing of the preovulatory LH surge in
rodents. First, female mice with mutated circadian
clock genes that are involved in encoding daily
biological time present alterations in reproductive
capacities (Miller et al., 2004; Pilorz and
Steinlechner, 2008; Ratajczak et al., 2009). For
example, the Clock/Clock mutant female mice
present irregular estrous cycles, and this alter-
ation is associated with a suppression of the LH
surge and ovulation (Miller et al., 2004). Second,
neuroanatomical studies have reported that SCN
ablation suppresses the LH surge (Antunes-
Rodrigues and McCann, 1967), impairs the
estrous cycle, and leads to an anovulatory condi-
tion characterized by a persistent estrous stage
(Brown-Grant and Raisman, 1977; Stetson and
Watson-Whitmyre, 1976; Wiegand et al., 1980).
Third, SCN outputs toward GnRH and Kp
neurons have been described (de la Iglesia and
Schwartz, 2006; Williams et al., 2011), and two
SCN transmitters, VIP and VP, have been
reported as putative circadian signals to the
reproductive system (Khan and Kauffman,
2012). The current concept is that VIP can
directly stimulate GnRH neurons’ firing in a
time-dependent manner, and VP neurons project
directly to the AVPV Kp neurons. About 50%
of the AVPV Kp neurons receive SCN VP fibers
(Vida et al., 2010) and the daily pattern of VP
synthesis and release matches Kp neuron activity
with a peak in the late afternoon (Shinohara
et al., 1994). Moreover, VP injection in the
AVPV area induces an LH surge, even in SCN-
lesioned animals (Palm et al., 1999), and

conversely, infusion of a VP antagonist on the day
of proestrus prevents the LH surge (Funabashi
et al., 1999). Although no experiments have yet
formally proven that VP regulates Kp synthesis
and/or release in the AVPV neurons, it is tempt-
ing to speculate that the Kp neurons of the
AVPV represent a key interface to integrate the
circadian signal, in addition to the estrogen signal,
to time the LH surge in the late afternoon of the
proestrus (Fig. 2).
A circadian oscillator in Kp neurons?
Interestingly, recent findings demonstrate that
AVPV Kp neurons also exhibit a robust circadian
pattern of gene expression and neuronal activa-
tion coincident with the circadian pattern of LH
(Robertson et al., 2009). Indeed, ovariectomized
female mice implanted with an estrogen implant
(OVX þ E2) showed a robust surge of LH 1 h
before the subjective night (CT11 and 12),
whereas low levels of LH were detected at the
other time points. In synchrony with this LH
secretion, a strong circadian pattern with higher
Kiss1 and c-Fos mRNA levels at CT11 and 12
was observed in the AVPV. Moreover, in intact
female mice, c-Fos and Kiss1 mRNA levels are
significantly increased in Kp neurons in the late
afternoon of the proestrus day coincident with
the LH surge (Bur et al., unpublished; Smith
et al., 2006). These findings demonstrate that cir-
cadian-timed neuronal activation of Kp neurons
in the AVPV is involved in the late afternoon
preovulatory LH surge. The circadian signal may
arise from VP-containing SCN outputs; however,
it may also come from an intrinsic autonomous
circadian clock located in AVPV Kp neurons.
Recent studies revealed that circadian clock gene
expression in neuroendocrine structures interferes
with their rhythmic behavior (Bonnefont, 2010).
Thus, clock genes and proteins are rhythmically
expressed in the immortalized GnRH-secreting
GT1-7 cell line, and these cells exhibit daily
changes in peptide expression and GnRH
427
secretion in response to Kp and VIP (Gillespie
et al., 2003; Zhao and Kriegsfeld, 2009). Interest-
ingly, a recent study reported that a circadian
transcription factor, albumin D-site binding pro-
tein (Dbp), is able to induce Kiss1 transcription
in AVPV Kp neurons (Xu et al., 2011). However,
further experiments are necessary to clarify the
existence of a circadian machinery in the AVPV
Kp neurons, which may govern the rhythmic
expression of Kiss1 and other rhythmic cues.
RFRP-3, another member of the RFamide family
of peptides, in the game
Recently, another member of the RFamide family
of peptides, RFRP-3, was proposed to be
involved in the circadian regulation of the female
reproductive cycle (Gibson et al., 2008). The Rfrp
gene, the mammalian ortholog of avian gonado-
tropin inhibitory hormone (GnIH), encodes a pre-
cursor that produces two peptides, RFRP-1 and
RFRP-3 (Clarke et al., 2008; Fukusumi et al.,
2001; Hinuma et al., 2000; Kriegsfeld et al., 2006;
Ukena and Tsutsui, 2005; Ukena et al., 2002;
Yoshida et al., 2003). RFRP neurons are located
in the dorsomedial nucleus of the hypothalamus
(DMH) and fiber networks are found in multiple
brain regions and particularly on GnRH neurons
(Johnson et al., 2007; Mason et al., 2010; Ukena
and Tsutsui, 2001). A large body of evidence
now indicates that both central and peripheral
administration of RFRP-3 inhibits LH secretion
(Anderson et al., 2009; Clarke et al., 2008; John-
son et al., 2007; Kadokawa et al., 2009; Kriegsfeld
et al., 2006; Pineda et al., 2010b). RFRP-3
neurons express ERa, suggesting that they may
mediate estrogen feedback (Kriegsfeld et al.,
2006). Estrogen downregulates Rfrp mRNA
expression in ovariectomized mice (Molnar et al.,
2011) and RFRP-3 cell numbers as well as c-FOS/
RFRP-3 coexpression are reduced in proestrus
Syrian hamsters, relative to diestrus (Gibson
et al., 2008). These results indicate that estrogen
downregulation of the RFRP-3 system alleviates
428
inhibition of GnRH release during the LH surge.
Interestingly, in rats and ewes, there are no
changes in Rfrp expression following alterations
in estradiol levels (Quennell et al., 2010; Smith
et al., 2008), indicating that there may be species
differences in the involvement of RFRP neurons
in estrogen-feedback mechanisms. Little is known
about the interaction between the SCN and the
RFRP-3 system. In rodents, both VP- and VIP-
positive fibers from the SCN project to the
DMH; however, it is not known which of these,
if any, innervate RFRP-3 cells (Abrahamson and
Moore, 2001; Gibson et al., 2008; Kalsbeek
et al., 1993). Taken together, these data indicate
that, in some species, RFRP-3 may play a role in
the regulation of the estrus cycle, notably in
the estrogen-dependent feedback mechanisms.
However, whether and how this action is con-
trolled by the circadian system remains to be
investigated.
The kisspeptinergic neurons of the ARC nucleus
integrate photoperiod/melatoninergic information
to time seasonal reproduction
Most animals living in the wild restrict fertility to a
particular time of the year to ensure the birth and
weaning of the offspring during the most favorable
season. Although various environmental factors
are important to take into consideration, a majority
of species use the highly predictable annual
variations of light duration (or photoperiod) to
establish the time of year. Since the pioneer studies
of Reiter and Hoffman in the 1960s, it is well
established that the annual/photoperiodic rhythm
in nocturnal melatonin is critical to synchronize
reproduction with the seasons (Bartness et al.,
1993; Hoffman and Reiter, 1965; Pitrosky and Pevet,
1997). Thus, Syrian hamsters raised in long day
(LD, 14 light/10 h dark) conditions are sexually
active and, upon exposure to short day (SD, 10
light/14 h dark) conditions for 8–10 weeks, undergo
a dramatic inhibition of reproductive activity with
low levels of circulating gonadotropins and a
marked involution of the gonads and accessory
organs resulting in low levels of circulating sex ster-
oids. Surgical removal of the pineal gland before
exposure to SD prevents hamsters from undergoing
sexual inactivation. Conversely, exogenous melato-
nin injections mimicking SD conditions in hamsters
raised in LD induce sexual inactivation. Finally,
when SD exposure is prolonged over 25–30 weeks,
hamsters become refractory to the inhibitory
melatoninergic signal and undergo a complete
endogenous reactivation of the reproductive system.
Strikingly, not all seasonal species respond to the
photoperiodic changes the same way, and this is
related to the length of gestation time. Thus, in
sheep, where gestation lasts approximately 5
months, reproduction is stimulated in SD conditions
and becomes quiescent as days become longer. As a
consequence, they are regarded as SD breeders,
while hamsters are LD breeders. However, in
both photoperiodic models, the seasonal rhythm
of reproduction is synchronized by changes in
nocturnal melatonin peak duration. In the Syrian
hamster, an increase in melatonin peak duration
inhibits reproductive activity, whereas in sheep,
the same melatoninergic information stimulates
reproduction.
Although the role of melatonin in synchronizing
seasonal reproduction is indisputable, the cellular
and molecular sites of action of melatonin are still
not well understood. It is clear that melatonin
does not act directly on GnRH neurons and
responsiveness to GnRH does not change with pho-
toperiod (Urbanski et al., 1991). Melatonin-binding
sites have been identified in a number of brain
structures but with considerable species differences
(Masson-Pevet et al., 1994). Lesion or melatonin-
infusion studies at various anatomical sites
suggested that the mediobasal hypothalamus is
involved in melatonin action (Malpaux et al., 1993;
Maywood and Hastings, 1995). Besides, a high den-
sity of melatonin receptors has been identified in the
pars tuberalis of the adenohypophysis of a number
of mammalian species (Masson-Pevet and Gauer,
1994), pointing to this structure as a critical photope-
riodic target for melatonin action.

Kp neurons as gatekeepers of seasonal
reproduction
The potent stimulatory effect of Kp on GnRH
and gonadotropin release, the observation that
the sexual phenotype of KiSS1R-mutated mice
resembles that of hamsters exposed to SD, and
the localization of Kiss1-expressing neurons in
the mediobasal hypothalamic area were all strong
arguments to test, in the Syrian hamster, the
hypothesis that photoperiod/melatonin could reg-
ulate seasonal reproduction via an action on
Kiss1-expressing neurons (Ansel et al., 2010,
2011; Revel et al., 2006a, 2007).
We first observed that Kiss1 mRNA and Kp-
immunoreactivity are found in neurons of the
ARC and AVPV of the adult male Syrian
hamsters, similar to other reports in rats and
mice. When Syrian hamsters were kept for 8–10
weeks in SD, in parallel to the expected impor-
tant reduction in testis size and circulating levels
of testosterone, the number of Kiss1-expressing
neurons was markedly reduced in both nuclei. In
the ARC, the SD-induced reduction in Kiss1
expression was not the result of decreased levels
of testosterone since castration of LD Syrian
hamsters led to a significant increase in Kiss1
mRNA levels in the ARC. This is in agreement
with an inhibitory effect of sex hormones on Kiss1
expression in the ARC, as already reported in
other rodent species. In contrast, we observed
that the removal of endogenous melatonin by
pinealectomy in SD-exposed Syrian hamsters
increased ARC Kiss1 mRNA and reactivated
reproductive activity within a few weeks (Fig. 3a).
Conversely, late afternoon melatonin injections in
sexually active Syrian hamsters kept in LD signif-
icantly reduced ARC Kiss1 mRNA levels together
with a marked reduction in testicular activity
(Fig. 3b). It is important to note that melatonin
treatment had to be carried out for at least 5
weeks to be effective. In the AVPV, the level of
Kiss1 mRNA was also reduced in SD compared
to LD, but the underlying mechanisms are differ-
ent. Indeed, in this structure, testosterone
429
(a)
Total number of Kiss1 neurons
250
200 *
% of control
150
100
50
0
SD SD + PinX
(b)
Total number of Kiss1 neurons
150
% of control
100
*
50
0
LD LD + Mel
Fig. 3. Melatonin regulation of Kiss1-expressing neurons in
the arcuate nucleus of male Syrian hamsters. (a) Hamsters
were pinealectomized (PinX) or sham-operated and kept in
short day conditions (SD) for 10 weeks. The number of
Kiss1-expressing neurons (1) was higher in PinX, sexually
active, hamsters. (b) Hamsters were raised in long day (LD)
conditions and injected daily with ringer or melatonin (50 mg,
100 ml/animals; 2 h before lights off) for 8 weeks. The
number of Kiss1-expressing neurons (measured by in situ
hybridization) was lower in melatonin-injected, sexually
inactive hamsters. Values are expressed as percent of control;
*p < 0.05 as compared to control hamsters.
stimulates Kiss1 gene expression; therefore, the
SD-induced decrease in Kiss1 mRNA is a conse-
quence of melatonin/SD-induced reduction in
the circulating level of testosterone. These
findings strongly suggest that the long nocturnal
peak of melatonin in SD inhibits reproductive
activity via a sex steroid-independent (ARC) or
a sex steroid-related (AVPV) inhibition of Kiss1
expression in the male Syrian hamster. Strikingly,
when Syrian hamsters became refractory to the
inhibitory melatoninergic signal (kept over 25
430
weeks in SD), the spontaneous sexual reactiva-
tion was associated with an increase in Kiss1
mRNA in the ARC, indicating that Kiss1 expres-
sion also escapes melatonin’s inhibitory signal in
photorefractory animals. We further tested the
hypothesis that the SD-driven reduction in hypo-
thalamic Kp may be responsible for the inhibition
of reproductive activity (Ansel et al., 2011; Revel
et al., 2006a). Thus, sexually inactive Syrian
hamsters, kept in SD, received a chronic intra-
cerebroventricular infusion of 0.25 nmol/h Kp10
or vehicle for 4 weeks. Remarkably, the Kp-
treated animals underwent a strong reactivation
of testicular activity and increased levels of cir-
culating testosterone, in marked contrast to the
vehicle-treated hamsters. This gonadal response
was of similar amplitude to that observed in SD
hamsters transferred back to LD on the day of
surgery. Acute peripheral (intraperitoneal, intra-
venous, or subcutaneous) administration of Kp is
known to stimulate reproductive activity almost
as efficiently as an acute central infusion
(Mikkelsen et al., 2009; Thompson et al., 2004).
In line with this observation, we found that twice
daily intraperitoneal injections of Kp54 in SD-
inhibited hamsters could restore testicular activity
as well. Altogether, our data demonstrate that, in
Syrian hamsters, melatonin in SD inhibits Kp
expression in the ARC and AVPV which in turn
downregulates reproductive activity (Fig. 4).
Photoperiodic variation of Kp expression was
reported in other seasonal mammals. In male
Siberian hamsters kept in SD, Kiss1 gene expression
is reduced in the AVPV but, unexpectedly, higher in
the ARC compared to LD (Mason et al., 2007;
Simonneaux et al., 2009). In Siberian, like in Syrian
hamsters, testosterone inhibits Kiss1 expression in
the ARC; thus it is possible that the reduced levels
of testosterone in SD are sufficient to explain the
increase in Kiss1 expression in the Siberian hamster,
whereas in the Syrian hamster, this effect would be
blunted by the strong inhibitory effect of melatonin.
Acute Kp injection in the male Siberian hamster
triggers LH release, but chronic subcutaneous
administration of Kp in photoinhibited hamsters
appears unable to reactivate the reproductive axis
(Greives et al., 2008). Whether this discrepancy is
due to differences in dose, route of administration
or species is being explored in our laboratory.
The effect of photoperiod/season on Kiss1
expression has also been examined in the ewe
(Caraty et al., 2007; Franceschini et al., 2006; Smith
et al., 2007, 2009). Kiss1-expressing neurons are
found in the ARC, and Kiss1 mRNA levels are
increased during the breeding season in SD. Kp
infusion during the anestrous season caused ovula-
tion in more than 80% of treated animals, whereas
less than 20% of control animals ovulated.
This suggests that, similar to hamsters, a steroid-
independent seasonal modulation of Kiss1 expres-
sion occurs in the ARC of sheep to drive seasonal
reproduction. However, whether this effect on Kp
neurons is also controlled by melatonin has not been
demonstrated. If melatonin is indeed driving sea-
sonal changes in Kiss1 expression in the sheep as it
does in the Syrian hamster, it will be a major chal-
lenge to determine why a long SD melatonin peak
inhibits Kiss1 expression in Syrian hamsters and
increases it in sheep. This may help to identify the
long-sought-after mechanisms underlying the differ-
ence between LD and SD breeders. Most probably,
melatonin is not acting directly on ARC neurons (Li
et al., 2011; Maywood et al., 1996), and therefore,
melatonin-sensitive system(s), operating upstream
of the Kiss1 neurons, have to be discovered. Two
candidates, the RFRP neurons, located in the
DMH, and the thyroid stimulating hormone
(TSH)-regulated deiodinases, located in the
tanycytes of the basal hypothalamus, may be the
missing link(s) between melatonin and the Kp-
regulated seasonal reproduction.
RFRP neurons: A missing link between
melatonin and Kiss1 neurons?
Because melatonin receptors have been identified
in the Syrian hamster DMH, and because lesions
of this same area prevent SD-induced testicular
regression (Maywood and Hastings, 1995), we
 431

RFRP neuron

DMH

T3 TSHR
Dio2/3
Kiss neuron T4
Kiss neuron

tanycytes 3V

AVPV ARC
cap

TSH
GnRH neuron

cap

cap
Melatonin
CH3
TSH
CH3O
CH2 CH2 N
H
C
O
MeIR pars tuberalis
N

Fig. 4. Schematic representation of a putative hypothalamic T3/RFRP/Kp network controlling seasonal reproduction in mammals.
Melatonin binding to its receptors located in the pars tuberalis regulates TSH release. TSH in turn binds to its receptors located on
the tanycyte cell bodies lining the basal part of the third ventricle to regulate diodinases 2/3 activities and therefore local metabolism
of the thyroid hormones T4 and T3. Kiss1 neurons located in the anteroventro-periventricular (AVPV) and arcuate (ARC) nuclei
project to GnRH cell bodies and nerve terminals to stimulate GnRH release in the portal blood at the median eminence. RFRP-
expressing neurons located in the dorsomedial hypothalamus (DMH) also project to the GnRH neurons, and possibly to the
Kiss1 neurons. Notably, melatonin in short photoperiod downregulates expression of Dio2, Rfrp, and Kiss1 genes in the Syrian
hamster. Additionally, melatonin regulates the plasticity of the tanycytes’ end feet in the median eminence.

hypothesized that RFRP neurons might be the
missing link between melatonin and Kiss1. We
subsequently demonstrated that, in Syrian and
Siberian hamsters, the level of Rfrp mRNA
and the number of RFRP-immunoreactive cell
bodies were reduced in sexually quiescent animals
acclimated to SD compared with sexually active
animals maintained under LD (Revel et al.,
2008). The reduction in Rfrp expression in
SD was independent from secondary changes
in gonadal steroids and was abolished in
pinealectomized hamsters, whereas injections of
LD hamsters with melatonin reduced Rfrp expres-
sion down to SD levels (Revel et al., 2008),
indicating that the regulation is dependent on mel-
atonin. We also found that central administration
of RFRP-3 in sexually active male Syrian hamsters
produced a significant increase in both LH and tes-
tosterone plasma concentrations (Ancel et al.,
2012). Whereas this result is in complete contra-
diction with the available data in the literature, it
is in accordance with the observation that Rfrp
432
expression is higher in sexually active Syrian
hamsters relative to sexually quiescent hamsters.
We also observed that a 5-week-long central infu-
sion of RFRP-3 to sexually quiescent SD male
Syrian hamsters restored testicular activity (Ancel
et al., 2012), similarly to Kp (Revel et al., 2006a).
Remarkably, we found that the administration of
RFRP-3 induced a significant increase in Kiss1
mRNA levels in the ARC (Ancel et al., 2012).
Taken together, these data indicate that RFRP-3
stimulates the reproductive function in the male
Syrian hamster, likely via the Kiss1 neurons of
the ARC (Fig. 4).
TSH-driven regulation of tanycyte deiodinases is
central for seasonal processes
Thyroid hormones have long been known to be
involved in seasonal reproduction (Karsch et al.,
1995), but only quite recently the underlying
mechanisms and their relationship with melatonin
started to be understood (Fig. 4). First, in European
hamsters, cells of the pars tuberalis containing mela-
tonin receptors synthesize TSH in a photoperiod-
dependent manner with lower levels in SD
conditions (Dardente et al., 2003) and the molecular
signaling mechanism of melatonin on TSH expres-
sion was recently described in sheep pars tuberalis
(Dardente et al., 2010). Second, deiodinases
2 (Dio2) and deiodinases 3 (Dio3), the enzymes
involved in the local metabolism of the thyroid hor-
mones T4 and T3, are expressed specifically in
tanycytes, a specific subset of glial cells lining the
basal part of the third ventricle, in a photoperiod-
dependent manner in seasonal breeders (quail
(Yoshimura et al., 2003), Syrian hamster (Revel
et al., 2006b), Siberian hamster (Barrett et al.,
2007; Watanabe et al., 2004), European hamster
(Hanon et al., 2010), sheep (Hanon et al., 2008),
and goat (Yasuo et al., 2006)). Notably, this photo-
periodic regulation is also present in some mice
strains, which are not seasonal breeders (Yasuo
et al., 2009). Third, TSH secreted by the pars
tuberalis stimulates Dio2 activity via TSH receptors
located on the tanycytes (Hanon et al., 2008; Nakao
et al., 2008). Fourth, chronic central infusion of T3
reactivates photoinhibited reproductive activity in
the quail (Yoshimura et al., 2003) and Siberian ham-
ster (Barrett et al., 2007). Altogether, these recent
findings point to the pars tuberalis TSH/tanycyte
T3 model as a pivotal system for the photoperiodic
control of reproduction. In mammals, the
mechanisms involved in the T3-driven regulation
of reproductive activity are yet unknown. Interest-
ingly, we recently reported in the Siberian hamster
that melatonin displays a profound remodeling of
the tanycyte structure, especially in the median emi-
nence area where the GnRH nerve fibers end
(Bolborea et al., 2011). Because GnRH secretion
is tightly controlled by the tanycytes at their nerve
terminals (Prevot et al., 2003, 2007), it can be
hypothesized that the local production of T3 by
Dio2 may regulate reproductive activity via a modu-
lation of GnRH release at the median eminence
(Yamamura et al., 2006).
Toward a seasonal T3/RFRP/Kp network in the
mediobasal hypothalamus?
In the recent years, the findings that (1) expres-
sion of Kp and RFRP in the mediobasal hypothal-
amus and local thyroid hormone metabolism by
the tanycytes are markedly regulated by melato-
nin/photoperiod in seasonal mammals and (2)
each of these transmitters can modulate repro-
ductive activity have led us to reconsider the neu-
roendocrine control of seasonal reproduction
(Fig. 4). Obviously, the three transmitters play a
critical role in this regulation, but whether they
act independently or not is yet to be established.
Remarkably, Kp is always stimulatory of repro-
ductive activity (regardless of the species tested),
but the lack of melatonin receptors in the rodent
and sheep ARC indicates that Kp neurons are
located downstream of the melatoninergic signal.
By contrast, the RFRP-3 effect on gonadotropin
and sex steroid production is species dependent,
being stimulatory in Syrian and Siberian hamsters

but inhibitory in sheep. RFRP-3 neurons may
therefore be located at a crucial node to translate
the photoperiodic melatoninergic message as
inhibitory or stimulatory of reproductive activity.
Although melatonin-binding sites have been
reported in the DMH area of the Syrian hamster,
whether melatonin receptors are localized on
RFRP-3 neurons remains to be established. Inter-
estingly, we have observed that the testicular
reactivation of photoinhibited Syrian hamsters
by a chronic central infusion of RFRP-3 is coinci-
dent with a significant increase in ARC Kiss1
expression, indicating that RFRP neurons could
be upstream of the Kp neurons for the mela-
toninergic regulation of reproductive activity in
these hamsters. Finally, the pars tuberalis/TSH
control of tanycyte T3 production is set at the first
position to translate the melatoninergic signal
toward the central control of reproduction, but it
is yet unknown where and how T3 exerts this con-
trol. In spite of the great advances made recently
in the understanding of the seasonal control of
reproduction, particularly in identifying the critical
transmitters involved, some pieces of the puzzle
are still lacking to fully understand how photope-
riodic changes in melatonin production synchro-
nize reproduction in seasonal mammals.
Conclusion
The 2003 milestone discovery of the pivotal role
of Kp as a potent stimulator of GnRH release
has helped to understand how the time of the
day and year is integrated in brain structures to
synchronize reproductive activity with the cycling
environment. The latest findings indicate that the
SCN VP output connection with AVPV Kp
neurons is critical to time the LH surge, at least
in rodents. In seasonal species, the ARC Kp
neurons appear to be the final gatekeeper to syn-
chronize reproduction with seasons. It remains to
be discovered what is(are) the system(s) acting
upstream to control the photoperiodic synthesis
of Kp.
433
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A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)
Progress in Brain Research, Vol. 199
ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 25

Circannual rhythm in the varied carpet beetle,

Anthrenus verbasci

Yosuke Miyazaki{, Tomoyosi Nisimura{ and Hideharu Numata},*

{
Faculty of Clinical Education, Ashiya University, Hyogo, Japan
{
College of Bioresource Sciences, Nihon University, Fujisawa, Japan
}
Graduate School of Science, Kyoto University, Kyoto, Japan

Abstract: Although circannual rhythms controlling different physiological processes and various aspects
of behavior have been reported in numerous organisms, our understanding of the underlying biological
mechanisms is still quite limited. We examined the mechanisms controlling the circannual pupation
rhythm of the varied carpet beetle, Anthrenus verbasci. This rhythm is self-sustainable, exhibits
temperature compensation of the periodicity, and is entrainable to environmental changes. In addition,
the circannual phase response curves to a photoperiod pulse display Type 0 or Type 1 resetting,
depending on the duration of the pulse. Thus, we infer that this rhythm is derived from a self-sustaining
biological oscillator with a period of about a year, that is, a circannual clock, analogous to the circadian
clock. Further, a circadian clock appears to mediate photoperiodic time measurement for phase resetting
of the circannual clock. Based on these results and previous research performed in other organisms, we
discuss the general characteristics of the physiological mechanisms underpinning circannual rhythmicity.

Keywords: Anthrenus verbasci; circannual clock; circannual rhythm; circadian clock; entrainment; phase
response curve; photoperiodism; temperature compensation; zeitgeber.

Introduction to enhance the rate of survival and reproduction.

Most organisms have evolved endogenous rhythms
corresponding to various environmental cycles,
including daily cycles, tidal cycles, and annual cycles,
*Corresponding author.
Tel.: þ81-75-753-4073; Fax: þ81-75-753-4113
E-mail: numata@ethol.zool.kyoto-u.ac.jp
When isolated from external temporal cues,
these biological rhythms free-run with an inherent
period close to, but significantly different from,
that of the environmental cycle. Appropriate envi-
ronmental cues, that is, zeitgebers, can entrain
these endogenous rhythms to the environmental
cycle (DeCoursey, 2004; Pittendrigh, 1981a;
Saunders, 2002). The circadian rhythm, which has

http://dx.doi.org/10.1016/B978-0-444-59427-3.00025-3 439
440
an endogenous period close to 24 h and entrains to
daily cycles of light and temperature, has been stud-
ied by many researchers in a variety of organisms.
Chronobiologists have started to uncover the
mechanisms behind the biological clocks that gener-
ate rhythmicity, and today, the basic principles of cir-
cadian clocks are understood in detail (Johnson
et al., 2004; Rosbash, 2009; Saunders, 2002; Stillman
et al., 2007). However, compared to circadian
rhythms, the sources of other endogenous rhythms
are only partly understood, owing to a considerable
lack of research. In particular, it is unclear whether
the mechanism that generates internal periodicity is
a self-sustaining biological oscillator similar to the
circadian clock.
A biological rhythm with an endogenous period
close to a year is called a circannual rhythm.
The circannual rhythms of various physiological
processes and behavioral traits have been reported
in a variety of organisms, including dinoflagellates,
brown algae, higher plants, coelenterates, annelids,
mollusks, crustaceans, insects, fish, reptiles, birds,
and mammals (Goldman et al., 2004; Gwinner, 1986;
Olive and Garwood, 1983). In vertebrates, Pengelley
and Fisher (1957, 1963) were the first to conclusively
demonstrate the circannual rhythm of the golden-
mantled ground squirrel, Spermophilus lateralis.
The seasonal changes in body weight and food con-
sumption, and the occurrence of hibernation, were
shown to have a period shorter than 12 months when
squirrels were maintained for 2 years under constant
photoperiod (12-h light and 12-h darkness, LD 12:12)
and temperature.
In invertebrates, Blake (1958, 1959) first
reported that a circannual rhythm regulates pupa-
tion of the varied carpet beetle, Anthrenus verbasci
(Insecta, Coleoptera, Dermestidae), which nor-
mally has a 2-year life cycle in southern England.
Moreover, she was the first to show the following
major properties of the circannual rhythm (Blake,
1958, 1959, 1960, 1963): (1) a self-sustaining oscilla-
tion with a period deviating significantly from that
of the environmental cycle, (2) temperature com-
pensation of the period length, and (3) the exis-
tence of a zeitgeber entraining the rhythm to the
environmental cycle and advance and delay phase
shifts induced by the stimulus (see also Gwinner,
1986; Saunders, 2002). The importance of Blake’s
work is comparable to the many studies of
circannual rhythms carried out later in other
animals but has not received the attention it
deserves (Saunders, 2002).
Since 1996, we have been examining the pupa-
tion rhythm in a Japanese population of A. verbasci.
In Japan, the duration of the life cycle in this species
is generally considered to be 1 year. In some cases,
however, 2 or 3 years are required to complete
the larval development (Kiritani, 1958; Miyazaki
et al., 2009a). We reevaluated Blake’s results and
introduced various new approaches based on our
current knowledge of circadian and circannual
rhythms and of photoperiodism. We found that
the circannual rhythm of A. verbasci is generated
by a circannual clock analogous to the circadian
clock in its phase response (Miyazaki et al., 2005,
2007; Nisimura and Numata, 2001) and that the
circannual rhythm, with a period of about 40 weeks,
is entrained to an exact year by naturally changing
photoperiods (Miyazaki et al., 2006; Nisimura
and Numata, 2003). Moreover, the circannual clock
is independent of the circadian clock (Nisimura
and Numata, 2002), although a circadian clock is
involved in the time measurement system for phase
resetting of the circannual clock in response to
photoperiodic changes (Miyazaki et al., 2009b).
Menaker (1974) stated that the major difficulty
in the study of circannual rhythms is the ratio of
the period length of a single circannual cycle to
the length of the productive life of the biologist.
Despite this difficulty, a number of researchers
have studied circannual rhythms in a variety
of species (Goldman et al., 2004; Gwinner,
1986, 2003; Lincoln and Hazlerigg, 2010; Paul
et al., 2008; Wikelski et al., 2008). In this review,
based on our results obtained in A. verbasci and
those from studies on various other organisms,
we discuss the general characteristics of the
physiological mechanism producing circannual
rhythmicity and its responsiveness to photope-
riodic changes.

Characteristics of the circannual rhythm
Self-sustainability
Biological rhythms exhibit self-sustainability
when examined under constant conditions with-
out input from environmental cues (Johnson
et al., 2004; Pittendrigh, 1981a; Saunders, 2002).
A constant 24-h light–dark cycle has been used
routinely to detect circannual rhythms because
unchanging photoperiods contain no information
about the duration of a year (Gwinner, 1986).
Figure 1a shows the frequency of pupation times
of A. verbasci when larvae were reared under
LD 12:12 at 20  C. These insects exhibited rhyth-
mic pupation. Larvae pupated 23–31 weeks after
hatching as the first pupation group. The second
group pupated 49–74 weeks after hatching.
80 (a)
60
40
Number of pupae
20
0
60 (b)
40
20
0 light in flies (Pittendrigh, 1966; Saunders, 2002).
0 20 40 60 80
Weeks after hatching
Fig. 1. Pupation of Anthrenus verbasci under LD 12:12 (a) or
constant dim light of 0.006 Wm 2 intensity (b) at 20  C. The
triangle indicates the median of each pupation group.
Modified from Nisimura and Numata (2001).
441
A third pupation group was observed 89–109
weeks after hatching. The interval between the
medians of the first and second groups was 37
weeks and that of the second and third groups
was 37.5 weeks. This periodicity can be explained
by the gate concept, which was originally
introduced for the allowed time for eclosion in
the circadian rhythm of flies (Pittendrigh, 1966).
Thus, the existence of an endogenous circannual
rhythm in the process of pupation was demonstra-
ted in a Japanese population of A. verbasci
(Nisimura and Numata, 2001). We repeated the
experiments under LD 12:12 at 20  C from various
times of the year and observed a similar rhythmic-
ity, although the third pupation group has not been
clearly observed in many cases (Miyazaki et al.,
2005, 2009b; Nisimura and Numata, 2001, 2003).
The timing of pupation in the first and second
cycles was negligibly affected by slowing larval
development with a diet of low-nutrient food,
although the percentage of pupae in each cycle
changed remarkably (Miyazaki et al., 2009a).
It has been reported that, for some species,
circannual rhythms persist under constant light
or darkness. For example, the circannual hiberna-
tion rhythm of S. lateralis persists with a period
shorter than 12 months, irrespective of whether
the lighting conditions are constant darkness, con-
stant light, or LD 12:12 (Pengelley et al., 1976; see
also Gwinner, 1986). Figure 1b shows the fre-
quency of pupation times of A. verbasci when lar-
vae were continuously maintained under constant
dim light. The periodic pattern was less clear than
that under LD 12:12 (Nisimura and Numata,
2001). When we first obtained these results, we
thought that this indistinct rhythm resulted from
the damping of the circannual rhythm, as observed
for the circadian eclosion rhythm under constant
100 120 However, later experiments revealed that the
circannual rhythm of A. verbasci clearly persists
under constant light after preexposure to LD 12:12
for 8 weeks. The pattern of pupation was similar to
that under continuous LD 12:12 (Fig. 2; Miyazaki
and Numata, 2010). The circannual rhythmicity
442
50 (a)
25
0 development and from 6 to 21 months (mean,
Number of pupae
50 (b)
25
0
50 (c)
25
0
0 20 40 60 80
Weeks after hatching
Fig. 2. The effect of preexposure to LD 12:12 on pupation of
Anthrenus verbasci under constant light of 0.9 Wm 2
intensity at 20  C. Larvae were maintained continuously
under LD 12:12 (a) or constant light (b), or transferred from
LD 12:12 to constant light at 8 weeks after hatching (c).
Filled bars indicate the duration of LD 12:12 exposure. The
triangle indicates the median of each pupation group.
Modified from Miyazaki and Numata (2010).
observed under constant light indicates that daily
light–dark cycles are not necessary to sustain this
endogenous rhythm, as observed in a British popu-
lation under constant darkness (Blake, 1958,
1959). As described later, the change in the photo-
period is a zeitgeber for this circannual rhythm.
It is likely that the circannual rhythm of A. verbasci
oscillates under constant light, but the initial
circannual phase of newly hatched larvae varies
among individuals. Therefore, for a circannual
rhythm to be clearly observed under constant light,
it is necessary to synchronize the phase of the larvae
by subjecting them to 24-h photoperiodic cycles.
The circannual period of A. verbasci is 8–10
months (Blake, 1959; Miyazaki et al., 2009b;
Nisimura and Numata, 2001). The circannual per-
iods under constant conditions are shorter than 1
year in many species, although longer periods
have also been reported (Gwinner, 1986). Individ-
ual circannual periods often show great variability,
even within a single species (Gwinner, 1986).
The circannual period under constant dim light in
the dark-eyed junco, Junco hyemalis, ranges from
6 to 20 months (mean, 13.7 months) in testicular
15.0 months) in the onset of postnuptial molts
(Holberton and Able, 1992). These deviations
from 12 months, of about  50% and þ 75% in
extreme cases, represent considerable variations,
compared to deviations from 24 h of approxi-
mately  13% (21 h) and þ 17% (28 h) in circadian
free-running periods (Aschoff, 1979). In addition,
some species display circannual rhythms only
under certain photoperiodic conditions (Gwinner,
100 1986, 1996; Paul et al., 2008; Schaffelke and
Lüning, 1994). For example, the European star-
ling, Sturnus vulgaris, shows a clear circannual
rhythm in testicular development under LD 12:12
but not under LD 11:13 or LD 13:11 (Schwab,
1971). The circannual rhythm of A. verbasci was
obscure under LD 15:9 and LD 16:8 and arrhyth-
mic under LD 14:10 (Nisimura and Numata,
2003). The influence of photoperiod on the
circannual period has also been reported
(Gwinner, 1986 for review). In the rainbow trout,
Oncorhynchus mykiss, the circannual period is sig-
nificantly shorter under LD 18:6 and constant light
than under LD 6:18 (Duston and Bromage, 1986).
The circannual period of A. verbasci was 5 weeks
longer under LD 13:11 than under LD 12:12
(Nisimura and Numata, 2003). Under LD 10:14 in
one experiment, the rhythm persisted with a much
longer period of 52 weeks (Nisimura and Numata,
2002), but in another experiment, pupation after
the first peak was asynchronous and the period
length could not be determined (Miyazaki and
Numata, 2009). Although circannual rhythms in
some species, including the African stonechat,
Saxicola torquatus, and the Siberian chipmunk,
Tamias sibiricus, have been reported to persist for
approximately 10 years (Gwinner, 1996; Kondo
et al., 2006), circannual rhythms in most organisms
tend to dampen within several cycles under constant
conditions in the laboratory (Gwinner, 1986).
In general, both inter- and intraindividual variability
in the endogenous period in the same species is
 443

considerably larger for most circannual rhythms
than for circadian rhythms. Thus, Gwinner (1986)
concluded that circannual rhythms have weaker
self-sustainability than circadian rhythms.
Temperature compensation
Biological oscillators are based on intricate bio-
chemical mechanisms. Although most biochemical
processes depend on temperature, temperature
compensation is required for accurate time
measurement in biological rhythms (Johnson
et al., 2004; Pittendrigh, 1981a; Saunders, 2002).
Aperiodic fluctuations in temperature perturb
time measurement and need to be eliminated by
buffering mechanisms, that is, compensation
mechanisms. In fact, period length is almost fully
temperature compensated in circadian rhythms
under steady-state conditions at various constant
temperatures (Balzer and Hardeland, 1988;
Saunders, 2002; Sweeney and Hastings, 1960). In
biological rhythms with longer periods, however,
the demonstration of temperature compensation
is sometimes difficult. First, it is difficult to main-
tain strict constant conditions for a long duration,
and second, a desired constant temperature under
These results suggest that the circannual period
is, at least to a certain extent, temperature com-
pensated (Gwinner, 1986).
More conspicuous examples of temperature-
compensated circannual rhythms are present in
poikilotherms (Gwinner, 1986). In our studies
of A. verbasci, when larvae were exposed under
LD 12:12, pupation began 21–27 weeks after hatch-
ing at constant temperatures between 17.5  C and
27.5  C, and the second group pupated about 40
weeks after the first. High temperatures did not
result in earlier pupation as might be expected
from observations of normal developmental pro-
cesses. In fact, the first pupation peaks were slightly
delayed by higher temperature (Fig. 3; Nisimura
and Numata, 2001). We concluded that the timing
of pupation displayed temperature compensation,
in line with the results of Blake (1958, 1959).
20
27.5 °C
10
0
20
25 °C
10
Number of pupae

0
experimental conditions often interferes with the 20
22.5 °C
regulation of seasonal physiological events (Balzer
10
and Hardeland, 1988; Gwinner, 1986). Neverthe-
less, temperature compensation has been reported 0
in some circannual rhythms (Gwinner, 1986). 20
20 °C
Temperature compensation was shown in the 10
circannual rhythm period of a hibernating homeo-
0
therm, S. lateralis, which spends many months 20
each year in deep torpor with a body temperature 17.5 °C
close to that of its environment. In this animal, 10
although the period of the first circannual cycle 0
during hibernation is slightly longer at 3  C than
at 12  C, this difference disappears in the second 0 20 40 60 80 100

and third cycles (Pengelley and Asmundson, Weeks after hatching

1969). Much larger differences would have been Fig. 3. Pupation of Anthrenus verbasci under LD 12:12 at
expected if the processes underlying circannual various constant temperatures. The triangle indicates the
rhythms showed a temperature dependence simi- median of each pupation group. Modified from Nisimura and
lar to that of many other physiological processes. Numata (2001).
444

Temperature compensation of the circannual When we exposed A. verbasci larvae to natural

period has also been shown in various other annual changes in photoperiod in Japan, at a con-
invertebrates, for example, oviposition of the slug, stant 20  C, pupation peaks were observed in Jan-
Limax flavus (10–20  C; Segal, 1960; see also uary or February in both the first and second
Gwinner, 1986); growth and development of cycles (Fig. 4a; Miyazaki et al., 2006; Nisimura
the hydroid, Campanularia flexuosa (10–24  C;
Brock, 1975a,b); and sexual maturation of the
polychaete, Nereis diversicolor (5–10  C; Olive and (a)
16
Garwood, 1983). Thus, in invertebrates, compensa- 10
40
tion mechanisms appear to modulate time measure-
ment in circannual rhythms under conditions of 0
(b)
changing environmental temperature. 16
40 10
Entrainability
0
(c)
Biological rhythms can be entrained by zeitgebers 16

Number of pupae
from environmental cycles, thereby maintaining a 40 10

Day length (h)

stable phase relationship to environmental cycles
0
(Johnson et al., 2004; Pittendrigh, 1981a; Saunders, (d)
2002). In many species, a change in photoperiod is 16
the dominant zeitgeber for entraining the circannual 40 10
rhythm to an annual cycle (Goldman et al., 2004; 0
Gwinner, 1986; Paul et al., 2008). For example, the (e)
16
circannual rhythms were entrained to sinusoidal
40 10
photoperiodic cycles when the zeitgeber periods
were 4–24 months and 2–12 months in antler 0
replacement of the sika deer, Cervus nippon, and (f)
16
in testicular development of S. vulgaris, respectively
40 10
(Goss, 1969; Gwinner, 1977; see also Gwinner,
1986). The circannual rhythms of growth in two kelp 0
species, Pterygophora californica and Laminaria J J A S ON D J FM A M J J A S ON D J FM A M

hyperborea, were also entrained to sinusoidal pho-

toperiodic cycles with a period of 3–12 months
(Lüning and Kadel, 1993; Schaffelke and Lüning,
1994). Although the endogenous period of cir-
cannual rhythms in many species often deviates
considerably from 12 months, entrainment to the
natural year is uneventfully accomplished by a
range of entrainment proportionally much larger
than that for circadian rhythms (Goldman et al.,
2004; Gwinner, 1986). Gwinner (1986) suggested
that at least some circannual rhythms with weaker
self-sustainability were more dependent on environ-
mental zeitgebers and more easily affected by them
than circadian rhythms.
Fig. 4. The effect of a 4-week long-day pulse on pupation of
Anthrenus verbasci under naturally changing day length at
20  C. Larvae maintained under naturally changing day
length were exposed to LD 16:8 for 4 weeks starting on
August 4 (b), September 1 (c), September 29 (d), October 27
(e), or November 24 (f). Nonexposed control larvae are
shown in (a). The triangle indicates the median of each
pupation group. A vertical dotted line crosses through the
median of each pupation group in the control experiment
(a). The solid curve in each panel indicates the natural day
length, including 1 h of twilight in Osaka, Japan (35 N), for
most of the experiment, and the artificial day length
produced by white fluorescent lamps during the LD 16:8
pulse. The shaded bar in (a) shows the critical photoperiodic
range for determining whether the day length is long or
short. Modified from Miyazaki et al. (2006).

and Numata, 2003), similar to the observations
by Blake (1960) of A. verbasci populations in
England. Therefore, a circannual pupation
rhythm in A. verbasci, displaying a periodicity of
about 40 weeks under LD 12:12, is entrained to
exactly 1 year by exposure to natural changes in
photoperiod.
Blake (1963) suggested that the first and sec-
ond cycles of the pupation rhythm in A. verbasci
were controlled differently. She argued that, in
the first cycle, pupation was advanced by increas-
ing day length but that, in the second cycle,
pupation was inhibited by decreasing day length.
However, our results obtained by altering contin-
uous short-day (LD 12:12) and long-day (LD
16:8) cycles at various time points indicate that
whether pupation is advanced or delayed depends
on the phase at which the photoperiodic stimulus
is applied, rather than the cycle of the circannual
rhythm (Miyazaki et al., 2005, 2007; Nisimura
and Numata, 2001). Phase-dependent shifts in
rhythm are important factors in entrainment and
have been observed in many different circadian
rhythms. This topic will be explored in detail
later.
Compared to the photoperiod, the role of non-
photoperiodic cues has not been sufficiently
examined, although seasonal changes in tempera-
ture, daytime light intensity, and social stimuli
have been suggested as potential zeitgebers in
various species (Goldman et al., 2004; Gwinner,
1986, 2003; Paul et al., 2008). In many cases,
nonphotoperiodic cues act by directly driving
or suppressing expression of the trait without
entrainment of the endogenous rhythm (Paul
et al., 2008). When A. verbasci larvae are exposed
to natural fluctuations in light and temperature,
they pupate each year in April, rather than in
January and February (Miyazaki et al., 2009a;
Nisimura and Numata, 2003). It is likely that
low temperatures between December and March
suppress pupation until April and that this
suppression also contributes to the synchrony
of pupation in the field. However, there
remains the possibility that natural temperature
445
changes act as an effective zeitgeber for the
circannual rhythm of A. verbasci, as suggested
by Blake (1960).
Three different hypotheses for circannual rhythm
generation
Circannual clock hypothesis
A simple hypothesis to explain the generation of
the circannual rhythm assumes the existence of a
self-sustaining biological oscillator with a period
of about a year, that is, a circannual clock,
analogous to the circadian clock (Gwinner, 1981a;
Mrosovsky, 1978; Pengelley and Asmundson,
1974). However, the most troublesome feature of
this hypothesis is the long-term nature of the oscil-
lator. Many animals exhibiting circannual rhythms,
including hibernating mammals and migratory
birds, have short life spans of a few years
(Gwinner, 1986), and therefore it is difficult to
examine the circannual clock hypothesis in these
organisms. Then, the other two hypotheses that
do not involve a circannual clock, as mentioned
below, may have arisen. However, for A. verbasci,
our data suggest that the pupation rhythm is gener-
ated by a circannual clock similar to the circadian
clock, because the phase response curves (PRCs)
obtained in this circannual rhythm share many
similar features with PRCs obtained in circadian
rhythms (Miyazaki et al., 2005, 2007; and see later
sections for detail).
Frequency demultiplication hypothesis
Gwinner (1973) proposed the frequency demulti-
plication hypothesis, which states that circannual
rhythms are derived from circadian rhythms
through a process of frequency demultiplication.
In other words, organisms generate circannual
rhythms by transforming the circadian periodicity
of about a day to one cycle of about a year. The
possibility that circadian rhythms are involved in
446
the production of circannual rhythms is appeal-
ing, because circadian rhythms are a ubiquitous
feature of animal physiology. Moreover, Gwinner
(1973) observed a positive correlation between
circadian and circannual periods under constant
dim light in S. vulgaris.
The frequency demultiplication hypothesis
requires that the period of the circannual rhythm
is proportional to the period of the entrained circa-
dian rhythms. Therefore, rigorous tests of this
hypothesis can be performed by exposing
individuals to light–dark cycles of different periods
(T). We reared A. verbasci larvae under different
constant photoperiods, ranging from T ¼ 20 to
26 h (Nisimura and Numata, 2002). Because the
range of entrainment in circadian rhythms is usu-
ally between 18 and 30 h (Aschoff and Pohl,
1978), the circadian rhythms of the larvae should
entrain to the LD 10:10, LD 10:14, and LD 10:16
photoperiods. Based on the result under LD
10:14 (T ¼ 24), in which the first group of larvae
pupated 25 weeks after hatching, the frequency
demultiplication hypothesis predicts that pupation
should occur 21 and 27 weeks after hatching under
LD 10:10 (T ¼ 20) and LD 10:16 (T ¼ 26), respec-
tively (Fig. 5). However, the first group of larvae
pupated 27 weeks after hatching under LD 10:10
and 25 weeks after hatching under LD 10:16.
Thus, there is no positive correlation between
the period of the light–dark cycle and the
timing of pupation in A. verbasci (Nisimura and
Numata, 2002). To date, similar experiments
have been performed in three species of birds
and one mammal, but results supporting the fre-
quency demultiplication hypothesis have not been
obtained (Carmichael and Zucker, 1986; Gwinner,
1981b; Wikelski et al., 2008).
Further, circannual rhythms are exhibited by
most individuals even after disruption of circadian
activity patterns by ablating the suprachiasmatic
nucleus in mammals or the pineal gland in birds,
which are the locations of the circadian pacemaker
(e.g., Pant and Chandola-Saklani, 1992; Zucker
et al., 1983). These results also do not support the
frequency demultiplication hypothesis.
30
Larval duration (weeks)
25
20
0
20 22 24 26
Period of the light–dark
cycle (h)
Fig. 5. Evaluation of the frequency demultiplication
hypothesis for the circannual pupation rhythm in Anthrenus
verbasci. Larvae were kept under LD 10:10 (T ¼ 20), 10:14
(T ¼ 24), and 10:16 (T ¼ 26) at 20  C. Open circles represent
medians of the first pupation group. Vertical lines represent
interquartiles. Closed circles and a broken line represent the
timing of the first pupation group predicted by the frequency
demultiplication hypothesis and the results under LD 10:14.
Based on the data of Nisimura and Numata (2002).
Sequence of linked stages hypothesis
There is the hypothesis that circannual rhythms
merely result from a sequence of linked physio-
logical stages, with each stage normally taking a
given amount of time to complete and then lead-
ing to the next, and so on (i.e., a chain of interval
timers). The last stage is linked back to the first
(Mrosovsky, 1978). Circannual rhythms in some
hibernators, for example, the edible dormouse,
Glis glis; the little pocket mouse, Perognathus
longimembris; and the 13-lined ground squirrel,
Spermophilus tridecemlineatus, can be explained
by this hypothesis, but those in others cannot
(Mrosovsky, 1978).
Mrosovsky (1974) suggested that a circannual
clock and a sequence of stages can be characterized
as a pendulum-type oscillator and a relaxation
oscillator, respectively. However, because the lat-
ter is one-dimensional, the sequence of linked
stages hypothesis cannot explain some features of
phase resetting of rhythms (see Lakin-Thomas,

1995 for details; see also Oda et al., 2000), for
example, Type 0 resetting and arrhythmicity
induced by a zeitgeber pulse in the circannual
rhythm of A. verbasci (Miyazaki et al., 2005,
2007), which we discuss further in the following
section.
PRCs and the biological oscillator
In circadian rhythms, the response to a zeitgeber
depends on the phase subjected to the zeitgeber.
PRCs are constructed to represent these phase-
dependent responses. A circadian PRC is a plot
of the magnitude of phase shifts (phase advances
and delays) obtained by giving a light pulse at vari-
ous phases of a free-running rhythm under con-
stant darkness (Johnson, 1999; Johnson et al.,
2004; Pittendrigh, 1981b). A light pulse in early
subjective night generally delays a phase, whereas
a light pulse in late subjective night advances a
phase. A pulse applied in subjective day has a little
or no effect on the phase. The strength of a light
pulse, that is, intensity, duration, or both, often
changes the amplitude of phase shifts in the PRC.
Strong pulses produce Type 0 PRCs with large
phase shifts and a distinct break point at the transi-
tion between delays and advances, whereas weak
pulses produce Type 1 PRCs with small phase
shifts and a continuous transition between phase
delays and advances (Fig. 6a and b).
In contrast to circadian rhythms, reports of
PRCs to zeitgeber pulses in circannual rhythms
are uncommon (Paul et al., 2008). In the
circannual spawning rhythm of the rainbow trout,
O. mykiss, a pulse of constant light for 2 months
induced a phase-dependent phase shift. However,
because the treatment was performed on the
rhythm entrained to naturally changing day
length, the response curve obtained did not satisfy
the definition of the PRC, in which a free-running
rhythm is perturbed under constant conditions
(Randall et al., 1998).
To construct a complete PRC to photoperiod
pulses for a circannual rhythm, we kept A. verbasci
447
larvae under LD 12:12 and exposed the larvae to
LD 16:8 for 4 weeks (4-week long-day pulse) dur-
ing various phases of the rhythm (Miyazaki et al.,
2005). When larvae were exposed to a 4-week
long-day pulse, 4 weeks after hatching, pupation
of the first and second groups was delayed by
9–9.5 weeks, compared to control insects (Fig. 7b).
However, when a 4-week long-day pulse was given
16 weeks after hatching, pupation was advanced
by 2–3 weeks in both groups (Fig. 7c). Thus,
whether a 4-week long-day pulse advances or delays
the phase of the circannual rhythm depends on the
phase in which the pulse is given. Based on these
observations, we constructed a circannual PRC
(Fig. 6c) where the period of the rhythm under con-
tinuous LD 12:12 (37 weeks; Fig. 7a) is shown in
terms of angle degrees (0–360 ). The initial phase
under LD 12:12, that is, the beginning of this exper-
iment, is represented as 180 . The range 0–180 is
considered subjective summer and 180–360 is con-
sidered subjective winter. This circannual PRC to
4-week long-day pulses closely resembles the Type
0 PRC of circadian rhythms (Fig. 6a and c).
We also constructed a circannual PRC to
2-week long-day pulses (Fig. 6d). The phase shifts
are relatively smaller than those caused by a
4-week long-day pulse, and a PRC to 2-week
long-day pulses has a continuous transition
between delays and advances in the middle of
the subjective winter. Therefore, this curve is cat-
egorized as Type 1 (Miyazaki et al., 2007).
In circadian rhythms, the two types of phase
resetting, dependent on the strength of the zeitge-
ber stimulus, can be theoretically explained by
the concept that a circadian clock is a biological
oscillator that has two or more state variables.
A number of different oscillator types have been
proposed, including an adjustable-amplitude oscil-
lator, a limit cycle oscillator, and a multioscillator
system (Johnson et al., 2004; Lakin-Thomas,
1995; Winfree, 2000). It is known that arrhy-
thmicity of a circadian rhythm can be evoked by
a single light pulse of a certain strength delivered
near the middle of the subjective night. This phe-
nomenon can be explained by the notion that the
448

(a) (b)
Subjective Subjective Subjective Subjective
day night day night
180

Phase shift (degree)

90

-90

-180

(c) (d)
Subjective Subjective Subjective Subjective
summer winter summer winter
180
Phase shift (degree)

90

-90

-180
0 90 180 270 360 0 90 180 270 360
Phase of pulse onset (degree) Phase of pulse onset (degree)

Fig. 6. Comparison of phase response curves for circadian and circannual rhythms. (a, b) Phase response curves in circadian
rhythms, Type 0 (a) and Type 1 (b). (c, d) Phase response curves in the circannual pupation rhythm of Anthrenus verbasci, a
curve to 4-week long-day pulses (c) and a curve to 2-week long-day pulses (d). Larvae were kept under LD 12:12 at 20  C and
exposed to LD 16:8 for 4 (c) or 2 (d) weeks at various phases in the circannual rhythm. The circannual period under continuous
LD 12:12 is shown in terms of angle degrees (0–360 ). Open and closed circles represent the phase shifts in the first and second
pupation group after pulse perturbation, respectively. Broken lines in (c) show the split into advanced and delayed groups. (c)
From Miyazaki et al. (2005). (d) From Miyazaki et al. (2007).

oscillator is driven to the phaseless singularity in be a biological oscillator with two or more state
the phase space or that the phases of individual variables with circannual variation (Johnson
oscillators of the multioscillator system are et al., 2004; Lakin-Thomas, 1995). This implies that
scattered (Johnson et al., 2004; Lakin-Thomas, the circannual rhythm of A. verbasci is generated
1995; Winfree, 1970, 2000). In the circannual by a circannual clock specialized for adaptation
rhythm of A. verbasci, arrhythmicity of pupation to annual cycles, much like the circadian clock
can also be evoked by a 4-week long-day pulse and its adaptation to daily cycles.
administered in the middle of the subjective winter Recently, a circannual PRC for reproduction in
(Miyazaki et al., 2007). According to the theoreti- the European hamster, Cricetus cricetus, was con-
cal explanations for Type 1 and Type 0 phase structed by exposing hamsters, normally maintained
resetting and the loss of rhythmicity induced under long days (LD 16:8), to short days (LD 10:14)
by a zeitgeber pulse, the mechanism behind for 1 month (Monecke et al., 2009). The shape of the
the circannual rhythm of A. verbasci is thought to PRC was nearly identical to the circannual PRC to
 449

100 cycles seems to be applicable to many diverse

(a)
organisms.
80

60
Photoperiodism for entrainment
40
Entrainment under natural day length
20
The circannual rhythm of A. verbasci shows endog-
0 enous periodicity of about 40 weeks under continu-
100
(b) ous LD 12:12 but is entrained to a strictly annual
80 period by changes in photoperiod. This entrain-
Number of pupae

ment can be explained as a phase delay induced

60
40
20
0
80
(c)
60
40
20
0 4-week long-day pulses given between August 4
0 20 40 60
Weeks after hatching
Fig. 7. The effect of a 4-week long-day pulse on pupation of
Anthrenus verbasci under LD 12:12 at 20  C. Larvae
maintained under LD 12:12 (filled bars) were exposed to LD
16:8 (rectangle bars) for 4 weeks, commencing 4 weeks after
hatching (b) or 16 weeks after hatching (c). Nonexposed
control larvae are shown in (a). The triangle indicates the
median of each pupation group. A vertical dotted line
crosses through the median of each pupation group in the
control experiment (a). Modified from Miyazaki et al. (2005).
LD 16:8 for 4 weeks in A. verbasci, a species taxo-
nomically quite distant from C. cricetus. Therefore,
these phase responses may signify a fundamental
and common characteristic of circannual rhythms,
and the concept that the circannual rhythm is pro-
duced by a circannual clock specialized for annual
by a decrease in day length in late summer or early
autumn (Nisimura and Numata, 2001, 2003). How-
ever, it is necessary to know the exact temporal
changes in the phase of the circannual clock under
natural day length to understand the biological
entrainment mechanisms operating under natural
conditions.
If we compare the phase shifts induced by the
photoperiod pulse under natural photoperiod
with those shown in the circannual PRC, we can
describe the temporal change in phase under nat-
ural day length in terms of the phase in the PRC.
Therefore, we examined the phase responses to
80 100 and November 24 under natural day length at a
constant temperature of 20  C (Miyazaki et al.,
2006). These pulses caused a phase shift in the
first pupation group, as under LD 12:12. A long-
day pulse given on August 4, September 1, or
September 29 caused a phase delay, and a pulse
given on October 27 or November 24 caused a
phase advance. Pupation was least synchronous
just before the transition from delay to advance
(Fig. 4). These phase responses were similar to
those in the subjective winter of a circannual
PRC to 4-week long-day pulses under LD 12:12
(Fig. 6c). Therefore, the circannual clock changes
its phase at least from early August to late
November under natural day length, as occurs in
the subjective winter under LD 12:12. In the sec-
ond pupation group, however, larvae pupated at
the same time as control larvae not subjected to
450
a long-day pulse, regardless of the phase response
of the first group (Fig. 4). This result can be
explained by entrainment to the geophysical year
by long days during spring and summer.
We also examined the range of photoperiodic
changes effective for circannual phase shifts in
A. verbasci by exposing larvae to various longer
photophases for 4 weeks against a background
of LD 12:12 or LD 10:14 (Miyazaki and Numata,
2009). Phase advances were smaller than
phase delays, and the magnitude of the change
depended on the amplitude of the photoperiodic
changes rather than the absolute photophase
duration. In contrast, a clear phase delay was
induced when the photoperiodic change exceeded
a critical value in the photophase between 13 and
14 h, regardless of the amplitude of the change.
It is likely that the response in phase delay is more
important in the seasonal timing of pupation under
natural conditions because the endogenous period
of this rhythm is considerably shorter than 1 year.
In photoperiodic responses of many species, an
important feature is the existence of a critical day
length above or below which behavioral and phys-
iological responses are observed (Goldman et al.,
2004; Nelson et al., 2010; Saunders, 2002). To
entrain a circannual period to the natural annual
cycle, A. verbasci in central Japan probably uses
a critical value in the photophase between 13 and
14 h for determining whether the day length is long
or short. It is thought that stable entrainment to
the natural year is established by giving long-term
exposure to a photophase longer than 13 h during
spring and summer, or during late subjective sum-
mer or early subjective winter of the circannual
rhythm, where phase delay responses to long days
are exhibited in the PRC (Figs. 4a and 6c).
An important role of long days in summer has
also been suggested in entrainment of the
circannual rhythm in luteinizing hormone secre-
tion of the sheep, Ovis aries (Woodfill et al.,
1994). In pinealectomized ewes, which do not
respond to changes of day length, photoperiodic
information was applied during one of the four
seasons each year via infusion of melatonin, a
hormone that mediates the effects of the photope-
riod. The infusion was designed to simulate sea-
son-specific melatonin secretion. Infusion during
the summer was most effective in entrainment to
annual cycles and maintained the proper relation-
ship between the phase of the rhythm and the
season during which the infusion was provided.
The response and phase setting of circannual
rhythms to photoperiodic changes likely vary,
depending on the species or subspecies (Gwinner,
1986; Helm et al., 2009). To better understand the
full diversity of responses of circannual rhythms
to photoperiodic changes, further research will
be required in a wide variety of species.
Involvement of a circadian clock
A wide variety of species have evolved photoperi-
odism without relation to circannual rhythms.
In such photoperiodism, the photoperiodic time
measurement system generally involves a circa-
dian clock (Goldman et al., 2004; Nelson et al.,
2010; Saunders, 2002). In phase responses in the
circannual rhythm of A. verbasci, photoperiodic
time measurement is also necessary, although
unlike regular photoperiodism, the effects of pho-
toperiod are indirect and are due to resetting of
the circannual rhythm. The Nanda–Hamner pro-
tocol has frequently been used to clarify the
involvement of a circadian system in photope-
riodic responses. In this protocol, groups of
organisms are subjected to light–dark cycles with
a fixed short photophase followed by a variable
scotophase to give cycle lengths (T) of up to
72 h or more. If organisms show short-day
responses when T is a multiple of 24 h, but not
when T is not a multiple of 24 h, the response is
considered positive, thus indicating the involve-
ment of a circadian system in photoperiodic time
measurement.
The Nanda–Hamner protocol has been used to
examine the reproductive activities of several ver-
tebrate species exhibiting circannual rhythms. In
O. mykiss, the effects of constant 48-h, 54-h, and
 451

60-h photoperiods were examined over 2 years, LD 12:12

50
but the circadian nature of the photoperiodic
responses was obscure (Duston and Bromage, 0
1986). Positive responses to the Nanda–Hamner LD 12:24
50
protocol were demonstrated for birds and
mammals (e.g., Almeida and Lincoln, 1982; 0
Gwinner and Eriksson, 1977), although these LD 12:36
50
observations were conducted over a period of less
than a year.

Number of pupae
0
We examined the effects of exposure to LD 12:48
50
the Nanda–Hamner protocol (LD 12:12, LD
12:24, LD 12:36, LD 12:48, and LD 12:60) on 0
the circannual rhythm of A. verbasci to examine LD 12:60
50
whether the circadian system is involved in
photoperiodic time measurement for entrainment 0
(Miyazaki et al., 2009b). Figure 8 shows the Continuous darkness
50
results when larvae were exposed to various
photoperiods for 120 days and then transferred 0
to LD 12:12. When larvae were exposed to LD LD 16:8
50
12:36 or LD 12:60, for which T is a multiple of
24 h, the pupation pattern was similar to that 0
under continuous LD 12:12. In contrast, under
LD 12:24 (T ¼ 36 h), the pupation pattern was 0 20 40 60 80 100
similar to that under LD 16:8, being considerably Weeks after hatching
delayed compared to that under LD 12:12. Under
Fig. 8. The effect of exposure to the Nanda–Hamner protocol for
LD 12:48 (T ¼ 60 h), pupation was also delayed 120 days on pupation of Anthrenus verbasci at 20  C. Larvae were
and was less synchronous than that of the first transferred to LD 12:12 after exposure to various photoperiods.
group under LD 12:60 (T ¼ 72 h) or constant Shaded areas represent exposure to various photoperiods. The
darkness. Thus, photoperiods for which T was triangle indicates the median of each pupation group. Modified
from Miyazaki et al. (2009b).
not a multiple of 24 h (i.e., LD 12:24 and LD
12:48) produced a large phase delay compared
to LD 12:12. These results clearly indicate that temperature compensation of the period, and
the circannual rhythm of A. verbasci shows a pos- entrainability to a zeitgeber (Nisimura and
itive Nanda–Hamner response in phase resetting Numata, 2001), all of which are key characteristics
to photoperiod. Therefore, a circadian clock is of a clock mechanism linked to environmental
most likely involved in photoperiodic entrainment cycles (Johnson et al., 2004; Pittendrigh, 1981a;
of this rhythm, although it is not responsible for Saunders, 2002). The characteristics of this rhythm
generating the circannual rhythm, as described indicate that the frequency demultiplication
above. hypothesis does not accurately describe the under-
lying mechanism (Nisimura and Numata, 2002).
The circannual PRCs were obtained by giving
Conclusions and future directions long-day pulses, and the shapes of the curves
closely resembled those of circadian PRCs to light
In the circannual pupation rhythm of A. verbasci, pulses (Miyazaki et al., 2005, 2007). Similarities to
we confirmed self-sustainability (i.e., persistence), circadian rhythms in the resetting patterns indicate
452
that the mechanism producing circannual rhyth-
micity in A. verbasci is a circannual clock analogous
to the circadian clock (Miyazaki et al., 2007).
The circannual clock has most likely evolved to
increase the chances of survival and reproductive
success, by allowing the organism to better pre-
dict and adapt to seasonal changes (DeCoursey,
2004; Gwinner, 1981a, 1986; Pittendrigh, 1981a).
To permit accurate- and consistent-seasonal
responses, A. verbasci refers not only to ambient
photoperiod but also to the phase of a circannual
clock (Miyazaki et al., 2006), and it refers to a cir-
cadian clock to judge photoperiod (Miyazaki
et al., 2009b). Figure 9 shows a schematic diagram
of the physiological system behind the circannual
rhythm of A. verbasci. Thus, in the field, an
annual rhythm of A. verbasci is established by a
combination of exogenous factors and endoge-
nous physiological mechanisms comprising circa-
dian and circannual clocks. It is likely that this
schema is also applicable to certain other
Photoreceptor
Circannual
Photoperiodic
clock
time
measurement
system
Phase
reset
Time-of-year
information
Effector
system
Seasonal
activity
Fig. 9. Schematic diagram of the physiological components of the circannual rhythm in Anthrenus verbasci. The relationship
between the circadian clock involved in photoperiodic time measurement and a circadian clock controlling daily activities
(dashed arrow) is unclear.
organisms, for example, O. aries (Lincoln and
Hazlerigg, 2010; Lincoln et al., 2006).
The neural and molecular mechanisms that
generate circannual rhythmicity remain unknown,
not only for A. verbasci but also for other species
as well. In S. lateralis, various neural tissues and
endocrine glands, including the suprachiasmatic
nucleus and the pineal gland, have been ablated,
but the anatomical location of the circannual
pacemaker is still unclear (Zucker, 2001). In
O. aries, however, Lincoln et al. (2006) suggested
that the adenohypophysis is the site of the pace-
maker for the circannual rhythm of prolactin
secretion. The adenohypophysis secrets prolactin
with circannual periodicity, without neural inputs
from the brain, and is a target of pineal melatonin
signals transmitting photoperiodic information.
Recently, a few models have been proposed to
generate the circannual rhythm in mammals.
In T. sibiricus, the complex of hibernation-specific
proteins is secreted by the liver. Before hibernation,
Light
Phase reset
Circadian Circadian
clock clock
Time-of-day
information
Effector
system
Daily
activity

this complex is transported to the brain where it
mediates the onset of hibernation (Kondo et al.,
2006). For circannual rhythm generation in this spe-
cies, Kondo (2007) proposed a model in which
levels of this hibernation-specific protein complex
in the brain and periphery form a negative-feed-
back loop. In the circannual rhythm of O. aries,
MacGregor and Lincoln (2008) proposed a physio-
logical model consisting of a negative-feedback
mechanism comprising the components of the pars
tuberalis and pars distalis within the adenohypoph-
ysis. These two models postulates a negative-feed-
back loop formed by a number of different tissues.
In contrast, Lincoln and Hazlerigg (2010) proposed
that circannual rhythmicity is autonomously gener-
ated by cyclical histogenesis in specific sites in
various tissues, such as the brain, pituitary, and
periphery.
Because circannual rhythms have also been
observed in coelenterates (Brock, 1975a), it is
possible that animals can exhibit circannual rhyth-
micity without the need for a central nervous sys-
tem. Further, circannual rhythms are observed
even in eukaryotic unicellular organisms, and
therefore, molecular-feedback loops within a sin-
gle cell, similar to those in a circadian clock,
may be involved in generating the circannual
rhythm (Anderson and Keafer, 1987; Matrai
et al., 2005). If circannual rhythms have evolved
independently several times and are of heteroge-
neous origin in different groups of organisms
(Farner, 1970; Gwinner, 1981a, 1986), entirely dif-
ferent mechanisms may drive the circannual
rhythm in different species.
Several approaches are available to uncover the
mechanism behind circannual rhythmicity in
A. verbasci. Because a photoperiodic response
involving a circadian system resets the phase of a
circannual clock in this species (Fig. 9), one way is
to identify the anatomical location of a circannual
clock by reference to the centers for photoperiod-
ism and circadian rhythmicity. One may proceed
by studying the expression and regulation of circa-
dian clock genes, similar to the approach used to
unravel the molecular mechanisms of insect
453
photoperiodism (Goto et al., 2010). Another
method is provided by studies in birds. In avian
circannual rhythms, there are clear differences in
the degree of rhythmicity, timing of behavior, and
responsiveness to photoperiod among populations
living in different areas. This geographic variation
is clearly attributable to genetic differences
because crossbreeding produces an intermediate
response in the hybrid progeny (Gwinner, 1986,
1996; Helm et al., 2009). A. verbasci is a geograph-
ically widespread species, and therefore, it proba-
bly also has genetic variation. The genetic
differences may help in elucidating the molecular
mechanisms of circannual rhythmicity in this
organism.
A direct way to identify molecular candidates
involved in the generation of circannual rhythmic-
ity is to use DNA microarrays, similar to
approaches used to identify candidates that play
a role in circadian rhythm generation (Akhtar
et al., 2002; McDonald and Rosbash, 2001).
Subsequently, the precise involvement of the can-
didates in the circannual rhythm may be revealed
by RNA interference (RNAi). In beetles, inhibi-
tion of gene expression in various physiological
responses has been performed by RNAi (e.g.,
Niimi et al., 2005; Tomoyasu and Denell, 2004).
Therefore, we propose DNA microarrays and
RNAi as the next step to clarify the biological
mechanism behind the circannual rhythm in
A. verbasci.
Since Konopka and Benzer (1971) first demon-
strated that a gene, called period, regulates the cir-
cadian behavior of the fruit fly, Drosophila
melanogaster, enormous advances in our knowl-
edge of the genetic and molecular aspects of circa-
dian rhythmicity have occurred. A clear picture
of the underlying molecular-feedback loops is
emerging for numerous organisms (Rosbash,
2009; Saunders, 2002). We anticipate that our
knowledge of the physiological and molecular
mechanisms behind circannual rhythmicity will
similarly advance greatly when researchers begin
to isolate the molecular components of this funda-
mental biological process.
454
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ISSN: 0079-6123
Copyright Ó 2012 Elsevier B.V. All rights reserved.

CHAPTER 26

Avian migration: Temporal multitasking and

a case study of melatonin cycles in waders

Barbara Helm{,{,},*, Eberhard Gwinner{,w, Anita Koolhaas}, Phil Battley||,

Ingrid Schwabl{, Anne Dekinga} and Theunis Piersma}

{
Max-Planck-Institut für Ornithologie, Andechs, Germany
{
Fachbereich Biologie, Universität Konstanz, Konstanz, Germany
}
Institute of Biodiversity, Animal Health and Comparative Medicine, University of
Glasgow, Glasgow, United Kingdom
}
Department of Marine Ecology, Royal Netherlands Institute for Sea Research (NIOZ), Den Burg, Texel,
The Netherlands
||
Ecology Group, Institute of Natural Resources, Massey University, Private Bag, Palmerston North, New Zealand

Abstract: Timing “in the real world” must cope with the temporal complexity of natural environments.
Extreme examples for the resultant “multitasking” are migratory birds, which precisely time movements
to remote areas. New field technologies highlight temporal accuracy, while captivity studies emphasize
underlying programs and plasticity of schedules. After reviewing these findings, we focus on waders,
which undertake spectacular long-distance migrations, have robust circannual clocks, and cope with diel,
tidal, and polar environments. To explore features that may facilitate such multitasking, we speculated
that melatonin amplitudes are low and damped during seasons when entrainment to subtle Zeitgebers
occurs. We measured melatonin profiles under European daylength in two species with different
ecologies and found low-amplitude melatonin cycles that changed over the year. Annual patterns neither
fully supported our hypothesis, nor simply reflected daylight availability. While migratory birds are
inspiring models for chronobiology, mechanistic understanding of their multitasking is still poor.

Keywords: clock; circannual; annual; tidal; circadian; moon; bird.

Temporal multitasking—Life in complex

*Corresponding author.
Tel.: þ497531884915; Fax: þ497531884916
temporal environments
E-mail: helm@orn.mpg.de
Chronobiology advances in great strides, as is
evident from this volume. Its predominant focus
w
Deceased

http://dx.doi.org/10.1016/B978-0-444-59427-3.00026-5 457
458
recently has been on circadian rhythms, owing to
their near-ubiquitous occurrence and to rapidly
developing molecular tools. While this work has
been immensely productive, we have gained com-
paratively little insight on how biological time
keeping functions under the natural conditions in
which it has evolved. This “real world” (Menaker,
2006) is structured by rhythmic processes on differ-
ent temporal scales, which are all of core interest to
chronobiology as a discipline concerned with “sys-
tematic study of adaptations to cope with regular
geophysical cycles” (Dunlap et al., 2004). Thus, its
domains are based on rotations of earth and moon
in relation to sun and comprise rhythms at the
scales of solar year, solar day, lunar month, and
tidal cycle, as well as shorter rhythms contained
within (e.g., ultradian rhythms). This wide scope
was emphasized early on (Chovnick, 1960;
Menaker, 1971), but time scales other than circa-
dian have received little further attention.
Organisms, however, cope with the different time
scales and their clocks must be constructed accord-
ingly. For example, daylength changes periodically
over the year in most global habitats, and many
species adjust daily activity patterns over the
annual cycle (Daan and Aschoff, 1975; Foster and
Kreitzman, 2005, 2009; Foster and Roenneberg,
2008; Hut et al., 2012). Likewise, the periodic
waxing and waning of the moon can have consider-
able effects on activity patterns, in particular for
nocturnal species (Fernández-Duque et al., 2010)
and in marine environments (Tessmar-Raible
et al., 2011). Further, cycles on different time scales
can impart conflicting benefits and demands on
scheduling. For example, in tidal habitats, bitidal
cycles have a period length of 24.8 h, while a
solar day takes 24 h. For some tidal organisms,
therefore, important aspects of the environment,
such as food availability or predation risk, may
fluctuate with different periodicity (Palmer, 2000;
Tessmar-Raible et al., 2011).
Organisms have internalized timing on differ-
ent scales as endogenous circadian, circatidal,
circalunar, or circannual rhythms (Gwinner, 1986;
Tessmar-Raible et al., 2011), which must interact
to accommodate temporal complexity. There
is increasing evidence that, while clocks on differ-
ent time scales may rely on different mechanisms,
there is cross talk, coordination, and mutual
adjustment (e.g., Miyazaki et al., 2012; Tessmar-
Raible et al., 2011). For example, fundamental
properties of circadian clocks, such as entrain-
ment mechanisms, deviation of the free-running
period from 24 h, and possibly the evolution of
its endogenous basis, are related to the annual
change in daylength (Pittendrigh and Daan,
1976). Further, circadian and circatidal rhythms
may interact in such a way that one suppresses
the other at certain phase angles (Palmer, 2000;
Tessmar-Raible et al., 2011). Accordingly, study-
ing clocks on different time scales could lead
to substantial advances in our understanding of
biological time keeping from an ecological as
well as mechanistic perspective. Based on such
developments, there has been an increasing call
to reactivate a broader perspective in chronobiol-
ogy with the help of new tools from molecular
and field biology (Hut et al., 2012; Miyazaki
et al., 2012; Tessmar-Raible et al., 2011).
Our contribution uses a classical model system
of chronobiology, avian long-distance migration
(Aschoff, 1955; Gwinner, 1986; Hamner and Stock-
ing, 1970; Rowan, 1926), as an extreme example of
temporal multitasking. We first provide an overview
of avian timing and then illustrate its complexity and
general characteristics in a taxon that stands out for
performance on different temporal scales, waders
(Order Charadriiformes). We complement the
review by empirical data from a study aimed at
identifying aspects of the circadian system that
may enhance such multitasking. To this end, we
examined diel profiles of the circadian hormone
melatonin across the annual cycle in two species of
wader selected for contrasting temporal habits.
Our overall intention is to promote thought about
timing as an integrated response to complex, tempo-
ral challenges. Although focused on birds, these
considerations are relevant for many kinds of
organisms, with the possible inclusion of our own
species for which cycles on other than daily scales

may be more relevant than sometimes acknowl-
edged (e.g., Foster and Kreitzman, 2009; Foster
and Roenneberg, 2008; Miyazaki et al., 2012;
Tessmar-Raible et al., 2011; Wirz-Justice et al., 2001).
Challenges of avian migration
Birds as a taxon have much to offer with respect
to timing in the real world because their ecology
and evolutionary biology are relatively well
explored. Being conspicuous by sight and sound,
the striking and sophisticated annual and daily
schedules can be readily observed and experi-
mentally tested. On an annual scale, birds as a
long-lived taxon show regular, full cycles of
diverse activities (e.g., Gwinner, 1996b; Newton,
2008). Within each annual cycle, almost all spe-
cies undergo at least one bout of reproduction
and regeneration (i.e., moult, the replacement of
body plumage and flight feathers), respectively.
There are countless variations of annual cycles.
For example, closely related taxa may differ
strikingly by complex patterns of additional, inter-
mittent moults (Newton, 2008). In general, such
seasonal activities have to be well timed with
respect to the environment and usually require
anticipation of environmental fluctuations well in
advance (Visser et al., 2010). Daily activities
are often “announced” by songs and calls so that
species-specific timing (i.e., phase of activity)
inspired the construction of “bird clocks” that
illustrate its sequence. The outstanding mobility
of birds entails a need to keep track of time
of day, for example, for the use of a time-
compensated sun compass as a navigational tool
(Kramer, 1959). Although being mostly diurnal
as a taxon, many species show occasional or fre-
quent nocturnal and crepuscular activities (Daan
and Aschoff, 1975; Martin, 1990) or keep track
of lunar and tidal cycles (Daan and Koene,
1981; Leyrer et al., 2006; Pinet et al., 2011).
Migration adds a layer of complexity to timing
on these different scales. Movements often have
substantial effects on the Zeitgeber conditions a
459
migrant experiences. In addition to light, other
temporal aspects of habitats like daily patterns
of food availability or predation risk may also
be changed by migration, and in extreme cases,
timing can shift from diel to tidal schedules.
Migration also complicates the annual cycle by
adding processes which birds undergo. Next to
migration periods per se, migrants add life-cycle
stages such as additional moult periods and pre-
paratory phases such as hyperphagia to build fuel
stores (Newton, 2008; Wingfield, 2005). Further,
migratory birds are a well-known model for close
links between time scales. Many species change
their activity patterns during migration periods
by assuming nocturnality in the wild and related,
spontaneous night activity (Zugunruhe) in captiv-
ity (Gwinner, 1996a). This temporal plasticity is
of wide interest to chronobiology and sleep
research (Mrosovsky and Hattar, 2005; Rattenborg
et al., 2004). Below, we give an overview of how
birds meet the temporal challenges of migration.
Annual time keeping
The phenology (i.e., periodic seasonal recurrence)
of bird migration has been closely observed
by humans from historical times until today
(Birkhead, 2008; Foster and Kreitzman, 2009).
For migrants, keeping track of the seasons is diffi-
cult because of their exposure to widely separated
global environments (Fig. 1). Their movements
nonetheless often show such striking “punctuality”
that they were used as calendrical information, for
example, for farming practices (so-called calendar
birds). Observers also noted considerable variation
between species in punctuality. So-called weather
birds differ from calendar birds by large variation
in spring arrival dates, often in response to exter-
nal conditions (Gwinner and Helm, 2003; Newton,
2008; Ramenofsky et al., 2012). The spectrum of
patterns found in nature indicates that long-term
timing in birds involves diverse internal and exter-
nal factors (Visser et al., 2010). For the purpose
of studying endogenous rhythms, the primary
460
Red Knot
Great Knot
Bar-Tailed Godwit
Black-Tailed Godwit
Ruff
Fig. 1. Map showing major southward migration pathways around the world of Red Knot, Great Knot, Ruff, Bar-Tailed Godwit,
and Black-Tailed Godwit. Different lines for Red Knots and Bar-Tailed Godwits represent subspecies with differing migration
patterns.
interest of chronobiologists has been in calendar
birds. Nonetheless, weather birds have a lot to offer,
as chronobiologists become increasingly interested
in plasticity of time keeping (Hut et al., 2012;
Ramenofsky et al., 2012).
The primary, external calendar that is available
to organisms is photoperiod, that is, the annual
cycle in length of the light fraction of the
day. In most global environments, daylength
markedly changes over the year so that, for a
given locality, it provides precise predictive infor-
mation (Bradshaw and Holzapfel, 2007; Foster
and Kreitzman, 2009). Birds are highly photope-
riodic (Dawson et al., 2001; Sharp, 2005) and
may adjust annual cycles in response to subtle
changes in daylength of half an hour or less
(Hau et al., 1998; Helm et al., 2009) or follow
accelerated photoperiodic cycles by undergoing
several annual cycles within a single year (Gwinner,
1986). However, the use of photoperiod as calendri-
cal information is greatly complicated by long-
distance migration (Aschoff, 1955; Hamner and
Stocking, 1970; Rowan, 1926). The very movements
of migrants, carried out predominantly on a
North–South axis, result in substantial modification
of the daylength they experience. In extreme cases,
transequatorial or interpolar migrations expose
birds to long days for most of the year (Egevang
et al., 2010). This puts high demands on internal
long-term timing mechanisms, including an ability
to correctly interpret temporal cues at a given
time and location. Photoperiodism must therefore

be complemented by refined mechanisms that regu-
late its use in a phase-specific way (Bradshaw
and Holzapfel, 2007; Miyazaki et al., 2012). Such
mechanisms have been documented in experimen-
tal and comparative studies. Birds usually become
temporarily unresponsive to previously stimulating
daylengths (photorefractoriness; Dawson et al.,
2001; Hahn et al., 2009; Sharp, 2005). The conditions
for regaining responsiveness to stimulatory day-
lengths differ greatly between species and show
clear links to migration (Gwinner, 1988, 1996a,b;
Hahn et al., 2009; Helm et al., 2009). In general,
long-distance migrants do not require short-day
conditions to regain responsiveness to long days
(i.e., photosensitivity) but recover it spontaneously
under a wide range of conditions. At the same
time, delayed recovery of long-day responsiveness
protects migrants from inappropriate seasonal
behavior like breeding on transequatorial winter
grounds (Gwinner, 1988, 1996a,b; Hamner and
Stocking, 1970). Such phase-specific and species-
specific responses to daylength point to a high
potential for evolutionary changes of biological time
keeping. Different populations within a species may
differ in the timing of seasonal activities under iden-
tical field and captive conditions, and similar varia-
tion can be observed even within populations
(Battley, 2006; Curry-Lindahl, 1963; Rees, 1989).
Several recent studies report substantial heritability
for the timing of migration, which could allow
rapid evolutionary adjustment to changing environ-
mental conditions (Pulido and Berthold, 2003; van
Noordwijk et al., 2006; Visser et al., 2010). Timing
differences between individuals can result from dif-
ferent responses to cues like daylength and ambient
temperature (Bradshaw and Holzapfel, 2007). Such
differential responses, if innate, can be interpreted
as “reaction norms” to environmental cues (i.e.,
range of phenotypes expressed by a single genotype
as a function of a given variable) and can be ana-
lyzed by quantitative genetic tools (van Noordwijk
et al., 2006; Visser et al., 2010).
In various species of migratory birds, annual pro-
cesses oscillate as self-sustained circannual rhythms
(Gwinner, 1986). The general characteristics of
461
circannual rhythms and their phase responses
to the main Zeitgeber, photoperiod, are elaborated
in greatest detail for invertebrates and are
summarized in this volume (Miyazaki et al., 2012).
Circannual rhythms in migratory birds belong to
the earliest, definitive experimental demonstrations
of such long-term timing mechanisms (Gwinner,
1967) and are often particularly robust. For
example, in Dark-eyed Juncos (Junco hyemalis),
circannual cycles in migratory restlessness and
related processes continued for several years even
under constant dim light (Holberton and Able,
1992). Palearctic long-distance migrants, such as
Sylvid warblers (Berthold et al., 1972), can express
robust circannual rhythms under a wide range of
constant daylengths from LD 10:14 h to 16:8 h.
In contrast, in other, less migratory birds, the range
of permissive conditions under which circannual
rhythms are expressed can be very narrow
(Dawson et al., 2001). Detailed data for geographic
differentiation of circannual rhythms are available
from studies of Stonechats (Saxicola torquata;
Gwinner, 1996b; Helm et al., 2009). In this songbird
taxon, local populations differ greatly in migratory
behavior and correspondingly in seasonal sched-
uling. A comparison of Siberian long-distance
migrants and European short-distance migrants
showed that these differences are partly hard-wired
in distinct circannual rhythms. Consequently, the
taxa differed markedly in response to a given
daylength: for example, slightly longer simulated
days of more southerly wintering grounds advanced
the annual cycle in European Stonechats but
delayed it in Siberian Stonechats (Helm et al.,
2009). Such data indicate that the specific ways in
which circannual systems respond to Zeitgeber
stimuli are presumably subject to strong selection
(Gwinner, 1996a,b).
Environmental factors modify or fine-tune the
timing and progress of migration (Newton, 2008;
Ramenofsky et al., 2012; Visser et al., 2010).
Within the time window set for migration, its prog-
ress may depend on conducive winds and weather
conditions, fuel stores, and feeding conditions
en route (Gwinner, 1999). In many species of
462
migrants, social factors also contribute to the fine-
tuning of timing, so that aggregations may be seen
as individuals’ compromises between the relative
benefits of joining a group and those of going by
own schedule (Helm et al., 2006). In contrast, a role
as Zeitgebers for circannual rhythms of migrants
appears unlikely for nonphotic factors like tropical
seasonality or fluctuations in food availability
(Gwinner and Helm, 2003). For example, while
tropical migrants show highly localized breeding
cycles, migrants depart from wintering sites with dif-
ferent rainfall patterns much more synchronously.
Further, in palearctic warblers, departure from
equatorial winter grounds coincides closely with
the onset of spring migratory restlessness in captive
conspecifics. Both observations support the idea
that spring preparations of equatorial migrants
are mainly timed by an endogenous circannual
mechanism (Gwinner and Helm, 2003). Con-
tributions of programmed timing at one extreme
and of environmental factors at the other differ not
only between calendar birds and weather birds but
sometimes also within species and over the course
of a migration period (Newton, 2011). Clearly,
the balance between control of annual cycles by
endogenous clocks versus environmental responses
must be finely adjusted to match the ecology of a
given population (Visser et al., 2010).
Daily time keeping
Like annual cycles, daily cycles of birds have long
been considered precise and reliable, as sym-
bolized by the wake-up call of the rooster in
rural life. The circadian basis of these cycles
and their precision and photosensitivity were
established by chronobiologists early on (Daan
and Aschoff, 1975; Foster and Kreitzman,
2005), and their physiological basis is known in
some detail (Brandstätter and Abraham, 2003;
Cassone et al., 2009; Gwinner, 2001; Kumar
et al., 2004). The circadian pacemaking system
of birds is complex and consists of several major
components that generally include the pineal
gland and hypothalamic structures and, in some
species, also the retina. The pineal gland pro-
duces and secretes the hormone melatonin into
the bloodstream in a 24-h rhythm, with generally
high levels at night and low levels during day-
time. Melatonin plays a major signaling role in
the synchronization of avian daily rhythmicity.
Its rhythmic secretion persists with circadian
periodicity even in pineal organs cultured
in vitro (Gwinner and Brandstätter, 2001). The
second major component of the pacemaking sys-
tem is a structure in the hypothalamus likened to
the mammalian suprachiasmatic nucleus (SCN)
that shows periodic expression of clock genes
and generates an autonomous circadian rhythm
with neuronal output. The rhythms created by
these main pacemakers interact with each other,
as detailed in models of “neuroendocrine feed-
back loop” and “internal resonance” (Cassone
and Menaker, 1984; Gwinner and Brandstätter,
2001). The internal-resonance model assumes
that the pineal rhythm of melatonin secretion
amplifies the rhythm in the SCN, and in turn,
the rhythm of the SCN amplifies that in the
pineal via the hypothalamo-spinal cord-superior
cervical ganglion tract. Such mutual augmenta-
tion would result in a high-amplitude circadian
rhythm that would be strongly self-sustained
(Abraham et al., 2010; Gwinner and Helm,
2003; Menaker et al., 1997). Contributions of the
oscillatory components to the overall pacemaking
system differ between the taxa studied in detail
(mostly songbirds and galliforms; Cassone et al.,
2009; Gwinner, 2001; Kumar et al., 2004; Menaker
et al., 1997). Likewise, the amplitude of the nightly
peak in melatonin differs substantially between
taxa. Rhythmicity of the pineal may be particularly
important in songbirds, in which elimination of
the melatonin rhythm severely disrupts activity
rhythms (Gwinner et al., 1997). Nocturnally active
owls, in contrast, display robust circadian rhythms
despite a rudimentary pineal gland and no signifi-
cant melatonin amplitude (Van’t Hof et al., 1998).
Further, some but not all Arctic bird species for
which melatonin profiles were examined during

polar midsummer had attenuated melatonin peaks
while still retaining rhythmicity (e.g., Miché et al.,
1991; Reierth et al., 1999; Silverin et al., 2009). Thus,
circadian organization is multifaceted and thereby
offers many levels of adjustment for meeting the
particular challenges posed by migration.
In migratory birds, the circadian system masters
at least two particular challenges. First, migrants
often experience substantial shifts in the phase
and length of the solar day, with implications for
daily timing and for the correct interpretation of
time-compensated navigational cues. Surprisingly,
even when moving quickly across time zones dur-
ing East–West flights, migrants can apparently
correctly use the sun compass (Kishkinev et al.,
2010; Kramer, 1959). A second major challenge
for the circadian system are regular switches of
daily activity patterns related to migration in diur-
nal species that migrate at night. Considering the
physiological challenges of migratory flight, the
avian capacity to carry out such additional “shift
work” seems quite remarkable (Rattenborg et al.,
2004). Research on migratory birds has been
motivated by a wish to understand how these
challenges are met.
Several lines of evidence show that the daily
recurrence of migratory behavior is under direct
control of a circadian timer. With the exception of
one study (Coverdill et al., 2008), nocturnal rest-
lessness of migrants remained clearly rhythmic
after transferring from synchronizing light–dark
cycles to constant dim light (e.g., Coppack et al.,
2008; Gwinner, 1996a; Kumar et al., 2006; Pohl,
2000). When free-running period lengths were
estimated separately, they were shorter for “day
activity” than for migration-specific “night activ-
ity,” indicating possible involvement of at least
two oscillators (Bartell and Gwinner, 2005). A cur-
rent conceptual idea therefore suggests that the
temporal flexibility of the avian circadian system
is partly achieved by two oscillators with variable
coupling. During diurnality, the night-activity oscil-
lator is suppressed by or phase-locked to the day-
activity oscillator; if coupling strength of the
oscillators is modified, night activity appears
463
and the two oscillators can stabilize in antiphase
(Bartell and Gwinner, 2005; Mukhin et al., 2009).
In addition, there is evidence for masking by light
that differs for the day and night components
of locomotor activity (Coverdill et al., 2008).
Switches to nocturnal restlessness can result from
internal and external factors. In many long-distance
migrants, periodic nocturnality is clearly driven by
circannual rhythms (Gwinner, 1996a; Holberton
and Able, 1992), but coupling of the oscillators is
also sensitive to environmental factors, in particu-
lar, food. This plasticity is not restricted to migra-
tion and can be triggered by nutritional and
social cues during nonmigratory periods and in
resident birds (Helm and Gwinner, 2006; Mukhin
et al., 2009; Terrill, 1987). The mechanisms
and sites of the putative oscillators governing
day–night-activity remain obscure. Some experi-
ments suggest that the pineal gland is involved
in the circadian regulation of migration-related
behavior, as evidenced, for example, by disrupted
rhythms of nocturnal restlessness after pinealec-
tomy (McMillan, 1972). The melatonin amplitude,
in turn, is modulated by conditions affecting
migratory activity. For example, refeeding of
birds after fasting temporarily increased peak
melatonin levels and interrupted nocturnal activ-
ity (Gwinner, 1996a). Recent experimental
manipulation of melatonin levels as well as food
availability indicated a primary effect of nutrition,
rather than of melatonin, on nocturnality (Fusani
et al., 2011).
A role for melatonin in avian migration is
also suggested by changes in its amplitude during
migration periods. For example, in one study,
the nocturnal melatonin peak was periodically
damped during migratory season even under con-
stant, circannual light conditions (Gwinner et al.,
1993). Such fluctuations have been interpreted
in the context of different pacemaker components
in birds (Gwinner, 2001; Gwinner et al., 1997).
As explained above for the internal-resonance
model, mutual augmentation of pacemakers
would result in a highly self-sustained oscillation,
which in turn makes the system relatively inert
464
in its ability to respond to phase shifts (Abraham
et al., 2010). Conversely, a reduction of amplitude
of the output of the pacemaker as a whole, as
indicated by a reduced peak in melatonin, would
make the system more responsive to Zeitgebers
(Gwinner et al., 1997). This has indeed been shown
by experiments that eliminated the pineal melatonin
rhythm by implants or pinealectomy and resulted
in greater sensitivity of the circadian system to
weak Zeitgebers such as T-cycles and periodic food
availability (Gwinner and Brandstätter, 2001;
Gwinner et al., 1997). Accordingly, temporary
reduction of the melatonin amplitude could enable
birds to modify their schedules rapidly during
migration and thereby help them to cope with aris-
ing phase shifts. This idea has been supported by
preliminary evidence for shorter resynchronization
times during than outside the migratory seasons
(Pohl, 2000). Adjustment of the melatonin ampli-
tude and its interactivity with light is therefore a can-
didate mechanism for adaptations of birds for
meeting migration-related challenges (Gwinner
and Helm, 2003).
Lunar and tidal cycles
In contrast to solar cycles, lunar and tidal rhythms
of birds are poorly known. Most terrestrial species
are diurnal for most of the year and are believed
to be relatively unaffected by moon-related cycles,
while in nocturnal species, effects of moonlight on
activity are evident (Martin, 1990). For example,
Nightjars (Order Caprimulgiformes) reduce activ-
ity and drop body temperature when moonlight
is unavailable, resulting in cyclical heterothermy
related to the lunar cycle (Smit et al., 2011). Com-
pared to terrestrial species, seabirds have generally
more flexible diel activity patterns, which may be
linked to moonlight (Martin, 1990). In Boobies
(Order Pelecaniformes) on the Galapagos Islands,
diel profiles of plasma melatonin and corticosterone
were indeed damped during moonlit nights (Tarlow
et al., 2003). A recent study of tropical Petrels
(Order Procellariiformes) confirmed substantial
lunar cycles of activity and additional coordination
of seasonal migration with lunar and solar cycles
(Pinet et al., 2011). A role of lunar cycles for sea-
sonal behavior has also been suggested for another
sea-dweller, the Sooty Tern (Sterna fuscata; Order
Charadriiformes) (Chapin and Wing, 1959). On a
tropical island, this species breeds approximately
every 9 months, displaying what resembles a free-
running circannual cycle in the wild. Closer analysis
of the data suggests that the precise intervals are
determined by lunar cycles, so that the birds initiate
reproduction during every 10th lunar cycle, but
long-term data from individual birds are still lacking
for consolidation of this idea (Chapin and Wing,
1959). Finally, tidal cycles are generally not well
understood but are clearly relevant for birds that
specialize on foraging along shorelines, that is,
waders, which are considered below in detail.
Temporal multitasking illustrated: Waders
Waders master outstanding challenges of biological
time keeping. Specialized on coastal and terrestrial
wetlands, they cope with daily as well as tidal cycles,
carry out extreme long-distance migrations, and
breed at high latitudes (van de Kam et al., 2004;
Fig. 1). On their polar breeding grounds, they not
only face an extremely short breeding season
but also encounter light conditions in which Zeitge-
ber information is greatly reduced in amplitude.
Waders are a diverse group that displays stark con-
trasts in lifestyles so that relationships between time
keeping, behavioral ecology, and physiology can be
readily explored.
Annual punctuality, daily flexibility
The group of waders contains many primary
examples of highly punctual calendar birds
(Piersma et al., 1990) as well as some weather birds
(e.g., Lapwing, Vanellus vanellus; Schüz, 1971).
Recent advances in animal tracking technology
and observation efforts involving citizen scientists
have boosted information on migratory schedules.

Remarkable insights into the rigidity of scheduling
of annual cycle events come from Bar-Tailed
Godwits (Limosa lapponica baueri). These birds
carry out the longest documented nonstop migra-
tory flights for any landbird—10,000 km from New
Zealand to Asia, where they refuel before migrating
to their Alaskan breeding grounds, and 11,700 km
from Alaska to New Zealand after breeding (Fig. 1;
Battley et al., 2012; Gill et al., 2009). Preparation
for migration from New Zealand begins as early as
late December, when birds may start increasing
mass (P.F. Battley and J.R. Conklin, Massey Univ.,
unpublished data) and soon afterward start to moult
into breeding plumage. Birds depart on migration
mostly from mid-March until early April (Battley,
1997; Conklin and Battley, 2011). The first data on
their repeatable schedules came from a color-
banding study conducted in the Firth of Thames
(Battley, 2006). Individuals were monitored across
2 or 3 years, and despite limitations in detecting
marked birds, there was a highly significant positive
relationship between last sighting in 1 year and the
equivalent date in a subsequent year. Most birds
departed within the same week each year, even
though the population as a whole departed across
a month, so that repeatability (Lessells and Boag,
1987) of apparent departure date was 0.83. This
work was followed up at an isolated estuary where
a smaller population could be monitored in great
detail (Conklin and Battley, 2011). There, individ-
ual departures were generally confirmed to the
day and mostly directly observed. Quite remark-
ably, the calculated repeatability was virtually iden-
tical (0.84) to that from the previous study. Across a
span of roughly a month, most birds left within the
same week across 3 years; most of those that did
not were delayed due to avoidance of winds that
were unsuitable for migration. Clearly, New
Zealand Godwits have strongly characterized indi-
vidual migration schedules.
One possible interpretation for individual varia-
tion was that birds of different schedules bred
in places with different breeding seasons, as
suspected for sympatrically wintering songbirds
(Curry-Lindahl, 1963). For waders like Godwits,
465
a crucial aspect of breeding habitat is thaw date,
which varies with breeding latitude. A study using
geolocators, which calculate position based on
recordings of local daylight (Conklin et al., 2010),
confirmed that the latitude the birds attempted to
breed at was closely associated with their schedul-
ing throughout migration (Fig. 1). Even upon first
departure from New Zealand, 45% of the variance
in the timing of migration was explained simply
by what latitude the bird was heading to. By
individuals spending varying amounts of time
staging in Asia, the relationship between departure
date and latitude became even stronger upon leav-
ing the Yellow Sea (r2 ¼ 0.80), and by the time
birds arrived on the breeding grounds, there was
almost no variation left to explain (r2 ¼ 0.91).
Hence, the timing of an individual’s migration
toward the arctic and subarctic breeding grounds,
even from deep into the southern hemisphere,
reflected a repeatable schedule relating to eventual
arrival time on the breeding grounds. Surprisingly,
differences in arrival time tended to carry over to
the end of the breeding season so that birds
from the more clement, lower-latitude locations
departed on their trans-Pacific migration earlier
than those from the colder high-latitude locations
(r2 ¼ 0.32). Taken together, the observed patterns
strongly suggest that these Godwits largely rely
on internal timing programs for their migratory
schedules and that these timing programs may dif-
fer between breeding populations, as reported for
songbirds (Curry-Lindahl, 1963; Helm et al., 2009).
A second, striking example of punctuality in
waders may give leads to how such local patterns
might evolve. In the closely related Icelandic
Black-Tailed Godwit (L. limosa islandica), the
mates of breeding pairs winter in separate locations
(Gunnarsson et al., 2004; Fig. 1). Surprisingly, previ-
ously mated birds nonetheless returned to the
breeding sites at very similar times as their mates
(r ¼ 0.97). Synchrony of arrival times was also
demonstrated in the lower-latitude sister subspecies
L. limosa limosa (Lourenço et al., 2011). This may
be highly consequential for mate choice and breed-
ing success because pair formation in migratory
466
birds can be strongly time-dependent. In the
Iceland-breeding subspecies, two out of the three
Black-Tailed Godwit pairs whose mates returned
8 days or more apart divorced, suggesting that syn-
chronous arrivals are an important factor in
mate retention. Similarly, time-dependent pair for-
mation is known from other migrants like penguins,
swans, and songbirds (summarized in Helm et al.,
2006). Based on such observations and on an
assumed partly genetic regulation, the evolution of
timing could be accelerated by assortative mating
(Bearhop et al., 2005).
In at least two species of wader, the precise
annual timing is based on robust circannual rhythms
(Piersma, 2002; Piersma et al., 2008). Birds that
were kept first under simulated natural daylength
and then under constant light–dark cycles displayed
clear circannual cycles of periodic fattening and
moult of body plumage and flight feathers. Strik-
ingly, however, these cycles differed from those in
songbirds by an endogenous period length that
was clearly longer, rather than shorter, than the
solar year (Gwinner, 1986; Piersma et al., 2008).
Daily activity patterns of waders are generally
reported to be relatively flexible and are thought
to depend only weakly on internal clocks (Krüll
et al., 1985; Martin, 1990). This is based on several
lines of evidence. Behavioral observations indicate
that waders can forage at night, aided by sophisti-
cated tactile sensing of prey in soft ground. When
light intensity is increased, for example, by artifi-
cial illumination along shorelines, the birds
take advantage of visual opportunities and extend
foraging into the night (Santos et al., 2010). Flexi-
ble timing is also strongly suggested by activity
patterns of waders in tidal habitats, for which rich
descriptive data and, to our knowledge, the only
experiment on avian tidal cycles are available.
For example, constant recording of Red Knots
(Calidris canutus) at a roost in the winter quarters
yielded clear bitidal patterns of attendance. Rest
times of the birds drifted straight across daytime
and nighttime without any evidence of modulation
or masking by daylight (Leyrer et al., 2006; see van
Gils et al., 2006). However, observations from
other species indicate that light conditions might
affect activity patterns via predation risk
(Beauchamp and Ruxton, 2008). In Oystercatchers
(Haematopus ostralegus), behavioral observations
strongly suggested periodic movements in anticipa-
tion of tidal food availability. An experimental
approach yielded findings that, although not con-
clusive, were suggestive of possible (circa-)tidal
clocks in these birds (Daan and Koene, 1981).
Temporal flexibility is also reported from
Arctic-breeding waders that frequently experi-
ence continuous polar light from arrival until
migratory departure. The annual time period dur-
ing which the birds can breed is extremely short so
that they should reproduce as early as possible after
arrival to maximize reproduction (Yohannes et al.,
2010). While seasonal time pressure is high, light
availability could be permissive for efficient timing
of activities around the clock. Preliminary data
from a single Alaskan study site support this idea
(Silke Steiger et al., unpublished data). The study
examined several sympatrically breeding species
that differed in mating system and in the ways the
sexes shared parental care (Székely et al., 2006).
Based on this diversity, species-specific activity
patterns were predicted and indeed discovered.
Under continuous light, the birds were either briefly
active around the clock or showed rhythmic activity,
most commonly while incubating (Cresswell et al.,
2003). However, only some species synchronized
their rhythmic activities to the 24 h cycle of subtle,
possible Zeitgebers such as changes in light intensity,
polarization pattern, or sun azimuth (Gwinner,
2001; Krüll et al., 1985; Pohl, 1999). Activity rhythms
of other species drifted across the 24-h day, and
in some of these cases, breeding partners showed
remarkably well-coordinated incubation shifts
which are suggestive of social synchronization (Silke
Steiger et al., unpublished data).
Melatonin cycles of captive waders
In view of their extensive temporal multitasking,
we used waders to explore mechanisms that may

enhance such flexibility. Based on concepts of
internal resonance, Daily time keeping and neuro-
endocrine feedback loops (see above), we specula-
ted that melatonin cycles of waders should have
low amplitudes, which could be further reduced
during seasons when the birds would entrain to
subtle Zeitgebers. To test predictions of circadian
modulation via adjustment of the melatonin ampli-
tude, we compared two wader species that share
many features of migration and the annual cycle
(Fig. 1) (Mendes et al., 2006; Piersma et al.,
1996). The Ruff (Philomachus pugnax) and Red
Knot are similarly sized basal waders (sandpipers,
family Scolopacidae). Both breed at mid to high
latitudes, winter in temperate to tropical zones,
have definitive periods of migration, and like to
flock. They show robust annual cycles in the wild
(Buehler and Piersma, 2008) and in captivity
(Piersma et al., 2008). However, the species also
represent prominent contrasts in ecology
(summarized in Piersma, 2003; Mendes et al.,
2006; Piersma et al., 2000). Whereas Ruffs stay at
inland freshwater wetlands and breed at mid
latitudes, Knots never leave the seashore except
to breed on the northernmost tundra (Fig. 1).
Ruffs are lek-breeding, highly sexual dimorphic
birds, while Knots are socially monogamous with
biparental care. Ruffs typically have strongly pat-
terned diel rhythms of diurnal feeding and noctur-
nal use of communal roosts, whereas Knots lead a
fully tidal life, foraging during day and night, and
gathering at roosts at high tides (Leyrer et al.,
2006), except for the brief time when they breed
in the High Arctic. Based on these differences,
we expected to find corresponding differences in
the daily and annual profiles of melatonin.
Specifically, we asked (1) whether melatonin
rhythms were low in these birds that perform on
different temporal scales and entrain to subtle
Zeitgebers and (2) whether melatonin profiles dif-
fered between the two species such that Ruffs
would show a clear, diel pattern. Knots should
have a lower melatonin amplitude than Ruffs as
an adaptation for, or a consequence of, tidal activ-
ity cycles to enhance entrainment to nonphotic
467
Zeitgebers. We also asked (3) whether melatonin
profiles changed across the year. We predicted
that, in both species, melatonin should be reduced
during life-cycle stages when birds entrain to rap-
idly shifting or subtle Zeitgebers, that is, during
migration and breeding. Alternatively, melatonin
profiles could vary seasonally, simply as a reflec-
tion of light conditions. (4) Lastly, we tested for
suppressive effects of moonlight.
Both species were studied in small flocks under
identical conditions at the Royal Netherlands
Institute for Sea Research (NIOZ) on the island of
Texel (53 050 N 4 500 E) in the Dutch Wadden Sea.
Data on annual cycles in various body traits, includ-
ing seasonally changing immune parameters, have
been published elsewhere (Piersma et al., 2000).
For some Red Knots, overall seasonal patterns of
nightly amplitude and duration of elevated plasma
melatonin were compared to immune parameters
to test for immuno-enhancement (Buehler et al.,
2009). Here, we newly report melatonin profiles
around the year for Ruffs and detailed diel profiles
for Knots. We relate these patterns to seasonal
activities and to tentative data on activity collected
from small groups because these gregarious birds
cannot be well studied individually.
Methods
Experiments involved eight Red Knots of the sub-
species islandica (four males, three females, one
sex unknown), which winters in Europe, and seven
Ruffs (all males), which use the Wadden Sea for
staging (Fig. 1; for details, see Buehler et al., 2009;
Piersma et al., 2000). In the Dutch Wadden Sea
Ruffs were caught on March 15, 1997 using a
“wilsternet,” while Knots were captured with
mistnets near a high-tide roost between 1994 and
1997. At capture, birds were ringed, aged as older
than 2 years, and transferred to outdoor aviaries
(4.5 m  1.5 m  2.3 m high) at the NIOZ. Knots
were kept in a single aviary, while Ruffs, which
are more territorial, were divided over two
aviaries. One-quarter of the surface contained a
shallow basin with sand and running seawater
468
(mudflat). Each aviary contained a small freshwa-
ter basin for drinking and bathing. The birds were
fed ad libitum portions of protein-rich trout-food
pellets and once per week were screened for physi-
cal condition, while the aviaries were disinfected
and the food replenished. Birds experienced local
photoperiod and temperature. Compared to wild
conspecifics, Ruffs were thus exposed to identical
conditions in spring and fall (Fig. 1). In summer,
they would have experienced slightly longer days
at more northerly latitudes and winter days would
also be longer on their African winter grounds.
In contrast, Knots were exposed to conditions
that were identical to those experienced by wild
conspecifics that stay in the Netherlands from late
summer until spring. During summer, daylength
was much shorter for the captive Knots than for
conspecifics on their Arctic-breeding grounds.
Motivation for migration should be similar in
spring but differ between the species in autumn
when Ruffs should be active migrants, whereas
Knots would be already at their wintering grounds.
Behavioral observations of Knots and other species
suggest that migration may indeed be suppressed
by site recognition (Ketterson and Nolan, 1988;
Schwabl et al., 1991). To delineate life-cycle stages
(Fig. 2), data on phenology were collected for all
birds once per week. Body mass was measured on
an electronic balance to the nearest gram. Moult
was scored on breast plumage for body moult
(0 ¼ no feathers growing, 1 ¼ feathers growing)
and on the primaries for flight feather moult
(scores from 1 ¼ feather dropped to 5 ¼ new
feather for all 10 primaries, thus reaching values
from 0 to 50; Ginn and Melville, 1983). Extent of
breeding plumage was scored on a scale between
1 ¼ basic, indicating full gray winter plumage, and
7 ¼ full alternate, indicating full rusty-red breeding
plumage. Experiments started for Knots on Febru-
ary 20 and for Ruffs on March 24, 1997 and were
completed on February 11, 1998. Melatonin was
sampled biweekly as explained in Fig. 3.
As a rough overview of activity patterns, in one
aviary group of each species wading in the
simulated mud flats was continuously recorded.
In Knots, feeding activity was also surveyed by
an additional sensor next to the single source of
food. Ruffs, in contrast, were equipped with sev-
eral feeders because of their higher aggression
so that foraging was not monitored. Locomotion
and feeding were surveyed by custom-made pho-
tocell systems. Beam breaks of an infrared beam
were recorded in 2-min bins on a computerized
data-logging system. Activity data originated
from initially eight Knots (six birds from August
1997) and from initially four Ruffs (three birds
from August 1997). Sensors yielded data through-
out experimentation, except for brief periods of
technical problems. Because of behavioral
differences between the species, we interpreted
their activity records from the artificial mudflats
differently. Red Knots forage on mudflats, and
even in our aviaries with permanent food supply
elsewhere, they spent 10–20% of each day actively
probing for food on the artificial mudflats. In con-
trast, they roost on dry land and not on mudflats.
Therefore, recordings from the mudflat in Knots
reflect feeding and general activity, and the addi-
tional feeding sensor narrowed down foraging
motivation. Ruffs, in contrast, usually do not feed
on mudflats, but they like to roost on wet ground
if available. Therefore, recordings from the mud-
flat combine general activity and resting behavior.
However, the group recordings of both species
yield only tentative information.
Results and discussion
Daily profiles of melatonin are shown for all sam-
pling periods and separately for life-cycle stages
(Figs. 3 and 4). We conducted two overview ana-
lyses (Table 1) that converged in all major points:
melatonin levels strongly differed between day-
time versus nighttime (LD) and depended greatly
on life-cycle stage. We also found some evidence
that effects of light differed over the annual cycle.
In general, melatonin peaks tended to be higher
during late moult and wintering, but there was
no direct relationship to daylength (Figs. 3 and
 469

(a) (b)
Knot Winter Spring Breeding state Moult Winter Ruff Winter Spring Breeding state Moult
Fuel Fly Transitory Fuel & fly Fuel & fly Postmigratory

16 16
Daylength (h)

Daylength (h)
14 1 2 3A 3B 4 1 1 2 3 4A 4B 1 14

12 12

10 10
8 8

Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1

240
180

Body mass (g)

Body mass (g)

220
160
200
140
180
120
160
100
140
Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1

50 50
Primary moult

Primary moult
40 40
30 30
20 20
10 10
0 0
Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1
8 8
Plumage color

Plumage color
6 6

4 4

2 2

0 0
Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1
4 4

Breast moult
3 3
Breast moult

2 2

1 1

0 0

Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1 Mar 1 Apr 1 May 1 Jun 1 Jul 1 Aug 1 Sep 1 Oct 1 Nov 1 Dec 1 Jan 1 Feb 1

Fig. 2. Derivation of life-cycle stages based on phenology of (a) Knots and (b) Ruffs. Top: natural daylength, with stars marking
days of blood sampling and letters indicating life-cycle stage; bottom: mean  SD for body mass and scores of primary moult,
plumage color, and breast moult; vertical lines indicate demarcations of life-cycle stages. Stage definitions Knot: (1) “winter”:
quiescent time. From end of autumn moult until start of spring preparations, that is, fattening and moult into breeding plumage;
(2) “spring”: fueling and prenuptial moult. From start of fattening, moult, and recoloration until reaching full breeding coloration
and peak body mass; (3) “breeding state”: defined by full breeding plumage. From reaching peak body mass and breeding
coloration until the onset of moult; (3A) “flight phase”: ca. 4 weeks of using up fuel stores, from maximum to trough of body
mass. Prediction: melatonin low; (3B) “transitory phase for nonbreeders”: corresponding to breeding in the wild, from end of
body mass peak until start of moult. Prediction: melatonin low; (4) “moult”: from start to completion of primary moult,
corresponding to moult of wild conspecifics on the winter grounds slightly later in the year. Stage definitions Ruff: (1) “winter”:
quiescent time. From end of autumn moult until start of spring preparations, that is, moult into breeding plumage; (2) “spring”:
fueling and prenuptial moult. In contrast to Knots, Ruffs overlap these activities with migratory flight. From start of moult and
recoloration until reaching full breeding coloration and peak body mass. Prediction: melatonin low; (3) “breeding state”: defined
by full breeding plumage. From peak body mass and breeding coloration until the onset of moult; (4) “moult”: renewal of
primaries. From start to completion of primary moult, corresponding to moult of wild birds started during migration and
completed on the African winter grounds; (4A) “fuel, flight and moult phase,” starting with a transitory phase in nonbreeders
with slow and then progressively faster moult and fueling. Unlike in captive Knots, this corresponds to flight phases in the wild.
From starting of color loss and increase of body mass until birds reach peak body mass. Prediction: melatonin low; (4B)
“postmigratory completion of moult”: From end of body mass peak until completion of moult, corresponding to finalizing of
moult in Africa.
470

Knots Ruffs
60
30

60 Mar 1
30

60 60
30 30
60 60
30 Apr 1 30

60 60
30 30

60 60
30 May 1 30

60 60
30 30

60 60
30 Jun 1 30

60 60
30 30

60 60
30 30
Jul 1
60 60
30 30

Sep 1
60 60
30 30

60 60
30 30
Oct 1
60 60
30 30

60 60
30 30
Nov 1
60
30

60 60
30 30
Dec 1
60 60
30 30

60
30
Jan 1 60
60
30 30

60 60
30 30

60 Feb 1 60
30 30

12:00 16:00 20:00 24:00 04:00 08:00 12:00 12:00 16:00 20:00 24:00 04:00 08:00 12:00
 471

4). Both models did not support species
differences. Results for damping effects of moon-
light on melatonin were equivocal: comparison of
elevated versus baseline melatonin by logistic
regression was more supportive of moonlight
effects than comparison of melatonin levels by lin-
ear mixed effect models. Within the species, sub-
stantial effects of light (LD) and life-cycle stage
were confirmed, while there was little evidence
for interactions between these factors. Effects of
moon were clearly more pronounced in Ruffs com-
pared to Knots. Predictions for stage-dependent
suppression of melatonin amplitudes were only
partly supported. In Fig. 4, the life-cycle stages
for which low melatonin was predicted are plotted
by solid lines. While melatonin was indeed low
during these times of year, similarly low
amplitudes also occurred at other times of year.
Group activity patterns were overall rhythmic
(Fig. 5; Table 2). In Knots, wading and feeding
recurred twice a day, with dominant periodicities
around 12 h (during the breeding flight phase, a
main peak around 22 h had only slightly higher
spectral power than one at 10.9 h). These peri-
odicities had relatively high spectral power but
were best supported during moult and least
supported during the breeding flight phase.
Actograms were also suggestive of additional,
longer periodicities in autumn, which, however,
could not be ascertained. Surprisingly, the main
feeding activity of Knots shifted into the night
after moult. In Ruffs, support for rhythmicity
was generally much weaker and primary peri-
odicities were more variable than in Knots. How-
ever, during postmigratory moult, Ruffs showed
pronounced activity cycles that recurred twice
per day.
Overall, our study showed rhythmic, but low
melatonin levels in the two wader species that
were of similar magnitude as in other seabirds
(Miché et al., 1991; Tarlow et al., 2003; Wikelski
et al., 2006). Melatonin cycles changed over the
annual cycle, but the seasonal course did not fully
support damping during particular life-cycle
stages as had been shown for migratory songbirds
(Gwinner and Brandstätter, 2001; Gwinner et al.,
1993), nor did it simply reflect the amount of
available daylight. Our findings add to heteroge-
neous evidence for links between light availability
and melatonin in birds (Buehler et al., 2009;
Gwinner and Brandstätter, 2001; Miché et al.,
1991; Reierth et al., 1999; Silverin et al., 2009).
Moonlight apparently exerted a mild, damping
influence on melatonin (Tarlow et al., 2003),

Fig. 3. Diel melatonin profiles across the annual cycle for Knot (left) and Ruff (right) (mean  SE; [pg/ml]). Plots are arranged by
corresponding time of year. Vertical curves show time of sunset and sunrise, respectively. The birds were sampled biweekly to
examine diel profiles over an entire year, alternating between phases of full moon and new moon. Sampling took place over a 24-h
session, starting and ending at mid-day with seven bleedings per bird at 4-h intervals (12:00, 16:00, 20:00, 00:00, 04:00, 08:00;
12:00 h; Piersma et al., 2000). On each sampling date, blood was collected for at least six birds of both species. The protocol
followed local time, implying that samples were collected an hour earlier relative to the solar day during summer savings time.
Blood was collected within 15 min after entering the aviary by lightly puncturing the brachial wing vein. We collected 150–250 ml of
blood into heparinized capillary tubes that were stored cool. Tubes were centrifuged within 2 h of sampling at 6900  g for 15 min,
and plasma was subsequently stored at -80  C until transfer on dry ice to the Max-Planck Institute for Ornithology in Andechs,
Germany. Data collection was briefly interrupted in August 1997, and for Ruffs, two sampling periods were missing (12 November;
19 December). Individual birds were sampled up to 22 times during a total of 23 sampling periods, and overall we analyzed 1667
samples. Simultaneous monitoring of health parameters indicated no harmful effects (Piersma et al., 2000). We quantified
melatonin by the established procedure at our laboratory (Silverin et al., 2009; Van’t Hof and Gwinner, 1996) as described for the
Knots (Buehler et al., 2009). Due to the sudden loss of Ebo Gwinner and subsequent complications, we unfortunately have only
long-term information about the melatonin assay at our institute. Accordingly, mean recoveries are at 90%, and intraassay and
interassay variation were 4.5% and 14%, respectively. For further details on Knots, see Buehler et al. (2009).
472

70
Knots Winter
Spring
Breed A
60 Breed B
Moult
Melatonin (pg/ml)

50

40

30

20

12:00 16:00 20:00 24:00 04:00 08:00 12:00

70
Ruffs
Winter
Spring
Breed
60
Moult A
Moult B
Melatonin (pg/ml)

50

40

30

20

12:00 16:00 20:00 24:00 04:00 08:00 12:00

Time of day

Fig. 4. Daily profiles of plasma melatonin (mean  SD; [pg/ml]) by species-specific life-cycle stage (cf. Fig. 2) for Knots (top) and
Ruffs (bottom). Solid lines highlight stages for which low melatonin amplitudes had been predicted.
 473

Table 1. Melatonin profiles in Knot and Ruff

Species comparison Knot Ruff

Logistic Linear mixed Logistic Linear mixed Logistic Linear mixed

Factors regression effects regression effects regression effects

Light/dark w42 ¼ 198.4; w42 ¼ 159.3; w52 ¼ 154.8; w52 ¼ 109.9; w52 ¼ 52.9; w52 ¼ 46.3;
(LD) p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001
Species w12 ¼ 0.007; w12 ¼ 2.32; – – – –
p ¼ 0.935 p ¼ 0.128
Life-cycle w62 ¼ 138.6; w62 ¼ 102.4; w82 ¼ 69.4; w82 ¼ 47.4; w82 ¼ 110.9; w82 ¼ 103.5;
stage p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001
Moon w12 ¼ 5.13; w12 ¼ 0.02; w12 ¼ 0.6; w12 ¼ 5.1; w12 ¼ 18.7; w12 ¼ 8.5;
p ¼ 0.02 p ¼ 0.88 p ¼ 0.449 p ¼ 0.024 p < 0.001 p ¼ 0.003
Light/dark* w32 ¼ 11.3; w32 ¼ 9.5; w42 ¼ 13.2; w42 ¼ 4.1; w42 ¼ 3.9; w42 ¼ 7.0;
Life-cycle p ¼ 0.010 p ¼ 0.023 p ¼ 0.010 p ¼ 0.392 p ¼ 0.426 p ¼ 0.134
stage

Results from analyses using logistic regression and linear mixed effect models; inference based on likelihood-ratio testing. Data on melatonin were not
normally distributed because about two-thirds of the values were at baseline concentrations (lower detection limit: 17 pg/ml). We analyzed these cen-
sored data in two ways: Distinguishing between baseline and elevated melatonin, we performed logistic regression with binominal error distribution,
using procedure GLMER in the software R (R Core Development Team, 2009). We analyzed melatonin grouped by individual bird by the factors
daytime versus nighttime (LD), “moon,” “species,” life-cycle stage (stage), and interaction of LD stage, (LD* stage, whereby asterisk indicate inter-
action). In this model, assumptions were met, but information content was reduced. We then analyzed original (log-converted) melatonin data using
linear mixed effect models (procedure LMER in R), using full information but deviating from distributional assumptions. Significance testing was per-
formed by likelihood-ratio testing (procedure ANOVA in R). In these overview analyses, life-cycle stages (see Fig. 2) were made comparable by
pooling data from stage “breed A” in Knots with those of “spring,” and by pooling data from stage “moult B” in Ruffs with those of winter. Within
species, we analyzed data over their defined, specific life-cycle stages.

particularly in Ruffs. Given the flexible schedules Concluding remarks

of waders and their entrainment to subtle
Zeitgebers, one may ask why waders retained diel
melatonin rhythmicity at all, while some noctur-
nal species have no significant melatonin ampli-
tude and may nonetheless show robust circadian
rhythms (Van’t Hof et al., 1998; Wikelski et al.,
2006). Presumably, the rise in melatonin in
waders could serve physiological functions other
than those involved in circadian rhythmicity, for
example, induction of sleep despite continuous
light and reduction in body temperature (Silverin
et al., 2009; Tarlow et al., 2003). An additional
possibility, immuno-enhancement, was found to
be unlikely for Red Knots (Buehler et al., 2009).
Experimental studies involving melatonin treat-
ment of different avian species did indeed indi-
cate separate adjustment of several regulatory
functions of melatonin (Murakami et al., 2001),
but such presumable, diverse effects of melatonin
cycles in birds still require further exploration.
We hope to have shown that migratory birds are an
exciting study system because their wide range of
temporal problem-solving offers opportunities for
addressing important questions in chronobiology.
As first suggested for birds, circannual systems
can now progressively be viewed as “periodically
changing dispositions to respond to environmental
cues” (Gwinner, 1999) that are fine-tuned by evo-
lution. New studies of migrant circadian systems
highlight remarkable plasticity and responsiveness
to external conditions, but mechanisms behind the
observed diversity of avian time keeping still
remain to be understood. The balance between
rigidity and flexibility of timing, as evident in
migratory birds, is of growing interest to chronobi-
ology but is also relevant for the general public.
Global change progresses at a disconcerting rate
and its implications for seasonal and daily timing
become increasingly evident. Organisms have
474

May 1

May 1
May 1

Jun 1

Jun 1
Jun 1

Jul 1

Jul 1
Jul 1

Aug 1

Aug 1
Aug 1

Sep 1

Sep 1
Sep 1

Oct 1

Oct 1
Oct 1

Nov 1

Nov 1
Nov 1

Dec 1

Dec 1
Dec 1

Jan 1

Jan 1
Jan 1

Feb 1

Feb 1
Feb 1

00:00 12:00 24:00 12:00 24:00 00:00 12:00 24:00 12:00 24:00 00:00 12:00 24:00 12:00 24:00
Time of day

Fig. 5. Actograms showing activities of groups of waders around the annual cycle; data are double-plotted across the day. Left:
wading of Knots; middle: feeding of Knots; and right: wading of Ruffs on a simulated mudflat.
 475

Table 2. Rhythmicity of wading activity of groups of Knots and Ruffs; tabulated data are time intervals of analysis, primary
periodicity, and corresponding maximum of spectral power separated by life-cycle stage

Life-cycle stage Start End Period Spectral power

Knot
1 Winter Nov 18, 1997 Feb 3, 1998 12.1 354.5
2 Spring Apr 5, 1997 May 19, 1997 13.1 953.5
3 Breed A “flight phase” May 20, 1997 Jun 23, 1997 21.8 148.2
3 Breed B “transitory” Jun 24, 1997 Jul 16, 1997 11.8 461.0
4 Moult (1) Aug 7, 1997 Aug 22, 1997 12.0 1851.2
4 Moult (2) Aug 29, 1997 Nov 17, 1997 11.9 1493.7
Ruff
1 Winter Apr 5, 1997 Apr 14, 1997 15.9 52.7
2 Spring Apr 15, 1997 May 26, 1997 46.4 46.3
3 Breed (1) May 27, 1997 Jul 16, 1997 60.5 78.4
3 Breed (2) Jul 25, 1997 Aug 5, 1997 12.2 87.7
4A Moult “fuel and flight” Aug 29, 1997 Nov 23, 1997 11.9 153.9
4B Moult “postmigratory” Nov 24, 1997 Feb 3, 1998 12.0 10082.4

Due to equipment failure, registration from the moult stage in Knots and the breeding stage in Ruffs were split into two parts. We used maximum
entropy spectral analysis (MESA) (Dowse, 2007) to identify the primary periods during which spectral power peaked.

altered their temporal behavior and natural sys-
tems begin to come out of synchrony in what
appears to be a puzzling, largely unexplained mix-
ture of organismic responses. While many species
cannot keep up with changes, others are surpris-
ingly apt to take advantage of new conditions.
Changes in temporal patterns, and as a conse-
quence, in behavior and species distribution, likely
affect not only biodiversity per se but also human
health and agricultural productivity. Understand-
ing changes in time keeping has therefore become
of pressing interest. Chronobiologists are in a
privileged position to address such predicted
changes and the mechanisms that might determine
organismic responses.
Acknowledgments
the DEC (Animal Experiment Committee) of the
Dutch Royal Academy of Sciences (KNAW).
Tom Van’t Hof provided valuable endocrine
expertise and ideas for the melatonin experiments.
Wolfgang Goymann and Josef Habersetzer helped
to complete analysis of melatonin and activity data.
Marina Lehmann, Fränzi Körner, and Dusty
Dowse gave friendly assistance on data handling
and analysis, and Edda Starck and Dick Visser cre-
ated graphics. We are most grateful to Joop
Jukema for catching the Ruffs, Piet Duiven for
catching the Knots, and Ciska Raaymakers, Timo
Verbeek, and Bernard Spaans for all sorts of prac-
tical help. The shorebird work on Texel was
supported by a PIONIER-grant to T. P. from the
Netherlands Organization for Scientific Research
(NWO). B. H. is supported by the European Social
Fund in Baden-Württemberg.

We greatly miss our coauthor Ebo Gwinner, who

has been the driving intellectual force behind the
empirical study but did not live to see its publica-
tion. We thank Helga Gwinner for kindly
supporting this late publication of his work.
Research was carried out under the auspices of
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 Subject Index

Note: Page numbers followed by “f” indicate figures, and “t” indicate tables.

ACTH. See Adrenocorticotrophin (ACTH) role, 120

Adenosine monophosphate-activated protein SCN, 121
kinase (AMPK) activation, 164–165, 187 VIP, 132–133
Adrenocorticotrophin (ACTH), 166, 171 Autonomic nervous system (ANS), 184, 185, 189
ANS. See Autonomic nervous system (ANS) Avian migration. See Temporal multitasking
Anteroventro-periventricular (AVPV) nucleus, AVP. See Arginine vasopressin (AVP)
Kp neurons
circadian oscillator, 427 BAC. See Bacterial artificial chromosome (BAC)
circadian signal, 425–427, 426f Bacterial artificial chromosome (BAC), 251,
estrogen signal, 425, 426f 257–258
female rodents, 425, 426f
RFRP-3, 427–428 cAMP. See Cyclic adenosine monophosphate
Anthrenus verbasci, circannual rhythm. (cAMP)
See Circannual rhythm, Anthrenus verbasci Cardiotrophin-like cytokine (CLC)
Arcuate (ARC) nucleus, Kp neurons PK2, 124
melatonin-binding sites, 428 receptor, 123
RFRP neurons, 430–432 rhythm, 123
seasonal reproduction role, 123
GnRH and and gonadotropin release, 429 TGF-a, 122–123
Kiss1 expression, 430 Cardiovascular function and circadian disruption
melatonin regulation, 429–430, 429f cardiomyopathic hamsters, 346
Siberian hamsters, 430 hypertension
T3/RFRP/Kp network, 429–430, 431f clock genes, 347
Syrian hamster, 428 SCN dysfunction, 346–347
T3/RFRP/Kp network, 432–433 misalignment and clock gene mutations,
TSH-driven regulation, tanycyte deiodinases, 345–346
432 shift work, 346
Arginine vasopressin (AVP) Tau mutant hamsters, 346
circadian rhythms, 133 Central and peripheral clock interaction
deficiency, 120–121 ACTH, 171
description, 120 communication routes, circadian timing system,
GnRH, 121 166–167, 167f

481
482

Central and peripheral clock interaction intersubject and intrasubject confound,

(Continued) 386–387
daily fluctuation, 166 PVT and recovery rate, 387–388
description, 163–164 tasks and sleep loss, 386–387
FEO, 169 driving performance, 386
fibroblasts, 170 human performance, regulation
GC secretion (see Glucocorticoid (GC) homeostatic and circadian process, 383
secretion) sleep inertia, 383
glucose metabolism waking performance and sleep propensity,
clock-deficient animal models, 173 383–384
euglycemia, 174 sleepiness and fatigue, 383
hepatic sympathetic denervation, 173 sleep propensity, 384
insulin sensitivity, 173–174 Circadian clocks
maintenance, 172 description, 41–42
neurons, 174 development
orexin, 172–173 aanat2 expression, 46
immortalized rat fibroblast (Rat-1) cells, 170 blastula, 47
MASCO, 169–170 bmal mRNA expression, 47
mediobasal hypothalamus (MBH), 168 circadian rhythms, 46–47
molecular clockworks DD, 46–47
AMPK activation, 164–165 per1b expression, 46–47
circadian pacemaker, SCN light-entrainable peripheral clocks, 47–53
(see Suprachiasmatic nuclei (SCN)) zebrafish (see Zebrafish)
orchestration, 165 Circadian output signals
redox status, 164–165 AVP, 120–121
TTL, 164 dye tracing experiments, 120
pathways, 167f, 170 neuronal firing, 123
Per gene expression, 171 PK2, 121–122
PVN, 166 SCN, 119–120
retina, 167–168 TGF-a and CLC, 122–123
reward system controls and animal behavior, Circadian rhythms
168–169 description, 184
tissue, transplantation, 165–166 nongenetic clock
TTL, 166 AMPK activation, 187
Chronic partial sleep restriction cAMP, 187
accidents and medical errors, 383 heme, 187–188
behavioral alertness, 384–386 NAD, 187
causes peroxiredoxins (PRX), 188
chronotype, 382–383 posttranslational modifications, 186–187
commute/travel, 382 oscillators, 184
leisure, 382 regulation, peripheral tissues, 184–185
paid work, 380–382 SCN (see Suprachiasmatic nuclei (SCN))
cognitive performance social interactions
attention and working memory, 388 abiotic factors, 267
executive function and deficits, 388 Acomys cahirinus and A. russatus, 269
 483

American mink, 269 constant routine and forced desynchrony

animals, 273 protocols, 340–341
assays, 274 coronary artery ischemia, 345
Coda, 276 epinephrine and norepinephrine, 344–345
description, 267–268 morning peak and behavioral changes, 340
environment, 273–274 parasympathetic nervous activity, markers,
experimental design, 274–275 345
field and laboratory, 271–272 platelets, 341–344
Gerbillus allenbyi and G. pyramidum, 269 relative frequency histogram, 339–340, 340f
honeybees, 270 resting heart rate, 341, 342f
maternal entrainment, 271 standardized bicycle exercise, 341, 343f
mixed sex interactions, 272 light exposure, 339
Mongolian gerbil, 269 metabolic function (see Metabolic function and
mutualism, 268 circadian disruption)
Nile grass rat, 275 and nutrition (see Nutrition and circadian
parasitism, 268–269 timing system)
predator–prey, 270 SCN and circadian oscillators, 338
wavelet transforms, 275 western societies, 337–338
transcriptional–translational feedback model, Circannual clock hypothesis, 445
185–186 Circannual rhythm, Anthrenus verbasci
WAT (see White adipose tissue (WAT), adenohypophysis, 452
circadian rhythms) biological clocks, mechanisms, 439–440
Circadian system characteristics and environmental cycles, 451–452
clocks, 84 circannual clock hypothesis, 445
distribution, ontogenesis, 97 description, 440
LD cycle, 83–84 DNA microarrays and RNAi, 453
peripheral clocks, 94–95 entrainability, 444–445
SCN, 84 environmental cycles, 439–440
utero, 84–92 frequency demultiplication hypothesis, 445–446
Circadian thermoenergetics (CTE) Japanese population, 440
hypothesis molecular mechanisms, 453
energy balance, 297–298 photoperiodism, entrainment
LD cycle, 298–299 circadian clock, 450–451
proximate mechanisms, 296–297 natural day length, 449–450
temperatures, 297–298, 298f, 299 physiological components, 452, 452f
ultimate mechanism PRCs and biological oscillator, 447–449
ambient temperatures, 297 properties, 440
mice, food intake, 297, 298f self-sustainability
Circadian timing system constant light/darkness, 441–442, 442f
cardiovascular function (see Cardiovascular frequency, pupation times, 441, 441f
function and circadian disruption) inter-and intraindividual variability, 442–443
core clock genes, 338–339 Junco hyemalis and Sturnus vulgaris, 442–443
description, 338 sequence of linked stages hypothesis, 446–447
internal clock and adverse cardiovascular Tamias sibiricus and Ovis aries, 452–453
events temperature compensation, 443–444
484

Clock gene functions, 139 synaptic overload, 221–222

Cryptochromes (CRYs) thalamus-driven process, 223
Drosophila Timeless protein, 234–236 topographical modifications, 222
PERs, 236 description, 220
CRYs. See Cryptochromes (CRYs) implication, medicine
Cyclic adenosine monophosphate (cAMP), 187 clinical practice, 229
dissociation and sleep inertia, 229
Deep brain photoreceptors, 418 hippocampus, 228
Disc1. See Disrupted-in-Schizophrenia-1 (Disc1) neuronal tiredness, 228
Disk-over water methodology, 377–378 NREM sleep, 229
Disrupted-in-Schizophrenia-1 (Disc1), performance, 227
259–260 sleep-like and wake-like activity, 229
Drosophila state-synchronization, 228–229
Clk-gal4, 62–64 Enhanced green fluorescence protein (EGFP),
CRY, 64 121–122
gal4 and uas, 62 EUMODIC. See European Mouse Disease Clinic
LNs and DNs, 62 (EUMODIC)
PDF, 62 European conditional mouse mutagenesis
Pdf-gal80, 64–65 (EUCOMM ) program, 256, 260–261
PER/TIM, 62–64 European Mouse Disease Clinic (EUMODIC),
Dual oscillator model 260–261
Drosophila, 61–65 Evening (E) cells
E cells, 75–76 dominance, 66
LD cycles, 61 light activates output, 66–67
M cells, 76 light decelerates, 67–70
phase angle between M and E peak, 61 oscillators, 70–72
photoperiods, 61
SCN, 61 Familial advanced sleep-phase syndrome
(FASPS), 237f, 241, 257–258
EGFP. See Enhanced green fluorescence protein FASPS. See Familial advanced sleep-phase
(EGFP) syndrome (FASPS)
Electroencephalographic (EEG), local sleep Fast Fourier Transform (FFT) analysis, 134–135,
behavioral transition 136f
cortical neurons, 221 FEO. See Food-entrainable oscillator (FEO)
deactivation, thalamus, 222 Food anticipatory activity (FAA), 50–53
delta activity, 222 Food-entrainable oscillator (FEO), 169
firing pattern, 221 Forward genetics, mouse mutant generation
intracerebral and scalp, recordings, 222–223, description, 248
224f ENU (see N-ethyl-N-nitrosourea (ENU),
NREM sleep (see Non-rapid eye movement mouse mutant generation)
(NREM) sleep) Mus musculus, 248
processes, 220–221 spontaneous mutations, 248–249
slow-delta and sigma frequency range, transversions, 249
222–223, 225f X-ray, 249
SWA and hippocampus, 221–222 Frequency demultiplication hypothesis, 445–446

GC secretion. See Glucocorticoid (GC) secretion
Gene knockout, mouse mutant generation
description, 254–255
inducible and tissue-specific
gene expression, liver, 256
loxP sites and Cre recombinase, 256
resources, 256–257, 257t
nonparalogous genes, 255–256
paralogous genes
Clock and Npas2, 255
Dec1 and Dec2, 255
Drosophila Per, 255
PAR bZIP transcription factors, 255
transgenics
Cry1 overexpression, 258
determination, 258
FASPS, 257–258
tetracycline transactivator (tTA), 258
Glucocorticoid (GC) secretion
Addison’s disease, 170–171
cortex and medulla, 171
disruption, 170–171
neuronal signals, 171
nycthemeral production, 171
receptors, 171–172
GnRH. See Gonadotropin-releasing hormone
(GnRH)
Gonadotropin-releasing hormone (GnRH)
and AVP, 121
description, 423
secretion and tanycytes, 432
stimulatory effect, Kp, 429
gpr50. See G protein-coupled receptor
50 (gpr50)
G protein-coupled receptor 50 (gpr50)
energy homeostasis, 420
melatonin receptors, evolution, 419–420
G-protein-coupled receptor kinases (GRKs), 32
G-protein signaling pathway
Gq/11-type G-proteins, 25–27, 26t
phospholipase C activation
PLC, 27
PLCb1 and PLCb3, 27–28
IGL. See Intergeniculate leaflet (IGL)
485
IKMC. See International Knockout Mouse
Consortium (IKMC)
IMPC. See International Mouse Phenotyping
Consortium (IMPC)
Individual shift-work load (ISL), 403–404
Inner plexiform layer (IPL), 20–21
Intergeniculate leaflet (IGL), 317–318
International Knockout Mouse Consortium
(IKMC), 257t, 261
International Mouse Phenotyping Consortium
(IMPC)
challenges, 262
description, 261
forward genetics, 261–262
Intracerebroventricular (ICV) delivery, 121
ISL. See Individual shift-work load (ISL)
Kisspeptin (Kp) neurons
ARC nucleus (see Arcuate (ARC) nucleus, Kp
neurons)
AVPV nucleus (see Anteroventro-
periventricular (AVPV) nucleus,
Kp neurons)
gonadotropic axis
GnRH, 423
Kiss1 gene, 423–424, 424f
photoneuroendocrine pathway, 424–425
sex steroids and metabolic factors, 424
Knockout mouse project (KOMP), 256, 260–261
KOMP. See Knockout mouse project (KOMP)
Lateral hypothalamic area (LHA), 320
Lateral neurons (LNs), 65
LHA. See Lateral hypothalamic area (LHA)
Light/dark (LD) cycle, 95–97
Light-entrainable peripheral clocks
bioluminescent, 47–48
cavefish
description, 49–50
FAA, 50–53
free-running period, 50–53
LD cycles, 50–53
opsin genes, 53
cycling clock gene expression, 47–48
gene expression
486

Light-entrainable peripheral clocks (Continued) glucocorticoid signaling, 320–321

D box-binding factors, 49, 50f molecular basis, 316–317
light input pathway, 49, 51f NPY and IGL, 317–318
MAPK signaling, 48–49 orexin and LHA, 320
Per2, 49 serotonin and raphe nuclei, 318–320
SCN, 48–49 photic entrainment
light exposure, 47–48 glutamate role, 308
peripheral photoreceptors, 48 negative masking, light, 308
photoreceptors, 48 phase shift data, laboratory rodent, 306–307,
Local sleep 307f
behavioral transition, 220–227 physiological correlates, 315–316
description, 220 research techniques, 324–325
EEG (see Electroencephalographic (EEG), species generalization, 324
local sleep) zeitgeber vs. zeitnehmer, 325–326
implication, medicine, 227–230 Luminance and temporal niche switching
Locomotor activity, feedback actions effects, moonlight, 288–289
aging, 322 Mediterranean lobster, 288
arousal and motivation, 315
benzodiazepine-induced circadian effects, 309 Mammalian circadian clock
circadian phase, alteration, 314–315 conceptual model
circadian responses, discrete “pulses,” 309–310 assumptions, implications, and uncertainties,
core clock gene mutations, 322–323 9–14
endogenous circadian oscillators, 305–306 cones, role, 10–11
entrainment to scheduled locomotor activity melanopsin, 11–12
free-running rhythms, 312 photoreceptor, 12–14
mouse model, 312–313, 314f rods, cones and melanopsin, 9, 10f
phase angle and nonphotic PRC, 313–314 cones, 7–8
running wheel and tau shortening, 312 description, 1
signaling mutants, 323–324 environment, 1–2
extra-SCN oscillators, 324 ipRGCs, 2
interactions, nonphotic and photic entraining melanopsin, 8–9, 11–12
stimuli photoentrainment pathway, 1–3, 2f
dark pulses, 310–311 photoreceptor, 3
light–dark (LD) cycle shift and conflict red-cone knockin, 4, 6f
studies, 311–312 RHT, 14
laboratory rodents, 309 SCN, 3
mammalian circadian system, 306, 306f Mammalian nocturnal bottleneck, temporal niche
nonphotic entrainment switching
activity/arousal-associated stimuli, 309 arguments, 285–286
description, 308 ectothermic predators and competitors, 285
diurnal species, 321–322 evidence, 287
restricted feeding and food-entrainable extinction, dinosaurs, 285
oscillator, 308–309 factors, 285
nonphotic phase shifting and entrainment MASCO. See Methamphetamine-sensitive
electrical output, SCN, 317 oscillator (MASCO)
 487

Meal patterns and human health gpr50, 419–420

breakfast, 367 subtypes, 419
clinical nutrition, 367–368 rhythmical gene expression, 417–418
day and night secretion, 416–417
NES and clinical trials, 366 Metabolic function and circadian disruption
shift workers and short sleepers, 366 adverse endocrine effects, night work, 351
wakefulness, food intake and anabolic human endogenous circadian rhythms, 347–348
metabolic processes, 365–366, 366f impaired glucose metabolism, 350
enteral tube feeding, 368 isocaloric meals and leptin, 348–349
meal frequency, 366–367 leptin, glucose and insulin, 348, 349f
parenteral nutrition, 368–369 lipopolysaccharide (LPS) challenge, 349–350
Melanopsin, 8–9 molecular clock genes, 350–351
Melanopsin phototransduction organ-specific clocks, 351
Gb subunits, 33 SCN, 347–348
GMP, 23 shift work, 348
G-protein signaling pathway, 25–27 Methamphetamine-sensitive oscillator (MASCO),
ion channels and potentials firing, 29–30 169–170
light absorption Morning and evening oscillator model
GPCR, 24–25 ablate clock neurons, 65
Opn4L and Opn4S, 25 complexity, 76–77
spectrophotometry analysis, 25 dominance, 66
PKA, 30–32 dual oscillator model, 61–62
pRGCs, 19–20 flies adaptation
rods and cones, 23 clock, 72
scaffold proteins, 33 dawn and dusk, 72
signaling pathway, 23, 24f PDF-positive l-LNvs, 72–73
TRPC6 and TRPC7 channels, 30 fruit flies, 65
TRP channel activation, 28–29 gal4, 65–66
Melatonin LNs, 65
actions, vertebrate lineages temperature and photoperiod influence, 59–60,
circadian coordination, 419 60f
extra-retinal photoreception, 418–419 temperature effects
receptor distribution, 419 flies, natural-like temperature cycles, 74
cycles, captive waders light and, 74–75
daily profiles, plasma melatonin, 468–471, Morning (M) cells
472f dominance, 66
diel melatonin profiles, 468–471, 470f inhibit output, 66–67
feeding activity, 468 light accelerates, 67–70
group activity patterns, 471, 474f, 475t oscillators, 70–72
immuno-enhancement, 467 Mouse mutant generation
life-cycle stages, derivation, 467–468, 469f description, 247–248
light availability and physiological functions, EUCOMM, KOMP and IKMC, 261
471–473 EUMODIC, 261
Ruff vs. Red Knot, 466–467 forward genetics (see Forward genetics, mouse
receptors, divergent evolution mutant generation)
488

Mouse mutant generation (Continued) cyclical appearance, 225–226, 227f

gene knockout, reverse genetics (see Gene description, 223–225
knockout, mouse mutant generation) intracerebral and scalp, recordings, 225–226,
IMPC, 261–262 226f
Multi-electrode array dish (MED), 129–130 sensory-motor processing, 226
Mutualism, 268 time–frequency analysis, 226, 228f
Process S, 210
NAD. See Nicotinamide adenine dinucleotide SCN neuronal activity, 208–210
(NAD) NPY. See Neuropeptide Y (NPY)
Nanda–Hamner protocol, 450–451 NREM sleep. See Non-rapid eye movement
NES. See Night eating syndrome (NES) (NREM) sleep
N-ethyl-N-nitrosourea (ENU), mouse mutant Nutrition and circadian timing system
generation central clock, 360
description, 249 digestion and metabolism
gene-driven approach, reverse genetics carbohydrates, 363–365
description, 259, 260f food intake, 361–363
Disc1 and Srr, 259–260 gastrointestinal system, 363
identification, 259 lipids, 365
mutation detection, DNA, 259, 260f disturbed rhythms and type 2 diabetes, 369
real-time monitoring assays, 259 food availability, 359–360
mapping and positional cloning meal patterns and human health (see Meal
BAC, 251 patterns and human health)
candidate region, 251 peripheral clocks
principles, 251 autonomic cycle, 361
redundancy and epistasis, investigation, 254 gene oscillations, 360
screening physiological function, 360–361
affect, clock circuitry, 254 SCN, 361, 362f
Drosophila, 253–254
Fbxl3, 253 Ontogenetic development
flybase, 253–254 circadian systems, 105–106
identification, Clock gene, 251–253 endogenous rhythm generator
phenodeviants, circadian phenotypes, AANAT activity, 108–109
250–251 adrenal gland, 111
strategies, 249–250 Avp expression, 108
Neuropeptide Y (NPY), 317–318 description, 108
Nicotinamide adenine dinucleotide (NAD), 187 environmental lighting, 110
Night eating syndrome (NES), 366 feeding and social interaction, 110
Non-rapid eye movement (NREM) sleep liver, 111
cortical neurons, 221 Per2 and MT1, 109–110
Cry1 and Cry2 double-knockout mice, 210–211 peripheral oscillators, 110
description, 204 PT, 110–111
2 h/2 h protocol, 212–214, 213f SCN, 108
homeostatic process, 214 Per and Cry genes, 105–106
motor cortex (Mc) local activations SCN, 105–106
cortical deactivation, 226–227 synchronization mechanisms
 489

HPA, 107–108 phase resetting and state variables, 447–448

melatonin, 107 2-week long-day pulse, 447
Per expression, 106–107 4-week long-day pulse, 447, 449f
PVN, 107 nonphotic, 313–314
SCN, 107 Phase-shifting capacity, SCN neurons
adjustment, 152–153
Parasitism, 268–269 constant darkness, 151
Paraventricular nucleus (PVN), 107–108, 166, coupled oscillator theory, 152
168, 171, 172, 184, 185, 190f curve fitting analysis, 151–152, 152f
Pars tuberalis (PT) desynchrony, advances, 153
melatonin, 110–111 differences, synchronization, 153
SCN, 110–111 GABA, 151
PCEs. See Photoreceptor conserved elements peaks, multiunit activity rhythm, 151
(PCEs) resynchronization, 153
PERIOD2 (PER2) protein shift work and jet lag, description, 150–151
brain, 239–240 Photoperiodism, vertebrates
circadian oscillator deep brain photoreceptors and birds, 418
histone deacetlyases (HDAC), 236–237 energy budgets, life history strategies and
nonrhythmic overexpression, 236–237 seasonality
protein–protein interaction regions, 236–237, breeding and offspring survival, 413–414
237f circadian rhythm generators, 414
CRYs, 236 environmental variability, 414
cyanobacteria, 233–234 species survival, 413
history, 234–236 gpr50 and energy homeostasis, 420
liver metabolism melatonin
blood glucose concentration, 238–239 actions, vertebrate lineages, 418–419
detoxification enzymes, 238–239 receptors, divergent evolution, 419–420
nuclear receptors, 239, 240f photoperiodic axis, mammals
mechanisms, 234 Eya3-dependent transcription, TSHb,
posttranslational modifications, 241 417–418, 417f
sleep, 240–241 hypothalamic deiodinase expression, 416–417
stabilizing loop, 238 melatonin secretion, 416–417
synchronization, 242 thyroid hormone (TH) signaling
Zeitgebers, 233–234 deiodinase gene expression, 416
PER2 protein. See PERIOD2 (PER2) protein endostyle and comparative genomics
Phase-resetting capacity, SCN neurons approaches, 415–416
description, 150 evolutionary appearance, elements, 415–416,
distribution, PRCs, 149–150, 150f 416f
low-and high-amplitude rhythm, 149 metabolism, hypothalamus, 414–415, 415f
NMDA receptors, 149 nutrient-sensing pathway, 415–416
short and long photoperiods, 149 Photoreceptor
Phase response curves (PRCs) function, 4
biological oscillator in vitro, 12
circadian and circannual rhythms, 447, 448f photoentrainment, 12
Cricetus cricetus, 448–449 rod phase, 13
490

Photoreceptor (Continued) circadian timing system, 400

saturation point, 13 description, 406–407
sensitivity ranges, 13–14 health problems, 399–400
Photoreceptor conserved elements (PCEs), 43–44 internal time, 400–401
Photosensitive retinal ganglion cell (pRGCs) laboratory studies
functional differences, 21–22 advantages, 404–406
IPL, 20–21 disadvantages, 406
melanopsin expressing, 20–21, 20f real-life settings
M1-type cells, 20–21 cortisol awakening response and stress,
physiological responses, 22–23 402–403
retinal connections, 22 ISL, 403–404
variable responses, 34–35 shift type and incidence rate, 404
PKA. See Protein kinases activity (PKA) stress and adverse effects, health, 401–402
PRCs. See Phase response curves (PRCs) “shift-clock-work,” 407f, 407
pRGCs. See Photosensitive retinal ganglion cell Sleep restriction
(pRGCs) chronic partial (see Chronic partial sleep
Primates, temporal niche switching, 287–288 restriction)
Prokineticins (PK2) description, 378
description, 121 disk-over water methodology, 377–378
EGFP, 121–122 healthy human sleep
ICV delivery, 122 circadian process, 379
rhythm, 122 duration, 379–380
Protein kinases activity (PKA) homeostasis, 378–379
cAMP pathway, 32 states and characteristics, 378
DAG, 30–32 interindividual differences
GRKs, 32 biomarkers and behavioral predictors, 390–391
PKCz, 32 genetic polymorphisms, 390
TRPC6 and TRPC7, 30–32 neurobehavioral performance, 388–389, 389f
Psychomotor Vigilance Test (PVT), 387–388 nocturnal sleep, 390–391, 391f
PVN. See Paraventricular nucleus (PVN) task dependent and cognitive performance,
PVT. See Psychomotor Vigilance Test (PVT) 389–390
neurobehavioral impairment, management,
Retinohypothalamic tract (RHT) activity, 8 391–392
RF-amide-related peptide (RFRP) Sleep, rodents
mediobasal hypothalamus, 432–433 circadian–homeostatic interactions
neurons, 430–432 conceptual model, 208–210
RFRP-3 and circadian regulation, 427–428 phase-shifting experiments, humans, 208
RFRP. See RF-amide-related peptide (RFRP) pressure, changes, 208
Rodents, temporal niche switching, 287 SCN neuronal activity, 208–210
time course, Process S, 210
SCN. See Suprachiasmatic nuclei (SCN) clock genes
Sequence of linked stages hypothesis, 446–447 Cry1 and Cry2, 210–211
Serine racemase (Srr), 259–260 description, 210
Shift-work research mPer3, 211
chronotype-specific differences, 400–401 mPer1 and mPer2, 211

clocks, photoperiod, running wheels and light,
207–208
comparison, mouse strains, 206t, 207
crepuscular behavior, 204
description, 204
12 h:12 h L:D cycle, 204, 205t
modeling, 214–215
protocols and techniques, 214
separation, circadian and homeostatic
processes
daily changes, 211
desynchrony protocol, 211–212
22-h dim light–dark cycle, 212
2 h/2 h short-sleep deprivation protocol, 212
“90-min day” protocol, 212
NREM sleep (see Non-rapid eye movement
(NREM) sleep)
SCN lesions, 211
TST (see Total sleep time (TST), rodents)
Srr. See Serine racemase (Srr)
Suprachiasmatic nuclei (SCN)
cAMP-signaling pathway, 87
cellular oscillators and coupling
behavioral rhythms, 130–131
spontaneous firing rhythms, 130, 131f
central and peripheral clock interaction (see
Central and peripheral clock interaction)
circadian molecular clock mechanism, 86
circadian rhythms, regulation, 338
circadian timing system
central clock, 360
peripheral clocks, 361, 362f
clock gene functions, 139
description, 84
development, 87–88
dysfunction and hypertension, 346–347
electrical output, suppression, 317
extra-SCN oscillators, 324
food intake, 363
functional network, 86
in vitro, 86–87
lesioned hamsters, 312–313
maternal entrainment
lactation, 93
LD cycle, 92–93
491
Per1 and Per2 expressions, 93
rabbit pups, 93–94
MED, 129–130
metabolic-related factors, 347–348
neuron, 105–106
ontogenesis, 85, 85f
oscillators and non-oscillator cells
AVP and VIP, 132–133
Per1-luc rhythms, 131–132, 132f
pacemaker phase, 321
periventricular cell groups, 85–86
population, 92
regional oscillators
bioluminescence images, 134–135, 135f
FFT, 134–135, 136f
LD cycle, 133–134
organotypic slice culture, 133
per-luc rhythms, 133–134, 134f
serotonin signaling, 319
sleep, rodents
Cry1,2 knockout mice, 210–211
neuronal activity, 208–210
subregions, 306
TTX, 136–138
VIP, 135–136
WAT, circadian rhythms (see White adipose
tissue (WAT), circadian rhythms)
Suprachiasmatic nuclei (SCN) neurons
aging and disease, effects, 153–154
constant light effects
coupling factors, 146–147
in vitro and in vivo recordings, 145–146, 146f
description, 143–144
generation, circadian rhythms, 144
intercellular coupling, circuit, 154–156
phase-resetting capacity (see Phase-resetting
capacity, SCN neurons)
phase-shifting capacity (see Phase-shifting
capacity, SCN neurons)
seasonal changes, waveform
brain slice preparation, 147–148
clock gene expression, in situ analysis, 147
C57 mice, 147
distribution, single-unit electrical activity
patterns, 147–148, 148f
492
Suprachiasmatic nuclei (SCN) neurons
(Continued)
longer duration activity, 148
photoperiodic encoding, 147
waveform, electrical activity
distribution, 145, 145f
in vitro and in vivo electrophysiological
recordings, 144
onset–offset, behavioral patterns, 144–145
Temporal multitasking
annual time keeping
circannual rhythms, 461
environmental factors, 461–462
long-distance migrants, 460–461
photoperiodism, 460–461
southward migration pathways, 459–460, 460f
Sylvid warblers, 461
avian long-distance migration, 458–459
challenges, avian migration, 459
chronobiology and activity patterns, 457–458
daily time keeping
challenges, 463
circadian pacemaking system, birds, 462–463
internal-resonance model, 462–463
melatonin role, 463–464
nocturnal restlessness, 463
entrainment mechanisms, 458
lunar and tidal cycles, 464
waders (see Waders and temporal multitasking)
Temporal niche switching
ambient temperature
colder water, 289
diurnal rhythms and locomotor activity, 289
effects, 289
behavioral programs, 282, 285f
CTE hypothesis (see Circadian
thermoenergetics (CTE) hypothesis)
description, 281–282
golden spiny mouse, 288, 288f
limitation, factors, 289–290
locomotor activity, 282
luminance, 288–289
lunar cycle and light properties, 299
mammals
nocturnal bottleneck (see Mammalian
nocturnal bottleneck, temporal niche
switching)
primates, 287–288
rodents, 287
mechanisms
circadian oscillator properties, 293–295, 295f
diurnal and nocturnal degus, 293–295
expression, behavioral program, 293–295,
295f
golden spiny mice, 293–295
locomotor activity, Drosophila melanogaster,
295–296
Zeitgebers, 285f, 296
microchiroptera bats, 299
species, description, 282, 283t
visual adaptations (see Visual adaptations,
temporal niche switching)
Tetrodotoxin (TTX)
effects, 136–138, 137f
resistant circadian rhythms, 138–139
treatment, 136–138
Total sleep time (TST), rodents
C57BL/6 mouse, 204–205, 205t
comparison, mouse strains, 206t, 207
description, 204
hourly distribution, activity, 207
light vs. dark period, 207
mice vs. rats, 206t, 207
photoperiods, 206t, 208
running wheel, 207–208
Transcriptional–translational feedback loop
(TTL), 164, 166
TSHb. See TSH beta subunit (TSHb)
TSH beta subunit (TSHb)
Eya3-dependent transcription, 417–418, 417f
photoperiodic signaling, birds, 418
TTL. See Transcriptional–translational feedback
loop (TTL)
Utero
clocks, entrainment
dopamine/ melatonin, 91–92
LD cycle, 91–92
molecular clock mechanism, 89–91
 493

synchronized circadian rhythms, 89–91, 90f Waders and temporal multitasking

peripheral clocks annual punctuality and daily flexibility
heart and kidney, 89 assortative mating, 465–466
hepatocytes, 88–89 behavioral observations, 466
liver, 88 breeding habitat and geolocators, 465
Rev-erba expression, 88–89 Calidris canutus and Haematopus ostralegus,
prefetal development 466
Bmall, 85 continuous polar light, 466
multipotent cells, 84–85 internal timing programs, 465
SCN, 85–88 Limosa lapponica baueri, 464–465
L. limosa islandica, 465–466
Varied carpet beetle. See Circannual rhythm, description, 464
Anthrenus verbasci melatonin cycles
Vasoactive intestinal peptide (VIP) daily profiles, plasma melatonin, 468–471,
AVP, 132–133 472f
SCN, 135–136 diel melatonin profiles, 468–471, 470f
Vertebrate core clock mechanism, zebrafish feeding activity, 468
multiple clock genes group activity patterns, 471, 474f, 475t
cry genes, 45–46 immuno-enhancement, 467
genome duplication, 45–46 life-cycle stages, derivation, 467–468, 469f
mammals, 45 light availability and physiological functions,
per genes, 45–46 471–473
new clock genes Ruff vs. Red Knot, 466–467
asparagine mutation, 45 White adipose tissue (WAT), circadian rhythms
bioluminescence, 44 adipokines, description, 194–195
Drosophila, 44 adiponectin, 194
melatonin, 44 catecholamines, 184–185
microsatellite markers, 44–45 depots, 189
Visual adaptations, temporal niche switching description, 183
ascorbic acid concentration, 292–293 development, 188
night-and day-adapted vision feeding–fasting cycle, 185
foraging, 290–291 gene expression, 192–193
mouse, cones, 286t, 291 innervation
physiological and anatomical determinants, lipodystrophy and hypothalamus, 189
290 parasympathetic, 191
reef fish, 290 sensory nerves, 191
rod-cone densities, visual acuity and sensitivity sympathetic nerve fibers, 189–191
golden spiny mice, 291–292 leptin, 193–194
ground squirrels, 291 lipogenesis, 192
macula and fovea, 292 lipolysis, 192
tapetum, 292 non-neural signals, 185
spectral filtering and UV protection obesity
coloration, lenses, 292 db/db mice, 195–196
ground squirrel, 292, 299 ob/ob mice, 195
properties, photoreceptors, 292, 293t, 294f subcutaneous WAT (sWAT), 195
494

White adipose tissue (WAT), circadian rhythms Zebrafish

(Continued) chronobiology
Zucker rat, 196 circadian clock genes, 42–43
oscillators, 184 circadian timing system, 43
pathways and timing information, 185, 186f PCEs, 43–44
plasma corticosterone concentrations, 185 pineal cells, 43
PVN and ANS, 184 embryology, 42
resistin, 194–195 vertebrate core clock mechanism,
types, adipose tissue, 188 44–46
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Volume 149: Cortical Function: A View from the Thalamus, by V.A. Casagrande, R.W. Guillery and S.M. Sherman (Eds.) – 2005
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Volume 150: The Boundaries of Consciousness: Neurobiology and Neuropathology, by Steven Laureys (Ed.) – 2005,
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Volume 152: Autonomic Dysfunction after Spinal Cord Injury, by L.C. Weaver and C. Polosa (Eds.) – 2006, ISBN 0-444-51925-4.
Volume 153: Hypothalamic Integration of Energy Metabolism, by A. Kalsbeek, E. Fliers, M.A. Hofman, D.F. Swaab, E.J.W. Van
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Volume 156: Understanding Emotions, by S. Anders, G. Ende, M. Junghofer, J. Kissler and D. Wildgruber (Eds.) – 2006,
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Volume 157: Reprogramming of the Brain, by A.R. Mller (Ed.) – 2006, ISBN 978-0-444-51602-2.
Volume 158: Functional Genomics and Proteomics in the Clinical Neurosciences, by S.E. Hemby and S. Bahn (Eds.) – 2006,
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Volume 159: Event-Related Dynamics of Brain Oscillations, by C. Neuper and W. Klimesch (Eds.) – 2006, ISBN 978-0-444-52183-5.
Volume 160: GABA and the Basal Ganglia: From Molecules to Systems, by J.M. Tepper, E.D. Abercrombie and J.P. Bolam
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Volume 163: The Dentate Gyrus: A Comprehensive Guide to Structure, Function, and Clinical Implications, by H.E. Scharfman
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Volume 164: From Action to Cognition, by C. von Hofsten and K. Rosander (Eds.) – 2007, ISBN 978-0-444-53016-5.
Volume 165: Computational Neuroscience: Theoretical Insights into Brain Function, by P. Cisek, T. Drew and J.F. Kalaska (Eds.) –
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Volume 166: Tinnitus: Pathophysiology and Treatment, by B. Langguth, G. Hajak, T. Kleinjung, A. Cacace and A.R. Mller
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Volume 167: Stress Hormones and Post Traumatic Stress Disorder: Basic Studies and Clinical Perspectives, by E.R. de Kloet,
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Volume 168: Models of Brain and Mind: Physical, Computational and Psychological Approaches, by R. Banerjee and
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Volume 169: Essence of Memory, by W.S. Sossin, J.-C. Lacaille, V.F. Castellucci and S. Belleville (Eds.) – 2008,
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Volume 170: Advances in Vasopressin and Oxytocin – From Genes to Behaviour to Disease, by I.D. Neumann and R. Landgraf
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Volume 171: Using Eye Movements as an Experimental Probe of Brain Function—A Symposium in Honor of Jean Büttner-
Ennever, by Christopher Kennard and R. John Leigh (Eds.) – 2008, ISBN 978-0-444-53163-6.
Volume 172: Serotonin–Dopamine Interaction: Experimental Evidence and Therapeutic Relevance, by Giuseppe Di Giovanni,
Vincenzo Di Matteo and Ennio Esposito (Eds.) – 2008, ISBN 978-0-444-53235-0.
Volume 173: Glaucoma: An Open Window to Neurodegeneration and Neuroprotection, by Carlo Nucci, Neville N. Osborne,
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Volume 174: Mind and Motion: The Bidirectional Link Between Thought and Action, by Markus Raab, Joseph G. Johnson and
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Volume 175: Neurotherapy: Progress in Restorative Neuroscience and Neurology — Proceedings of the 25th International
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The Netherlands, August 25–28, 2008, by J. Verhaagen, E.M. Hol, I. Huitinga, J. Wijnholds, A.A. Bergen, G.J. Boer
and D.F. Swaab (Eds.) –2009, ISBN 978-0-12-374511-8.
Volume 176: Attention, by Narayanan Srinivasan (Ed.) – 2009, ISBN 978-0-444-53426-2.
Volume 177: Coma Science: Clinical and Ethical Implications, by Steven Laureys, Nicholas D. Schiff and Adrian M. Owen (Eds.) –
2009, 978-0-444-53432-3.
Volume 178: Cultural Neuroscience: Cultural Influences On Brain Function, by Joan Y. Chiao (Ed.) – 2009, 978-0-444-53361-6.
Volume 179: Genetic models of schizophrenia, by Akira Sawa (Ed.) – 2009, 978-0-444-53430-9.
Volume 180: Nanoneuroscience and Nanoneuropharmacology, by Hari Shanker Sharma (Ed.) – 2009, 978-0-444-53431-6.
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Volume 181: Neuroendocrinology: The Normal Neuroendocrine System, by Luciano Martini, George P. Chrousos, Fernand
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Volume 182: Neuroendocrinology: Pathological Situations and Diseases, by Luciano Martini, George P. Chrousos, Fernand
Labrie, Karel Pacak and Donald W. Pfaff (Eds.) – 2010, 978-0-444-53616-7.
Volume 183: Recent Advances in Parkinson's Disease: Basic Research, by Anders Björklund and M. Angela Cenci (Eds.) – 2010,
978-0-444-53614-3.
Volume 184: Recent Advances in Parkinson's Disease: Translational and Clinical Research, by Anders Björklund and M. Angela
Cenci (Eds.) – 2010, 978-0-444-53750-8.
Volume 185: Human Sleep and Cognition Part I: Basic Research, by Gerard A. Kerkhof and Hans P.A. Van Dongen (Eds.) –
2010, 978-0-444-53702-7.
Volume 186: Sex Differences in the Human Brain, their Underpinnings and Implications, by Ivanka Savic (Ed.) – 2010, 978-0-444-
53630-3.
Volume 187: Breathe, Walk and Chew: The Neural Challenge: Part I, by Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta
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Volume 188: Breathe, Walk and Chew; The Neural Challenge: Part II, by Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta
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Volume 189: Gene Expression to Neurobiology and Behaviour: Human Brain Development and Developmental Disorders, by
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Volume 190: Human Sleep and Cognition Part II: Clinical and Applied Research, by Hans P.A. Van Dongen and
Gerard A. Kerkhof (Eds.) – 2011, 978-0-444-53817-8.
Volume 191: Enhancing Performance for Action and perception: Multisensory Integration, Neuroplasticity and Neuroprosthetics:
Part I, by Andrea M. Green, C. Elaine Chapman, John F. Kalaska and Franco Lepore (Eds.) – 2011, 978-0-444-
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Volume 192: Enhancing Performance for Action and Perception: Multisensory Integration, Neuroplasticity and Neuroprosthetics:
Part II, by Andrea M. Green, C. Elaine Chapman, John F. Kalaska and Franco Lepore (Eds.) – 2011, 978-0-444-
53355-5.
Volume 193: Slow Brain Oscillations of Sleep, Resting State and Vigilance, by Eus J.W. Van Someren, Ysbrand D. Van Der Werf,
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Volume 194: Brain Machine Interfaces: Implications For Science, Clinical Practice And Society, by Jens Schouenborg, Martin
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Volume 195: Evolution of the Primate Brain: From Neuron to Behavior, by Michel A. Hofman and Dean Falk (Eds.) – 2012,
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Volume 196: Optogenetics: Tools for Controlling and Monitoring Neuronal Activity, by Thomas Knöpfel and Edward S. Boyden
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Volume 197: Down Syndrome: From Understanding the Neurobiology to Therapy, by Mara Dierssen and Rafael De La Torre
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Volume 198: Orexin/Hypocretin System, by Anantha Shekhar (Ed.) – 2012, 978-0-444-59489-1.