Behavioural Brain Research 332 (2017) 164–171

Contents lists available at ScienceDirect

Behavioural Brain Research

journal homepage: www.elsevier.com/locate/bbr

Research report

Habenula and interpeduncular nucleus differentially modulate predator T

odor-induced innate fear behavior in rats
⁎
Daniel Vincenza,c,1, Kerstin E.A. Werneckeb,c,1, Markus Fendtb,c, Jürgen Goldschmidta,c,
a
Leibniz Institute for Neurobiology, Department Systems Physiology of Learning, 39118 Magdeburg, Germany
b
Institute for Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Germany
c
Center for Behavioral Brain Sciences, Magdeburg, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Fear is an important behavioral system helping humans and animals to survive potentially dangerous situations.
Avoidance Fear can be innate or learned. Whereas the neural circuits underlying learned fear are already well investigated,
Risk assessment the knowledge about the circuits mediating innate fear is still limited. We here used a novel, unbiased approach
Defensive strategy to image in vivo the spatial patterns of neural activity in odor-induced innate fear behavior in rats. We in-
Lesion
travenously injected awake unrestrained rats with a 99m-technetium labeled blood flow tracer (99mTc-HMPAO)
Habenulo-interpeduncular system
during ongoing exposure to fox urine or water as control, and mapped the brain distribution of the trapped tracer
using single-photon emission computed tomography (SPECT). Upon fox urine exposure blood flow increased in a
number of brain regions previously associated with odor-induced innate fear such as the amygdala, ventromedial
hypothalamus and dorsolateral periaqueductal grey, but, unexpectedly, decreased at higher significance levels in
the interpeduncular nucleus (IPN). Significant flow changes were found in regions monosynaptically connected
to the IPN. Flow decreased in the dorsal tegmentum and entorhinal cortex. Flow increased in the habenula (Hb)
and correlated with odor effects on behavioral defensive strategy. Hb lesions reduced avoidance of but increased
approach to the fox urine while IPN lesions only reduced avoidance behavior without approach behavior. Our
study identifies a new component, the IPN, of the neural circuit mediating odor-induced innate fear behavior in
mammals and suggests that the evolutionarily conserved Hb-IPN system, which has recently been implicated in
cued fear, also forms an integral part of the innate fear circuitry.

1. Introduction In laboratory rodents, predator-related odors are often used to elicit

The emotion fear promotes the survival of humans and animals as
they enable them to appropriately respond to threatening stimuli [1].
However, too much fear or inappropriate fear is maladaptive, and al-
tered activation patterns in brain regions mediating fear behavior in
rodents are known to be related to anxiety disorders in humans [2].
During the last decades, numerous studies revealed the neural cir-
cuitry underlying fear and demonstrated that the brain fear system is
highly conserved [1,3,4]. Notably, the focus of these studies was most
often on learned fear which has led to a much better understanding of
learned fear than of innate fear. The neuronal circuits mediating innate
fear behaviors are still incompletely understood, and the knowledge
about the evolution of these circuits, their relationship to circuits in-
volved in learned fear as well as their potential role in psychiatric
diseases in humans is limited.
innate fear behaviors. Upon exposure to these odors (e.g., fur, urine or
feces) naive laboratory rodents show a number of defensive responses
[5,6]. Evidence has been provided that the rodent amygdala, tradi-
tionally studied in paradigms of learned fear [1,7,8], also participates in
odor-induced innate fear behavior [9–11]. Studies in zebrafish, in
contrast, have pointed to an involvement of the habenulo-inter-
peduncular system in odor-induced fear behavior [12,13]. In teleosts,
the medial division of the dorsal pallium is discussed as a homologue of
the mammalian amygdala [14,15], but an elaborated telencephalic
structure like the mammalian amygdala is lacking. In line with these
comparative anatomical considerations a recent study in mice has re-
vealed an involvement of the projection of the lateral habenula (LHb) to
the laterodorsal tegmentum (LDTg) in innate fear [16]. The projection
from the Hb to the interpeduncular nucleus (IPN) was not of interest in
this study but was shown to be involved in cued fear in mice [17].

⁎
Corresponding author at: Leibniz-Institute for Neurobiology, Brenneckestraße 6, 39118 Magdeburg, Germany.
E-mail addresses: daniel.vincenz@lin-magdeburg.de (D. Vincenz), kerstin.wernecke@yahoo.de (K.E.A. Wernecke), markus.fendt@med.ovgu.de (M. Fendt),
juergen.goldschmidt@lin-magdeburg.de (J. Goldschmidt).
1
Authors contributed equally.

http://dx.doi.org/10.1016/j.bbr.2017.05.053
Received 17 February 2017; Received in revised form 9 May 2017; Accepted 24 May 2017
Available online 26 May 2017
0166-4328/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
D. Vincenz et al.
These findings raise the question how extensive extra-amygdalar routes
are for the processing of innate fear in mammals.
We here aimed at elucidating the neuronal circuits underlying
predator odor-induced innate fear behavior using a novel, unbiased in
vivo imaging approach [18]. We intravenously injected the blood flow
tracer 99m-technetium hexamethylpropylene amine oxime (99mTc-
HMPAO) in awake behaving rats during exposure to fox urine or water
as control condition. 99mTc-HMPAO is a lipophilic complex that, after
crossing the blood-brain barrier, is rapidly converted to a hydrophilic
99m
Tc-compound which is trapped in the brain [19]. We mapped the
brain distribution of the trapped tracer using single-photon emission
computed tomography (SPECT) in anesthetized animals after stimula-
tion. 99mTc-HMPAO-SPECT is similar in rationale to 18F-2-fluoro-2-
deoxyglucose positron emission tomography (18F-FDG-PET) but, due to
the short plasma half-life of 99mTc-HMPAO, the temporal resolution can
be higher with 99mTc-HMPAO-SPECT. In addition, in rodents SPECT can
provide higher spatial resolution than PET [20].
In contrast to 18F-FDG, which can be injected as a bolus before the
behavioral experiments start, 99mTc-HMPAO must be injected con-
tinuously during stimulation of the animals or ongoing behavior, re-
spectively. We developed an approach for repeated continuous tracer
injections in awake behaving mice via jugular vein catheters [19,21],
and here introduce this approach in rats. We found that the blood flow
patterns differed markedly between fox urine exposure and the control
condition. In particular, we observed highly significant changes in
blood flow in the IPN and Hb suggesting that both nuclei take part in a
network of brain structures mediating odor-induced innate fear beha-
vior in mammals. We confirmed these findings by correlating regional
cerebral blood flow (rCBF) changes with behavioral variables and
performing behavioral lesion studies.
2. Materials and methods
2.1. Animals
Eighty-eight male rats were used (SPECT: 13 Wistar rats, 20
Sprague-Dawley (SD) rats, individual housing; lesions: 55 SD rats,
group housing). We started our SPECT study with Wistar rats and
continued the study with SD rats since this rat strain shows a more
robust behavioral response to predator odors [22,23]. All rats
(200–350 g) were housed in temperature- and humidity-controlled
rooms (22 ± 2 °C; 50–55%) with a 12 h light/dark cycle (lights on at
6:00 a.m.). Tap water and food pellets were available ad libitum. All
experiments were conducted during the light phase and were in ac-
cordance with International guidelines for the care and use of labora-
tory animals in experiments (2010/63/EU) and approved by the local
ethical committee.
2.2. Odor exposure test
Behavioral testing took place in identical testing boxes
(45 cm × 45 cm × 60 cm) constructed from opaque PVC as previously
described [11]. The boxes were equipped with overhead observation
cameras for video recording and contained four glass bowls (44 mm
diameter, 25 mm high) one in each corner. The testing boxes were
thoroughly cleaned with soapy water after each test and ventilated with
clean air.
99m
2.3. Tc-HMPAO injection during ongoing behavior
Rats used in the SPECT-imaging experiments were habituated in
daily 10 min sessions to the testing box with empty glass bowls and to
the experimental procedure. Following three days of habituation, each
rat was implanted with a silicon catheter (Gaudig Laborfachhandel
GbR, Sülzetal-Osterwedding, Germany; OD: 1.3 mm, ID: 0.5 mm, total
catheter length 11 cm) into the right external jugular vein [24]. The
165
Behavioural Brain Research 332 (2017) 164–171
catheter was filled with catheter lock solution (Cath-Loc HGS, SAI In-
fusion Technologies, USA) to prevent clogging. After implantation,
animals were given at least one day to recover from surgery.
For tracer injections during ongoing behavior the jugular vein ca-
theter was connected to a rotatable swivel (Quick Connect Balance
Swivel, SAI Infusion Technologies, USA) via a saline-filled teflon tube
(“Tefzel-tube”, CS-Chromatographie Service GmbH, Langerwehe,
Germany, OD: 1/16 inch ID: 0.5 mm, length 55 cm). A syringe con-
taining the 99mTc-HMPAO solution was placed in a syringe pump
(Harvard Instruments, Holliston, Massachusetts, USA) and connected to
the swivel using a second 40 cm long 0.9% NaCl filled teflon tube.
Injections were made at flow rates of 80 μl per min. Since the tube
system was filled with ca. 200 μl saline, it took ca. 2.5 min for the tracer
to reach the ratś circulation after switching on the syringe pump. After
5 min continuous 99mTc-HMPAO injection the teflon tube and the ca-
theter were cleared with saline for 2.5 min. The images thus represent
the average blood flow from minute 2.5 to ca. 7.5 within the ten minute
period of ongoing behavior.
During testing, rats wore a harness (Covance Infusion Harness,
Instech Laboratories Inc., Plymouth Meeting, Pennsylvania, USA) to
which they had been habituated. This harness served as catheter pro-
tection to prevent twisting or blocking of teflon tube or catheter.
All rats were injected and imaged twice. In the first session rats were
exposed for 10 min to 1 ml water, in the second session, at least 48 h
later, to 1 ml fox urine (Maine Odor Solutions, Maine, USA). The re-
spective odor sample was pseudorandomly pipetted into one of the
glass bowls.
The syringes for injection were filled with 400 μl 250 MBq freshly
prepared [18] 99mTc-HMPAO (CeretecTM, GE Healthcare, Buchler,
Braunschweig, Germany). After injection the remaining 99mTc in the
syringe and in the teflon tube was measured with a radionuclide cali-
brator (Aktivimeter Isomed 2010, Nuklear Medizin-Technik Dresden
GmbH, Germany). On average 200.3 ± 1.8 MBq were injected. 6.6%
of the initial dose remained within the syringes and 3.5% within the
teflon tubes.
After completion of each experiment, rats were immediately an-
esthetized with 4% isoflurane, transferred to the SPECT/CT (computed
tomography)-scanner and scanned under gas anesthesia (1.5–2.0%
isoflurane in 2:1 O2:N2O volume ratio).
2.4. SPECT/CT scanning
A four-head NanoSPECT/CT™ scanner (Mediso, Hungary) was used
for co-registered SPECT/CT scanning. SPECT scans of the rat head were
made using nine-pinhole apertures with 1.5 mm pinhole diameters with
a nominal resolution ≤ 1.2 mm. In total, 24 projections were acquired
during a scan time of 2 h. Axial field of view (FOV) was 38.9 mm.
Measured photopeaks were set to the default values of the NanoSPECT/
CT™ (140 keV +/− 5%, for 99mTc). SPECT-images were reconstructed
at an isotropic voxel size of 333 μm using the aperture manufacturer’s
software (HiSPECT™, SCIVIS, Goettingen). CT scans (45 kVp, 177 μA,
270 projections, 500 msec per projection, 96 μm pixel size) were ac-
quired from the same FOV as the SPECT-images. CTs were re-
constructed with the manufactureŕs software (InVivoScope™ 1.43) with
isotropic voxel-sizes of 200 μm. All animals received two CT-scans per
SPECT-imaging session, one before and one after the SPECT-scans to
control for movements between start and end of the imaging session.
2.5. Lesion studies
After one day of habituation, baseline behavioral responses to 1 ml
water and 1 ml fox urine were assessed for 10 min in two pre-surgery
test sessions. Only one odor was tested per day (odor order and odor
corner were pseudorandomized). Then, rats underwent stereotactic
surgeries (see below). The animals were randomly assigned either to a
sham-operated control group or to a Hb/IPN-lesioned group. Post-
D. Vincenz et al.
surgery test sessions began earliest three days after surgery. These
sessions were identical to the pre-surgery test sessions.
The behavioral data analysis was limited to the first five min of
testing in the lesion study and to five min tracer injection (min 2.5–7.5)
in the SPECT experiments. Using EthoVision XT software for video
tracking (Noldus Information Technologies, Wageningen, Netherlands),
the percentage of time an animal spent in the odor quadrant (as a
measure of avoidance behavior) was calculated. Numbers of contacts
with the glass bowl containing the odor sample (as a measure of risk
assessment behavior) were scored manually. Extremely inactive ani-
mals (> 75% time in one quadrant) were excluded from behavioral
analysis.
2.6. Electrolytic lesions
Rats were anesthetized with 2.0–2.5% isoflurane (5% for induction)
in oxygen (2.4 l/min) and fixed in a stereotactic frame. Electrolytic
lesions of the Hb or the IPN were made using stainless steel electrodes
(TeWa acupuncture needle, B-type, Suhl, Germany, 0.30 mm in dia-
meter) insulated with varnish except for the ∼500 μm long tip. The
electrode was lowered to the Hb (−3.7 mm rostral, ± 0.6 mm lateral,
−5.4 mm ventral to bregma) or to the IPN (-6.3 mm rostral, −9.4 mm
ventral to bregma). Bilateral lesions were then generated by passing a
∼0.1 mA anodal current through the electrode tip for 10–15 s. The
current was provided by a pulse stimulator (A-M Systems, Model 2100,
Sequim, USA) and monitored by a digital multimeter (Metex
Corporation, M-3610B, Seoul, Korea). At the end of the surgery, the
incision was closed by a suture and the animals were allowed to recover
for at least three days. For the sham-operated control animals, the
electrode was lowered to the same coordinates in the brain, but no
current was applied.
2.7. Histology
After completion of behavioral testing, sham-operated and Hb/IPN-
lesioned animals were sacrificed. Brains were rapidly removed and
fixed. Coronal sections corresponding to the Hb and IPN were sliced
and Nissl-stained. The extent of complete tissue destruction or gliosis
was verified under a light microscope and mapped on schematic
Paxinos and Watson [25] brain atlas sections.
2.8. Statistical analysis
2.8.1. SPECT-data
SPECT/CT images were aligned to a rat brain MR-template using the
MPI-Tool™-Software (Advanced Tomo Vision, Germany). The MR-
template was an in-house template that had been tested for alignment
with CTs in ongoing studies and in the present study (Supplementary
Fig. 1). It was manually segmented from a rat head MR data set ob-
tained with a 4.7 T Bruker Biospec 47/20 scanner equipped with a BGA
09 (400 mT/m) gradient system (40 axial T2-weighted spin-echo
images were measured using a RARE sequence, TR 5150 ms, TE 15 ms,
slice thickness 0.8 mm, inter slice distance 0.8 mm, FOV 40 × 40 mm,
matrix 256 × 256, RARE factor 8, averages 6).
CT-images of every rat were aligned to the template MR-images
based on skull-landmarks. The coordinates of the aligned CTs were used
to align the SPECT-data to the MR-image. Our MR template has a
slightly different angle in the rostrocaudal axis compared to the Paxinos
and Watson atlas [25] brain, i.e. dorsal and ventral parts of a section do
not fit exactly to one single bregma level in the Paxinos and Watson
atlas. We indicate in the figures the bregma levels according to the
regions of interest (e.g. Hb or IPN). The SPECT-brain data were seg-
mented using a whole-brain VOI (volume of interest) and cut out of the
SPECT-data with the Osirix™ software (Pixmeo, Switzerland). All brain
SPECT-data sets were intensity-normalized to the same global mean
using the MPI-Tool™ software. A voxelwise paired t-test was performed
166
Behavioural Brain Research 332 (2017) 164–171
comparing water versus fox urine exposure. Statistical analysis and
smoothing (median filter with a 3 × 3 × 3 kernel) was done with the
Magnan™-Software (version 2.4, BioCom GbR, Germany). In ac-
cordance with previous small-animal radionuclide imaging studies,
uncorrected P-values were used [26–28]. SPECT/MR fusion images
were made in Osirix™ and arranged for illustration using Photoshop™
(version CS6). For the analysis of correlations between tracer content
and behavioral data the significant (P < 0.01) areas of the P-map were
used as VOIs to obtain the average tracer contents of the different brain
structures. The tracer content in Hb and IPN were correlated with the
percentage of time spent in the odor quadrant and the number of
contacts to the odor source. Correlations were calculated and illustrated
with GraphPad Prism (Version 6, GraphPad Software Inc., La Jolla,
USA).
2.8.2. Lesion studies
The behavior (avoidance of the odor sample, contacts with the odor
sample) of the sham-operated and Hb/IPN-lesioned groups was com-
pared separately for the pre- and post-operative test sessions by two-
way repeated measures analyses of variance (ANOVA) with group as a
between-subject factor and odor (water vs. fox urine) as a within-sub-
ject factor. Significant ANOVAs were followed by Holm-Sidak’s mul-
tiple comparisons tests. Behavioral data were expressed as means
+ standard errors of the mean. For all statistical evaluations, a
P < 0.05 was considered statistically significant. All analyses were
carried out using GraphPad Prism.
3. Results
3.1. Fox urine induced changes in regional cerebral blood flow
Voxel-wise statistical analysis of global-mean normalized 99mTc-
content revealed highly significant changes in tracer-distributions in a
number of different brain areas. In the pooled data from both strains
(Fig. 1) as well as in the SD rats (Supplementary Fig. 2) highly con-
spicuous reductions in tracer content at very high significance levels
were present in the IPN (Fig. 1K, arrowhead) as well as increases in the
Hb (Fig. 1G). Significant decreases in tracer content in the IPN and
increases in the Hb were also found in Wistar rats, but significance
levels were generally lower in this group of thirteen animals.
P-values differed for left and right side in the Hb and other nuclei. In
SD rats voxels below threshold at P < 0.05 were found bilaterally with
slightly different locations within left and right Hb. Left-right differ-
ences in significance levels are regularly reported in the Hb in human
18F-FDG-PET and fMRI studies [29–31]. The reasons for these asym-
metries have remained unclear.
Besides the significant differences in the IPN and Hb, we found, in
the pooled data, significant changes in the following brain regions:
Tracer contents increased at significance levels of P < 0.01 and peaks
of P < 0.001 in the right olfactory bulb (Fig. 1A), in a region centered
on the glomerular layer of the right accessory olfactory bulb extending
into the frontal association cortex (Fig. 1B), left prelimbic cortex
(Fig. 1C), right anterior insular cortex/piriform cortex (Fig. 1D), left
infralimbic cortex, a region at the border of left caudatoputamen and
ventral pallidum most closely corresponding to the interstitial nucleus
of the posterior limb of the anterior commissure (IPAC) (Fig. 1F), right
ventromedial hypothalamus (VMH) (Fig. 1H), right amygdalopiriform
transition area (APir) and adjacent posterior cortical (PCo) amygdala
(Fig. 1J, arrowhead), right parietal association cortex (Fig. 1J, arrow)
and right reticular formation/brainstem. Further activations without
peaks of P < 0.001 were found in the right dorsolateral periaqueductal
gray (dlPAG) extending into the adjacent superior colliculus (Fig. 1L).
Significant decreases in tracer content at levels of P < 0.01, again
with peaks of P < 0.001, were found in the right striatum (Fig. 1E),
left primary somatosensory cortex, right primary somatosensory cortex,
left ventral posteromedial thalamic nucleus (Fig. 1I), right primary
D. Vincenz et al.
visual cortex (Fig. 1L), left lateral entorhinal cortex (Fig. 1M), a region
peaking on the left dorsal tegmentum (DTg) extending into the adjacent
dorsal raphe and rostral laterodorsal tegmentum (LDTg) (Fig. 1N) and
in the left ventral cochlear nucleus. Further deactivations, without
peaks of P < 0.001, were found in primary and secondary motor
cortices, right nucleus accumbens core, right lateral posterior thalamic
nucleus and in the cerebellar vermis peaking at bregma −10.8.
We did not analyze in detail the patterns of voxels below threshold
at lower significance levels (P < 0.05). In both data sets the area of
decreased tracer content in the IPN extended at these significance levels
into peri-interpeduncular regions covering the interfascicular nucleus
rostrally, the ventral tegmental area (VTA) laterally and the linear
nucleus of the raphe dorsally. A significant increase with few voxels
below threshold was found in the right medial amygdala.
3.2. Habenular activity correlates with predator odor-induced fear
responses
We next examined whether rCBF changes in the Hb and the IPN,
respectively, correlated with the defensive behaviors observed during
exposure to fox urine. The correlation analysis showed that fox urine-
induced rCBF changes in Hb were negatively correlated with fox urine-
induced avoidance behavior (Fig. 2). Thus, the more rCBF in the Hb
increased during exposure to fox urine the less strongly the odor
quadrant was avoided (r2 = 0.56, P = 0.05). Vice versa, rCBF changes
in Hb were positively correlated with the number of contacts with the
fox urine odor source, i.e. approach behavior (Fig. 2; r2 = 0.64,
P = 0.03). For rCBF changes in the IPN, no such correlations were
found (avoidance: r2 = 0.03, P = 0.73; contacts: r2 = 0.27, P = 0.23;
data not shown).
167
Behavioural Brain Research 332 (2017) 164–171
Fig. 1. Probability maps (P-maps) indicating sig-
nificant changes in normalized 99mTc-content in rats
(n = 33) under fox urine exposure versus water ex-
posure (control condition). P-maps (uncorrected) are
shown at two different significance levels in order to
illustrate locations of significance peaks and spatial
extent of significant voxels below threshold. Sections
are arranged from rostral (A) to caudal (N). Numbers
indicate coordinates relative to bregma corre-
sponding to the Paxinos and Watson atlas (Paxinos
and Watson, 2009). For each section, P-maps at
P < 0.01 are shown on the right and P-maps at
P < 0.001 on the left. Color look-up tables for both
maps are shown in A. Significant decreases in nor-
malized 99mTc content during fox urine exposure in
comparison to the control condition are color-coded
from blue to violet, significant increases in red to
yellow. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.)
3.3. Lesions of the Hb induce a shift from avoidance to approach behavior
We next addressed whether electrolytic lesions of either the Hb or
the IPN alter the defensive strategy chosen by the rats during exposure
to fox urine. Avoidance and approach behavior in response to the odor
was assessed before and after lesioning the Hb or the IPN. A sham-
operated control group was submitted to the same testing procedure to
control for general effects of the surgical procedure and behavioral
changes occurring due to habituation to the test situation. Rats in the
lesion groups typically encompassed complete lesions of intermediate
and posterior parts of either the Hb or the IPN with the most anterior
part of these structures being barely damaged (Fig. 3D, Fig. 4D, Sup-
plementary Fig. 3, Supplementary Fig. 4).
Before lesioning the Hb or the IPN, rats assigned to either the sham
or lesion groups avoided the odor quadrant containing fox urine
(Fig. 3A and Fig. 4A; ANOVAs, factor odor: Ps = 0.0003) and there
were no group differences (Ps > 0.59). After the surgery and recovery
for a few days, rats that received sham operations of either the Hb or
the IPN still expressed avoidance behavior towards fox urine
(Ps < 0.05). In contrast, rats with electrolytic lesions of either the Hb
or the IPN showed a significant reduction in avoidance behavior to-
wards fox urine (ANOVA, group x odor interaction: Hb: P = 0.04; IPN:
P = 0.02; Figs. Fig. 33A and Fig. 44A).
Furthermore, fox urine and water evoked similar levels of odor
contacts, i.e. approach behavior to the odor sample, in the different
lesion groups and their respective sham-operated control groups before
performing the surgery (Figs. Fig. 33B and Fig. 44B; Ps > 0.24). This
was still the case after the surgery in sham-lesioned animals
(Ps > 0.43). However, rats with lesions of the Hb (P = 0.0009) but
not of the IPN (P = 0.79) expressed more approach behavior, i.e.
contacted the fox urine sample more often in comparison to the water
sample (group x odor interaction: Hb: P = 0.02, IPN: P = 0.33). These
Fig. 2. Fox urine-induced fear responses correlate with rCBF changes
in the Hb. During 99mTc-HMPAO injections, avoidance behavior (%
time rats occupied the odor quadrant) and approach behavior
(number of odor contacts) to fox urine or to the water control was
analyzed (only in 7 SD rats with active behavioral profiles). For all
parameters, percent difference values for the two exposure conditions
were calculated. The dots represent individual animals and the lines
the linear fit with 95% confidence intervals.
D. Vincenz et al.
experimental data indicate that the Hb and the IPN differently con-
tribute to the processing of innate defensive responses in rats: Hb le-
sions induced a switch from an avoidance strategy to an approach
strategy whereas IPN lesions only blocked avoidance behavior but had
no effects on approach behavior.
4. Discussion
In the present study, we used 99mTc-HMPAO SPECT-imaging of rCBF
for analyzing the spatial patterns of neural activity in the brains of rats
exposed to the predator odor fox urine. We continuously injected the
168
Behavioural Brain Research 332 (2017) 164–171
Fig. 3. Hb lesions changed the defensive strategy in rats. Results are
shown for both sham-operated (n = 18) and Hb-lesioned (n = 12)
rats for pre-surgery and post-surgery testing during exposure to water
(white bars) and fox urine (blue/red bars). *P < 0.05, **P < 0.01,
Holm-Sidak’s multiple comparisons tests after significant effects in
two-way repeated measures ANOVAs. Representative photo-
micrographs showing Nissl-stained coronal sections through the Hb of
a sham-operated (C) and a Hb-lesioned rat (D).
flow tracer during a five-minute period within sessions of ten minutes
duration. The SPECT-images can be seen as representing, using blood
flow as a proxy, the average spatial patterns of neural activity from
minute 2.5 to min 7.5 within these ten-minute exposure times. We
found significant increases in 99mTc-content upon fox urine exposure as
compared to the control condition in the VMH, parts of the amygdala
(APir and PCo), Hb and dlPAG, as well as decreases in the IPN, dorsal
tegmentum (DTg and LDTg) and entorhinal cortex.
To the best of our knowledge, our study is the first in vivo imaging
study providing data on brain-wide activation patterns in awake, be-
having rodents exposed to a predator odor. Previous studies have used
Fig. 4. IPN lesions impaired fox urine-induced avoidance behavior.
Results are shown for both sham-operated (n = 14) and IPN-lesioned
(n = 11) rats for the pre-surgery and post-surgery testing during ex-
posure to water (white bars) and fox urine (blue/red bars).
*P < 0.05, Holm-Sidak’s multiple comparisons tests after significant
effects in two-way repeated measures ANOVAs. Representative pho-
tomicrographs showing Nissl-stained coronal sections through the IPN
of a sham-operated (C) and a IPN-lesioned rat (D).
D. Vincenz et al.
immediate early gene mapping techniques [32–34] or functional mag-
netic resonance imaging (fMRI) in restrained rats [35–37]. Our findings
of activations in the VMH, APir, PCo and dlPAG are in agreement with
behavioral studies using local pharmacological or optogenetic manip-
ulations [8,11,38,39]. The participation of the Hb in the innate fear
circuitry has very recently been suggested [16] and is strongly sup-
ported by our data. However, the deactivation of the IPN upon exposure
to a predator odor is, to the best of our knowledge, a novel finding.
Notably, in some brain areas described to be involved in predator
odor processing, e.g. the dorsal premammillary nucleus and the medial
amygdala [9,38], significant changes in blood flow could not be de-
tected or significance levels and number of voxels below threshold were
low. Limitations in sensitivity and spatial resolution may account for
this, e.g. partial volume effects masking flow responses in small groups
of neurons or lower contrasts between stimulus and control conditions.
A reason for these negative findings could, however, also be that neural
responses were absent or smaller and/or variances higher due to dif-
ferences in study design. A critical difference between our study and
previous studies investigating predator odor effects on rodent behavior
is that different odor samples were used. Many of the previous studies
[10,16,24,32,34,37,40] used trimethylthiazoline (TMT), a component
of the fox anal gland secretion (but see ref. [8] for the use of bobcat
urine and ref. [33] for the use of cat urine). Partly, TMT was used in
very high doses (e.g., 100 μl) and these high TMT concentrations have
been discussed to have noxious in addition to the fear-inducing prop-
erties [41–43]. Consequently, the then observed brain-wide activation
pattern may not only reflect odor-induced fear but also pain perception.
TMT can induce fear behavior at very low doses (5 nl [44]) but un-
fortunately such low doses were never tested in c-fos or other brain
imaging experiments. We are confident that these low doses represent
much more natural threat situations, as is also the case if natural pre-
dator odors like urine samples or cat fur odor [33,45,46] are used. An
additional advantage of the more physiological concentrations and of
the use of natural odor samples is that a larger repertoire of behavioral
defensive responses can be observed [5]. These defensive responses can
range from escape and avoidance behavior via freezing and hiding
behavior to risk assessment behavior like stretched back attends or
contacting the odor [e.g., [5,9–11,22,33,38,44]]. In the present ex-
periment, two main defensive strategies in response to fox urine were
seen: (a) avoidance of the odor source and (b) approach to the odor
source which is considered to be risk assessment behavior [5,47,48].
We found that with increasing blood flow in the Hb approach behavior
increased whereas avoidance behavior decreased. It is noteworthy that
such correlation analyses between behavioral parameters and neural
activity in post-hoc selected brain regions are not easily done in rodents
with fMRI studies since the behavioral repertoire of rodents in the MRI
scanner is severely limited even in the awake state.
To further characterize the role of the Hb in innate fear processing,
we tested whether a functional Hb is necessary to express the two de-
fensive strategies (approach/avoidance) we observed during behavioral
testing. We chronically lesioned the Hb and exposed the rats to fox
urine. Sham-lesioned animals expressed mainly avoidance behavior to
the fox urine samples which was occasionally interrupted by approach
behavior. Notably, animals with Hb lesions no longer avoided fox urine
but instead increased approach behavior. This indicates that Hb lesions
obviously induce a change in the defensive strategy of rats, a shift from
an avoidance strategy to an approach strategy. To the best of our
knowledge, this is the first evidence that the Hb is involved in the
choice of behavioral defensive strategies. As with Hb lesions, IPN le-
sions blocked avoidance behavior to fox urine. However, IPN lesions
did not affect approach behavior. This implies that the Hb-IPN axis
seems to be only involved in avoidance behavior to a predator odor,
while approach behavior might be regulated by other Hb projections.
Our findings agree with data showing that optogenetic stimulation
of the pathway from the Hb to the rostromedial tegmental nucleus
(RMTg) induces acute and learned avoidance behavior to the
169
Behavioural Brain Research 332 (2017) 164–171
compartment of the experimental arena in which the Hb was stimulated
[49]. The RMTg has recently been described as a “GABA brake for the
dopamine systems”, i.e. it inhibits the dopamine neurons in the VTA
and the substantia nigra [50]. Of interest for the present study is that
lesions of the RMTg impair avoiding (freezing) but increase ap-
proaching (defensive burying) behavioral responses to TMT [51]. This
RMTg lesion effect is of very similar quality to the Hb lesion effect
observed in the present study.
In our study target regions of both the MHb and LHb are char-
acterized by decreases in rCBF upon exposure to fox urine. CBF and
neural activity are closely coupled [52], but it is still unclear to what
degree the flow changes in a given region reflect changes in presynaptic
metabolism or postsynaptic activities of local interneurons or projection
neurons. The interpretation of flow changes in Hb and IPN is further
complicated by the fact that both nuclei are anatomically complex
structures composed of several subnuclei [53,54] that may differen-
tially contribute to the flow changes in the entire nucleus.
Both the MHb and LHb send excitatory projections to their target
regions [16,55]. It is generally assumed that the well-established in-
hibitory effect of LHb-stimulations on dopaminergic VTA neurons is
primarily mediated by excitatory projections to GABAergic neurons in
the VTA or RMTg [50,55]. This pathway has been implicated in reward,
aversion and more recently in risk assessment [56]. Anatomically si-
milar, an excitatory LHb projection to inhibitory interneurons in the
LDTg was found to be involved in innate fear processing [16]. The rCBF
decrease in the DTg and VTA that we observe in our study may thus
reflect the net effect of an activation of GABAergic neurons in these
regions. The LHb receives afferents from the prelimbic cortex [57]
where we found an increase in rCBF, suggesting an involvement of a
cortico-habenular-tegmental pathway in odor-induced innate fear be-
havior. Tegmental nuclei, both dorsal and ventral, send efferents to the
IPN [58–61]. The reduction in rCBF in the IPN may thus be an indirect
effect of LHb projections on tegmental areas. Alternatively or in addi-
tion, it might also be explained by a reduction of excitatory cholinergic
input from the MHb that has recently been shown to increase freezing
to conditioned stimuli [17].
The anatomical connectivity makes it likely that the highly inter-
connected Hb, IPN and dorsal and ventral tegmental nuclei influence
each other and act as a complex network in odor-induced innate fear
behavior. It has been emphasized previously [17] that the Hb-IPN
pathway provides an extra-amygdalar route in the processing of con-
ditioned fear as there are no known direct connections between either
Hb or IPN and the amygdala. The present data support this view but it
should be noted, that a number of brain regions, where we found sig-
nificant changes in blood flow, are connected to both, the amygdala-
hypothalamus-PAG pathway as well as to the Hb or IPN, making it
unlikely that information flows in amygdalar and extra-amygdalar
routes are independent of each other. The prelimbic cortex, for in-
stance, as one input source of the LHb receives afferent projections from
the amygdala [62], and the VTA as an LHb target projects to the
amygdala [63]. LHb output can indirectly also affect PAG activity via
modulation of serotonergic neurons [64]. The PAG in turn receives
afferents from the LTDg [65] that is reciprocally connected with the IPN
[58,60,61]. In view of ipsilateral glutamatergic and contralateral G-
ABAergic projections of different IPN-subnuclei to the LTDg, Quina and
colleagues in a recent tracing study [61] hypothesized that the MHb-
IPN system may be involved in directional locomotion. This hypothesis
remains to be tested but it illustrates how subsets of nuclei within the
network of brain regions that we detected in our study might pre-
ferentially mediate or influence a particular behavioral component in
odor-induced innate fear behavior.
In summary, we here used SPECT to image in vivo the brain-wide
spatial patterns of the average cerebral blood flow in awake, freely
moving rats exposed to a predator-odor. Our study demonstrates the
value of this novel approach as it confirms established brain regions
known to be involved in odor-induced innate fear behavior but also
D. Vincenz et al.
identifies regions that were found only very recently or were hitherto
unknown. We present the first data demonstrating that rCBF within the
Hb and the IPN are affected by exposure to a predator odor.
Furthermore, rCBF changes within the Hb were correlated with a
change in the behavioral defensive strategy. In line with this observa-
tion, Hb lesions induced a switch from an avoidance to an approach
defensive strategy whereas IPN lesions blocked only avoidance defen-
sive behavior.
Acknowledgements
This work was funded by the federal state of Saxony-Anhalt, Project:
Center for Behavioral Brain Sciences (CBBS). The authors wish to thank
Ines Bodewald, Kristin Böttger, Kathrin Freke and Dana Mayer for ex-
cellent technical assistance and Frank Angenstein for providing the MR
data set. We are further grateful to Dr. Bertram Gerber for critical
comments to the manuscript and Timothy French for language editing.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bbr.2017.05.053.
References
[1] J.E. LeDoux, Evolution of human emotion: a view through fear, Prog. Brain Res. 195
(2012) 431–442.
[2] L.M. Shin, I. Liberzon, The neurocircuitry of fear, stress, and anxiety disorders,
Neuropsychopharmacology 35 (2010) 169–191.
[3] A. Etkin, T.D. Wager, Functional neuroimaging of anxiety: a meta-analysis of
emotional processing in PTSD, social anxiety disorder, and specific phobia, Am. J.
Psychiatry 164 (2007) 1476–1488.
[4] S. Maren, Pavlovian fear conditioning as a behavioral assay for hippocampus and
amygdala function: cautions and caveats, Eur. J. Neurosci. 28 (2008) 1661–1666.
[5] R.A. Dielenberg, I.S. McGregor, Defensive behavior in rats towards predatory odors:
a review, Neurosci. Biobehav. Rev. 25 (2001) 597–609.
[6] M. Fendt, T. Endres, C.A. Lowry, R. Apfelbach, I.S. McGregor, TMT-induced auto-
nomic and behavioral changes and the neural basis of its processing, Neurosci.
Biobehav. Rev. 29 (2005) 1145–1156.
[7] M. Fendt, M.S. Fanselow, The neuroanatomical and neurochemical basis of condi-
tioned fear, Neurosci. Biobehav. Rev. 23 (1999) 743–760.
[8] K. Kondoh, Z. Lu, X. Ye, D.P. Olson, B.B. Lowell, L.B. Buck, A specific area of ol-
factory cortex involved in stress hormone responses to predator odours, Nature 532
(2016) 103–106.
[9] M. Muller, M. Fendt, Temporary inactivation of the medial and basolateral amyg-
dala differentially affects TMT-induced fear behavior in rats, Behav. Brain Res. 167
(2006) 57–62.
[10] C.M. Root, C.A. Denny, R. Hen, R. Axel, The participation of cortical amygdala in
innate, odour-driven behavior, Nature 515 (2014) 269–273.
[11] K.E.A. Wernecke, D. Vincenz, S. Storsberg, W. D'Hanis, J. Goldschmidt, M. Fendt,
Fox urine exposure induces avoidance behavior in rats and activates the amygdalar
olfactory cortex, Behav. Brain Res. 279 (2015) 76–81.
[12] M. Agetsuma, H. Aizawa, T. Aoki, R. Nakayama, M. Takahoko, M. Goto, et al., The
habenula is crucial for experience-dependent modification of fear responses in
zebrafish, Nat. Neurosci. 13 (2010) 1354–1356.
[13] S. Jesuthasan, Fear, anxiety, and control in the zebrafish, Dev. Neurobiol. 72 (2012)
395–403.
[14] R.G. Northcutt, Connections of the lateral and medial divisions of the goldfish
telencephalic pallium, J. Comp. Neurol. 494 (2006) 903–943.
[15] M.F. Wullimann, E. Rink, The teleostean forebrain: a comparative and develop-
mental view based on early proliferation, Pax6 activity and catecholaminergic or-
ganization, Brain Res. Bull. 57 (2002) 363–370.
[16] H. Yang, J. Yang, W. Xi, S. Hao, B. Luo, X. He, et al., Laterodorsal tegmentum
interneuron subtypes oppositely regulate olfactory cue-induced innate fear, Nat.
Neurosci. 19 (2016) 283–289.
[17] J. Zhang, L. Tan, Y. Ren, J. Liang, R. Lin, Q. Feng, et al., Presynaptic excitation via
GABAB receptors in habenula cholinergic neurons regulates fear memory expres-
sion, Cell 166 (2016) 716–728.
[18] A. Kolodziej, M. Lippert, F. Angenstein, J. Neubert, A. Pethe, O.S. Grosser, et al.,
SPECT-imaging of activity-dependent changes in regional cerebral blood flow in-
duced by electrical and optogenetic self-stimulation in mice, Neuroimage 103
(2014) 171–180.
[19] R.D. Neirinckx, L.R. Canning, I.M. Piper, D.P. Nowotnik, R.D. Pickett, R.A. Holmes,
et al., Technetium-99 m d,l-HM-PAO: A new radiopharmaceutical for SPECT ima-
ging of regional cerebral blood perfusion, J. Nucl. Med. 28 (1987) 191–202.
[20] D.J. Rowland, S.R. Cherry, Small-animal preclinical nuclear medicine in-
strumentation and methodology, Semin. Nucl. Med. 38 (2008) 209–222.
[21] S. Bhattacharya, R. Herrera-Molina, V. Sabanov, T. Ahmed, E. Iscru, F. Stöber, et al.,
170
Behavioural Brain Research 332 (2017) 164–171
Genetically-induced retrograde amnesia of associative memories after neuroplastin
ablation, Biol. Psychiatry 81 (2017) 124–135.
[22] J.B. Rosen, E.A. West, M.P. Donley, Not all rat strains are equal: differential un-
conditioned fear responses to the synthetic fox odor 24,5-trimethylthiazoline in
three outbred rat strains, Behav. Neurosci. 120 (2006) 290–297.
[23] L.G. Staples, I.S. McGregor, Defensive responses of Wistar and Sprague-Dawley rats
to cat odour and TMT, Behav. Brain Res. 172 (2006) 351–354.
[24] J. Goldschmidt, T. Wanger, A. Engelhorn, H. Friedrich, M. Happel, A. Ilango, et al.,
High-resolution mapping of neuronal activity using the lipophilic thallium chelate
complex TlDDC: protocol and validation of the method, Neuroimage 49 (2010)
303–315.
[25] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates. Compact, 6th
edition, Elsevier, UK, 2009.
[26] H. Endepols, S. Sommer, H. Backes, D. Wiedermann, R. Graf, W. Hauber, Effort-
based decision making in the rat: an [18F]fluorodeoxyglucose micro positron
emission tomography study, J. Neurosci. 30 (2010) 9708–9714.
[27] M. Michaelides, S.A. Anderson, M. Ananth, D. Smirnov, P.K. Thanos, J.F. Neumaier,
et al., Whole-brain circuit dissection in free-moving animals reveals cell-specific
mesocorticolimbic networks, J. Clin. Invest. 123 (2013) 5342–5350.
[28] P.K. Thanos, L. Robison, E.J. Nestler, R. Kim, M. Michaelides, M.K. Lobo, et al.,
Mapping brain metabolic connectivity in awake rats with μPET and optogenetic
stimulation, J. Neurosci. 33 (2013) 6343–6349.
[29] P.J. Carlson, N. Diazgranados, A.C. Nugent, L. Ibrahim, D.A. Luckenbaugh,
N. Brutsche, et al., Neural correlates of rapid antidepressant response to ketamine in
treatment-resistant unipolar depression: a preliminary positron emission tomo-
graphy study, Biol. Psychiatry 73 (2013) 1213–1221.
[30] K. Hennigan, K. D'Ardenne, S.M. McClure, Distinct midbrain and habenula path-
ways are involved in processing aversive events in humans, J. Neurosci. 35 (2015)
198–208.
[31] R.R. Lawson, B. Seymour, E. Loh, A. Lutti, R.J. Dolan, P. Dayan, et al., The habenula
encodes negative motivational value associated with primary punishment in hu-
mans, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 11858–11863.
[32] A. Asok, L.W. Ayers, B. Awoyemi, J. Schulkin, J.B. Rosen, Immediate early gene and
neuropeptide expression following exposure to the predator odor 25-dihydro-2,4,5-
trimethylthiazoline (TMT), Behav. Brain Res. 248 (2013) 85–93.
[33] R.A. Dielenberg, G.E. Hunt, I.S. McGregor, When a rat smells a cat: the distribution
of Fos immunoreactivity in rat brain following exposure to a predatory odor,
Neuroscience 104 (2001) 1085–1097.
[34] H.E. Day, C.V. Masini, S. Campeau, The pattern of brain c-fos mRNA induced by a
component of fox odor2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, sug-
gests both systemic and processive stress characteristics, Brain Res. 1025 (2004)
139–151.
[35] M. Febo, A.S. Pira, Increased BOLD activation to predator stressor in subiculum and
midbrain of amphetamine-sensitized maternal rats, Brain Res. 1382 (2011)
118–127.
[36] W. Huang, M.E. Heffernan, Z. Li, N. Zhang, D.H. Overstreet, J.A. King, Fear induced
neuronal alterations in a genetic model of depression: an fMRI study on awake
animals, Neurosci. Lett. 489 (2011) 74–78.
[37] M.S. Kessler, S. Debilly, S. Schoppenthau, T. Bielser, A. Bruns, B. Kunnecke, et al.,
fMRI fingerprint of unconditioned fear-like behavior in rats exposed to tri-
methylthiazoline, Eur. Neuropsychopharmacol. 22 (2012) 222–230.
[38] N.S. Canteras, E. Pavesi, A.P. Carobrez, Olfactory instruction for fear: neural system
analysis, Front. Neurosci. 9 (2015) 276.
[39] P.S. Kunwar, M. Zelikowsky, R. Remedios, H. Cai, M. Yilmaz, M. Meister, et al.,
Ventromedial hypothalamic neurons control a defensive emotion state, eLife (2015)
4, http://dx.doi.org/10.7554/eLife.06633.
[40] L.G. Staples, I.S. McGregor, R. Apfelbach, G.E. Hunt, Cat odor, but not tri-
methylthiazoline (fox odor), activates accessory olfactory and defense-related brain
regions in rats, Neuroscience 151 (2008) 937–947.
[41] L.W. Ayers, A. Asok, F.D. Heyward, J.B. Rosen, Freezing to the predator odor 24,5
dihydro 2,5 trimethylthiazoline (TMT) is disrupted by olfactory bulb removal but
not trigeminal deafferentation, Behav. Brain Res. 253 (2013) 54–59.
[42] M. Fendt, T. Endres, 23,5-Trimethyl-3-thiazoline (TMT), a component of fox odor −
just repugnant or really fear-inducing? Neurosci. Biobehav. Rev. 32 (2008)
1259–1266.
[43] J.B. Rosen, A. Asok, T. Chakraborty, The smell of fear: innate threat of 25-dihydro-
2,4,5-trimethylthiazoline, a single molecule component of a predator odor, Front.
Neurosci. 9 (2015) 292.
[44] T. Endres, R. Apfelbach, M. Fendt, Behavioral changes induced in rats by exposure
to trimethylthiazoline, a component of fox odor, Behav. Neurosci. 119 (2005)
1004–1010.
[45] D.T. Hubbard, D.C. Blanchard, M. Yang, C.M. Markham, A. Gervacio, I.L. Chun,
et al., Development of defensive behavior and conditioning to cat odor in the rat,
Physiol. Behav. 80 (2004) 525–530.
[46] M.D. Reed, K.E. Price, J. Archbold, A. Moffa, M. Febo, Predator odor-evoked BOLD
activation in the awake rat: modulation by oxytocin and V(1)a vasopressin receptor
antagonists, Brain Res. 1494 (2013) 70–83.
[47] R.J. Blanchard, D.C. Blanchard, J. Rodgers, S.M. Weiss, The characterization and
modelling of antipredator defensive behavior, Neurosci. Biobehav. Rev. 14 (1990)
463–472.
[48] N. McNaughton, Fear, anxiety and their disorders: past, present and future neural
theories, Psychol. Neurosci. 4 (2011) 173–181.
[49] A.M. Stamatakis, G.D. Stuber, Activation of lateral habenula inputs to the ventral
midbrain promotes behavioral avoidance, Nat. Neurosci. 15 (2012) 1105–1107.
[50] M. Barrot, S.R. Sesack, F. Georges, M. Pistis, S. Hong, T.C. Jhou, Braking dopamine
systems: a new GABA master structure for mesolimbic and nigrostriatal functions, J.
D. Vincenz et al.
Neurosci. 32 (2012) 14094–14101.
[51] T.C. Jhou, H.L. Fields, M.G. Baxter, C.B. Saper, P.C. Holland, The rostromedial
tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons,
encodes aversive stimuli and inhibits motor responses, Neuron 61 (2009) 786–800.
[52] P.T. Fox, M.E. Raichle, Stimulus rate dependence of regional cerebral blood flow in
human striate cortex, demonstrated by positron emission tomography, J.
Neurophysiol. 51 (1984) 1109–1120.
[53] K.H. Andres, M. von During, R.W. Veh, Subnuclear organization of the rat habe-
nular complexes, J. Comp. Neurol. 407 (1999) 130–150.
[54] H.J. Groenewegen, S. Ahlenius, S.N. Haber, N.W. Kowall, W.J. Nauta,
Cytoarchitecture, fiber connections, and some histochemical aspects of the inter-
peduncular nucleus in the rat, J. Comp. Neurol. 249 (1986) 65–102.
[55] K. Brinschwitz, A. Dittgen, V.I. Madai, R. Lommel, S. Geisler, R.W. Veh,
Glutamatergic axons from the lateral habenula mainly terminate on GABAergic
neurons of the ventral midbrain, Neuroscience 168 (2010) 463–476.
[56] S.B. Floresco, The nucleus accumbens: an interface between cognition, emotion,
and action, Annu. Rev. Psychol. 66 (2015) (2015) 25–52.
[57] U. Kim, T. Lee, Topography of descending projections from anterior insular and
medial prefrontal regions to the lateral habenula of the epithalamus in the rat, Eur.
J. Neurosci. 35 (2012) 1253–1269.
[58] J. Cornwall, J.D. Cooper, O.T. Phillipson, Afferent and efferent connections of the
laterodorsal tegmental nucleus in the rat, Brain Res. Bull. 25 (1990) 271–284.
[59] R. Zhao-Shea, S.R. DeGroot, L. Liu, M. Vallaster, X. Pang, Q. Su, G. Gao, O.J. Rando,
171
Behavioural Brain Research 332 (2017) 164–171
et al., Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-
medial habenula circuit induces anxiety during nicotine withdrawal, Nat. Commun.
6 (2015) 6770, http://dx.doi.org/10.1038/ncomms7770.
[60] L.B. Lima, D. Bueno, F. Leite, S. Souza, L. Gonçalves, I.C. Furigo, J. Donato Jr.,
M. Metzger, Afferent and efferent connections of the interpeduncular nucleus with
special reference to circuits involving the habenula and raphe nuclei, J. Comp.
Neurol. (Mar (24)) (2017), http://dx.doi.org/10.1002/cne.24217 Epub ahead of
print.
[61] L.A. Quina, J. Harris, H. Zeng, E.E. Turner, Specific connections of the inter-
peduncular subnuclei reveal distinct components of the habenulopeduncular
pathway, J. Comp. Neurol (Apr (7)) (2017), http://dx.doi.org/10.1002/cne.24221
Epub ahead of print.
[62] W.B. Hoover, R.P. Vertes, Anatomical analysis of afferent projections to the medial
prefrontal cortex in the rat, Brain Struct. Funct. 212 (2007) 149–179.
[63] L. Yetnikoff, A.Y. Cheng, H.N. Lavezzi, K.P. Parsley, D.S. Zahm, Sources of input to
the rostromedial tegmental nucleus, ventral tegmental area, and lateral habenula
compared: a study in rat, J. Comp. Neurol. 523 (2015) 2426–2456.
[64] R.L. Pobbe, H. Zangrossi Jr, The lateral habenula regulates defensive behaviors
through changes in 5-HT-mediated neurotransmission in the dorsal periaqueductal
gray matter, Neurosci. Lett. 479 (2010) 87–91.
[65] K. Satoh, H.C. Fibiger, Cholinergic neurons of the laterodorsal tegmental nucleus:
efferent and afferent connections, J. Comp. Neurol. 253 (1986) 277–302.