Fluoxetine: Pharmacology, Mechanisms of Action and Potential Side Effects

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Fluoxetine: Pharmacology, mechanisms of action and potential side effects
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PHARMACOLOGY - RESEARCH, SAFETY TESTING AND REGULATION
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FLUOXETINE
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PHARMACOLOGY, MECHANISMS OF
ACTION AND POTENTIAL SIDE EFFECTS
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PHARMACOLOGY - RESEARCH, SAFETY
TESTING AND REGULATION
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PHARMACOLOGY - RESEARCH, SAFETY TESTING AND REGULATION
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FLUOXETINE
PHARMACOLOGY, MECHANISMS OF
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ACTION AND POTENTIAL SIDE EFFECTS
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GRAZIANO PINNA, PHD, DR. RER. MEDIC.
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EDITOR
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New York
Copyright © 2015 by Nova Science Publishers, Inc.
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Additional color graphics may be available in the e-book version of this book.
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Library of Congress Cataloging-in-Publication Data
ISBN: 978-1-63482-076-9
Library of Congress Control Number: 2015930863
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Published by Nova Science Publishers, Inc. † New York

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Contents
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Preface vii
Chapter 1 Activity of Fluoxetine in Animal Models of Depression and Anxiety 1
Trevor R. Norman
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Chapter 2 The Neurosteroidogenic Action of Fluoxetine Unveils the
Mechanism for the Anxiolytic Property of SSRIs 25
Graziano Pinna
Chapter 3 Developmental Fluoxetine Exposure and Neuroendocrine Outcomes 43
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Jodi L. Pawluski, Mary Gemmel, Eszter Császár,
Harry W. M. Steinbusch and Ine Rayen
Chapter 4 The Impact of Fluoxetine Treatment on BDNF
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Signaling and Cellular Plasticity in the Brain 71
Jochen De Vry, Fabien Boulle, Bart P. F. Rutten,
Daniel L. A. van den Hove and Jos Prickaerts
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Chapter 5 Fluoxetine and Its Novel Effect on Adult Neurogenesis 97

Koji Ohira
Chapter 6 The Role of Neurotrophins in Fluoxetine‘s Mechanism of Action 107
Maria Ladea and Mihai Bran
Chapter 7 The Role of Epigenetic Mechanisms in the Therapeutic
Effects of Fluoxetine and Other Selective Serotonin
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Reuptake Inhibitors 125

Kristen Ashley Horner
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Chapter 8 microRNAs As Novel Players in Depression Pathogenesis

and in the Mechanisms of Action of Fluoxetine and
Other Antidepressants 147
Guangxing Bai, Michael Dunbar, Levi D. Miller,
Bhaskar Roy, Ian J. Soller, Richard C. Shelton
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and Yogesh Dwivedi

vi Contents
Chapter 9 Preclinical and Clinical Aspects of a Combination

Therapy with Antagonists to N-Methyl-D-Aspartate
Receptor to Enhance Fluoxetine‘s Antidepressant Effects 167
Gislaine Z. Réus, Helena M. Abelaira,
Fabricia Petronilho and João Quevedo
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Chapter 10 Fluoxetine and Glutamate Release and Transmission 187
Laura Musazzi, Paolo Tornese, Giulia Treccani
and Maurizio Popoli
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Chapter 11 Fluoxetine and Other SSRI Antidepressants Potentiate
Addiction-Related Gene Regulation by
Psychostimulant Medications 207
Vincent Van Waes and Heinz Steiner
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Chapter 12 Role of Fluoxetine on Depression-Related
Pathophysiological Mechanisms 227
Miroslav Adzic, Milos Mitic, Iva Lukic,
Jelena Djordjevic and Marija B. Radojcic
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Chapter 13 Clinical Implications of Fluvoxamine and Fluoxetine
with Sigma-1 Receptor Chaperone Activity in the
Treatment of Neuropsychiatric Disorders 279
Yakup Albayrak and Kenji Hashimoto
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Chapter 14 Fluoxetine: Pharmacological Analysis of
Depression-Like Responses in Zebrafish 295
Julian Pittman, Roaa Hadi and Katie Ichikawa
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Chapter 15 How Could Fluoxetine Exert Therapeutic Effects in Stroke? 307
Virginie Beray-Berthat, Michel Plotkine
and Raymond Mongeau
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Index 329
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Preface
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The introduction of the iconic antidepressant, fluoxetine, best known in the drug market
as Prozac®, for the pharmacotherapy of major depression in 1987 marked a major milestone
in the development of new and more efficacious antidepressant medications. Fluoxetine
quickly became the most prescribed psychotropic drug worldwide and its success in
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improving mood disorders has triggered the development of a large number of congener
molecules, which reached a market high of nearly $15 billion in 2003. These drugs,
commonly known as SSRIs were developed for their common ability to selectively inhibit
serotonin reuptake, thus, increasing the levels of this neurotransmitter in the synaptic milieu
and enhancing serotonergic neurotransmission. This neurochemical effect of fluoxetine and
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congeners was regarded as the mechanism of action for the improvement of mood and
depressive symptoms. Over a quarter of a century after its FDA approval, studies in the field
have recently provided scientific evidence that fluoxetine‘s pharmacological effects may be
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mediated by a number of other important and more impelling mechanisms rather than just by
the ability of this drug to act as a ―selective‖ serotonin reuptake inhibitor. Accordingly,
fluoxetine has often been used as a prototypic drug to advance understanding into the as yet
unknown mechanism of action of antidepressants.
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Hence, this book reviews several preclinical and clinical reports suggesting that the
beneficial behavioral effects of fluoxetine may be mediated by regulating gene expression,
modifying epigenetic mechanisms as well as altering microRNAs. One of the most prominent
mechanisms for the therapeutic relevance of fluoxetine relates to influencing neuroplasticity
by enhancing neurotropic factors, including BDNF signaling and altering adult neurogenesis.
The ability of fluoxetine to increase neurosteroid levels accounts for the anxiolytic effects of
this drug. Fluoxetine action at sigma-1 receptor or modulating glutamatergic
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neurotransmission as well as the combination of fluoxetine with other psychotropic drugs is

discussed in relation to its therapeutic effects. While fluoxetine was primarily prescribed as an
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antidepressant, this drug currently represents a gold standard for a broad spectrum of
psychiatric disorders, including post-traumatic stress disorder (PTSD) and a range of anxiety
disorders. This drug even possesses analgesic actions and is a valuable therapy for stroke.
This book also highlights emerging evidence on the gender-specific effects of fluoxetine,
its potential adverse features, the addiction liability in combination with psychostimulants,
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and the impact of perinatal fluoxetine exposure.

I greatly appreciate the mentoring of Professor Emeritus, Erminio Costa (1923-2009),
who introduced me to the study of fluoxetine and its neurosteroidogenic action. These studies
viii Graziano Pinna
resulted in discovering a novel and more selective mechanism by which fluoxetine at non-
serotonergic doses enhances GABAergic neurosteroid biosynthesis. The preclinical
observation that low-doses of fluoxetine primarily enhance brain allopregnanolone levels now
offers a therapeutic strategy for several new clinical applications for psychiatric disorders
ranging from anxiety disorders to PTSD and premenstrual dysphoria devoid of
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benzodiazepine-like side-effects.
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In: Fluoxetine ISBN: 978-1-63482-076-9
Editor: Graziano Pinna © 2015 Nova Science Publishers, Inc.
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Chapter 1
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Activity of Fluoxetine in Animal Models
of Depression and Anxiety
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Trevor R. Norman
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Department of Psychiatry, University of Melbourne,
Austin Hospital, Victoria, Australia
Abstract
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In the life cycle of a potential antidepressant medication, activity in animal models is
usually an initial step indicating potential clinical activity. Several test systems have
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become de rigueur in identifying molecules for further clinical evaluation as
antidepressants. In particular, the forced swim test (FST) and the tail suspension test
(TST) have found almost universal acceptance as rapid, efficient methods for screening
compounds for ―antidepressant-like‖ activity, albeit with a number of caveats. More time
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consuming models, such as the chronic mild stress (CMS) and olfactory bulbectomy
(OB) models are not as frequently used but may have more clinical relevance since
activity in these models is only apparent after chronic administration of the drugs, a
feature of all clinically marketed antidepressants to date. In general, activity in several
models is evaluated for new agents to avoid the issue of false negatives. Further, it is now
apparent, in clinical populations, that antidepressant agents can be regarded as ―broad
spectrum‖ with activity in several psychiatric diagnoses. While the drugs are primarily
employed clinically as antidepressants, many possess analgesic actions as well as
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demonstrating efficacy in a range of anxiety disorders. Evaluation of new molecules in

appropriate animal models of anxiety is often conducted to indicate potential activity.
Across the range of Selective Serotonin Reuptake Inhibitors (SSRIs) evaluation in animal
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models has indicated antidepressant activity but demonstration of anxiolytic effects in

widely utilised tests has been problematic. The activity of fluoxetine, a unique SSRI by
virtue of its pharmacokinetic profile, in animal models of depression and anxiety is
reviewed.
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Keywords: models, depression, anxiety, behaviour, fluoxetine

2 Trevor R. Norman
Introduction
Modern psychopharmacology has reached a cross roads. Increasing development costs to
bring new molecules to market has caused a re-think by major pharmaceutical companies
about investment in this domain. Part of the increased investment costs can be ascribed to the
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inability of clinical psychopharmacological trials to clearly establish efficacy against placebo.
For example, it is often contended that the gap in clinical response between an antidepressant
and a placebo appears to be diminishing. However, from the first introduction of
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antidepressant medications in the 1950‘s the problem of the clinical distinction of a putative
antidepressant from a placebo was already apparent (Horden, 1965). Many factors identified
then are still in evidence today: diagnostic issues, homogeneity of the patient population with
respect to previous exposure to antidepressant medications, adequate dosage of medication,
compliance, inter-rater reliability for symptomatology, severity and number of previous
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episodes; all remain as contemporary potential confounders in the assessment of medications.
Commonly used and accepted animal behavioural tests for detecting antidepressant
activity have been identified as a potential obstacle in bringing forward new molecules.
Activity in pre-clinical tests has not always translated well to the clinic leading some to
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question their utility (Borsini et al., 2002). Many compounds identified pre-clinically have
been abandoned after an extensive and expensive clinical trial program. A case in point was
that of the substance P antagonist, aprepitant (MK 869), as an antidepressant. The compound
showed antidepressant-like activity in animal behavioural tests as well antidepressant effects
in an early phase clinical trial, but was abandoned for this indication when subsequent trials
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failed to show separation from placebo (Keller et al., 2006).
Against this existing background, it is sobering to note that many of the pharmacological
tools, the subject of current criticisms, are the very ones which were applied, successfully, to
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add fluoxetine to the antidepressant armamentarium. A review of the activity of fluoxetine in
animal behavioural tests to assess antidepressant-like activity is presented. While the pre-
clinical testing of fluoxetine for detecting antidepressant activity provided a largely uniform
picture, the same cannot be said of studies conducted for anxiolytic activity. As such a mixed
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picture has emerged, it is arguable that fluoxetine may have been overlooked if it was to be
developed de novo as an antianxiety agent. A brief overview of the pharmacology of
fluoxetine is presented to place the compound in the context of contemporary antidepressant /
anxiolytic agents.
Fluoxetine
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The historical development of the fluoxetine molecule and its emergence as a pre-
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eminent antidepressant of its time has been well chronicled in the literature (Wong et al.,
1995, 2005). The molecule with the chemical description (±) (3-(p-trifluoromethylphenoxy)-
N-methyl-3-phenylpropylamine Hydrochloride (LY110140) (Figure 1) was first described in
the 1970‘s (Wong et al., 1975). The principal pharmacological property described for the
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compound was the ability to act as a selective reuptake inhibitor of serotonin into rat brain
synaptosomes (Wong et al., 1975). While this activity was thought, at the time, to confer the
compound with potential antidepressant effects, it was not until the 1980‘s that fluoxetine was
Activity of Fluoxetine in Animal Models of Depression and Anxiety 3
widely available as an antidepressant agent. Following its introduction as an antidepressant,

fluoxetine has been shown to have additional therapeutic efficacy in anxiety disorders such as
obsessive compulsive disorder and panic disorder, where extensive trials have been conducted
(Boyer et al., 1991). In other anxiety disorders, such as Post Traumatic Stress Disorder
(Davidson and Connor, 1998) and Social Anxiety Disorder (Lydiard, 1998), there is some
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evidence for efficacy from small open evaluations.
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Figure 1. Chemical structure of (±)-N-Methyl-3-phenyl-3-[(α, α, α -trifluoro-p-tolyl)oxy]propylamine,
hydrochloride (fluoxetine Hydrochloride).
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Fluoxetine exists as a racemic mixture of equivalent amounts of the R and the S
enantiomers. Both enantiomers inhibit synaptosomal uptake of 5HT with similar potencies
(Ki value of 21nM and 16nM for R- and S-fluoxetine respectively) (Wong et al., 1985; Wong
et al., 1991). The major metabolites in humans and animals of the enantiomers are the de-
methylated compounds, R- and S-nor-fluoxetine. Both S-nor-fluoxetine (Ki ~20nM) and R-
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nor-fluoxetine (Ki ~268nM) inhibit 5-HT reuptake, but with the S-enantiomer clearly more
potent (Wong, et al., 1993).
Fluoxetine and its enantiomers have low or negligible affinity in vitro for a variety of
common receptor subtypes: muscarinic, adrenergic, opioid and histaminic, which
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distinguishes the compound from some other antidepressants notably, the tricyclic class of
agent. In clinical practice, this distinction confers some advantages in terms of an improved
side effect profile experienced by patients receiving the drug relative to those experienced by
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patients taking tricyclic compounds. There is some suggestion that this profile may also result
in a better cardiovascular safety profile after intentional overdose (Borys et al., 1990).
Fluoxetine binds relatively weakly to 5HT1, 5HT2, and 5HT3 receptor subtypes (Wong et al.,
1991; Table 1). On the other hand, fluoxetine enantiomers exhibit stereospecific binding to
the 5HT2C recognition site in the bovine choroid plexus (Table 1). The R (-) enantiomer of
fluoxetine is about 20 times more potent than the S (+) enantiomer (Ki values 150 and
3600nM, respectively) with respect to 5HT2C binding.
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Table 1. Binding of fluoxetine and its enantiomers to serotonin receptor subtypes

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Serotonin Receptor Subtype

Compound 5HT1A 5HT1B 5HT1D 5HT2A 5HT2C 5HT3
Fluoxetine 11000 6200 4300 1800 270 19000
R(-) Fluoxetine 16000 11000 17000 620 150 11000
S(+) Fluoxetine 5900 3400 3900 1700 3600 >10,000
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Data are expressed as Ki values nM; from Wong et al., 1991.

4 Trevor R. Norman
In line with its in vitro pharmacology as a serotonin reuptake inhibitor, fluoxetine also
demonstrated an in vivo pharmacology supporting this mechanism of action. Both fluoxetine
and its enantiomers were able to prevent the depletion of serotonin content in brain induced
by the administration of p-chloro-amphetamine (Fuller et al., 1975). Similar results were
noted when other 5HT depleting agents such as methamphetamine or fenfluramine were
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administered (Wong et al., 1975). Selectivity for serotonin was demonstrated as there were no
effects of fluoxetine on the depletion of catecholamines following administration of agents
(e.g., 6-OH-DOPA) known to rapidly deplete brain stores of these neurotransmitters (Wong et
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al., 1975).
While acute in vivo and in vitro pharmacology of an agent can provide some useful
insights to the mechanism of action of medications, effects on chronic administration are
more likely to be relevant to the explanation of clinical effects. In the case of antidepressant
medications important clinical effects do not emerge until after repeated dosing. Similarly,
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inhibition of 5HT uptake by fluoxetine occurs rapidly (Wong et al., 1974; 1975; 1985) but the
antidepressant action is delayed (Wernicke et al., 1987). In line with this notion the effects of
repeated dosing of fluoxetine on brain receptors in rodents was investigated. The prevailing
paradigm at the time was that antidepressant medications exerted their clinical effects by the
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down-regulation of central β1-adrenergic receptors (Banerjee et al., 1977). Further, the role of
the 5HT2A receptor subtype was also regarded as an important determinant of antidepressant
action (de Montigny and Aghajanian, 1978).
Studies with fluoxetine have generally failed to show a statistically significant effect on
either β1 or 5HT2A receptors number or affinity following chronic administration of the drug
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(see Wong et al., 1995 for review). On the other hand, chronic treatment with fluoxetine
down-regulated 5HT1A receptor numbers without an alteration of receptor affinity (Wong and
Bymaster, 1980). With respect to β-adrenergic function, chronic administration of fluoxetine
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(10mg/kg for two weeks) to rats failed to alter the noradrenaline stimulated cAMP response in
a cerebral cortex tissue slice preparation (Mishra et al., 1979). Subsequent notions of the
action of antidepressant medications have focused on the role of neutrophic factors, in
particular Brain Derived Neurotrophic Factor (BDNF), and the ability of repeated
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antidepressant administration to increase BDNF in the hippocampus (Nibuya et al., 1995).

Fluoxetine, like all other antidepressants examined, has been shown to possess this property
(Malberg and Duman, 2003). Whether such a mechanism is relevant to clinical antidepressant
effects is a moot point.
A distinguishing feature of fluoxetine, relevant to its clinical use, is the pharmacokinetic
profile of the drug. The plasma elimination half-life of fluoxetine is from 2-4 days while that
of the principal, active metabolite, nor-fluoxetine, is from 7-15 days (Harvey and Preskorn,
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2001). For other SSRI medications elimination half-life is generally of the order of 24 hours.
Consequently steady-state concentrations of the drug are reached slowly (from 30-60days)
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and the drug might be administered on a once a week basis and still achieve steady state. In
terms of patient compliance, missing a regular dose of the drug is likely to have only minor
clinical consequences.
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Activity in Depression Models

Overview of Models
It is apparent that there are no laboratory animals that can mimic all of the features of
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clinical depression: some symptoms of the human disorder are incapable of being ‗modeled‘
in other species e.g., guilt, depressed mood. Nevertheless some features can be modeled and
are probably entirely analogous (from a neurochemical perspective at least) with the human
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symptoms. Thus animal models may be used under circumscribed conditions to understand
the neurochemical aetiology of individual symptoms or symptom clusters arising in man. It
can be claimed for these symptoms that such an animal model has face and construct validity.
Such an approach has been termed endophenotypic (Cryan et al., 2002). A more detailed
critique of specific animal models is beyond the scope of this limited review and is provided
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elsewhere (Cryan et al., 2002). In the situation of drug discovery this aspect of a model is less
important than predictive validity: does activity in this behavioural test reliably and robustly
predict that a compound(s) active in the test will be active in the clinic? It is at this juncture
that it has been suggested that animal models have provided false positive results for many
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compounds taken through to clinical development. Evaluation of molecules in several models
may provide a more reliable set of data from which to gauge molecules to bring forward to
clinical testing. Assessment of activity in several models may also diminish the possibility of
false negatives as it is often the case that a molecule is not active in all tests conducted.
Fluoxetine has been tested therefore, in several models indicative of potential antidepressant
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activity in the clinic.
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Fluoxetine Studies
Overview of Studies Reviewed

While some animal tests have assumed a pre-eminence with respect to the evaluation of
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antidepressant-like activity of molecules, others are not as widely used due to their labor
intensive requirements. Fluoxetine has been most frequently evaluated in the forced swim and
tail suspension tests. These tests provide a relatively quick indication of antidepressant-like
activity and benefit from the fact that they are generally responsive to acute doses of the
agent. Thus, it is possible to investigate dose response relationships in a timely manner for the
selection of doses for potential further pre-clinical testing as well as gain some indication of
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activity relative to doses of comparative agents. The route of administration used in most
testing has been intra-peritoneal (i.p.) as this is more likely to correspond to later clinical use
as oral formulations. Nevertheless, oral (p.o.) and subcutaneous (s.c.) administration to
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rodents is occasionally utilized. In the studies described here fluoxetine has been administered
i.p. in the majority of studies. Further, both tests have been used to evaluate drug effects after
chronic administration. Results from acute and chronic administration are not always
concordant, the findings dependent on strain of animal, route of administration and conditions
under which the test is conducted (e.g., environmental lighting, size of test chambers,
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background noise, ambient temperature, temperature of the water (forced swim)). Many of
the conditions are standardized and the molecule of choice compared readily to other
6 Trevor R. Norman
antidepressants at known effective doses of those agents in the test. Most frequently
imipramine has been used as a comparator although drugs from other classes have been
occasionally been used (moclobemide, an MAO-A inhibitor, other SSRIs, clomipramine a
moderately selective serotonergic tricyclic antidepressant). While imipramine is a well-
known, clinically effective antidepressant, there have been concerns expressed that the forced
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swim test particularly may only detect agents with similar pharmacological properties (Detke
et al., 1997). Thus modifications to this test which quantitate other behaviours (swimming,
escape, climbing) in the test have increased its reliability for the detection of many different
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classes of antidepressant (Cryan et al., 2005).
Tests which detect antidepressant-like effects only after chronic administration of drugs
have also been investigated after administration of fluoxetine. Arguably such models are
more representative of the proposed clinical use of the drug while at the same time may
reflect neuro-adaptive changes taking place. These latter changes are possibly more relevant
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to the mechanism of action of the drug (see above). There is less information about the
activity of fluoxetine in these tests, but generally the results are consistent in indicating an
antidepressant-like activity as well as effectiveness equivalent to comparators.
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Activity in Specific ‘Antidepressant-Like’ Tests
The forced swim test (FST) is a rapid screening paradigm for potential antidepressant
activity (Porsolt et al., 1977). The test is based on the observation that rats or mice when
forced to swim in a restricted space from which there is no possibility of an escape,
eventually cease to struggle (behavioural despair or helplessness). Single doses of fluoxetine
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were shown to dose dependently diminish immobility time (increase swim time in the
chamber) in mice over the range 2 to 40mg/kg after a single intra-peritoneal dose (Kulkani
and Dhir, 2007). Both imipramine and venlafaxine were also active in the test over
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comparable dose ranges. Neither mianserin nor trazodone was active in the test at the doses
studied. Similar results in the FST were observed for fluoxetine over the dose range 3-30
mg/kg following acute and sub-chronic (1 week) administration (De Vry et al., 1999). Sub-
cutaneous administration of fluoxetine for 14days also prolonged the swimming time and
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increased escape attempts in the rat FST (Chau et al., 2011). Similarly mice directly infused
with fluoxetine in the raphe nuclei were also observed to have increased swimming time
(Baudry et al., 2010).
Clearly, the findings reviewed above are consistent and fluoxetine would be expected to
have antidepressant activity in the clinic. The findings however need to be nuanced by the
influence of various factors affecting behavioural outcome in animals. Thus, while fluoxetine
was shown to be active in the FST, Dulawa and colleagues (2004) showed that the results can
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be dependent on the strain of animal used for the study. Thus, four inbred mouse strains
(C57BL/6, 129SvEv, BALB/c and DBA/2) were investigated for their behavioural response
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to chronic treatment (24days) with fluoxetine (5, 10 mg/kg/day in drinking water). Only the
highly anxious BALB/c strain was sensitive to the effects of fluoxetine in the FST. Sub-
chronic (~4 days) and chronic (~24 days) fluoxetine treatment (10, 18, 25 mg/kg/day) were
further investigated in BALB/C mice in the FST. Only chronic treatment with 10 and 18
mg/kg/day increased swimming and reduced immobility, while sub-chronic treatment was
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inactive. These results may not translate across species as Wistar rats classified behaviourally
as either high or low anxiety did not demonstrate differences in behaviour in the FST (Ho et
al., 2002). In this study, the response to medications was not measured. In contrast, Roman
High (RHA) and Low avoidance (RLA) rats did demonstrate differences in behavioural
responses in the FST (Piras et al., 2014). RLA rats demonstrate greater immobility and fewer
climbing behaviours when compared to RHA animals. Subacute (1 day) treatment with
desipramine, fluoxetine, and chlorimipramine (2.5–5 mg/kg) did not affect the behaviour of
RLA rats. On the other hand, chronic treatment (15days) of all three agents demonstrated
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antidepressant-like behaviour (decreased immobility; increased climbing (desipramine):
increased swimming (fluoxetine)). The behavior of RHA rats in the FST was not affected by
either subacute or chronic treatments with these agents. These results are broadly in line with
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those of Dulawa (2004) described above, but in a different species. Thus not only strain, but
species, can affect the results of animal behavioural tests adding a further layer of complexity
to tests designed to identify potential molecules of interest for development as
antidepressants.
Although there are some inconsistencies between studies, due mostly to the strain of
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animal chosen to undertake the investigation, the results are generally supportive of a
response in the test consistent with known clinically effective antidepressants.
A somewhat similar paradigm to the FST is the tail suspension test (TST) which also
involves an inescapable stress and the development of immobility (Steru et al., 1985).
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Haemodynamic stress is provided by the mouse being hung in an uncontrollable fashion by
the tail. When administered prior to the test, antidepressant drugs cause the animal to persist
in escape-directed behaviours for longer periods of time than their vehicle-treated
counterparts. The amount of time spent immobile is recorded manually or using an automated
method. Acute antidepressant treatments decrease these immobility scores (Cryan et al.,
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2005). The limitations and nuances of the test have been reviewed in detail elsewhere (Cryan
et al., 2005). In an early study, fluoxetine (4-32 mg/kg) was shown to dose dependently
decrease the immobility time of mice in the TST comparably with that of four other SSRI
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drugs (Perrault et al., 1992). Reduction of immobility was reported even at the low dose of
0.5 mg/kg of fluoxetine in mice (Ismail et al., 2009). In other studies, reduction of immobility
time was noted with doses up to 64 mg/kg (Teste et al., 1993). The TST is generally
conducted with acute doses of the test agent but some sub-chronic and chronic treatments
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with fluoxetine have reported. Thus 14 days of treatment up to 20 mg/kg of fluoxetine was
active in the test (Ukai et al., 1998) while a 7day treatment with 20 mg/kg was also effective
(Yu et al., 2002). Strain of mice used did not appear to alter the results of studies performed
with fluoxetine (Cryan et al., 2005). Mice with genetic backgrounds of putative importance in
the aetiology of depression (e.g., 5HT1A knock outs) have also served as a method of
investigating the effects of medications in these ―depressed models‖. Under baseline
conditions, 5-HT1A receptor mutant mice had significantly lower immobility than wild-type
a
mice (Mayorga et al., 2001). Fluoxetine (20 mg/kg and paroxetine (20 mg/kg) had no effect
on immobility in these mutant mice whereas desipramine (20 mg/kg) significantly decreased
ov
immobility. It was suggested that the presence of 5-HT1A receptors is possibly critical for the
expression of the antidepressant-like behaviour of SSRIs in this test.
In summary it can be concluded that fluoxetine has been extensively evaluated in the TST
paradigm and generally found to be active.
The foregoing tests primarily rely on acute or subacute administration of molecules to
N
assess antidepressant-like activity. As antidepressants require chronic administration for

clinical efficacy, animal tests which also depend on repeated administration to produce their
activity are reputedly more reliable in assessing potential utility in the clinic. One such test
8 Trevor R. Norman
relies on the development of hyperactivity of rats or mice in an open field following bilateral
removal of the olfactory bulbs (Cairncoss et al., 1978). This test has been extensively
evaluated with respect to its predictive and face validity, although its construct validity
remains elusive (Song and Leonard, 2005). Chronic, but not acute, administration of
antidepressant drugs reverses the hyper-locomotion in a novel arena that is observed
c.
following olfactory bulbectomy. A wide variety of antidepressants have been investigated in
the model and the predictive value is high even for molecules which seemingly do not rely on
monoamine mechanisms for their antidepressant effects (e.g., agomelatine; Norman et al.,
In
2012). Fluoxetine (10 mg/kg once daily for 5 weeks) has been shown to significantly
attenuate the hyper-locomotor activity in bulbectomised male Sprague–Dawley rats compared
to vehicle treated animals (Roche et al., 2007). Similar results were obtained in female Swiss
mice following a 14 day administration of 10 mg/kg fluoxetine (Freitas et al., 2013). These
changes in behavioural activity were accompanied by alterations in hippocampal cell
rs
signaling pathways widely thought to be involved in synaptic plasticity. Thus, while
bulbectomy induced significant increases in ERK1 and CREB (Ser133) phosphorylation and in
the expression of BDNF immuno-content, chronic fluoxetine treatment attenuated these
increases. Attenuation of hyperactivity of bulbectomised rats in a highly illuminated open
he
field by fluoxetine (10 mg/kg for 21 days) was reported by increasing the rate of habituation
of the animals (Mar et al., 2002). The open field test is usually conducted in a dimly
illuminated apparatus. The highly illuminated field is believed to impart a greater stress to the
animals (Mar et al., 2002).
Disturbances of circadian rhythms have long been associated with depressive disorders
is
(Norman, 2010). In different species of rodents bulbectomy has been shown to alter circadian
rhythms manifest as lengthening the free-running period in mice and hamsters and delaying
the phase of activity onset (Bittman et al., 1989; Possidentte et al., 1990; Pieper et al., 1991).
bl
Chronic administration of fluoxetine (8mg/kg for 14days) reversed the effects of bulbectomy
on the mean locomotor activity while significantly shortening the free-running period of the
activity rhythm; the phase of the activity rhythm was not significantly altered. The data are
consistent with an antidepressant-like effect of fluoxetine in the model and further suggest
Pu
that circadian alterations in depression are also subjected to fluoxetine induced changes.
Studies in bulbectomised animals are consistent in that fluoxetine reverses the hyper-
locomotor activity induced by this procedure on repeated, but not acute, administration. This
effect is consistently found with other the clinically effective antidepressants with which
fluoxetine has been compared and strongly suggests an antidepressant-like effect for the drug.
Chronic stress is considered a precipitating factor for depression in humans. Chronic
exposure of rodents to unpredictable mild stressors (chronic mild stress, CMS) produces
a
anhedonic behaviour that resembles certain aspects of human depression. It has been
proposed as a valid model of depression (Willner, 1992; Willner et al., 1997). This model
ov
relies on measuring a decrease in the preference for a sucrose solution in animals previously
trained to prefer a 1% sucrose solution instead of water. This is suggested as a model of the
symptom of anhedonia. Restoration of sucrose preference can be taken as a measure of the
extent to which a novel agent possesses antidepressant-like behaviour. The model is generally
regarded as having good face validity, although its reproducibility in different laboratories
N
along with other aspects of the model has been strongly criticised (Reid et al., 1997). The
model is responsive only to chronic drug administration.
Administration of fluoxetine (10 mg/kg) has been shown to restore sucrose preference in
stressed animals compared to vehicle over a four week treatment period (Grippo et al., 2006).
Treatment of male inbred BALB/c mice with fluoxetine (15 mg/kg) for seven weeks also
reversed stress-induced changes in the mice such as grooming behaviour in the splash test and
attack frequency in the resident-intruder paradigm (Mutlu et al., 2012). The effects on sucrose
c.
preference were not reported. Treatment of male rats for 21 days with fluoxetine (10 mg/kg)
was able to reverse the decrease in sucrose consumption induced by the CMS procedure
(Rong et al., 2010).
In
Antidepressant treatment attenuated the decreases in the expression of the astroglial
protein S100B and its receptor RAGE (receptor for advanced glycation end products) in the
hippocampus and cerebrospinal fluid induced by stress. The glia-derived neurotrophic marker
S100B, produced mainly by astrocytes, has been shown to be increased in the serum of
patients with melancholia (Arolt et al., 2003) and there is some evidence for a reduction
rs
following antidepressant treatment (Schroeter et al., 2002). Thus antidepressants are able to
modify the expression of this protein in the animal paradigm, although the specificity of the
marker to depression remains debatable (Schroeter et al., 2002).
While monoamine mechanisms have dominated ideas about the aetiology of major
he
depressive disorder, a cholinergic super-sensitivity hypothesis has been proposed based on
responses in depressed patients (Janowsky et al., 1994). Based on this notion, the Flinders
Line rats were developed by selective breeding for differential responses to the
anticholinesterase agent, di-isopropyl fluorophosphate (DFP) (Overstreet, 2002). Flinders
Sensitive Line (FSL) rats are more sensitive to the hypothermic effects of DFP, whereas
is
Flinders Resistant Line (FRL) rats are not more resistant than an outbred control (Overstreet
et al., 1979). FSL animals show an exaggerated immobility in the forced swim test which is
responsive to chronic antidepressant treatment. A standard protocol for the testing of any
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potential antidepressant drug in this model consists of 14 days once-daily treatment and a
single 5-min swim test conducted 22–26 h after last treatment. Fluoxetine (5 mg/kg) in this
protocol was found to be active (Overstreet, 2002).
Activity for fluoxetine has been demonstrated in a limited number of studies utilizing
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olfactory bulbectomy, chronic mild stress and the Flinders Sensitive Line of animals. The
activity of fluoxetine in these various animal models attests to the antidepressant-like effect
for the molecule in pre-clinical paradigms.
The clinical trial data base has demonstrated antidepressant activity of the drug in
humans (Benfield et al., 1986).
a
Activity in Anxiety Models

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Overview of Models
Multiple animal behavioural paradigms have historically been used to evaluate the
potential of a molecule for use an antianxiety agent. These range from the classical conflict
tests, such as the Geller-Seifter punished responding, to ethological models based on the
N
natural aversion of rodents for open brightly-lit spaces, to more sophisticated genetic models
targeting so-called ‗endophenotypes‘.
10 Trevor R. Norman
A discussion of the nuances of each of these models is beyond the scope of this review
but a detailed critical evaluation has been presented elsewhere (Cryan and Sweeney, 2011).
This review focuses on the use of ethologically based models such as behaviour in the
elevated plus maze, and the extent to which fluoxetine demonstrates responses indicative of
anxiolytic or anxiogenic effects.
c.
There are several issues which may complicate the interpretation of data obtained from
these non-conditioned responses in animals:
In
1) most models have been validated with GABAergic anxiolytic agents
(benzodiazepines, barbiturates) and their usefulness for the detection of drugs
working via alternate mechanisms of action has been questioned;
2) behaviour is often influenced by extraneous factors which may be difficult to control
on a day to day basis;
rs
3) animals cannot be repeatedly tested in the apparatus as they habituate to the test
environment;
4) agents tested may affect motor performance and the majority of these tests depend on
locomotor activity to derive the primary outcome variable;
he
5) because of inherent variability in animal responses relatively large numbers of
animals are required to achieve statistical significance;
6) reliability of tests between laboratories may be poor.
Other factors may also be important determinants of behavioural outcome. One

is
consideration is the influence of circadian variation on behaviour. Because of their nocturnal
nature, rodent behaviour is significantly greater at night and can be reflected in anxiety testing
paradigms. For example, light–dark crossing behaviour under the influence of diazepam is
bl
significantly higher when animals are tested at the beginning of their dark period compared to
testing at the start of the light period (unpublished data, see Figure 2 for experimental details).
On the other hand, in the social interaction test there was no influence of diazepam on
behaviour between day and night-time testing.
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Furthermore, as with the antidepressant paradigms discussed above strain, species

(Ramos, et al., 1997) and gender (Palanza, 2001) of the animals used can be an important
consideration. Some paradigms are subject to floor effects, particularly when considering
anxiogenic behaviour. In the elevated plus maze the putative anxiogenic compound isatin was
not distinguishable from vehicle whereas oxazepam was clearly differentiated from vehicle
and isatin for open arm time (unpublished data; see Figure 3 for experimental details).
Despite these shortcomings and technical complications, several tests have been
a
developed which are widely used in assessing anxiolytic potential of new compounds. These
include the elevated plus maze, light-dark crossings, open field behaviour, social interaction
ov
test, novelty-induced hypophagia and defensive burying (also known as marble burying test).
Fluoxetine has been tested in many of these paradigms for anxiolytic activity.
N
TIME IN THE LIGHT AREA (SECONDS)

SOCIAL INTERACTION TEST LIGHT DARK BOX
TIME IN INTERACTION (SECONDS)

150 DIAZEPAM 40
DIAZEPAM
VEHICLE
VEHICLE
30
c.
100
20
50
10
In
0 0
PM
PM
AM
AM
TIME OF DAY TIME OF DAY
rs
The social interaction test was The light-dark test was conducted in a
conducted in an open-topped box box measuring 60cm X 60cm X 35cm
measuring 60cm X 60cm X 35cm divided by a partition 15cm high by
illuminated by a 40watt bulb (200lux at 60cm wide. Half the box was painted
he
floor level). Two rats from the same test black and half white. The white side was
group (diazepam or vehicle) were placed illuminated by a 40watt bulb (200lux at
in the middle of the arena and the time floor level). Time spent in the light area
interacting (sniffing, grooming or was defined as consecutive periods
crawling over each other) during a 5 spent with all four paws in the white half
is
minute test period was recorded. Testing of the test arena during a 5minute
was videotaped and scored by testperiod. Testing was videotaped and
independent scorers blind to the scored by independent scorers blind to
bl
treatment. the treatment.
Two-way ANOVA showed no effect of Two-way ANOVA showed a
time of day on the results (F 1, 16 = 0.16) statistically significant effect of time (F
but there was an effect of treatment (F 1, 1, 16 = 7.37; P<0.05) and treatment (F 1, 16
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16 = 8.38; P<0.05). There was no = 6.28; P<0.05). There was no

interaction effect (F 1, 16 = 0.89) interaction effect (F 1, 16 = 3.53)
Experiments were conducted in male Sprague-Dawley rats weighing between 220-
320g and housed individually under constant 12:12 lighting conditions. Animals
were handled daily for 7 days before the experiments. Rats were injected with
vehicle or diazepam (2mg/kg) 30 minutes before testing. The behaviour was
a
measured between 0800H and 1030H for the AM condition and between 2000H and
2230H for the PM condition. Separate groups of animals were used for each test.
(Unpublished data from the laboratory of the author).
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Figure 2. Diurnal variation of performance on anxiety tests.

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12 Trevor R. Norman
200
150
TIME (SEC)
c.
100
In
50
)
LE
G
/K
/K
rs
IC
G
H
0M
M
VE
(3
(2
M
N
PA
TI
A
ZE
IS
XA
he
O
Male Sprague-Dawley rats (150-250g) were housed in plastic cages in groups of four with food and
water available ad lib. Animals were maintained on a 12:12 light dark cycle. Isatin and oxazepam
were obtained from Sigma-Aldrich, Sydney, Australia. Rats (n=9 per group) received either
vehicle (DMSO), isatin (20mg/kg) or oxazepam (3mg/kg) dissolved in vehicle in an injection
is
volume of 1ml/kg fifteen minutes before testing in the elevated plus maze. Animals were placed in
the dead space of the maze facing a closed arm and the behaviour videotaped for five minutes
scored later by observers blind to treatments Entry to and time in an arm were recorded when all
four paws were in the arm. Data were analysed by ANOVA and post-hoc comparisons between all
bl
groups performed with a Student-Newman-Keuls multiple comparison test using Graph Pad Prism
5 software. There was a statistically significant difference between groups for time on the open
arms (F 2, 26= 6.95; P<0.005). Post-hoc tests showed that both vehicle (P<0.005) and isatin
(P<0.005) were different from oxazepam but that isatin and vehicle were not significantly
different. The data suggest a ‗floor effect‘ with respect to the detection of anxiogenic-like effects.
Pu
(Unpublished data from the laboratory of the author).
Figure 3. Behaviour of a putative anxiogenic agent, isatin, on the elevated plus maze.
Fluoxetine Studies
a
One of the most widely used models to study effects of antidepressants on anxiety-like
behaviour is the elevated plus maze. The maze uses the natural fear of rodents to avoid open
and elevated places. The apparatus consists of two enclosed and two opposite open arms in
ov
the form of a plus sign and is elevated 50cm above the floor. Animals naïve to the apparatus,
spend about 30% of the test time on the open arms, while treatment with anxiolytic agents
increases open-arm exploration (Pellow et al. 1985; Lister 1987). Modifications of the maze
have also been employed in some studies, but the interpretation of results is essentially the
N
same. The initial clinical use of SSRIs is often characterised by the emergence of an
anxiogenic response leading to the development of akathisia and is experienced by a
significant number of patients (Leo, 1996). This may arise due by indirect stimulation of 5-
HT2A receptors, resulting in inhibition of DA release (Hansen, 2001). Continued treatment

with the medication generally results in an anxiolytic effect. This differential temporal
response is also apparent in some studies with the elevated plus maze. For example, acute
administration of fluoxetine (1-10 mg/kg) produced a dose dependent diminution in the ratio
of open arm to total arm entries in male Wistar rats i.e., an anxiogenic response (Handley and
c.
McBlane 1993). Yet, the same doses produced anxiolytic effects in the Vogel conflict
paradigm (punished drinking behaviour). In other studies with acute doses of fluoxetine using
Sprague-Dawley and Wistar rats as well as Swiss mice either no effect of fluoxetine has been
In
observed or an increased anxiogenic response is noted. Acute administration studies are
summarised in Table 2. The results therefore somewhat mimic the clinical observations.
Repeated administration of fluoxetine also has not produced a consistent anxiolytic response
in either rats or mice. Indeed some studies have recorded anxiogenic responses similar to that
of acute doses (see Table 3 for summary of studies). This has also been the case with other
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members of the SSRI class of antidepressants (Borsini et al., 2002). This may be a drawback
of the test system and its limitation to detect only anxiolytic agents acting at GABA-A
receptors (Rodgers et al., 1997).
he
Table 2. Acute effects of fluoxetine in various animal tests of anxiolytic-like activity
Animal Species /Strain Dose (mg/kg) * Outcome Reference

Elevated Plus Maze
SD rats 1-10 No effect Griebel et al. 1997
Wistar rats 1.25-20 Anxiogenic Handley and McBlane 1992
is
Wistar rats 5 Anxiogenic Silva et al. 1999
Wistar rats 10 Anxiogenic Kshama et al. 1990
Wistar rats 5.6-10 No effect or Silva and Brandao 2000
anxiogenic
bl
WKY rats 5-20 No effect Griebel et al. 1999
Swiss mice 2.5-20 No effect Durcan et al. 1988
Swiss mice 5-20 No effect Rodriguez-Filho and Takahashi 1999
ICR mice 1-10 No effect Nakamura and Kurasawa 2001
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Light Dark Box

Wistar rats 0.01-10 s.c. No effect Sanchez and Meier 1997
CD1 female mice 5-20 U dose-response de Angelis 1996
Swiss mice 10 Anxiogenic Artaiz et al. 1998
Swiss mice 1-16 No effect Bourin et al. 1996
Social interaction test
SD rats 5 Anxiogenic To and Bagdy 1999; To et al. 1999
SD rats 2.5–10 Anxiogenic Bristow et al. 2000
a
HSD rats 10 Anxiogenic File et al. 1999

LSD rats 10 Anxiogenic File et al. 1999
Gerbils 10 Anxiogenic Cheeta et al. 2001
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Marble Burying Test

Mice Various Anxiolytic Brocco et al. 2000
Novelty Suppressed Feeding
Long Evans rats 10 Anxiogenic i.p. Bodnoff et al. 1989
Hole Board Apparatus/ Free Exploration
Swiss mice 2.5-20 No effect Durcan et al. 1988
N

BALBc mice 5-20 Anxiogenic (20) Belzung et al. 2001
* All doses by intra-peritoneal injection except where indicated; s.c. sub-cutaneous.
14 Trevor R. Norman
Table 3. Effects of chronic fluoxetine in various animal tests of anxiolytic-like activity
Animal Species /Strain Dose (mg/kg)* Outcome Reference

(Treatment length)
Elevated Plus Maze
Wistar rats 10 p.o. No effect Silva and Brandao 2000
c.
(14 days)
WKY rats 5, 20 No effect Griebel et al. 1999
(23-24 days)
Wistar rats 5 Anxiogenic Silva et al. 1999
In
(22 days)
SHR rats 5, 10 No effect Durand et al. 1999
(13 days)
WKY rats 5, 10 Anxiogenic? Durand et al. 1999
(13 days)
LSD rats 10 No effect File et al. 1999
(14 days)
rs
HSD rats 10 Anxiogenic File et al. 1999
(14 days)
Mice 10 Anxiogenic at 7days Kurt et al. 2000
(7 or 14 days)
he
Social Interaction test
SD rats 5 No effect To et al. 1999
(21 days)
SD rats 10 Anxiolytic (14 days) Bristow et al. 2000
(14 or 21 days) No effect (21 days)
HSD rats 10 No effect File et al. 1999
(14 days)
is
LSD rats 10 No effect File et al. 1999
(14 days)
Novelty Suppressed Feeding
Long Evans rats 10 Anxiolytic Bodnoff et al. 1989
bl
(21 days)
* All doses in these experiments were administered by intra-peritoneal injection except where noted.
p.o. oral administration.
Pu
A variant of the plus maze is the Unstable Elevated Exposed Plus Maze (UEEPM) which
consists of a plus-shaped maze elevated 50 cm above the floor, with four exposed arms
measuring 35 X 12 cm and a centre square measuring 12 X 12 cm. The apparatus is oscillated
at 85 rpm in the horizontal plane by a motor. Doses of fluoxetine (1–10 mg/kg p.o.) were
administered to male Brown Norway rats as a single dose 30 min prior to maze exposure or
for 21 days in the diet, before being tested in the UEEPM. Acute fluoxetine had no effect on
behaviour but chronic fluoxetine (10 mg/kg) significantly attenuated the animals‘ propensity
a
to escape from the UEEPM (Jones et al., 2002). This paper also noted that chronic handling
of animals could decrease escape behaviour i.e., be anxiolytic per se. Prior handling of
ov
animals has been shown to decrease anxiety (Andrews and File, 1991). Handling regimens
also affected the outcome of fluoxetine treatment in the elevated plus maze (Robert et al.,
2011). Fluoxetine (5 mg/kg), when administered acutely or repeatedly for 15 days, induced an
anxiogenic-like behaviour. A modest variation in the prior experience of the rats, daily
handling plus an injection, modified the response to fluoxetine in the elevated plus maze
N
increasing the anxiogenic-like behaviour.

The effect of acute and chronic administration of fluoxetine in the elevated T-maze was
investigated in male Wistar rats (Poltronieri et al., 2003). The T-maze apparatus consists of
three elevated arms, one enclosed and two open. Learned fear is measured by recording the
time taken to leave the enclosed arm in three consecutive trials. Unconditioned fear is
evaluated by recording the time to escape from the open arm Graeff et al., 1998). In contrast
to the plus maze chronic, but not acute fluoxetine in the dose range 5-15 mg/kg was active as
an anxiolytic agent in this test. Similar results were found in other studies where acute
c.
fluoxetine administration caused an increase in anxiety-like behaviour in the elevated T-maze
(Dygalo et al., 2006; Gomes et al., 2009).
The effect of an acute administration of fluoxetine has been tested in the light-dark box
In
(DeAngelis, 1996). The "light-dark" box is based on the aversive properties of light and uses
as its index of anxiety the time spent in the lit area. Fluoxetine was administered by intra-
peritoneal injection to female mice at doses of 5, 10 or 20 mg/kg 30 minutes before the test.
Fluoxetine (10 and 20 mg/kg) significantly reduced the aversive behaviour of mice for the lit
area suggesting an anxiolytic-like effect. Locomotor activity data showed that fluoxetine did
rs
not increase in activity in this test. The anxiolytic effects of the drug then are not due to an
overall increase in the activity of the mice but rather due to anxiolytic-like effect. These data
are in sharp contrast to the majority of reports using this apparatus which show either no
anxiolytic-like activity of fluoxetine or an anxiogenic-like effect (see Table 2). The reasons
he
for the discrepancy between studies are not clear but might be due to the use of different
species or strains of animals as well as the choice of fluoxetine dose. Thus, in Wistar rats
doses of fluoxetine from 0.1 to 1mg/kg were not active (Sanchez and Meier, 1997) while
doses up to 16 mg/kg were also inactive in Swiss mice (Bourin et al. 1996). Using a dose of
10 mg/kg fluoxetine was anxiogenic-like in Wistar rats (Kshama et al. 1990) and Swiss mice
is
(Artaiz et al. 1998).
Both acute and chronic doses of fluoxetine have been tested in the social interaction test.
This test measures the time spent in active social interaction by a pair of unfamiliar
bl
conspecifics under conditions of high illumination and a novel environment. Such conditions
are generally anxiogenic to the rat while anxiolytic drugs such as benzodiazepines increase
the time spent in social interaction (File 1980; see Figure 2). Consistent results for fluoxetine
administration have usually been observed in the social interaction test after acute doses.
Pu
Male Sprague-Dawley rats treated with acute doses of 2.5 to 10 mg/kg had anxiogenic
responses (To and Bagdy, 1999; To et al., 1999; Bristow et al., 2000). Treatment with 5
mg/kg for 3 weeks had no effect on interaction time (To et al., 1999). In contrast, Bristow and
co-workers (2000) showed an anxiolytic response after a two or three week treatment with 10
mg/kg fluoxetine. A variant strain of Sprague-Dawley rats bred for high and low sensitivity to
the hypothermic response induced by the 5-HT1A receptor agonist 8- OH-DPAT was used to
investigate the effects of fluoxetine (File et al. 1999). The HDS line has been shown to have a
a
higher level of anxiety. These HDS and LDS animals did not differ in their response to
fluoxetine 10 mg/kg after either acute or chronic (14 day) treatment with the drug. Similarly,
ov
in gerbils acute treatment with 10 mg/kg induced an anxiogenic response (Cheeta et al. 2001).
The social interaction test data confirm the anxiogenic-like nature of single doses of
fluoxetine found in other paradigms. Although some chronic dosing studies suggest an
anxiolytic-like profile most suggest that fluoxetine is without effect.
The suppression of feeding in food-deprived rats when exposed to a novel environment,
N
hyponeophagia, has been taken as a model of anxiety and is reversed by benzodiazepines

(Shephard and Broadhurst 1982). Acute treatment of Long-Evans rats with fluoxetine was
anxiogenic-like i.e., increased the latency to feed, but anxiolytic-like after 21days
16 Trevor R. Norman
administration of 10 mg/kg i.e., reduced the latency to begin feeding (Bodnoff et al., 1989). In
male Balb/cJ mice subchronic (4–5 days) treatment with fluoxetine had no effect on
neophagia but chronic treatment (23 days) with 10 and 18 mg/kg was anxiolytic (Dulawa and
Hen, 2005).
The hole-board apparatus consists of an enclosed arena with holes in the floor into which
c.
an animal can poke its‘ head (head-dipping). Frequency and duration of head-dipping provide
a measure of neophilia (or directed exploration) which is independent of the general
locomotor activity of the animal (File and Wardill, 1975). As with the other apparatus
In
surveyed, mixed results have been found for the behaviour of fluoxetine in this test. Acute
administration of 2.5, 5, 10 or 20 mg/kg to Swiss mice was without behavioural effects
(Durcan et al., 1988). On the other hand, acute administration of 10mg/kg to Wistar rats had
an anxiogenic-like effect (Kshama, et al., 1990).
In a free exploration test, where mice are confronted with a familiar and a novel
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compartment, acute doses of fluoxetine (5, 10, 20mg/kg) in BALB/c mice was found to
exhibit anxiogenic-like behaviours (Belzung et al., 2001).
Defensive burying of noxious stimuli or unusual objects like glass marbles under bedding
materials is observed in rodents. The behaviour is thought to be an expression of anxiety. Rats
he
or mice placed in a cage in which glass marbles are laying on sawdust are buried by the
animal. The marble burying behaviour is sensitive to anxiolytic and anxiogenic compounds
(Broekkamp et al. 1986). When administered to mice fluoxetine was effective in reducing
marble burying behaviour suggesting an anxiolytic-like effect (Brocco et al., 2000). The
minimum effective dose of fluoxetine in this test was ~7.5mg/kg.
is
Conclusion
bl
The behavioural effects of fluoxetine have been investigated in various animal paradigms
used to detect antidepressant and anxiolytic activity. A uniformity of results has been
observed in the antidepressant-like models where fluoxetine has been found to be active.
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However, in tests used to detect anxiolytic-like activity fluoxetine has presented a mixed
picture. Following acute doses the drug is generally anxiogenic-like in the responses
observed, although a few studies have reported no behavioural changes or anxiolytic-like
effects. In this respect, the pre-clinical data are in accordance with observations in clinical
practice. Patients presenting with anxiety disorders can often experience an initial
exacerbation of their anxiety at the start of treatment. Where the data are not congruent is
a
following repeated dosing which, in the clinic, is usually anxiolytic, but in pre-clinical studies
mostly induces no changes or anxiogenic-like behaviour. It is not clear why such
discrepancies between clinical and pre-clinical data are evident. It may reflect the nature of
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‗anxiety‘ in animals which in model system is based on innate or conditioned fear

(conditioned fear models have not been reviewed here). In humans, anxiety is more than fear
and encompasses a morbid dread of the future. Furthermore anxiety may arise in the absence
of any recognisable threat (Lewis, 1980). Thus it may be that fluoxetine acts on these aspects
of anxiety in humans to relieve symptoms overall, aspects which are clearly not definable in
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animals. This speaks to the adequacy of animal paradigms as predictive tests for identifying
molecules to bring forward for clinical development.
References
Andrews, N., File, S.E., (1991). Handling of rats modifies behavioural effects of drugs in the
elevated plus-maze test of anxiety, Eur. J. Pharmacol. 235, 109–112.
Arolt, V., Peters, M., Erfurth, A., Wiesmann, M., Missler, U., Rudolf, S., Kirchner, H.,
c.
Rothermundt, M., (2003). S100B and response to treatment in major depression: a pilot
study. Eur. Neuropsychopharmacol. 13, 235–239.
Artaiz, I., Zazpe, A., del Rio, J., (1998). Characterization of serotonergic mechanism involved
In
in the behavioural inhibition induced by 5-hydroxytryptophan in a modified light-dark
test in mice, Beh. Pharmacol. 9, 103–112.
Banerjee, S.P, Kung, L.S., Chanda, S.K., (1977). Development of beta-adrenergic receptor
sub-sensitivity by antidepressants. Nature. 268, 455–456.
Belzung, C., Le Guisquet, A.M., Barreau, S., Calatayud, F., (2001). An investigation of the
rs
mechanisms responsible for acute fluoxetine-induced anxiogenic-like effects in mice,
Behav. Pharmacol. 12,151–162.
Benfield, P., Heel, R.C., Lewis, S.P., (1986). Fluoxetine: a review of its pharmacodynamic
and pharmacokinetic properties, and therapeutic efficacy in depressive illness, Drugs. 32,
he
481-508.
Bittman, E.L., Crandell, R.G., Lehman, M.N., (1989). Influences of the paraventricular and
suprachiasmatic nuclei and olfactory bulbs on melatonin responses in the Golden
Hamster, Brain Res. 40, 118-126.
Bodnoff, S.R., Suranyi Cadotte, B., Quirion. R., Meaney, M.J., (1989). A comparison of the
is
effects of diazepam versus several typical and atypical anti-depressant drugs in an animal
model of anxiety, Psychopharmacol. 97, 277–279.
Borsini, F., Podhorna, J., Marazziti, D., (2002). Do animal models of anxiety predict
bl
anxiolytic-like effects of antidepressants? Psychopharmacol. 163, 121–141.
Borys, D.J., Setzer, S.C., Ling, L.J., Reisdorf, J.J., Day, L.C., Krenzelok, E.P., (1990). The
Effects of Fluoxetine in the Overdose Patient, Clin. Toxicol. 28, 331-340.
Bourin, M., Redrobe, J.P., Hascoet, M., Baker, G.B., Colombel, M.C., (1996). A schematic
Pu
representation of the psychopharmacological profile of antidepressants, Prog.

Neuropsychopharmacol. Biol. Psychiat. 20, 1389–1402.
Boyer, W.F., McFadden, G.A., Feighner, J.P., (1991). The efficacy of selective serotonin re-
uptake inhibitors in anxiety and obsessive compulsive disorder, In: Feighner, J.P., Boyer,
W.F., (Eds) Selective serotonin re-uptake inhibitors: Perspectives in Psychiatry, Volume
1, Wiley, New York, pp. 109-117,
Bristow, L.J., O‘Connor, D., Watts, R., Duxon, M.S., Hutson, P.H., (2000). Evidence for
a
accelerated desensitization of 5-HT(2C) receptor following combined treatment with

fluoxetine and the 5-HT1A receptor antagonists, WAY 100,635, in the rat,
ov
Neuropharmacol. 39, 1222–1236.

Brocco, M., Dekeyne, A., Gobert, A., de Nanteuil, G., Millan, M.J., (2000). Actions of the
neurokinin1 antagonists, GR205,171 and CP99,994, in behavioral and neurochemical
models of potential antidepressant activity, Int. J. Neuropsychopharmacol. 3, S376.
Broekkamp, C.L., Rijk, H.W., Joly-Gelouin, D., Lloyd, K.L., (1986). Major tranquillizers can
N
be distinguished from minor tranquillizers on the basis of effects on marble burying and
swim-induced grooming in mice. Eur. J. Pharmacol. 126, 223–229.
18 Trevor R. Norman
Cairncross, K.D., Cox, B., Forster, C., Wren, A. F., (1978). A new model for the detection of
antidepressant drugs: Olfactory bulbectomy in the rat compared with existing models. J.
Pharmacol. Meth. 1, 131-143.
Chau, D.T., Rada, P.V., Kim, K., Kosloff, R.A., Hoebel, B.G., (2011). Fluoxetine alleviates
behavioral depression while decreasing acetylcholine release in the nucleus accumbens
c.
shell, Neuropsychopharmacol. 36, 1729–1737.
Cheeta, S., Tucci, S., Sandhu, J., Williams, A.R., Rupniak, N.M., File, S.E., (2001).
Anxiolytic actions of the substance P (NK1) receptor antagonist L-760735 and the 5-
In
HT1A agonist 8-OH-DPAT in the social interaction test in gerbils, Brain Res. 915, 170–
175.
Cryan, J.F., Markou, A., Lucki, I., (2002). Assessing antidepressant activity in rodents: recent
developments and future needs, Trends. Pharmacol. Sci. 23, 238-245.
Cryan, J.F., Mombereau, C., Vassout, A., (2005). The tail suspension test as a model for
rs
assessing antidepressant activity: Review of pharmacological and genetic studies in mice,
Neurosci. Biobehav. Rev. 29, 571–625.
Cryan, J.F., Sweeney, F.F., (2011). The age of anxiety: role of animal models of anxiolytic
action in drug discovery, Br. J. Pharmacol. 164, 1129–1161.
he
Cryan, J.F., Valentino, R.J., Lucki, I., (2005). Assessing substrates underlying the behavioral
effects of antidepressants using the modified rat forced swimming test, Neurosci.
Biobehav. Rev. 29, 547–569.
Davidson, J.R.T., Connor K.M., (1998). Serotonin and serotonergic drugs in post-traumatic
stress disorder, In: Montgomery, S.A. and den Boer, J.A, (Eds) SSRIs in Depression and
is
Anxiety: Perspectives in Psychiatry, Volume 7, Wiley, New York, pp. 173-188.
De Angelis, L., (1996). Experimental anxiety and antidepressant drugs: the effects of
moclobemide, a selective reversible MAO-A inhibitor, fluoxetine and imipramine in
bl
mice, Naunyn-Schmiedeberg's. Archiv. Pharmacol. 354, 379-383.
de Montigny, C., Aghajanian, G.K., (1978). Long-term antidepressant treatment increases
responsivity of forebrain neurons to serotonin, Science, 202, 1303-1306.
De Vry, J., Maurel, S., Schreiber, R., Beun, R., Jentzsch, K.R., (1999). Comparison of
Pu
hypericum extracts with imipramine and fluoxetine in animal models of depression and
alcoholism Eur. Neuropsychopharmacol. 9, 461–468.
Detke, M.J., Johnson, J., Lucki, I., (1997). Acute and chronic antidepressant drug treatment in
the rat forced swimming test model of depression, Expt. Clin. Psychopharmacol. 5,107–
112.
Dulawa, S.C., Hen, R., (2005). Recent advances in animal models of chronic antidepressant
effects: The novelty-induced hypophagia test, Neurosci. Biobehav. Rev. 29, 771–783.
a
Dulawa, S.C., Holick, K.A., Gundersen, B., Hen, R., (2004). Effects of chronic fluoxetine in
animal models of anxiety and depression. Neuropsychopharmacol. 29, 1321–1330.
ov
Durand, M., Berton, O., Aguerre, S., Edno, L., Combourien, I., Mormede, B., Chaouloff, F.,
(1999). Effects of repeated fluoxetine on anxiety related behaviours, central serotonergic
system, and the corticotropic axis in SHR and WKY rats, Neuropharmacol. 38, 893–907.
Durcan, M.J., Lister, R.G., Eckardt, M.J.,Linnoila, M., (1988). Behavioral interactions of
fluoxetine and other 5-hydroxytryptamine uptake inhibitors with ethanol in tests of
N
anxiety, locomotion and exploration, Psychopharmacol. 96, 528–533.

Dygalo, N., Shishkina, G., Kalinina, T., Yudina, A., Ovchinnikova, E., (2006). Effect of
repeated treatment with fluoxetine on tryptophan hydroxylase-2 gene expression in the
rat brainstem, Pharmacol. Biochem. Behav. 85, 220-227.
File S.E., Wardill A.G., (1975). The reliability of the hole-board apparatus,
Psychopharmacol. 44, 47–51.
c.
File, S.E., (1980). The use of social interaction as a method of detecting anxiolytic activity of
chlordiazepoxide-like drugs, J. Neurosci. Meth. 2, 219–238.
File, S.E., Ouagazzi, A-E., Gonzalez, L.E., Overstreet, D.H., (1999). Chronic fluoxetine in
In
tests of anxiety in rat lines selectively bred for differential 5-HT1A receptor function,
Pharmacol. Biochem. Behav. 62, 695–701.
Freitas, A.E., Machado, D.G., Budni, J., Neis, V.B., Balen, G.O., Lopes, M.W., de Souza,
L.F., Dafre, A.L., Leal, R.B., Rodrigues, A.L.S., (2013). Fluoxetine modulates
hippocampal cell signalling pathways implicated in neuroplasticity in olfactory
rs
bulbectomized mice, Behav. Brain. Res. 237, 176– 184.
Fuller, R.W., Perry, K.W., Molloy, B.B., (1975). Effect of 3-(p-trifluoromethylphenoxy)-N,
N-methyl-3-phenylpropylamine on the depletion of brain serotonin by 4-
chloroamphetamine, J. Pharm. Exp. Ther. 193, 793–803.
he
Gomes, K.S., de Carvalho-Netto, E.F., Monte, K.C.D., Acco, B., Nogueira, P.J., Nunes de-
Souza, R.L., (2009). Contrasting effects of acute and chronic treatment with imipramine
and fluoxetine on inhibitory avoidance and escape responses in mice exposed to the
elevated T-maze. Brain. Res. Bull. 78, 323-327.
Graeff, F.G., Netto, C.F., Zangrossi, H., (1998). The elevated T-maze as an experimental
is
model of anxiety, Neurosci. Biobehav. Rev. 23,237-246.
Griebel, G., Cohen, C., Perrault, G., Sanger, D.J., (1999). Behavioral effects of acute and
chronic fluoxetine in Wistar-Kyoto rats, Physiol. Behav. 67, 315–320.
bl
Griebel, G., Rodgers, R.J., Perrault, G., Sanger, D.J., (1997). Risk assessment behaviour:
evaluation of utility in the study of 5-HT-related drugs in the rat elevated plus-maze test.
Pharmacol. Biochem. Behav. 57, 817–827.
Grippo, A.J., Beltz, T.G., Weiss R.M., Johnson, A.K., (2006). The effects of chronic
Pu
fluoxetine treatment on chronic mild stress-Induced cardiovascular changes and

anhedonia, Biol. Psychiat. 59, 309 –316.
Handley, S.L., McBlane, J.W., (1992). Opposite effects of fluoxetine in two animal models of
anxiety, Br. J. Pharmacol. 107(S2), 446P.
Hansen, L., (2001). A critical review of akathisia, and its possible association with suicidal
behaviour, Hum. Psychopharmacol. 16, 495-505.
Harvey, A.T., Preskorn, S.H., (2001). Fluoxetine pharmacokinetics and effect on CYP2C19
a
in young and elderly volunteers, J. Clin. Psychopharmacol. 21, 161-166.

Hoa, Y.J., Eichendorff, J., Rainer, K., Schwarting, K., (2002). Individual response profiles of
ov
male Wistar rats in animal models for anxiety and depression, Behav. Brain. Res. 136, 1-
12.
Horden, A., (1965). The antidepressant drugs, New. Engl. J. Med. 22, 1159-1169.
Ismail, M.O., Dar, A., (2009). Comparison of the efficacy of fluoxetine, phenelzine and
moclobemide in rodents using animal models of depression, Pak. J. Pharmacol. 26, 19-
N
23.
Janowsky, D.S., Overstreet, D.H., Nurnberger, J.I., (1994). Is cholinergic sensitivity a genetic
marker for the affective disorders? Amer. J. Med. Gen. 54, 335–344.
20 Trevor R. Norman
Jones, N., King, S.M., Duxon, M.S., (2002). Further evidence for the predictive validity of
the unstable elevated exposed plus-maze, a behavioural model of extreme anxiety in rats:
Differential effects of fluoxetine and chlordiazepoxide, Behav. Pharmacol. 13, 525–535.
Keller, M., Montgomery, S., Ball, W., Morrison, M., Snavely, D., Lui, G., Hargreaves, R.,
Hietala, J., Lines, C., Beebe, K., Reines, S., (2006). Lack of efficacy of the substance P
c.
(neurokinin 1 receptor) antagonist aprepitant in the treatment of major depressive
disorder, Biol. Psychiat. 59, 216-223.
Kshama, D., Hrishikeshavan, H.J., Shanbhogue, R., (1990). Modulation of baseline behavior
In
in rats by putative serotonergic agents in three etho-experimental paradigms, Behav.
Neural. Biol. 54, 234– 253.
Kulkarni, S.K., Dhir, A., (2007). Effect of various classes of antidepressants in behavioural
paradigms of despair Prog. Neuro-Psychopharmacol. Biol. Psychiat. 31, 1248–1254.
Kurt, M., Arik, A.C., Celik, S., (2000). The effects of sertraline and fluoxetine on anxiety in
rs
the elevated plus-maze test in mice, J. Basic. Clin. Physiol. Pharmacol. 11, 173–180.
Leo, R.J., (1996). Movement disorders associated with the serotonin selective reuptake
inhibitors. J. Clin. Psychiat. 57, 449-454.
Lewis, A.J., (1980). Problems presented by the ambiguous word ‗anxiety‘ as used in
he
psychopathology, In: Handbook of Studies on Anxiety, G.D. Burrows and B.M. Davies
(Eds), Elsevier, Amsterdam, pp 1-15.
Lister, R.G., (1987). The use of a plus-maze to measure anxiety in the mouse,
Psychopharmacol. 92,180–185.
Lydiard, R.B., (1998). SSRIs in social phobia with or without major depression, In:
is
Montgomery, S.A. and den Boer, J.A, (Eds) SSRIs in Depression and Anxiety:
Perspectives in Psychiatry, Volume 7, Wiley, New York, pp. 135-147.
Malberg, J., Duman, R.S., (2003). Cell proliferation in adult hippocampus is decreased by
bl
inescapable stress: Reversal by fluoxetine treatment. Neuropsychopharmacol. 28, 1562–
1571.
Mar, A., Spreekmeester, E., Rochford, J., Fluoxetine-induced increases in open-field
habituation in the olfactory bulbectomized rat depend on test aversiveness but not on
Pu
anxiety, Pharmacol. Biochem. Behav. 73, 703–712.

Mayorga, A.J., Dalvi, A., Page, M.E., Zimov-Levinson, S., Hen, R., Lucki, I., (2001).
Antidepressant-like behavioural effects in 5-hydroxytryptamine (1A) and 5-
hydroxytryptamine (1B) receptor mutant mice. J. Pharm. Exp. Ther. 298, 1101–1107.
Mishra, R., Janowsky, A., Sulser, F., (1979). Subsensitivity of the norepinephrine receptor
coupled adenylate cyclase system in the brain. Effects of nisoxetine versus fluoxetine,
Eur. J. Pharmacol. 60, 379-382.
a
Mutlu, O., Gumuslu, E., Ulak, G., Celikyurt, I.K., Kokturk, S., Kır, H.M., Akar, F., Erden, F.,
(2012). Effects of fluoxetine, tianeptine and olanzapine on unpredictable chronic mild
ov
stress-induced depression-like behaviour in mice, Life. Sci. 91, 1252–1262.

Nakamura, K., Kurasawa, M., (2001). Anxiolytic effects of aniracetam in three different
mouse models of anxiety and the underlying mechanism, Eur. J. Pharm. 420, 33–43.
Nibuya, M., Morinobu, S., Duman, R.S., (1995). Regulation of BDNF and trkB mRNA in rat
brain by chronic electroconvulsive seizure and antidepressant drug treatments, J.
N
Neurosci. 15, 7539 –7547.

Norman, T.R., (2010). Dysfunctional circadian rhythms and mood disorders: Opportunities
for novel therapeutic approaches. In: Depression: From Psychopathology to
Pharmacotherapy, J.F.Cryan and B.E.Leonard (eds), Modern Trends in

Pharmacopychiatry, Basel, Krager, pp 32-52.
Norman, T.R., Cranston, I., Irons, J.A., Gabriel, C., Dekeyne, A., Millan, M.J., Mocaër, E.,
(2012). Agomelatine suppresses locomotor hyperactivity in olfactory bulbectomised rats:
A comparison to melatonin and to the 5-HT2C antagonist, S32006, Eur. J. Pharmacol.
c.
674, 27-32.
Overstreet, D. H., Russell, R. W., Helps, S. C., Messenger, M., (1979). Selective breeding for
sensitivity to the anticholinesterase, DFP. Psychopharmacol. 65, 15–20.
In
Overstreet, D.H., (2002). Behavioural characteristics of rat lines selected for differential
hypothermic responses to cholinergic or serotonergic agonists, Behav. Gen. 32, 335-348.
Palanza, P., (2001). Animal models of anxiety and depression: how are females different?
Neurosci. Biobehav. Rev. 25, 219-233.
Pellow, S., Chopin, P., File, S.E., Briley, M., (1985). Validation of open: closed arm entries in
rs
an elevated plus-maze as a measure of anxiety in the rat, J. Neurosci. Meth. 14, 149–167.
Perrault, G.H., Morel, I.E., Zivkovic, B., Sanger, D.J., (1992). Activity of litoxetine and other
serotonin uptake inhibitors in the tail suspension test in mice, Pharmacol. Biochem.
Behav. 42, 45-47.
he
Pieper, D.R., Lobocki, C.A., (1991). Olfactory bulbectomy lengthens circadian period of
locomotor activity in golden hamsters, Amer. J. Physiol. 30, R973-R978.
Piras, G., Piludu, M.A., Giorgi, O., Corda, M.G., (2014).Effects of chronic antidepressant
treatments in a putative genetic model of vulnerability (Roman low-avoidance rats) and
resistance (Roman high-avoidance rats) to stress-induced depression, Psychopharmacol.
is
231, 43–53.
Poltronieri, S.C., Zangrossi, H., de Barros Viana, M., (2003). Anti-panic-like effect of
serotonin reuptake inhibitors in the elevated T-maze, Behav. Brain. Res. 147, 185–192.
bl
Porsolt, R.D., Bertin, A., Jalfre, M., (1977). Behavioral despair in mice: a primary screening
test for antidepressants. Archiv. Int. Pharmacodyn. Ther. 229, 327–336.
Possidente, B., Lumia, A.R., McGinnis, M.Y., Rapp, M., McEldowney, S., (1996). Effects of
fluoxetine and olfactory bulbectomy on mouse circadian activity rhythms, Brain. Res.
Pu
713, 108-113.
Possidente, B., Lumia, A.R., McGinnis, M.Y., Teicher, M.H., de Lemos, E., Sterner, L.,
Deros, L., (1990). Olfactory bulb control of circadian activity rhythm in mice, Brain. Res.
513, 325-328.
Ramos, A., Berton, O., Mormède, P., Chaouloff, F., (1997). A multiple-test study of anxiety-
related behaviours in six inbred rat strains, Behav. Brain. Res. 85, 57-69.
Reid, I., Forbes, N., Stewart, C., Matthews, K., (1997). Chronic mild stress and depressive
a
disorder: a useful new model? Psychopharmacol. 134, 365–367.

Robert, G., Drapier, D., Bentué-Ferrerb, D., Renault, A., Reymann, J-M., (2011). Acute and
ov
chronic anxiogenic-like response to fluoxetine in rats in the elevated plus-maze:

Modulation by stressful handling, Behav. Brain. Res. 220, 344–348.
Roche, M., Harkin, A., Kelly, J.P., (2007). Chronic fluoxetine treatment attenuates stressor-
induced changes in temperature, heart rate, and neuronal activation in the olfactory
bulbectomized rat, Neuropsychopharmacol. 32, 1312–1320 (2007).
N
Rodgers, R.J., Cao, B-J., Dalvi, A., Holmes, A., (1997). Animal models of anxiety: an
ethological perspective. Braz. J. Med. Biol. Res. 30, 289–304.
22 Trevor R. Norman
Rodriguez-Filho, R., Takahashi, R.N., (1999). Antinociceptive effects induced by

desipramine and fluoxetine are dissociated from their antidepressant or anxiolytic action
in mice, Int. J. Neuropsychopharmacol. 2, 263–269.
Rong, H., Wang, G., Liu, T., Wang, H., Wan, Q., Weng, S., (2010). Chronic mild stress
induces fluoxetine-reversible decreases in hippocampal and cerebrospinal fluid levels of
c.
the neurotrophic factor S100B and its specific receptor, Int. J. Mol. Sci. 11, 5310-5322.
Sanchez, C., Meier, E., (1997). Behavioural profiles of SSRIs in animal models of depression,
anxiety and aggression. Are they all alike? Psychopharmacol. 129, 197–205.
In
Schroeter, M.L., Abdul-Khaliq, H., Diefenbacher, A., Blasig, I. E., (2002). S100B is
increased in mood disorders and may be reduced by antidepressive treatment, Neurorep.
13, 1675-1678.
Shephard, R.A., Broadhurst, P.L., (1982). Hyponeophagia and arousal in rats: effects of
diazepam, 5-methoxy-N,N-dimethyltryptamine, d-amphetamine and food deprivation,
rs
Psychopharmacol. 78,368–372.
Silva, M.T., Alves, C.R., Santarem, E.M., (1999). Anxiogenic-like effect of acute and chronic
fluoxetine on rats tested on the elevated plus-maze, Braz. J. Med. Biol. Res. 32, 333–339.
Silva, R.C.B., Brandao, M.L., (2000). Acute and chronic effects of gepirone and fluoxetine in
he
rats tested in the elevated plus-maze: an ethological analysis. Pharmacol. Biochem.
Behav. 65, 209–216.
Song, C., Leonard, B.E., (2005). The olfactory bulbectomised rat as a model of depression.
Steru, L., Chermat, R., Thierry, B., Simon, P., (1985). The tail suspension test: a new method
is
for screening antidepressants in mice, Psychopharmacol. 85, 367–70.
Teste, J.F., Pelsy-Johann, I., Decelle, T., Boulu, R.G., (1993). Anti-immobility activity of
different antidepressant drugs using the tail suspension test in normal or reserpinized
bl
mice, Fund. Clin. Pharmacol. 7, 219–226.
To, C.T., Anheuer, Z.E., Bagdy, G., (1999). Effects of acute and chronic fluoxetine treatment
of CRH-induced anxiety, Neurorep. 10, 553–555.
To, C.T., Bagdy, G., (1999). Anxiogenic effect of central CCK administration is attenuated
Pu
by chronic fluoxetine or ipsapirone treatment, Neuropharmacol. 38, 279–282.

Ukai, M., Maeda, H., Nanya, Y., Kameyama, T., Matsuno, K., (1998). Beneficial effects of
acute and repeated administrations of sigma receptor agonists on behavioural despair in
mice exposed to tail suspension, Pharmacol. Biochem. Behav. 61, 247–252.
Wernicke, J.F., Dunlop, S.R., Dornseif, B.E., Zerbe, R.L., (1987). Fixed-dose fluoxetine
therapy for depression. Psychopharmacol. Bull. 23, 164-168.
Willner, P., (1997). Validity, reliability and utility of the chronic mild stress model of
a
depression: A 10-year review and evaluation. Psychopharmacol. 134, 319 –329.

Willner, P., Muscat, R., Papp, M., (1992). Chronic mild stress-induced anhedonia: A realistic
ov
animal model of depression. Neurosci. Biobehav. Rev. 16, 525–534.

Wong D.T., Bymaster, F.P., Horng, J.S., Molloy, B.B., (1975). A new selective inhibitor for
uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluorometimylphenoxy)-N-
methyl-3-phenylpropylamine. J. Pharm. Exp. Ther. 193, 804-811.
Wong, D.T., Bymaster, F.P., (1980). Sub-sensitivity of serotonin receptors after long-term
N
treatment of rats with fluoxetine. Res. Comm. Chem. Path. Pharmacol. 32, 41-51.
Wong, D.T., Bymaster, F.P., and Engleman, E.A., (1995). Prozac (Fluoxetine, Lilly 110140),
the first selective serotonin uptake inhibitor and an antidepressant drug: Twenty years
since its first publication, Life. Sci. 57, 411-441.
Wong, D.T., Bymaster, F.P., Reid, L.R., Fuller, R.W., Perry, K.W., (1985). Inhibition of
serotonin
c.
Wong, D.T., Bymaster, F.P., Reid, L.R., Mayle, D.A., Krushinski, J.H., Robertson, D.W.,
(1993). Norfluoxetine enantiomers as inhibitors of serotonin uptake in rat brain,
Neuropsychopharmacol. 8: 337-344.
In
Wong, D.T., Horng, J.S., Bymaster, F.P., Hauser, K.L., Molloy, B.B., (1974). A selective
inhibitor, Life. Sci. 15, 471-479.
Wong, D.T., Perry, K.W., Bymaster, F.P., (2005). The discovery of fluoxetine hydrochloride
(Prozac), Nature. Rev. Drug. Dis. 4, 764-774.
Wong, D.T., Threlkeld. P.G., Robertson, D.W., (1991). Affinities of fluoxetine, its
rs
enantiomers, and other inhibitors of serotonin uptake for subtypes of serotonin receptors,
Neuropsychopharmacol. 5,43–47.
Yu, Z.F., Kong, L.D., Chen, Y., (2002). Antidepressant activity of aqueous extracts of
curcuma longa in mice. J. Ethnopharmacol. 83, 161–165.
he
Conflict of Interest Statement
In the past five years the author has been or is currently a member of advisory boards for
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Eli Lilly and Lundbeck. He has been in the receipt of travel grants, research funds or has been
a paid speaker for Eli Lilly, Lundbeck, Servier, Astra Zeneca, Wyeth (Pfizer), Organon
(Merck Sharpe Dohme), Bristol Myers Squibb.
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Chapter 2
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The Neurosteroidogenic Action of
Fluoxetine Unveils the Mechanism
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for the Anxiolytic Property of SSRIs
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Graziano Pinna*
The Psychiatric Institute, Department of Psychiatry, College of Medicine,
University of Illinois at Chicago, US
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Abstract
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In the past decade, clinical studies have suggested that the pharmacological action of
SSRIs, such as fluoxetine may include the ability of these drugs to normalize decreased
brain levels of neurosteroids in patients with depression and post-traumatic stress
disorder (PTSD), in particular the progesterone derivative allopregnanolone, which
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potently and allosterically modulates the action of GABA at GABAA receptors. Thus, by
this mechanism these drugs may induce anxiolytic effects and have therefore become the
drug of choice to treat anxiety disorders and PTSD avoiding the benzodiazepine-like side
effects. Furthermore, neurosteroidogenic doses of fluoxetine and congeners represent a
suitable treatment for a number of other psychiatric conditions, including premenstrual
dysphoria and catamenial epilepsy, which are the result of a rapid drop of progesterone
and its metabolite levels in the late luteal phase.
Using the socially isolated mouse as an animal model of PTSD, preclinical studies
a
have demonstrated that fluoxetine and norfluoxetine ameliorate anxiety-like behavior,

fear responses, and aggressive behavior expressed by such mice through increasing
corticolimbic levels of allopregnanolone. This is a novel and more selective mechanism
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than 5-HT reuptake inhibition, which for several decades has been thought to be the main
molecular mechanism for the therapeutic action of SSRIs. Thus, fluoxetine acting as a
selective brain steroidogenic stimulant (SBSS) at low non-serotonergic doses may
stimulate allopregnanolone biosynthesis in corticolimbic neurons and thereby offer a
novel non-traditional mechanism by which this drug exert potent non-sedating
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pharmacological effects for the treatment of anxiety, depression, and PTSD.
*The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor Street,
Chicago IL 60612; Tel.: 312-355-1464; Email: gpinna@psych.uic.edu; graziano_pinna@yahoo.com.
26 Graziano Pinna
Keywords: allopregnanolone; 5α-reductase type I; selective brain steroidogenic stimulants

(SBSSs); aggressive behavior; fear responses; GABAA receptors; social isolation;
anxiety, PTSD
c.
Introduction
The downregulation of neurosteroid biosynthesis has been implicated as a possible
In
contributor to the development of anxiety and depressive disorders (Uzunova et al., 1998;
Romeo et al., 1998; Rasmusson et al., 2006; Pinna et al., 2006a; Longone et al., 2008).
Clinical studies demonstrated that decreases in the serum, plasma, and CSF content of
neuroactive steroids, particularly the GABAA receptor-active progesterone derivative,
allopregnanolone, are associated with a number of psychiatric conditions, such as depression,
rs
anxiety disorders, PTSD, premenstrual dysphoria, and impulsive aggression (Rasmusson et
al., 2006; Nappi et al., 2001; Ströhle et al., 2002; Rapkin et al., 1997; Marx et al., 2009;
Pearlstein, 2010; Amin et al., 2007; Bäckström et al., 2003; Bloch et al., 2000; Uzunova et al.,
1998; Romeo et al., 1998). These clinical findings suggest that downregulation of
he
allopregnanolone levels could be a cause for these psychiatric disorders, and have stimulated
investigation into new neurosteroidogenic ligands to elevate allopregnanolone biosynthesis as
future treatment for anxiety, PTSD, and depression (Serra et al., 1999; Rupprecht et al., 2010;
Schüle et al., 2011; Costa et al., 2011).
We measured allopregnanolone levels in the cerebrospinal fluid (CSF) and brain of
is
patients with PTSD and depression (Rassmusson et al., 2006; Agis-Balboa et al., 2014). In
depressed patients, the concentration of allopregnanolone in the CSF was approximately half
of that measured in the CSF of nonpsychiatric patients (Uzunova et al., 1998). To test the
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hypothesis that this decrease in the CSF allopregnanolone levels of depressed patients reflects
a decrease of brain allopregnanolone content, we compared the expression of 5α-reductase
type I mRNA in the prefrontal-cortex (area BA9) between depressed patients and age-
matched non-psychiatric subjects (Agis-Balboa et al., 2014). In depressed patients, the
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cortical but not cerebellum expression levels of 5α-reductase type I mRNA was decreased to
about 50% of the levels measured in non-psychiatric comparison subjects (Agis-Balboa et al.,
2014).
Furthermore, CSF allopregnanolone levels in premenopausal women with PTSD were
40% of levels seen in healthy comparison subjects and were inversely correlated with PTSD
re-experiencing and comorbid depressive symptoms (Rasmusson et al., 2006). In fact, CSF
a
allopregnanolone levels were lowest in those patients with PTSD and comorbid depression. In
addition, the ratio of allopregnanolone to its steroid precursor, 5α-dihydroprogesterone, was
decreased among the PTSD patients, suggesting the presence of a block in allopregnanolone
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synthesis (Rasmusson et al., 2006; reviewed in Pinna et al., 2006a).

Taken together, these data suggest that a deficit of GABAergic neurotransmission, likely
caused by a downregulation of brain allopregnanolone biosynthesis, must be among the
molecular mechanisms considered in the etiology of major depression and PTSD.
Remarkably, treatment with fluoxetine and fluvoxamine normalized the CSF and cortical
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allopregnanolone levels of the depressed patients (Uzunova et al., 1998). Moreover, there was
a statistically significant correlation between the improvement in depressive symptoms and
The Neurosteroidogenic Action of Fluoxetine Unveils the Mechanism … 27
the increase of CSF allopregnanolone elicited by fluoxetine or fluvoxamine. Similar results

were reported when allopregnanolone or levels of 5α-tetrahydrodeoxycorticosterone, another
positive modulator of GABAA receptor function, were measured in the plasma of depressed
patients treated with SSRIs (van Broekhoven et al., 2003). In support of these data, a pilot
study conducted in our laboratory, has recently demonstrated that various selective serotonin
c.
reuptake inhibitor (SSRI) antidepressants elevated the frontocortical levels of
allopregnanolone in depressed patients (Agis-Balboa et al., 2014).
Given the seminal discovery that fluoxetine and its congeners increase the content of
In
allopregnanolone in the CSF and prefrontal cortex of depressed patients (Uzunova et al.,
1998) and in neurons of various rodent brain areas (Uzunov et al., 1996), we hypothesized
that normalization of brain allopregnanolone levels may underlie the therapeutic effects of
SSRIs in depression and PTSD. This hypothesis has since been supported by preclinical
research using the socially isolated mouse as an animal model of behavioral deficits that
rs
resemble symptoms of human anxiety disorders and PTSD (Pinna et al., 2003; 2004;
reviewed in Pinna et al., 2006a; 2009; and in Pinna, 2010) and in studies using the
bulbectomized rat as a model of depression (Uzunova et al., 2004; 2006). Corticolimbic
allopregnanolone levels become markedly decreased in association with the development of
he
anxiety-like behaviors, resistance to sedation, and heightened aggression in the socially
isolated mouse (Pinna et al., 2006b; 2003; Nin Schuler et al., 2011). Administration of
fluoxetine and its congeners upregulate the downregulation of corticolimbic allopregnanolone
levels and abolish the emergence of such behaviors. Further, the behavioral effects of
fluoxetine were found to be unrelated to the serotonin reuptake blocking activity for which
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this class of psychotropic drugs was originally developed (Pinna et al., 2003; 2004).
These data are thus in support of a novel mechanism whereby fluoxetine acting as a
selective brain steroidogenic stimulant (SBSS) increases brain corticolimbic
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allopregnanolone levels and improves behavioral deficits characteristic of PTSD, anxiety, and
depression.
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Physiological Role of Neurosteroids

at GABAA Receptors
Allopregnanolone is synthesized in a region-specific way in primary GABAergic and
glutamatergic neurons, including pyramidal neurons, granular cells, reticulo-thalamic
neurons, medium spiny neurons of the striatum and nucleus accumbens, and Purkinje cells
a
(Agis-Balboa et al., 2006; 2007). Allopregnanolone synthesized in glutamatergic cortical or

hippocampal pyramidal neurons or in granular cells of the dentate gyrus may be secreted in
an autocrine fashion to act locally by binding post-synaptic or extra-synaptic GABAA
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receptors located on the same dendrites or cell bodies of the cortical or hippocampal
pyramidal neuron in which it was produced (Figure 1) (Agis-Balboa et al., 2006).
Alternatively, allopregnanolone might diffuse laterally into synaptosome membranes of the
cell bodies or dendritic arborization of glutamatergic neurons to attain intracellular access to
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specific neurosteroid binding sites of GABAA receptors (Figure 1) (Agis-Balboa et al., 2006;
Akk et al., 2005).
28 Graziano Pinna
c.
In
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he
is
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Figure 1. Therapeutic strategies to increase neurosteroidogenesis and improve PTSD and anxiety by
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enhancing GABAergic neurotransmission. Fluoxetine (FLX)‘s improvement of PTSD and anxiety

symptoms may be achieved by increasing corticolimbic allopregnanolone levels. TSPO ligands induce
an upstream regulation of neurosteroidogenesis by gating the entry of cholesterol into the inner
mitochondrial membranes of glial cells, and its conversion into pregnenolone. Pregnenolone can then
be taken up by pyramidal neurons (Costa and Guidotti, 1991) where a cascade of enzymatic processes
takes place in the cytosol resulting in the production of allopregnanolone. FLX induces a downstream
activation of neurosteroidogenesis likely by inhibiting at the level of 3α-HSD the reconversion of
allopregnanolone into its precursor, 5-DHP (Fry et al., 2014). Neurosteroidogenesis is not globally
a
expressed in the brain but relies on rate-limiting step enzymes, which guard allopregnanolone
availability and thereby normalize its physiological levels in the required corticolimbic areas (e.g., after
activation of TSPO or after FLX). Allopregnanolone, synthesized in glutamatergic cortical or
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hippocampal pyramidal neurons, may improve PTSD and anxiety symptoms after being secreted in an
autocrine fashion and act locally by binding post-synaptic or extra-synaptic GABAA receptors located
on the same neuron in which it was produced (arrow 1) (Agis-Balboa et al., 2006, 2007).
Allopregnanolone may also diffuse into synaptosome membranes of the cell bodies or dendritic
arborization to attain intracellular access to specific neurosteroid binding sites of GABA A receptors
(arrow 2) (Akk et al., 2005). TSPO, translocate protein (18 kDa); 5α-DHP, 5α-dihydroprogesterone; 5α-
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RI, 5α-reductase type I; 3α-HSD, 3α-hydroxysteroid dehydrogenase; FLX, fluoxetine.

Allopregnanolone is thus synthesized in the brain independently from peripheral sources

(Baulieu et al., 1981; Cheney et al., 1995; Guidotti et al., 2001; Stoffel-Wagner 2001) and
reach physiologically relevant levels that modulate neurotransmission (Puia et al., 1990;
1991; 2003; Pinna et al., 2000; Lambert et al., 2003; 2009; Belelli et al., 2005; Majewska,
1992). Application of allopregnanolone resulted in a potent (nM affinity) positive allosteric
c.
modulation of the action of GABA at GABAA receptors (Puia et al., 1990; 1991; Lambert et
al., 2003; 2009), which result in pharmacological actions, including anticonvulsant,
anxiolytic, antidepressant, and at the highest dosage, sedative-hypnotic actions (D‘Aquila et
In
al., 2010; Nin et al., 2008; Rodriguez-Landa et al., 2009; Kita and Furukawa, 2008; Lonsdale
and Burnham, 2007; Mares et al., 2006; Martin-Garcia and Pallares, 2005; Jain et al., 2005).
The finding that allopregnanolone permits, facilitates, and fine-tunes the efficacy of
direct GABAA receptor activators and positive allosteric modulators of GABA effects at
GABAA receptors substantiates its endogenous physiological relevance (Guidotti et al., 2001;
rs
Puia et al., 2003; Pinna et al., 2000; Matsumoto et al., 1999; 2003). Allopregnanolone
facilitates GABAergic neurotransmission via two binding sites in the GABAA receptor that,
respectively, mediate the potentiation and direct activation effects of allopregnanolone. Direct
GABAA receptor activation is initiated by binding of allopregnanolone at a site formed by
he
interfacial residues between the  and  subunits (Hosie et al., 2006). Binding of
allopregnanolone at the potentiation site located in a cavity within the -subunit results in
marked enhancement of GABAA receptor activation (Hosie et al., 2006). Allopregnanolone
binds with higher affinity at GABAA receptors incorporating δ subunits in combination with
α4 and α6 (Belelli et al., 2002; Belelli and Lambert, 2005; Maguire and Mody, 2009; Mihalek
is
et al., 1999; Stell et al., 2003). The affinity of allopregnanolone for these GABAA receptor
subtypes is in the low nM range (Puia et al., 1990; 1991). Importantly, this GABAA receptor
conformation is benzodiazepine insensitive (Puia et al., 1990; 1991; Vicini, 1991). Thus,
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allopregnanolone or analogs are more advantageous than benzodiazepines because they
improve anxiety, fear, and aggressiveness when benzodiazepines are inactive. These
observations suggest that drugs designed to selectively increase neurosteroidogenesis may
alleviate PTSD and anxiety disorders by facilitating GABAA receptor neurotransmission.
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PTSD-Like Behavioral Deficits Are Modeled in

Mice by Social Isolation
Social isolation in rodents is a potent stressful condition that results in decreased
a
neurosteroid biosynthesis and the onset of several behavioral deficits that resemble behavioral
abnormalities observed in patients with anxiety/PTSD/depressive disorders (Schüle et al.,
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2011; Pinna, 2010; Matsumoto et al., 1999). Our laboratory and others have indeed
determined that exposure of mice or rats to protracted social isolation stress for 4-8 weeks
induces a decrease in allopregnanolone levels in several corticolimbic structures as a result of
a downregulation of the mRNA and protein expression of 5α-reductase type I (Pinna et al.,
2003; Matsumoto et al., 1999; Serra et al., 2000; Bortolato et al., 2011; reviewed in Pinna
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2010; Matsumoto et al., 2007). The largest decrease of 5α-reductase was found in the
amygdala and hippocampus, followed by the olfactory bulb and the frontal cortex (Pibiri et
al., 2008). The expression of 5α-reductase failed to change in the cerebellum and striatum
30 Graziano Pinna
(Pibiri et al., 2008). 5α-reductase type I is specifically decreased in cortical pyramidal

neurons of layers V-VI, in hippocampal CA3 pyramidal neurons and glutamatergic granular
cells of the dentate gyrus, and in the pyramidal-like neurons of the basolateral amygdala
(Agis-Balboa et al., 2007).
Behaviorally, fear responses and aggressive behavior increased during the four weeks of
c.
social isolation to reach a plateau between four and eight weeks of isolation (Pibiri et al.,
2008; Pinna et al., 2008). Socially isolated mice also exhibited impaired and incomplete fear
extinction (Pibiri et al., 2008). We had previously observed that socially isolated mice
In
exhibited higher levels of anxiety-like behavior, determined by the elevated plus maze and in
the open field (Pinna et al., 2006a; Nin Schuler et al., 2011).
Pharmacological experiments have demonstrated that corticolimbic allopregnanolone
levels play a pivotal role in the regulation of contextual fear responses and aggression.
Treatment with allopregnanolone dose-dependently decreased aggression in a manner that
rs
correlated with an increase in corticolimbic allopregnanolone content (Pinna et al., 2003).
Furthermore, allopregnanolone also normalized the exaggerated contextual fear responses and
anxiety of socially isolated mice (Pibiri et al., 2008). Administration of the potent 5α-
reductase competitive inhibitor SKF 105,111 to normal group-housed mice (Cheney et al.,
he
1995; Pinna et al., 2000; 2008) rapidly (~1 h) decreased levels of allopregnanolone in the
olfactory bulb, frontal cortex, hippocampus, and amygdala by 80-90% (Pinna et al., 2008;
Pibiri et al., 2008) in association with a dose-dependent increase of conditioned contextual
fear responses (Pibiri et al., 2008). The effects of SKF 105,111 on conditioned contextual fear
responses were reversed by administering allopregnanolone doses that normalized
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hippocampus allopregnanolone levels (Pibiri et al., 2008). These results were confirmed by
many other investigators who have observed that allopregnanolone elicits anxiolytic and
antidepressant effects (Nin Schuler et al., 2011; Nin et al., 2008; Rodgers and Johnson, 1998;
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Frye and Rhodes, 2006; Bitran et al., 1991; Wieland et al., 1991; Deo et al., 2010; Engin and
Treit, 2007; Frye et al., 2009).
The expression of GABAA receptor subunits in emotional brain circuits has been
associated with different physiological responses as well as the mediation of the
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pharmacological effects of several GABAA receptor ligands that target specific subsets of
GABAA receptor subunits (Rudolph and Möhler, 2004; Rudolph et al., 1999). Socially
isolated mice express changes in the mRNA and protein expression of several GABAA
receptor subunits in the frontal cortex and hippocampus (Pinna et al., 2006b). The mRNA for
, and γ2 GABAA receptor subunit subtypes were reduced by about 50%, while the
mRNAs encoding α4 and α5 subunits were increased by about 130% compared to levels
measured in group-housed mice (Pinna et al., 2006b). These results were confirmed in
a
experiments in which α1 and α5 protein levels were assessed by Western blot in synaptic
membrane preparations in the frontal cortex and hippocampus (Pinna et al., 2006b).
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We further studied whether the decline of GABAA receptor 1 subunit expression in the
frontal cortex during social isolation is layer- or cell-specific (Pinna et al., 2006b). We
determined the expression levels of 1 subunit mRNA in frontal cortex layer I, which is
enriched in GABAergic interneurons and neuropil formed by the convergence of afferent
thalamic fibers with apical dendrites of pyramidal neurons. We found that 1 mRNA levels
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were decreased by 50% in layer I neuropil, whereas the expression of 1 subunit mRNA in
the pyramidal neurons of layer V was unchanged by social isolation (Pinna et al., 2006b).
The altered GABAA receptor subunit composition in socially isolated mice resulted in an
altered pharmacological responses to a range of GABAA receptor ligands. Socially isolated
mice showed a sustained resistance to the sedative and anxiolytic properties of diazepam and
zolpidem, positive allosteric GABAA receptor modulators that bind with high affinity to α1,
α2, α3, or α5 subunit-containing GABAA receptors (diazepam) and to subunit-containing
c.
GABAA receptors (zolpidem) (Pinna et al., 2006). Hence, our results suggests that the
resistance of socially isolated mice to the anxiolytic activity of diazepam may result from the
formation of benzodiazepine-insensitive GABAA receptors in neuronal circuits that play a
In
pivotal role in regulating anxiety behaviors (Nin Schuler et al., 2011; Pinna et al., 2006b).
Fluoxetine Acting As a Selective Brain

Steroidogenic Stimulant (SBSS), Corrects
rs
the Behavioral Deficits in Socially Isolated Mice
In socially isolated mice, aggressive behavior toward a same-sex intruder is correlated
he
with the extent of allopregnanolone level downregulation in several corticolimbic structures
(Pinna et al., 2003). When the corticolimbic levels of allopregnanolone in socially isolated
mice were normalized by a systemic administration with allopregnanolone, there was a dose-
dependent decrease of aggressive behavior (Pinna et al., 2003; 2006a). Allopregnanolone‘s
antiaggressive effects were confirmed by experiment in which allopregnanolone was directly
is
infused into the basolateral amygdala, which increased the levels of allopregnanolone in the
basolateral amygdala and hippocampus (Nelson and Pinna, 2011). Based on these results we
concluded that in socially isolated mice the decrease of corticolimbic allopregnanolone levels
is responsible for the expression of aggression.
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The groundbreaking discovery of Uzunov and collaborators in 1996 that SSRI
antidepressants, including fluoxetine increase the brain levels of allopregnanolone opened a
new exciting field of neuropsychopharmacology. This first observation in rodents was
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followed by the clinical finding in depressed patients by Uzunova and collaborators that
fluoxetine and fluvoxamine pharmacological action could result in an elevation of CSF
allopregnanolone levels. Fluoxetine-induced allopregnanolone biosynthesis correlated with
improved depressive symptoms (Uzunova et al., 1998). Since then, these results have been
observed in plasma and serum of depressed patients treated with fluoxetine and other SSRIs
(Romeo et al., 1998; Eser et al., 2006; Longone et al., 2008; 2011).
In a recent study, in a cohort of SSRI-treated and non-treated depressed patients, we
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observed a significant increase of prefrontal cortex BA9 allopregnanolone levels in absence

of changes of progesterone and 5α-dihydroprogesterone levels (Agis-Balboa et al., 2014).
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Hence, these first findings obtained in a pilot study in the human brain encourage further
investigation on a larger cohort of treated and non-treated depressed patients. Our results in
the human brain are in line with previous findings that have observed and increase of
allopregnanolone levels in CSF and blood of treated depressed patients. Also, this study
further suggests that the mechanism underlying unipolar major depression may involve a
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brain deacrease of allopregnanolone biosynthesis, which may trigger a downregulation of

GABAergic neurotransmission.
32 Graziano Pinna
In line with these clinical studies, several other SSRI antidepressants have been shown to
potently increase the levels of allopregnanolone in the brain of rodents. Paroxetine and
fluoxetine increased the levels of allopregnanolone in rats and mice without affecting the
brain levels of the allopregnanolone precursors, pregnenolone and progesterone (Uzunov et
al., 1996). When a racemic mixture of the R- and S-isomers of fluoxetine was administered in
c.
mice or rats, the corticolimbic allopregnanolone levels were elevated at the same time that a
normalization of the righting reflex loss induced by pentobarbital in mice was observed
(Pinna et al., 2003; 2004; 2009). Remarkably, these fluoxetine doses failed to affect the
In
righting reflex response and allopregnanolone levels in group housed mice (Pinna et al., 2003;
2004). Furthermore, serotonin synthesis inhibition by a treatment with p-chlorophenylalanine
failed to block the behavioral effects of fluoxetine, which further suggested that the action of
fluoxetine was independent from the ability of this drug to affect serotonin mechanisms. On
the other hand, these findings further supported a role for corticolimbic allopregnanolone
rs
levels as the primary mechanism of action of fluoxetine (Matsumoto et al., 1999).
In another study we hypothesized that fluoxetine and congeners could ameliorate the
behavioral deficits of socially isolated mice by upregulating corticolimbic allopregnanolone
levels rather than by inhibiting serotonin reuptake. These experiments were performed using
he
the R- and S-stereoisomers of fluoxetine and norfluoxetine as pharmacological tools. We
expected that these drugs would stereospecifically upregulate corticolimbic allopregnanolone
levels but have no stereoselectivity with regard to inhibition of 5-HT reuptake. We also
hypothesized that doses of fluoxetine and norfluoxetine stereoisomers that increase
corticolimbic allopregnanolone content might differ from those that inhibit 5-HT reuptake.
is
Remarkably, fluoxetine dose-dependently and stereospecifically normalized the duration of
pentobarbital-induced sedation and reduced aggressive behavior, fear responses, and anxiety-
like behavior at the same submicromolar doses that normalized the downregulation of brain
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allopregnanolone content in socially isolated mice (Pinna et al., 2003; 2004; 2006a; 2008;
2009; Pinna, 2010). Importantly, the S-stereoisomers of fluoxetine or norfluoxetine appeared
to be 3 to 7 fold more potent than their respective R-stereoisomers and S-norfluoxetine was
about 5-fold more potent than S-fluoxetine. The effective concentrations (EC50s) of S-
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fluoxetine and S-norfluoxetine that normalize the brain allopregnanolone content were found
to be 10- (S-fluoxetine) and 50-fold (S-norfluoxetine) lower than their respective EC50s
required to inhibit 5-HT reuptake (Pinna et al., 2003; 2004; 2009; Pinna, 2010). Furthermore,
the SSRI activity of S or R-fluoxetine and of S or R-norfluoxetine was devoid of
stereospecificity (Pinna et al., 2003; 2004).
Our experiments hence provided a strong support to the hypothesis that elevation of
allopregnanolone biosynthesis is a primary mechanism for the antiaggressive, anxiolytic, and
a
anti-fear action of fluoxetine and that neither the behavioral action nor the normalization of
corticolimbic allopregnanolone content by fluoxetine is related to its intrinsic SSRI activity.
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These studies demonstrated that fluoxetine with high potency and stereospecificity is
capable of reducing behavioral deficits by upregulating corticolimbic allopregnanolone
content and suggested that this drug and its congeners may affect specific targets involved in
the regulation of neurosteroidogenesis. As far as the fine mechanisms involving the
neurosteroidogenic action of fluoxetine, our group and other collaborators have demonstrated
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that protracted social isolation in rodents affects the expression of 5α-reductase type I in
specific corticolimbic structures and fails to change the expression of 3α-HSD. Also, the
finding that brain progesterone levels do not change in socially isolated mice and that
progesterone levels in group-housed and socially isolated mice are not affected by fluoxetine
administration, suggests that a mechanism downstream from 5α-reductase is responsible for
the neurosteroidogenic action of selective brain steroidogenic stimulants (SBSSs), including
SSRIs (Uzunov et al., 1996; Matsumoto et al., 1999). Furthermore, the finding that low doses
of the S isomers of fluoxetine or norfluoxetine increase corticolimbic levels of
c.
allopregnanolone in socially isolated mice, but fail to change levels in group-housed mice,
suggests that 5α-reductase and/or 3α-HSD may become more susceptible to the effects of
SBSSs during isolation (reviewed in Pinna, 2010). More investigations at the molecular
In
enzymatic level using subcellular fraction obtained from brains of male and female rats in
diestrus treated with fluoxetine recently established that a short-term treatment with
fluoxetine caused a significant increase in brain allopregnanolone concentrations in female
but not in male rats (Fry et al., 2014). Enzyme assay performed on native rat brain fractions
and on activities expressed in HEK cells demonstrated fluoxetine to exert no effects on the
rs
aldo-keto reductase, which is responsible for the production of allopregnanolone from its
precursor, 5α-dihydroprogesterone. However, fluoxetine appeared to inhibit the microsomal
dehydrogenase oxidating allopregnanolone to 5α-dihydroprogesterone and thereby resulting
in an accumulation of brain allopregnanolone levels (Figure 1) (Fry et al., 2014). These
he
results are in contrast with a previous in vitro study that had showed that SSRIs increase the
concentrations of allopregnanolone by stimulating the conversion of 5α-dihyprogesterone into
allopregnanolone (Griffin and Mellon, 1999). In support with the finding that fluoxetine
increase the levels of allopregnanolone by inhibiting the reconversion of allopregnanolone
into 5α-dihyprogesterone are findings obtained by Rupprecht and collaborators who
is
investigated the neurosteroidogenic effects of mirtazapine, a noradrenergic and specific
serotonergic antidepressant. These researchers found that mirtazapine in in vitro studies
inhibited dose-dependently the activity of the microsomal 3α-HSD toward the oxidative
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direction (Schüle et al., 2014). Notwithstanding the fine mechanism of action regulating the
neurosteroidogenic effects of fluoxetine, the finding that low non-serotonergic doses of this
drug potently increase allopregnanolone levels in corticolimbic areas provides an important
new therapeutic strategy for the treatment of psychiatric disorders, which arise from a
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downregulation of allopregnanolone biosynthesis. These disorders include PTSD and

depression as well as premenstrual dysphoria and catamenial epilepsy, which are triggered by
an abrupt fall of allopregnanolone levels. Hence, banking on the rapid neurosteroid-enhancing
properties of SSRIs, this class of drugs and other SBSSs offer an important short-term
treatment opportunity in women affected by menstrual cycle-associated psychopathologies
(Pinna et al., 2006; Lovick, 2013).
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Fluoxetine’s Mechanism of Action My Include

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a Stimulation of Brain Derived Neurotrophic

Factor (BDNF) Mediated by Allopregnanolone in
Corticolimbic Neurons
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PTSD and depression have consistently been associated with decreased hippocampal
volume with no differences in total cerebral volume and with functional impairments (Sheline
34 Graziano Pinna
et al., 1996; 2002; Schmahl et al., 2009; Acheson et al., 2012). The hippocampal volume loss
appears to have functional significance including an association with memory loss (Nagahara
and Tuszynski, 2011). These studies suggest that depression and PTSD are associated with
hippocampal atrophy. A decrease of brain derived neurotrophic factor (BDNF) in
hippocampus and plasma has been consistently inversely associated with the severity of
c.
symptoms of PTSD and depression as well as with an impairment of cognitive function in
other psychiatric disorders (Sen et al., 2008). Treatment with neurosteroidogenic
antidepressants, including fluoxetine resulted in an improvement of PTSD and depressive
In
symptoms associated with a significant improvement in hippocampal volume (Nagahara and
Tuszynski, 2011, Vermetten et al., 2003). Importantly, the effects of neurosteroidogenic
antidepressants have been reported in several studies to upregulate serum and hippocampal
BDNF levels in a manner that correlated with improved symptoms (Sen et al., 2008, Shimizu
et al., 2003, Chen et al., 2001). These findings suggest that fluoxetine and other
rs
antidepressant mechanisms of action may include an upregulation of BDNF expression,
which may counteract the hippocampal atrophy by stimulating dendritic spine arborization
and morphology as well as supporting neurogenesis and the survival of existing neurons.
On the other hand, progesterone and its metabolites including allopregnanolone play an
he
important role in neurogenesis and neuronal survival as demonstrated in several recent
preclinical investigations. In adult male mice, administration of progesterone increased by
two-fold the number of cells, likely by enhancing cell survival of newborn neurons in the
hippocampal dentate gyrus (Zhang et al., 2010). In an in vitro study, both progesterone and
allopregnanolone promoted human and rat neural progenitor cell proliferation with
is
allopregnanolone showing greater efficacy (Wang et al., 2005). In in vivo studies
allopregnanolone increased the BrdU incorporation in 3-month-old mouse hippocampal
subgranular zone (SGZ) as well as the subventricular zone (SVZ), (Wang et al., 2010; Brinton
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and Wang, 2006). These data suggested that for both neuroprotection and neurogenesis,
allopregnanolone may play a pivotal role.
Collectively, these and other investigations led us to hypothesis that in PTSD and
depressed patients who show a decrease of CSF and brain allopregnanolone biosynthesis,
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antidepressant-induced allopregnanolone level upregulation was involved in the

antidepressant mechanisms underlying the elevation of BDNF levels, which then triggers
neurotrophism and neurogenesis. We specifically hypothesized that allopregnanolone
regulates BDNF expression. Studies conducted in our laboratory in socially isolated mice
have shown that a decrease of corticolimbic allopregnanolone levels is associated with
decreased levels of corticolimbic BDNF mRNA expression (Nin et al., 2011). These
neurochemical deficits are associated with behavioral dysfunctions, including exaggerated
a
contextual fear conditioning and impaired extinction, anxiety-like behavior, and

aggressiveness (Nelson et al., 2010; Nin et al., 2011). Importantly, allopregnanolone levels
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and BDNF mRNA expression are downregulated in the same brain areas, such as the medial
frontal cortex, hippocampus, and BLA (Pibiri et al., 2008; Nin et al., 2011). Furthermore, in
socially isolated mice the mean of spine density along the entire extent of the apical and basilar
dendrites was decreased as well as the percentage of mature spines in layer III from the frontal
cortex. The greatest decrease in percentage of mature spines for isolated mice was the proximal
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portion of the apical dendrite, while for the basilar dendrite the greatest decrease in percentage of
mature spines was the mid and distal portion of the dendrite (Tueting and Pinna, 2002).
Further, we found that a one week treatment with allopregnanolone or low S-

norfluoxetine doses sufficient to increase the levels of allopregnanolone in corticolimbic areas
normalized BDNF mRNA expression in socially isolated to the levels of group-housed mice
(Nin et al., 2011). This treatment also improved dendritic spine morphology and behavioral
deficits. S-norfluoxetine and allopregnanolone treatment improved contextual fear
c.
conditioning and facilitated fear extinction, suggesting that allopregnanolone- or S-
norfluoxetine-induced BDNF upregulation in corticolimbic areas strengthens extinction
processing (Pibiri et al., 2008, Nin et al., 2011; Nelson et al., 2010). Importantly, S-
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norfluoxetine and allopregnanolone actions on conditioned fear responses were mimicked by
a single BDNF (0.75-0.075 ng/l) bilateral microinfusion into the BLA, which dose-
dependently facilitated fear extinction and abolished the reinstatement of fear responses in the
absence of locomotion impairment (Nin et al., 2011; Martinez et al., 2011). Hence, our
investigation suggests that by increasing allopregnanolone levels, SBSS drugs such as S-
rs
norfluoxetine may regulate corticolimbic BDNF expression and may induce a long-term
improvement in the behavioral dysfunctions that relate to PTSD.
he
Conclusion
Fluoxetine has served as a pharmacological prototypic agent to study the
neurosteroidogenic action of SSRIs. This effect appears to occur at concentrations far below
those required to inhibit serotonin reuptake. Consistently, using an animal model of
is
anxiety/PTSD/depression, the behavioral effects of fluoxetine appear to correlate with the
ability of this drug to upregulate allopregnanolone biosynthesis rather than by affecting
serotonergic mechanisms. Hence, our investigation suggests that the elevation of neurosteroid
bl
biosynthesis is a fast, primary and more selective mechanism by which fluoxetine and
congeners improve behavioral deficits that result from protracted stress and include increased
aggressiveness, inappropriate exaggerated fear, and anxiety-like behavior.
Hence, the finding that fluoxetine at low doses primarily enhances neurosteroidogenesis,
Pu
led us to suggest that fluoxetine and congeners, and other neurosteroidogenic drugs, including
the TSPO (18 kDa translocase protein) ligands (see Figure 1) (Papadopoulos et al., 2006;
Rupprecht et al., 2009, 2010; Schüle et al., 2011, 2014), formally known as the peripheral
benzodiazepine receptor (Costa and Guidotti, 1991; Costa et al., 1994), be part of a new class
of pharmacological agents, the SBSSs (selective brain steroidogenic stimulants). These
neurosteroidogenic drugs are now emerging as a new promising therapeutic strategy for the
a
treatment of psychiatric disorders associated with a downregulation of brain allopregnanolone

biosynthesis, including anxiety disorders, premenstrual dysphoria, depression, and PTSD.
Importantly, the SBSSs appear to be more efficacious than benzodiazepines, as well as devoid
ov
of the unwanted side-effects induced by benzodiazepines.

The molecular mechanisms for the anxiolytic and anti-PTSD effects of fluoxetine appear
to include an upregulation of neurosteroidogenesis by inhibiting the reconversion of
allopregnanolone into its precursor, 5α-dihydroprogesterone, thus inhibiting the activity of
3α-HSD toward the oxidative direction. Given the brain region- and neuron-specific
N
expression of 3α-HSD, the action of fluoxetine on neurosteroid biosynthesis is likely to be

neuron and site-specific. Hence, SBSSs such as fluoxetine and its congeners, that specifically
36 Graziano Pinna
target key enzymes downstream in allopregnanolone neosynthesis, may be devoid of

unwanted side effects induced by activation of a broader neurosteroidogenic cascade within a
broader array of brain targets.
Furthermore, long-term administration of allopregnanolone or low S-norfluoxetine doses
sufficient to increase the levels of allopregnanolone in corticolimbic areas appeared to
c.
regulate BDNF expression. Remarkably, S-norfluoxetine and allopregnanolone improvement
of dysfunctional emotional behavior were mimicked by a single BDNF bilateral
microinfusion into the BLA in the absence of locomotion impairment (Nin et al., 2011;
In
Martinez et al., 2011). This treatment also improved dendritic spine morphology and
behavioral deficits.
Hence, our investigation suggest that by increasing allopregnanolone levels, SBSS drugs
may regulate corticolimbic BDNF expression and may induce a long-term improvement in
the behavioral dysfunctions that relate to psychiatric disorders.
rs
In conclusion, novel SBSS drugs that specifically increase corticolimbic
allopregnanolone biosynthesis appear to be a promising new pharmacological class of non-
sedative drugs, devoid of benzodiazepine-like side effects and more efficacious than
benzodiazepines in the treatment of PTSD, premenstrual dysphoria, and anxiety spectrum
he
disorders.
References
is
Acheson, D.T., Gresack, J.E., Risbrough, V.B., (2012). Hippocampal dysfunction effects on
context memory: Possible etiology for posttraumatic stress disorder. Neuropharmacology
62(2), 674-85.
bl
Agis-Balboa, R.C., Guidotti, A., Pinna, G., (2014). 5α-reductase type I expression is
downregulated in the prefrontal cortex/Brodmann's area 9 (BA9) of depressed patients.
Psychopharmacology 231(17), 3569-80.
Agis-Balboa, R.C., Pinna, G., Kadriu, B., Costa, E., Guidotti, A., (2007). Downregulation of
Pu
5α-reductase type I mRNA expression in cortico-limbic glutamatergic circuits of mice

socially isolated for four weeks. Proc. Natl. Acad. Sci. U.S.A. 104, 18736-41.
Agis-Balboa, R.C., Pinna, G., Zhubi, A., Maloku, E., Veldic, M., Costa, E., Guidotti, A.,
(2006). Characterization of brain neurons that express enzymes mediating neurosteroid
biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 103, 14602-14607.
Akk, G., Shu, H.J., Wang, C., Steinbach, J.H., Zorumski, C.F., Covey, D.F., Mennerick, S.,
a
(2005). Neurosteroid access to the GABAA receptor. J. Neurosc. 25, 11605-11613.

Amin, Z., Mason, G.F., Cavus, I., Krystal, J.H., Rothman, D.L., Epperson, C.N., (2007). The
interaction of neuroactive steroids and GABA in the development of neuropsychiatric
ov
disorders in women. J. Psychopharmacol. 21, 414-20.

Bäckström, T., Andreen, L., Birzniece, V., Björn, I., et al., (2003). The role of hormones and
hormonal treatments in premenstrual syndrome. C.N.S. Drugs. 17, 325-42.
Baulieu, E.E., (1981). Steroid hormones in the brain: several mechanisms. In: Steroid
hormone regulation of the brain. Fuxe K, Gustafson JA, Wettenberg L (editors).
N
Elmsford, NY: Pergamon; pp. 3-14.

Belelli, D., Casula, A., Ling, A., Lambert, J.J., (2002). The influence of subunit composition
on the interaction of neurosteroids with GABAA receptors. Neuropharmacology 43, 651-
61.
Belelli, D., Lambert, J.J., (2005). Neurosteroids: endogenous regulators of the GABAA
receptor. Nat. Rev. Neurosci. 6, 565-575.
c.
Bitran, D., Hilvers, R.J., Kellogg, C.K., (1991). Anxiolytic effects of 3alpha-hydroxy-
5alpha(beta)-pregnan-20-one: endogenous metabolites of progesterone that are active at
the GABAA receptor. Brain Res. 561, 157-61.
In
Bloch, M., Schmidt, P.J., Danaceau, M., Murphy, J., Nieman, L., Rubinow, D.R., (2000).
Effects of gonadal steroids in women with a history of postpartum depression. Am. J.
Psychiatry 157, 924-30.
Bortolato, M., Devoto, P., Roncada, P., Frau, R., Flore, G., Saba, P., Pistritto, G., Soggiu, A.,
Pisanu, S., Zappala, A., Ristaldi, M.S., Tattoli, M., Cuomo, V., Marrosu, F., Barbaccia,
rs
M.L., (2011). Isolation rearing induced reduction of brain 5α-reductase expression:
Relevance to dopaminergic impairments. Neuropharmacology 60(7-8), 1301-8.
Brinton, R.D., Wang, J.M., (2006). Preclinical analyses of the therapeutic potential of
allopregnanolone to promote neurogenesis in vitro and in vivo in transgenic mouse model
he
of Alzheimer's disease. Curr. Alzheimer Res. 3, 11-7.
Chen, B., Dowlatshahi, D., MacQueen, G.M., Wang, J.F., Young, L.T., (2001). Increased
hippocampal BDNF immunoreactivity Biol. Psychiatry. 50, 260-5.
Cheney, D.L., Uzunov, D., Costa, E., Guidotti, A., (1995). Gas chromatographic-mass
fragmentographic quantitation of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) and
is
its precursors in blood and brain of adrenalectomized and castrated rats. J. Neurosci. 15,
4641–4650.
Costa, B., Da Pozzo, E., Chelli, B., Simola, N., Morelli, M., Luisi, M., Maccheroni, M.,
bl
Taliani, S., Simorini, F., Da Settimo, F., Martini, C., (2011). Anxiolytic properties of a 2-
phenylindolglyoxylamide TSPO ligand: Stimulation of in vitro neurosteroid production
affecting GABAA receptor activity. Psychoneuroendocrinology 36, 463-472.
Costa, E., Cheney, D.L., Grayson, D.R., Korneyev, A., Longone, P., Pani, L., Romeo, E.,
Pu
Zivkovich, E., Guidotti, A., (1994). Pharmacology of neurosteroid biosynthesis. Role of

the mitochondrial DBI receptor (MDR) complex. Ann. N.Y. Acad. Sci. 746, 223-42.
Costa, E., Guidotti, A., (1991). Diazepam binding inhibitor (DBI): a peptide with multiple
biological actions. Life Sci. 49(5), 325-44. Review.
D‘Aquila, P.S., Canu, S., Sardella, M., Spanu, C., Serra, G., Franconi, F., (2010). Dopamine
is involved in the antidepressant-like effect of allopregnanolone in the forced swimming
test in female rats. Behav. Pharmacol. 21, 21-8.
a
Deo, G.S., Dandekar, M.P., Upadhya, M.A., Kokare, D.M., Subhedar, N.K., (2010).
Neuropeptide Y Y1 receptors in the central nucleus of amygdala mediate the anxiolytic-
ov
like effect of allopregnanolone in mice: Behavioral and immunocytochemical evidences.

Brain Res. 1318, 77-86.
Engin, E., Treit, D., (2007). The anxiolytic-like effects of allopregnanolone vary as a function
of intracerebral microinfusion site: the amygdala, medial prefrontal cortex, or
hippocampus. Behav. Pharmacol. 18, 461-70.
N
Eser, D., Schüle, C., Baghai, T.C., Romeo, E., Uzunov, D.P., Rupprecht, R., (2006).
Neuroactive steroids and affective disorders. Pharmacol. Biochem. Behav. 84(4), 656-66.
38 Graziano Pinna
Fry, J.P., Li, K.Y., Devall, A.J., Cockcroft, S., Honour, J.W., Lovick, T.A., (2014).
Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal
dehydrogenase rather than activating an aldo-keto reductase. Br J Pharmacol. doi:
10.1111/bph.12891.
Frye, C.A., Paris, J.J., Rhodes, M.E., (2009). Increasing 3alpha,5alpha-THP following
c.
inhibition of neurosteroid biosynthesis in the ventral tegmental area reinstates anti-
anxiety, social, and sexual behavior of naturally receptive rats. Reproduction 137, 119-
28.
In
Frye, C.A., Rhodes, M.E., (2006). Infusions of 5alpha-pregnan-3alpha-ol-20-one
(3alpha,5alpha- THP) to the ventral tegmental area, but not the substantia nigra, enhance
exploratory, antianxiety, social and sexual behaviours and concomitantly increase
3alpha,5alpha-THP concentrations in the hippocampus, diencephalon and cortex of
ovariectomised oestrogenprimed rats. J. Neuroendocrinol. 18, 960-75.
rs
Griffin, L.D., Mellon, S.H., (1999). Selective serotonin reuptake inhibitors directly alter
activity of neurosteroidogenic enzymes. Proc. Natl. Acad. Sci. U.S.A. 96, 13512-7.
Guidotti, A., Dong, E., Matsumoto, K., Pinna, G., Rasmusson, A.M., Costa, E., (2001). The
socially-isolated mouse: a model to study the putative role of allopregnanolone and 5α-
he
dihydroprogesterone in psychiatric disorders. Brain Res. Rev. 37, 110-115.
Hosie, A.M., Wilkins, M.E., da Silva, H.M., Smart, T.G., (2006). Endogenous neurosteroids
regulate GABAA receptors through two discrete transmembrane sites. Nature 444, 486-9.
Jain, N.S., Hirani, K., Chopde, C.T., (2005). Reversal of caffeine-induced anxiety by
neurosteroid 3-alpha-hydroxy-5-alpha-pregnane-20-one in rats. Neuropharmacology 48,
is
627-38.
Kita, A., Furukawa, K., (2008). Involvement of neurosteroids in the anxiolytic-like effects of
AC-5216 in mice. Pharmacol. Biochem. Behav. 89(2), 171-8.
bl
Lambert, J.J., Belelli, D., Peden, D.R., Vardy, A.W., Peters, J.A., (2003). Neurosteroid
modulation of GABAA receptors. Prog. Neurobiol. 71, 67–80.
Lambert, J.J., Cooper, M.A., Simmons, R.D., Weir, C.J., Belelli, D., (2009). Neurosteroids:
endogenous allosteric modulators of GABAA receptors. Psychoneuroendocrinology 34,
Pu
Suppl 1, S48-58.
Longone, P., di Michele, F., D'Agati, E., Romeo, E., Pasini, A., Rupprecht, R., (2011).
Neurosteroids as neuromodulators in the treatment of anxiety disorders. Front.
Endocrinol. (Lausanne). 2, 55.
Longone, P., Rupprecht, R., Manieri, G.A., Bernardi, G., Romeo, E., Pasini, A., (2008). The
complex roles of neurosteroids in depression and anxiety disorders. Neurochem. Int.
52(4-5), 596-601.
a
Lonsdale, D., Burnham, W.M., (2007). The anticonvulsant effects of allopregnanolone

against amygdalakindled seizures in female rats. Neurosci. Lett. 411, 147-51.
ov
Lovick, T., (2013). SSRIs and the female brain--potential for utilizing steroid-stimulating
properties to treat menstrual cycle-linked dysphorias. J. Psychopharmacol. 27(12), 1180-
5.
Maguire, J., Mody, I., (2009). Steroid hormone fluctuations and GABAA Receptor plasticity.
Psychoneuroendocrinology 34, Suppl 1, S84-90.
N
Majewska, M.D., (1992). Neurosteroids: endogenous bimodal modulators of the GABAA

receptor. Mechanism of action and physiological significance. Prog. Neurobiol. 38, 379–
395.
Mares, P., Mikulecká, A., Haugvicová, R., Kasal, A., (2006). Anticonvulsant action of
allopregnanolone in immature rats. Epilepsy Res. 70, 110-7.
Martinez, L.A., Pibiri, F., Nelson, M., Nin, M.S., Pinna, G., (2011). Selective brain
steroidogenic stimulant (SBSS)-induced neurosteroid upregulation facilitates fear
extinction and reinstatement of fear memory by a BDNF-mediated mechanism in a
c.
mouse model of PTSD. Society for Neuroscience Abstract. Washington, DC. November
12-16, 2011.
Martin-Garcia, E., Pallares, M., (2005). The intrahippocampal administration of the
In
neurosteroid allopregnanolone blocks the audiogenic seizures induced by nicotine. Brain
Res. 1062, 144-50.
Marx, C.E., Keefe, R.S., Buchanan, R.W., Hamer, R.M., Kilts, J.D., Bradford, D.W., et al.,
(2009). Proof-of concept trial with the neurosteroid pregnenolone targeting cognitive and
negative symptoms in schizophrenia. Neuropsycopharmacology 34, 1885-903.
rs
Matsumoto, K., Nomura, H., Murakami, Y., Taki, K., Takahata, H., Watanabe, H., (2003).
Long-term social isolation enhances picrotoxin seizure susceptibility in mice: up-
regulatory role of endogenous brain allopregnanolone in GABAergic systems. Pharm.
Biochem. Behav. 75, 831-835.
he
Matsumoto, K., Puia, G., Dong, E., Pinna, G., (2007). GABAA receptor neurotransmission
dysfunction in a mouse model of social isolation-induced stress: possible insights into a
nonserotonergic mechanism of action of SSRIs in mood and anxiety disorders. Stress 10,
3-12.
Matsumoto, K., Uzunova, V., Pinna, G., Taki, K., Uzunov, D.P., Watanabe, H., Mienvielle,
is
J.- M., Guidotti, A., Costa, E., (1999). Permissive role of brain allopregnanolone content
in the regulation of pentobarbital-induced righting reflex loss. Neuropharmacology 38,
955-963.
bl
Mihalek, R.M., Banerjee, P.K., Korpi, E.R., Quinlan, J.J., Firestone, L.L., Mi, Z.P., Lagenaur,
C., Tretter, V., Sieghart, W., Anagnostaras, S.G., Sage, J.R., Fanselow, M.S., Guidotti,
A., Spigelman, I., Li. Z., DeLorey, T.M., Olsen, R.W., Homanics, G.E., (1999).
Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor
Pu
delta subunit knockout mice. Proc. Natl. Acad. Sci. U.S.A. 96(22), 12905-10.
Nagahara, A.H., Tuszynski, M.H., (2011). Potential therapeutic use Nat. Rev. Drug Discov.
10, 209-19.
Nappi, R.E., Petraglia, F., Luisi, S., Polatti, F., Farina, C., Genazzani, A.R., (2001). Serum
allopregnanolone in women with postpartum ―blues‖. Obstet. Gynecol. 97, 77-80.
Nelson, M., Pibiri, F., Pinna, G., (2010). Relationship between allopregnanolone (Allo) and
reduced brain BDNF and reelin expression in a mouse model of posttraumatic stress
a
disorders (PTSD). Society for Neuroscience 2010 Abstract, 666.22

Nelson, M., Pinna, G., (2011). S-norfluoxetine infused into the basolateral amygdala
ov
increases allopregnanolone levels and reduces aggression in socially isolated mice.

Neuropharmacology 60, 1154-1159.
Nin Schuler, M., Martinez, L.A., Thomas, R., Nelson, M., Pinna, G., (2011).
Allopregnanolone and S-norfluoxetine decrease anxiety-like behavior in a mouse model
of anxiety/depression. Trabajos del Instituto Cajal 83, 215-216.
N
Nin, M.S., Salles, F.B., Azeredo, L.A., Frazon, A.P., Gomez, R., Barros, H.M., (2008).
Antidepressant effect and changes of GABAA receptor gamma2 subunit mRNA after
hippocampal administration of allopregnanolone in rats. J. Psychopharmacol. 22, 477-85.
40 Graziano Pinna
Papadopoulos, V., Baraldi, M., Guilarte, T.R., Knudsen, T.B., Lacapère, J.J., Lindemann, P.,
Norenberg, M.D., Nutt, D., Weizman, A., Zhang, M.R., Gavish, M., (2006). Translocator
protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor
based on its structure and molecular function. Trends Pharmacol. Sci. 27, 402-9.
Pearlstein, T., (2010). Premenstrual dysphoric disorder: out of the appendix. Arch. Womens
c.
Ment. Health 13, 21-3.
Pibiri, F., Nelson, M., Guidotti, A., Costa, E., Pinna, G., (2008). Decreased allopregnanolone
content during social isolation enhances contextual fear: a model relevant for
In
posttraumatic stress disorder. Proc. Natl. Acad. Sci. U.S.A. 105, 5567-5572.
Pinna, G., (2010). In a mouse model relevant for post-traumatic stress disorder, selective
brain steroidogenic stimulants (SBSS) improve behavioral deficits by normalizing
allopregnanolone biosynthesis. Behav. Pharmacol. 21, 438-50.
Pinna, G., Agis-Balboa, R., Pibiri, F., Nelson, M., Guidotti, A., Costa, E., (2008).
rs
Neurosteroid biosynthesis regulates sexually dimorphic fear and aggressive behavior in
mice. Neurochemical Research 33, 1990-2007.
Pinna, G., Agis-Balboa, R.C., Zhubi, A., Matsumoto, K., Grayson, D.R., Costa, E., Guidotti,
A., (2006B). Imidazenil and diazepam increase locomotor activity in mice exposed to
he
protracted social isolation. Proc. Natl. Acad. Sci. U.S.A. 103, 4275-4280.
Pinna, G., Costa, E., Guidotti, A., (2004). Fluoxetine and norfluoxetine stereospecifically
facilitate pentobarbital sedation by increasing neurosteroids. Proc. Natl. Acad. Sci. U.S.A.
101, 6222-6225.
Pinna, G., Costa, E., Guidotti, A., (2006A). Fluoxetine and norfluoxetine stereospecifically
is
and selectively increase brain neurosteroid content at doses that are inactive on 5-HT
reuptake. Psychopharmacology 186, 362-372.
Pinna, G., Costa, E., Guidotti, A., (2009). SSRIs act as selective brain steroidogenic
bl
stimulants (SBSSs) at low doses that are inactive on 5-HT reuptake. Curr. Opin.
Pharmacol. 9, 24-30.
Pinna, G., Dong, E., Matsumoto, K., Costa, E., Guidotti, A., (2003). In socially isolated mice,
the reversal of brain allopregnanolone down-regulation mediates the anti-aggressive
Pu
action of fluoxetine. Proc. Natl. Acad. Sci. U.S.A. 100, 2035-2040.

Pinna, G., Uzunova, V., Matsumoto, K., Puia, G., Mienville, J.-M., Costa, E., Guidotti, A.,
(2000). Brain allopregnanolone regulates the potency of the GABAA receptor agonist
muscimol. Neuropharmacology 39, 440-448.
Puia, G., Mienville, J.-M., Matsumoto, K., Takahata, H., Watanabe, H., Costa, E., Guidotti,
A., (2003). On the putative physiological role of allopregnanolone on GABAA receptor
function. Neuropharmacology 44, 49–55.
a
Puia, G., Santi, M.R., Vicini, S., Pritchett, D.B., Purdy, R.H., Paul, S.M., Seeburg, P.H.,
Costa, E., (1990). Neurosteroids act on recombinant human GABAA receptors. Neuron 4,
ov
759-765.
Puia, G., Vicini, S., Seeburg, P.H., Costa, E., (1991). Influence of recombinant gamma-
aminobutyric acid—a receptor subunit composition on the action of allosteric modulators
of gammaaminobutyricacid-gated Cl-currents. Mol. Pharmacol. 39, 691–696.
Rapkin, A.J., Morgan, M., Goldman, L., Brann, D.W., Simone, D., Mahesh, V.B., (1997)
N
Progesterone metabolite allopregnanolone in women with presmenstrual syndrome.

Obstet. Gynecol. 90, 709-14.
Rasmusson, A.M., Pinna, G., Paliwal, P., Weisman, D., Gottschalk, C., Charney, D., et al.,
(2006). Decreased cerebrospinal fluid allopregnanolone levels in women with
posttraumatic stress disorder. Biol. Psychiatry 60, 704-13.
Rodgers, R.J., Johnson, N.J., (1998). Behaviorally selective effects of neuroactive steroids on
plus-maze anxiety in mice. Pharmacol. Biochem. Behav. 59, 221-32.
c.
Rodríguez-Landa, J.F., Contreras, C.M., García-Ríos, R.I., (2009). Allopregnanolone
microinjected into the lateral septum or dorsal hippocampus reduces immobility in the
forced swim test: participation of the GABAA receptor. Behav. Pharmacol. 20, 614-622.
In
Romeo, E., Ströhle, A., Spalletta, G., di Michele, F., Hermann, B., Holsboer, F., Pasini, A.,
Rupprecht, R., (1998). Effects of antidepressant treatment on neuroactive steroids in
major depression. Am. J. Psychiatry. 155(7), 910-3.
Rudolph, U., Crestani, F., Benke, D., Brünig, I., Benson, J.A., Fritschy, J.M., Martin, J.R.,
Bluethmann, H., Möhler, H., (1999). Benzodiazepine actions mediated by specific
rs
gamma-aminobutyric acid(A) receptor subtypes. Nature 401, 796-800.
Rudolph, U., Möhler, H., (2004). Analysis of GABAA receptor function and dissection of the
pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu.
Rev. Pharmacol. Toxicol. 44, 475-98.
he
Rupprecht, R., Papadopoulos, V., Rammes, G., Baghai, T.C., Fan, J., Akula, N., Groyer, G.,
Adams, D., Schumacher, M., (2010). Translocator protein (18 kDa) (TSPO) as a
therapeutic target for neurological and psychiatric disorders. Nat. Rev. Drug Discov.
9(12), 971-88.
Rupprecht, R., Rammes, G., Eser, D., Baghai, T.C., Schüle, C., Nothdurfter, C., Troxler, T.,
is
Gentsch, C., Kalkman, H.O., Chaperon, F., Uzunov, V., McAllister, K.H., Bertaina-
Anglade, V., La Rochelle, C.D., Tuerck, D., Floesser, A., Kiese, B., Schumacher, M.,
Landgraf, R., Holsboer, F., Kucher, K., (2009). Translocator protein (18 kD) as target for
bl
anxiolytics without benzodiazepine-like side effects. Science. 325(5939), 490-3.
Schmahl, C., Berne, K., Krause, A., Kleindienst, N., Valerius, G., Vermetten, E., Bohus, M.,
(2009). Hippocampus and amygdala J. Psychiatry Neurosci. 34, 289-95.
Schüle, C., Eser, D., Baghai, T.C., Nothdurfter, C., Kessler, J.S., Rupprecht, R., (2011).
Pu
Neuroactive steroids in affective disorders: target for novel antidepressant or anxiolytic

drugs? Neuroscience 191, 55-77.
Schüle, C., Nothdurfter, C., Rupprecht, R., (2014). The role of allopregnanolone in depression
and anxiety. Prog. Neurobiol. 113, 79-87.
Sen, S., Duman, R., Sanacora, G., (2008). Serum brain-derived neurotrophic factor,
depression, and antidepressant medications: meta-analyses and implications. Biol.
a
Serra, M., Madau, P., Chessa, M.F., Caddeo, M., Sanna, E., Trapani, G., Franco, M., Liso, G.,
Purdy, R.H., Barbaccia, M.L., Biggio, G., (1999). 2-Phenyl-imidazo(1,2-a)pyridine
ov
derivatives Br. J. Pharmacol. 127, 177-87.

Serra, M., Pisu, M.G., Littera, M., Papi, G., Sanna, E., Tuveri, F., Usala, L., Purdy, R.H.,
Biggio, G., (2000) Social isolation-induced decreases in both the abundance of
neuroactive steroids and GABA(A) receptor function in rat brain. J. Neurochem. 75, 732-
40.
N
Sheline, Y.I., Mittler, B.L., Mintun, M.A., (2002). The hippocampus Eur. Psychiatry Suppl 3,
300-5.
42 Graziano Pinna
Sheline, Y.I., Wang, P.W., Gado, M.H., Csernansky, J.G., Vannier, M.W., (1996).
Hippocampal atrophy Proc. Natl. Acad. Sci. U.S.A. 93, 3908-13.
Shimizu, E., Hashimoto, K., Okamura, N., Koike, K., Komatsu, N., Kumakiri, C., Nakazato,
M., Watanabe, H., Shinoda, N., Okada, S., Iyo, M., (2003). Alterations of serum Biol.
c.
Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M., Mody, I., (2003). Neuroactive steroids
reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta
subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. U.S.A. 100(24), 14439-44.
In
Stoffel-Wagner, B., (2001). Neurosteroid metabolism in the human brain. European Journal
of Endocrinology 145, 669–679.
Ströhle, A., Romeo, E., di Michele, F., Pasini, A., Yassouridis, A., Holsboer, F., Rupprecht,
R., (2002). GABAA receptor-modulationg neuroactive steroid composition in patients
with panic disorder before and during paroxetine treatment. Am. J. Psychiatry 159, 145-7.
rs
Tueting, P., Pinna, G., (2002). Behavior associated with an enriched environment and with
social isolation in mice. Society for Neuroscience Abstract. Poster. Orlando, FL.
November 2-7, 2002. 106.12
Uzunov, D.P., Cooper, T.B., Costa, E., Guidotti, A., (1996). Fluoxetine elicited changes in
he
brain neurosteroid content measured by negative ion mass fragmentography. Proc. Natl.
Acad. Sci. U.S.A. 93, 12599-12604.
Uzunova, V., Sampson, L., Uzunov, D.P., (2006). Relevance of endogenous 3alpha-reduced
neurosteroids to depression and antidepressant action. Psychopharmacology 186(3), 351-
61.
is
Uzunova, V., Sheline, Y., Davis, J.M., Rasmusson, A., Uzunov, D.P., Costa, E., Guidotti, A.,
(1998). Increase in the cerebrospinal fluid content of neurosteroids in patients with
unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc. Natl.
bl
Acad. Sci. U.S.A. 95, 3239-44.
Uzunova, V., Wrynn, A.S., Kinnunen, A., Ceci, M., Kohler, C., Uzunov, D.P., (2004).
Chronic antidepressants reverse cerebrocortical allopregnanolone decline in the olfactory-
bulbectomized rat. Eur. J. Pharmacol. 486(1), 31-4.
Pu
van Broekhoven, F., Verkes, R.J., (2003). Neurosteroids in depression: a review.

Psychopharmacology 165, 97-110.
Vermetten, E., Vythilingam, M., Southwick, S.M., Charney, D.S., Bremner, J.D., (2003)
Long-term treatment Biol. Psychiatry 54, 693-702.
Vicini, S., (1991). Pharmacologic significance of the structural heterogeneity of the GABAA
receptor-chloride ion channel complex. Neuropsychopharmacology 4, 9-15.
Wang, J.M., Johnston, P.B., Ball, B.G., Brinton, R.D., (2005). The neurosteroid
a
allopregnanolone promotes proliferation J. Neurosci. 25, 4706-18.

Wang, J.M., Singh, C., Liu, L., Irwin, R.W., Chen, S., Chung, E.J., Thompson, R.F., Brinton,
ov
R.D., (2010). Allopregnanolone reverses neurogenic and cognitive deficits in mouse

model of Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 107, 6498-503.
Wieland, S., Lan, N.C., Mirasedeghi, S., Gee, K.W., (1991). Anxiolytic activity of the
progesterone metabolite 5alpha-pregnan-3alpha-o1-20-one. Brain Res. 565, 263-8.
Zhang, Z., Yang, R., Zhou, R., Li, L., Sokabe, M., Chen, L., (2010). Progesterone promotes
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the survival Hippocampus 20, 402-12.

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Chapter 3
In
Developmental Fluoxetine Exposure
and Neuroendocrine Outcomes
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Jodi L. Pawluski1,2,3*, Mary Gemmel3, Eszter Császár3,4,
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Harry W. M. Steinbusch1 and Ine Rayen1
1
School for Mental Health and Neuroscience, Department of Neuroscience,
Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands
2
GIGA-Neurosciences, University of Liège, Belgium
3
Department of Biological Sciences, Ohio University, Athens, OH, US
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4
The Institute of Experimental Pharmacology and Toxicology,
Slovak Academy of Sciences, Department of Reproductive Toxicology,
Bratislava, Slovak Republic
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Abstract
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Selective serotonin reuptake inhibitor (SSRI) medications are the most common
antidepressant treatment used to treat maternal mood disorders during pregnancy and the
postpartum period. Up to 10% of pregnant women are prescribed SSRIs, with fluoxetine
being one of the most popular SSRIs for women. These medications cross the placenta
raising questions about their role in fetal development. In turn, serotonin itself plays an
important role in stress regulation and sexual differentiation of the brain. Recent clinical
work shows that prenatal SSRI medications act to ‗program‘ the developing HPA system.
a
Preclinical research points to a long term effect of perinatal exposure to SSRIs on

neuroendocrine outcomes related to both the hypothalamic-pituitary-adrenal (HPA) and
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hypothalamic-pituitary-gonadal (HPG) systems. Not surprisingly, these effects markedly

differ in male and female offspring. This chapter will highlight emerging evidence from
clinical and preclinical studies investigating the impact of perinatal fluoxetine exposure
on the developing HPA and HPG systems, investigating both neuroendocrine effects and
behavioral outcomes. Given the significant impact that maternal adversity can have on
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*
Corresponding Author: Jodi L. Pawluski, Ph.D., Department of Biological Sciences, Ohio University, Athens,
Ohio, 45701. e-mail: pawluski@ohio.edu / j.pawluski@gmail.com.
44 Jodi L. Pawluski, Mary Gemmel, Eszter Császár et al.
the development of the HPA and HPG systems, the potential role of maternal adversity,
in combination with SSRI treatment, will also be discussed.
Keywords: fluoxetine, development, stress, sexual differentiation, SSRI, depression,

hippocampus, neural plasticity, steroid hormones
c.
Introduction
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It is well known that women are more vulnerable to develop depression during their
childbearing years (Burke et al. 2005). Depression occurring during pregnancy and the
postpartum period affects up to 20% of the women (Almond 2009; Leung and Kaplan 2009;
Limlomwongse and Liabsuetrakul 2006; Marcus 2009). Risk factors for developing
depression during the perinatal period include having a history of depression, anxiety,
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stressful life events, lacking social support, an unplanned pregnancy, and being of lower
socio-economic status (Lancaster et al. 2010; Stewart 2011). Women suffering from stress,
depression, or anxiety during gestation can put their infant at risk for poor physical and
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mental development (Huizink et al. 2003; Oberlander et al. 2009; Talge et al. 2007). Children
exposed to maternal stress or depression during gestation often show impairments in
cognitive abilities, such as language development (2 year old), impulsivity in cognitive tasks
(14 year old and 15 year old), lower school marks (6 year old), and also have an increased
risk to develop neuropsychiatric disorders such as autism (4 to 12 year old) (Beversdorf et al.
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2005; Huizink et al. 2003; Huizink et al. 2004; D. P. Laplante et al. 2004; Niederhofer and
Reiter 2004; Van den Bergh et al. 2005b; Van den Bergh et al. 2008).
Numerous antidepressant treatments are available to treat depression, serotonin–
norepinephrine reuptake inhibitors, and selective serotonin reuptake inhibitor (SSRI). SSRI
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medications, with the exception of paroxetine, are considered to be safe for prenatal use, as
neuroteratogenic effects have not been reported with these medications (Gentile 2005).
Therefore, SSRI medications are most often prescribed to treat maternal mood disorders
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during the perinatal period, with up to 10% of pregnant women prescribed SSRI medications
(Cooper et al. 2007; Fleschler and Peskin 2008; Oberlander et al. 2006; Ververs et al. 2006).
The most popular SSRIs used by pregnant women are fluoxetine, sertraline and citalopram
(Andrade et al. 2008; Colvin et al. 2011).
Twenty-five percent of women already using antidepressant medications prior to
conception continue this treatment during pregnancy, while 0.5 percent of women start using
antidepressant medications during pregnancy (Ververs et al. 2006). SSRIs can affect fetal and
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neonatal development, as they pass through the placental barrier and are present in breast
milk (Kristensen et al. 1999). Perinatal SSRI exposure increases extracellular serotonin
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concentrations in the brain by inhibiting the serotonin transporter, and, as a result, increases
serotonin (5-HT) signalling in the fetus (Baumann and Rochat 1995; Oberlander et al. 2009).
Clinical data has shown that developmental changes in serotonin concentration, after prenatal
SSRI exposure, may be related to reduced fetal head growth, serotonin-related behavioral and
physiological symptoms in neonates in the first weeks of life, lower birth weight, younger
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gestational age, reduced heart rate variability, and behavioral abnormalities (El Marroun et al.
2012; Moses; Nulman et al. 2002; Oberlander et al. 2009). Recent research has also shown
that developmental exposure to SSRI medications, such as fluoxetine, can ‗program‘
Developmental Fluoxetine Exposure and Neuroendocrine Outcomes 45
neuroendocrine pathways such as the developing hypothalamic-pituitary-adrenal (HPA) and

hypothalamic-pituitary-gonadal (HPG) axes (Olivier et al. 2013; Pawluski 2011; Pawluski et
al. 2012b; I. Rayen et al. 2013, 2014a). This is perhaps not surprising as serotonin plays an
integral role in stress regulation and sexual differentiation of the brain.
This chapter will highlight emerging evidence from clinical and preclinical studies
c.
investigating the impact of perinatal fluoxetine exposure on the developing HPA and HPG
systems, neuroendocrine effects and behavioral outcomes. In this chapter, we will briefly
describe: 1) the role of serotonin in development, 2) developmental effects of fluoxetine
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exposure on the HPA system and mood, 3) developmental effects of fluoxetine on the HPG
system and reproductive behaviors, 4) Conclusions and Future directions. Knowledge of the
long-term developmental implications of antidepressant medication exposure, in appropriate
animal models of maternal adversity, is important in order to select the appropriate treatment
and interventions for the mother, and improve the outcome for the child.
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Role of Serotonin in Development
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Serotonin is a neurotransmitter derived from the amino acid tryptophan. Serotonergic
neurons are localized in the midbrain, pineal gland, substantia nigra, hypothalamus and in the
raphe nucleus (Herlenius and Lagercrantz 2004). From these regions neural projections
extend throughout the brain, enabling management of complex sensory and motor sensations
during different life stages. Serotonin, in addition, also influences proliferation,
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differentiation, and migration of neurons as well as synaptogenesis (Herlenius and
Lagercrantz 2004). Serotonin is known to play a neurotransmitter role in the adult brain by
modulating emotion, learning, cognition, sleep and stress responses (Ansorge et al. 2007).
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During development, serotonergic cells are among the earliest appearing cells; appearing
in the 5-12th week after fertilization in humans (Herlenius and Lagercrantz 2004). After their
generation in the raphe nucleus, serotonergic neurons project diffusely into the spinal cord
and the forebrain, and are the basis of neuronal progenitor cells (Whitaker-Azmitia et al.
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1996; Whitaker-Azmitia 2001). There is a relatively high concentration of serotonin neurons

until 2 years of age in children, with a gradual decrease until 5 years of age, at which time the
concentration of serotonergic neurons remain relatively stable (Hadjikhani 2010; Herlenius
and Lagercrantz 2004; Whitaker-Azmitia 2001). Serotonin is implicated in several processes
during development, such as cell division, migration, differentiation, growth cone elongation,
myelination, dendritic pruning and synaptogenesis (Gaspar et al. 2003). Furthermore,
a
serotonin modulates the development of its own, and other, neuronal systems, including the
HPA axis (Andrews and Matthews 2004; Whitaker-Azmitia et al. 1990). One key player in
the actions of serotonin during development is the high affinity serotonin transporter SERT
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(Lebrand et al. 1996). SERT expression is limited in the human fetus during the 12th to 14th
week after fertilization; the period in which thalamocortical tracts and cortical sensory map
formation occurs (Gaspar et al. 2003; Herlenius and Lagercrantz 2004; Verney et al. 2002).
The subsequent task of SERT in the body is to maintain the homeostasis of serotonin levels in
the cells.
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Pharmaceuticals such as SSRI medications inhibit SERT, thereby limiting the uptake of
serotonin from the synaptic cleft and increasing the concentration of serotonin in the
intracellular space. Altered serotonin levels and serotonergic activity occur with depression in
adulthood. Exposure to SSRIs during development results in an acute increase in serotonergic
tone (Homberg et al. 2010; Oberlander et al. 2009) and preclinical studies have shown that
perinatal SSRI exposed animals have suppressed serotonergic tone during adulthood, possibly
through increased auto-inhibitory feedback signalling (Kinney et al. 1997; Maciag et al.
c.
2006c; Simpson et al. 2011; Weaver et al. 2010).
The effects of developmental exposure to SSRIs, such as citalopram, on the serotonin
circuitry during adulthood are evident in the dorsal and medial raphe via reductions in TPH
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expression (Maciag et al. 2006c; Maciag et al. 2006b), and in the cortex and hippocampus via
reductions in serotonin transporter expression (Maciag et al. 2006c; Simpson et al. 2011;
Weaver et al. 2010). These studies clearly indicate a suppression of serotonergic tone in
adulthood, after increased serotonergic tone during development. Interestingly, others have
shown that altering the HPA system of the mother, via dexamethasone treatment, suppresses
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the developing serotonergic system and developmental exposure to fluoxetine can normalize
serotonergic transmission (Nagano et al. 2012). Therefore, changing the serotonergic tone, via
perinatal SSRI exposure, has long term effects on the serotonergic system which may or may
not be beneficial.
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The Developmental Effects of Fluoxetine Exposure
on the HPA System
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Neuroendocrine Effects
The limbic-hypothalamic-pituitary-adrenal axis (HPA axis) regulates the stress response

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and plays an important role in the development of mood disorders. When an organism is
exposed to stress, several processes in the central nervous system (CNS) are initiated,
primarily through the HPA axis. Activation of the HPA axis begins when catecholamines,
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such as epinephrine and norepinephrine, are released from the sympathetic nervous system.
This release triggers secretion of corticotrophin-releasing hormone (CRH) by neurosecretory
cells in the hypothalamus. These two neuroendocrine processes enhance the activation of
each other during stress response. Subsequently, the anterior pituitary secretes and releases
adrenocorticotrophic hormone (ACTH) in response to increased CRH levels. Glucocorticoids,
such as cortisol (in humans) or corticosterone (CORT; in rodents), are released from the
adrenal cortex in response to stress and increased ACTH (Palkovits 1987). These
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glucocorticoids feedback to the hippocampus and hypothalamus, via binding to

glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), to further regulate the
HPA system (Herman et al. 2012; Palkovits 1987; Rothman and Mattson 2010). Chronic
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stress commonly results in glucocorticoid hypersecretion and, in susceptible individuals,

chronic stress can lead to long lasting HPA responses which can result in mood disorders
such as depression and anxiety (McEwen and Stellar 1993). Furthermore dysregulation of the
HPA system is evident in depression and anxiety. For example, depressed individuals show
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abnormal cortisol rhythms, with lower cortisol levels in the morning and higher cortisol levels
in the evening (Burke et al. 2005).
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Figure 1. The HPA axis in the fetus is controlled by both positive and negative feedback. Stress
stimulates the paraventricular nucleus (PVN) of the hypothalamus and corticotropin-releasing hormone
(CRH) and arginine-vasopressin (AVP) are released. CRH and AVP reach the anterior pituitary gland,
a
where adrenocorticotrophin hormone (ACTH) is released into circulation. ACTH is transported to the
adrenal cortex, where it stimulates the production of glucocorticoids into circulation. Glucocorticoids
increase the negative feedback signal by binding with mineralocorticoid receptors (MR) and
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glucocorticoid receptors (GR) at the different levels of the HPA system. Ascending serotonergic
pathways from the dorsal raphe nucleus innervate the hippocampus and hypothalamus. Increases in
serotonin levels lead to an up regulation of GR expression.
Serotonin plays a role in the development and function of the HPA axis (Andrews and
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Matthews 2004; Erdeljan et al. 2005; P. Laplante et al. 2002; Meaney et al. 1994), and not
surprisingly a growing body of clinical and preclinical data is showing that developmental
SSRI exposure plays a key role on HPA axis activity throughout varying developmental
stages.
Although limited, emerging clinical data shows that prenatal SSRI exposure alters HPA
function in infants by reducing umbilical cord blood cortisol levels (J. S. Davidson et al.
2006; S. Davidson et al. 2009), early evening cortisol levels at 3 months of age (Oberlander et
c.
al. 2008), and basal cortisol levels in saliva at 6 months of age (Brennan et al. 2008). This
reduction in basal cortisol levels may be caused by sequestration of increased levels in
corticosterone-binding globulin (CBG) in neonatal serum after prenatal SSRI exposure
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(Pawluski et al. 2012a). Prenatal SSRI exposure has also been shown to increase serum CBG
levels after vaginal delivery and is associated with decreased diurnal salivary cortisol changes
at three months of age (Pawluski et al. 2012a). Therefore SSRI exposure may ―buffer‖ HPA
system development and future response to stress exposure.
With regards to stress reactivity, neonates (soon after birth) and infants (2 months old)
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prenatally exposed to SSRIs display less facial reactivity and a lower heart rate during
recovery from a heel lance compared to non SSRI-exposed infants (Oberlander et al. 2002;
Oberlander et al. 2005). Similarly, antenatal depression exposure and fetal effects of SSRIs
have been associated with internalizing behavior in children 3 years of age (Oberlander et al.
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2010). These findings suggest that prenatal SSRI exposure blunts the response to acute pain
and the HPA response in newborns. Clinical results, therefore, highlight the importance of
further investigation into the long term effects of SSRI medication on the developing HPA
system and subsequent neurobehavioral modifications.
Emerging preclinical data has shown that prenatal SSRI exposure has a host of effects on
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the developing HPA system. Prenatal SSRI exposure to fluoxetine increases plasma cortisol
levels in fetal lambs (Morrison et al. 2004) and increases ACTH and cortisol levels in infant
rhesus monkeys exposed to fluoxetine and maternal separation (Clarke et al. 1998). Early
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postnatal fluoxetine treatment attenuates the serum corticosterone response to stress in
juvenile male mouse offspring exposed to maternal stress (Ishiwata et al. 2005). We have also
shown that developmental exposure to fluoxetine significantly affects the HPA axis primarily
in adolescent male, but not female, offspring (Pawluski et al. 2012b), regardless of maternal
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stress exposure. In addition, increased CBG levels were evident in prenatally stressed male
offspring exposed to maternal fluoxetine (Knaepen et al. 2013a; Pawluski et al. 2012b).
Alterations in CBG and corticosterone may suggest a more regulated response of the HPA
system in SSRI exposed offspring, as an increase in CBG levels decreases free active
corticosterone levels. Adolescent male offspring also exhibited decreased expression of
glucocorticoid receptors (GR) and expression of the co-activator, GR interacting protein
(GRIP1), in the hippocampal region (Pawluski et al. 2012b) (these effects were regardless of
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exposure to maternal stress). Decreased GRIP1 expression may result from initial decreased
GR expression or from direct modification by serotonin reuptake inhibition (Pawluski et al.
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2012b). Previous in vitro work has indicated that serotonin can increase GR mRNA in the
hippocampus (Erdeljan et al. 2001; Erdeljan et al. 2005). It was also shown that chronic use
of SSRIs during adulthood generally increased GR expression in the rat hypothalamus and
hippocampus (Yau et al. 2004), changes which were associated with a decrease in HPA axis
reactivity (Flandreau et al. 2013). However, we found a decrease in GR expression and its
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coactivator, GRIP1, in adolescent males (but not females)(Pawluski et al. 2012b). This
indicates that exposure to SSRIs during development regulates the HPA axis in a very
different way than exposure during adulthood.
Recent literature also shows that the effect of developmental SSRI exposure on the HPA
system plays a role in the developing nociceptive system and pain perception. We have
shown that post-operative pain was decreased in adult male offspring subject to prenatal
stress alone and increased in offspring developmentally exposed to fluoxetine alone (Knaepen
et al. 2013b). Moreover, post-operative pain was normalized in prenatally stressed offspring
c.
exposed to fluoxetine (Knaepen et al. 2013b). This was paralleled by a decrease in
corticosteroid binding globulin (CBG) levels in prenatally stressed offspring and a
normalization of serum CBG levels in prenatally stressed offspring developmentally exposed
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to fluoxetine (Knaepen et al. 2013b). Thus, these findings indicate that developmental
fluoxetine exposure normalizes the long-term effects of maternal adversity on post-operative
pain in offspring and these effects may be due, in part, to the involvement of the HPA system.
Hippocampal Plasticity
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The hippocampus has long been implicated in the etiology of mood disorders and
regulation of stress (Eisch et al. 2008; Kempermann 2002; Kempermann and Kronenberg
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2003; Lucassen et al. 2010; McEwen 2005). Interestingly, the hippocampus is one of two
brain regions where there is a high rate of neurogenesis throughout the lifespan (the other
region being the subventricular zone) (Eriksson et al. 1998; Pawluski et al. 2009b). These
new neurons and plasticity in the hippocampus are thought to play an important role in stress
regulation, depression and the efficacy of antidepressant medications (Dranovsky and Hen
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2006; Santarelli et al. 2003).
Serotonin is an important neurotransmitter involved in regulating the rate of hippocampal
neurogenesis during adulthood (Banasr et al. 2001; Brezun and Daszuta 1999b; Djavadian
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2004). Depletion of serotonin reduces the number of newly formed neurons in the
hippocampus, whereas increased levels of serotonin promote the rate of cell proliferation, via
actions on the 5-HT1a receptor (Brezun and Daszuta 1999b, 1999a; Malberg et al. 2000;
Varrault et al. 1992). Furthermore, increases in serotonin levels, after SSRI exposure,
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significantly upregulate hippocampal neurogenesis during adulthood (Malberg et al. 2000;

Perera et al. 2007) and it has also been suggested that hippocampal neurogenesis is important
for behavioral effects obtained after antidepressant medication exposure (Eisch and Petrik
2012; Santarelli et al. 2003; Wainwright and Galea 2013). Moreover, the effects of stress on
hippocampal neurogenesis, and the associated affect-related behaviors, may be reversed by
antidepressant medication in adulthood (Belzung and Lemoine 2011; Wainwright and Galea
2013).
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SSRI exposure during development has a very different effect on hippocampal

neurogenesis than SSRI administration during adulthood. For example, prenatal SSRI
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exposure decreases neonatal S100B levels, a protein which stimulates glial cell proliferation,
neuronal survival, and induces neurite outgrowth (Bhattacharyya et al. 1992; Gonzalez-
Martinez et al. 2003; Pawluski et al. 2009a; Winningham-Major et al. 1989). To date, only a
few studies have investigated how developmental SSRI exposure affects hippocampal
plasticity (Ishiwata et al. 2005; I. Rayen et al. 2011): early treatment of pups with fluoxetine
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reverses the prenatally stress-induced decrease in CA3 spine and synapse density in juvenile
and adolescent male mouse offspring (Ishiwata et al. 2005), and reverses hippocampal cell
proliferation and cell death in juvenile rat offspring prematurely separated from their mother
(Lee et al. 2001). We have also found that developmental exposure to fluoxetine reverses the
reduction in hippocampal cell proliferation and hippocampal neurogenesis in adolescent male
and female offspring exposed to prenatal stress (I. Rayen et al. 2011). When offspring were
exposed to fluoxetine treatment alone, hippocampal cell proliferation was significantly
decreased in adolescence (I. Rayen et al. 2011). We have also shown that developmental
c.
fluoxetine exposure to prenatally stressed offspring reverses the effect of prenatal stress or
fluoxetine exposure alone on the number of immature neurons in adulthood (I. Rayen et al.
under review). In addition, postnatal citalopram treatment can lead to a decrease in serotonin
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transporter levels in the hippocampus of rat offspring (Weaver et al. 2010) and postnatal SSRI
treatment, again, can lead to upregulation of BDNF mRNA in the hippocampus of adult
offspring (Karpova et al. 2009). These findings point to a long-term impact of developmental
fluoxetine exposure on hippocampal neurogenesis.
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Figure 2. A) Representative photomicrographs of new surviving cells (BrdU-ir), as well as B) mean
(±SEM) total number new surviving cells in the granule cell layer of the hippocampus. B) For total
number of new surviving cells, developmental fluoxetine exposure, after maternal stress, significantly
reduced the number of new surviving cells in adult female offspring compared to adult females
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developmentally exposed to fluoxetine only (p=.04). There were no significant differences in males.
‗*‘denotes significantly different. CV = Control + Vehicle, CF = Control + Fluoxetine, PSV = Prenatal
Stress + Vehicle, and PSF = Prenatal Stress + Fluoxetine. (n=5-7/sex/group) (Rayen et al.,2014).
Furthermore, we have found that in adult female, but not male, offspring, developmental
fluoxetine exposure greatly increases new cell survival and significantly decreases
synaptophysin density in the granule cell layer of the hippocampus (I. Rayen et al. 2014b).
a
When SSRIs are administered to adult female mice, a rise in hippocampal cell proliferation is
observed and a dose dependent effect on new cell survival is observed with either no effect or
a decrease in new cell survival (with a 10mg/kg dose in adulthood: (Hodes et al. 2010a;
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Hodes et al. 2010b)). However, the effect of SSRI administration on developing offspring
differs markedly from adult exposure to these medications. As mentioned previously,
exposure to SSRIs during development results in an acute increase in serotonergic tone
(Oberlander et al. 2009) and preclinical studies have shown that perinatal SSRI exposed
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animals have suppressed serotonergic tone during adulthood, possibly through increased auto-
inhibitory feedback signalling (Kinney et al. 1997; Maciag et al. 2006c; Simpson et al. 2011;
Weaver et al. 2010).
It is also possible that changing steroid hormone levels during puberty may lead to
structural changes in the hippocampus, as a result of altered expression of estrogen and
androgen receptors (Galea et al. 2006; Galea 2008; Tabori et al. 2005; Weiland et al. 1997),
and therefore may differentially affect cell proliferation and new cell survival. The fact that
developmental fluoxetine exposure affects hippocampal new cell survival differently in male
c.
and in female adult offspring may also be the result of the role of serotonin in sexual
differentiation of the brain, as it is implicated in the development of the hypothalamic-
pituitary-gonadal axis (Dohler et al. 1991; Jarzab and Dohler 1984) and can potentially
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change estrogen and androgen receptor expression permanently. Much more work is needed
to determine underlying mechanisms responsible for the sex differences in hippocampal
neurogenesis after developmental exposure to fluoxetine.
Mood and Affect-Related Behaviors
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It is generally considered that stress and alterations in the HPA system can play a
prominent role in affect-related behaviors such as depression. Early-life stressors appear to be
he
one of the best predictors of mood disorder development (Eiland and Romeo 2013). Children
exposed to maternal mood disorders early during development are at an increased risk for
cognitive, behavioral and emotional disturbances (Glover et al. 2010; D. P. Laplante et al.
2004; Talge et al. 2007; Van den Bergh et al. 2005a). Preclinical findings parallel clinical
work and show that adult offspring exposed to prenatal stress are more likely to display
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affect-related behaviors, such as anxiety and depressive-like behavior, as well as learning
deficits and vulnerability to drug addiction (Louvart et al. 2005; Maccari and Morley-Fletcher
2007; Van den Hove et al. 2005; Weinstock 2007, 2008).
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Developmental exposure to SSRI medications also impacts affect-related behaviors in
children (Misri et al. 2006; Oberlander et al. 2007; Oberlander et al. 2010). Children 4 years
of age, exposed prenatally to SSRIs, have higher scores of externalizing behaviors, such as
aggression, attention/hyperactivity, and oppositional or defiant behaviors, than the clinical
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cutoff (Oberlander et al. 2007). On the other hand, exposure to both prenatal SSRIs and
maternal depression results in increased internalizing behaviors (depression, anxiety, and
withdrawal) in 3 and 4 year old children (Oberlander et al. 2010). Preclinical studies have
also demonstrated that developmental SSRI exposure has an impact on affective-like
behaviors in offspring. Developmental SSRI exposure impairs social play and response to
novelty in juvenile offspring (Simpson et al. 2011), increases anxiety-like behavior in adult
offspring (Ansorge et al. 2004; Ansorge et al. 2008; Lisboa et al. 2007; Simpson et al. 2011),
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and increases depressive-like behavior in adult offspring (Hansen et al. 1997; Karpova et al.
2009; Popa et al. 2008)(Table 1). Others have also reported a decrease in anxiety-like
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behavior and depressive-like behavior in offspring (McAllister et al. 2012) (For an overview
of these preclinical findings see Table 1; also see (Kiryanova et al. 2013; Olivier et al. 2013).
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Table 1. Preclinical data investigating perinatal SSRI exposure on depression-related behaviors in offspring.
P=postnatal day, G=gestational day
SSRI
Age investigated Species SSRI (dose/day) Results Comments Reference
admin
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P73-P83 C57BL/6 mice Fluoxetine G15-P12 Fluoxetine decreases anxiety- In drinking water of McAllister et al.
(25 mg/kg) like behavior and decreases dams 2012
depressive-like behavior
P33-39 Sprague-Dawley rat Fluoxetine (5mg/kg) P1-21 Fluoxetine exposure reverses Osmotic minipump to Rayen et al. 2011
the effects of maternal stress the dam; male and
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on immobility in the forced female offspring used;
swim test model of maternal
stress
P25 (novel tone), P32-34 Long Evans rats Citalopram P8-21 Citalopram impairs social play s.c. injections to Simpson et al.
(juvenile play) P30 and P82 (20 mg/kg) and response to novelty, with offspring 2011
(novel object) males being more affected than
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females
P65 Wistar rat Fluoxetine G11-21 Fluoxetine increases anxiety in Oral gavage to dams; Olivier et al. 2011
(12 mg/kg) the novelty-suppressed males only
feeding, the footshock-induced
conditioned place aversion test,
P110 C57BL/6J mice
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Fluoxetine
(10 mg/kg)
P4-21
and the elevated plus maze
Fluoxetine decreases
behavioral despair in the
forced swim test
i.p. injections to
offspring; males only
Karpova et al.
2009
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P61-75 Swiss albino CD1 Escitalopram P5-19 Escitalopram increases s.c. injections to Popa et al. 2008
mouse (10 mg/kg) depressive-like behavior by offspring; females only
increasing sleep anomalies,
anhedonia and helplessness
P90-104 129S6/SvEv mice Fluoxetine P4-21 Fluoxetine, clomipramine, and i.p. injections to Ansorge et al.
(5HTT+/+ and (10 mg/kg), citalopram reduce novelty offspring 2008
5HTT+/- offspring) clomipramine exploration, and increases
(20 mg/kg), and stress reactivity
citalopram
a
(10 mg/kg)
ov
N
c.
In
Age investigated Species SSRI (dose/day) SSRI Results Comments Reference
admin
P30 and P70 (females), and Swiss mice Fluoxetine G0-P21 Fluoxetine increases Oral gavage to dams Lisboa et al. 2007
P40 and P70 (males) (7,5 mg/kg) immobility on the forced swim
test in females at P30 and P70,
and decreases ambulation in
rs
the open field test at P40 in
male offspring
P84 Mice Fluoxetine P4-21 Fluoxetine increases anxiety- i.p. injections to Ansorge et al.
(10 mg/kg) like behavior offspring; mouse 2004
species not reported
he
P140-154 Wistar rat LU 10-134-C P8-21 Lu-10-134-C increases the s.c. injections to Hansen et al. 1997
(2.5, 5, 10 mg/kg immobility time on the forced offspring; males only
twice a day) swim test
is
bl
Pu
a
ov
N
Furthermore, work we have done using a model of maternal adversity shows that
developmental exposure to maternal fluoxetine reverses the decrease in depression-related
behavior seen after prenatal stress exposure (I. Rayen et al. 2011). The mechanism behind this
result is unclear; thus how fluoxetine may act to reverse the effect of prenatal stress on
depression-related behavior in adolescence remains unknown. Evidence suggests that prenatal
c.
maternal stress programs the HPA axis as well as behavior, and plasticity of the developing
monoamine system in the brain is partly responsible for these changes (Charil et al. 2010;
Glover et al. 2010). Developmental fluoxetine exposure also impacts HPA function (Ishiwata
In
et al. 2005; Morrison et al. 2004; Morrison et al. 2005; Oberlander et al. 2009; Pawluski et al.
2012b; Pawluski et al. 2012a). For example, postnatal fluoxetine exposure reverses the high
corticosterone levels associated with prenatal stress in juvenile mice offspring (Ishiwata et al.
2005) and reduces serum corticosterone levels, hippocampal GR, and GRIP1 expression in
adolescent male offspring (Pawluski et al. 2012b). Thus, early exposure to SSRIs may
‗modulate‘ physiological systems, such as the HPA system, which are influenced by exposure
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to maternal adversity and play an important role in the development of mood disorders.
Interestingly, the effects of developmental exposure to fluoxetine on the HPA system,
neural plasticity, and related behaviors are, at times, sexually differentiated with particularly
he
more evident effects in male, but not female, offspring. Although, it is not known why there is
such a sex difference in the HPA system after exposure to developmental fluoxetine, it is
likely that sex steroids modulate these sex differences, as estradiol can influence HPA
function (Atkinson and Waddell 1997; Viau and Meaney 1991; Viau 2002). Serotonin
concentrations also differ in many brain regions in male and female offspring (Duchesne et al.
is
2009) and, therefore, changing the serotonin concentration via SSRI exposure during
development may alter the serotonergic system in a sexually dimorphic way.
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The Developmental Effects of Fluoxetine Exposure
on the HPG Axis
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As mentioned previously, depression, function of the HPA system, and hippocampal

plasticity differ in males and females (Fernandez-Guasti et al. 2012; Galea et al. 2006; Vega
et al. 2011). Not surprisingly, serotonin plays a key role in sexual differentiation through its
role in the development of the hypothalamic-pituitary-gonadal (HPG) axis (Dohler et al.
1991; Dohler 1991; Jarzab and Dohler 1984). Sexual differentiation of the brain depends on
actions of estrogens and androgens (Dohler 1991; Segarra et al. 1991). In male rats, plasma
a
testosterone levels peak on days 18-19 of gestation (Ward and Weisz 1980; Weisz and Ward
1980) and a second peak in testosterone occurs during the first few hours after birth (Baum et
al. 1988; Corbier et al. 1978; Lalau et al. 1990; Slob et al. 1980). Testosterone, and mainly its
ov
estrogenic metabolites synthesized from local aromatization, is essential for masculinization

and defeminization of the brain during this critical period of sexual differentiation (Rhees et
al. 1997). In females, α-fetoprotein (AFP), an important plasma binding globulin during fetal
development, and sequestered systemic estradiol prevents the masculinization effect of
N
systemic estradiol on the brain (Bakker and Brock 2010).

Developmental SSRIs and Sexual Differentiation
Preclinical data has shown that developmental exposure to SSRIs may have a long-term
effects on the HPG system in both male and female offspring (Harris et al. 2012; Maciag et
al. 2006c; I. Rayen et al. 2013, 2014a) (Table 2).
c.
Male Offspring
Recent work has shown that perinatal fluoxetine exposure can inhibit and delay testicular
In
development, adversely affecting several testicular parameters important for the establishment
of sperm production in adulthood (de Oliveira et al. 2013; Muller et al. 2013; Vieira et al.
2013). We have also found reduction in anogenital distance after developmental fluoxetine
exposure in juvenile male offspring. This reduction in anogenital distance may be due, in part,
to increases in serotonin levels during the organizational stage of sexual brain differentiation,
rs
which occurs early during development (Arnold and Breedlove 1985). Interestingly,
alterations of anogenital distance can be an early indicator of diminished sexual behavior in
adulthood (Keshet and Weinstock 1995), supporting findings that adult male offspring have
diminished sexual behavior after exposure to SSRI medications during development (Maciag
he
et al. 2006c; I. Rayen et al. 2013).
Mirmiran et al. (1981) were perhaps the first to report that early postnatal exposure to the
tricyclic antidepressant, chlorimipramine, leads to dimished sexual behavior in adult male rats
(Mirmiran et al. 1981). Others also replicated these results showing that neonatal exposure to
the tricyclic antidepressant, clomipramine (15 mg/kg, s.c., twice daily), leads to diminished
is
sexual behavior in male rat offspring, including decreases in mounting, intromission and
ejaculatory behavior (Vogel et al. 1990). Recent work on the developmental impact of SSRI
medications on sexual behavior in male offspring has shown that only postnatal exposure to
bl
SSRI medication affects sexual behavior; Olivier et al. (2011) and Cagliano et al. (2008)
report no effect of exposure to fluoxetine during gestation on copulatory behavior in adult
male offspring (Cagiano et al. 2008; Olivier et al. 2011). Postnatal injection of citalopram to
pups results in impaired mounting behavior and a decrease in the number of intromissions and
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ejaculations (Harris et al. 2012; Maciag et al. 2006c). This impairment in sexual behavior in
male rats neonatally exposed to citalopram is mimicked by the stimulation of the 5-HT1B
receptor and partly by stimulation of the 5-HT1A and the 5HT2 receptor (Harris et al. 2012;
Maciag et al. 2006a; Maciag et al. 2006c; C.A. Wilson et al. 1998), indicating a contribution
of the serotonin autoreceptors during development on male sexual behavior.
Postnatal exposure to fluoxetine also results in impairments in male sexual behavior;
perinatal exposure to fluoxetine impairs sexual motivation in male mice (Gouvea et al. 2008)
a
and postnatal administration of fluoxetine to pups directly disrupts male sexual behavior with
fewer mounts, intromissions and ejaculations in adult male offspring (Rodriguez-Porcel et al.
ov
2011). We have also found that postnatal fluoxetine exposure, via suckling, impairs male
sexual behavior (I. Rayen et al. 2013), with a decrease in the number of intromissions, a
longer latency to the first intromission, and a longer latency to the first ejaculation. However,
we have shown that if fluoxetine exposure occurs after maternal stress (more analogous to the
clinical situation) than there is no effect of fluoxetine on male sexual behavior. This suggests
N
that the effects of developmental exposure to SSRI medications on sexual behavior in male
offspring appears only when given to healthy dams and/or offspring, and that maternal stress
can alter sexual differentiation in such a way that limits the effects of fluoxetine on
copulatory behaviors.
These changes in male sexual behavior with postnatal SSRI exposure have been
associated with changes in the medial preoptic area (MPOA) (I. Rayen et al. 2013; Soga et al.
2012). We have shown that developmental exposure to both fluoxetine and maternal stress
c.
significantly decreases the area of the SDN-POA, a sexually differentiated brain area. In
addition, serotonin levels are significantly reduced in males between the 2nd and 3rd
postnatal week (Ladosky and Gaziri 1970). The drop in serotonin activity appears essential
In
for full masculinization of the brain, given that administration of the serotonin precursor 5-
hydroxytryptophan or serotonin receptor 5-HT2 agonist (K)[2,5-dimethoxy-4-iodophenyl]-2-
amino propane to males or androgenized females, over a period that covers the transient fall
in endogenous serotonin, reduces the size of the SDN-POA, as well as the AVPv (Murray et
al. 2004). Conversely, treatment with parachlorophenylalanine, a serotonin synthesis
rs
inhibitor, enhances masculinization and defeminization (C.A. Wilson et al. 1998).
Interestingly, manipulation of serotonin synthesis during the first week after birth in males
has no effect on subsequent adult reproductive function or behavior (Jarzab and Dohler 1984;
C. A. Wilson et al. 1986; C.A. Wilson et al. 1998). Therefore, inhibition of the natural
he
serotonin drop, resulting from maternal fluoxetine exposure, likely antagonizes the perinatal
masculinization effects of testosterone during the second and/or third week postpartum. It
should also be noted that developmental exposure to another SSRI, citalopram, led to a
significant reduction in the number of androgen receptor-immunoreactive cells in the MPOA
(Soga et al. 2012). Although estrogen receptor activation by estrogenic metabolites of
is
testosterone is of primary importance to induce masculinization and defeminization of the
brain and male sexual behavior, androgen receptors are required to allow perinatal
testosterone to induce complete masculinization of brain and behavior (Sato et al. 2004). The
bl
effect of SSRI medications and serotonin receptor activation on androgen receptors therefore,
requires further investigation.
Female Offspring
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In contrast to the effects in male offspring, developmental exposure to SSRI medications

has no effect on reproductive organ weight, vaginal opening (Muller et al. 2013) or estrous
cyclicity in female offspring (I. Rayen et al. 2014a). Furthermore, developmental exposure to
fluoxetine facilitates proceptive and receptive behaviors in adult female offspring with a
significant increase in the number of proceptive behaviors, a significant increase in the
lordosis quotient, and a significant decrease in the rejection quotient (I. Rayen et al. 2014a).
Previous research has shown that the actions of serotonin on sexual differentiation in the
a
developing female are dependent on the timing of serotonergic manipulations, and often show
a down regulation of female sexual behavior if the serotonergic system is manipulated during
ov
the first week of life. For example, stimulation of serotonin synthesis by injection of l-
tryptophan on postnatal days 1–7 results in an inhibition of female lordosis behavior (Dohler
et al. 1991; Jarzab and Dohler 1984), and treatment with p-chlorophenylalanine (PCPA), a
serotonin synthesis inhibitor, between postnatal days 1 to 7 reduces ear wiggling (Dakin et al.
2008). In addition, 5-hydroxytryptophan (5-HTP), a serotonin precursor administered on
N
postnatal days 1 to 7 to female offspring, stimulates male-type behavior by reducing the

latency to the first mount made by the female (C. A. Wilson et al. 1986).
c.
In
Table 2. Preclinical data investigating perinatal SSRI exposure on sexual differentiation. P=postnatal day, G=gestational day
SSRI
Sex Species SSRI admin Results Comments Reference
(dose/day)
Female Sprague-Dawley Fluoxetine P1-21 Fluoxetine facilitates sexual behaviors, Osmotic minipump to Rayen et al. 2014
rs
rat (5mg/kg) regardless of maternal stress exposure, the dam; model of
and does not affect SDN-POA, AVPv and maternal stress
pBST brain areas
Fluoxetine does not affect estrous
cyclicity
he
Male/Female Sprague-Dawley Fluoxetine P1-21 Fluoxetine exposure reduces anogenital Osmotic minipump to Rayen et al. 2013
rat (5mg/kg) distance in males only, regardless of the dam; model of
maternal stress maternal stress
Male Sprague-Dawley Fluoxetine P1-21 Fluoxetine exposure inhibits sexual Osmotic minipump to Rayen et al. 2013
rat (5mg/kg) behavior. the dam; model of
Fluoxetine exposure after maternal stress maternal stress
is
decreases the area of the SDN-POA
Male Wistar rat Fluoxetine G13-P21 Fluoxetine reduces the body and testis Oral gavage to dam de Oliveira et al.
(5, 10, and 20 weights, volume of the seminiferous 2013
mg/kg)
bl tubules and epithelium

Fluoxetine affects the length of the
seminiferous tubules and the population of
Sertoli cells
Pu
Male/Female Wistar rat Fluoxetine G7-P21 Females: no effect of fluoxetine on organ Oral gavage to dam Müller et al. 2013
(17 mg/kg) weight or vaginal opening
Males: fluoxetine exposure tended to
reduce daily sperm production
Male Wistar rat Fluoxetine G0-P21 Fluoxetine decreases weight of the full Oral gavage to dam Vieira et al. 2012
(7.5mg/kg) seminal vesicles, decreases the number of
spermatozoa, reduces seminiferous
a
epithelium height and diameter of

seminiferous tubules
ov
N
c.
In
Table 2. (Continued)
SSRI
Sex Species SSRI admin Results Comments Reference
(dose/day)
Male Long Evans rat Citalopram (5, P8-21 Citalopram exposure results in dose- s.c. injections to Harris et al., 2012
rs
10 or dependent reductions mounting, offspring
20mg/kg/d) intromissions and ejaculations
Male Wistar rat Flouxetine G11-21 Fluoxetine does not affect male sexual s.c. injections to dam Olivier et al. 2011
(12 mg/kg) behavior
Male Long-Evans rat Fluoxetine P8–21 Fluoxetine disrupts male sex behavior: s.c. injections to Rodriguez Porcel
he
(5mg/kg) fewer mounts, intromissions and offspring et al. 2011
ejaculations
Male Wistar Fluoxetine G13-20 Fluoxetine has no effect on male sex s.c. injections to dam Cagliano et al.
rat (5 and 10 behavior 2008
mg/kg)
Male Swiss mice Flouxetine G0-P21 Fluoxetine impairs sexual motivation Oral gavage to dam Gouvea et al. 2008
(7.5 mg/kg)
is
Male Long Evans rat Citalopram P8-21 Citalopram decreases sexual activity: s.c. injections to Maciag et al. 2006
(5 mg/kg) fewer mounts, intromissions and offspring
ejaculations
bl
Pu
a
ov
N
Our work on the effects of developmental exposure to SSRI medications on female

sexual behavior expands previous work by showing that continual exposure to the SSRI,
fluoxetine, during postnatal day 1–21 increases proceptivity and receptivity in adult female
offspring. This suggests that the timing of exposure to the increased serotonin levels is
important for the effect on female sexual behavior. Interestingly, sex differences in brain
c.
serotonin levels appear between days 11 and 16 of life, suggesting that manipulation of the
serotonin levels during this period has a different effect on male and female sexual behavior
(Dohler et al. 1991; Jarzab and Dohler 1984). More research is needed to determine the
In
mechanisms behind the effects of developmental SSRI exposure on sex differences in male
and female offspring.
General Conclusions and Future Directions
rs
The present chapter highlights research investigating effects of developmental fluoxetine
exposure on the emerging HPA and HPG systems. In summary, preclinical research points to
an active role of perinatal SSRI exposure on many lasting neurobehavioral outcomes in male
he
and female offspring. This is not surprising as serotonin is a key player in the development
and modulation of the HPA system (Andrews and Matthews 2004; Erdeljan et al. 2005),
hippocampal plasticity (Brezun and Daszuta 1999b), affect-related behaviors (Ansorge et al.
2004; Ansorge et al. 2008; Hansen et al. 1997; Lisboa et al. 2007; I. Rayen et al. 2011) and
sexual differentiation of the brain and behavior (Dohler et al. 1991; Jarzab and Dohler 1984;
is
Ladosky and Gaziri 1970). For example, serotonin is involved in HPA programming during
development, as serotonin increases hippocampal GR transcription in a dose-dependent
manner (Erdeljan et al. 2001). In addition, there appears to be a bidirectional relationship
bl
between serotonin and glucocorticoids, with glucocorticoids altering neurotransmission
mediated by serotonin (Joels et al. 1995; Meijer and de Kloet 1998). As mentioned
previously, serotonin also plays an important role in sexual differentiation of the brain and
behavior (Dohler et al. 1991; Jarzab and Dohler 1984; Ladosky and Gaziri 1970). For
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example, in male offspring, developmental exposure to SSRIs inhibits sexual behavior

(Maciag et al. 2006c; I. Rayen et al. 2013), while in female offspring it facilitates sexual
behavior (I. Rayen et al. 2014a).
In addition, it should be highlighted that much of the preclinical research on the effects of
early exposure to SSRIs has primarily investigated the effects of SSRI treatment in healthy
offspring, i.e., offspring that have not been subjected to maternal adversity. However, the
a
effects of developmental exposure to SSRI medications can vary in the presence of maternal
adversity (Ishiwata et al. 2005; Knaepen et al. 2013b; Pawluski 2011). Given that clinical
studies focus on effects of SSRIs in mothers suffering from mood disorders, further
ov
preclinical research is needed to understand the impact of SSRIs on neurobehavioral

outcomes in offspring exposed to maternal adversity.
The use of SSRI antidepressant medications, such as fluoxetine, to treat maternal mood
disorders during the perinatal period is of increasing concern, as up to 10 % of women are on
SSRIs during pregnancy and in the postpartum period (Cooper et al. 2007; Oberlander et al.
N
2006). These medications are capable of passing through the placental barrier and are also
evident in breast milk (Gentile et al. 2007; Gentile 2007). It is, therefore, important to
understand the long-term implications of developmental exposure to fluoxetine, and other

SSRIs, on offspring outcomes. With greater information on the risks and benefits of
developmental SSRI exposure, appropriate recommendations can be made on the use of these
medications during pregnancy and in the postpartum period.
c.
References
In
Almond, P. (2009), 'Postnatal depression: a global public health perspective', Perspect Public
Health, 129 (5), 221-7.
Andrade, S. E., Raebel, M. A., Brown, J., Lane, K., Livingston, J., Boudreau, D., Rolnick, S.
J., Roblin, D., Smith, D. H., Willy, M. E., Staffa, J. A., and Platt, R. (2008), 'Use of
antidepressant medications during pregnancy: a multisite study', Am J Obstet Gynecol,
rs
198 (2), 194 e1-5.
Andrews, M. H. and Matthews, S. G. (2004), 'Programming of the hypothalamo-pituitary-
adrenal axis: serotonergic involvement', Stress, 7 (1), 15-27.
Ansorge, M. S., Hen, R., and Gingrich, J. A. (2007), 'Neurodevelopmental origins of
he
depressive disorders', Curr Opin Pharmacol, 7 (1), 8-17.
Ansorge, M. S., Morelli, E., and Gingrich, J. A. (2008), 'Inhibition of serotonin but not
norepinephrine transport during development produces delayed, persistent perturbations
of emotional behaviors in mice', J Neurosci, 28 (1), 199-207.
Ansorge, M. S., Zhou, M., Lira, A., Hen, R., and Gingrich, J. A. (2004), 'Early-life blockade
is
of the 5-HT transporter alters emotional behavior in adult mice', Science, 306 (5697),
879-81.
Arnold, A. P. and Breedlove, S. M. (1985), 'Organizational and activational effects of sex
bl
steroids on brain and behavior: a reanalysis', Horm Behav, 19 (4), 469-98.
Atkinson, H. C. and Waddell, B. J. (1997), 'Circadian variation in basal plasma corticosterone
and adrenocorticotropin in the rat: sexual dimorphism and changes across the estrous
cycle', Endocrinology, 138 (9), 3842-8.
Pu
Bakker, J. and Brock, O. (2010), 'Early oestrogens in shaping reproductive networks:

evidence for a potential organisational role of oestradiol in female brain development', J
Neuroendocrinol, 22 (7), 728-35.
Banasr, M., Hery, M., Brezun, J. M., and Daszuta, A. (2001), 'Serotonin mediates oestrogen
stimulation of cell proliferation in the adult dentate gyrus', Eur J Neurosci, 14 (9), 1417-
24.
a
Baum, M. J., Brand, T., Ooms, M., Vreeburg, J. T., and Slob, A. K. (1988), 'Immediate
postnatal rise in whole body androgen content in male rats: correlation with increased
testicular content and reduced body clearance of testosterone', Biol Reprod, 38 (5), 980-6.
ov
Baumann, P. and Rochat, B. (1995), 'Comparative pharmacokinetics of selective serotonin

reuptake inhibitors: a look behind the mirror', Int Clin Psychopharmacol, 10 Suppl 1, 15-
21.
Belzung, C. and Lemoine, M. (2011), 'Criteria of validity for animal models of psychiatric
disorders: focus on anxiety disorders and depression', Biol Mood Anxiety Disord, 1 (1), 9.
N
Beversdorf, D. Q., Manning, S. E., Hillier, A., Anderson, S. L., Nordgren, R. E., Walters, S.
E., Nagaraja, H. N., Cooley, W. C., Gaelic, S. E., and Bauman, M. L. (2005), 'Timing of
prenatal stressors and autism', J Autism Dev Disord, 35 (4), 471-8.
Bhattacharyya, A., Oppenheim, R. W., Prevette, D., Moore, B. W., Brackenbury, R., and
Ratner, N. (1992), 'S100 is present in developing chicken neurons and Schwann cells and
c.
promotes motor neuron survival in vivo', J Neurobiol, 23 (4), 451-66.
Brennan, P. A., Pargas, R., Walker, E. F., Green, P., Newport, D. J., and Stowe, Z. (2008),
'Maternal depression and infant cortisol: influences of timing, comorbidity and treatment',
In
J Child Psychol Psychiatry, 49 (10), 1099-107.
Brezun, J. M. and Daszuta, A. (1999a), 'Serotonin depletion in the adult rat produces
differential changes in highly polysialylated form of neural cell adhesion molecule and
tenascin-C immunoreactivity', J Neurosci Res, 55 (1), 54-70.
Brezun, J. M. and Daszuta, A. (1999b), 'Depletion in serotonin decreases neurogenesis in the
rs
dentate gyrus and the subventricular zone of adult rats', Neuroscience, 89 (4), 999-1002.
Burke, H. M., Davis, M. C., Otte, C., and Mohr, D. C. (2005), 'Depression and cortisol
responses to psychological stress: a meta-analysis', Psychoneuroendocrinology, 30 (9),
846-56.
he
Cagiano, R., Flace, P., Bera, I., Maries, L., Cioca, G., Sabatini, R., Benagiano, V., Auteri, P.,
Marzullo, A., Vermesan, D., Stefanelli, R., and Ambrosi, G. (2008), 'Neurofunctional
effects in rats prenatally exposed to fluoxetine', Eur Rev Med Pharmacol Sci, 12 (3), 137-
48.
Charil, A., Laplante, D. P., Vaillancourt, C., and King, S. (2010), 'Prenatal stress and brain
is
development', Brain Res Rev, 65 (1), 56-79.
Clarke, A. S., Kraemer, G. W., and Kupfer, D. J. (1998), 'Effects of rearing condition on HPA
axis response to fluoxetine and desipramine treatment over repeated social separations in
bl
young rhesus monkeys', Psychiatry Res, 79 (2), 91-104.
Colvin, L., Slack-Smith, L., Stanley, F. J., and Bower, C. (2011), 'Dispensing patterns and
pregnancy outcomes for women dispensed selective serotonin reuptake inhibitors in
pregnancy', Birth Defects Res A Clin Mol Teratol, 91 (3), 142-52.
Pu
Cooper, W. O., Willy, M. E., Pont, S. J., and Ray, W. A. (2007), 'Increasing use of
antidepressants in pregnancy', Am J Obstet Gynecol, 196 (6), 544 e1-5.
Corbier, P., Kerdelhue, B., Picon, R., and Roffi, J. (1978), 'Changes in testicular weight and
serum gonadotropin and testosterone levels before, during, and after birth in the perinatal
rat', Endocrinology, 103 (6), 1985-91.
Dakin, C. L., Wilson, C. A., Kallo, I., Coen, C. W., and Davies, D. C. (2008), 'Neonatal
stimulation of 5-HT(2) receptors reduces androgen receptor expression in the rat
a
anteroventral periventricular nucleus and sexually dimorphic preoptic area', Eur J

Neurosci, 27 (9), 2473-80.
ov
Davidson, J. S., Bolland, M. J., Croxson, M. S., Chiu, W., and Lewis, J. G. (2006), 'A case of
low cortisol-binding globulin: use of plasma free cortisol in interpretation of
hypothalamic-pituitary-adrenal axis tests', Ann Clin Biochem, 43 (Pt 3), 237-9.
Davidson, S., Prokonov, D., Taler, M., Maayan, R., Harell, D., Gil-Ad, I., and Weizman, A.
(2009), 'Effect of exposure to selective serotonin reuptake inhibitors in utero on fetal
N
growth: potential role for the IGF-I and HPA axes', Pediatr Res, 65 (2), 236-41.
de Oliveira, W. M., de Sa, I. R., de Torres, S. M., de Morais, R. N., Andrade, A. M., Maia, F.
C., Tenorio, B. M., and da Silva Junior, V. A. (2013), 'Perinatal exposure to fluoxetine
via placenta and lactation inhibits the testicular development in male rat offspring', Syst
Biol Reprod Med, 59 (5), 244-50.
Djavadian, R. L. (2004), 'Serotonin and neurogenesis in the hippocampal dentate gyrus of
adult mammals', Acta Neurobiol Exp (Wars), 64 (2), 189-200.
Dohler, K. D. (1991), 'The pre- and postnatal influence of hormones and neurotransmitters on
c.
sexual differentiation of the mammalian hypothalamus', Int Rev Cytol, 131, 1-57.
Dohler, K. D., Jarzab, B., Sickmoller, P. M., Kokocinska, D., Kaminski, M., Gubala, E.,
Achtelik, W., and Wagiel, J. (1991), 'Influence of neurotransmitters on sexual
In
differentiation of brain structure and function', Exp Clin Endocrinol, 98 (2), 99-109.
Dranovsky, A. and Hen, R. (2006), 'Hippocampal neurogenesis: regulation by stress and
antidepressants', Biol Psychiatry, 59 (12), 1136-43.
Duchesne, A., Dufresne, M. M., and Sullivan, R. M. (2009), 'Sex differences in corticolimbic
dopamine and serotonin systems in the rat and the effect of postnatal handling', Prog
rs
Neuropsychopharmacol Biol Psychiatry, 33 (2), 251-61.
Eiland, L. and Romeo, R. D. (2013), 'Stress and the developing adolescent brain',
Neuroscience, 249, 162-71.
Eisch, A. J. and Petrik, D. (2012), 'Depression and hippocampal neurogenesis: a road to
he
remission?', Science, 338 (6103), 72-5.
Eisch, A. J., Cameron, H. A., Encinas, J. M., Meltzer, L. A., Ming, G. L., and Overstreet-
Wadiche, L. S. (2008), 'Adult neurogenesis, mental health, and mental illness: hope or
hype?', J Neurosci, 28 (46), 11785-91.
El Marroun, H., Jaddoe, V. W., Hudziak, J. J., Roza, S. J., Steegers, E. A., Hofman, A.,
is
Verhulst, F. C., White, T. J., Stricker, B. H., and Tiemeier, H. (2012), 'Maternal use of
selective serotonin reuptake inhibitors, fetal growth, and risk of adverse birth outcomes',
Arch Gen Psychiatry, 69 (7), 706-14.
bl
Erdeljan, P., MacDonald, J. F., and Matthews, S. G. (2001), 'Glucocorticoids and serotonin
alter glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR) mRNA levels
in fetal mouse hippocampal neurons, in vitro', Brain Res, 896 (1-2), 130-6.
Erdeljan, P., Andrews, M. H., MacDonald, J. F., and Matthews, S. G. (2005),
Pu
'Glucocorticoids and serotonin alter glucocorticoid receptor mRNA levels in fetal guinea-
pig hippocampal neurons, in vitro', Reprod Fertil Dev, 17 (7), 743-9.
Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A. M., Nordborg, C., Peterson, D.
A., and Gage, F. H. (1998), 'Neurogenesis in the adult human hippocampus', Nat Med, 4
(11), 1313-7.
Fernandez-Guasti, A., Fiedler, J. L., Herrera, L., and Handa, R. J. (2012), 'Sex, stress, and
mood disorders: at the intersection of adrenal and gonadal hormones', Horm Metab Res,
a
44 (8), 607-18.
Flandreau, E. I., Bourke, C. H., Ressler, K. J., Vale, W. W., Nemeroff, C. B., and Owens, M.
ov
J. (2013), 'Escitalopram alters gene expression and HPA axis reactivity in rats following
chronic overexpression of corticotropin-releasing factor from the central amygdala',
Psychoneuroendocrinology, 38 (8), 1349-61.
Fleschler, R. and Peskin, M. F. (2008), 'Selective serotonin reuptake inhibitors (SSRIs) in
pregnancy: a review', MCN Am J Matern Child Nurs, 33 (6), 355-61; quiz 62-3.
N
Galea, L. A. (2008), 'Gonadal hormone modulation of neurogenesis in the dentate gyrus of

adult male and female rodents', Brain Res Rev, 57 (2), 332-41.
Galea, L. A., Spritzer, M. D., Barker, J. M., and Pawluski, J. L. (2006), 'Gonadal hormone
modulation of hippocampal neurogenesis in the adult', Hippocampus, 16 (3), 225-32.
Gaspar, P., Cases, O., and Maroteaux, L. (2003), 'The developmental role of serotonin: news
from mouse molecular genetics', Nat Rev Neurosci, 4 (12), 1002-12.
Gentile, S. (2005), 'The safety of newer antidepressants in pregnancy and breastfeeding',
c.
Drug Saf, 28 (2), 137-52.
Gentile, S. (2007), 'Use of contemporary antidepressants during breastfeeding: a proposal for
a specific safety index', Drug Saf, 30 (2), 107-21.
In
Gentile, S., Rossi, A., and Bellantuono, C. (2007), 'SSRIs during breastfeeding: spotlight on
milk-to-plasma ratio', Arch Womens Ment Health, 10 (2), 39-51.
Glover, V., O'Connor, T. G., and O'Donnell, K. (2010), 'Prenatal stress and the programming
of the HPA axis', Neurosci Biobehav Rev, 35 (1), 17-22.
Gonzalez-Martinez, T., Perez-Pinera, P., Diaz-Esnal, B., and Vega, J. A. (2003), 'S-100
rs
proteins in the human peripheral nervous system', Microsc Res Tech, 60 (6), 633-8.
Gouvea, T. S., Morimoto, H. K., de Faria, M. J., Moreira, E. G., and Gerardin, D. C. (2008),
'Maternal exposure to the antidepressant fluoxetine impairs sexual motivation in adult
male mice', Pharmacol Biochem Behav, 90 (3), 416-9.
he
Hadjikhani, N. (2010), 'Serotonin, pregnancy and increased autism prevalence: is there a
link?', Med Hypotheses, 74 (5), 880-3.
Hansen, H. H., Sanchez, C., and Meier, E. (1997), 'Neonatal administration of the selective
serotonin reuptake inhibitor Lu 10-134-C increases forced swimming-induced immobility
in adult rats: a putative animal model of depression?', J Pharmacol Exp Ther, 283 (3),
is
1333-41.
Harris, S. S., Maciag, D., Simpson, K. L., Lin, R. C., and Paul, I. A. (2012), 'Dose-dependent
effects of neonatal SSRI exposure on adult behavior in the rat', Brain Res, 1429, 52-60.
bl
Herlenius, E. and Lagercrantz, H. (2004), 'Development of neurotransmitter systems during
critical periods', Exp Neurol, 190 Suppl 1, S8-21.
Herman, J. P., McKlveen, J. M., Solomon, M. B., Carvalho-Netto, E., and Myers, B. (2012),
'Neural regulation of the stress response: glucocorticoid feedback mechanisms', Braz J
Pu
Med Biol Res, 45 (4), 292-8.

Hodes, G. E., Hill-Smith, T. E., and Lucki, I. (2010a), 'Fluoxetine treatment induces dose
dependent alterations in depression associated behavior and neural plasticity in female
mice', Neurosci Lett, 484 (1), 12-6.
Hodes, G. E., Hill-Smith, T. E., Suckow, R. F., Cooper, T. B., and Lucki, I. (2010b), 'Sex-
specific effects of chronic fluoxetine treatment on neuroplasticity and pharmacokinetics
in mice', J Pharmacol Exp Ther, 332 (1), 266-73.
a
Homberg, J. R., Schubert, D., and Gaspar, P. (2010), 'New perspectives on the
neurodevelopmental effects of SSRIs', Trends Pharmacol Sci, 31 (2), 60-5.
ov
Huizink, A. C., Mulder, E. J., and Buitelaar, J. K. (2004), 'Prenatal stress and risk for
psychopathology: specific effects or induction of general susceptibility?', Psychol Bull,
130 (1), 115-42.
Huizink, A. C., Robles de Medina, P. G., Mulder, E. J., Visser, G. H., and Buitelaar, J. K.
(2003), 'Stress during pregnancy is associated with developmental outcome in infancy', J
N
Child Psychol Psychiatry, 44 (6), 810-8.

Ishiwata, H., Shiga, T., and Okado, N. (2005), 'Selective serotonin reuptake inhibitor
treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction',
Neuroscience, 133 (4), 893-901.
Jarzab, B. and Dohler, K. D. (1984), 'Serotoninergic influences on sexual differentiation of
the rat brain', Prog Brain Res, 61, 119-26.
c.
Joels, M., Hesen, W., and de Kloet, E. R. (1995), 'Long-term control of neuronal excitability
by corticosteroid hormones', J Steroid Biochem Mol Biol, 53 (1-6), 315-23.
Karpova, N. N., Lindholm, J., Pruunsild, P., Timmusk, T., and Castren, E. (2009), 'Long-
In
lasting behavioural and molecular alterations induced by early postnatal fluoxetine
exposure are restored by chronic fluoxetine treatment in adult mice', Eur
Neuropsychopharmacol, 19 (2), 97-108.
Kempermann, G. (2002), 'Regulation of adult hippocampal neurogenesis - implications for
novel theories of major depression', Bipolar Disord, 4 (1), 17-33.
rs
Kempermann, G. and Kronenberg, G. (2003), 'Depressed new neurons--adult hippocampal
neurogenesis and a cellular plasticity hypothesis of major depression', Biol Psychiatry, 54
(5), 499-503.
Keshet, G. I. and Weinstock, M. (1995), 'Maternal naltrexone prevents morphological and
he
behavioral alterations induced in rats by prenatal stress', Pharmacol Biochem Behav, 50
(3), 413-9.
Kinney, G. G., Vogel, G. W., and Feng, P. (1997), 'Decreased dorsal raphe nucleus neuronal
activity in adult chloral hydrate anesthetized rats following neonatal clomipramine
treatment: implications for endogenous depression', Brain Res, 756 (1-2), 68-75.
is
Kiryanova, V., McAllister, B. B., and Dyck, R. H. (2013), 'Long-term outcomes of
developmental exposure to fluoxetine: a review of the animal literature', Dev Neurosci,
35 (6), 437-9.
bl
Knaepen, L., Pawluski, J. L., Patijn, J., van Kleef, M., Tibboel, D., and Joosten, E. A.
(2013a), 'Perinatal maternal stress and serotonin signaling: Effects on pain sensitivity in
offspring', Dev Psychobiol.
Knaepen, L., Rayen, I., Charlier, T. D., Fillet, M., Houbart, V., van Kleef, M., Steinbusch, H.
Pu
W., Patijn, J., Tibboel, D., Joosten, E. A., and Pawluski, J. L. (2013b), 'Developmental
fluoxetine exposure normalizes the long-term effects of maternal stress on post-operative
pain in Sprague-Dawley rat offspring', PLoS One, 8 (2), e57608.
Kristensen, J. H., Ilett, K. F., Hackett, L. P., Yapp, P., Paech, M., and Begg, E. J. (1999),
'Distribution and excretion of fluoxetine and norfluoxetine in human milk', Br J Clin
Pharmacol, 48 (4), 521-7.
Ladosky, W. and Gaziri, L. C. (1970), 'Brain serotonin and sexual differentiation of the
a
nervous system', Neuroendocrinology, 6 (3), 168-74.

Lalau, J. D., Aubert, M. L., Carmignac, D. F., Gregoire, I., and Dupouy, J. P. (1990),
ov
'Reduction in testicular function in rats. II. Reduction by dexamethasone in fetal and

neonatal rats', Neuroendocrinology, 51 (3), 289-93.
Lancaster, C. A., Gold, K. J., Flynn, H. A., Yoo, H., Marcus, S. M., and Davis, M. M. (2010),
'Risk factors for depressive symptoms during pregnancy: a systematic review', Am J
Obstet Gynecol, 202 (1), 5-14.
N
Laplante, D. P., Barr, R. G., Brunet, A., Galbaud du Fort, G., Meaney, M. L., Saucier, J. F.,
Zelazo, P. R., and King, S. (2004), 'Stress during pregnancy affects general intellectual
and language functioning in human toddlers', Pediatr Res, 56 (3), 400-10.
Laplante, P., Diorio, J., and Meaney, M. J. (2002), 'Serotonin regulates hippocampal
glucocorticoid receptor expression via a 5-HT7 receptor', Brain Res Dev Brain Res, 139
(2), 199-203.
Lebrand, C., Cases, O., Adelbrecht, C., Doye, A., Alvarez, C., El Mestikawy, S., Seif, I., and
Gaspar, P. (1996), 'Transient uptake and storage of serotonin in developing thalamic
c.
neurons', Neuron, 17 (5), 823-35.
Lee, H. J., Kim, J. W., Yim, S. V., Kim, M. J., Kim, S. A., Kim, Y. J., Kim, C. J., and Chung,
J. H. (2001), 'Fluoxetine enhances cell proliferation and prevents apoptosis in dentate
In
gyrus of maternally separated rats', Mol Psychiatry, 6 (6), 610, 725-8.
Leung, B. M. and Kaplan, B. J. (2009), 'Perinatal depression: prevalence, risks, and the
nutrition link--a review of the literature', J Am Diet Assoc, 109 (9), 1566-75.
Limlomwongse, N. and Liabsuetrakul, T. (2006), 'Cohort study of depressive moods in Thai
women during late pregnancy and 6-8 weeks of postpartum using the Edinburgh
rs
Postnatal Depression Scale (EPDS)', Arch Womens Ment Health, 9 (3), 131-8.
Lisboa, S. F., Oliveira, P. E., Costa, L. C., Venancio, E. J., and Moreira, E. G. (2007),
'Behavioral evaluation of male and female mice pups exposed to fluoxetine during
pregnancy and lactation', Pharmacology, 80 (1), 49-56.
he
Louvart, H., Maccari, S., and Darnaudery, M. (2005), 'Prenatal stress affects behavioral
reactivity to an intense stress in adult female rats', Brain Res, 1031 (1), 67-73.
Lucassen, P. J., Meerlo, P., Naylor, A. S., van Dam, A. M., Dayer, A. G., Fuchs, E., Oomen,
C. A., and Czeh, B. (2010), 'Regulation of adult neurogenesis by stress, sleep disruption,
exercise and inflammation: Implications for depression and antidepressant action', Eur
is
Neuropsychopharmacol, 20 (1), 1-17.
Maccari, S. and Morley-Fletcher, S. (2007), 'Effects of prenatal restraint stress on the
hypothalamus-pituitary-adrenal axis and related behavioural and neurobiological
bl
alterations', Psychoneuroendocrinology, 32 Suppl 1, S10-5.
Maciag, D., Coppinger, D., and Paul, I. A. (2006a), 'Evidence that the deficit in sexual
behavior in adult rats neonatally exposed to citalopram is a consequence of 5-HT1
receptor stimulation during development', Brain Res, 1125 (1), 171-5.
Pu
Maciag, D., Williams, L., Coppinger, D., and Paul, I. A. (2006b), 'Neonatal citalopram
exposure produces lasting changes in behavior which are reversed by adult imipramine
treatment', Eur J Pharmacol, 532 (3), 265-9.
Maciag, D., Simpson, K. L., Coppinger, D., Lu, Y., Wang, Y., Lin, R. C., and Paul, I. A.
(2006c), 'Neonatal antidepressant exposure has lasting effects on behavior and serotonin
circuitry', Neuropsychopharmacology, 31 (1), 47-57.
Malberg, J.E., Eisch, A.J., Nestler, E.J., and Duman, R.S. (2000), 'Chronic antidepressant
a
treatment increases neurogenesis in adult rat hippocampus', J Neurosci, 20 (24), 9104-10.

Marcus, S. M. (2009), 'Depression during pregnancy: rates, risks and consequences--
ov
Motherisk Update 2008', Can J Clin Pharmacol, 16 (1), e15-22.

McAllister, B. B., Kiryanova, V., and Dyck, R. H. (2012), 'Behavioural outcomes of perinatal
maternal fluoxetine treatment', Neuroscience, 226, 356-66.
McEwen, B. S. (2005), 'Glucocorticoids, depression, and mood disorders: structural
remodeling in the brain', Metabolism, 54 (5 Suppl 1), 20-3.
N
McEwen, B. S. and Stellar, E. (1993), 'Stress and the individual. Mechanisms leading to
disease', Arch Intern Med, 153 (18), 2093-101.
Meaney, M. J., Diorio, J., Francis, D., LaRocque, S., O'Donnell, D., Smythe, J. W., Sharma,
S., and Tannenbaum, B. (1994), 'Environmental regulation of the development of
glucocorticoid receptor systems in the rat forebrain. The role of serotonin', Ann N Y Acad
Sci, 746, 260-73; discussion 74, 89-93.
Meijer, O. C. and de Kloet, E. R. (1998), 'Corticosterone and serotonergic neurotransmission
c.
in the hippocampus: functional implications of central corticosteroid receptor diversity',
Crit Rev Neurobiol, 12 (1-2), 1-20.
Mirmiran, M., van de Poll, N. E., Corner, M. A., van Oyen, H. G., and Bour, H. L. (1981),
In
'Suppression of active sleep by chronic treatment with chlorimipramine during early
postnatal development: effects upon adult sleep and behavior in the rat', Brain Res, 204
(1), 129-46.
Misri, S., Reebye, P., Kendrick, K., Carter, D., Ryan, D., Grunau, R. E., and Oberlander, T. F.
(2006), 'Internalizing behaviors in 4-year-old children exposed in utero to psychotropic
rs
medications', Am J Psychiatry, 163 (6), 1026-32.
Morrison, J. L., Riggs, K. W., and Rurak, D. W. (2005), 'Fluoxetine during pregnancy: impact
on fetal development', Reprod Fertil Dev, 17 (6), 641-50.
Morrison, J. L., Riggs, K. W., Chien, C., Gruber, N., McMillen, I. C., and Rurak, D. W.
he
(2004), 'Chronic maternal fluoxetine infusion in pregnant sheep: effects on the maternal
and fetal hypothalamic-pituitary-adrenal axes', Pediatr Res, 56 (1), 40-6.
Moses-Kolko, E. L., Bogen, D., Perel, J., Bregar, A., Uhl, K., Levin, B., and Wisner, K. L.
(2005), 'Neonatal signs after late in utero exposure to serotonin reuptake inhibitors:
literature review and implications for clinical applications', JAMA, 293 (19), 2372-83.
is
Muller, J. C., Boareto, A. C., Lourenco, E. L., Zaia, R. M., Kienast, M. F., Spercoski, K. M.,
Morais, R. N., Martino-Andrade, A. J., and Dalsenter, P. R. (2013), 'In utero and
lactational exposure to fluoxetine in Wistar rats: pregnancy outcomes and sexual
bl
development', Basic Clin Pharmacol Toxicol, 113 (2), 132-40.
Murray, J. F., Dakin, C. L., Siddiqui, A., Pellatt, L. J., Ahmed, S., Ormerod, L. J., Swan, A.
V., Davies, D. C., and Wilson, C. A. (2004), 'Neonatal 5HT activity antagonizes the
masculinizing effect of testosterone on the luteinizing hormone release response to
Pu
gonadal steroids and on brain structures in rats', Eur J Neurosci, 19 (2), 387-95.
Nagano, M., Liu, M., Inagaki, H., Kawada, T., and Suzuki, H. (2012), 'Early intervention with
fluoxetine reverses abnormalities in the serotonergic system and behavior of rats exposed
prenatally to dexamethasone', Neuropharmacology, 63 (2), 292-300.
Niederhofer, H. and Reiter, A. (2004), 'Prenatal maternal stress, prenatal fetal movements and
perinatal temperament factors influence behavior and school marks at the age of 6 years',
Fetal Diagn Ther, 19 (2), 160-2.
a
Nulman, I., Rovet, J., Stewart, D. E., Wolpin, J., Pace-Asciak, P., Shuhaiber, S., and Koren,
G. (2002), 'Child development following exposure to tricyclic antidepressants or
ov
fluoxetine throughout fetal life: a prospective, controlled study', Am J Psychiatry, 159

(11), 1889-95.
Oberlander, T. F., Gingrich, J. A., and Ansorge, M. S. (2009), 'Sustained neurobehavioral
effects of exposure to SSRI antidepressants during development: molecular to clinical
evidence', Clin Pharmacol Ther, 86 (6), 672-7.
N
Oberlander, T. F., Warburton, W., Misri, S., Aghajanian, J., and Hertzman, C. (2006),
'Neonatal outcomes after prenatal exposure to selective serotonin reuptake inhibitor
antidepressants and maternal depression using population-based linked health data', Arch
Gen Psychiatry, 63 (8), 898-906.
Oberlander, T. F., Grunau, R. E., Fitzgerald, C., Papsdorf, M., Rurak, D., and Riggs, W.
(2005), 'Pain reactivity in 2-month-old infants after prenatal and postnatal serotonin
reuptake inhibitor medication exposure', Pediatrics, 115 (2), 411-25.
c.
Oberlander, T. F., Reebye, P., Misri, S., Papsdorf, M., Kim, J., and Grunau, R. E. (2007),
'Externalizing and attentional behaviors in children of depressed mothers treated with a
selective serotonin reuptake inhibitor antidepressant during pregnancy', Arch Pediatr
In
Adolesc Med, 161 (1), 22-9.
Oberlander, T. F., Papsdorf, M., Brain, U. M., Misri, S., Ross, C., and Grunau, R. E. (2010),
'Prenatal effects of selective serotonin reuptake inhibitor antidepressants, serotonin
transporter promoter genotype (SLC6A4), and maternal mood on child behavior at 3
years of age', Arch Pediatr Adolesc Med, 164 (5), 444-51.
rs
Oberlander, T. F., Eckstein Grunau, R., Fitzgerald, C., Ellwood, A. L., Misri, S., Rurak, D.,
and Riggs, K. W. (2002), 'Prolonged prenatal psychotropic medication exposure alters
neonatal acute pain response', Pediatr Res, 51 (4), 443-53.
Oberlander, T. F., Grunau, R., Mayes, L., Riggs, W., Rurak, D., Papsdorf, M., Misri, S., and
he
Weinberg, J. (2008), 'Hypothalamic-pituitary-adrenal (HPA) axis function in 3-month old
infants with prenatal selective serotonin reuptake inhibitor (SSRI) antidepressant
exposure', Early Hum Dev.
Olivier, J. D., Akerud, H., Kaihola, H., Pawluski, J. L., Skalkidou, A., Hogberg, U., and
Sundstrom-Poromaa, I. (2013), 'The effects of maternal depression and maternal selective
is
serotonin reuptake inhibitor exposure on offspring', Front Cell Neurosci, 7, 73.
Olivier, J. D., Valles, A., van Heesch, F., Afrasiab-Middelman, A., Roelofs, J. J., Jonkers, M.,
Peeters, E. J., Korte-Bouws, G. A., Dederen, J. P., Kiliaan, A. J., Martens, G. J.,
bl
Schubert, D., and Homberg, J. R. (2011), 'Fluoxetine administration to pregnant rats
increases anxiety-related behavior in the offspring', Psychopharmacology (Berl).
Palkovits, M. (1987), 'Organization of the stress response at the anatomical level', Prog Brain
Res, 72, 47-55.
Pu
Pawluski, J. L. (2011), 'Perinatal Selective Serotonin Reuptake Inhibitor Exposure: Impact on

Brain Development and Neural Plasticity', Neuroendocrinology.
Pawluski, J. L., Galea, L. A., Brain, U., Papsdorf, M., and Oberlander, T. F. (2009a),
'Neonatal S100B protein levels after prenatal exposure to selective serotonin reuptake
inhibitors', Pediatrics, 124 (4), e662-70.
Pawluski, J. L., Brummelte, S., Barha, C. K., Crozier, T. M., and Galea, L. A. (2009b),
'Effects of steroid hormones on neurogenesis in the hippocampus of the adult female
a
rodent during the estrous cycle, pregnancy, lactation and aging', Front Neuroendocrinol,
30 (3), 343-57.
ov
Pawluski, J. L., Brain, U. M., Underhill, C. M., Hammond, G. L., and Oberlander, T. F.
(2012a), 'Prenatal SSRI exposure alters neonatal corticosteroid binding globulin, infant
cortisol levels, and emerging HPA function', Psychoneuroendocrinology, 37 (7), 1019-
28.
Pawluski, J. L., Rayen, I., Niessen, N. A., Kristensen, S., van Donkelaar, E. L., Balthazart, J.,
N
Steinbusch, H. W., and Charlier, T. D. (2012b), 'Developmental fluoxetine exposure

differentially alters central and peripheral measures of the HPA system in adolescent
male and female offspring', Neuroscience, 220, 131-41.
Perera, T. D., Coplan, J. D., Lisanby, S. H., Lipira, C. M., Arif, M., Carpio, C., Spitzer, G.,
Santarelli, L., Scharf, B., Hen, R., Rosoklija, G., Sackeim, H. A., and Dwork, A. J.
(2007), 'Antidepressant-induced neurogenesis in the hippocampus of adult nonhuman
primates', J Neurosci, 27 (18), 4894-901.
Popa, D., Lena, C., Alexandre, C., and Adrien, J. (2008), 'Lasting syndrome of depression
c.
produced by reduction in serotonin uptake during postnatal development: evidence from
sleep, stress, and behavior', J Neurosci, 28 (14), 3546-54.
Rayen, I, Gemmel, M, Pauley, G, Steinbusch, H., and Pawluski, J. L. (under review),
In
'Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-
dependent effects on hippocampal plasticity', Psychopharmacology.
Rayen, I., Steinbusch, H. W., Charlier, T. D., and Pawluski, J. L. (2013), 'Developmental
fluoxetine exposure and prenatal stress alter sexual differentiation of the brain and
reproductive behavior in male rat offspring', Psychoneuroendocrinology.
rs
Rayen, I., Steinbusch, H. W., Charlier, T. D., and Pawluski, J. L. (2014a), 'Developmental
fluoxetine exposure facilitates sexual behavior in female offspring', Psychopharmacology
(Berl), 231 (1), 123-33.
Rayen, I., van den Hove, D. L., Prickaerts, J., Steinbusch, H. W., and Pawluski, J. L. (2011),
he
'Fluoxetine during development reverses the effects of prenatal stress on depressive-like
behavior and hippocampal neurogenesis in adolescence', PLoS One, 6 (9), e24003.
Rayen, I., Gemmel, M., Pauley, G., Steinbusch, H. W., and Pawluski, J. L. (2014b),
'Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-
dependent effects on hippocampal plasticity', Psychopharmacology (Berl).
is
Rhees, R. W., Kirk, B. A., Sephton, S., and Lephart, E. D. (1997), 'Effects of prenatal
testosterone on sexual behavior, reproductive morphology and LH secretion in the female
rat', Dev Neurosci, 19 (5), 430-7.
bl
Rodriguez-Porcel, F., Green, D., Khatri, N., Harris, S. S., May, W. L., Lin, R. C., and Paul, I.
A. (2011), 'Neonatal exposure of rats to antidepressants affects behavioral reactions to
novelty and social interactions in a manner analogous to autistic spectrum disorders',
Anat Rec (Hoboken), 294 (10), 1726-35.
Pu
Rothman, S. M. and Mattson, M. P. (2010), 'Adverse stress, hippocampal networks, and

Alzheimer's disease', Neuromolecular Med, 12 (1), 56-70.
Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee,
J., Duman, R., Arancio, O., Belzung, C., and Hen, R. (2003), 'Requirement of
hippocampal neurogenesis for the behavioral effects of antidepressants', Science, 301
(5634), 805-9.
Sato, T., Matsumoto, T., Kawano, H., Watanabe, T., Uematsu, Y., Sekine, K., Fukuda, T.,
a
Aihara, K., Krust, A., Yamada, T., Nakamichi, Y., Yamamoto, Y., Nakamura, T.,
Yoshimura, K., Yoshizawa, T., Metzger, D., Chambon, P., and Kato, S. (2004), 'Brain
ov
masculinization requires androgen receptor function', Proc Natl Acad Sci U S A, 101 (6),
1673-8.
Segarra, A. C., Luine, V. N., and Strand, F. L. (1991), 'Sexual behavior of male rats is
differentially affected by timing of perinatal ACTH administration', Physiol Behav, 50
(4), 689-97.
N
Simpson, K. L., Weaver, K. J., de Villers-Sidani, E., Lu, J. Y., Cai, Z., Pang, Y., Rodriguez-
Porcel, F., Paul, I. A., Merzenich, M., and Lin, R. C. (2011), 'Perinatal antidepressant
exposure alters cortical network function in rodents', Proc Natl Acad Sci U S A, 108 (45),
18465-70.
Slob, A. K., Ooms, M. P., and Vreeburg, J. T. (1980), 'Prenatal and early postnatal sex
differences in plasma and gonadal testosterone and plasma luteinizing hormone in female
and male rats', J Endocrinol, 87 (1), 81-7.
c.
Soga, T., Wong, D. W., Putteeraj, M., Song, K. P., and Parhar, I. S. (2012), 'Early-life
citalopram-induced impairments in sexual behavior and the role of androgen receptor',
In
Stewart, D. E. (2011), 'Clinical practice. Depression during pregnancy', N Engl J Med, 365
(17), 1605-11.
Tabori, N. E., Stewart, L. S., Znamensky, V., Romeo, R. D., Alves, S. E., McEwen, B. S., and
Milner, T. A. (2005), 'Ultrastructural evidence that androgen receptors are located at
extranuclear sites in the rat hippocampal formation', Neuroscience, 130 (1), 151-63.
rs
Talge, N. M., Neal, C., and Glover, V. (2007), 'Antenatal maternal stress and long-term
effects on child neurodevelopment: how and why?', J Child Psychol Psychiatry, 48 (3-4),
245-61.
Van den Bergh, B. R., Mulder, E. J., Mennes, M., and Glover, V. (2005a), 'Antenatal
he
maternal anxiety and stress and the neurobehavioural development of the fetus and child:
links and possible mechanisms. A review', Neurosci Biobehav Rev, 29 (2), 237-58.
Van den Bergh, B. R., Van Calster, B., Smits, T., Van Huffel, S., and Lagae, L. (2008),
'Antenatal maternal anxiety is related to HPA-axis dysregulation and self-reported
depressive symptoms in adolescence: a prospective study on the fetal origins of depressed
is
mood', Neuropsychopharmacology, 33 (3), 536-45.
Van den Bergh, B. R., Mennes, M., Oosterlaan, J., Stevens, V., Stiers, P., Marcoen, A., and
Lagae, L. (2005b), 'High antenatal maternal anxiety is related to impulsivity during
bl
performance on cognitive tasks in 14- and 15-year-olds', Neurosci Biobehav Rev, 29 (2),
259-69.
Van den Hove, D. L., Blanco, C. E., Aendekerk, B., Desbonnet, L., Bruschettini, M.,
Steinbusch, H. P., Prickaerts, J., and Steinbusch, H. W. (2005), 'Prenatal restraint stress
Pu
and long-term affective consequences', Dev Neurosci, 27 (5), 313-20.

Varrault, A., Bockaert, J., and Waeber, C. (1992), 'Activation of 5-HT1A receptors expressed
in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis',
Mol Biol Cell, 3 (9), 961-9.
Vega, P., Barbeito, S., Ruiz de Azua, S., Martinez-Cengotitabengoa, M., Gonzalez-Ortega, I.,
Saenz, M., and Gonzalez-Pinto, A. (2011), 'Bipolar disorder differences between genders:
special considerations for women', Womens Health (Lond Engl), 7 (6), 663-74; quiz 75-6.
a
Verney, C., Lebrand, C., and Gaspar, P. (2002), 'Changing distribution of monoaminergic
markers in the developing human cerebral cortex with special emphasis on the serotonin
ov
transporter', Anat Rec, 267 (2), 87-93.

Ververs, T., Kaasenbrood, H., Visser, G., Schobben, F., de Jong-van den Berg, L., and
Egberts, T. (2006), 'Prevalence and patterns of antidepressant drug use during pregnancy',
Eur J Clin Pharmacol, 62 (10), 863-70.
Viau, V. (2002), 'Functional cross-talk between the hypothalamic-pituitary-gonadal and -
N
adrenal axes', J Neuroendocrinol, 14 (6), 506-13.

Viau, V. and Meaney, M. J. (1991), 'Variations in the hypothalamic-pituitary-adrenal
response to stress during the estrous cycle in the rat', Endocrinology, 129 (5), 2503-11.
Vieira, M. L., Hamada, R. Y., Gonzaga, N. I., Bacchi, A. D., Barbieri, M., Moreira, E. G.,
Mesquita Sde, F., and Gerardin, D. C. (2013), 'Could maternal exposure to the
antidepressants fluoxetine and St. John's Wort induce long-term reproductive effects on
male rats?', Reprod Toxicol, 35, 102-7.
Vogel, G., Neill, D., Hagler, M., and Kors, D. (1990), 'A new animal model of endogenous
c.
depression: a summary of present findings', Neurosci Biobehav Rev, 14 (1), 85-91.
Wainwright, S. R. and Galea, L. A. (2013), 'The Neural Plasticity Theory of Depression:
Assessing the Roles of Adult Neurogenesis and PSA-NCAM within the Hippocampus',
In
Neural Plast, 2013, 805497.
Ward, I. L. and Weisz, J. (1980), 'Maternal stress alters plasma testosterone in fetal males',
Science, 207 (4428), 328-9.
Weaver, K. J., Paul, I. A., Lin, R. C., and Simpson, K. L. (2010), 'Neonatal exposure to
citalopram selectively alters the expression of the serotonin transporter in the
rs
hippocampus: dose-dependent effects', Anat Rec (Hoboken), 293 (11), 1920-32.
Weiland, N. G., Orikasa, C., Hayashi, S., and McEwen, B. S. (1997), 'Distribution and
hormone regulation of estrogen receptor immunoreactive cells in the hippocampus of
male and female rats', J Comp Neurol, 388 (4), 603-12.
he
Weinstock, M. (2007), 'Gender differences in the effects of prenatal stress on brain
development and behaviour', Neurochem Res, 32 (10), 1730-40.
Weinstock, M. (2008), 'The long-term behavioural consequences of prenatal stress', Neurosci
Biobehav Rev, 32 (6), 1073-86.
Weisz, J. and Ward, I. L. (1980), 'Plasma testosterone and progesterone titers of pregnant rats,
is
their male and female fetuses, and neonatal offspring', Endocrinology, 106 (1), 306-16.
Whitaker-Azmitia, P. M. (2001), 'Serotonin and brain development: role in human
developmental diseases', Brain Res Bull, 56 (5), 479-85.
bl
Whitaker-Azmitia, P. M., Murphy, R., and Azmitia, E. C. (1990), 'Stimulation of astroglial 5-
HT1A receptors releases the serotonergic growth factor, protein S-100, and alters
astroglial morphology', Brain Res, 528 (1), 155-8.
Whitaker-Azmitia, P. M., Druse, M., Walker, P., and Lauder, J. M. (1996), 'Serotonin as a
Pu
developmental signal', Behav Brain Res, 73 (1-2), 19-29.

Wilson, C. A., Pearson, J. R., Hunter, A. J., Tuohy, P. A., and Payne, A. P. (1986), 'The effect
of neonatal manipulation of hypothalamic serotonin levels on sexual activity in the adult
rat', Pharmacol Biochem Behav, 24 (5), 1175-83.
Wilson, C.A., Gonzalez, M.I., Albonetti, M.E., and Farabollini, F. (1998), 'The Involvement
of Neonatal 5-HT Receptor-Mediated Effects on Sexual Dimorphism of Adult Behavior
in the Rat. Males, femalesand behavior.', Praeger, Westport, USA, pp. 109–27.
a
Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989),
'Neurite extension and neuronal survival activities of recombinant S100 beta proteins that
ov
differ in the content and position of cysteine residues', J Cell Biol, 109 (6 Pt 1), 3063-71.
Yau, J. L., Noble, J., Chapman, K. E., and Seckl, J. R. (2004), 'Differential regulation of
variant glucocorticoid receptor mRNAs in the rat hippocampus by the antidepressant
fluoxetine', Brain Res Mol Brain Res, 129 (1-2), 189-92.
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Chapter 4
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The Impact of Fluoxetine Treatment
on BDNF Signaling and Cellular
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Plasticity in the Brain
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Jochen De Vry, Fabien Boulle, Bart P. F. Rutten,
Daniel L. A. van den Hove and Jos Prickaerts*
Department of Psychiatry and Neuropsychology,
School for Mental Health and Neuroscience,
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Maastricht University, Maastricht, the Netherlands
European Graduate School of Neuroscience (EURON),
Maastricht University, Maastricht, the Netherlands
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Abstract
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The neurotrophin hypothesis of depression, which states that the pathophysiology of

depression is caused by a lack of neurotrophic support, has gained much interest over the
past years. Chronic exposure to stress, a major determinant of depression decreases
neurotrophin production in the brain. These neurotrophins, like brain-derived
neurotrophic factor (BDNF), are critically involved in the proliferation, survival and
differentiation of new neurons in the subgranular zone of the hippocampus. Besides their
role in neurogenesis, neurotrophins play a pivotal role in synaptogenesis, synaptic
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plasticity, dendritic arborization and long-term potentiation. As such, neurotrophins are a

crucial factor regulating plasticity in the brain. The functional effects of neurotrophins are
evident from their important role in memory formation, cognitive function, and resilience
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to stress and depression. Not only do neurotrophins like BDNF have intrinsic
antidepressant properties, studies show that antidepressants, including selective serotonin
reuptake inhibitors (SSRI‘s) such as fluoxetine, critically depend on functional BDNF
signaling. The neurotrophin hypothesis of depression helps explaining why
antidepressants require a few weeks of administration before they become therapeutically
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effective, although they acutely increase neurotransmitter levels in the brain.

Antidepressants increase the expression of BDNF, and as a consequence of its
* Corresponding Author address, Email: jos.prickaerts@maastrichtuniversity.nl.

72 Jochen De Vry, Fabien Boulle, Bart P. F. Rutten et al.
neurotrophic properties, they gradually increase cellular plasticity and connectivity over
time. This slow repair mechanism is needed to counteract the atrophy in limbic structures
of the ―depressed‖ brain such as the hippocampus. This chapter focusses on the role of
BDNF in cellular plasticity in relation to affective disorders. Special attention is given as
to how BDNF signaling and its role in plasticity is implicated in the mechanism of action
of antidepressants, SSRI‘s in particular.
c.
Keywords: brain-derived neurotrophic factor, SSRI, plasticity, nucleus accumbens,
epigenetics
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The Neurotrophin Hypothesis of Depression
Major depression (MD) is a devastating mental disorder, expected to be the second
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leading cause of disability worldwide by 2020 (Murray and Lopez 1997). The neurotrophin
hypothesis of depression states that the pathophysiology of depression is caused by stress-
induced reductions in neurotrophic support (Duman et al. 2000, Jacobs et al. 2000).
Neurotrophins are a family of growth factors including brain-derived neurotrophic factor
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(BDNF) that are involved in the proliferation, survival and differentiation of new neurons
(i.e., neurogenesis) (Benraiss et al., 2001; Lee et al., 2002; Linnarsson et al., 2000; Sairanen et
al., 2005). Neurotrophic factors have been shown to increase hippocampal plasticity as is
evident from their neurogenic properties and their implications in neuronal survival and
synaptic plasticity (Figurov et al. 1996, Korte et al. 1995, Lee et al. 2002, Sairanen et al.
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2005). Loss of neurotrophic support following long-lasting exposure to stress is thought to be
a major mechanism causing atrophy and reduced plasticity in the brain (McEwen 1999, Smith
et al. 1995). The neurotrophin hypothesis has gained acceptance from observations showing
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lower hippocampal BDNF levels in post-mortem brains from suicide victims (Karege et al.
2005b). When suicide victims were treated with antidepressants at the time of death, only
slight decreases in BDNF levels are seen (Chen et al. 2001b). Different classes of
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antidepressants also augment hippocampal BDNF levels in various animal models for
depression (Calabrese et al. 2010, Nibuya et al. 1995, Xu et al. 2006), and evidence is
accumulating that BDNF itself contains antidepressant properties. When directly injected in
the hippocampus, BDNF exerts antidepressant effects in rodent models and tests of
depression (Gourley et al. 2008, Shirayama et al. 2002), while a reduction or loss of BDNF in
the hippocampus attenuates antidepressant effects in mice (Adachi et al. 2008, Monteggia et
al. 2007). These studies confirm that BDNF signaling in the hippocampus is highly involved
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in the etiology of depression and in antidepressant functioning. In addition, neurotrophins

explain the delayed behavioural response of antidepressant treatment. Although classical
antidepressants quickly increase neurotransmitter levels, it takes 2-3 weeks of antidepressant
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treatment before they become effective. This delay is attributed to required changes in
plasticity that are stimulated by increased neurotrophin levels.
Importantly, the involvement of neurotrophins in the onset of depression and in
antidepressant efficacy is not explained by functional changes in neurons only. Glial cells in
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the hippocampus decrease in number in depressed patients and in animal models for
depression (Czeh et al. 2007, Muller et al. 2001), and antidepressants increase the amount of
glial cells in the hippocampus and frontal cortex (Czeh et al. 2007, Malberg et al. 2000,
The Impact of Fluoxetine Treatment on BDNF Signaling and Cellular Plasticity … 73
Santarelli et al. 2003). Importantly, exposing rodents to stress or antidepressant treatment

does not alter the ratio between glial and neuronal cells which suggests that glial cells can be
equally important in the pathophysiology of depression and in antidepressant functioning
(Czeh et al. 2007, Malberg et al. 2000, Santarelli et al. 2003). Indeed, deleting basal forebrain
BDNF specifically in astrocytes induces depression-like behaviour and attenuates
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antidepressant response in mice, which is similar to a neuron-specific deletion of BDNF
(Monteggia et al. 2007). Taken together, these studies show that the neurotrophin hypothesis
of depression is not necessarily limited to neurons. Despite their name, neurotrophins affect
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other cell populations as well.
BDNF Signaling Cascades
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The rodent BDNF gene has a complex structure consisting of multiple 5‘ non-coding
exons, each driven by a specific promoter and one 3‘ coding exon. Multiple transcription
variants have been described, all containing one of the 5‘ non-coding exons combined with
the final 3‘ coding exon. Initially, 5 exons were identified and they were named exon I
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through V with exon V being the coding exon (Metsis et al. 1993, Nair et al. 2007, Timmusk
et al. 1993). Later, 4 additional non-coding exons were identified and the exons were renamed
as follows: exon I and II (unaltered), exon III (new exon), exon IV (formerly exon III), exon
V (new exon), exon VI (formerly exon IV), exon VII and VIII (new exons), and coding exon
IX (formerly coding exon V) (Aid et al. 2007, Liu et al. 2006).
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BDNF is produced as a 32 kDa precursor protein (proBDNF) consisting of a pro-domain
(18 kDa) and a mature domain (14 kDa). After translation, BDNF protein is secreted either
constitutively or in a regulated activity-dependent way (Lu et al. 2005). The activity-
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dependent release of BDNF is a particularly important mechanism for enhancing neuronal
communication specifically in active neurons of the brain. BDNF, released by active neurons,
increases synaptic strength with adjacent neurons by mechanisms like long-term potentiation
(LTP), thus ameliorating their connectivity (Lu et al. 2008, Minichiello 2009). The pro-
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domain can be proteolytically cleaved at specific cleavage sites yielding different forms of
proBDNF (ranging from 16 kDa to 32 kDa) or mature BDNF (mBDNF, 14 kDa). This
proteolytic processing occurs either intracellularly by furin (PC3) or PC1, or extracellularly
by plasmin or other matrix metalloproteases (e.g., MMP-3 or MMP-7) (Lee et al. 2001). Both
proBDNF and mBDNF can exert very different functions by binding specific receptors, as is
described in the next section. The conversion of proBDNF to mBDNF therefore offers an
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important regulatory mechanism to control BDNF action.

Tropomyosin-related kinase B (TrkB), a member of the Trk family of receptor tyrosine
kinases, is a high-affinity receptor for BDNF. Various Trk receptors exist that specifically
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bind different neurotrophins with high affinity: TrkA binds nerve growth factor (NGF), TrkB
binds BDNF and neurotrophin-4, while TrkC is specifically activated by neurotrophin-3.
Activation of TrkB is linked to signaling cascades that ultimately lead to increased
neurogenesis, synaptogenesis and neuronal survival (Figures 1 and 2). Thus, TrkB contributes
to overall plasticity in the brain, particularly in the hippocampus where TrkB is abundantly
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expressed (Donovan et al. 2008, Krause et al. 2008, Yan et al. 1997). The p75 neurotrophin
receptor (p75NTR) is able to bind all proneurotrophins with low affinity, but unlike TrkB,
this receptor is linked with apoptotic c-Jun N-terminal kinase signaling which ultimately
results in cell death (Kenchappa et al. 2006, Lee et al. 2001, Wang et al. 2000). This ―Yin
Yang‖ theory of neurotrophins, with proBDNF-p75NTR signaling inducing apoptosis and
mBDNF-TrkB signaling enhancing plasticity, has been challenged though (Lu et al. 2005). It
has been reported that proBDNF can activate TrkB and mBDNF may activate p75NTR, albeit
c.
to a lesser extent (Fayard et al. 2005). In addition, co-expression of p75NTR and Trk
receptors within the same cell leads to interaction of both receptors and conformational
changes in the Trk receptor. These conformational changes create high-affinity binding sites
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for neurotrophins which lead to greater responsiveness to their respective neurotrophins
(Chao and Hempstead 1995) (Figure 1). This dual receptor mechanism enables cells to
regulate neurotrophin action very accurately by altering the balance of these receptors.
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Figure 1. proBDNF specifically binds p75NTR and its co-receptor sortilin to induce apoptotic
signaling. Proteolytic cleavage of proBDNF yields mBDNF which specifically binds TrkB and
enhances plasticity in the brain. Cross-reaction between both BDNF forms and the neurotrophin
receptors is possible to a low extent, i.e., proBDNF may also bind TrkB and mBDNF can bind
p75NTR. When cells co-express both receptors, interaction between p75NTR and TrkB enhances the
affinity of TrkB for BDNF.
Binding of BDNF to TrkB induces conformational changes and dimerization of this

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receptor, leading to autophosphorylation of tyrosine residues in its cytoplasmic domain and

recruitment of adaptor proteins that are linked to different signaling cascades (Figure 2).
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These signaling cascades include the PI-3K/Akt, Erk/MAPK and PLC-γ cascades. PI-3K
activation stimulates PDK-1 and PDK-2 activity, which phosphorylates and activates Akt
(Chan et al. 1999). Via interaction with targets including GSK-3β and BAD, Akt triggers anti-
apoptotic effects and increases neuronal survival. Erk/MAPK signaling is activated via the
TrkB docking proteins Shc, Grb-2, SOS and Ras (Kaplan and Miller 1997). Eventually, the
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transcription factor cyclic AMP response element binding protein (CREB) can be activated,
which regulates the transcription of genes involved in proliferation, survival and neuronal
differentiation (e.g., BDNF, Bcl-2). Via activation of Rsk, Erk also phosphorylates and
inactivates the pro-apoptotic factor BAD (Shaltiel et al. 2007), which enhances cell survival.
Finally, PLC-γ binds specific phosphotyrosine sites in the intracellular TrkB domain.
Activated PLC-γ hydrolyses phosphatidyl inositides to generate IP3 and DAG and activates
PKCδ, which is required for proper Erk/MAPK signaling (Corbit et al. 1999). Depending on
the activation of these signaling cascades, TrkB signaling induces specific molecular
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responses that increase different aspects of plasticity like neuronal survival, neurogenesis and
synaptogenesis (Huang and Reichardt 2003, Kaplan and Miller 2000).
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Figure 2. Schematic overview of the different BDNF-TrkB signaling cascades. Following binding of
BDNF, homodimerization and autophosphorylation of TrkB takes place and different binding proteins
linked to specific signaling cascades are recruited. Phosphorylation of Tyr785 in the cytoplasmic
domain of TrkB triggers activation of PLCγ, which on its turn hydrolyses phosphatidyl inositides
(PIP2) into IP3 and DAG. IP3 causes an intracellular release of Ca 2+ which, together with DAG,
activates PKCδ, required for proper Erk/MAPK signaling. In addition, the increase in intracellular Ca2+
activates CaM and CaMKK which results in phosphorylation and activation of the transcription factor
a
CREB. This transcription factor regulates expression of genes involved in plasticity (e.g., neurogenesis,
synaptogenesis and neuronal survival). Phosphorylation of the Tyr490 residue of TrkB recruits the Shc-
Grb2-SOS binding complex which on its turn activates Ras or PI-3K. Activated Ras triggers
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phosphorylation and activation of the Erk/MAPK cascade which eventually results in activation of
RSK. RSK phosphorylates and activates CREB, but it also phosphorylates the pro-apoptotic factor
BAD. This leads to its dissociation from the anti-apoptotic factor Bcl-2, thus increasing survival.
Phosphorylation of PI-3K activates the PI-3K/Akt cascade which results in phosphorylation and
dissociation of BAD from Bcl-2 and in phosphorylation and inhibition of GSK3β. Inhibition of GSK3β
results in accumulation of the transcription factor β-catenin and activation of Wnt-regulated gene
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expression. Arrows indicate activation of signaling molecules, blunt-end arrows represent inhibitory
activity.
BDNF Enhances Plasticity in the Brain and

Has Antidepressant Properties
BDNF signaling plays a role in several mechanisms that influence overall plasticity in the
c.
brain. These include among others neurogenesis, synaptic plasticity, cellular survival and
synaptogenesis. Enhanced plasticity is beneficial for the organism as it improves memory,
increases resistance to stress and produces antidepressant-like effects. We will provide a short
overview of the literature supporting the involvement of BDNF in plasticity processes in
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different in vivo and in vitro models.
BDNF, an Essential Mediator of Brain Plasticity
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Neurogenesis in the brain entails the generation of new neurons and their integration in
neuronal networks. Critical processes include the proliferation of progenitor cells,
differentiation into functional neurons and survival of these newborn cells which allows their
migration and integration in complex neuronal networks. A plethora of studies have shown
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the necessity and sufficiency of BDNF-TrkB signaling in these different aspects of
neurogenesis. BDNF infusion or overexpression in the brain for instance increases
neurogenesis in different areas of the brain including the hippocampus (Benraiss et al. 2001,
Pencea et al. 2001, Scharfman et al. 2005, Zigova et al. 1998). BDNF+/- heterozygous mice
have approximately half the normal BDNF levels and show decreased proliferation in the
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hippocampus (Lee et al. 2002, Rossi et al. 2006). When these heterozygous mice are exposed
to an enriched environment, the expected increase in hippocampal neurogenesis is abolished
(Rossi et al. 2006). These studies illustrate the sufficiency and requirement of BDNF for
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neurogenesis. In a different study, BDNF was shown to play a critical role in neuronal
differentiation, but not in proliferation in rats (Taliaz et al. 2010). Interestingly, BDNF+/-
transgenic mice and mice expressing TrkB.T1, a truncated form of TrkB lacking tyrosine
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kinase activity and intracellular signaling, have increased proliferation in the hippocampus
(Sairanen et al. 2005). Survival of newborn neurons however is greatly diminished in these
transgenic animals showing that BDNF signaling may be required for survival of newborn
hippocampal neurons, rather than neurogenesis per se (Sairanen et al. 2005). Indeed, BDNF
has been shown to promote survival of cultured rat embryo septal cholinergic neurons
(Alderson et al. 1990). BDNF also plays a critical role in the survival of cultured cortical
neurons from newborn rats. The majority of these cells die after application of an anti-BDNF
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antibody, and this can be rescued by addition of excess BDNF (Vutskits et al. 2001). In
addition, BDNF can protect the neonatal rat brain from hypoxic-ischemic insults by
increasing neuronal survival (Han and Holtzman 2000). BDNF is also directly implicated in
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the differentiation of progenitor cells to adult neurons. Cultured rodent progenitor cells from
different areas of the brain quickly differentiate into neurons after supplementing BDNF
(Ahmed et al. 1995, Palmer et al. 1997), and adding BDNF to human neuroblastoma cells
stimulates TrkB phosphorylation and neuronal differentiation (Kaplan et al. 1993). Finally,
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BDNF is critically involved in synaptic function, as it regulates important aspects of synaptic

physiology. It protects and repairs existing synapses and stimulates new synapse formation,
even in the presence of various toxins (Lu et al. 2013). BDNF overexpression increases
dendrite complexity in the dentate gyrus (DG), with increased first order dendrites, total
dendritic length and total number of branch points (Tolwani et al. 2002). In conclusion,
BDNF can be regarded as an essential mediator of plasticity in the brain.
c.
The Antidepressant Nature of BDNF
BDNF infusions in the brain generally produce antidepressant effects in rodent models of
depression, although these effects are restricted to specific brain regions. Antidepressant
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effects have been found when BDNF was injected in the midbrain, cerebroventricular system
and hippocampus (Hoshaw et al. 2005, Shirayama et al. 2002, Siuciak et al. 1997). The
antidepressant actions of BDNF in the hippocampus are limited though to the DG and the
cornu ammonis 3 (CA3), and do not include the CA1 (Shirayama et al. 2002). Contrasting
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results have complicated the view that loss of BDNF may be sufficient to induce depressive-
like behaviour. Studies with heterozygous BDNF+/- knockout mice for example show that
results seemingly depend on the specific depression test used and the extent of the BDNF
knockout throughout the brain (Chourbaji et al. 2004, MacQueen et al. 2001, Saarelainen et
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al. 2003). Depressive phenotypes as a consequence of BDNF knockdown have been reported
in the learned helplessness test, but not in the forced swim test (FST) (MacQueen et al. 2001).
A lentiviral knockdown of BDNF by RNA interference in specific subregions of the rat
hippocampus has shown that a reduction of BDNF expression in the DG, but not the CA3,
reduces neurogenesis and induces behaviours that are associated with depression (Taliaz et al.
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2010). A specific BDNF knockdown in the ventral subiculum induces anhedonic-like
behaviour, a symptom of depression. In contrast, selective loss of BDNF in the DG or the
CA1 by RNA interference did not induce anxiety- or depression-related behaviour in mice
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(Adachi et al. 2008). Rather than producing a depressive-like phenotype, BDNF knockdown
in BDNF+/- mice only attenuates responses to antidepressant treatment (Saarelainen et al.
2003). This was not confirmed in another study where hippocampal BDNF knockdown did
not block the antidepressant effects of electroconvulsive therapy (ECT) (Taliaz et al. 2012).
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Because homozygous BDNF-/- knockout mice are not viable, conditional BDNF knockout
mice have been generated. In such animals, antidepressant efficacy of desipramine is blocked
in the FST (Monteggia et al. 2004), while depression-like behaviour is only observed in
female but not male mice (Monteggia et al. 2007). This demonstrates important gender
differences in depression-related behaviour in animal models, possibly reflecting the sexual
dimorphism seen in humans. Also, the age of the animals greatly affects the impact of BDNF
on behaviour. For instance, it was found that hippocampal BDNF expression plays a critical
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role in resilience to chronic stress and that a reduction of hippocampal BDNF expression in
young, but not adult rats induces prolonged elevations in corticosterone secretion (Taliaz et
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al. 2011).
Transgenic mice overexpressing full-length TrkB, with increased TrkB activity in the
hippocampus and cortex, show increased latency to immobility in the FST, facilitated
learning and reduced anxiety (Koponen et al. 2004a, Koponen et al. 2005, Koponen et al.
2004b). TrkB conditional knockout mice, like BDNF, do not show depressive-like behaviour
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(Zorner et al. 2003). In contrast, transgenic mice overexpressing truncated TrkB in these brain
regions are resistant to the effects of antidepressants and show impaired long-term memory
(Saarelainen et al. 2003, Saarelainen et al. 2000). Summarizing these studies, it is clear that
BDNF/TrkB plays a pivotal role in the molecular mechanisms regulating mood. However, it
remains to be clarified whether a reduction or loss of BDNF or TrkB in selective brain areas
may be sufficient to induce depressive-like behaviour or to attenuate antidepressant efficacy.
c.
The Mesolimbic Area Complicates the BDNF Story
In contrast to the antidepressant effects of BDNF in the hippocampus (Gourley et al.

2008, Shirayama et al. 2002), injection of BDNF in the ventral tegmental area (VTA)-nucleus
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accumbens (NAc) results in depressive-like behaviours in rodents (Eisch et al. 2003).
Likewise, viral overexpression of BDNF in the VTA, a major source of BDNF for the NAc,
induces depressive-like behaviour and attenuates antidepressant efficacy in rats (Taliaz et al.
2012). Also, viral-mediated deletion of BDNF in the VTA reduces social avoidance induced
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by social defeat stress in mice and elicits antidepressant effects in the FST and sucrose
preference test in rats (Berton et al. 2006, Taliaz et al. 2012).
The opposing effects of BDNF in the hippocampus compared to the mesolimbic area
have not been explained yet, but answers may be found in the dual receptor system for
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BDNF. It has been shown that TrkB is highly expressed in the hippocampus (Donovan et al.
2008, Krause et al. 2008, Yan et al. 1997). p75NTR is predominantly expressed in the basal
forebrain including the cholinergic neurons of the medial septum (MS), the horizontal and
vertical diagonal band of Broca (HDB and VDB), and the nucleus basalis of Meynert (Gibbs
and Pfaff 1994, Koh et al. 1989, Lee et al. 1998, Sobreviela et al. 1994). These areas of the
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basal forebrain, with neuronal afferents projecting towards the hippocampus, may be a source
for hippocampal p75NTR. Nevertheless, several studies have shown little or no p75NTR
expression in the hippocampus (Krause et al. 2008, Lee et al. 1998, Yan et al. 1997, Young et
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al. 2007). Therefore, infusion of BDNF in the hippocampus may predominantly activate TrkB
mediated signaling, resulting in increased plasticity and an antidepressant outcome. A
possible explanation for the pro-depressant effects of BDNF in the NAc-VTA system may be
the relatively high p75NTR/TrkB ratio in this brain region, thus favoring BDNF-induced
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apoptosis (Martinowich et al. 2007).

In the NAc, knockdown of BDNF or overexpression of truncated TrkB, which lacks the
intracellular domain and effectively blocks TrkB signaling, exerts antidepressant effects in
rodents (Berton et al. 2006, Eisch et al. 2003). Likewise, ANA-12, a selective TrkB
antagonist which primarily binds TrkB in the striatum following systemic administration,
exerts antidepressant effects in the FST and tail suspension test in mice (Cazorla et al. 2011).
As TrkB itself produces opposite behavioural outcomes in the hippocampus and the NAc, this
a
suggests that TrkB-related signaling events mediate the behavioural effects of BDNF. In other
words, BDNF itself does not produce these behavioural effects, but only indirectly via TrkB
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signaling. A similar picture emerges from studies with the downstream transcription factor
CREB. Overexpression of CREB in the hippocampus exerts an antidepressant-like effect
(Chen et al. 2001a). Viral vector-mediated elevations of CREB in the NAc however reduce
the rewarding effects of cocaine, morphine and sucrose, which is indicative for anhedonia
(Barrot et al. 2002, Carlezon et al. 1998, Pliakas et al. 2001). Interestingly, upregulation of
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CREB in the NAc also reduces responses to aversive stimuli (Barrot et al. 2002). Together,
this shows that excess CREB in the NAc produces a generalized numbing of responses to
emotional stimuli, both rewarding and aversive in nature. This may explain why
overexpression of CREB in the NAc produces effects that appear pro-depressant (Newton et
al. 2002, Pliakas et al. 2001). Conversely, a reduction of CREB in the NAc produces
antidepressant-like effects (Conti et al. 2002, Green et al. 2010, Newton et al. 2002, Pliakas et
al. 2001). It may therefore appear contradictory that prolonged social isolation during
adulthood decreases CREB activity in the NAc in rats (Wallace et al. 2009). This was
c.
associated with emotional hyperactivity, as evidenced by increased anxiety-like behaviour.
Disrupting CREB function in the NAc by means of a dominant negative form of CREB or by
RNA interference also induces anxiety-like effects (Barrot et al. 2002, Barrot et al. 2005,
In
Green et al. 2010), although this was not replicated by global knockdown of CREB in mutant
mice (Valverde et al. 2004). Since BDNF, TrkB and CREB all produce opposite behaviours
in the VTA-NAc compared to the hippocampus, it is more likely that the involved signaling
cascades and the resulting gene transcription explain the behavioural outcome, rather than
different ratios of the BDNF receptors TrkB and p75NTR.
rs
The different neurochemical environment of the NAc and hippocampus are likely
responsible for the different target genes that are regulated by CREB in these structures
(Carlezon et al. 2005, Nestler and Carlezon 2006, Yu and Chen 2011). Target genes of CREB
in the hippocampus like BDNF and Bcl-2 are mainly involved in plasticity-enhancing
he
processes like neurogenesis, synaptogenesis, cell survival and cell differentiation. In contrast,
an important target gene regulated by CREB in the NAc is dynorphin, a selective ligand for
the қ opioid receptor which is linked to a negative emotional state (Carlezon et al. 2005, Cole
et al. 1995, Green et al. 2010, McClung and Nestler 2003, Muschamp et al. 2011). Different
co-activators of transcription may be present in different brain regions, which together with
is
CREB coordinate the expression of distinct target genes (Zhang et al. 2005). Also on an
epigenetic level, differential methylation of CpG islands in the promoter region of these target
genes, which alters its affinity for transcription factors, offers a likely explanation (Carlezon
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et al. 1998). Indeed, it has been shown that cAMP response element (CRE) methylation
silences CREB target genes in a tissue-specific manner (Iannello et al. 2000, Mancini et al.
1999).
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Fluoxetine Increases Hippocampal Plasticity

Chronic stress has a detrimental impact on plasticity in the brain and affects processes
like neurogenesis, neuronal survival, and synaptogenesis (Duman 2004, Malberg et al. 2000).
In the following section, we will show that antidepressants probably improve affect by
a
stimulating overall plasticity and reversing neuronal damage inflicted by chronic stress.
Chronic, but not acute antidepressant administration including fluoxetine increases
neurogenesis in the adult rat hippocampus (Malberg et al. 2000). Transgenic Huntington‘s
ov
disease mice show depressive-like phenotypes and decreased hippocampal neurogenesis, both
of which can be reversed by administration of fluoxetine (Grote et al. 2005). In addition,
fluoxetine treatment rescues the observed volume loss in the DG in these mice. Also atypical
antidepressants like ECT and lithium increase neurogenesis (Chen et al. 2000, Duman et al.
2000, Jacobs et al. 2000, Madsen et al. 2000, Malberg et al. 2000). These findings suggest
N
that the behavioural effects of chronic antidepressant treatment may be mediated by

stimulating neurogenesis in the hippocampus. Indeed, chronic antidepressant treatment,
including fluoxetine, reverses the anxiety/depressive-like state and inhibition of hippocampal

neurogenesis induced by corticosterone administration in mice (David et al. 2009). In
BALB/cJ mice however, chronic fluoxetine treatment does not increase neurogenesis,
showing that distinct mechanisms of action may be present in different mouse strains (Holick
et al. 2008). Importantly, blocking hippocampal neurogenesis also blocks the behavioural
c.
effects of different classes of antidepressants including fluoxetine, showing that intact
neurogenesis is required for antidepressant efficacy (Dranovsky and Hen 2006, Santarelli et
al. 2003). Interestingly, when hippocampal neurogenesis is abolished by X-irradiation, the
In
efficacy of fluoxetine is blocked in some, but not all, behavioural assays, suggesting
neurogenesis-dependent and -independent mechanisms of antidepressant action (David et al.
2009). This was confirmed in another study where ablation of hippocampal neurogenesis in
BALB/cJ mice did not attenuate antidepressant responses to fluoxetine in the FST and
novelty-induced hypophagia test (Holick et al. 2008).
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As stated before, neurogenesis is a complex process consisting of several subsequent
phases. Studies have been conducted to elucidate whether neurogenesis in general, or precise
parts of it like neuronal survival, are specifically influenced by antidepressants. For example,
it was shown that the selective serotonin reuptake inhibitor (SSRI) fluoxetine does not affect
he
division of stem-like cells in the DG, but rather increases symmetric divisions of an early
progenitor cell class (Encinas et al. 2006). It was further demonstrated that these amplifying
neuronal progenitors are the sole class of neuronal progenitors targeted by fluoxetine in the
adult brain, suggesting that the fluoxetine-induced increase in new neurons arises as a result
of the expansion of this cell type. In vitro, fluoxetine treatment increases cell proliferation and
is
differentiation of cultured rat postnatal cerebellar neural progenitors via activation of the
signaling cascade Erk-CREB, which is linked to BDNF-TrkB activation (Zusso et al. 2008).
Chronic treatment with imipramine and fluoxetine produce a similar increase in markers for
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proliferation and apoptosis in the DG, indicating that these drugs increase both neurogenesis
and neuronal elimination simultaneously (Sairanen et al. 2005). These results suggest that
antidepressants might increase turnover of hippocampal neurons rather than neurogenesis per
se. Antidepressant treatment has also been shown to stimulate survival of new neurons with
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chronic but not sub-chronic fluoxetine treatment increasing hippocampal cell proliferation
and survival of newborn cells in mice (Wu and Castren 2009).
Besides neurons, astrocytes are also implicated in the pathophysiology of depression and
in neurotrophic action. It is therefore interesting to note that chronic fluoxetine treatment in
tree shrews prevents a stress-induced numerical decrease of astrocytes in the hippocampal
formation (Czeh et al. 2006). Therefore, glial changes may contribute to the cellular actions
of antidepressants. Indeed, we have found that sub-chronic treatment with the SSRI
a
citalopram and the tricyclic antidepressant (TCA) imipramine increases differentiation and
neurite length, but not neurite numbers in neuronal and astrocytic cell lines (data not
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published). The increased neurite outgrowth following antidepressant treatment is in line with
the general view that antidepressants induce plastic changes in the brain (Duman et al. 1997,
Manji et al. 2001, Nestler et al. 2002). A few studies, however, failed to show the neurite
enhancing properties of citalopram and imipramine. For example, citalopram and imipramine
inhibit neuronal sprouting from goldfish retina explants (Lima et al. 1994). Also, imipramine
N
was found to enhance the NGF-induced neurite sprouting in PC12 cells in a dose-dependent
manner although it was ineffective by itself (Jang et al. 2009, Takebayashi et al. 2002).
Antidepressants also increase neuronal structural plasticity in the brain. Chronic

fluoxetine administration in rats induces changes in glutamate receptor subunits and increases
dendritic spine density and large, mushroom-type spine numbers in the cerebral cortex
(Ampuero et al. 2010). These changes stabilize and strengthen neuronal networks and
probably precede the anxiolytic and antidepressant effects seen in these animals. Fluoxetine
c.
has also been shown to increase the expression of polysialylated neural cell adhesion
molecule, which is involved in neuronal structural plasticity in the CA3, medial prefrontal,
visual and somatosensory cortex (Varea et al. 2007a, Varea et al. 2007b). In addition, this
In
treatment increases spine density in the principal apical dendrite of pyramidal neurons
(Guirado et al. 2009). Sub-chronic administration of fluoxetine induces a robust increase in
pyramidal cell dendritic spine synapse density in the CA1 and CA3 of ovariectomized rats
(Hajszan et al. 2005). Finally, antidepressants alter the electrophysiological characteristics of
neurons. For example, chronic fluoxetine treatment restores neuronal plasticity in amblyopic
rs
rats, measured by an increased visual evoked potential in the primary visual cortex (Maya
Vetencourt et al. 2008). Like ECT, fluoxetine produces equivalent increases in dentate field
excitatory post-synaptic potentials, while LTP induction is attenuated in both groups (Stewart
and Reid 2000).
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Fluoxetine Increases BDNF Signaling
Chronic antidepressant treatment increases BDNF expression in different subregions of
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the hippocampus including the DG, CA1 and CA3. This includes typical antidepressants like
SSRI‘s and monoamine oxidase inhibitors, but also atypical antidepressant treatment such as
ECT (Dias et al. 2003, Holoubek et al. 2004, Newton et al. 2003, Nibuya et al. 1995, Nibuya
bl
et al. 1996, Russo-Neustadt et al. 1999, Smith et al. 1997, Xu et al. 2003). This is in stark
contrast with the effects of different stressors, which typically decrease hippocampal BDNF
expression (Duman and Monteggia 2006). We have confirmed these findings and found that
chronic treatment of rats with the dual serotonin/noradrenaline reuptake inhibitor duloxetine
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and the triple serotonin/noradrenaline/dopamine reuptake inhibitor DOV 216,303 restores

BDNF protein levels in the rat hippocampus, which was initially decreased due to injection
stress (Prickaerts et al. 2012). Other brain structures besides the hippocampus show changes
in BDNF expression following antidepressant treatment as well. For instance, chronic
fluoxetine treatment restores neuronal plasticity in the visual cortex of adult rats through local
increases in BDNF protein levels (Maya Vetencourt et al. 2008). Treatment of healthy rats
a
with desipramine and phenelzine increases BDNF mRNA in the frontal cortex and
hippocampus (Dwivedi et al. 2006). ECT has been shown to cause robust changes in BDNF
levels in the VTA and hippocampus, while manipulation of BDNF expression in the VTA,
ov
but not in the hippocampus, blocks the antidepressant-like effect of ECT (Taliaz et al. 2012).
This suggests that BDNF in the VTA is essential in the mechanism of action of ECT, while
hippocampal BDNF changes may only reflect a secondary effect. With regard to the SSRI
fluoxetine, some contrasting results have been reported with chronic fluoxetine treatment
either increasing (Coppell et al. 2003, De Foubert et al. 2004, Dwivedi et al. 2006, Nibuya et
N
al. 1996, Vinet et al. 2004) or not affecting (Altar et al. 2003, Conti et al. 2002, Dias et al.
2003) hippocampal BDNF levels. Interestingly, increased BDNF expression is only apparent
after chronic fluoxetine administration. Sub-chronic administration or a single injection of

fluoxetine even decreases hippocampal BDNF expression in rats (Coppell et al. 2003, De
Foubert et al. 2004). Moreover, antidepressants have been shown to regulate specific BDNF
transcription variants. Desipramine for example increases expression of BDNF transcription
variants I and III, while fluoxetine only increases variant II (Dwivedi et al. 2006). This could
c.
explain why some studies don‘t show overall changes in BDNF levels because general BDNF
measurements reflect a net effect on all transcriptions variants. We have already highlighted
the importance of glial cells in the pathophysiology of depression and in antidepressant
In
action. This holds true for the effects antidepressants have on BDNF signaling as well, which
is not limited to neurons. In differentiated astroglial cells, treatment with imipramine and
citalopram increases expression of BDNF and CREB (unpublished data), confirming previous
findings with primary cortical mouse astrocyte cultures treated with the SSRI‘s fluoxetine and
paroxetine (Allaman et al. 2011). Also, DOV 216,303 and duloxetine increase BDNF protein
rs
levels in rat C62B astrocytomas (Prickaerts et al. 2012). In murine HT22 neuronal cells
however, only DOV 216,303 increases intracellular BDNF and this antidepressant even
decreases BDNF release. This stresses the complex nature of the BDNF response to
antidepressant treatment and indicates that it is a cell type and antidepressant specific
he
mechanism.
Antidepressants do not only affect BDNF itself, but also key players involved in BDNF-
TrkB signaling. Chronic ECT and antidepressant drugs such as fluoxetine increase TrkB
expression in the frontal cortex and hippocampus in rats (Nibuya et al. 1995, Nibuya et al.
1996). Acute as well as chronic treatment with antidepressants including fluoxetine induces
is
TrkB autophosphorylation in the prefrontal cortex, anterior cingulate cortex and hippocampus
(Rantamaki et al. 2007, Saarelainen et al. 2003). This shows that the expression, as well as the
activity of TrkB can be stimulated by antidepressants. Proteins within the different TrkB
signaling cascades have been linked to antidepressant actions in rodents including PLCγ,
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Ca2+/CaMKIV, Erk1/2, and Bcl-2 (Murray and Hutson 2007, Rantamaki et al. 2007,
Tiraboschi et al. 2004). We have seen in previous in vitro studies that antidepressants activate
different TrkB signaling cascades in neuronal and glial cells and that pathway activation also
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depends on the type of antidepressant used (unpublished data). Imipramine for instance
affects more signaling cascades compared to citalopram. This is likely as TCA‘s affect
additional monoaminergic systems compared to the serotonergic system, which is the only
neurotransmitter affected by SSRI‘s. Chronic and, in some cases, acute administration of
fluoxetine increases CREB phosphorylation in the rodent prefrontal cortex, anterior cingulate
cortex and hippocampus (Pinnock et al. 2010, Rantamaki et al. 2007, Saarelainen et al. 2003,
Tiraboschi et al. 2004). This was not replicated in another study though, as only repeated but
a
not acute treatment with citalopram activated hippocampal CREB in mice (Mombereau et al.
2010). In the same study however, the effects of citalopram were abolished in CREB mutant
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mice showing that this transcription factor is essential in the mechanism of action of
citalopram.
Many antidepressants increase BDNF mRNA and protein after a few days of treatment.
Interestingly, increased phosphorylation of TrkB and subsequent activation of the
transcription factor CREB takes place within an hour after drug administration (Rantamaki et
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al. 2007, Saarelainen et al. 2003). This temporal discrepancy indicates that initial activation of
TrkB may be initiated without involvement of endogenous BDNF. Indeed, imipramine is able
to rapidly induce TrkB phosphorylation after a single administration, even in BDNF-/-
knockout mice (Rantamaki et al. 2011). BDNF is critically involved though in the long-term
effects of fluoxetine on plasticity. Chronic fluoxetine treatment in transgenic mice carrying
the BDNF Val66Met mutation, which impairs proper processing and secretion of BDNF,
does not increase BDNF protein levels (Bath et al. 2012). As a consequence, fluoxetine
treatment does not attenuate impaired survival of newborn neurons and impaired LTP in these
c.
mice.
Evidence also supports the involvement of BDNF-TrkB signaling in antidepressant action
in humans. Post-mortem analysis has revealed decreased BDNF and TrkB expression in the
In
hippocampus of depressed suicide patients, which are elevated in patients medicated with
antidepressants before death (Chen et al. 2001b, Dwivedi et al. 2003, Karege et al. 2005b).
Furthermore, serum BDNF in living depressed patients is abnormally low, but can be restored
by antidepressant treatment (Aydemir et al. 2005, Karege et al. 2005a, Karege et al. 2002,
Shimizu et al. 2003). Supporting evidence has also been found for fluoxetine specifically, as
rs
depressed patients treated for 8 weeks with fluoxetine show increased serum BDNF levels
(Gonul et al. 2005).
he
Epigenetic Regulation of BDNF in Depression and
Antidepressant Treatment
Recent evidence shows that epigenetic processes, involving e.g., DNA methylation and
chromatin modifications, represent key mechanisms by which environmental factors induce
is
enduring changes in gene expression, and, as such, may play an important role in the onset of
various psychiatric disorders including MD (see review by (Boulle et al. 2012)). More
specifically, various environmental factors, particularly those occurring during early
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development, have been shown to produce long-lasting epigenetic changes at the BDNF gene,
affecting the availability and function of the BDNF protein. Furthermore, such stable
epigenetic imprints on the BDNF gene have been postulated to explain, at least in part, the
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delayed efficacy of treatments as well as the high degree of relapse observed in psychiatric
disorders. For example, the methyl-CpG binding protein 2 (MeCP2) is known for its
repression of BDNF gene transcription (Im et al. 2010, Klose and Bird 2003). MeCP2 binds
selectively to methylated DNA at the rat's BDNF promoter IV, where it is associated with the
co-repressor molecules Sin3a and histone deacetylase 1 (HDAC1) to form a complex that
maintains the repressed state of the BDNF gene (Martinowich et al. 2003). Other proteins
such as the growth arrest and DNA-damage-inducible protein b (Gadd45b) have been shown
a
to be required for activity-induced DNA demethylation at BDNF promoter IX, which is

associated with increased hippocampal neurogenesis in mice (Ma et al. 2009). In addition to
changes in DNA-methylation, post-translational modifications of histone tails have been
ov
shown to regulate BDNF gene expression. For example, methylation at lysine (K) 27 on
histone H3 (H3K27) is usually associated with transcriptional repression, whereas acetylation
on histone H3 and H4 is associated with transcriptional activation of BDNF. In a
groundbreaking study by Tsankova and colleagues, chronic social defeat stress decreased
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levels of BDNF mRNA IV and V in the hippocampus of stressed mice (Tsankova et al.
2007). Interestingly, this robust decrease in expression was associated with long-lasting
H3K27 hypermethylation at the corresponding promoters. Another study, using perinatal
exposure to methylmercury as a mouse model for depression, demonstrated that the observed
decrease in hippocampal BDNF mRNA levels following developmental methylmercury
exposure was mediated by H3K27 hypermethylation and H3 hypoacetylation at BDNF
promoter IV (Onishchenko et al. 2008). Interestingly, BDNF promoter IV also contains a
CREB binding site (Dias et al. 2003, Tao et al. 2002, West et al. 2001), while CREB-binding
c.
protein (CBP) has a central role in chromatin remodeling due to its histone acetyl-transferase
(HAT) properties (Chan and La Thangue 2001). In humans, levels of BDNF promoter
methylation in the blood were significantly associated with suicidal behavior in MD patients
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(Kang et al. 2013). In addition, increased peripheral BDNF methylation was observed after
childhood maltreatment and predicted childhood depression scores (Weder et al. 2014),
suggesting that blood BDNF DNA methylation profiles may serve as a potent diagnostic
biomarker in MD (Fuchikami et al. 2011, Song et al. 2014).
Similarly, various antidepressants, including SSRI‘s, have been shown to normalize
rs
BDNF gene expression by means of epigenetic mechanisms. For example, in mice, chronic
imipramine administration could reverse the social defeat-induced repression of BDNF
expression at promoter IV and V by inducing H3-K9 and H3-K14 acetylation (Tsankova et al.
2007), whereas chronic treatment with fluoxetine in mice has been shown to upregulate H3
he
acetylation at BDNF promoter IV (Onishchenko et al. 2008) (for a more complete overview,
see Boulle et al. 2012).
As epigenetic regulation of the BDNF gene is hypothesized to underlie the
abovementioned differential exon regulation and usage - resulting in subcellular- and brain
region-specific distribution -, developing drugs that modify epigenetic regulation at specific
is
BDNF exons represents a promising strategy for the treatment of psychiatric disorders
(Gourley et al. 2008). Evidently, it is yet to be determined whether these epigenetic changes
are the cause or the consequence of the associated pathology. The fact that epidemiologically
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relevant environmental risk factors such as chronic stress modulate BDNF levels in animal
models through epigenetic mechanisms, and that pharmacological interventions are able to
restore BDNF levels via changes in the epigenome, suggests that epigenetic changes at the
BDNF gene are rather causal in the pathology than merely representing an epiphenomenon.
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Conclusion
In this chapter, we showed that antidepressants including SSRI‘s like fluoxetine have a
positive effect on plasticity in the brain, stimulating neurogenesis, synaptogenesis and cellular
a
survival. It is thought that increased hippocampal plasticity counteracts stress-induced

atrophy in the brain and increases neuronal connectivity, thus mediating beneficial effects on
mood. BDNF was identified as a critical player in this process, (epigenetically) regulated by
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chronic antidepressant treatment and a central mediator of hippocampal plasticity.

Importantly, BDNF-TrkB-CREB signaling influences mood in a region-specific manner, with
a general antidepressant effect in broad regions of the brain and pro-depressant effects in the
mesolimbic area specifically. This is most likely explained by regional differences in
regulated target genes. Finally, glial cells are often underestimated and neglected but appear
N
to play an equally important role in depression and in antidepressant action.

References
Adachi, M., Barrot, M., Autry, A.E., Theobald, D., Monteggia, L.M., (2008). Selective loss of
brain-derived neurotrophic factor in the dentate gyrus attenuates antidepressant efficacy.
Biol. Psychiatry 63, 642-649.
c.
Ahmed, S., Reynolds, B.A., Weiss, S., (1995). BDNF enhances the differentiation but not the
survival of CNS stem cell-derived neuronal precursors. J. Neurosci. 15, 5765-5778.
Aid, T., Kazantseva, A., Piirsoo, M., Palm, K., Timmusk, T., (2007). Mouse and rat BDNF
In
gene structure and expression revisited. J. Neurosci. Res. 85, 525-535.
Alderson, R.F., Alterman, A.L., Barde, Y.A., Lindsay, R.M., (1990). Brain-derived
neurotrophic factor increases survival and differentiated functions of rat septal
cholinergic neurons in culture. Neuron 5, 297-306.
Allaman, I., Fiumelli, H., Magistretti, P.J., Martin, J.L., (2011). Fluoxetine regulates the
rs
expression of neurotrophic/growth factors and glucose metabolism in astrocytes.
Psychopharmacology (Berl.) 216, 75-84.
Altar, C.A., Whitehead, R.E., Chen, R., Wortwein, G., Madsen, T.M., (2003). Effects of
electroconvulsive seizures and antidepressant drugs on brain-derived neurotrophic factor
he
protein in rat brain. Biol. Psychiatry 54, 703-709.
Ampuero, E., Rubio, F.J., Falcon, R., Sandoval, M., Diaz-Veliz, G., Gonzalez, R.E., Earle,
N., Dagnino-Subiabre, A., Aboitiz, F., Orrego, F., Wyneken, U., (2010). Chronic
fluoxetine treatment induces structural plasticity and selective changes in glutamate
receptor subunits in the rat cerebral cortex. Neuroscience 169, 98-108.
is
Aydemir, O., Deveci, A., Taneli, F., (2005). The effect of chronic antidepressant treatment on
serum brain-derived neurotrophic factor levels in depressed patients: a preliminary study.
Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 261-265.
bl
Barrot, M., Olivier, J.D., Perrotti, L.I., DiLeone, R.J., Berton, O., Eisch, A.J., Impey, S.,
Storm, D.R., Neve, R.L., Yin, J.C., Zachariou, V., Nestler, E.J., (2002). CREB activity in
the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli.
Proc. Natl. Acad. Sci. U. S. A. 99, 11435-11440.
Pu
Barrot, M., Wallace, D.L., Bolanos, C.A., Graham, D.L., Perrotti, L.I., Neve, R.L.,
Chambliss, H., Yin, J.C., Nestler, E.J., (2005). Regulation of anxiety and initiation of
sexual behavior by CREB in the nucleus accumbens. Proc. Natl. Acad. Sci. U. S. A. 102,
8357-8362.
Bath, K.G., Jing, D.Q., Dincheva, I., Neeb, C.C., Pattwell, S.S., Chao, M.V., Lee, F.S.,
Ninan, I., (2012). BDNF Val66Met impairs fluoxetine-induced enhancement of adult
a
hippocampus plasticity. Neuropsychopharmacology 37, 1297-1304.

Benraiss, A., Chmielnicki, E., Lerner, K., Roh, D., Goldman, S.A., (2001). Adenoviral brain-
derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment
ov
from endogenous progenitor cells in the adult forebrain. J. Neurosci. 21, 6718-6731.
Berton, O., McClung, C.A., Dileone, R.J., Krishnan, V., Renthal, W., Russo, S.J., Graham,
D., Tsankova, N.M., Bolanos, C.A., Rios, M., Monteggia, L.M., Self, D.W., Nestler, E.J.,
(2006). Essential role of BDNF in the mesolimbic dopamine pathway in social defeat
stress. Science 311, 864-868.
N
Boulle, F., van den Hove, D.L., Jakob, S.B., Rutten, B.P., Hamon, M., van Os, J., Lesch,
K.P., Lanfumey, L., Steinbusch, H.W., Kenis, G., (2012). Epigenetic regulation of the
BDNF gene: implications for psychiatric disorders. Mol. Psychiatry 17, 584-596.
Calabrese, F., Molteni, R., Cattaneo, A., Macchi, F., Racagni, G., Gennarelli, M., Ellenbroek,
B.A., Riva, M.A., (2010). Long-Term duloxetine treatment normalizes altered brain-
c.
derived neurotrophic factor expression in serotonin transporter knockout rats through the
modulation of specific neurotrophin isoforms. Mol. Pharmacol. 77, 846-853.
Carlezon, W.A., Jr., Duman, R.S., Nestler, E.J., (2005). The many faces of CREB. Trends
In
Neurosci. 28, 436-445.
Carlezon, W.A., Jr., Thome, J., Olson, V.G., Lane-Ladd, S.B., Brodkin, E.S., Hiroi, N.,
Duman, R.S., Neve, R.L., Nestler, E.J., (1998). Regulation of cocaine reward by CREB.
Science 282, 2272-2275.
Cazorla, M., Premont, J., Mann, A., Girard, N., Kellendonk, C., Rognan, D., (2011).
rs
Identification of a low-molecular weight TrkB antagonist with anxiolytic and
antidepressant activity in mice. J. Clin. Invest. 121, 1846-1857.
Chan, H.M., La Thangue, N.B., (2001). p300/CBP proteins: HATs for transcriptional bridges
and scaffolds. J. Cell Sci. 114, 2363-2373.
he
Chan, T.O., Rittenhouse, S.E., Tsichlis, P.N., (1999). AKT/PKB and other D3
phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent
phosphorylation. Annu. Rev. Biochem. 68, 965-1014.
Chao, M.V., Hempstead, B.L., (1995). p75 and Trk: a two-receptor system. Trends Neurosci.
18, 321-326.
is
Chen, A.C., Shirayama, Y., Shin, K.H., Neve, R.L., Duman, R.S., (2001a). Expression of the
cAMP response element binding protein (CREB) in hippocampus produces an
antidepressant effect. Biol. Psychiatry 49, 753-762.
bl
Chen, B., Dowlatshahi, D., MacQueen, G.M., Wang, J.F., Young, L.T., (2001b). Increased
hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication.
Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N., Manji, H.K., (2000). Enhancement of
Pu
hippocampal neurogenesis by lithium. J. Neurochem. 75, 1729-1734.

Chourbaji, S., Hellweg, R., Brandis, D., Zorner, B., Zacher, C., Lang, U.E., Henn, F.A.,
Hortnagl, H., Gass, P., (2004). Mice with reduced brain-derived neurotrophic factor
expression show decreased choline acetyltransferase activity, but regular brain
monoamine levels and unaltered emotional behavior. Brain Res. Mol. Brain Res. 121, 28-
36.
Cole, R.L., Konradi, C., Douglass, J., Hyman, S.E., (1995). Neuronal adaptation to
a
amphetamine and dopamine: molecular mechanisms of prodynorphin gene regulation in

rat striatum. Neuron 14, 813-823.
ov
Conti, A.C., Cryan, J.F., Dalvi, A., Lucki, I., Blendy, J.A., (2002). cAMP response element-
binding protein is essential for the upregulation of brain-derived neurotrophic factor
transcription, but not the behavioral or endocrine responses to antidepressant drugs. J.
Neurosci. 22, 3262-3268.
Coppell, A.L., Pei, Q., Zetterstrom, T.S., (2003). Bi-phasic change in BDNF gene expression
N
following antidepressant drug treatment. Neuropharmacology 44, 903-910.

Corbit, K.C., Foster, D.A., Rosner, M.R., (1999). Protein kinase Cdelta mediates neurogenic
but not mitogenic activation of mitogen-activated protein kinase in neuronal cells. Mol.
Cell. Biol. 19, 4209-4218.
Czeh, B., Muller-Keuker, J.I., Rygula, R., Abumaria, N., Hiemke, C., Domenici, E., Fuchs,
E., (2007). Chronic social stress inhibits cell proliferation in the adult medial prefrontal
c.
cortex: hemispheric asymmetry and reversal by fluoxetine treatment.
Neuropsychopharmacology 32, 1490-1503.
Czeh, B., Simon, M., Schmelting, B., Hiemke, C., Fuchs, E., (2006). Astroglial plasticity in
In
the hippocampus is affected by chronic psychosocial stress and concomitant fluoxetine
treatment. Neuropsychopharmacology 31, 1616-1626.
David, D.J., Samuels, B.A., Rainer, Q., Wang, J.W., Marsteller, D., Mendez, I., Drew, M.,
Craig, D.A., Guiard, B.P., Guilloux, J.P., Artymyshyn, R.P., Gardier, A.M., Gerald, C.,
Antonijevic, I.A., Leonardo, E.D., Hen, R., (2009). Neurogenesis-dependent and -
rs
independent effects of fluoxetine in an animal model of anxiety/depression. Neuron 62,
479-493.
De Foubert, G., Carney, S.L., Robinson, C.S., Destexhe, E.J., Tomlinson, R., Hicks, C.A.,
Murray, T.K., Gaillard, J.P., Deville, C., Xhenseval, V., Thomas, C.E., O'Neill, M.J.,
he
Zetterstrom, T.S., (2004). Fluoxetine-induced change in rat brain expression of brain-
derived neurotrophic factor varies depending on length of treatment. Neuroscience 128,
597-604.
Dias, B.G., Banerjee, S.B., Duman, R.S., Vaidya, V.A., (2003). Differential regulation of
brain derived neurotrophic factor transcripts by antidepressant treatments in the adult rat
is
brain. Neuropharmacology 45, 553-563.
Donovan, M.H., Yamaguchi, M., Eisch, A.J., (2008). Dynamic expression of TrkB receptor
protein on proliferating and maturing cells in the adult mouse dentate gyrus.
bl
Hippocampus 18, 435-439.
Dranovsky, A., Hen, R., (2006). Hippocampal neurogenesis: regulation by stress and
antidepressants. Biol. Psychiatry 59, 1136-1143.
Duman, R.S., (2004). Depression: a case of neuronal life and death? Biol. Psychiatry 56, 140-
Pu
145.
Duman, R.S., Heninger, G.R., Nestler, E.J., (1997). A molecular and cellular theory of
depression. Arch. Gen. Psychiatry 54, 597-606.
Duman, R.S., Malberg, J., Nakagawa, S., D'Sa, C., (2000). Neuronal plasticity and survival in
mood disorders. Biol. Psychiatry 48, 732-739.
Duman, R.S., Monteggia, L.M., (2006). A neurotrophic model for stress-related mood
disorders. Biol. Psychiatry 59, 1116-1127.
a
Dwivedi, Y., Rizavi, H.S., Conley, R.R., Roberts, R.C., Tamminga, C.A., Pandey, G.N.,
(2003). Altered gene expression of brain-derived neurotrophic factor and receptor
ov
tyrosine kinase B in postmortem brain of suicide subjects. Arch. Gen. Psychiatry 60, 804-
815.
Dwivedi, Y., Rizavi, H.S., Pandey, G.N., (2006). Antidepressants reverse corticosterone-
mediated decrease in brain-derived neurotrophic factor expression: differential regulation
of specific exons by antidepressants and corticosterone. Neuroscience 139, 1017-1029.
N
Eisch, A.J., Bolanos, C.A., de Wit, J., Simonak, R.D., Pudiak, C.M., Barrot, M., Verhaagen,
J., Nestler, E.J., (2003). Brain-derived neurotrophic factor in the ventral midbrain-nucleus
accumbens pathway: a role in depression. Biol. Psychiatry 54, 994-1005.
Encinas, J.M., Vaahtokari, A., Enikolopov, G., (2006). Fluoxetine targets early progenitor
cells in the adult brain. Proc. Natl. Acad. Sci. U. S. A. 103, 8233-8238.
Fayard, B., Loeffler, S., Weis, J., Vogelin, E., Kruttgen, A., (2005). The secreted brain-
derived neurotrophic factor precursor pro-BDNF binds to TrkB and p75NTR but not to
TrkA or TrkC. J. Neurosci. Res. 80, 18-28.
c.
Figurov, A., Pozzo-Miller, L.D., Olafsson, P., Wang, T., Lu, B., (1996). Regulation of
synaptic responses to high-frequency stimulation and LTP by neurotrophins in the
hippocampus. Nature 381, 706-709.
In
Fuchikami, M., Morinobu, S., Segawa, M., Okamoto, Y., Yamawaki, S., Ozaki, N., Inoue, T.,
Kusumi, I., Koyama, T., Tsuchiyama, K., Terao, T., (2011). DNA methylation profiles of
the brain-derived neurotrophic factor (BDNF) gene as a potent diagnostic biomarker in
major depression. PLoS ONE 6, e23881.
Gibbs, R.B., Pfaff, D.W., (1994). In situ hybridization detection of trkA mRNA in brain:
rs
distribution, colocalization with p75NGFR and up-regulation by nerve growth factor. J.
Comp. Neurol. 341, 324-339.
Gonul, A.S., Akdeniz, F., Taneli, F., Donat, O., Eker, C., Vahip, S., (2005). Effect of
treatment on serum brain-derived neurotrophic factor levels in depressed patients. Eur.
he
Arch. Psychiatry Clin. Neurosci. 255, 381-386.
Gourley, S.L., Kiraly, D.D., Howell, J.L., Olausson, P., Taylor, J.R., (2008). Acute
hippocampal brain-derived neurotrophic factor restores motivational and forced swim
performance after corticosterone. Biol. Psychiatry 64, 884-890.
Green, T.A., Alibhai, I.N., Roybal, C.N., Winstanley, C.A., Theobald, D.E., Birnbaum, S.G.,
is
Graham, A.R., Unterberg, S., Graham, D.L., Vialou, V., Bass, C.E., Terwilliger, E.F.,
Bardo, M.T., Nestler, E.J., (2010). Environmental enrichment produces a behavioral
phenotype mediated by low cyclic adenosine monophosphate response element binding
bl
(CREB) activity in the nucleus accumbens. Biol. Psychiatry 67, 28-35.
Grote, H.E., Bull, N.D., Howard, M.L., van Dellen, A., Blakemore, C., Bartlett, P.F., Hannan,
A.J., (2005). Cognitive disorders and neurogenesis deficits in Huntington's disease mice
are rescued by fluoxetine. Eur. J. Neurosci. 22, 2081-2088.
Pu
Guirado, R., Varea, E., Castillo-Gomez, E., Gomez-Climent, M.A., Rovira-Esteban, L.,
Blasco-Ibanez, J.M., Crespo, C., Martinez-Guijarro, F.J., Nacher, J., (2009). Effects of
chronic fluoxetine treatment on the rat somatosensory cortex: activation and induction of
neuronal structural plasticity. Neurosci. Lett. 457, 12-15.
Hajszan, T., MacLusky, N.J., Leranth, C., (2005). Short-term treatment with the
antidepressant fluoxetine triggers pyramidal dendritic spine synapse formation in rat
hippocampus. Eur. J. Neurosci. 21, 1299-1303.
a
Han, B.H., Holtzman, D.M., (2000). BDNF protects the neonatal brain from hypoxic-
ischemic injury in vivo via the ERK pathway. J. Neurosci. 20, 5775-5781.
ov
Holick, K.A., Lee, D.C., Hen, R., Dulawa, S.C., (2008). Behavioral effects of chronic
fluoxetine in BALB/cJ mice do not require adult hippocampal neurogenesis or the
serotonin 1A receptor. Neuropsychopharmacology 33, 406-417.
Holoubek, G., Noldner, M., Treiber, K., Muller, W.E., (2004). Effect of chronic
antidepressant treatment on beta-receptor coupled signal transduction cascade. Which
N
effect matters most? Pharmacopsychiatry 37 Suppl 2, S113-119.

Hoshaw, B.A., Malberg, J.E., Lucki, I., (2005). Central administration of IGF-I and BDNF
leads to long-lasting antidepressant-like effects. Brain Res. 1037, 204-208.
Huang, E.J., Reichardt, L.F., (2003). Trk receptors: roles in neuronal signal transduction.
Annu. Rev. Biochem. 72, 609-642.
Iannello, R.C., Gould, J.A., Young, J.C., Giudice, A., Medcalf, R., Kola, I., (2000).
Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs
through selective targeting of an activating transcription factor/cAMP-responsive
c.
element-binding site. J. Biol. Chem. 275, 19603-19608.
Im, H.I., Hollander, J.A., Bali, P., Kenny, P.J., (2010). MeCP2 controls BDNF expression and
cocaine intake through homeostatic interactions with microRNA-212. Nat. Neurosci. 13,
In
1120-1127.
Jacobs, B.L., van Praag, H., Gage, F.H., (2000). Adult brain neurogenesis and psychiatry: a
novel theory of depression. Mol. Psychiatry 5, 262-269.
Jang, S.W., Liu, X., Chan, C.B., Weinshenker, D., Hall, R.A., Xiao, G., Ye, K., (2009).
Amitriptyline is a TrkA and TrkB receptor agonist that promotes TrkA/TrkB
rs
heterodimerization and has potent neurotrophic activity. Chem. Biol. 16, 644-656.
Kang, H.J., Kim, J.M., Lee, J.Y., Kim, S.Y., Bae, K.Y., Kim, S.W., Shin, I.S., Kim, H.R.,
Shin, M.G., Yoon, J.S., (2013). BDNF promoter methylation and suicidal behavior in
depressive patients. J. Affect. Disord. 151, 679-685.
he
Kaplan, D.R., Matsumoto, K., Lucarelli, E., Thiele, C.J., (1993). Induction of TrkB by
retinoic acid mediates biologic responsiveness to BDNF and differentiation of human
neuroblastoma cells. Eukaryotic Signal Transduction Group. Neuron 11, 321-331.
Kaplan, D.R., Miller, F.D., (1997). Signal transduction by the neurotrophin receptors. Curr.
Opin. Cell Biol. 9, 213-221.
is
Kaplan, D.R., Miller, F.D., (2000). Neurotrophin signal transduction in the nervous system.
Curr. Opin. Neurobiol. 10, 381-391.
Karege, F., Bondolfi, G., Gervasoni, N., Schwald, M., Aubry, J.M., Bertschy, G., (2005a).
bl
Low brain-derived neurotrophic factor (BDNF) levels in serum of depressed patients
probably results from lowered platelet BDNF release unrelated to platelet reactivity. Biol.
Karege, F., Perret, G., Bondolfi, G., Schwald, M., Bertschy, G., Aubry, J.M., (2002).
Pu
Decreased serum brain-derived neurotrophic factor levels in major depressed patients.

Psychiatry Res. 109, 143-148.
Karege, F., Vaudan, G., Schwald, M., Perroud, N., La Harpe, R., (2005b). Neurotrophin
levels in postmortem brains of suicide victims and the effects of antemortem diagnosis
and psychotropic drugs. Brain Res. Mol. Brain Res. 136, 29-37.
Kenchappa, R.S., Zampieri, N., Chao, M.V., Barker, P.A., Teng, H.K., Hempstead, B.L.,
Carter, B.D., (2006). Ligand-dependent cleavage of the P75 neurotrophin receptor is
a
necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron
50, 219-232.
ov
Klose, R., Bird, A., (2003). Molecular biology. MeCP2 repression goes nonglobal. Science
302, 793-795.
Koh, S., Oyler, G.A., Higgins, G.A., (1989). Localization of nerve growth factor receptor
messenger RNA and protein in the adult rat brain. Exp. Neurol. 106, 209-221.
Koponen, E., Lakso, M., Castren, E., (2004a). Overexpression of the full-length neurotrophin
N
receptor trkB regulates the expression of plasticity-related genes in mouse brain. Brain
Res. Mol. Brain Res. 130, 81-94.
Koponen, E., Rantamaki, T., Voikar, V., Saarelainen, T., MacDonald, E., Castren, E., (2005).
Enhanced BDNF signaling is associated with an antidepressant-like behavioral response
and changes in brain monoamines. Cell. Mol. Neurobiol. 25, 973-980.
Koponen, E., Voikar, V., Riekki, R., Saarelainen, T., Rauramaa, T., Rauvala, H., Taira, T.,
Castren, E., (2004b). Transgenic mice overexpressing the full-length neurotrophin
c.
receptor trkB exhibit increased activation of the trkB-PLCgamma pathway, reduced
anxiety, and facilitated learning. Mol. Cell. Neurosci. 26, 166-181.
Korte, M., Carroll, P., Wolf, E., Brem, G., Thoenen, H., Bonhoeffer, T., (1995). Hippocampal
In
long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor.
Proc. Natl. Acad. Sci. U. S. A. 92, 8856-8860.
Krause, S., Schindowski, K., Zechel, S., von Bohlen und Halbach, O., (2008). Expression of
trkB and trkC receptors and their ligands brain-derived neurotrophic factor and
neurotrophin-3 in the murine amygdala. J. Neurosci. Res. 86, 411-421.
rs
Lee, J., Duan, W., Mattson, M.P., (2002). Evidence that brain-derived neurotrophic factor is
required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis
by dietary restriction in the hippocampus of adult mice. J. Neurochem. 82, 1367-1375.
Lee, R., Kermani, P., Teng, K.K., Hempstead, B.L., (2001). Regulation of cell survival by
he
secreted proneurotrophins. Science 294, 1945-1948.
Lee, T.H., Kato, H., Pan, L.H., Ryu, J.H., Kogure, K., Itoyama, Y., (1998). Localization of
nerve growth factor, trkA and P75 immunoreactivity in the hippocampal formation and
basal forebrain of adult rats. Neuroscience 83, 335-349.
Lima, L., Matus, P., Urbina, M., (1994). Serotonin inhibits outgrowth of goldfish retina and
is
impairs the trophic effect of taurine. J. Neurosci. Res. 38, 444-450.
Liu, Q.R., Lu, L., Zhu, X.G., Gong, J.P., Shaham, Y., Uhl, G.R., (2006). Rodent BDNF
genes, novel promoters, novel splice variants, and regulation by cocaine. Brain Res.
bl
1067, 1-12.
Lu, B., Nagappan, G., Guan, X., Nathan, P.J., Wren, P., (2013). BDNF-based synaptic repair
as a disease-modifying strategy for neurodegenerative diseases. Nat. Rev. Neurosci. 14,
401-416.
Pu
Lu, B., Pang, P.T., Woo, N.H., (2005). The yin and yang of neurotrophin action. Nat. Rev.
Neurosci. 6, 603-614.
Lu, Y., Christian, K., Lu, B., (2008). BDNF: a key regulator for protein synthesis-dependent
LTP and long-term memory? Neurobiol. Learn. Mem. 89, 312-323.
Ma, D.K., Jang, M.H., Guo, J.U., Kitabatake, Y., Chang, M.L., Pow-Anpongkul, N., Flavell,
R.A., Lu, B., Ming, G.L., Song, H., (2009). Neuronal activity-induced Gadd45b promotes
epigenetic DNA demethylation and adult neurogenesis. Science 323, 1074-1077.
a
MacQueen, G.M., Ramakrishnan, K., Croll, S.D., Siuciak, J.A., Yu, G., Young, L.T.,
Fahnestock, M., (2001). Performance of heterozygous brain-derived neurotrophic factor
ov
knockout mice on behavioral analogues of anxiety, nociception, and depression. Behav.

Neurosci. 115, 1145-1153.
Madsen, T.M., Treschow, A., Bengzon, J., Bolwig, T.G., Lindvall, O., Tingstrom, A., (2000).
Increased neurogenesis in a model of electroconvulsive therapy. Biol. Psychiatry 47,
1043-1049.
N
Malberg, J.E., Eisch, A.J., Nestler, E.J., Duman, R.S., (2000). Chronic antidepressant
treatment increases neurogenesis in adult rat hippocampus. J. Neurosci. 20, 9104-9110.
Mancini, D.N., Singh, S.M., Archer, T.K., Rodenhiser, D.I., (1999). Site-specific DNA
methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB
and SP1 transcription factors. Oncogene 18, 4108-4119.
Manji, H.K., Drevets, W.C., Charney, D.S., (2001). The cellular neurobiology of depression.
Nat. Med. 7, 541-547.
c.
Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y., Fan, G., Sun, Y.E., (2003).
DNA methylation-related chromatin remodeling in activity-dependent BDNF gene
regulation. Science 302, 890-893.
In
Martinowich, K., Manji, H., Lu, B., (2007). New insights into BDNF function in depression
and anxiety. Nat. Neurosci. 10, 1089-1093.
Maya Vetencourt, J.F., Sale, A., Viegi, A., Baroncelli, L., De Pasquale, R., O'Leary, O.F.,
Castren, E., Maffei, L., (2008). The antidepressant fluoxetine restores plasticity in the
adult visual cortex. Science 320, 385-388.
rs
McClung, C.A., Nestler, E.J., (2003). Regulation of gene expression and cocaine reward by
CREB and DeltaFosB. Nat. Neurosci. 6, 1208-1215.
McEwen, B.S., (1999). Stress and hippocampal plasticity. Annu. Rev. Neurosci. 22, 105-122.
Metsis, M., Timmusk, T., Arenas, E., Persson, H., (1993). Differential usage of multiple
he
brain-derived neurotrophic factor promoters in the rat brain following neuronal
activation. Proc. Natl. Acad. Sci. U. S. A. 90, 8802-8806.
Minichiello, L., (2009). TrkB signalling pathways in LTP and learning. Nat. Rev. Neurosci.
10, 850-860.
Mombereau, C., Gur, T.L., Onksen, J., Blendy, J.A., (2010). Differential effects of acute and
is
repeated citalopram in mouse models of anxiety and depression. Int. J.
Neuropsychopharmacol. 13, 321-334.
Monteggia, L.M., Barrot, M., Powell, C.M., Berton, O., Galanis, V., Gemelli, T., Meuth, S.,
bl
Nagy, A., Greene, R.W., Nestler, E.J., (2004). Essential role of brain-derived
neurotrophic factor in adult hippocampal function. Proc. Natl. Acad. Sci. U. S. A. 101,
10827-10832.
Monteggia, L.M., Luikart, B., Barrot, M., Theobold, D., Malkovska, I., Nef, S., Parada, L.F.,
Pu
Nestler, E.J., (2007). Brain-derived neurotrophic factor conditional knockouts show

gender differences in depression-related behaviors. Biol. Psychiatry 61, 187-197.
Muller, M.B., Lucassen, P.J., Yassouridis, A., Hoogendijk, W.J., Holsboer, F., Swaab, D.F.,
(2001). Neither major depression nor glucocorticoid treatment affects the cellular
integrity of the human hippocampus. Eur. J. Neurosci. 14, 1603-1612.
Murray, C.J., Lopez, A.D., (1997). Alternative projections of mortality and disability by
cause 1990-2020: Global Burden of Disease Study. Lancet 349, 1498-1504.
a
Murray, F., Hutson, P.H., (2007). Hippocampal Bcl-2 expression is selectively increased
following chronic but not acute treatment with antidepressants, 5-HT(1A) or 5-
ov
HT(2C/2B) receptor antagonists. Eur. J. Pharmacol. 569, 41-47.

Muschamp, J.W., Van't Veer, A., Parsegian, A., Gallo, M.S., Chen, M., Neve, R.L., Meloni,
E.G., Carlezon, W.A., Jr., (2011). Activation of CREB in the nucleus accumbens shell
produces anhedonia and resistance to extinction of fear in rats. J. Neurosci. 31, 3095-
3103.
N
Nair, A., Vadodaria, K.C., Banerjee, S.B., Benekareddy, M., Dias, B.G., Duman, R.S.,
Vaidya, V.A., (2007). Stressor-specific regulation of distinct brain-derived neurotrophic
factor transcripts and cyclic AMP response element-binding protein expression in the
postnatal and adult rat hippocampus. Neuropsychopharmacology 32, 1504-1519.
Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J., Monteggia, L.M., (2002).
Neurobiology of depression. Neuron 34, 13-25.
Nestler, E.J., Carlezon, W.A., Jr., (2006). The mesolimbic dopamine reward circuit in
c.
depression. Biol. Psychiatry 59, 1151-1159.
Newton, S.S., Collier, E.F., Hunsberger, J., Adams, D., Terwilliger, R., Selvanayagam, E.,
Duman, R.S., (2003). Gene profile of electroconvulsive seizures: induction of
In
neurotrophic and angiogenic factors. J. Neurosci. 23, 10841-10851.
Newton, S.S., Thome, J., Wallace, T.L., Shirayama, Y., Schlesinger, L., Sakai, N., Chen, J.,
Neve, R., Nestler, E.J., Duman, R.S., (2002). Inhibition of cAMP response element-
binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like
effect. J. Neurosci. 22, 10883-10890.
rs
Nibuya, M., Morinobu, S., Duman, R.S., (1995). Regulation of BDNF and trkB mRNA in rat
brain by chronic electroconvulsive seizure and antidepressant drug treatments. J.
Neurosci. 15, 7539-7547.
Nibuya, M., Nestler, E.J., Duman, R.S., (1996). Chronic antidepressant administration
he
increases the expression of cAMP response element binding protein (CREB) in rat
hippocampus. J. Neurosci. 16, 2365-2372.
Onishchenko, N., Karpova, N., Sabri, F., Castren, E., Ceccatelli, S., (2008). Long-lasting
depression-like behavior and epigenetic changes of BDNF gene expression induced by
perinatal exposure to methylmercury. J. Neurochem. 106, 1378-1387.
is
Palmer, T.D., Takahashi, J., Gage, F.H., (1997). The adult rat hippocampus contains
primordial neural stem cells. Mol. Cell. Neurosci. 8, 389-404.
Pencea, V., Bingaman, K.D., Wiegand, S.J., Luskin, M.B., (2001). Infusion of brain-derived
bl
neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the
parenchyma of the striatum, septum, thalamus, and hypothalamus. J. Neurosci. 21, 6706-
6717.
Pinnock, S.B., Blake, A.M., Platt, N.J., Herbert, J., (2010). The roles of BDNF, pCREB and
Pu
Wnt3a in the latent period preceding activation of progenitor cell mitosis in the adult
dentate gyrus by fluoxetine. PLoS ONE 5, e13652.
Pliakas, A.M., Carlson, R.R., Neve, R.L., Konradi, C., Nestler, E.J., Carlezon, W.A., Jr.,
(2001). Altered responsiveness to cocaine and increased immobility in the forced swim
test associated with elevated cAMP response element-binding protein expression in
nucleus accumbens. J. Neurosci. 21, 7397-7403.
Prickaerts, J., De Vry, J., Boere, J., Kenis, G., Quinton, M.S., Engel, S., Melnick, L.,
a
Schreiber, R., (2012). Differential BDNF Responses of Triple Versus Dual Reuptake
Inhibition in Neuronal and Astrocytoma Cells as well as in Rat Hippocampus and
ov
Prefrontal Cortex. J. Mol. Neurosci. 48, 167-175.

Rantamaki, T., Hendolin, P., Kankaanpaa, A., Mijatovic, J., Piepponen, P., Domenici, E.,
Chao, M.V., Mannisto, P.T., Castren, E., (2007). Pharmacologically diverse
antidepressants rapidly activate brain-derived neurotrophic factor receptor TrkB and
induce phospholipase-Cgamma signaling pathways in mouse brain.
N

Rantamaki, T., Vesa, L., Antila, H., Di Lieto, A., Tammela, P., Schmitt, A., Lesch, K.P.,
Rios, M., Castren, E., (2011). Antidepressant drugs transactivate TrkB neurotrophin
receptors in the adult rodent brain independently of BDNF and monoamine transporter
blockade. PLoS ONE 6, e20567.
Rossi, C., Angelucci, A., Costantin, L., Braschi, C., Mazzantini, M., Babbini, F., Fabbri,
M.E., Tessarollo, L., Maffei, L., Berardi, N., Caleo, M., (2006). Brain-derived
neurotrophic factor (BDNF) is required for the enhancement of hippocampal
c.
neurogenesis following environmental enrichment. Eur. J. Neurosci. 24, 1850-1856.
Russo-Neustadt, A., Beard, R.C., Cotman, C.W., (1999). Exercise, antidepressant
medications, and enhanced brain derived neurotrophic factor expression.
In
Saarelainen, T., Hendolin, P., Lucas, G., Koponen, E., Sairanen, M., MacDonald, E.,
Agerman, K., Haapasalo, A., Nawa, H., Aloyz, R., Ernfors, P., Castren, E., (2003).
Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is
required for antidepressant-induced behavioral effects. J. Neurosci. 23, 349-357.
rs
Saarelainen, T., Pussinen, R., Koponen, E., Alhonen, L., Wong, G., Sirvio, J., Castren, E.,
(2000). Transgenic mice overexpressing truncated trkB neurotrophin receptors in neurons
have impaired long-term spatial memory but normal hippocampal LTP. Synapse 38, 102-
104.
he
Sairanen, M., Lucas, G., Ernfors, P., Castren, M., Castren, E., (2005). Brain-derived
neurotrophic factor and antidepressant drugs have different but coordinated effects on
neuronal turnover, proliferation, and survival in the adult dentate gyrus. J. Neurosci. 25,
1089-1094.
is
J., Duman, R., Arancio, O., Belzung, C., Hen, R., (2003). Requirement of hippocampal
neurogenesis for the behavioral effects of antidepressants. Science 301, 805-809.
Scharfman, H., Goodman, J., Macleod, A., Phani, S., Antonelli, C., Croll, S., (2005).
bl
Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF
infusion in adult rats. Exp. Neurol. 192, 348-356.
Shaltiel, G., Chen, G., Manji, H.K., (2007). Neurotrophic signaling cascades in the
pathophysiology and treatment of bipolar disorder. Curr. Opin. Pharmacol. 7, 22-26.
Pu
Shimizu, E., Hashimoto, K., Okamura, N., Koike, K., Komatsu, N., Kumakiri, C., Nakazato,
M., Watanabe, H., Shinoda, N., Okada, S., Iyo, M., (2003). Alterations of serum levels of
brain-derived neurotrophic factor (BDNF) in depressed patients with or without
antidepressants. Biol. Psychiatry 54, 70-75.
Shirayama, Y., Chen, A.C., Nakagawa, S., Russell, D.S., Duman, R.S., (2002). Brain-derived
neurotrophic factor produces antidepressant effects in behavioral models of depression. J.
Neurosci. 22, 3251-3261.
a
Siuciak, J.A., Lewis, D.R., Wiegand, S.J., Lindsay, R.M., (1997). Antidepressant-like effect
of brain-derived neurotrophic factor (BDNF). Pharmacol. Biochem. Behav. 56, 131-137.
ov
Smith, M.A., Makino, S., Kvetnansky, R., Post, R.M., (1995). Stress and glucocorticoids
affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in
the hippocampus. J. Neurosci. 15, 1768-1777.
Smith, M.A., Zhang, L.X., Lyons, W.E., Mamounas, L.A., (1997). Anterograde transport of
endogenous brain-derived neurotrophic factor in hippocampal mossy fibers. Neuroreport
N
8, 1829-1834.
Sobreviela, T., Clary, D.O., Reichardt, L.F., Brandabur, M.M., Kordower, J.H., Mufson, E.J.,
(1994). TrkA-immunoreactive profiles in the central nervous system: colocalization with
neurons containing p75 nerve growth factor receptor, choline acetyltransferase, and
serotonin. J. Comp. Neurol. 350, 587-611.
Song, Y., Miyaki, K., Suzuki, T., Sasaki, Y., Tsutsumi, A., Kawakami, N., Shimazu, A.,
Takahashi, M., Inoue, A., Kan, C., Kurioka, S., Shimbo, T., (2014). Altered DNA
methylation status of human brain derived neurotrophis factor gene could be useful as
c.
biomarker of depression. Am. J. Med. Genet. B Neuropsychiatr. Genet. 165, 357-364.
Stewart, C.A., Reid, I.C., (2000). Repeated ECS and fluoxetine administration have
equivalent effects on hippocampal synaptic plasticity. Psychopharmacology (Berl.) 148,
In
217-223.
Takebayashi, M., Hayashi, T., Su, T.P., (2002). Nerve growth factor-induced neurite
sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants. J.
Pharmacol. Exp. Ther. 303, 1227-1237.
Taliaz, D., Loya, A., Gersner, R., Haramati, S., Chen, A., Zangen, A., (2011). Resilience to
rs
chronic stress is mediated by hippocampal brain-derived neurotrophic factor. J. Neurosci.
31, 4475-4483.
Taliaz, D., Nagaraj, V., Haramati, S., Chen, A., Zangen, A., (2012). Altered Brain-Derived
Neurotrophic Factor Expression in the Ventral Tegmental Area, but not in the
he
Hippocampus, Is Essential for Antidepressant-Like Effects of Electroconvulsive Therapy.
Taliaz, D., Stall, N., Dar, D.E., Zangen, A., (2010). Knockdown of brain-derived neurotrophic
factor in specific brain sites precipitates behaviors associated with depression and reduces
neurogenesis. Mol. Psychiatry 15, 80-92.
is
Tao, X., West, A.E., Chen, W.G., Corfas, G., Greenberg, M.E., (2002). A calcium-responsive
transcription factor, CaRF, that regulates neuronal activity-dependent expression of
BDNF. Neuron 33, 383-395.
bl
Timmusk, T., Palm, K., Metsis, M., Reintam, T., Paalme, V., Saarma, M., Persson, H.,
(1993). Multiple promoters direct tissue-specific expression of the rat BDNF gene.
Neuron 10, 475-489.
Tiraboschi, E., Tardito, D., Kasahara, J., Moraschi, S., Pruneri, P., Gennarelli, M., Racagni,
Pu
G., Popoli, M., (2004). Selective phosphorylation of nuclear CREB by fluoxetine is

linked to activation of CaM kinase IV and MAP kinase cascades.
Tolwani, R.J., Buckmaster, P.S., Varma, S., Cosgaya, J.M., Wu, Y., Suri, C., Shooter, E.M.,
(2002). BDNF overexpression increases dendrite complexity in hippocampal dentate
gyrus. Neuroscience 114, 795-805.
Tsankova, N., Renthal, W., Kumar, A., Nestler, E.J., (2007). Epigenetic regulation in
a
psychiatric disorders. Nat. Rev. Neurosci. 8, 355-367.

Valverde, O., Mantamadiotis, T., Torrecilla, M., Ugedo, L., Pineda, J., Bleckmann, S., Gass,
ov
P., Kretz, O., Mitchell, J.M., Schutz, G., Maldonado, R., (2004). Modulation of anxiety-
like behavior and morphine dependence in CREB-deficient mice.
Varea, E., Blasco-Ibanez, J.M., Gomez-Climent, M.A., Castillo-Gomez, E., Crespo, C.,
Martinez-Guijarro, F.J., Nacher, J., (2007a). Chronic fluoxetine treatment increases the
N
expression of PSA-NCAM in the medial prefrontal cortex. Neuropsychopharmacology

32, 803-812.
Varea, E., Castillo-Gomez, E., Gomez-Climent, M.A., Blasco-Ibanez, J.M., Crespo, C.,
Martinez-Guijarro, F.J., Nacher, J., (2007b). Chronic antidepressant treatment induces
contrasting patterns of synaptophysin and PSA-NCAM expression in different regions of
the adult rat telencephalon. Eur. Neuropsychopharmacol. 17, 546-557.
Vinet, J., Carra, S., Blom, J.M., Brunello, N., Barden, N., Tascedda, F., (2004). Chronic
c.
treatment with desipramine and fluoxetine modulate BDNF, CaMKKalpha and
CaMKKbeta mRNA levels in the hippocampus of transgenic mice expressing antisense
RNA against the glucocorticoid receptor. Neuropharmacology 47, 1062-1069.
In
Vutskits, L., Djebbara-Hannas, Z., Zhang, H., Paccaud, J.P., Durbec, P., Rougon, G., Muller,
D., Kiss, J.Z., (2001). PSA-NCAM modulates BDNF-dependent survival and
differentiation of cortical neurons. Eur. J. Neurosci. 13, 1391-1402.
Wallace, D.L., Han, M.H., Graham, D.L., Green, T.A., Vialou, V., Iniguez, S.D., Cao, J.L.,
Kirk, A., Chakravarty, S., Kumar, A., Krishnan, V., Neve, R.L., Cooper, D.C., Bolanos,
rs
C.A., Barrot, M., McClung, C.A., Nestler, E.J., (2009). CREB regulation of nucleus
accumbens excitability mediates social isolation-induced behavioral deficits. Nat.
Neurosci. 12, 200-209.
Wang, S., Bray, P., McCaffrey, T., March, K., Hempstead, B.L., Kraemer, R., (2000).
he
p75(NTR) mediates neurotrophin-induced apoptosis of vascular smooth muscle cells.
Am. J. Pathol. 157, 1247-1258.
Weder, N., Zhang, H., Jensen, K., Yang, B.Z., Simen, A., Jackowski, A., Lipschitz, D.,
Douglas-Palumberi, H., Ge, M., Perepletchikova, F., O'Loughlin, K., Hudziak, J.J.,
Gelernter, J., Kaufman, J., (2014). Child abuse, depression, and methylation in genes
is
involved with stress, neural plasticity, and brain circuitry. J. Am. Acad. Child Adolesc.
Psychiatry 53, 417-424 e415.
West, A.E., Chen, W.G., Dalva, M.B., Dolmetsch, R.E., Kornhauser, J.M., Shaywitz, A.J.,
bl
Takasu, M.A., Tao, X., Greenberg, M.E., (2001). Calcium regulation of neuronal gene
expression. Proc. Natl. Acad. Sci. U. S. A. 98, 11024-11031.
Wu, X., Castren, E., (2009). Co-treatment with diazepam prevents the effects of fluoxetine on
the proliferation and survival of hippocampal dentate granule cells. Biol. Psychiatry 66,
Pu
5-8.
Xu, H., Chen, Z., He, J., Haimanot, S., Li, X., Dyck, L., Li, X.M., (2006). Synergetic effects
of quetiapine and venlafaxine in preventing the chronic restraint stress-induced decrease
in cell proliferation and BDNF expression in rat hippocampus. Hippocampus 16, 551-
559.
Xu, H., Steven Richardson, J., Li, X.M., (2003). Dose-related effects of chronic
antidepressants on neuroprotective proteins BDNF, Bcl-2 and Cu/Zn-SOD in rat
a
hippocampus. Neuropsychopharmacology 28, 53-62.

Yan, Q., Radeke, M.J., Matheson, C.R., Talvenheimo, J., Welcher, A.A., Feinstein, S.C.,
ov
(1997). Immunocytochemical localization of TrkB in the central nervous system of the

adult rat. J. Comp. Neurol. 378, 135-157.
Young, K.M., Merson, T.D., Sotthibundhu, A., Coulson, E.J., Bartlett, P.F., (2007). p75
neurotrophin receptor expression defines a population of BDNF-responsive neurogenic
precursor cells. J. Neurosci. 27, 5146-5155.
N
Yu, H., Chen, Z.Y., (2011). The role of BDNF in depression on the basis of its location in the
neural circuitry. Acta Pharmacol. Sin. 32, 3-11.
Zhang, X., Odom, D.T., Koo, S.H., Conkright, M.D., Canettieri, G., Best, J., Chen, H.,
Jenner, R., Herbolsheimer, E., Jacobsen, E., Kadam, S., Ecker, J.R., Emerson, B.,
Hogenesch, J.B., Unterman, T., Young, R.A., Montminy, M., (2005). Genome-wide
analysis of cAMP-response element binding protein occupancy, phosphorylation, and
target gene activation in human tissues. Proc. Natl. Acad. Sci. U. S. A. 102, 4459-4464.
c.
Zigova, T., Pencea, V., Wiegand, S.J., Luskin, M.B., (1998). Intraventricular administration
of BDNF increases the number of newly generated neurons in the adult olfactory bulb.
Mol. Cell. Neurosci. 11, 234-245.
In
Zorner, B., Wolfer, D.P., Brandis, D., Kretz, O., Zacher, C., Madani, R., Grunwald, I., Lipp,
H.P., Klein, R., Henn, F.A., Gass, P., (2003). Forebrain-specific trkB-receptor knockout
mice: behaviorally more hyperactive than "depressive". Biol. Psychiatry 54, 972-982.
Zusso, M., Debetto, P., Guidolin, D., Barbierato, M., Manev, H., Giusti, P., (2008).
Fluoxetine-induced proliferation and differentiation of neural progenitor cells isolated
rs
from rat postnatal cerebellum. Biochem. Pharmacol. 76, 391-403.
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Chapter 5
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Fluoxetine and Its Novel Effect
on Adult Neurogenesis
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Koji Ohira*
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Division of Systems Medical Science, Institute for Comprehensive Medical Science,
Fujita Health University, Toyoake, Japan
Abstract
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The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat
depression and anxiety disorders. However, the cellular mechanisms underlying the
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antidepressant effects of FLX remain unclear. Previous studies have demonstrated that
FLX treatment increases hippocampal adult neurogenesis, which is the most notable
finding and is hypothesized to mediate the antidepressant effects of this substance. As
alternative candidate mechanisms, recent studies have found that chronic treatment with
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FLX alters adult neurogenesis in two other regions of the adult brain: the subventricular
zone, where adult neurogenesis continues throughout life in the mammal, and the cortex,
where neural progenitor cells exist and their neurogenic capacity can be evoked by
stimuli, including traumatic brain injuries. In this chapter, I will provide an overview of
the evidence for adult neurogenesis in these regions and discuss its functional/therapeutic
relevance for the treatment of neurological and psychiatric disorders.
Keywords: adult neurogenesis, antidepressant, neocortex, progenitor cell, subventricular zone

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Introduction
The antidepressant fluoxetine (FLX) is one of the most widely used drugs for treating
depression and anxiety disorders. FLX is classified into the group of selective serotonin
reuptake inhibitors (SSRIs), and it inhibits the reuptake of serotonin into pre-synaptic
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* Corresponding Author address: Division of Systems Medical Science, Institute for Comprehensive Medical
Science, Fujita Health University, Toyoake 470-1192, Japan. Email: ohira@fujita-hu.ac.jp
98 Koji Ohira
neurons. Consequently, FLX seems to increase the level of serotonin in the synaptic regions,
in which serotonin stays longer than it normally would, and may repeatedly stimulate the
serotonin receptors on post-synaptic neurons. SSRIs immediately change extracellular levels
of serotonin in the central nervous system (CNS), but their therapeutic effects usually require
weeks of treatment (Stahl, 1998). Some of the adverse psychiatric effects of SSRIs also
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emerge after a delay during chronic treatment, or even after withdrawal of the drugs (Ali &
Milev, 2003; Goldberg & Truman, 2003). These findings suggest that adaptive or plastic
changes in the CNS may be associated with the adverse effects as well as the therapeutic
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effects of SSRIs.
The most notable effect of FLX in the CNS may be hippocampal adult neurogenesis. It
has been reported that chronic FLX treatment for 2–4 weeks results in increased neurogenesis
and cell proliferation in the adult dentate gyrus (DG) (Kodama, Fujioka, & Duman, 2004;
Malberg, Eisch, Nestler, & Duman, 2000; Santarelli et al., 2003), a response that has been
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linked to the behavioral effects of FLX (Santarelli et al., 2003). In this chapter, I will focus on
the effects of FLX on adult neurogenesis in regions other than the hippocampus.
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FLX and Adult Neurogenesis in
the Subventricular Zone
The subventricular zone (SVZ) is the other main region where adult neurogenesis occurs
in adult brains under healthy conditions. In the SVZ of adult brains, neural stem/progenitor
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cells produce granule or periglomerular neurons of the olfactory bulb throughout life in
mammals (Zhao, Deng, & Gage, 2008). A large number of studies concerning the effects of
FLX on hippocampal neurogenesis are available, whereas there are only a few reports on the
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effects of FLX on neurogenesis in the SVZ (Encinas, Vaahtokari, & Enikolopov, 2006;
Hodes, Hill-Smith, Suckow, Cooper, & Lucki, 2010; Kodama et al., 2004; Malberg et al.,
2000; Nasrallah, Hopkins, & Pixley, 2010; Santarelli et al., 2003; Surget et al., 2008). Almost
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all of the studies in the literature have revealed no influence of FLX on neurogenesis in the
SVZ. In these experiments, FLX was administered for 2–4 weeks as a chronic treatment
model, and the time course in the experiments on neurogenesis in the SVZ were similar to
those for the DG (Encinas et al., 2006; Hodes et al., 2010; Kodama et al., 2004; Malberg et
al., 2000; Nasrallah et al., 2010; Santarelli et al., 2003; Surget et al., 2008). Fibers containing
5-hydroxytryptamine (5-HT) and 5-HT receptor subtypes can be found in the SVZ (Banasr,
Hery, Printemps, & Daszuta, 2004). In addition, a pharmacological experiment using agonists
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and antagonists of 5-HT receptor subtypes has suggested that 5-HT regulates neurogenesis in
the SVZ (Banasr et al., 2004). These findings highlight the probability that FLX has a late-
onset effect on adult neurogenesis in the SVZ. In fact, a recent study reported that chronic
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treatment with FLX for more than 6 weeks decreased adult neurogenesis in the SVZ of mice
(Figure 1) (Ohira & Miyakawa, 2011), which shows that the effect of FLX on neurogenesis in
the SVZ is opposite to its effect in the DG. In a study of postmortem brains from
unmedicated, depressed suicide cases and healthy controls with no psychiatric problems,
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Maheu et al. (Maheu, Devorak, Davoli, Turecki, & Mechawar, 2012) reported that the former
displayed a tendency toward increased expression of doublecortin, which is a marker of
immature neurons, compared with the latter (p = 0.074).
Fluoxetine and Its Novel Effect on Adult Neurogenesis 99
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Figure 1. Effects of FLX on adult neurogenesis in the olfactory bulb.

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(A) Survival of new neurons 3 weeks after BrdU treatment in the olfactory bulbs of mice treated with
FLX for 9 weeks. Tissue sections were double-stained with anti-BrdU (green) and anti-NeuN
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(magenta). Higher magnifications of the boxed-in areas in (A) are shown in (B). (B) High power
images of BrdU and NeuN double-positive cells (indicated by arrowheads) in the olfactory bulbs in
control (top) and FLX-treated mice (bottom). (C) Quantification of BrdU and NeuN double-
positive cells in the olfactory bulb. The values are means ± SEM of 4–5 animals in each group. ** p <
0.01, compared with sham-operated group. Ex, external plexiform layer; Gr, granule cell layer; Gm,
glomerular layer; Su, subependymal zone. Figure from Ref. 13.
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Furthermore, those receiving antidepressant treatment at the time of death displayed

significantly lower levels of cell proliferation compared with patients not receiving
100 Koji Ohira
antidepressant treatment (p = 0.036); on the other hand, unmedicated patients displaying

higher levels of cell proliferation in the SVZ than either the medicated patients (p = 0.069) or
the controls (p = 0.049). This study in humans suggests that adult neurogenesis in the SVZ
may be increased in unmedicated patients with depression, and that antidepressants reverse
the increase in adult neurogenesis in the SVZ, findings consistent with the results of murine
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experiments. Recent studies have shown that treatment with antidepressants containing FLX
increases adult neurogenesis in the SVZ. However, since there is little evidence supporting a
role for FLX in adult SVZ neurogenesis, it will be necessary to further characterize the effects
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of FLX on adult neurogenesis in the SVZ.
Chronic FLX Treatment Increases Cortical

Adult Neurogenesis
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FLX not only has antidepressant effects, but also neuroprotective functions, following
ischemia/stroke in human subjects and animal models. The cortex seems to be a non-
neurogenic region in adult mammals under healthy conditions (Gould, Reeves, Graziano, &
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Gross, 1999; Kornack & Rakic, 2001), although there is considerable controversy as to
whether adult neurogenesis occurs in healthy adult mammals. Numerous studies have shown
that adult neurogenesis occurs in the cortex of adult mammals under pathologic conditions,
such as ischemia, aspiration of tissue, and laser-induced lesions (Ohira, 2011). These results
suggest that neural stem/progenitor cells (NSCs/NPCs), which can produce new neurons
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depending on pathological stimuli, may exist in the adult cortex or around brain tissues.
Previous studies have found NSCs/NPCs in the SVZ (Gould et al., 1999; Jiang, Gu,
Brännström, Rosqvist, & Wester, 2001; Jin et al., 2003; Magavi, Leavitt, & Macklis, 2000;
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Sundholm-Peters, Yang, Goings, Walker, & Szele, 2005), white matter (Nunes et al., 2003),
gray matter (Dayer, Cleaver, Abouantoun, & Cameron, 2005), and marginal zone (Ohira et
al., 2010; Sirko et al., 2009; Xue et al., 2009). Of these areas, NPCs in the marginal zone at
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least have been shown to respond to FLX treatment (Ohira, Takeuchi, Shoji, & Miyakawa,
2013). Chronic FLX treatment reportedly increased the number of NPCs in the cortex in a
dose-dependent manner and up-regulated production of inhibitory interneurons (Figure 2).
Furthermore, the newborn neurons provided neuroprotection against ischemic brain injury.
The neuroprotective effect of newborn neurons was examined using the cell death marker,
activated caspase-3. The number of active caspase-3-positive cells within 20–110 μm of the
soma of newborn neurons was significantly less than the number within the corresponding
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area around control cells (Figure 3), suggesting that the neuroprotective functions of newly
generated interneurons are localized. The control cells were defined as active-casapse-3-
negative and distributed in the same layers, areas, and locations in the cortex as the newborn
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neurons were in the cortex. In ischemic brains, excitotoxicity is induced by enhanced

excitatory neurotransmission (Blaesse, Airaksinen, Rivera, & Kaila, 2009), and upregulation
of inhibitory mechanisms limits neuronal hyperexcitability in mammalian models of ischemic
damage (Ovbiagele, Kidwell, Starkman, & Saver, 2003). Thus, the increase in the number of
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inhibitory interneurons may enhance the inhibitory effect on excitatory neurotransmission in

ischemic brains. Regardless of the fact that the neuroprotective/therapeutic effects of FLX
under ischemic conditions in animals (Chang et al., 2006; Lim et al., 2009; Li et al., 2009)
and human subjects (Chollet et al., 2011; Dam et al., 1996; Pariente et al., 2001; Yi, Liu, &
Zhai, 2010) have already been demonstrated, the underlying mechanisms are still unclear.
FLX-induced neurogenesis of inhibitory interneurons in the adult cortex might be a candidate
mechanism for the neuroprotective effect of FLX against ischemia, as well as the
antidepressant effect in depression.
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Figure 2. FLX increases in the number of GABAergic interneurons in the adult cortex.
(A) Brain sections were stained with anti-GABA (green) and anti-BrdU (magenta) antibodies.
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Arrowheads indicate the same cell body. Scale bars, 500 μm. (B) In all layers of the cortical regions
examined, FLX treatment significantly increased the number of GABA/BrdU-double-positive
interneurons (*p < 0.05, **p < 0.01, two-way analysis of variance, n = 8 each). In layer 6 particularly,
the number of GABA/BrdU double-positive interneurons was significantly elevated. Moreover, layer-
specific increases in GABA/BrdU double-positive interneurons were also found in layer 3 of the frontal
cortex and layer 5 of the motor cortex. Figure from Ref. 27.
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102 Koji Ohira
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Figure 3. New neurons in the cortex coincide with the suppression of ischemia-induced active caspase-3
expression.
(A, B) Representative cell images are shown. New neurons were labeled using retroviral vectors
expressing the fluorescent protein Venus. Top: a control neuron (A). Bottom: a newly formed neuron
(B). Scale bars, 100 μm (A, B). (C) The number of cells positive for activated caspase-3 within 20–110
μm of a Venus-expressing cell soma was significantly less than the number within the corresponding
area around control cells (*p < 0.05, **p < 0.01, repeated-measures analysis of variance). (D) Hoechst
staining showed that there are many surviving neurons uniformly distributed around both control and
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newly-generated neurons. Figure from Ref. 27.

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Discussion
Until recently, FLX has been considered to up-regulate adult neurogenesis only in the
hippocampus of the adult nervous system. However, it has also been found that FLX has an
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important role in adult neurogenesis in the SVZ and cortex. Interestingly, although FLX can
regulate adult neurogenesis in these regions, the direction of the effect changes depending on
the regions: upregulation in the hippocampus and cortex and downregulation in the SVZ.
Although the molecular mechanisms that result in different effects of FLX on adult
neurogenesis in these regions are not known, a possibility is the composition of serotonin
receptors. It has been shown that each subtype of receptors can up-regulate or down-regulate
adult neurogenesis. For examples, 5-HT2C receptors increase cell proliferation in the SVZ
(Banasr et al., 2004), whereas 5-HT1B receptors decrease cell proliferation in the SVZ
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(Banasr et al., 2004). In the hippocampus, intense expression of 5-HT1A receptors has been
detected and increases cell proliferation in the DG (Banasr et al., 2004; Klempin et al., 2010).
Several subtypes of 5-HT receptors, such as 5-HT1A, 1E, 1F, 2A, 2C, are distributed in the
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cortex (Barnes & Sharp, 1999), but their effects on adult neurogenesis are currently unknown.
Further studies are needed in the future to reveal the underlying mechanisms by which FLX
determines the direction of adult neurogenesis.
It remains unknown whether or not neurogenesis provides a therapeutic benefit for
depression and/or causes the side effects of FLX treatment. Treatment with SSRIs containing
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FLX seems to cause side effects, such as aggression, violence, and psychosis (Healy,
Herxheimer, & Menkes, 2006). Some of the side effects of FLX might be mediated by
regulation of adult neurogenesis in these regions. It would be of interest to determine whether
and how the regulation of adult neurogenesis by FLX in these regions is responsible for its
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therapeutic effects in depression and/or the side effects of FLX.
The last point is that the existence of endogenous NSCs/NPCs in the hippocampus, SVZ,
and cortex, which can be regulated by FLX, offers the hope that these cells may be utilized to
treat a variety of neurological and perhaps psychiatric disorders. We have noted that drugs
that are orally administrated can regulate neurogenesis in the SVZ, hippocampus, and cortex.
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Although certain drugs are prescribed for specific conditions, they may be used for diverse
purposes. For instances, FLX was originally used for the treatment of depression, but might
also be used for neuroprotection against stroke. Although obstacles remain regarding their
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clinical application, NSCs/NPCs-based therapies for neuropsychological diseases are an area
of intense research interest.
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References
Ali, S., & Milev, R. (2003). Switch to mania upon discontinuation of antidepressants in
patients with mood disorders: a review of the literature. Canadian Journal of Psychiatry,
48(4), 258–264.
Banasr, M., Hery, M., Printemps, R., & Daszuta, A. (2004). Serotonin-induced increases in
a
adult cell proliferation and neurogenesis are mediated through different and common 5-
HT receptor subtypes in the dentate gyrus and the subventricular zone.
Neuropsychopharmacology, 29(3), 450–460.
ov
Barnes, N. M., & Sharp, T. (1999). A review of central 5-HT receptors and their function.
Neuropharmacology, 38(8), 1083–1152.
Blaesse, P., Airaksinen, M. S., Rivera, C., & Kaila, K. (2009). Cation-chloride cotransporters
and neuronal function. Neuron, 61(6), 820–838.
Chang, Y.-C., Tzeng, S.-F., Yu, L., Huang, A.-M., Lee, H.-T., Huang, C.-C., & Ho, C.-J.
N
(2006). Early-life fluoxetine exposure reduced functional deficits after hypoxic-ischemia

brain injury in rat pups. Neurobiology of Disease, 24(1), 101–113.
104 Koji Ohira
Chollet, F., Tardy, J., Albucher, J.-F., Thalamas, C., Berard, E., Lamy, C., Bejot, Y., Deltour,
S., Jaillard, A., Niclot, P., Guillon, B., Moulin, T., Marque, P., Pariente, J., Arnaud, C., &
Loubinoux, I. (2011). Fluoxetine for motor recovery after acute ischaemic stroke
(FLAME): a randomised placebo-controlled trial. Lancet Neurology, 10(2), 123–130.
Dam, M., Tonin, P., De Boni, A., Pizzolato, G., Casson, S., Ermani, M., Freo, U., Piron, L.,
c.
&Battistin, L. (1996). Effects of fluoxetine and maprotiline on functional recovery in
poststroke hemiplegic patients undergoing rehabilitation therapy. Stroke, 27(7), 1211–
1214.
In
Dayer, A. G., Cleaver, K. M., Abouantoun, T., & Cameron, H. A. (2005). New GABAergic
interneurons in the adult neocortex and striatum are generated from different precursors.
The Journal of Cell Biology, 168(3), 415–427.
Encinas, J. M., Vaahtokari, A., & Enikolopov, G. (2006). Fluoxetine targets early progenitor
cells in the adult brain. Proceedings of the National Academy of Sciences of the United
rs
States of America, 103(21), 8233–8238.
Goldberg, J. F., & Truman, C. J. (2003). Antidepressant-induced mania: an overview of
current controversies. Bipolar Disorders, 5(6), 407–420.
Gould, E., Reeves, A. J., Graziano, M. S., & Gross, C. G. (1999). Neurogenesis in the
he
neocortex of adult primates. Science (New York, N.Y.), 286(5439), 548–552.
Healy, D., Herxheimer, A., & Menkes, D. B. (2006). Antidepressants and violence: problems
at the interface of medicine and law. PLoS Medicine, 3(9), e372.
Hodes, G. E., Hill-Smith, T. E., Suckow, R. F., Cooper, T. B., & Lucki, I. (2010). Sex-
specific effects of chronic fluoxetine treatment on neuroplasticity and pharmacokinetics
is
in mice. The Journal of Pharmacology and Experimental Therapeutics, 332(1), 266–273.
Jiang, W., Gu, W., Brännström, T., Rosqvist, R., & Wester, P. (2001). Cortical neurogenesis
in adult rats after transient middle cerebral artery occlusion. Stroke, 32(5), 1201–1207.
bl
Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X. O., Batteur, S., & Greenberg, D. A. (2003).
Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum.
Molecular and Cellular Neurosciences, 24(1), 171–189.
Klempin, F., Babu, H., De Pietri Tonelli, D., Alarcon, E., Fabel, K., & Kempermann, G.
Pu
(2010). Oppositional effects of serotonin receptors 5-HT1a, 2, and 2c in the regulation of

adult hippocampal neurogenesis. Frontiers in Molecular Neuroscience, 3, 14.
Kodama, M., Fujioka, T., & Duman, R. S. (2004). Chronic olanzapine or fluoxetine
administration increases cell proliferation in hippocampus and prefrontal cortex of adult
rat. Biological Psychiatry, 56(8), 570–580.
Kornack, D. R., & Rakic, P. (2001). Cell proliferation without neurogenesis in adult primate
neocortex. Science (New York, N.Y.), 294(5549), 2127–30.
a
Lim, C.-M., Kim, S.-W., Park, J.-Y., Kim, C., Yoon, S. H., & Lee, J.-K. (2009). Fluoxetine
affords robust neuroprotection in the postischemic brain via its anti-inflammatory effect.
ov
Journal of Neuroscience Research, 87(4), 1037–1045.

Li, W.-L., Cai, H.-H., Wang, B., Chen, L., Zhou, Q.-G., Luo, C.-X., Liu, N., Ding, X.-S., &
Zhu, D.-Y. (2009). Chronic fluoxetine treatment improves ischemia-induced spatial
cognitive deficits through increasing hippocampal neurogenesis after stroke. Journal of
Neuroscience Research, 87(1), 112–122.
N
Magavi, S. S., Leavitt, B. R., & Macklis, J. D. (2000). Induction of neurogenesis in the
neocortex of adult mice. Nature, 405(6789), 951–955.
Maheu, M., Devorak, J., Davoli, M., Turecki, G., & Mechawar, N. (2012). Human olfactory
bulb neurogenesis in medicated and unmedicated depressed suicides. (Program No.
358.05/X4). Presented at the Society for Neuroscience, New Orleans, LA.
Malberg, J. E., Eisch, A. J., Nestler, E. J., & Duman, R. S. (2000). Chronic antidepressant
treatment increases neurogenesis in adult rat hippocampus. Journal of Neuroscience,
c.
20(24), 9104–9110.
Nasrallah, H. A., Hopkins, T., & Pixley, S. K. (2010). Differential effects of antipsychotic
and antidepressant drugs on neurogenic regions in rats. Brain Research, 1354, 23–29.
In
Nunes, M. C., Roy, N. S., Keyoung, H. M., Goodman, R. R., McKhann, G., 2nd, Jiang, L.,
Kang, J., Nedergaard, M., & Goldman, S. A. (2003). Identification and isolation of
multipotential neural progenitor cells from the subcortical white matter of the adult
human brain. Nature Medicine, 9(4), 439–447.
Ohira, K. (2011). Injury-induced neurogenesis in the mammalian forebrain. Cellular and
rs
Molecular Life Sciences, 68(10), 1645–1656.
Ohira, K., Furuta, T., Hioki, H., Nakamura, K. C., Kuramoto, E., Tanaka, Y., Funatsu, N.,
Shimizu, K., Oishi, T., Hayashi, M., Miyakawa, T., Kaneko, T., & Nakamura, S. (2010).
Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells. Nature
he
Neuroscience, 13(2), 173–179.
Ohira, K., & Miyakawa, T. (2011). Chronic treatment with fluoxetine for more than 6 weeks
decreases neurogenesis in the subventricular zone of adult mice. Molecular Brain, 4, 10.
Ohira, K., Takeuchi, R., Shoji, H., & Miyakawa, T. (2013). Fluoxetine-induced cortical adult
neurogenesis. Neuropsychopharmacology, 38(6), 909–920.
is
Ovbiagele, B., Kidwell, C. S., Starkman, S., & Saver, J. L. (2003). Neuroprotective agents for
the treatment of acute ischemic stroke. Current Neurology and Neuroscience Reports,
3(1), 9–20.
bl
Pariente, J., Loubinoux, I., Carel, C., Albucher, J. F., Leger, A., Manelfe, C., Rascol, O., &
Chollet, F. (2001). Fluoxetine modulates motor performance and cerebral activation of
patients recovering from stroke. Annals of Neurology, 50(6), 718–729.
Pu
J., Duman, R., Arancio, O., Belzung, C., & Hen, R. (2003). Requirement of hippocampal
neurogenesis for the behavioral effects of antidepressants. Science (New York, N.Y.),
301(5634), 805–809.
Sirko, S., Neitz, A., Mittmann, T., Horvat-Bröcker, A., von Holst, A., Eysel, U. T., &
Faissner, A. (2009). Focal laser-lesions activate an endogenous population of neural
stem/progenitor cells in the adult visual cortex. Brain, 132, 2252–2264.
Stahl, S. M. (1998). Mechanism of action of serotonin selective reuptake inhibitors. Serotonin
a
receptors and pathways mediate therapeutic effects and side effects. Journal of Affective
Disorders, 51(3), 215–235.
ov
Sundholm-Peters, N. L., Yang, H. K. C., Goings, G. E., Walker, A. S., & Szele, F. G. (2005).
Subventricular zone neuroblasts emigrate toward cortical lesions. Journal of
Neuropathology and Experimental Neurology, 64(12), 1089–1100.
Surget, A., Saxe, M., Leman, S., Ibarguen-Vargas, Y., Chalon, S., Griebel, G., Hen, R., &
Belzung, C. (2008). Drug-dependent requirement of hippocampal neurogenesis in a
N
model of depression and of antidepressant reversal. Biological Psychiatry, 64(4), 293–

301.
106 Koji Ohira
Xue, J.-H., Yanamoto, H., Nakajo, Y., Tohnai, N., Nakano, Y., Hori, T., Iihara, K., &
Miyamoto, S. (2009). Induced spreading depression evokes cell division of astrocytes in
the subpial zone, generating neural precursor-like cells and new immature neurons in the
adult cerebral cortex. Stroke, 40(11), e606–613.
Yi, Z. M., Liu, F., & Zhai, S. D. (2010). Fluoxetine for the prophylaxis of poststroke
c.
depression in patients with stroke: a meta-analysis. International Journal of Clinical
Practice, 64(9), 1310–1317.
Zhao, C., Deng, W., & Gage, F. H. (2008). Mechanisms and functional implications of adult
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neurogenesis. Cell, 132(4), 645–660.
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Chapter 6
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The Role of Neurotrophins in
Fluoxetine’s Mechanism of Action
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Maria Ladea1* and Mihai Bran2
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1
University of Medicine and Pharmacy ―Carol Davila‖, Bucharest, Romania
Clinical Hospital of Psychiatry ―Prof. Dr. Alexandru Obregia‖
2
Coltea Hospital, Bucharest, Romania
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Abstract
Neurotrophins are involved in many functions, from differentiation and neuronal
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survival to synaptogenesis and activity-dependent forms of synaptic plasticity. Four
neurotrophins have been identified so far: nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4). Among
neurotrophins, BDNF is the most abundant and it has been more extensively studied and
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it has been associated with several disorders, such as mood disorders, schizophrenia,
substance-related disorders, eating disorders, and also with pain modulation. BDNF was
related to neuronal survival, synaptic signaling and synaptic consolidation. BDNF
enhances the growth and maintenance of several neuronal systems, serves as a
neurotransmitter modulator, and participates in plasticity mechanisms. Studies have been
performed assessing BDNF levels in depressive disorder and showed important
correlations between Major depressive disorder (MDD) and BDNF levels. The
substitution of valine (Val) to methionine (Met) in the BDNF gene has been shown to
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influence the activity of the BDNF protein with the Met allele of the BDNF being linked
with reduced BDNF activity and high risk for affective disorders. Patients with major
depression who are otherwise medically healthy have been observed to have activated
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inflammatory pathways and some studies suggest that inflammatory cytokines may
influence neuroplasticity by interfering with BDNF signaling. Stress and antidepressant
treatment exert opposing actions on the expression of BDNF in limbic brain regions
involved in the regulation of mood and cognition.
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*
Corresponding author address: Clinical Hospital of Psychiatry ―Prof. Dr. Alexandru Obregia‖, Berceni 10-12,
Sector 4, Bucharest, Romania; E-mail: marialadea@gmail.com.
108 Maria Ladea and Mihai Bran
Moreover, the functional significance of altered BDNF expression is highlighted by

studies demonstrating that stress and depression can lead to neuronal atrophy and cell
loss in hippocampus and other key limbic brain regions implicated in depression, and that
antidepressant treatment, including fluoxetine can block or reverse these effects. The
mechanisms underlying the actions of antidepressant treatment are still under
investigation, but the upregulation of BDNF and other neurotrophic factors by selective
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serotonin reuptake inhibitors and norepineprhine selective reuptake inhibitors
antidepressants could contribute to the long-term adaptations that are required for the
therapeutic actions of these treatments.
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Keywords: neurotrophins, antidepressants, depression
Introduction
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Neurotrophins are very important in central nervous system function, from differentiation
and neuronal survival to synaptogenesis and activity-dependent forms of synaptic plasticity.
Neurotrophins belong to a family of structurally and functionally related proteins and are a
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subset of neurotrophic factors. In humans there are four members of the neurotrophin family:
nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-
3) and neurotrophin-4 (NT-4) (Dawbarn et al., 2003).
Neurotrophins are involved in many functions both in central nervous system and
peripheral nervous system: differentiation and neuronal survival, synaptogenesis and activity-
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dependent forms of synaptic plasticity. Neurotrophins are responsible for various actions in
the developing peripheral and central nervous systems and once released by target cells,
regulate the type and the number of afferent synapses by promoting the survival of discrete
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neuronal sub-populations. Neurotrophins regulate cell death and survival in development as
well as in pathophysiological states, they affect neuronal survival and growth, they regulate
differentiation influencing cell fate choices and regulating neurite morphology. Neurotrophins
and their receptors are expressed in areas of the brain that undergo plasticity, indicating that
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they are able to modulate synaptic plasticity. The various effects of different neurotrophins on
neurons can be attributed to their selective binding of receptors: the Trk family of receptor
tyrosine kinases and the p75 neurotrophin receptor (p75NTR) (Jiang et al., 2013).
Neurotrophins Structure
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Neurotrophins arise from precursors, pro-neurotrophins with a molecular weight of 30–

35 kDa, which are proteolytically cleaved to produce mature proteins with a molecular weight
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of 12–13 kDa. Neurotrophins are processed either inside the cell by furin or pro-hormone
convertases, or extracellularly by plasmin or matrix metalloproteinases to produce mature
neurotrophins. Pro-NGF, pro-NT-3, and pro-NT-4 are packaged into constitutive vesicles
before secretion. Pro-BDNF is mainly packaged into regulated secretory pathway vesicles
processed and secreted in an activity-dependent manner. Zhou et al., showed that pro-BDNF
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is expressed in many regions of the rat CNS, including the spinal cord, substantia nigra,
amygdala, hypothalamus, cerebellum, hippocampus and cortex (Zhou et al., 2004). Schwann
The Role of Neurotrophins in Fluoxetine‘s Mechanism of Action 109
cells secrete predominantly pro-NGF and pro-BDNF, indicating that intracellular cleavage of
pro-neurotrophins is less efficient in these cells (Mowla et al., 1999). The pro-forms seem to
be up-regulated in some pathological conditions such as Alzheimer‘s disease, brain injury and
retinal dystrophy.
Structures of NGF, BDNF, NT-3 and NT-4 are quite similar. NGF is a homodimer
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consisting of two strands each 120 amino acids, which non-covalently dimerize to form a 26
kDa protein (Allen et al., 2006).
The pro-domains of all neurotrophins have substantial sequence homology and are
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conserved among vertebrate species. This indicates the fact that the pro-domains have an
important role in intracellular processing and recent studies show that the pro-domain of
neurotrophins is crucial for intracellular trafficking and secretion, and could significantly
affect neurotrophin function (Lu et al., 2005). A single-nucleotide polymorphism in the pro-
domain of the human BDNF gene affects the trafficking and secretion of BDNF. The
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nucleotide change that occurs at nucleotide 196, produce an amino acid substitution (Valine
to Methionine) at codon 66 in the pro-domain. Humans with the Met allele have impairments
in episodic memory, lower levels of hippocampal N-acetyl aspartate and abnormal
hippocampal function, as recorded by functional MRI (Lu et al., 2005).
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Neurotrophin Synthesis
Neurotrophins are synthesized in the endoplasmic reticulum as pro-neurotrophins. After
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being synthesized, pro-neurotrophins may follow different ways:
1) intracellular cleavage followed by secretion;

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2) secretion followed by extracellular cleavage;
3) secretion without cleavage.
Pro-neurotrophins signal by binding and activating the p75NTR receptor and in addition
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to interacting with p75NTR the secreted pro-neurotrophins might be degraded extracellular.
The Synthesis and Sorting of BDNF
Like all neurotrophins pro-BDNF is synthesized in the endoplasmic reticulum. Then it

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binds to intracellular sortilin in the Golgi to facilitate proper folding of the mature domain. A
motif in the mature domain of BDNF binds to carboxypeptidase E, an interaction that sorts
BDNF into large dense core vesicles, which are a component of the regulated secretory
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pathway. In the absence of this motif, BDNF is sorted into the constitutive pathway. After the
binary decision of sorting, BDNF is transported to the appropriate site of release, in dendrites
or in axons. There are cases when the pro-domain is not cleaved intracellular by furin or
protein convertases so pro-BDNF is released by neurons. Extracellular proteases, such as
metalloproteinase and plasmin, can subsequently cleave the pro-region to mature BDNF (Lu
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et al., 2005).
Neurotrophin Receptors
The neurotrophins act via two different receptor types: the p75 neurotrophin receptor,
(p75NTR), and the family of tyrosine kinase receptors (Trk). All the neurotrophins bind with
similar affinity to p75NTR, but each binds specifically with high affinity to different Trk
c.
receptors: NGF binds to TrkA, BDNF and NT-4 bind to TrkB, NT-3 to TrkC (Kaplan et al.,
2000). Pro-neurotrophins bind with high affinity to p75NTR, which was considered to be a
‗low-affinity‘ neurotrophin receptor.
Interaction of mature neurotrophins with Trk receptors leads to cell survival. Binding of
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pro-NGF and pro-BDNF to p75NTR leads to apoptosis. Secreted pro-neurotrophins may
serve as signalling molecules and the cleavage of pro-neurotrophins is an important
mechanism that governs the neurotrophin action.
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Figure 1. Neurotrophin receptors and functions (adapted after Lu B, Pang PT, Woo NH (2005) The
yin and yang of neurotrophin action. Nat Rev Neurosci.6, 603-614).
P75NTR is a member of the tumour necrosis factor family and it has a single
transmembrane region. The extracellular portion contains four cysteine-rich domains and it
has an intracellular Type II death domain which is implicated in apoptosis (Barker et al.,
2004). The intracellular Trk receptor domains are all very similar. The extracellular portion of
each of these Trk receptors can be subdivided into five domains D1–D5 with D5 region
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closest to the membrane, being able to bind its neurotrophin ligand without the other domains
being present (Robertson et al., 2001).
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Neurotrophins exist as homodimers and they pull together two tyrosine kinase receptors
resulting in transphosphorylation of tyrosine residues in the intracellular kinase domain of the
receptors. After being phosphorylated the tyrosines act as docking sites for signaling
molecules which regulate cell growth and survival through the Ras, phosphatidylinositol 3-
kinase and Phospholipase C-γ pathways (Kaplan et al., 2000). BDNF activity on the TrkB
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receptor has potent neuroprotective properties: activation of the mitogen-activated protein

kinase pathway increases expression of bcl-2, a protein family involved in caspase-regulated
apoptosis. BDNF is able to regulate neuronal survival via the phosphatidylinositol 3-
kinase/Akt pathway and BDNF–TrkB interaction results in phosphatidylinositol 3-kinase

generating phosphatidyl inositides which activate the protein kinase Akt. Akt is able to
phosphorylate, and therefore regulate a number of cell survival-related proteins, including
IkB, the forkhead transcription factor FKHRL1, glycogen synthase kinase-3B and Bad, a pro-
apoptotic member of the Bcl-2 family. The p75 neurotrophin receptor is able to modify the
c.
selectivity of binding of the neurotrophins to Trk receptors and it also signals independently
through nuclear factor κB, Akt c-Jun N-terminal kinase (Barker et al., 2004). It interacts with
several adaptor proteins associated with the induction of apoptosis and cell survival.
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Neutrophins and Cell Survival
Sometimes the P75NTR and Trk family receptors are present in the same cell, so it is
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important to establish under what circumstances neurotrophins have a survival or apoptotic
effect. Mature neurotrophins bind preferentially to Trk receptors to promote cell survival,
while pro-neurotrophins bind to p75NTR receptor to induce cell apoptosis. The survival of
neurons depends on the presence of optimal amounts of neurotrophins. The administration of
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neurotrophins to specific brain regions has shown to rescue neuronal loss associated with
ageing or induced by chemical or mechanical insults and the deletion of specific neurotrophin
genes results in the selective loss of certain populations of neurons that depend on the
neurotrophin for survival (Snider et al., 1994). The survival effects of neurotrophins are
mediated by Trk receptors because the pro survival effects of neurotrophins are absent in cells
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that lack Trk receptors. Two signalling pathways important for cell survival have been
identified:
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• the phosphatidylinositol 3-kinase pathway, which suppresses cell death by inhibiting
the apoptotic activities of forkhead56 and bcl2-associated death protein (del Peso et
al., 1997; Datta et al., 1997)
• the mitogen-activated protein kinase signaling cascade, which stimulates the activity
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and/or expression of antiapoptotic proteins, including bcl2 and the transcription

factor cyclic AMP responsive element binding protein (Aloyz et al., 1998; Riccio et
al., 1999).
Neurotrophins and Cell Death

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Binding of pro-neurotrophins to p75NTR determines cell death in various cell types

(oligodendrocytes, Schwann cells, corticospinal neurons, photoreceptors, smooth muscle
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cells, superior cervical ganglion cells) an effect opposite to that of mature neurotrophins
binding to Trk receptors. Sortilin which specifically binds to the pro-domain of NGF or
BDNF serves as a co-receptor with p75NTR in mediating cell death and the presence or
absence of sortilin in a cell determines whether or not the p75NTR receptor function as a
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death receptor. P75NTR receptor is dynamically regulated after insult or injury in the nervous
system and a possible function of pro-neurotrophins could be the elimination of the damaged
cells that express p75NTR (Beattie et al., 2002).
Neurotrophins and Depression

Synaptic Plasticity
In the adult brain, neurotrophins have a key role in synaptic plasticity. In culture cells,
c.
BDNF applied to neurons and muscle cells, potentiates neurotransmitter release at presynaptic
terminals and potentiates excitatory synaptic transmission in hippocampal neurons (Kang et
al., 1995). BDNF plays a critical role in learning and has a role in hippocampal-dependent
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cognitive performance. Disruption of BDNF expression by use of antisense oligonucleotides
in heterozygous BDNF knockout animals or by use of anti-BDNF antibodies causes severe
impairment in spatial memory and learning (Linnarsson et al., 1997; Mu et al., 1999).
Impaired learning was evident in the conditional TrkB knockout mice and in mice
overexpressing the truncated form of TrkB. Long-term potentiation (LTP) is associated with
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memory formation and the consolidation of long-term memory and it is used as a measure of
increase in synaptic efficacy at the synapse. Long-term potentiation occurs with concomitant
depolarization of the postsynaptic membrane, in conjunction with the binding of the
excitatory neurotransmitter glutamate to the N-methyl-d-aspartate receptor, a calcium
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permeable ion channel. BDNF mRNA levels are increased with induction of long-term
potentiation. BDNF knockout mice show impaired LTP in hippocampal slices which can be
rescued by application of exogenous BDNF. Mutational analysis of phosphorylation sites of
TrkB shows that inhibition of phosphorylation at the PLC-γ -binding site results in reduction
in LTP in the hippocampus, whereas mutation of the Shc site, essential for activation of
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mitogen activated protein kinase and PI3K, does not affect this. BDNF binds to the generic
p75NTR and inhibition of p75NTR does not stop BDNF-related long-term potentiation.
BDNF cause TrkB dependent suppression of AMPA-receptor-mediated currents in
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cultured hippocampal cells. BDNF and TrkB receptors also potentiates γ –aminobutyric acid-
receptor mediated responses, via TrkB, and PLC-γ. BDNF also have an effect on modulation
of voltage-dependent potassium channels, by protein phosphorylation and alters the activity
of G-protein-activated potassium channels. At nanomolar concentrations, BDNF was able to
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excite neurons in the hippocampus, cortex and cerebellum. This was reported to be associated
with TrkB-mediated gating of the sodium channels, suggesting a direct activation of NMDA
receptors.
In order to fulfil a regulatory role in synaptic function, it is essential that BDNF itself
should be able to be regulated in an activity-dependent manner. BDNF has been shown to
contain a motif (Ile16, Glu18, Ile105 and Asp106) within the mature protein which is able to
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interact with two basic residues in the sorting receptor carboxypeptidase E. It has been shown
that mutation of these residues in BDNF resulted in missorting of pro-BDNF to the
constitutive pathway in AtT-20 cells. Wild-type BDNF co-localized with pro-
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opiomelanocortin in a punctate pattern along cell processes, whereas mutated BDNF was
mostly perinuclear. Pro-BDNF is reported to be processed less efficiently by intracellular
proteases than the other neurotrophins; however, the amounts released either pro- or mature
BDNF, will presumably affect the binding of the neurotrophin to the receptors, and with the
pro-form binding preferentially to p75NTR rather than TrkB. Increased secretion of pro-
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BDNF may therefore have a negative effect on synaptic plasticity. Since increased neuronal
activity would result in an increase in BDNF synthesis and probably an increase in the pro-
form due to less time for processing, then the processing of pro-BDNF by extracellular
proteases such as plasmin may be important for regulation of LTP in the hippocampus.
BDNF polymorphism (Val66Met) in the pro-region is associated with a marked
impairment in regulated secretion of BDNF. Hippocampal neurons in culture transfected with
the Met66 form of BDNF, unlike the Val66 form, did not show localization in dendritic
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processes and secretogranin II-positive vesicles. Thus BDNF sorting from the Golgi complex
was affected. Met66 BDNF formed clusters in perinuclear regions, whereas Val66 BDNF
colocalized with synaptic markers. There was a consequent marked reduction in activity-
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dependent secretion of BDNF from neurons after potassium depolarization.
Structural Alterations of Synapses
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Long-term changes in synaptic efficacy (long-term potentiation and long-term
depression) are accompanied by morphological alterations of synapses, especially in the
structure and density of dendritic spines. Numerous studies support the idea that structural
modifications of synapses are associated with the bidirectional expression of long-term
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synaptic plasticity: long-term potentiation is accompanied by new synaptic formation and
long-term depression is accompanied by synapse retraction. The actions of mature BDNF
raise the possibility that bidirectional structural changes of synapses could be mediated by
BDNF. Exposure to BDNF leads to axonal branching, dendritic growth and the activity-
dependent refinement of synapses. BDNF regulation of spine formation in the dendrites of
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hippocampal neurons is controlled by cyclic AMP, a signaling molecule that is involved in
long-term potentiation and long-term memory. cAMP controls dendritic spine growth through
two mechanisms: the ‗cAMP gating‘ of BDNF signaling through TrkB phosphorylation, and
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the cAMP regulation of TrkB trafficking to the postsynaptic density. These indicate that
BDNF/Trk signaling is an active mechanism that transforms molecular signals into structural
modifications to elevate the synaptic efficacy. Pro-BDNF seems to promote synaptic
retraction and elimination through the p75NTR receptor. P75NTR receptor can negatively
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modulate the dendrite morphology and dendritic spine number of hippocampal pyramidal
neurons and long-term depression expression is enhanced by exogenous pro-BDNF but
impaired by p75NTR mutation 83. These findings could indicate that pro-BDNF–p75NTR
signaling is important in inducing activity-dependent signals into negative modulation of the
structural plasticity of hippocampal synapses.
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Neurogenesis and Depression

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A correlation between neurogenesis and depression was first established by Malberg and
Duman who reported that fluoxetine could reverse the stress-induced reduction in
hippocampal neurogenesis (Malberg et al., 2003). In vivo studies suggested that BDNF is
required for antidepressant-induced hippocampal neurogenesis. Heterozygous BDNF
knockout mice showed reduced proliferation and survival of hippocampal neuronal progenitor
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cells, and reduced hippocampal neurogenesis induced by dietary restriction or environmental

modification (Lee et al., 2002). Intraventricular and intrahippocampal BDNF infusions
increased neurogenesis in rat hippocampus, while intravenous BDNF administration in a rat
stroke model increased neurogenesis in the dentate gyrus and improved recovery (Zigova et
al., 1998; Scharfman et al., 2005; Schabitz et al., 2007).
NT-3 deletion in dentate gyrus reduced the number of differentiated hippocampal
progenitor cells (Shimazu et al., 2006), while intracerebroventricular NGF infusion promoted
survival of newborn neurons in the dentate gyrus of adults (Frielingsdorf et al., 2007).
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Downstream of Trk receptors, PI3K/Akt and Ras/ERK signaling pathways have been
suggested to converge on nuclear transcription factor cAMP response element-binding protein
(Fournier et al., 2012), which is responsible for regulating expression of genes involved in
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neurogenesis, such as cyclin A and cyclin D2 (Peltier et al., 2007).
Stress and Depression
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Research on animals models provide good information regarding the potential
pathological alterations that occur in humans and the theories resulting from preclinical work
are validated by studies with human patients. There are consistent results proving the
hypothesis that there is a decreased neurotrophic factor support, neuronal atrophy, and cell
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loss in patients who suffer from depression.
BDNF depression hypothesis originates from observations that acute and chronic stress in
humans and mammals decrease endogenous neurotrophin levels and can lead to atrophy of
the hippocampus. There are numerous studies reporting that stress can cause damage and
atrophy of neurons in certain brain structures. This atrophy of neurons was observed
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especially in the hippocampus, which is one of the limbic structures implicated in mood
disorders and which expresses high levels of receptors for glucocorticoids, the major stress
reactive adrenal steroid. Also included in the functions of hippocampal circuitry are control of
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learning and memory and regulation of the hypothalamic-pituitary-adrenal axis, both altered
in depression.
Alterations of hippocampal structure and function in response to stress leaded to an
analysis of neurotrophic factors and their function. There are many studies, which
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demonstrate that many different types of acute and chronic stress decrease the expression of
BDNF in the hippocampus. Decreased expression of BDNF in response to stress could occur
via one or more pathways:
• Administration of glucocorticoids (naturally increased by stress), decrease BDNF

expression, possibly via direct actions of the glucocorticoid receptor on BDNF gene
expression and this could occur via a glucocorticoid response element, although the
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BDNF gene has not been completely characterized.

• Increased 5-HT neurotransmission activates γ-amino butyric acid interneurons in the
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hippocampus, resulting in inhibitory postsynaptic potentials and hyperpolarization

that opposes the activity-dependent regulation of BDNF gene expression. Activity-
dependent gene expression is believed to occur via increased glutamate
neurotransmission that results in activation of Ca2+-dependent kinases, such as
Ca2+/calmodulin dependent protein kinase (CaMK). CaMK phosphorylates the
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cAMP response element binding protein, a transcription factor that activates BDNF
gene expression via binding to a Ca2+/cAMP response element in the exon III-
specific promoter of the BDNF gene.
• Increased expression of interleukin-1β is responsible for the down-regulation of

BDNF expression. IL-1β decreases activity dependent to glutamate release, as well
as to Ca2+ influx, effects that could decrease basal expression of BDNF and
contribute to the actions of stress (Duman 2004).
c.
There is evidence that the damaging effects of stress could be mediated by decreased
expression of other types of neurotrophic factors, not only BDNF. The nerve growth factor
and neurotrophin-3 levels are reported to be decreased in the hippocampus by exposure to
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long-term immobilization stress (Ueyama et al., 1997). Nerve growth factor and NT-3 couple
to the same signal transduction pathways as BDNF through their respective receptors, and
decreased expression of these factors could lead to alterations in the structure and function of
subpopulations of hippocampal neurons, depending on the complements of receptors that are
expressed in each cell type. Stress decreases the expression of another class of growth factor,
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vascular endothelial growth factor.
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Figure 2. Role of stress and antidepressant therapy on hippocampal BDNF functions (adapted after
Groves JO. (2007) Is it time to reassess the BDNF hypothesis of depression? Mol Psychiatry. 12, 1079-
1088).
Stress is reported to produce at least two different effects in hippocampus: a reduction in

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the number and length of apical dendrites of CA3 pyramidal cells and a decrease in neuronal
proliferation in the granule cell layer. Antidepressant administration can block these stress
effects and increase the proliferation of granule cells in adult hippocampus. The effects of
stress occur via increased levels of adrenal glucocorticoids and activation of N-methyl-D-
aspartate receptors. The actions of antidepressant treatment are believed to occur via
increased levels of 5-HT and/or norepinephrine and increased levels of brain derived
neurotrophic factor.
Dwivedi et al., demonstrate in a study that levels of BDNF and TrkB receptors are
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decreased in the prefrontal cortex and hippocampus of suicide victims who were depressed
relative to matched controls or patients taking an antidepressant at the time of death (Dwivedi
et al., 2003).
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Table 1. Postmortem studies of depressed patients that found low levels of BDNF
Population Effect on BDNF Reference

Suicide Depressed Decrease Chen et al., 2001
Suicide Depressed Decrease Dwivedi et al., 2003
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Suicide Depressed Decrease Karege et al., 2005
In another study Chen et al. reports that BDNF levels are increased in patients receiving
antidepressants at the time of death (Chen et al., 2001). Shimizu et al., reports that serum
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BDNF levels are decreased in patients who are depressed and didn‘t receive medication
relative to controls or patients receiving antidepressant treatment, and the severity of
depression is negatively correlated with BDNF levels (Shimizu et al., 2003).
Table 2. Results of studies assessing BDNF levels in depressed patients

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Population Efect on BDNF Reference
Depressed Decrease Karege et al., 2002
Depressed Decrease Shimizu et al., 2003
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Depressed Decrease Karege et al., 2005
Depressed Decrease Ladea et al., 2013
Depressed + Antidepressant Increase Shimizu et al., 2003
Depressed + Antidepressant Increase Gervasoni et al., 2005
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Depressed + Antidepressant Increase Aydemir et al., 2004

Depressed + Antidepressant Increase Ladea et al., 2013
BDNF Val66Met Polymorphism
The Val66Met BDNF polymorphism is the most studied variation in the BDNF gene and
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has been correlated with BDNF function, hippocampal activity, and depression. This single
nucleotide polymorphism at nucleotide 196, leads to substitution of methionine (Met) for
valine (Val) at codon 66, and interferes with BDNF protein sorting, secretion, and BDNF
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mRNA trafficking to dendrites. Some studies suggest a pro-depressive role of the BDNF Met-
allele, which has been also correlated with smaller hippocampal volumes (Frodl et al., 2007)
and patients carrying the Met BDNF are more likely to suffer from suicidal ideation and to
attempt suicide (Sarchiapone et al., 2008).
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Antidepressant Treatment and Neurotrophins

Long-term, chronic antidepressant treatment, including treatment with fluoxetine has led
to the hypothesis that alterations in functional and structural plasticity are necessary for a
therapeutic response (Duman et al., 1997). Due to the fact that neurotrophic factors action
c.
during development phase, contribute to regulation of neuronal survival and function in the
adult brain, members of the nerve growth factor family were considered potential targets for
antidepressant treatment. Many studies confirmed this hypothesis and found that different
In
classes of antidepressants increase the expression of BDNF in the major subfields of the
hippocampus, including the granule cell layer and the CA1, CA3 pyramidal cell layers.
In all classes of antidepressants: selective serotonin reuptake inhibitors, norepinephrine
selective reuptake inhibitors, monoamine oxidase inhibitors, atypical antidepressants, and
electroconvulsive seizures or transcranial magnetic stimulation, the up-regulation of BDNF is
rs
observed with the greatest effect on regulation of the electroconvulsive seizures and the
monoamine oxidase inhibitors. The increase of BDNF expression in the hippocampus was not
observed after the administration of other psychotropic drugs, such as opiates, antipsychotics,
psychostimulants and by this the pharmacological specificity of antidepressants was
he
demonstrated.
The up-regulation of BDNF by antidepressant treatment has been confirmed by a large
number of studies. There have been some inconsistent reports with certain classes of
antidepressants. Electroconvulsive seizures and monoamine oxidase inhibitor antidepressants
are consistently reported to increase levels of BDNF expression in the hippocampus. In most
is
studies, selective serotonin reuptake inhibitors and serotonin and norepinephrine reuptake
inhibitors antidepressants are also reported to increase the expression of BDNF, although
there are some studies that have not observed this effect. This could be due to the dose of
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drug or time of treatment. Brain-derived neurotrophic factor expression is activity dependent
and regulated by a variety of endocrine and environmental stimuli, and it is imperative that
conditions are closely monitored when conducting in vivo drug studies on the expression of
this neurotrophic factor.
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BDNF plays an important role in the plasticity of 5-HT neurons and could contribute to
the regulation of 5-HT neurotransmission in response to stress and antidepressant treatment
(Duman 2004). In combination with reports that serotonin reuptake inhibitors antidepressants
increase BDNF, a positive reciprocal interaction between the 5-HT system and BDNF
regulation could be confirmed.
Chronic antidepressant treatment increases neurogenesis in the adult hippocampus,
a
including increased proliferation and survival of new neurons. Some of the factors that could
underlie the actions of antidepressant treatment are serotonin and the 5-HT1A receptor,
neurotrophins, including brain-derived neurotrophic factor, fibroblast growth factor-2,
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insulin-like growth factor, and the cyclic adenosine monophosphate (cAMP)-cAMP response
element binding protein cascade.
The influence of antidepressants on other neurotrophic factors that may have roles in
survival and function of neurons in the central nervous system has been investigated in
different studies. A study of Newton et al. suggest that administration of electroconvulsive
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seizures increases the expression of VEGF and thereby opposes the actions of stress (Newton
et al., 2003), and some research by Duman suggests that antidepressants also increase the
expression of VEGF in the hippocampus (Duman et al., 2006). Postmortem studies in

depressed subjects report altered expression of fibroblast growth factor 2 and fibroblast
growth factor receptors and depressed patients receiving antidepressants had fibroblast
growth factor transcript levels more similar to control subjects than patients with no
medication (Evans et al., 2004). Mallei et al. demonstrate in a study that antidepressants up
c.
regulate levels of fibroblast growth factor-2. Chronic but not acute antidepressant treatment
with: fluoxetine, desipramine and mianserin increase the expression of fibroblast growth
factor-2 mRNA and also increase immune reactivity in the entorhinal cortex and
In
hippocampus (Mallei et al., 2002).
Table 3. Review of studies that examined the influence on BDNF regulation by different
types of antidepressants
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Treatment Effect on Reference
BDNF
Paroxetine, fluoxetine (SSRI) Increase Nibuya et al., 1996; Coppell et al., 2003; De Foubert et al.,
2004; Vinet et al., 2004
Sertraline (SSRI) Increase Nibuya et al., 1995; Coppell et al., 2003
he
Citalopram (SSRI) Increase Holoubek et al., 2004
Escitalopram (SSRI) Increase Ladea et al., 2013
Venlafaxine (NE/SSRI) Increase Xu et al., 2003
Mianserin (atypical) Increase Nibuya et al., 1995
Tranylcypromine (MAOI) Increase Nibuya et al., 1995, 1996; Russo-Neustadt et al.,
1999;Coppell et al., 2003; Dias et al., 2003; Garza et al.,
2004
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Imipramine, amitriptyline Increase Van Hoomissen et al., 2003; Xu et al., 2003
(tricyclic)
Desipramine (NESRI) Increase Nibuya et al., 1995; Dias et al., 2003; Vinet et al., 2004;
Russo-Neustadt et al., 1999
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Reboxetine (NESRI) Increase Russo-Neustadt et al., 2004
Memantine (NMDA antagonist) Increase Marvanova et al., 2001
AMP Akines Increase Lauterborn et al., 2003
Exercise Increase Neeper et al., 1996; Adlard et al., 2004; Russo-Neustadt et
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al., 1999, 2000, 2004

Electroconvulsive seizures Increase Nibuya et al., 1995; Smith et al., 1997; Newton et al., 2003;
Altar et al., 2003, 2004
Transcranial Magnetic Increase Muller et al., 2000
Stimulation
Desipramine, maprotiline No effect Coppell et al., 2003; Altar et al., 2003
(NESRI)
Tranylcypromine(MAOI) No effect Altar et al., 2003
Fluoxetine (SSRI) No effect Dias et al., 2003; Conti et al., 2002; Altar et al., 2003; Miro
a
et al., 2002
Mianserin, tianeptine (atypical) No effect Coppell et al., 2003; Kuroda and McEwen 1998
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Mechanisms for Antidepressant Regulation of BDNF
Duman demonstrates that antidepressant treatment up-regulates the cyclic adenosine

N
monophosphate (cAMP), cAMP response element binding protein (CREB) cascade and
increases BDNF expression. One of the intracellular pathways regulated by antidepressants is
the cAMP signal transduction cascade. Repeated antidepressant treatment up-regulates the
coupling of the stimulatory G protein (Gs) to adenylyl cyclase (AC), increases levels of
cAMP dependent protein kinase, and increases the function and expression of CREB. cAMP
dependent protein kinase, as well as Ca2+-dependent protein kinases or the mitogen activated
protein (MAP) kinase activate CREB via phosphorylation of specific amino acids residues
(P). Activation of the cAMP, Ca2+ or MAP kinase signaling pathways may occur via
c.
neurotransmitter receptors, including the β-adrenergic receptor (βAR) or 5-HT7 receptors,
which couple to Gs, or the α1-adrenergic receptor (α1AR) or 5-HT1A receptor, which couple
via the Gi/o protein subtype. CREB is a transcription factor, and one of the genes regulated by
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the cAMP-CREB cascade and antidepressant administration is brain-derived neurotrophic
factor gene. The exon III-specific promoter of the BDNF gene contains a Ca2+/cAMP
response element (CaRE) that binds CREB and is responsible for the induction of BDNF
expression. Increased CREB and BDNF could contribute to trophic effects of antidepressants,
including synaptic remodeling, increased neurogenesis, and neuronal survival (Duman 2004).
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Conclusion
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BDNF As a Possible Biomarker of Depression
Identifying biomarkers that could be used for diagnosing the depressive disorder has
remained a goal for all scientists and clinicians. Despite the uncertainty surrounding the
source of BDNF, the simplicity of the test and the consistent findings of reduced BDNF
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levels in subjects with depressive disorder suggest that the BDNF level may be a clinically
useful biomarker. However, the apparent lack of diagnostic specificity is likely to be a major
limitation for the true clinical utility of this measure. In addition to major depression, reduced
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BDNF levels have been reported in several other disorders: schizophrenia, bipolar disorder,
eating disorders, Huntington‘s disease, Alzheimer‘s disease, autism. Another important use of
the neurotrophin levels measurements could be to assess and monitor the response rate and
efficacy of the antidepressant treatment but again we have to face the specificity issue of the
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neurtrophins measurements.
References
Adlard, P., Perreau, V.M., Engesser-Cesar, C., Cotman, C.W. (2004). The time course of
a
induction of brain-derived neurotrophic factor nRNA and protein in the rat hippocampus
following voluntary exercise. Neurosci Lett. 363, 43–48
Allen, S.J., Dawbarn, D., (2006). Clinical relevance of the neurotrophins and their receptors.
ov
Clinical Science. 110, 175–191

Aloyz, R.S, Bamji, S.X., Pozniak, C.D., Toma, J.G., Atwal, J., Kaplan, D.R., Miller, F.D.
(1998). p53 is essential for developmental neuron death as regulated by the TrkA and p75
neurotrophin receptors. J. Cell Biol. 143, 1691–1703
N
Altar, C,, Laeng. P., Jurata, L.W., Brockman, J.A., Lemire, A., Bullard, J., Bukhman, Y.V.,
Young, T.A., Charles, V., Palfreyman, M.G. (2004). Electroconvulsive seizures regulate
gene expression of distinct neurotrophic signaling pathways. J Neurosci. 24, 2667–2677
Altar, C., Whitehead, R.E., Chen, R., Wortwein, G., Madsen, T.M. (2003). Effects of
electroconvulsive seizures and antidepressant drugs on brain-derived neurotrophic factor
protein in rat brain. Biol Psychiatry. 54, 703–709
Aydemir, O., Deveci, A., Taneli, F. (2004). The effect of chronic antidepressant treatment on
serum brain-derived neurotrophic factor levels in depressed patients: A preliminary
c.
study. Prog Neuropsychopharmacol Biol Psychiatry. 29, 261–265
Barker, P.A. (2004). p75NTR is positively promiscuous: novel partners and new insights.
Neuron. 42, 29–533
In
Beattie, M.S., Harrington, A.W., Lee, R., Kim, J.Y., Boyce, S.L., Longo, F.M., Bresnahan,
J.C., Hempstead, B.L., Yoon, S.O. (2002). ProNGF induces p75-mediated death of
oligodendrocytes following spinal cord injury. Neuron. 36, 375–386
Chen, B., Dowlatshahi, D., MacQueen, G.M., Wang, J.F., Young, L.T. (2001). Increased
hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication.
rs
Biol Psychiat. 50, 260–265
Conti, A., Cryan, J.F., Dalvi, A., Lucki, L., Blendy, J.A. (2002). CREB is essential for the
upregulation of BDNF transcription, but not the behavioral or endocrine responses to
antidepressant drugs. J Neurosci. 22, 3262–3268
he
Coppell, A., Pei, Q., Zetterstron, T.S.C. (2003). Bi-phasic change in BDNF gene expression
following antidepressant drug treatment. Neuropharmacology. 44, 903–910
Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., Greenberg, M.E. (1997). Akt
phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
Cell. 91, 231–241
is
Dawbarn, D., Allen, S.J. (2003) Neurotrophins and neurodegeneration. Neuropathol. Appl.
eurobiol. 29, 211–230
Dias, B., Banerjee, S.B., Duman, R.S., Vaidya, V.A. (2003). Differentiation regulation of
bl
brain derived neurotrophic factor transcripts by antidepressant treatments in the adult rat
brain. Neuropharmacology. 45, 553–563
Duman, R.S. (2004). Role of neurotrophic factors in the etiology and treatment of mood
disorders. Neuromolecular Med. 5, 11-25
Pu
Duman, R.S., Heninger, G.R., Nestler, E.J. (1997). A molecular and cellular theory of
depression. Arch Gen Psychiatry. 54, 597– 606
Duman, R.S., Monteggia, L.M. (2006). A neurotrophic model for stress-related mood
disorders. Biol Psychiatry. 59, 1116-1127
Dwivedi, Y., Rao, J.S., Rizavi, H.S., Kotowski, J., Conley, R.R., Roberts, R.C., Tamminga,
C.A., Pandey, G.N. (2003). Abnormal expression and functional characteristics of cyclic
adenosine monophosphate response element binding protein in postmortem brain of
a
suicide subjects. Arch. Gen. Psychiatry. 60, 273–282

De Foubert, G., Carney, S.L., Robinson, C.S., Destexhe, E.J., Tomlinson, R., Hicks, C.A.,
ov
Murray, T.K., Gaillard, J.P., Deville, C., Xhenseval, V., Thomas, C.E., O'Neill, M.J.,
Zetterström, T.S. (2004). Fluoxetine-induced change in rat brain expression of brain
derived neurotrophic factor varies depending on length of treatment. Neuroscience. 128,
597– 604
del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., Nunez, G. (1997). Interleukin-3-
N
induced phosphorylation of BAD through the protein kinase Akt. Science. 278, 687–689
Evans, S., Choudary, P.V., Neal, C.R., Li, J.Z., Vawter, M.P., Tomita, H., Lopez, J.F.,
Thompson, R.C., Meng, F., Stead, J.D., Walsh, D.M., Myers, R.M., Bunney, W.E.,
Watson, S.J., Jones, E.G., Akil, H. (2004). Dysregulation of the fibroblast growth factor
system in major depression. Proc Natl Acad Sci. 101,15506–15511
Frielingsdorf, H., Simpson, D.R., Thal, L.J., Pizzo, D.P. (2007). Nerve growth factor
c.
promotes survival of new neurons in the adult hippocampus. Neurobiol Dis. 26, 47–55
Fournier, N.M., Lee, B., Banasr, M., Elsayed, M., Duman, R.S. (2012). Vascular endothelial
growth factor regulates adult hippocampal cell proliferation through MEK/ERK- and
In
PI3K/Akt-dependent signaling. Neuropharmacology. 63, 642–652
Frodl, T., Schüle, C., Schmitt, G., Born, C., Baghai, T., Zill, P., Bottlender, R., Rupprecht, R.,
Bondy, B., Reiser, M., Möller, H.J., Meisenzahl, E.M. (2007). Association of the brain-
derived neurotrophic factor Val66Met polymorphism with reduced hippocampal volumes
in major depression. Arch Gen Psychiatry. 64, 410–416
rs
Garza, A., Ha, T.G., Chen, M.J., Russo-Neustadt, A.A. (2004) Exercise, antidepressant
treatment, and BDNF mRNA expression in the aging brain. Pharmacol Biochem Behav.
77, 209 –220
Gervasoni, N., Aubry, J.M., Bondolfi, G., Osiek, C., Schwald, M., Bertschy, G., Karege, F.
he
(2005). Partial normalization of serum brain-derived neurotrophic factor in remitted
patients after a major depressive episode. Neuropsychobiology. 51, 234 –238
Groves, J.O. (2007) Is it time to reassess the BDNF hypothesis of depression? Mol
Psychiatry. 12, 1079-1088
Holoubek, G., Noldner, M., Treiber, K., Muller, W.E. (2004) Effect of chronic antidepressant
is
treatment in beta-receptor coupled signal transduction cascade. Which effect matters
most? Pharmacopsychiatry. 37, S113– S119
Jiang, C., Salton, S.R. (2013). The Role of Neurotrophins in Major Depressive Disorder.
bl
Transl Neurosci. 4, 46–58
Kang, H.J., Schuman, E.M. (1995). Neurotrophin induced modulation of synaptic
transmission in the adult hippocampus. J. Physiol. 89, 11–22
Kaplan, D.R., Miller, F.D. (2000). Neurotrophin signal transduction in the nervous system.
Pu
Curr. Opin. Neurobiol. 10, 381–391

Karege, F., Vaudan, G., Schwald, M., Perroud, N., La Harpe, R. (2005). Neurotrophin levels
in postmortem brains of suicide victims and the effects of antemortem diagnosis and
psychotropic drugs. Brain Res Mol Brain Res. 136, 29–37
Karege, F., Perret, H., Bondolfi, G., Schwald, M., Bertschv, G., Aubrey, J.M. (2002).
Psychol Res. 109, 143–148
a
Kuroda, Y., McEwen, B.S. (1998). Effect of chronic restraint stress and tianeptine on growth
factors, growth-associated protein-43 and microtubule-associated protein 2 mRNA
ov
expression in the rat hippocampus. Brain Res Mol Brain Res. 59, 35–39
Ladea, M., Bran, M. (2013). Brain derived neurotrophic factor (BDNF) levels in depressed
women treated with open-label escitalopram. Psychiatr Danub. 25, 128-132
Lauterborn, J., Truong, G.S., Baudry, M., Bi, X., Lynch, G., Gall, C.M. (2003). Chronic
elevation of brain-derived neurotrophic factor by ampakines. J Pharmacol Exp Ther 307,
N
297–305
Lee, J., Duan, W., Mattson, M.P. (2002). Evidence that brain-derived neurotrophic factor is
by dietary restriction in the hippocampus of adult mice. J Neurochem. 82, 1367–1375
Linnarsson, S., Bjorklund, A., Ernfors, P. (1997). Learning deficit in BDNF mutant mice.
Eur. J. Neurosci. 9, 2581–2587
c.
Lu, B., Pang, P.T., Woo, N.H. (2005). The yin and yang of neurotrophin action. Nat Rev
Neurosci.6, 603-614
Lee, J., Duan, W., Mattson, M.P. (2002). Evidence that brain-derived neurotrophic factor is
In
by dietary restriction in the hippocampus of adult mice. J Neurochem. 82, 1367–1375
Malberg, J.E., Duman, R.S. (2003) Cell proliferation in adult hippocampus is decreased by
inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology. 28,
1562–1571
rs
Mallei, A., Shi, B., Mocchetti, I. (2002). Antidepressant treatments induce the expression of
basic fibroblast growth factor in cortical and hippocampal neurons. Mol. Pharmacol. 61,
1017–1024
Marvanova, M., Lakso, M., Pirhonen, J., Nawa, H., Wong, G., Castren, E. (2001). The
he
neuroprotective agent memantine induces brain-derived neurotrophic factor and trkB
receptor expression in rat brain. Mol Cell Neurosci. 18, 247–258
Miro, X., Perez-Torres, S., Puigdomenech, P., Palacios, J.M., Mengod, G. (2002). Differential
distribution of PDE4D splice variant mRNAs in rat brain suggests association with
specific pathways and presynaptical localization. Synapse. 45, 259 –269
is
Mowla, S.J., Pareek, S., Farhadi, H.F., Petrecca, K., Fawcett, J.P., Seidah, N.G., Morris, S.J.,
Sossin, W.S., Murphy, R.A. (1999). Differential sorting of nerve growth factor and brain-
derived neurotrophic factor in hippocampal neurons. J. Neurosci. 19, 2069–2080
bl
Mu, J.S., Li, W.P., Yao, Z.B., Zhou, X.F. (1999). Deprivation of endogenous brain-derived
neurotrophic factor results in impairment of spatial learning and memory in adult rats.
Brain Res. 835, 259–265
Muller, M., Tosci, N., Kresse, A.E., Poat, A., Keck, M.E. (2000). Long-term repetitive
Pu
transcranial magnetic stimulation increases the expression of brainderived neurotrophic

factor and cholecystokinin mRNA, but not neuropeptide tyrosinemRNAin specific areas
of rat brain. Neuropsychopharmacology, 23, 205–215
Neeper, S., Gomez-Pinilla, F., Choi, J., Cotman, C.W. (1996). Physical activity increases
mRNA for brain-derived neurotrophic factor and nerve growth factor in rat brain. Brain
Res. 726, 49 –56
Newton, S., Collier, E., Hunsberger, J., Adams, D., Salvanayagam, E., Duman, R.S. (2003).
a
Gene profile of electroconvulsive seizures: Induction of neurogenic and angiogenic

factors. J Neurosci. 23,10841–10851
ov
Nibuya, M., Nestler, E.J., Duman, R.S. (1996). Chronic antidepressant administration
increases the expression of Camp response element binding protein (CREB) in rat
hippocampus. J Neurosci. 16, 2365–2372
Nibuya, M., Morinobu, S., Duman, R.S. (1995). Regulation of BDNF and trkB mRNA in rat
brain by chronic electroconvulsive seizure and antidepressant drug treatments. J
N
Neurosci. 15, 7539 –7547

Riccio, A., Ahn, S., Davenport, C.M., Blendy, J.A., Ginty, D.D. (1999). Mediation by a
CREB family transcription factor of NGF-dependent survival of sympathetic neurons.
Science. 286, 2358–2361
Robertson, A.G., Banfield, M.J., Allen, S.J., Dando, J.A., Mason, G.G., Tyler, S.J., Bennett,
G.S., Brain, S.D., Clarke, A.R., Naylor, R.L., Wilcock, G.K., Brady, R.L., Dawbarn, D.
c.
(2001). Identification and structure of the nerve growth factor binding site on TrkA.
Biochem. Biophys. Res. Commun. 282, 131–141
Russo-Neustadt, A., Beard, R.C., Cotman, C.W. (1999). Exercise, antidepressant
In
medications, and enhanced brain derived neurotrophic factor expression.
Neuropsychopharmacology. 21, 679–682.
Russo-Neustadt, A., Alejandre, H., Garcia, C., Ivy, A.S., Chen, M.J. (2004). Hippocampal
brain-derived neurotrophic factor expression following treatment with reboxetine,
citalopram, and physical exercise. Neuropsychopharmacology. 29, 2189 –2199
rs
Russo-Neustadt, A., Beard, R.C., Huang, Y.M., Cotman, C.W. (2000). Physical activity and
antidepressant treatment potentiate the expression of specific brain-derived neurotrophic
factor transcripts in the rat hippocampus. Neuroscience. 101, 305–312
Peltier, J., O‘Neill, A., Schaffer, D.V. (2007). PI3K/Akt and CREB regulate adult neural
he
hippocampal progenitor proliferation and differentiation. Dev Neurobiol. 67, 1348–1361
Sarchiapone, M., Carli, V., Roy, A., Iacoviello, L., Cuomo, C., Latella, M.C., di
Giannantonio, M., Janiri, L., de Gaetano, M., Janal, M.N. (2008). Association of
polymorphism (Val66Met) of brain-derived neurotrophic factor with suicide attempts in
depressed patients. Neuropsychobiology. 57, 139–145
is
Scharfman, H., Goodman, J., Macleod, A., Phani, S., Antonelli, C., Croll, S. (2005).
Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF
infusion in adult rats. Exp Neurol. 192, 48–356
bl
Schabitz, W.R., Steigleder, T., Cooper-Kuhn, C.M., Schwab, S., Sommer, C, Schneider, A.,
Kuhn, H.G. (2007). Intravenous brain-derived neurotrophic factor enhances poststroke
sensorimotor recovery and stimulates neurogenesis. Stroke. 38, 2165–2172
Shimizu, E., Hashimoto, K., Olamura, N., (2003). Alterations of serum levels of brain-derived
Pu
neurotrophic factor (BDNF) in depressed patients with or without antidepressants. Biol.

Psychiat. 54, 70–75
Shimazu, K., Zhao, M., Sakata, K., Akbarian, S., Bates, B., Jaenisch, R., Lu, B. (2006). NT-3
facilitates hippocampal plasticity and learning and memory by regulating neurogenesis.
Learn Mem. 13, 307–315
Smith, M., Zhang, L.X., Lyons, W.E., Mamounas, L.A. (1997). Anterograde transport of
endogenous brain-derived neurotrophic factor in hippocampal mossy fibers. Neuroreport.
a
8, 1829 –1834.
Snider, W.D. (1994). Functions of neurotrophins during nervous system development: what
ov
the nockouts are teaching us. Cell. 77, 627–636

Ueyama, T., Kawai, Y., Nemoto, K., Sekimoto, M., Tone, S., Senba, E. (1997).
Immobilization stress educed the expression of neurotrophins and their receptors in the
rat brain. Neurosci Res. 28, 103–110
Van Hoomissen, J.D., Chambliss, H.O., Holmes, P.V., Dishman, R.K. (2003). Effects of
N
chronic exercise and imipramine on mRNA for BDNF after olfactory bulbectomy in rat.
Brain Res. 974, 228 –235
Vinet, J., Cara, S., Blom, J.M., Brunello, N., Barden, N., Tascedda, F. (2004). Chronic
treatment with desipramine and fluoxetine modulate BDNF, CaMKKalpha and
CaMKKbeta mRNA levels in the hippocampus of transgenic mice expressing antisense
RNA against the glucocorticoid receptor. Neuropharmacology. 47, 1062–1069
Zigova, T., Pencea, V., Wiegand, S.J., Luskin, M.B. (1998). Intraventricular administration of
c.
BDNF increases the number of newly generated neurons in the adult olfactory bulb. Mol
Cell Neurosci. 11 234–245
Zhou, X.F., Song, X.Y., Zhong, J.H., Barati, S., Zhou, F.H., Johnson, S.M. (2004).
In
Distribution and localization of pro-brain derived neurotrophic factor-like
immunoreactivity in the peripheral and central nervous system of the adult rat. J.
Neurochem. 91, 704–715
Xu, B., Goulding, E.H., Zang, K., Cepoi, D., Cone, R.D., Jones, K.R., Tecott, L.H.,
Reichardt, L.F. (2003). Brain derived neurotrophic factor regulates energy balance
downstream of melanocortin-4 receptor. Nat Neurosci. 6, 736 –742
rs
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Chapter 7
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The Role of Epigenetic Mechanisms
in the Therapeutic Effects of Fluoxetine
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and Other Selective Serotonin
Reuptake Inhibitors
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Kristen Ashley Horner*
Mercer University School of Medicine, US
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Abstract
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Epigenetics is the regulation of DNA transcription by DNA methylation, histone
modification and non-coding RNAs, in the absence of alterations to the original DNA
sequence that can be transmitted through generations. It is thought that epigenetic
mechanisms play a role in the pathogenesis, as well as the treatment of major depression.
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SSRIs are among the most common compounds used for the treatment of major
depression, and are also used as pharmacotherapy for several other psychiatric disorders.
However, little is known about the potential epigenetic changes that are induced by SSRI
treatment, how SRRI treatment may ameliorate the epigenetic alterations that are thought
to underlie the pathogenesis of major depression or if these changes contribute to
symptom relief. This chapter will review the recent studies on the epigenetic
modifications that are thought to contribute to major depressive disorder and may occur
a
as a result of SSRI treatment. For example, recent data show that SSRI treatment induces
epigenetic modulation of gene expression including histone acetylation in the
hippocampus, which is a region that is thought to be involved in the pathogenesis of
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major depression. In addition, these data suggest that epigenetic mechanisms may
contribute to the persistent therapeutic effects of SSRI treatment.
Keywords: major depressive disorder, antidepressants, limbic system, brain derived

neurotrophic factor, DNA methylation
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*
1550 College Street, Macon, GA 31207; horner_ka@mercer.edu.
126 Kristen Ashley Horner
Introduction
Epigenetics has recently emerged as a mechanism by which exposure to stressors or
stimuli in the environment can lead to enduring and stable changes in gene expression
(Onishchenko et al., 2008; Roth et al., 2009). The term epigenetics refers to modulatory
c.
mechanisms that can function contemporaneously with the nucleotides that compose the
DNA sequence of a gene. Epigenetic mechanisms that are responsible for changing the
function and expression of a gene are not encoded by the DNA sequence of the gene.
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Epigenetic modifications to a gene can result in long-term changes in the expression and
function of that gene, without altering the actual DNA sequence of the gene (Schroeder et al.,
2012). These changes in gene expression and function may be incurred by exposure to
environmental factors and are potentially heritable (Franklin et al., 2010; Roth et al., 2009).
Importantly, epigenetic mechanisms may play a role in the development or susceptibility to a
rs
number of psychiatric conditions, including major depressive disorder (MDD). And as
discussed below, epigenetic mechanisms may also mediate the responses to treatment with
selective serotonin reuptake inhibitors (SSRIs).
DNA Methylation
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Epigenetic Mechanisms
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DNA methylation is one of the most commonly studied mechanisms of epigenetic
modulation, and is thought to affect gene expression at the transcriptional level. Cytosine-
phosphate-guanine dinucleotide (CpG) sites are common targets for the addition of a methyl
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group (Szyf, 2006; Weber and Schubeler, 2007). Covalent modification of cytosine residues
by methylation at 5‘ carbon position leads to the projection of a methyl group into the major
groove of DNA (Newell-Price et al., 2000). This modification commonly occurs in mammals
at the palindromic sequence 5‘-CpG-3‘ without interfering the formation of hydrogen bonds
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with the guanine bases. Roughly 3% of all cytosine resides in the human genome are
methylated and this methylation is necessary for normal cellular function. In mammalian
DNA, the distribution of CpG bases is irregular, with the highest concentrations occurring at
what is known as CpG islands. There is a high concentration of CpG islands in the promoter
regions of genes, and these sites are typically less methylated than CpG sites that lie outside
of the islands (Wang and Leung, 2004).
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Enzymes known as DNA methyltransferases (DNMTs) mediate the addition of methyl

groups to CpG sites within the promoter region of the target gene using S-adenosyl
methionine as a substrate for transmethylation (Frieling and Tadić, 2013; Szyf, 2006; Weber
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and Schubeler, 2007). Several isoforms of DNMTs have been identified: DNMT1, DNMT2,
DNMT3a and DNMT3b (Weber and Schubeler, 2007). Each of these DNMTs mediate
specific enzymatic functions. For example, DNMT1 is thought to sustain methylation patters
during DNA replication, while DNMT3a and DNMT3b may catalyze the methylation of
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previously unmethylated double-stranded DNA (Kim et al., 2009; Newell-Price et al., 2000).
Importantly, methylation of CpG sites in the promoter region can directly suppress gene
expression by preventing the binding of transcription factors and enzymes necessary for the
The Role of Epigenetic Mechanisms in the Therapeutic Effects of Fluoxetine … 127
transcription of RNA (Hervouet et al., 2009; Szyf, 2006; Weber and Schubeler, 2007). The
methylation of CpG sites within the promoter region can also indirectly inhibit gene
expression via the recruitment of enzymes and proteins that result in the remodeling of
chromatin structure to a repressive configuration, leading to an overall decrease in protein
production. Methylated DNA attracts methyl CpG binding (MDB) proteins that associate
c.
with other proteins, including histone deacetylases and form a repressor complexes at the
promoter site of the gene resulting in a closed, inactive conformation of chromatin and a
repression of gene expression (Frieling and Tadić, 2013). Moreover, MDBs can recruit co-
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repressor complexes that interfere with transcriptional machinery, which also represses gene
expression (Vialou et al., 2013). These mechanisms of DNA methylation can effectively
silence a gene for long periods of time and potentially over generations (Rodenhiser and
Mann, 2006).
It is important to note that DNA methylation and the state of chromatin activation often
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work together to control gene expression. Often the methylation pattern of DNA will correlate
with the activation state of chromatin, as active chromatin correlates with unmethylated DNA,
while inactive chromatin correlates with methylated DNA (Szyf, 2006; Tachibana et al.,
2008). As will be discussed below, DNA methylation, which was once thought to be
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completed prior to birth and irreversible in the adult organism, can be influenced by and is
reactive to environmental factors, as well as antidepressant treatment (Dalton et al., 2014;
Vialou et al., 2013).
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Histone Modifications
Chromatin, which is found in the nucleus of the cell, consists genomic DNA wrapped
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around a core of histones. Chromatin serves a way to package and condense large amounts of
DNA, with chromatin existing in its most compacted state during cell division and in its least
condensed state during interphase (Becker et al., 1996). The basic unit of chromatin is the
nucleosome, and the core of the nucleosome consists of a histone octomer that contains one
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dimer, each of four histone isoforms (H2A, H2B, H3 and H4) around which a 147 bp strand
of DNA is wrapped (Borrelli et al., 2008). Post-translational modification of histones or
variations in the histone proteins, can result in chromatin remodeling and changes in
condensation (Berger, 2007). Chromatin exists in one of two functional states: open (active,
decondensed) or closed (inactive, condensed). These states mediate accessibility of the DNA
to enzymatic transcription factors. Specifically, chromatin in the open configuration allows
transcription factors access to the DNA thereby promoting the transcription of mRNA and
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gene expression, while the closed conformation limits the access of transcription factors to the
DNA, impeding mRNA transcription and resulting in a repression of gene expression (Szyf,
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2006; Tachibana et al., 2008).

Histones can be modified by the addition of an acetyl or methyl group at lysine residues
on the tail of the histone. These modifications can alter the interaction between genomic DNA
and histone core and are thought to play a role in the control of gene expression (Berger,
2007; Hayes and Hansen, 2001). Enzymes known as histone acetyltransferases (HATs),
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which use acetyl coenzyme A as a substrate mediate the addition of acetyl groups
(hyperacetylation) to lysine residues on the tails of both H3 and H4 histones, leading to
chromatin entering an active state (Vialou et al., 2013). The addition of acetyl groups negates
the positive charge of the lysine on the histone tail resulting in a weakening of the interaction
of DNA strand with the histone tail and gene activation (Borrelli et al., 2008; Cheung et al.,
2000). Gene expression is promoted by histone hyperacetylation via the subsequent unfolding
of the histone unit and chromatin decondensation, which then allows for the interaction of
transcription initiating factors with the DNA and mRNA synthesis (Crosio et al., 2003a;
c.
Eberharter and Becker, 2002; Grunstein, 1997; Hebbes et al., 1988; Korzus et al., 2004).
Alternatively, acetyl groups can by removed from the histone core by a group of enzymes
known as histone deacetylases (HDACs). HDACs are a family of enzymes that are divided
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into three classes. Class I and II HDACs regulate histone deacetylation at for most genes in
the brain, while class III HDACs appear to deacetylate several nuclear and cytoplasmic
proteins, as well as histones (Bellet and Sassone-Corsi, 2010; Kaeberlein et al., 1999;
Nakahata et al., 2009; Sassone-Corsi, 2012). Overall, histone deacetylation leads to chromatin
condensation and a repression of gene expression (Grunstein, 1997; Pile et al., 2003).
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Methyl groups may also be added to lysine and arginine groups located on the histone
tails by various histone methyltransferases (HMTs), which use S-adenosyl methionine as a
co-substrate (Rice and Allis, 2001). Methylation, unlike acetylation does not alter the charge
of the lysine residues in the histone tail, but does change the steric profile of the histone tail,
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via the addition of mono-, di- or trimethyl groups, which can potentially affect molecular
interactions with the DNA (Dillon et al., 2005, Morgunkova and Barlev, 2006). The status of
chromatin activation relies upon not only the particular lysine residues that are methylated,
but also the number of methyl groups added to the residue (Dillon et al., 2005; Morgunkova
and Barlev, 2006). As mentioned above, the patterns of histone modification and the patterns
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of DNA methylation are linked. It is also important to note that chromatin activity is linked to
methylation, as unmethylated chromatin is typically associated with the permissive state,
while methylated chromatin is usually associated with the repressed state (Szyf, 2006;
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Tachibana et al., 2008; Tsankova et al., 2007; Vialou et al., 2013). MDBs, such methyl-CpG-
binding protein 2 (MeCP2) can be recruited to methylated DNA, and are associated with large
protein complexes, containing HDACs and HMTs, which will repress gene expression
(Lachner and Jenuwein, 2002). Since repression of certain genes, such as brain-derived
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neurotrophic factor (BDNF) may be associated with MDD, then it is possible that differences
in the number and location of histone methylation could mean the difference between a pro-
depressive versus an anti-depressive phenotype (Duman and Newton, 2007; Frieling and
Tadić, 2013; Krishnan and Nestler, 2010).
Interestingly, it is has recently been established that intracellular mechanisms in the brain
can regulate chromatin remodeling (Tsankova et al., 2007). One of the most commonly
studied mechanisms involves the transcription factor cyclic AMP-response-element binding
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(CREB) protein, which is activated by a number of signaling pathways. Increases in cAMP

activity, intracellular Ca2+ and extracellular signal regulated kinase (ERK) all lead to the
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phosphorylation of CREB, which results in CREB activation and the recruitment of CREB
binding protein (CBP). CBP is a transcriptional co-activator that has intrinsic histone
acetyltransferase activity. Acetylation of histones by CBP leads to a loosening of the
chromatin structure, thereby allowing for increased transcriptional activation. This
mechanism of transcriptional activation has been described for a number of genes including
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the immediate early gene c-fos and BDNF, a neurotrophin whose dysregulation is thought to
be associated with MDD (Brami-Cherrier et al., 2005; Crosio et al., 2003b; Huang et al.,
2002; Kumar et al., 2005; Li et al., 2004; Tsankova et al., 2007; Tsankova et al., 2004;
Tsankova et al., 2006).
MicroRNA
c.
As of late, microRNAs (miRNAs) have emerged as an important epigenetic regulator of
gene expression. miRNAs are non-protein coding RNA molecules that are approximately 21-
23 bases long, and regulate the translation of protein from mRNA by binding to
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complementary regions of the target mRNA (Kolshus et al., 2014). The biosynthesis of
miRNAs can occur via several different pathways. For example, in the canonical pathway,
primary miRNAs are be enzymatically transcribed from independent miRNA genes located
within the genomic DNA. Within the nucleus, the primary miRNA is then processed by a
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protein complex to produce a double-stranded precursor miRNA that is approximately 70-110
nucleotides in length (Havens et al., 2012). Alternatively, precursor mRNAs can be derived
via direct transcription from introns, known as mirtrons (Chong et al., 2010; Krol et al.,
2010). Once the precursor miRNA has been synthesized it is transported from the nucleus to
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the cytoplasm where it is further processed to create a 22-bp miRNA that consists of two
complementary strands (Chendrimada et al., 2005). One of the strands of the miRNA is then
loaded into a RNA-induced silencing complex (RISC), and the complex is guided to the
target mRNA transcripts (Kawamata and Tomari, 2010). The miRNA may then regulate the
translation of the mRNA via enzymatic degradation and destruction of the target mRNA or by
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sterically hindering the protein synthesis machinery necessary for translation (Carthew and
Sontheimer, 2009; Krol et al., 2010; Wu et al., 2006).
Over 50% of mammalian mRNAs are potential targets for miRNA binding (Friedman et
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al., 2009), and to date, over 1500 miRNA sequences have been reported in the human
(Kozomara and Griffiths-Jones, 2011). A single miRNA can bind to and regulate several
target mRNAs or alternatively, several miRNAs can bind to and regulate a single mRNA
target. It is commonly accepted that the binding of miRNAs to target mRNAs suppresses
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gene expression; however, recent data indicates that this may not always be the case. For
example, reciprocal relationships can exist whereby the mRNA targets themselves regulate
the function and levels of miRNAs (Pasquinelli, 2012). Importantly, several studies have
shown that miRNAs contribute to the regulation of a number of neurobiological processes
including synaptic plasticity, apoptosis, cellular differentiation and neurogenesis (Bredy et al.,
2011; Kloosterman and Plasterk, 2006; Luikart et al., 2012; Magill et al., 2010; Saba and
Schratt, 2010). As such, dysregulation of miRNA expression has been linked to a number of
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pathological conditions, including MDD (Esteller, 2011; Im and Kenny, 2012).

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Epigenetics and MDD

As mentioned above, epigenetic mechanisms may play a role in the expression of a
number of psychiatric conditions, including MDD. For example, DNA methylation and
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changes in chromatin activation have been reported in humans suffering from MDD. In
particular, a large amount of research has focused on the epigenetic modifications to the
BDNF gene in depression (Boulle et al., 2012). Alterations in BDNF gene expression and its
target, the tropomyosin-receptor-kinase B (trkB) receptor within the brain‘s limbic circuitry
are associated with MDD (Dwivedi et al., 2003; Kim et al., 2007; Lee and Kim, 2010a; Lee
and Kim, 2010b; Thompson Ray et al., 2011). Studies in experimental animals suggest that
repression of bdnf expression in the hippocampus contributes to the pathophysiology of
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MDD, while increased activation of bdnf expression in this region is thought to contribute to
the efficacy of antidepressant treatment (Duman and Monteggia, 2006). On the other hand,
increases in BDNF levels in the nucleus accumbens are associated with depression-like
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behaviors in experimental animals (Krishnan et al., 2007), while reversal of bdnf mRNA
induction in this region results in an antidepressant-like effect (Berton et al., 2006). Several
animal studies indicate that stress, which can exacerbate and precipitate depression, can alter
the bdnf gene via epigenetic mechanisms. The structure of the bdnf gene is complicated as
several different transcripts can be generated via alternatively splicing at eight different
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noncoding exons, each with its own independent promoter (exons I-VIII; (Aid et al., 2007;
Boulle et al., 2012; Liu et al., 2005; Marini et al., 2004; Pruunsild et al., 2007; Tapia-
Arancibia et al., 2004; Timmusk et al., 1993). All eight of these transcripts give rise to an
identical BDNF protein, but each noncoding exon has a unique promoter with distinct
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chromatin architecture, although there is a single polymorphism in the BDNF gene at codon
66 where a valine to methionine substitution can occur that alters BDNF mRNA and protein
trafficking (Aid et al., 2007; Chen et al., 2006; Chiaruttini et al., 2009; Dmitrzak-Weglarz et
al., 2008; Egan et al., 2003; Liu et al., 2005; Marini et al., 2004; Pruunsild et al., 2007;
Rybakowski, 2008; Timmusk et al., 1993). Recent data suggests that in experimental animals,
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exposure to stress can induce differential epigenetic modifications to the promoters of specific
bdnf splice variants. In mice, exposure to chronic social defeat stress resulted in depression-
like behaviors (e.g., behavioral abnormalities, such as social avoidance) and dimethylation of
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lysine 27 on the H3 histone, which is a repressive modification on the promoters for the III
and IV splice variants of the bdnf gene, and was associated with down-regulation of bdnf III
and bdnf IV mRNA expression in the hippocampus (Tsankova et al., 2006). That is to say, in
the hippocampus after chronic social defeat stress, dimethylation of the H3 histone at the
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promoters for the bdnf III and bdnf IV splice variants resulted in a closed chromatin
conformation, leading to a repression of gene expression. Interestingly, these changes were
long-lasting, as the hypermethylation of H3 histone was evident one month after the cessation
of social defeat stress, as were social avoidance behaviors. Hypermethylation of lysine 27 on
the H3 histone has also been observed at the promoter of the bdnf IV splice variant in the
hippocampus in a mouse model of prenatal stress, which results in depressive-like behavior in
offspring (Onishchenko et al., 2008). These data indicate that exposure to stress can result in
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the formation of molecular scars on the hippocampus and long-lasting chromatin remodeling
in bdnf promoters, which could then lead to an increased susceptibility to developing MDD.
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Other studies also indicate that epigenetic modifications in the BDNF pathway are
associated with MDD. Patients with MDD show alterations in DNA methylation in the
promoter regions of the BDNF gene in peripheral blood, while increased methylation of the
DNA at a BDNF gene promoter was associated with decreased BDNF mRNA levels in the
brains of those with suicidal behavior, some of which also suffered from MDD (Fuchikami et
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al., 2009; Keller et al., 2010). Epigenetic modifications have also been observed in TrkB
receptors in association with MDD, as decreased mRNA expression of TRKB-T1, a truncated
isoform of the TrkB receptor present in neurons and glia (Baxter et al., 1997; Klein et al.,
1991; Middlemas et al., 1991; Rose et al., 2003), was associated with increased methylation
of CpG sites and lysine 27 on the H3 histone in the promoter region of TRKB-T1 gene, in
those who had committed suicide, some of whom had also suffered from MDD (Ernst et al.,
2009a; Ernst et al., 2009b). In line with this observation is a recent study by Kang and co-
workers (Kang et al., 2013) found a significant association between higher levels of BDNF
c.
promoter methylation and suicidal behavior, while Dell‘Osso and co-workers (Dell'Osso et
al., 2014) found that there were higher levels of BDNF promoter methylation in the peripheral
blood monocytes of depressed patients and patients with bipolar disorder II, which is
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characterized by depressive episodes and hypomanic episodes, as compared to patients with
bipolar disorder I, which is characterized by depressive episodes and full manic episodes
(Sadock and Sadock, 2007). Thus, widespread epigenetic modifications to the BDNF pathway
including the targets of BDNF binding, could underlie the pathophysiology of MDD.
Epigenetic modification of the P11 gene (also known as S100A10) may also contribute to
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the pathophysiology of MDD. P11 plays a role in the intracellular trafficking of
transmembrane proteins to the cell surface, and has been recognized as a key modulator of the
function of neurons (Rescher and Gerke, 2008). P11 has also been shown to interact with
serotonin receptors, whose function may also be altered in MDD (Melas et al., 2012). P11
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may have a particular contribution the pathophysiology of MDD, as P11 mRNA levels have
been shown to be decreased in the brain tissue of depressed patients, and p11 knock-out mice
show depressive-like behaviors (Anisman et al., 2008; Svenningsson et al., 1997). A recent
study by Melas and co-workers (2012), indicates that in Flinders Sensitive Line (FSL) rats,
which are thought to serve as an animal model of depression, p11 mRNA and P11 protein
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levels are decreased in the prefrontal cortex (Melas et al., 2012). The prefrontal cortex is a
region that shows diminished function with MDD, which is thought to underlie the cognitive
deficits that are observed in association with this disorder (Baxter et al., 1989). An analysis of
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DNA methylation patterns in FSL rats revealed hypermethylation in the promoter region of
the p11 gene. As mentioned above, hypermethylation of CpG sites in the promoter region of a
gene can prevent the interaction of transcription factors with DNA, resulting in a repression
of gene expression. The authors posited that hypermethylation of CpG sites on the p11 gene
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promoter could possibly prevent the binding of the androgen receptor. The androgen receptor
is a transcription factor that inhibits corticotrophin-releasing hormone that leads to the
dampening of the hypothalamic-pituitary-adrenal axis (Bao et al., 2006). The androgen
receptor has been shown to be down-regulated in depressed patients, although in the Melas
study, there was no difference in androgen receptor expression in FSL versus normal rats.
Nevertheless, these data suggest that epigenetic modulation of the P11 gene could contribute
to the expression of depressive disorders.
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Several studies have shown that miRNAs may play a role in a number of biological
pathways, such as synaptic plasticity and neurogenesis and may be involved in a number of
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mental disorders (Zeng et al., 2011). For example, post-mortem studies in humans have
shown that a number of miRNAs regulate the molecular mechanisms that underlie neural
plasticity and neural development which may be altered in those who suffer from
neuropsychiatric disorders, such as MDD (Beveridge et al., 2010; Kim et al., 2010; Mellios et
al., 2011; Smalheiser et al., 2012). Recently, the miRNA, MIR-16 has emerged as a specific
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epigenetic modulator whose expression could be related to the dysfunction of neurochemical

substrates that underlie the development of a depressive phenotype (Launay et al., 2011).
Enhanced neurogenesis in the hippocampus is thought to contribute to the therapeutic effects
of antidepressant treatment (Drzyzga et al., 2009; Duman, 2004a; MacQueen and Frodl, 2011;
Perera et al., 2011; Santarelli et al., 2003), while decreased hippocampal neurogenesis is
observed in association with MDD (Mendez-David et al., 2013). The protein B-cell chronic
lymphocytic lymphoma 2 (BCL-2) is a target of MIR-16, as determined from studies using
human tissues (Cimmino et al., 2005). BCL-2 has a well-established role as an anti-apoptotic
c.
protein, but has also been shown to exhibit neurotrophic effects (Drzyzga et al., 2009) as
overexpression of the bcl-2 gene results in increased neurogenesis in the hippocampus of
adult transgenic mice (Kuhn et al., 2005). mir-16 inhibits bcl-2 mRNA expression, and
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therefore, it is possible that increases in the expression of this miRNA in the hippocampus
may contribute to MDD due to diminished neurogenesis in this region (Launay et al., 2011).
As discussed below, SSRI-induced changes in BCL-2 activity may be mediated by
changes in MIR-16 expression, suggesting that this epigenetic mechanism may be a viable
target for antidepressant treatment.
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Another target of MIR-16 appears to be the serotonin transporter (SERT). Alterations in
SERT have been associated with MDD (Arango et al., 2001; Owens and Nemeroff, 1994),
and SERT is a target for a number of antidepressant therapies, particularly SSRIs. Studies
utilizing a murine cell line have shown that mir-16 is complementary to the 3‘ untranslated
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region of the sert mRNA (Baudry et al., 2010) and recent data indicates that in mouse, rat and
human brain, SERT expression is controlled by MIR-16 (Moya et al., 2013). Indeed, MIR-16
has been shown to be a negative regulator of SERT expression in humans, and in rodents,
overexpression of mir-16 leads to a decrease in the number of SERT binding sites, while
inhibition of mir-16 expression leads to an appearance of SERT binding sites (Baudry et al.,
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2010; Moya et al., 2013). Of note, mir-16 is expressed in low levels within the SERT-
expressing serotonergic neurons of the dorsal raphe, while mir-16 levels are higher in the
noradrenergic neurons of the locus coeruleus (Baudry et al., 2010; Launay et al., 2011; Moya
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et al., 2013). Interestingly, inhibition of mir-16 expression increased the number of SERT
binding sites in the locus coeruleus (Baudry et al., 2010; Launay et al., 2011). These data
suggest that blocking mir-16 expression can promote SERT protein production in
serotonergic cells, while enabling noradrenergic neurons to produce SERT protein, which
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indicates that these neurons could serve as a potential target during SSRI administration. In
line with this observation, emerging data indicates that SSRI treatment results in changes in
mir-16 expression, leading to a dorsal-raphe mediated mobilization of the locus coeruleus,
and will be discussed in further detail below (Baudry et al., 2010; Launay et al., 2011).
Epigenetics and SSRIs

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SSRIs are one of the most commonly prescribed antidepressants for the treatment of
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MDD (Millan, 2006). In recent years, it has become evident that epigenetic mechanisms,
along with their role in the pathogenesis of MDD, are also involved in the mechanisms of
action and therapeutic effects of SSRI treatment. Interestingly, a paradox exists between the
blockade of SERT and the therapeutic effects of SSRIs. Administration of SSRIs results in a
rapid increase in the levels of serotonin in the synapse in both rodents and primates, but
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SSRIs require at least a two-week administration time in order to see therapeutic effects
(Anderson et al., 2005; Kreiss and Lucki, 1995; Rutter et al., 1994; Wong and Licinio, 2001).
This discrepancy suggests that structural or functional changes must take place over time in
order for the therapeutic effects of SSRI treatment to be observed. This has lead to postulation
that epigenetic modifications may be a mechanism of action that underlies the lag in time
between the initial administration of SSRI and the alleviation of the symptoms of MDD. It is
important to note, however that a number of studies have shown that epigenetic modulation of
c.
gene expression can occur rapidly (e.g., within 24 hours) in response to stress or
antidepressant treatment, raising the possibility that epigenetic modifications may not
underlie the time lag between SSRI administration and symptom relief (Dyrvig et al., 2012;
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Fuchikami et al., 2009; Tsankova et al., 2004; Tsankova et al., 2006). Nevertheless, a number
of studies in both animals and humans have shown that chronic treatment with SSRIs results
in a several epigenetic modifications.
Several studies have investigated methylation of DNA as an epigenetic mechanism
utilized by SSRIs. One of the first studies that investigated epigenetic modification of gene
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expression in response to SSRI treatment by Cassel and co-workers (2006) examined the
effect of chronic fluoxetine treatment on mecp2 expression in adult rat brain. As described
above, MeCP2 can bind to methylated sites on DNA, leading to a silencing of gene
expression via recruitment of a histone deacetylase, which is will lead to a closed chromatin
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configuration. Repeated treatment with fluoxetine resulted in an increase in MeCP2 and
MBD1 (which binds to MeCP) protein and mecp2 and mbd1 mRNA, and was accompanied
by increased HDAC labeling and hdac expression and decreased H3 acetylation (Cassel et al.,
2006). These effects were seen in the caudate putamen, hippocampus and frontal cortex,
which are three regions that receive dense serotonergic inputs from the dorsal raphe nucleus.
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Interestingly, in the hippocampus, these changes were observed in the GABAergic neurons of
the dentate gyrus. The authors posit that SSRIs through serotonin (5-HT)-induced epigenetic
modifications results in a silencing of gene expression in post-mitotic neurons. It is unclear,
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however, how this observed effect might contribute to the mitigation of the symptoms of
MDD by SSRIs. More recent studies have focused on other aspects of SSRI-mediated DNA
methylation in both animals and humans. Paroxetine has been shown to reduce DNMT1
enzymatic activity in rat cortical astrocytes, possibly as a result of decreased expression of the
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HMT, G9a, which is a known activator of DNMT1 (Zimmermann et al., 2012). As mentioned
above, DNA hypermethylation of the promoter of p11 has been observed in the FSL rat and
was associated with diminished levels of P11 protein. Repeated treatment of FSL rats with
escitalopram resulted in a decrease in the methylation of the promoter region of the p11 gene,
which was coupled with an increase in p11 mRNA expression in the forebrain (Melas et al.,
2012). These changes in p11 promoter methylation and expression in escitalopram-treated
FSL rats were accompanied by a decrease in dnmt1 and dnmt2 mRNA expression indicating
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that diminished activity of these enzymes were most likely responsible for p11 promoter
hypomethylation in response to escitalopram.
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Another system that may be modulated epigenetically by SSRIs is the BDNF pathway.
Overall, effective antidepressant treatment seems to be associated with in an increase in
BDNF levels (Duman, 2004b; Duman and Monteggia, 2006). However, a recent study shows
that in patients suffering from MDD, treatment with fluoxetine did not significantly alter the
methylation of the BDNF exon IV promoter, suggesting that changes in methylation status in
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this particular region of DNA may not be involved in the response to SSRI treatment (Tadić
et al., 2014). On the other hand, recent studies by D‘Addario and co-workers found that
patients suffering from MDD or bipolar disorder and treated with antidepressants, some of
which were SSRIs, showed increased levels of methylation of the BDNF promoter
(D'Addario et al., 2013; Marini et al., 2004). It is unclear what the functional significant of
these findings might mean, in terms of the ability of SSRIs to modulate BDNF expression and
relieve the symptoms of depression, however, the authors suggest that the methylation state of
the BDNF gene may serve as a potential biomarker in mood disorders (Marini et al., 2004).
c.
Few studies have examined the impact of epigenetic modifications to the SERT gene on
the treatment response to antidepressants, which is somewhat surprising, given that SERT is
the primary target of SSRIs. However, a recent study by Domshke and co-workers has
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examined whether methylation patterns in the promoter region on the SERT gene are related
to treatment outcome with SSRIs in patients suffering from MDD (Domschke et al., 2014).
Interestingly, the study revealed that hypomethylation of the SERT promoter was associated
with poor treatment outcome after a 6-week trial of escitalopram. This was an unexpected
result, since, as described above, promoter hypermethylation is typically associated with
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suppression of gene expression (Hebbes et al., 1988; Szyf, 2006; Tachibana et al., 2008;
Weber and Schubeler, 2007), and the authors had originally hypothesized that
hypermethylation of the SERT promoter would lead to a suppression of SERT protein
expression and therefore, a poor outcome in response to SSRI treatment. However, in light of
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their surprising data the authors posited that the hypomethylation of the SERT promoter
reflects greater SERT transcription and SERT activity, which would result in decreased 5-HT
availability in the synaptic cleft and an impaired response to escitalopram treatment. While
additional studies are clearly needed to determine the precise role of methylation of the SERT
promoter and the response to SSRI treatment, this study nonetheless suggests that epigenetic-
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based information could be someday used to develop individually tailored pharmacotherapies
for MDD.
It should be duly noted that several of the human studies discussed above examined DNA
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methylation patterns following SSRI exposure in peripheral blood monocytes (PBMC), rather
than examining DNA methylation patterns in the brain (D'Addario et al., 2013; Dell'Osso et
al., 2014; Domschke et al., 2014; Kang et al., 2013; Marini et al., 2004). The examination of
markers in PBMC is a commonly used and well-established model of epigenetic regulation in
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the brain, especially in studies involving human participants, as there is no need for post-
mortem tissue in these studies, and changes in epigenetic markers can be compared with
behavioral or treatment outcomes. Indeed, several studies in humans, as well as rodents and
rhesus monkeys indicate that there is a degree of comparability between DNA methylation
levels in PBMCs and brain (Davies et al., 2012; Provencal et al., 2012; Ursini et al., 2011;
Wang et al., 2012), suggesting that methylation levels in the peripheral blood may mirror
changes in methylation levels that occur in the brain. However, it must be kept in mind that
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these studies do not allow for direct conclusions to be drawn regarding the methylation state
of DNA in the brain.
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Histone modifications have also been the focus of several recent studies involving the
epigenetic impact of SSRI treatment. One such modification involves changes in histone
acetylation. Histone acetylation is an important mechanism in the transcriptional regulation
and neural plasticity (Crosio et al., 2003a; Guan et al., 2002; Levenson and Sweatt, 2006;
Miller et al., 2010). In line with this observation, a recent study by Wang and co-workers
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(2011) found that chronic treatment with fluoxetine increased the level of acetylation of the
H3 histone and enhanced hippocampal neurogenesis (Wang et al., 2011), while Oobkubo and
co-workers (2013) found that escitalopram treatment increased H3 acetylation in the nucleus
accumbens (Ookubo et al., 2013). Onishchenko and colleagues (2008) found that fluoxetine
treatment promoted acetylation of the H3 histone in mice who exhibited depressive-like
behaviors following in utero exposure to methyl mercury (Onishchenko et al., 2008). In mice,
changes in hdac expression have also been observed after SSRI treatment, as escitalopram has
been shown to increase hdac2 and hdac3 expression in the caudate putamen, and hdac5
c.
expression in the amygdala (Ookubo et al., 2013), supporting the notion that histone
acetylation may contribute to the therapeutic effects of SSRI treatment, although the exact
mechanism by which this occurs is not completely clear.
In
Modulation of histone methylation has received attention as a potential epigenetic
mechanism induced by SSRI treatment, particularly with regards to the BDNF gene. As
mentioned above, in animal studies, methylation of lysine 27 on H3 results in a repression of
bdnf exon IV expression, and it is thought that this repression of bdnf contributes to the
depressive phenotype (Tsankova et al., 2007; Tsankova et al., 2006). Thus, decreased
rs
methylation of lysine 27 on H3 should correlate with an increase in bdnf expression and by
extension a positive treatment response. Indeed, recent data indicates that patients who were
suffering from MDD and responded successfully to citalopram therapy exhibited decreased
levels of methylation of on lysine 27 of histone 3, coupled with increased circulating BDNF
he
levels (Lopez et al., 2013), while patients who were currently being treated with a variety
antidepressants, including fluoxetine and citalopram also showed reduced methylation of
lysine 27 of histone 3 (Chen et al., 2011). Together, these findings suggest that
hypomethylation of H3 may be an important mechanism of action for SSRIs.
It is thought that the pathophysiology of depression could be the result of alterations in
is
neural plasticity processes, which encompasses the molecular and cellular responses of
neurons to inputs from other neurons or circulating factors (Duman and Charney, 1999;
Duman, 2004b). One of the most commonly studied mechanisms of neural plasticity is long-
bl
term potentiation (LTP), which is thought to subserve learning and memory processes. In
models of stress and depression, mechanisms of neural plasticity (such as LTP) are altered or
impaired, possibly as a result of an inability to make the appropriate neuronal adaptations in
response to an aversive stimulus (Duman and Charney, 1999; Duman, 2004b).
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Antidepressants may alleviate the symptoms of depression by resorting normal function to the
pathways that underlie neural plasticity or by facilitating the induction of appropriate
neuronal adaptations (Duman and Charney, 1999; Duman, 2004b). Transcriptional regulation
also figures prominently into the pathophysiology of depression, as transcription factor
function, and thus gene expression, may be impaired in depression, an effect that can be
reversed by treatment with antidepressants (Dowlatshahi et al., 1998; Duman and Charney,
1999; Duman, 2004b). Indeed, a recent study from Robison and co-workers (Robison et al.,
a
2013) indicate that antidepressants can epigenetically modify the activity of transcription
factors, and may influence synaptic plasticity. For example, SSRI-induced histone
ov
methylation can regulate the activity of the calmodulin-dependent protein kinase II

(CamKII) and binding of the transcription factor FosB, revealing a novel
epigenetic mechanism whereby SSRIs can alleviate the symptoms of MDD. In the nucleus
accumbens, which is an important region for the production of affective states (Drevets, 2001;
Milak et al., 2005; Nestler et al., 2002), chronic social defeat stress results in an increase in
N
FosB binding to the camkII promoter. However, when animals that were exposed to
chronic social defeat stress were treated with fluoxetine, in the nucleus accumbens there was
decreased binding of FosB to the camkII promoter and decreased camkII expression,
despite the fact that FosB levels were increased under these conditions (Robison et al.,
2013). Further analysis revealed that fluoxetine treatment increased dimethylation of lysine 9
on histone 3 the camkII
to the promoter, resulting in diminished camkII expression (Robison et al., 2013).
c.
Interestingly, this effect was also observed in the nucleus accumbens of medicated-depressed
patients (Robison et al., 2013). As of yet, it is unclear exactly how epigenetic repression of
camkIIexpression in the nucleus accumbens might relate to the therapeutic effects of SSRIs.
In
Acute treatment with SSRIs results in the potentiation of synapses in the hippocampus, in a
process that involves CamKII (Colbran and Brown, 2004). Therefore, it could be that
with chronic SSRI treatment, camkII expression is diminished as a compensatory
mechanism to prevent over-strengthening of certain synaptic connections within different
regions of the brain. However, additional studies are clearly needed in order to fully
rs
understand the role that epigenetic modulation of camkII expression by SSRI treatment
plays in the therapeutic effects of these drugs.
Several recent studies have examined miRNAs, with a focus on mir-16 as a potential
epigenetic modification by induced by SSRIs treatment. An initial study by Baudry and co-
he
workers (2010) found an interesting role for mir-16 in the epigenetic effects of SSRIs (Baudry
et al., 2010). As mentioned above, mir-16 is a negative regulator of sert expression, and is
expressed in higher levels in the locus coeruleus versus the dorsal raphe nucleus. When mice
were treated chronically with fluoxetine, mir-16 levels increased in the dorsal raphe, which
lead to a decrease in SERT levels in this region. This was coupled with an increase in the
is
release of the neurotrophic factor S100 from the dorsal raphe nucleus, which then acted on
the noradrenergic neurons of the locus coeruleus. S100 was able to suppress mir-16
expression in the locus coeruleus, which turned on serotonergic functions in these
bl
noradrenergic neurons. In essence, fluoxetine treatment was able to epigenetically create
additional 5-HT sites within the brain, giving this compound more regions in which to exert
its effects. In an expansion of this work, Launay and co-workers (2011) found that fluoxetine
Pu
acting on the serotonergic neurons of the dorsal raphe decreases the amount of mir-16 in the
hippocampus, which in turn increases the levels of SERT and BCL-2 (which, as discussed
earlier, is thought to mediate neurogenesis) within this region (Launay et al., 2011).
Interestingly, the release of S100 from the dorsal raphe to the locus coeruleus is responsible,
in part for the fluoxetine-induced hippocampal response. The study by Launay and co-
workers also revealed that fluoxetine-induced hippocampal neurogenesis was mediated by
mir-16 (Launay et al., 2011). This is of particular relevance, as hippocampal atrophy is has
a
been observed in patients suffering from depression (Hastings et al., 2004; Sapolsky, 2000;
Sheline et al., 1998; Sheline et al., 2003), while antidepressant treatment reverses
ov
hippocampal atrophy in humans suffering from depression or post-traumatic stress disorder

(Sheline et al., 2003; Vermetten et al., 2003), and induces hippocampal neurogenesis in
animals (Klomp et al., 2014; Mateus-Pinheiro et al., 2013). Furthermore, it has been
suggested that remission from depressive behaviors requires neurogenesis within the
hippocampus (Mateus-Pinheiro et al., 2013). Thus, epigenetic modifications to genes that
N
mediate neurogenesis in the hippocampus, which may be necessary for successful treatment
of depression, could underlie the beneficial effects of antidepressant treatment. Recently,
Bocchio-Chiavetto and co-workers have screened human patients that suffered from MDD
and who were receiving treatment with escitalopram for changes in miRNA expression. They
found that at least 30 different miRNAs were modulated by escitalopram treatment. At least
13 of these miRNAs have been shown to play a role in neural plasticity and stress responses,
and the pathophysiology of several neuropsychiatric diseases, which underscores the
importance of further investigation into the role that miRNA modulation may play in the
c.
therapeutic effects of SSRI therapy (Bocchio-Chiavetto et al., 2013).
Finally, there are a number of in vitro studies that have utilized human cell lines, which
have provided additional information on epigenetic changes that can occur in response to
In
SSRI treatment, particularly with regards to miRNA expression. A recent study by Angelucci
and co-workers (Becker et al., 1996) found that treatment of the human
glioblastoma/astrocytoma cell line U87 with paroxetine resulted in an increase in BDNF
mRNA and protein levels, as well as an increase in levels of the microRNA mir-30-5p.
Interestingly, mir-30-5p is an inhibitor of BDNF transcription, and its overexpression
rs
following paroxetine treatment could limit the effects of this drug on the BDNF system and
therefore could have the potential to diminish symptom relief in response to SSRI treatment.
Rodrigues and colleagues (Rodrigues et al., 2011) examined the effects of fluoxetine
treatment on miRNA expression BE(2)-M17 and SH-SY5Y cell lines and discovered that
he
mir-124a expression was diminished following fluoxetine administration. Previous work has
shown that mir-124a is a regulator of neuronal identity (Maisel et al., 2010; Pruunsild et al.,
2007), and the decrease in its expression following fluoxetine treatment suggests neuroplastic
changes may occur in response to this drug, although the specific nature of these changes and
how they might relate to the amelioration of the symptoms of depression are not yet clear.
is
Oved and co-workers (Aid et al., 2007) exposed human lymphoblastoid cells (LCL) from
healthy adults to paroxetine to determine if alterations in miRNA expression could be used as
a potential biomarker for sensitivity to SSRI treatment. The LCLs that were the most sensitive
bl
to paroxetine treatment, as measured by paroxetine-induced growth inhibition were found to
have almost 7-fold higher basal levels of mir-151-3p, suggesting that this miRNA may serve
as a useful biomarker for predicting positive treatment outcomes in response to SSRIs. While
these studies may have limitations in that that the results cannot necessarily be extrapolated to
Pu
the potential changes seen in the whole organism, they nevertheless provide important insight
in the epigenetic alterations that occur following SSRI treatment that can then be tested and
applied to experimental animals and humans.
Summary and Conclusion

a
Epigenetic mechanisms function along side genomic DNA and can promote or suppress
gene expression, ultimately impacting protein production. Epigenetic modifications that can
ov
influence gene expression include DNA methylation, modification of histones, which

influence chromatin remodeling and the expression of miRNAs. Epigenetics represent a
significant new direction for depression research and provides a mechanism for connecting
the long-term effects of adverse life events with the changes in gene expression that are
associated with MDD. For example, many studies have shown that BDNF, a neurotrophic
N
factor whose impairment may underlie the pathophysiology of MDD, can be epigenetically
modified, indicating that epigenetic mechanisms may contribute to the expression of MDD.
Importantly, epigenetic mechanisms are beginning to emerge as a novel mode of action for
antidepressants, in particular the SSRIs. Several lines of research indicate that treatment with
SSRIs can modulate or even in some cases reverse the epigenetic factors that may underlie
the expression of MDD. For instance, recent studies indicate that treatment with SSRIs can
reduce the methylation of the BDNF gene, which may result in its de-repression and
c.
amelioration of MDD symptoms. Continued investigation into the epigenetic effects of SSRI
treatment will aid in the development of more targeted therapies to treat depression and
possibly predict the treatment outcome.
In
References
Aid, T., et al., 2007. Mouse and rat BDNF gene structure and expression revisited. J Neurosci
rs
Res. 85, 525-35.
Anderson, G.M., et al., 2005. Time course of the effects of the serotonin-selective reuptake
inhibitor sertraline on central and peripheral serotonin neurochemistry in the rhesus
monkey. Psychopharmacology (Berl). 178, 339-46.
he
Anisman, H., et al., 2008. Serotonin receptor subtype and p11 mRNA expression in stress-
relevant brain regions of suicide and control subjects. J Psychiatry Neurosci. 33, 131-41.
Arango, V., et al., 2001. Serotonin 1A receptors, serotonin transporter binding and serotonin
transporter mRNA expression in the brainstem of depressed suicide victims.
Neuropsychopharmacology. 25, 892-903.
is
Bao, A.M., et al., 2006. A direct androgenic involvement in the expression of human
corticotropin-releasing hormone. Mol Psychiatry. 11, 567-76.
Baudry, A., et al., 2010. miR-16 targets the serotonin transporter: a new facet for adaptive
bl
responses to antidepressants. Science. 329, 1537-41.
Baxter, G.T., et al., 1997. Signal transduction mediated by the truncated trkB receptor
isoforms, trkB.T1 and trkB.T2. J Neurosci. 17, 2683-90.
Baxter, L.R., Jr., et al., 1989. Reduction of prefrontal cortex glucose metabolism common to
Pu
three types of depression. Arch Gen Psychiatry. 46, 243-50.

Becker, W.M., Reece, J.B., Peonie, M.F., 1996. The World of the Cell, Vol.,
Benjamin/Cummings, Menlo Park, CA.
Bellet, M.M., Sassone-Corsi, P., 2010. Mammalian circadian clock and metabolism - the
epigenetic link. J Cell Sci. 123, 3837-48.
Berger, S.L., 2007. The complex language of chromatin regulation during transcription.
a
Nature. 447, 407-12.

Berton, O., et al., 2006. Essential role of BDNF in the mesolimbic dopamine pathway in
social defeat stress. Science. 311, 864-8.
ov
Beveridge, N.J., et al., 2010. Schizophrenia is associated with an increase in cortical

microRNA biogenesis. Mol Psychiatry. 15, 1176-89.
Bocchio-Chiavetto, L., et al., 2013. Blood microRNA changes in depressed patients during
antidepressant treatment. Eur Neuropsychopharmacol. 23, 602-11.
Borrelli, E., et al., 2008. Decoding the epigenetic language of neuronal plasticity. Neuron. 60,
N
961-74.
Boulle, F., et al., 2012. Epigenetic regulation of the BDNF gene: implications for psychiatric
disorders. Mol Psychiatry. 17, 584-96.
Brami-Cherrier, K., et al., 2005. Parsing molecular and behavioral effects of cocaine in
mitogen- and stress-activated protein kinase-1-deficient mice. J Neurosci. 25, 11444-54.
Bredy, T.W., et al., 2011. MicroRNA regulation of neural plasticity and memory. Neurobiol
c.
Learn Mem. 96, 89-94.
Carthew, R.W., Sontheimer, E.J., 2009. Origins and Mechanisms of miRNAs and siRNAs.
Cell. 136, 642-55.
In
Cassel, S., et al., 2006. Fluoxetine and cocaine induce the epigenetic factors MeCP2 and
MBD1 in adult rat brain. Molecular pharmacology. 70, 487-92.
Chen, E.S., Ernst, C., Turecki, G., 2011. The epigenetic effects of antidepressant treatment on
human prefrontal cortex BDNF expression. Int J Neuropsychopharmacol. 14, 427-9.
Chen, Z.Y., et al., 2006. Genetic variant BDNF (Val66Met) polymorphism alters anxiety-
rs
related behavior. Science. 314, 140-3.
Chendrimada, T.P., et al., 2005. TRBP recruits the Dicer complex to Ago2 for microRNA
processing and gene silencing. Nature. 436, 740-4.
Cheung, P., Allis, C.D., Sassone-Corsi, P., 2000. Signaling to chromatin through histone
he
modifications. Cell. 103, 263-71.
Chiaruttini, C., et al., 2009. Dendritic trafficking of BDNF mRNA is mediated by translin and
blocked by the G196A (Val66Met) mutation. Proc Natl Acad Sci U S A. 106, 16481-6.
Chong, M.M., et al., 2010. Canonical and alternate functions of the microRNA biogenesis
machinery. Genes Dev. 24, 1951-60.
is
Cimmino, A., et al., 2005. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc
Natl Acad Sci U S A. 102, 13944-9.
Colbran, R.J., Brown, A.M., 2004. Calcium/calmodulin-dependent protein kinase II and
bl
synaptic plasticity. Current opinion in neurobiology. 14, 318-27.
Crosio, C., et al., 2003a. Chromatin remodeling and neuronal response: multiple signaling
pathways induce specific histone H3 modifications and early gene expression in
hippocampal neurons. Journal of cell science. 116, 4905-14.
Pu
Crosio, C., et al., 2003b. Chromatin remodeling and neuronal response: multiple signaling
pathways induce specific histone H3 modifications and early gene expression in
hippocampal neurons. J Cell Sci. 116, 4905-14.
D'Addario, C., et al., 2013. Epigenetic modulation of BDNF gene in patients with major
depressive disorder. Biol Psychiatry. 73, e6-7.
Dalton, V.S., Kolshus, E., McLoughlin, D.M., 2014. Epigenetics and depression: return of the
repressed. J Affect Disord. 155, 1-12.
a
Davies, M.N., et al., 2012. Functional annotation of the human brain methylome identifies
tissue-specific epigenetic variation across brain and blood. Genome Biol. 13, R43.
ov
Dell'Osso, B., et al., 2014. Epigenetic modulation of BDNF gene: Differences in DNA
methylation between unipolar and bipolar patients. Journal of affective disorders. 166C,
330-333.
Dillon, S.C., et al., 2005. The SET-domain protein superfamily: protein lysine
methyltransferases. Genome Biol. 6, 227.
N
Dmitrzak-Weglarz, M., et al., 2008. Association studies of the BDNF and the NTRK2 gene
polymorphisms with prophylactic lithium response in bipolar patients.
Pharmacogenomics. 9, 1595-603.
Domschke, K., et al., 2014. Serotonin transporter gene hypomethylation predicts impaired
antidepressant treatment response. Int J Neuropsychopharmacol. 17, 1167-76.
Dowlatshahi, D., et al., 1998. Increased temporal cortex CREB concentrations and
antidepressant treatment in major depression. Lancet. 352, 1754-5.
Drevets, W.C., 2001. Neuroimaging and neuropathological studies of depression:
c.
implications for the cognitive-emotional features of mood disorders. Curr Opin
Neurobiol. 11, 240-9.
Drzyzga, L.R., Marcinowska, A., Obuchowicz, E., 2009. Antiapoptotic and neurotrophic
In
effects of antidepressants: a review of clinical and experimental studies. Brain Res Bull.
79, 248-57.
Duman, R.S., Charney, D.S., 1999. Cell atrophy and loss in major depression. Biol
Psychiatry. 45, 1083-4.
Duman, R.S., 2004a. Depression: a case of neuronal life and death? Biol Psychiatry. 56, 140-
rs
5.
Duman, R.S., 2004b. Neural plasticity: consequences of stress and actions of antidepressant
treatment. Dialogues Clin Neurosci. 6, 157-69.
Duman, R.S., Monteggia, L.M., 2006. A neurotrophic model for stress-related mood
he
disorders. Biol Psychiatry. 59, 1116-27.
Duman, R.S., Newton, S.S., 2007. Epigenetic marking and neuronal plasticity. Biol
Dwivedi, Y., et al., 2003. Abnormal expression and functional characteristics of cyclic
adenosine monophosphate response element binding protein in postmortem brain of
is
suicide subjects. Arch Gen Psychiatry. 60, 273-82.
Dyrvig, M., et al., 2012. Epigenetic regulation of Arc and c-Fos in the hippocampus after
acute electroconvulsive stimulation in the rat. Brain Res Bull. 88, 507-13.
bl
Eberharter, A., Becker, P.B., 2002. Histone acetylation: a switch between repressive and
permissive chromatin. Second in review series on chromatin dynamics. EMBO Rep. 3,
224-9.
Egan, M.F., et al., 2003. The BDNF val66met polymorphism affects activity-dependent
Pu
secretion of BDNF and human memory and hippocampal function. Cell. 112, 257-69.
Ernst, C., Chen, E.S., Turecki, G., 2009a. Histone methylation and decreased expression of
TrkB.T1 in orbital frontal cortex of suicide completers. Mol Psychiatry. 14, 830-2.
Ernst, C., et al., 2009b. Alternative splicing, methylation state, and expression profile of
tropomyosin-related kinase B in the frontal cortex of suicide completers. Arch Gen
Esteller, M., 2011. Non-coding RNAs in human disease. Nat Rev Genet. 12, 861-74.
a
Franklin, T.B., et al., 2010. Epigenetic transmission of the impact of early stress across
generations. Biol Psychiatry. 68, 408-15.
ov
Friedman, R.C., et al., 2009. Most mammalian mRNAs are conserved targets of microRNAs.
Genome Res. 19, 92-105.
Frieling, H., Tadić, A., 2013. Value of genetic and epigenetic testing as biomarkers of
response to antidepressant treatment. Int Rev Psychiatry. 25, 572-8.
Fuchikami, M., et al., 2009. Single immobilization stress differentially alters the expression
N
profile of transcripts of the brain-derived neurotrophic factor (BDNF) gene and histone
acetylation at its promoters in the rat hippocampus. Int J Neuropsychopharmacol. 12, 73-
82.
Grunstein, M., 1997. Histone acetylation in chromatin structure and transcription. Nature.
389, 349-52.
Guan, Z., et al., 2002. Integration of long-term-memory-related synaptic plasticity involves
bidirectional regulation of gene expression and chromatin structure. Cell. 111, 483-93.
Hastings, R.S., et al., 2004. Volumetric analysis of the prefrontal cortex, amygdala and
c.
hippocampus in major depression. Neuropsychopharmacology. 29, 952-959.
Havens, M.A., et al., 2012. Biogenesis of mammalian microRNAs by a non-canonical
processing pathway. Nucleic Acids Res. 40, 4626-40.
In
Hayes, J.J., Hansen, J.C., 2001. Nucleosomes and the chromatin fiber. Curr Opin Genet Dev.
11, 124-9.
Hebbes, T.R., Thorne, A.W., Crane-Robinson, C., 1988. A direct link between core histone
acetylation and transcriptionally active chromatin. EMBO J. 7, 1395-402.
Hervouet, E., et al., 2009. Folate supplementation limits the aggressiveness of glioma via the
rs
remethylation of DNA repeats element and genes governing apoptosis and proliferation.
Clin Cancer Res. 15, 3519-29.
Huang, Y., Doherty, J.J., Dingledine, R., 2002. Altered histone acetylation at glutamate
receptor 2 and brain-derived neurotrophic factor genes is an early event triggered by
he
status epilepticus. J Neurosci. 22, 8422-8.
Im, H.I., Kenny, P.J., 2012. MicroRNAs in neuronal function and dysfunction. Trends
Neurosci. 35, 325-34.
Kaeberlein, M., McVey, M., Guarente, L., 1999. The SIR2/3/4 complex and SIR2 alone
promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes
is
Dev. 13, 2570-80.
Kang, H.J., et al., 2013. BDNF promoter methylation and suicidal behavior in depressive
patients. J Affect Disord. 151, 679-85.
bl
Kawamata, T., Tomari, Y., 2010. Making RISC. Trends Biochem Sci. 35, 368-76.
Keller, S., et al., 2010. Increased BDNF promoter methylation in the Wernicke area of suicide
subjects. Arch Gen Psychiatry. 67, 258-67.
Kim, A.H., et al., 2010. MicroRNA expression profiling in the prefrontal cortex of
Pu
individuals affected with schizophrenia and bipolar disorders. Schizophr Res. 124, 183-
91.
Kim, J.K., Samaranayake, M., Pradhan, S., 2009. Epigenetic mechanisms in mammals. Cell
Mol Life Sci. 66, 596-612.
Kim, Y.K., et al., 2007. Low plasma BDNF is associated with suicidal behavior in major
depression. Prog Neuropsychopharmacol Biol Psychiatry. 31, 78-85.
Klein, R., et al., 1991. The trkB tyrosine protein kinase is a receptor for brain-derived
a
neurotrophic factor and neurotrophin-3. Cell. 66, 395-403.

Klomp, A., et al., 2014. Effects of chronic fluoxetine treatment on neurogenesis and
ov
tryptophan hydroxylase expression in adolescent and adult rats. PLoS One. 9, e97603.
Kloosterman, W.P., Plasterk, R.H., 2006. The diverse functions of microRNAs in animal
development and disease. Dev Cell. 11, 441-50.
Kolshus, E., et al., 2014. When less is more--microRNAs and psychiatric disorders. Acta
Psychiatr Scand. 129, 241-56.
N
Korzus, E., Rosenfeld, M.G., Mayford, M., 2004. CBP histone acetyltransferase activity is a
critical component of memory consolidation. Neuron. 42, 961-72.
Kozomara, A., Griffiths-Jones, S., 2011. miRBase: integrating microRNA annotation and
deep-sequencing data. Nucleic Acids Res. 39, D152-7.
Kreiss, D.S., Lucki, I., 1995. Effects of acute and repeated administration of antidepressant
drugs on extracellular levels of 5-hydroxytryptamine measured in vivo. J Pharmacol Exp
Ther. 274, 866-76.
c.
Krishnan, V., et al., 2007. Molecular adaptations underlying susceptibility and resistance to
social defeat in brain reward regions. Cell. 131, 391-404.
Krishnan, V., Nestler, E.J., 2010. Linking molecules to mood: new insight into the biology of
In
depression. Am J Psychiatry. 167, 1305-20.
Krol, J., Loedige, I., Filipowicz, W., 2010. The widespread regulation of microRNA
biogenesis, function and decay. Nat Rev Genet. 11, 597-610.
Kuhn, H.G., et al., 2005. Increased generation of granule cells in adult Bcl-2-overexpressing
mice: a role for cell death during continued hippocampal neurogenesis. Eur J Neurosci.
rs
22, 1907-15.
Kumar, A., et al., 2005. Chromatin remodeling is a key mechanism underlying cocaine-
induced plasticity in striatum. Neuron. 48, 303-14.
Lachner, M., Jenuwein, T., 2002. The many faces of histone lysine methylation. Curr Opin
he
Cell Biol. 14, 286-98.
Launay, J.M., et al., 2011. Raphe-mediated signals control the hippocampal response to SRI
antidepressants via miR-16. Transl Psychiatry. 1, e56.
Lee, B.H., Kim, Y.K., 2010a. BDNF mRNA expression of peripheral blood mononuclear
cells was decreased in depressive patients who had or had not recently attempted suicide.
is
J Affect Disord. 125, 369-73.
Lee, B.H., Kim, Y.K., 2010b. The roles of BDNF in the pathophysiology of major depression
and in antidepressant treatment. Psychiatry Investig. 7, 231-5.
bl
Levenson, J.M., Sweatt, J.D., 2006. Epigenetic mechanisms: a common theme in vertebrate
and invertebrate memory formation. Cellular and molecular life sciences: CMLS. 63,
1009-16.
Li, J., et al., 2004. Dopamine D2-like antagonists induce chromatin remodeling in striatal
Pu
neurons through cyclic AMP-protein kinase A and NMDA receptor signaling. J

Neurochem. 90, 1117-31.
Liu, Q.R., et al., 2005. Human brain derived neurotrophic factor (BDNF) genes, splicing
patterns, and assessments of associations with substance abuse and Parkinson's Disease.
Am J Med Genet B Neuropsychiatr Genet. 134B, 93-103.
Lopez, J.P., et al., 2013. Epigenetic regulation of BDNF expression according to
antidepressant response. Mol Psychiatry. 18, 398-399.
a
Luikart, B.W., Perederiy, J.V., Westbrook, G.L., 2012. Dentate gyrus neurogenesis,
integration and microRNAs. Behav Brain Res. 227, 348-55.
ov
MacQueen, G., Frodl, T., 2011. The hippocampus in major depression: evidence for the
convergence of the bench and bedside in psychiatric research? Mol Psychiatry. 16, 252-
64.
Magill, S.T., et al., 2010. microRNA-132 regulates dendritic growth and arborization of
newborn neurons in the adult hippocampus. Proc Natl Acad Sci U S A. 107, 20382-7.
N
Maisel, M., et al., 2010. Genome-wide expression profiling and functional network analysis
upon neuroectodermal conversion of human mesenchymal stem cells suggest HIF-1 and
miR-124a as important regulators. Experimental cell research. 316, 2760-78.
Marini, A.M., et al., 2004. Role of brain-derived neurotrophic factor and NF-kappaB in
neuronal plasticity and survival: From genes to phenotype. Restor Neurol Neurosci. 22,
121-30.
Mateus-Pinheiro, A., et al., 2013. Sustained remission from depressive-like behavior depends
on hippocampal neurogenesis. Transl Psychiatry. 3, e210.
c.
Melas, P.A., et al., 2012. Antidepressant treatment is associated with epigenetic alterations in
the promoter of P11 in a genetic model of depression. Int J Neuropsychopharmacol. 15,
669-79.
In
Mellios, N., et al., 2011. miR-132, an experience-dependent microRNA, is essential for visual
cortex plasticity. Nat Neurosci. 14, 1240-2.
Mendez-David, I., et al., 2013. Adult hippocampal neurogenesis: an actor in the
antidepressant-like action. Ann Pharm Fr. 71, 143-9.
Middlemas, D.S., Lindberg, R.A., Hunter, T., 1991. trkB, a neural receptor protein-tyrosine
rs
kinase: evidence for a full-length and two truncated receptors. Mol Cell Biol. 11, 143-53.
Milak, M.S., et al., 2005. Neuroanatomic correlates of psychopathologic components of major
depressive disorder. Arch Gen Psychiatry. 62, 397-408.
Millan, M.J., 2006. Multi-target strategies for the improved treatment of depressive states:
he
Conceptual foundations and neuronal substrates, drug discovery and therapeutic
application. Pharmacol Ther. 110, 135-370.
Miller, C.A., et al., 2010. Cortical DNA methylation maintains remote memory. Nat
Neurosci. 13, 664-6.
Morgunkova, A., Barlev, N.A., 2006. Lysine methylation goes global. Cell Cycle. 5, 1308-12.
is
Moya, P.R., et al., 2013. miR-15a and miR-16 regulate serotonin transporter expression in
human placental and rat brain raphe cells. Int J Neuropsychopharmacol. 16, 621-9.
Nakahata, Y., et al., 2009. Circadian control of the NAD+ salvage pathway by CLOCK-
bl
SIRT1. Science. 324, 654-7.
Nestler, E.J., et al., 2002. Neurobiology of depression. Neuron. 34, 13-25.
Newell-Price, J., Clark, A.J., King, P., 2000. DNA methylation and silencing of gene
expression. Trends in endocrinology and metabolism: TEM. 11, 142-8.
Pu
Onishchenko, N., et al., 2008. Long-lasting depression-like behavior and epigenetic changes
of BDNF gene expression induced by perinatal exposure to methylmercury. Journal of
neurochemistry. 106, 1378-87.
Ookubo, M., et al., 2013. Antidepressants and mood stabilizers effects on histone deacetylase
expression in C57BL/6 mice: Brain region specific changes. J Psychiatr Res. 47, 1204-
14.
Owens, M.J., Nemeroff, C.B., 1994. Role of serotonin in the pathophysiology of depression:
a
focus on the serotonin transporter. Clin Chem. 40, 288-95.

Pasquinelli, A.E., 2012. MicroRNAs and their targets: recognition, regulation and an
ov
emerging reciprocal relationship. Nat Rev Genet. 13, 271-82.

Perera, T.D., et al., 2011. Necessity of hippocampal neurogenesis for the therapeutic action of
antidepressants in adult nonhuman primates. PLoS One. 6, e17600.
Pile, L.A., et al., 2003. The SIN3 deacetylase complex represses genes encoding
mitochondrial proteins: implications for the regulation of energy metabolism. J Biol
N
Chem. 278, 37840-8.

Provencal, N., et al., 2012. The signature of maternal rearing in the methylome in rhesus
macaque prefrontal cortex and T cells. J Neurosci. 32, 15626-42.
Pruunsild, P., et al., 2007. Dissecting the human BDNF locus: bidirectional transcription,
complex splicing, and multiple promoters. Genomics. 90, 397-406.
Rescher, U., Gerke, V., 2008. S100A10/p11: family, friends and functions. Pflugers Arch.
455, 575-82.
Rice, J.C., Allis, C.D., 2001. Histone methylation versus histone acetylation: new insights
c.
into epigenetic regulation. Curr Opin Cell Biol. 13, 263-73.
Robison, A.J., et al., 2013. Fluoxetine Epigenetically Alters the CaMKIIalpha Promoter in
Nucleus Accumbens to Regulate DeltaFosB Binding and Antidepressant Effects.
In
Neuropsychopharmacology. 39, 1178-86.
Rodenhiser, D., Mann, M., 2006. Epigenetics and human disease: translating basic biology
into clinical applications. CMAJ. 174, 341-8.
Rodrigues, A.C., et al., 2011. MicroRNA expression is differentially altered by xenobiotic
drugs in different human cell lines. Biopharmaceutics & drug disposition. 32, 355-67.
rs
Rose, C.R., et al., 2003. Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling
in glia cells. Nature. 426, 74-8.
Roth, T.L., et al., 2009. Lasting epigenetic influence of early-life adversity on the BDNF
gene. Biol Psychiatry. 65, 760-9.
he
Rutter, J.J., Gundlah, C., Auerbach, S.B., 1994. Increase in extracellular serotonin produced
by uptake inhibitors is enhanced after chronic treatment with fluoxetine. Neurosci Lett.
171, 183-6.
Rybakowski, J.K., 2008. BDNF gene: functional Val66Met polymorphism in mood disorders
and schizophrenia. Pharmacogenomics. 9, 1589-93.
is
Saba, R., Schratt, G.M., 2010. MicroRNAs in neuronal development, function and
dysfunction. Brain Res. 1338, 3-13.
Sadock, B.J., Sadock, V.A., 2007. Synopsis of Psychiatry, Vol., Lippincott, Williams and
bl
Wilkins, Philadelphia, PA.
Santarelli, L., et al., 2003. Requirement of hippocampal neurogenesis for the behavioral
effects of antidepressants. Science. 301, 805-9.
Sapolsky, R.M., 2000. The possibility of neurotoxicity in the hippocampus in major
Pu
depression: a primer on neuron death. Biol Psychiatry. 48, 755-765.

Sassone-Corsi, P., 2012. Minireview: NAD+, a circadian metabolite with an epigenetic twist.
Endocrinology. 153, 1-5.
Schroeder, M., et al., 2012. The epigenetic code in depression: implications for treatment.
Clin Pharmacol Ther. 91, 310-4.
Sheline, Y.I., Gado, M.H., Price, J.L., 1998. Amygdala core nuclei volumes are decreased in
recurrent major depression. Neuroreport. 9, 2023-2028.
a
Sheline, Y.I., Gado, M.H., Kraemer, H.C., 2003. Untreated depression and hippocampal
volume loss. Am J Psychiatry. 160, 1516-8.
ov
Smalheiser, N.R., et al., 2012. MicroRNA expression is down-regulated and reorganized in

prefrontal cortex of depressed suicide subjects. PLoS One. 7, e33201.
Svenningsson, P., et al., 1997. Cellular expression of adenosine A2A receptor messenger
RNA in the rat central nervous system with special reference to dopamine innervated
areas. Neuroscience. 80, 1171-85.
N
Szyf, M., 2006. Targeting DNA methylation in cancer. Bulletin du cancer. 93, 961-72.
Tachibana, M., et al., 2008. G9a/GLP complexes independently mediate H3K9 and DNA
methylation to silence transcription. EMBO J. 27, 2681-90.
Tadić, A., et al., 2014. Methylation of the promoter of brain-derived neurotrophic factor exon
IV and antidepressant response in major depression. Mol Psychiatry. 19, 281-3.
Tapia-Arancibia, L., et al., 2004. Physiology of BDNF: focus on hypothalamic function.
Front Neuroendocrinol. 25, 77-107.
Thompson Ray, M., et al., 2011. Decreased BDNF, trkB-TK+ and GAD67 mRNA expression
c.
in the hippocampus of individuals with schizophrenia and mood disorders. J Psychiatry
Neurosci. 36, 195-203.
Timmusk, T., et al., 1993. Multiple promoters direct tissue-specific expression of the rat
In
BDNF gene. Neuron. 10, 475-89.
Tsankova, N., et al., 2007. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci.
8, 355-67.
Tsankova, N.M., Kumar, A., Nestler, E.J., 2004. Histone modifications at gene promoter
regions in rat hippocampus after acute and chronic electroconvulsive seizures. J
rs
Neurosci. 24, 5603-10.
Tsankova, N.M., et al., 2006. Sustained hippocampal chromatin regulation in a mouse model
of depression and antidepressant action. Nat Neurosci. 9, 519-25.
Ursini, G., et al., 2011. Stress-related methylation of the catechol-O-methyltransferase Val
he
158 allele predicts human prefrontal cognition and activity. J Neurosci. 31, 6692-8.
Vermetten, E., et al., 2003. Long-term treatment with paroxetine increases verbal declarative
memory and hippocampal volume in posttraumatic stress disorder. Biol Psychiatry. 54,
693-702.
Vialou, V., et al., 2013. Epigenetic mechanisms of depression and antidepressant action. Annu
is
Rev Pharmacol Toxicol. 53, 59-87.
Wang, D., et al., 2012. Peripheral SLC6A4 DNA methylation is associated with in vivo
measures of human brain serotonin synthesis and childhood physical aggression. PLoS
bl
One. 7, e39501.
Wang, Y., Leung, F.C., 2004. DNA structure constraint is probably a fundamental factor
inducing CpG deficiency in bacteria. Bioinformatics. 20, 3336-45.
Wang, Y., et al., 2011. Fluoxetine increases hippocampal neurogenesis and induces
Pu
epigenetic factors but does not improve functional recovery after traumatic brain injury. J
Neurotrauma. 28, 259-68.
Weber, M., Schubeler, D., 2007. Genomic patterns of DNA methylation: targets and function
of an epigenetic mark. Current opinion in cell biology. 19, 273-80.
Wong, M.L., Licinio, J., 2001. Research and treatment approaches to depression. Nat Rev
Neurosci. 2, 343-51.
Wu, L., Fan, J., Belasco, J.G., 2006. MicroRNAs direct rapid deadenylation of mRNA. Proc
a
Natl Acad Sci U S A. 103, 4034-9.

Zeng, L., et al., 2011. MicroRNA-210 as a novel blood biomarker in acute cerebral ischemia.
ov
Front Biosci (Elite Ed). 3, 1265-72.

Zimmermann, N., et al., 2012. Antidepressants inhibit DNA methyltransferase 1 through
reducing G9a levels. Biochem J. 448, 93-102.
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Chapter 8
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microRNAs As Novel Players in
Depression Pathogenesis and in the
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Mechanisms of Action of Fluoxetine
and Other Antidepressants
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Guangxing Bai, Michael Dunbar, Levi D. Miller, Bhaskar Roy,
Ian J. Soller, Richard C. Shelton and Yogesh Dwivedi
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Department of Psychiatry and Behavioral Neurobiology
University of Alabama at Birmingham
Birmingham, AL, US
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Abstract
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Major depressive disorder (MDD) is a major public health concern. Although much
work has been done to characterize MDD, a large number of MDD patients do not
respond to the currently available medications and the relapse rate for depression is quite
high. Thus, there is an urgent need to fully understand the neurobiological abnormalities
associated with MDD and develop target-based therapeutic approaches. In this context,
microRNAs (miRNAs) have emerged as important gene regulators which are involved in
many higher brain functions. Because miRNAs show a highly regulated expression, they
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contribute in the development and maintenance of a specific transcriptome and thus have
the unique ability to influence a wide range of physiological and disease phenotypes.
Recent studies demonstrating involvement of miRNAs in several aspects of neural
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plasticity, stress response, and more direct studies in human postmortem brain, peripheral
blood cells - provide strong evidence that miRNAs can not only play a critical role in
MDD pathogenesis but can also open novel avenues for the development of therapeutic
targets. In this chapter, these aspects are discussed in a comprehensive manner.
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
Corresponding Author: Yogesh Dwivedi, Ph.D. Elesabeth Ridgely Shook Endowed Chair in Psychiatry. Professor
of Psychiatry. Director of Translational Research. UAB Mood Disorder Program. Department of Psychiatry
and Behavioral Neurobiology. University of Alabama at Birmingham. SC711 Sparks Center. 1720 2nd
Avenue South. Birmingham, AL 35294-0017. Email: ydwivedi@uab.edu.
148 Guangxing Bai, Michael Dunbar, Levi D. Miller et al.
Keywords: miRNA, depression, antidepressant, stress, gene regulation, synaptic plasticity,

neurogenesis
Introduction
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Major depressive disorder (MDD) is the most prevalent of psychiatric disorders. It affects
about 150 million people worldwide at any point in time (Wang et al., 2007) and about 17%
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of the US population during their lifetime (Kessler 2005). MDD is associated with poor
quality of life and premature death, as over 60% of suicides are related to MDD. Although
much work has been done to characterize MDD, about 40% of MDD patients do not respond
to the currently available medications (Fava et al., 1996). This is partially a result of poor
understanding of the molecular pathophysiology underlying MDD.
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miRNAs, a class of small non-coding RNAs, are the major regulators of neural plasticity
and higher brain functioning (Leistedt et al., 2013; Ota et al., 2013). By regulating gene
expression in a highly a coordinated and cohesive fashion (Malphettes, 2006), miRNAs can
participate in the development and maintenance of the transcriptome and thereby can
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influence physiological and disease phenotypes. The roles of miRNAs in various
neuropsychiatric diseases are being vigorously investigated as miRNAs can not only play a
direct role in disease pathogenesis, but they can also help in identifying the nature of
disordered pathways implicated in such pathogenesis (Choi et al., 2005; Mo et al., 2009; Xu
et al., 2011; Stäehler et al., 2012). This is also crucial in the development of novel therapeutic
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drugs.
Although studies examining the role of miRNAs in MDD are in early stages, several
studies from human postmortem brain and animal model systems have provided evidence
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suggesting that miRNAs may be crucial in the etiopathogenesis of MDD. Also, there are
multitudes of studies which points to the involvement of miRNAs in synaptic plasticity and
neurogenesis; crucial factors in MDD pathophysiology. The aim of this chapter is to review
the current status of our understanding of the role of miRNAs in MDD. We have focused on
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studies showing involvement of miRNAs in neuroplasticity, neurogenesis, and in stress-

related behavioral response. We have also summarized findings from human postmortem
brain and blood cell studies of MDD patients as well as the role of miRNAs in the mechanism
of action of antidepressants.
miRNAs: Synthesis and Mechanisms

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of Post-Transcriptional Repression
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miRNAs belong to the eukaryotic family of small non-coding RNAs comprised of more
than 200 family members per species and building a largest family accounting for 1% of the
genome (Kim, 2005). Recently released 20th version of the mirBase database shows 24521
entries representing hairpin precursor miRNAs expressing 30424 mature miRNA products in
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206 species (www.miRBase.org). In humans, so far, 2578 mature miRNAs and 1872
precursor miRNAs have been annotated (Kozomara and Griffiths-Jones, 2014).
microRNAs As Novel Players in Depression Pathogenesis … 149
miRNAs are single stranded molecules with mature length of 20-22 nucleotides that
control the activity of about 50% of protein-coding genes (Friedman and Jones 2009). These
miRNAs are transcribed by RNA polymerase II or III as a primary transcript followed by
processing with a set of enzymes belonging to the RNase III family, giving rise to an effector
molecule with impact on post-transcriptional gene regulation by either directly mediating
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mRNA decay or repressing translation with a precise mechanism (Bartel, 2004)
Conversion of Primary miRNA to Precursor miRNA: Single stranded mature miRNAs
are derived from longer double stranded molecules with atypical hairpin stem-loop structure
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known as primary miRNA (pri-miRNA). Generally, both transcriptional and post
transcriptional regulation act upon miRNA biogenesis pathway to produce the mature
transcript, (Ameres & Zamore, 2013). However, a few of them use a non-canonical pathway
to become mature. They are mainly derived from intronic regions of protein-coding genes
without recruiting splicing machinery for their biogenesis and popularly known as ‗Mirtron‘
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(Westholm et al., 2012). After transcription by RNA polymerase II (Lee et al., 2004), pri-miR
is converted to precursor miRNA (pre-miR) by the catabolic activity of RNase III family
member Drosha. Multiple forms of ~60 base long hairpin structure of precursor miRNA has
been reported to be produced from a single pri-miRNA (Gregory et al., 2004). In mammalian
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cells, Drosha forms a microprocessor complex with another protein named Digeorge
Syndrome at Critical Region 8 (DGCR8) (Han et al., 2006). This complex structure helps
increase activity of Drosha by modulating its affinity for the substrate and by increasing
cleavage site accuracy (Ameres & Zamore, 2013).
After Drosha mediated processing in nucleus, pre-miRNAs are transported to cytosol.
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This transportation is mediated by the nuclear transporter protein Exportin 5 and energized by
Ran-GTP complex (Lund et al., 2004). Once pre-miRNAs reach cytoplasm, another RNase III
enzyme Dicer cleaves pre-miRNA into a shorter form, producing single stranded miRNAs
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with a length of ~22 nucleotides (Zhang et al., 2002). However, this is not a unifying feature
in miRNA biogenesis. In certain cases, such as in zebra fish, pre-miR -451 is processed
without being acted on by Dicer enzyme (Yang et al., 2010). Due to its small size, precursor
miRNA-451 is directly recruited on the silencing complex for further action (Cheloufi et al.,
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2010).
Mechanisms of Gene Silencing by miRNA: One of the miRNA/miRNA* duplexes is
loaded onto an Argonaute homologue protein (Ago, isoforms of eIF2c) to generate the
effector complex, known as RNA-induced silencing complex (RISC). The other miRNA*
strand is degraded. RISC complex assembly is mainly comprised of Dicer, double stranded
RNA binding domain protein TRBP (TAR-binding protein), PACT (Protein Activator of
PKR) and Argonaut 2 (Ago2). In this complex the requirement of TRBP and PACT are not
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indispensable for Dicer activity for pre-miRNA processing but they do intensify the
functionality of Dicer (Chendrimada et al., 2005; Lee et al., 2006).
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RISC binds to specific ―short-seed‖ sequences located predominantly within the 3‘

untranslated region (3‘ UTR) of target mRNAs and either interferes with translation of the
mRNA or reduces mRNA levels by degradation. miRNA-mediated translational inhibition
depends upon the 5‘cap region of the target mRNA. Ago proteins can stimulate miRNA-
dependent inhibition of translation by competing with eIF4E for the 5‘cap binding site, thus
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preventing circularization of mRNA and lowering initiation efficiency (Mathonnet et al.,

2007). Because RISC/miRNA complex recognizes target mRNA based on a seed region
containing 2-8 nucleotides at the 5‘end of miRNA, it provides a mechanism by which one
miRNA can target several mRNAs (Brodersen et al., 2009). RISC can also associate with 60S
ribosome and eIF6 (Chendrimada et al., 2007). eIF6 regulates the formation of translationally
active 80S subunit; so by regulating association with eIF6, miRNAs can modify polysome
formation and expose target mRNAs for degradation (Chendrimada et al., 2007). MiRNA-
mediated regulation of mRNA stability is another mechanism by which miRNAs suppress
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expression of specific mRNA. Using miR-125b and let-7 as representative miRNAs, Wu et al.
(2006) showed that in mammalian cells, miRNA initiates the reduction in mRNA abundance
through accelerated deadenylation, which leads to rapid mRNA decay.
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Besides regulating translational and mRNA decay processes, it has been shown that
miRNA can also regulate gene transcription by targeting transcription factors. In this case,
levels of transcription factors are downregulated by miRNAs, which in turn cause less
expression of mRNA, leading to reduced protein synthesis (Kosik, 2006; Michalak, 2006)
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Role of miRNAs in MDD Pathogenesis
Multiple lines of investigation suggest that MDD is associated with altered
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neuroplasticity (Leistedt and Linkowski, 2013; Ota and Duman, 2013). In addition,
neurogenesis plays a crucial role in depression and in the mechanism of action of
antidepressants. Specifically, we and other investigators have shown that transactivation of
transcription factor CREB is altered (Dowlatshahi et al., 1998; Dwivedi et al., 2003), along
with expression of neuroplasticity genes such BDNF, NGF, NT-3 and their cognate TRK
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receptors in depressed brain (Dwivedi et al., 2003, 2009a). Hypoactivation of genes involved
in ERK2 and PI3 kinase pathways, such as ERK1/2, MAP kinase kinase1, ERK5, Rap-1, B-
Raf, and Epac (Dwivedi et al., 2001; 2006a; 2006b; 2007; Gourley et al., 2008;; Qi et al.,
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2008; Dwivedi et al., 2009; Todorovic, Dwivedi et al., 2009b; Yuan et al., 2010) has also
been found in postmortem brain of depressed subjects. In addition, the expression of protein
kinase A and protein kinase C, which are integral parts of the adenylyl cyclase-cyclic
adenosine monophosphate and phosphoinositide signaling systems and which are regulators
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of neural plasticity, are down regulated in depressed suicide subjects (Pandey et al., 1997;
Dwivedi and Pandey, 2008).
Interestingly, miRNAs play a critical role in both neurogenesis and neuroplasticity either
by directly impeding the translation process of target mRNAs with a post-transcriptional gene
silencing mechanism or indirectly by targeting transcription factors. In the following sections
we describe the role of miRNAs in neurogenesis and neural plasticity. In addition, miRNA
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modulations in brain and blood cells of MDD patients have been discussed in greater detail.
Regulation of Neurogenesis by miRNAs: Several studies indicate that MDD is
associated with decreased hippocampal neurogenesis and that mechanisms of action of
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antidepressants are associated with increased neurogenesis. Interestingly, ~20% to 40% of

miRNAs in the brain are developmentally regulated. miRNAs are not only important during
embryonic development but they are also critical in regulating adult neurogenesis (Miska et
al., 2004; Sempere et al., 2004). One of the most important miRNAs that regulate
neurogenesis is miR-124. miR-124a is the most abundant miRNAs in the mammalian brain
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and accounts for 25-48% of all brain-expressed miRNAs (Lagos-Quintana et al. 2002).
Transfection of mouse neuronal stem cells with miR-124a stimulates neuron-like
differentiation by promoting neuron-specific class III beta-tubulin 1 and MAP2 expression,

causing G0/G1 cell cycle arrest (Silber et al. 2008).
In addition, knockdown of this miRNA maintains subventricular zone stem cells as
dividing precursors, whereas its ectopic expression leads to precocious and increased neuron
formation in mice (Cheng et al. in 2009). The primary target of miR-124 is SRY-box
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transcription factor Sox9. miR-124-mediated overexpression of Sox9 abolishes neuronal
differentiation, whereas Sox9 knockdown leads to increased neuron formation (Chen et al.,
2009). Recently, Szulwach et al. (2010) showed that MeCP2, a DNA methyl-CpG-binding
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protein, can epigenetically regulate miR-137, which is regulated by Sox2, a core transcription
factor in stem cells. These investigators showed that overexpression of miR-137 promoted the
proliferation of adult neural stem cells, whereas a reduction of miR-137 enhanced adult neural
stem cell differentiation. In addition, miR-137 post-transcriptionally repressed the expression
of Ezh2, a histone methyltransferase and polycomb group protein. The miR-137-mediated
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repression of Ezh2 resulted in a global decrease in histone (H3) trimethylation at lysine 27
residue. Coexpression of Ezh2 rescued phenotypes associated with miR-137 overexpression,
thus demonstrating a fascinating cross-talk between miRNA and epigenetic regulation in the
modulation of adult neurogenesis with a feedback and feed forward loop model (Szulwach et
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al., 2010).
Regulation of Neural Plasticity by miRNAs: A growing body of evidence indicates that
a specific population of miRNAs is expressed within dendrites and in dendritic spines, where
they contribute in the regulation of local protein synthesis, and thus dendritic spine
morphogenesis. In fact, Lugli et al. (2008) have demonstrated that the entire synthesis
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machinery for miRNAs is localized at the synapse in mouse forebrain. Similarly, Kye et al.
(2007) showed that most neuronal miRNAs are detectable in the dendrites. Amongst them,
the most striking miRNA is miR-26a, which targets microtubule-associated protein 2, a
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protein involved in neural reorganization (Kye et al., 2007). Earlier, Schratt et al. (2006)
showed that expression of Limk1, a protein that controls dendritic spine development, is
regulated by miR-134 in the synaptodendritic compartment. They also found that exposure of
the hippocampal neurons to BDNF relieves miR-134 inhibition of Limk1 translation, causing
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an increase in the size of dendritic spines. Siegal et al. (2009) showed that miR-138, which
regulates the expression of Acyl protein thioesterase 1 (APT1), is highly localized within
dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons.
They found that palmitoylation of Gα13 by APT1 is critical in dendritic spine morphogenesis.
BDNF, a critical player in neural plasticity has been shown to regulate miR-132
(Kawashima et al., 2011). MiR-132 induces neurite outgrowth and modulates dendritic
morphology of cortical and hippocampal neurons by repressing p250GAP (Vo et al. 2005;
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Wayman et al., 2008). Deletion of miR-132 leads to a dramatic decrease in dendrite length,
arborization, and spine density (Magill et al., 2010). MiR-132 expression has also been shown
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to promote neuronal maturation and synapse formation by regulating MeCP2 expression

(Fukada et al., 2005; Jugloff et al., 2005; Klien et al., 2007). Interestingly, MeCP2 controls
BDNF expression, which itself leads to induction of miR-212/132 expression. With this, miR-
132 can take part in a feedback mechanism involved in the homeostatic control of MeCP2
expression (Nudelman et al., 2010). Besides, CREB, which modulates the transcription of
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several genes with cAMP responsive elements in their promoter regions (Silva et al., 1998;
Benito and Barco 2010; Sakamoto et al. 2011), also targets miR-132. In addition, miR-212 is
also targeted by CREB and plays a role in neuronal development and function (Vo et al.,
2005; Impey et al., 2004).
miRNAs and Stress Response: Stress, a major factor in MDD, differentially regulates
miRNA expression in hippocampus, amygdala (Meearson et al., 2010) and frontal cortex
(Rinaldi et al., 2010), key brain regions involved in emotion and cognitive processes. Early
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life stress, another critical factor in development of MDD (Raabe, 2013), has also been shown
to impact miRNAs in mice prefrontal cortex (Uchida et al., 2010). Genetic differences in
miRNA expression can influence an individual‘s coping response to a stressor. In this regard,
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it has been shown that stress-sensitive F344 rats, which release excessive corticosterone
(CORT) in response to a stressor (Uchida et al., 2010), express higher hypothalamic miR-18a.
Mechanistically, miR-18 binds to 3‘UTR of glucocorticoid receptor and reduces its
expression (Vreugdenhi, 2009). This results in reduced negative feedback leading to
increased CORT release. The importance of this increased release was demonstrated by
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exposing of neurons to excess CORT, which results in decreased BDNF-dependent neuronal
functions via suppression of miR-132 expression (Kawashima et al., 2010).
Recently we studied the expression of a large set of miRNA in frontal cortex of rats who
developed behavior (learned helpless [LH]) that resembles stress-induced depression vs. those
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that did not develop depression-like symptoms (non-learned helpless [NLH]) despite similar
exposure to inescapable shock (Smalheiser et al., 2011). We found that NLH rats show a
robust adaptive miRNA response to inescapable shocks whereas LH rats show a markedly
blunted miRNA response. An impressive number of miRNAs exhibited down regulated
expression in NLH rats when compared with control group. These include: miR-96, miR-141,
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miR-182, miR-183, miR-183*, miR-198, miR-200a, miR-200a*, miR-200b, miR-200b*,
miR-200c, and miR-429. These miRNAs were encoded at a few shared polycistronic loci,
suggesting that their down regulation was coordinately controlled at the level of transcription.
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Creb1 mRNA was identified as one of potential targets of several miRNAs and reciprocally
Creb1 was found to be a trans-activating factor on the upstream region of miR-96, miR-182,
miR-183, miR-200a, miR-200b, miR-200c, miR-220a*, and miR-200b.*
miRNA Modulation in MDD Postmortem Brain: We are the first to study miRNAs in
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the human postmortem brain of subjects who had major depression and died by suicide
(Smalheiser et al., 2013, 2014). We found that 21 miRNA were significantly downregulated
in the prefrontal cortex of MDD subjects-(listed in Table 1). When analyzed individually,
almost half of the down-regulated miRNAs were encoded at chromosomal loci near another
miRNA and are possibly transcribed by the same pri-miRNA gene transcripts (miR-142-5p
and 142-3p; miR-494, 376a*, 496, and 369-3p; miR-23b, 27b and 24-1*; miR-34b* and 34c;
miR-17* and 20a). In addition, three pairs of miRNAs were encoded at distances greater than
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100 kb but still found to lie within the same chromosomal region (miR-424 and 20b at
Xq26.2-3, 377 kb apart; miR-142 and 301a at 17q22, 820 kb apart; miR-324-5p and 497 at
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17p13.1, 205 kb apart). This suggests that at least some of the down-regulated miRNA
expression is due to decreased transcription. Many of the down-regulated miRNAs also
shared 5‘-seed sequences that are involved in target recognition. For example, identical seed
sequences are shared by a) miR-20a and 20b; b) miR-301a and 130a; and c) miR-424 and
497. Additionally, a 6-mer nucleotide motif is shared by miR-34a, 34b* and 34c, and
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strikingly, a 5-mer motif (AGUGC) within the 5‘-seed is shared by 5 of the affected miRNAs
(miR-148b, 301a, 130a, 20a, 20b) that is predicted to bind Alu sequences within the 3‘-UTR
region of target mRNAs. This suggests that the down-regulated miRNAs should exhibit
extensive overlap among their mRNA targets.
When pair-wise correlations were made (a complementary method of analyzing the
miRNA expression data to identify pairs of miRNAs that are co-regulated in their expression,
up or down, across individuals within a single group), a set of 29 miRNAs were identified,
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none of which were pair-wise correlated in the normal control group, but which formed a very
extensive inter-connected network in the depressed group. Several of the miRNAs (let-7b,
miR-132, 181b, 338-3p, 486-5p, and 650) were ―hubs‖ correlated with four to nine other
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miRNAs in the network. Target analysis revealed that many of the targets are transcription
factors, and nuclear, transmembrane and signaling proteins. Intriguingly, 4 different down-
regulated miRNAs target VEGFA (miR-20b, 20a, 34a, 34b*), a molecule implicated in
depression in both humans and in animal models. Other validated targets include BCL2 (miR-
34a), DNMT3B (miR-148b), and MYCN (miR-101, 34a). Among predicted targets, estrogen
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receptor alpha, ESR1, was predicted to be targeted by 3 different down-regulated miRNAs
(miR-148b, 301a, 496). Others targeted by 3 or more affected miRNAs include ubiquitin
ligases (UBE2D1 and UBE2W), signal transduction mediators (CAMK2G, AKAP1), the
splicing factor NOVA1 that regulates brain-specific alternative splicing; the GABA-A
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receptor sub-unit GABRA4; calcium channel CACNA1C; and brain-active transcription
factors including SMAD5, MITF, BACH2, MYCN, and ARID4A. Several of these predicted
targets interact with validated targets in MDD; for example, ARIA4A binds E2F1; SMAD5
binds RUNX1; and estradiol treatment decreases E2F1 levels in prefrontal cortex (Wang et
al., 2004). BACH2 transcription factor binding sites have been identified upstream of many
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brain-expressed miRNAs (Wu and Xie, 2006). Retinoblastoma binding protein 1 (ARIA4A)
is of interest because it recruits histone deacetylases and regulates gene expression via
chromatin-based silencing.
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Selected target proteins such as DMNT3b, VEGFA, and BCL2 were studied by
examining their expression in depressed suicide brain. DMNT3b was strongly up-regulated
in the depressed suicide group, whereas BCL2 was downregulated. Several miRNAs that
were co-regulated with their targets showed a strong positive correlation with DMNT3b and
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BCL2. A variety of factors such as transcription factor activity and turnover rate, as well
as possible regulatory effects of other miRNAs may also be responsible for changes in mean
expression levels of these target proteins. In addition, DNMT3b levels showed an extremely
strong positive correlation with miR-148b across subjects (r = 0.91 in controls, r = 0.94
in the depressed suicide group). Similarly, BCL2 was strongly and positively correlated
with miR-34a (r = 0.92 in healthy controls, r = 0.82 in the depressed suicide group). The
correlation of miR-34a was positive in healthy controls, but inverse in the depressed suicide
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group, presumably reflecting a reorganization of miRNA-target networks (Smalheiser et al.,

2012).
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Previous studies indicate that TrkB.T1, a BDNF receptor lacking a tyrosine kinase
domain that is highly expressed in astrocytes and regulates BDNF-evoked calcium transients,
is downregulated in frontal cortex of suicide subjects (Ernst et al., 2009). In a recent study,
Maussion et al. (2012) examined whether this TrkB.T1 gene is regulated by miRNAs. The
investigators found that Hsa-miR-185* and Hsa-miR-491-3p were upregulated in suicide
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completers with low expression of TrkB.T1; FDR.1. Bioinformatic analyses revealed five
putative binding sites for the DiGeorge syndrome linked miRNA Hsa-miR-185*in the 3'UTR
of TrkB.T1, but none for Hsa-miR-491-3P. The increase of Hsa-miR-185* in frontal cortex of
suicide completers was validated then confirmed in a larger, randomly selected group of
suicide completers, where an inverse correlation between Hsa-miR-185* and TrkB.T1
expression was observed. Silencing and overexpression studies performed in human cell lines
confirmed the inverse relationship between hsa-mir-185* and trkB-T1 expression.
Furthermore, luciferase assays demonstrated that Hsa-miR-185* binds to sequences in the
c.
3'UTR of TrkB.T1.These results suggest that an increase of Hsa-miR-185* expression levels
regulates, at least in part, the TrkB.T1 decrease observed in the frontal cortex of suicide
completers and further implicate the 22q11 region in psychopathology.
In
Alterations in metabolic enzymes of the polyamine system have been reported to play a
role in predisposition to suicidal behavior (Fiori et al., 2011). Recently, Lopez et al. (2013)
examined whether dysregulation of polyamine genes in depressed suicide completers could
be influenced by miRNA-mediated post-transcriptional regulation. These investigators
identified several miRNAs that target the 3'UTR of polyamine genes SAT1 and SMOX.
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When the expression of 10 miRNAs in the prefrontal cortex of suicide completers and
controls using qRT-PCR were profiled, they found that several miRNAs showed significant
up-regulation in the prefrontal cortex of suicide completers compared to psychiatrically-
healthy controls (miR-124, miR-139-5p, miR-195, miR-198, miR-320c, miR-33b, miR-34a,
he
miR-34c-5p, miR-497, miR-873). However, they found that only miR-139-5p and miR-320c
were inversely correlated with polyamine gene SAT1 whereas miR34c-5p and miR-320c
were inversely correlated with polyamine gene SMOX. These results suggest a relationship
between miRNAs and polyamine gene expression in the suicide brain, and postulate a
mechanism for SAT1 and SMOX down-regulation by post-transcriptional activity of
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miRNAs.
Polymorphism in genes responsible for microRNA processing has also been documented
to play a role in depressive disorder. For example, miRNA processing genes DGCR8, Ago1,
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and GEMIN4 were found to harbor polymorphism and exhibited a strong correlation with
pathophysiology of depression (He et al., 2012). Variant allele of DGCR8 rs3757 was
associated with increased risk of suicidal tendency and improved response to antidepressant
treatment, whereas the variant of AGO1 rs636832 showed decreased risk of suicidal
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tendency, suicidal behavior, and recurrence (He et al., 2012). This information could be very
useful and can be used as a diagnostic tool for early detection of depression pathophysiology.
MiRNA Modulation and Antidepressant Response
MDD Patient Population Studies

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Recently, several studies have indicated that mechanisms of action of antidepressants

could be associated with modulation in expression of miRNAs. Belzeaux et al. (2012)
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profiled miRNAs in peripheral blood mononuclear cells collected from 16 severe MDD
patients and 13 matched controls at baseline, at 2 and 8 weeks after antidepressant treatment.
Comparison of miRNA expression between MDD patients and controls at baseline and at 8
weeks showed a similar number of dysregulated miRNAs (14 miRNAs, with 9 miRNAs
upregulated and 5 downregulated, Table 1). Two miRNAs showed stable overexpression in
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MDD patients during the 8-week follow-up compared with controls (miR-941 and miR-589).
They also identified miRNAs exhibiting significant variations of expression among patients
with clinical improvement (7 upregulated and 1 downregulated). Fourteen dysregulated
miRNAs had putative mRNA targets that were differentially expressed in MDD, suggesting
that a common RNA regulatory network functions in MDD. These results suggest the
potential utility of miRNA signatures as markers of MDD evolution.
In a whole miRnome analysis, Bocchio-Chiavetto et al. (2013) examined blood miRNAs
from 10 MDD subjects after 12 weeks of treatment with escitalopram. They found that 30
c.
miRNAs were differentially expressed after the escitalopram treatment: 28 miRNAs were up-
regulated, and 2 miRNAs were down-regulated. Thirteen of them (let-7d, let-7e, miR-26a,
miR-26b, miR-34c-5p, miR-103, miR-128, miR-132, miR-183, miR-192, miR-335, miR-494
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and miR-22) play a role in the neural plasticity and stress response and in the pathogenetic
mechanisms of several neuropsychiatric diseases. As mentioned previously, miR-132 exerts
critical functions in the biological circuits implicated in neurogenesis and synaptic plasticity,
stimulating axonal and dendritic outgrowth in different brain areas (Mellios, 2011). This
miRNA, together with miR-26a, miR-26b and miR-183, widely contributes to the action of
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the neurotrophin BDNF in the brain (Wayman, 2008; Rinaldi et al., 2010; Caputo et al., 2011;
Kawashima et al., 2010). miR-132, miR-26a, miR-26b, miR-183, let-7d, let-7e, miR-26b,
miR-103, miR-128, miR-494 and miR-22 (Dwivedi et al., 2011; Serafini et al., 2012).play a
role in the pathogenesis of psychiatric disorders and in the mechanism of action of
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antipsychotic drugs and mood stabilizers.
To differentiate responders vs. non-responders, Oved et al. (2012) examined the growth
inhibition response of SSRI paroxetine in lymphoblastoid cell lines obtained from healthy
female individuals. Out of 80, they selected 8 LCLs which exhibited high or low sensitivities
to paroxetine. These LCLs were chosen for miRNA profiling. They found that miR-151-3p
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had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. miRNAs miR-212, miR-
132, miR-30b*, let-7b and let-7c also differed by >1.5-fold between the two LCL groups.
These findings suggested that profiling these specific miRNAs can differentiate responders
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vs. non-responders specifically for paroxetine treatment.
Animal Model Studies

Response to ECT, fluoxetine and ketamine: In rat model of electroconvulsive therapy
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(ECT) Ryan et al. (2013) examined expression of BDNF-associated miRNAs in rat brain and
blood following either acute (x1) or chronic (x10) ECS. They focused primarily on those
miRNAs that targeted BDNF. They found that level of one of the BDNF-related miRNAs
miR-212 was significantly increased in rat dentate gyrus following both acute and chronic
ECS. MiR-212 level was also increased in whole blood following chronic ECS and this was
positively correlated with miR-212 levels in the dentate gyrus. These results suggest that
alterations in miR-212 could be associated with BDNF modulation by ECS and that altered
a
miR-212 expression in both blood and brain can be used as an indicator of ECS response.
O‘Connor et al. (2013) investigated changes in hippocampal miRNAs induced by early-
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life stress (maternal separation) in rats and whether antidepressant treatments such as
selective serotonin reuptake inhibitor (SSRI) fluoxetine, the rapid acting N-methyl-d-aspartate
receptor antagonist ketamine, and electroconvulsive shock therapy (ECT) can reverse these
changes. Microarray analysis revealed that early-life stress affected levels of 24 hippocampal
miRNAs. When given to non-stressed animals, chronic fluoxetine treatment, repeated ECT,
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and acute ketamine treatment significantly altered the expression levels of 2, 10 and 14
miRNAs, respectively. One of these increases was common to all three antidepressants,
namely miR-598-5p. Chronic fluoxetine treatment significantly decreased 4 miRNAs
following maternal separation with 3 of these representing a partial normalization of stress-

induced changes. Repeated ECT altered 86 miRNAs (48 decreased, 38 increased); 16 of these
were a normalization of stress-induced changes. Acute ketamine treatment altered 55
miRNAs (32 decreased, 23 increased); 11 of these changes were a reversal to stress-induced
changes. ECT and ketamine treatment shared 43 common miRNA targets following maternal
c.
separation with seven being a reversal to stress-induced changes. All three antidepressants
shared one common miRNA target in maternal separated animals, i.e., miR-451, which
showed reversal following stress-induced change. These results suggest that changes to
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hippocampal miRNA expression may represent an important component of stress-induced
pathology and antidepressant action may reverse these changes.
In a PTSD mouse model, Schmidt et al. (2013) assessed miRNA profiles in prefrontal
cortices dissected from either fluoxetine or control-treated wild-type C57BL/6N mice 74 days
after their subjection to either a single traumatic electric foot-shock or mock-treatment.
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Screening for differences in the relative expression levels of all potential miRNA target
sequences in PFC resulted in identification of 5 miRNA candidate molecules. Validation of
these miRNAs revealed that the therapeutic action of fluoxetine in shocked mice was
associated with a significant reduction in mmu-miR-1971 expression, suggesting that
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traumatic stress and fluoxetine interact to cause distinct alterations in the mouse PFC miRNA
signature in the long-term. Although the functional significance of miR-1971 is not known at
present, however, gene ontology analysis of predicted miR-1971 target genes suggested that
this miRNA might be involved in basic metabolic processes like heterocyclic and organic
substance metabolism.
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MiR-16: Regulator of Serotonin Transporter and its Involvement in Fluoxetine-
induced Antidepressant Action: It has been shown that SSRI antidepressant treatment
reduces serotonin transporter (SERT) expression at protein level but does not affect SERT
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mRNA level (Benmansour et al.,, 2002). This suggests that SSRIs may interfere with SERT
expression at the translation level. This led to the examination of miRNA-regulated
expression of SERT. It was found that SERT is regulated by miR-16 (Baudry et al., 2010;
Hansen and Obrietan, 2013). SERT is under inhibitory control of miR-16, which binds to its
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3‘UTR region and inhibits its expression. Interestingly, in mouse brain, Baudry et al. (2010)
and Hansen and Obrietan (2013) demonstrated that miR-16 targets SERT in both raphe and
locus coeruleus (LC). Treatment of mice with fluoxetine elevated the levels of miR-16 in
serotonergic raphe nuclei and reduced SERT expression. Furthermore, the fluoxetine-
mediated increase in
miR-16 level in raphe was accompanied by a decrease in pre/pri–miR-16 supporting the
hypothesis that fluoxetine-induced up-regulation of miR-16 in raphe nuclei involved
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enhanced maturation from pre/pri-miR-16. How fluoxetine affects miR-16 maturation is not
clear, however, it has recently been shown that Wnt signaling may play a crucial role in such
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maturation (Millan, 2011). Surprisingly, fluoxetine decreased the level of miR-16 in the
noradrenergic LC, which was associated with the action of neurotrophic protein S100β
released by raphe in response to SSRI treatment. These findings were further confirmed in the
unpredictable chronic mild stress model (UCMS) such that mice exposed to a 6-week UCMS
alleviated behavioral response to the same extent when infused with fluoxetine or miR-16
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into raphe or by anti-miR-16 into the locus coeruleus (Launay et al., 2011). Fluoxetine-
exposed serotonergic neurons also secreted BDNF, Wnt2 and 15-Deoxy-delta12,14-
prostaglandin J2. These molecules were unable to mimic the action of fluoxetine on their own
but they acted synergistically to regulate miR-16 in hippocampus (Launay et al., 2011).
Putative miRNA binding sites in human SERT 3‘UTR have also been predicted by
bioinformatics analysis and validated by luciferase reporter assay experiments (Moya et al.,
2012). Wild type, antisense, and various mutants of the 3‘UTR were cloned downstream to
c.
the luciferase gene of reporter system. Then the luciferase-3‘UTR constructs were co-
transfected into rat raphe medullary raphe cells and human choriocarcinoma cells (expressing
SERT) combined with miRNA mimics, inhibitors or negative miRNA as control. The data
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obtained from those reporter assay experiments indicate that not only miR-16 but miR-15a
also plays a role in regulation of SERT expression in raphe nuclei. The effect of miR-15a was
comparable to those of miR-16 reported in mice by Baudry et al. (2010). These findings
represent a novel model for SERT expression regulation exerted by miR-15a/16 cluster
adjacently located at human chromosome 13q14.3.
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Possible Therapeutic Application of miRNAs in Depression and other
Psychiatric Illnesses
As mentioned above, it appears that miRNAs are significantly altered in depressed
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individuals. Complementary studies come from animal model system, where individual
miRNAs and their functional responses have been shown to be associated with many
physiological functions in brain. Interestingly, circulating miRNAs are being studied in many
psychiatric illnesses including depression as possible diagnostic tools. Although various
psychoactive drugs, including fluoxetine and other antidepressants alter the expression of
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miRNAs, how these miRNAs are involved in their therapeutic response is not clear at the
present time. But these studies provide at least reasonable proof that miRNAs can eventually
be used as therapeutic targets. Several strategies have been shown to utilize miRNAs for
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therapeutic approaches, particularly in cancer biology. For example, miRNA oligonucleotides
have been generated, which can directly compete with endogenous miRNAs. This strategy
has been successfully in targeting specific miRNA (miRNA-21) and reducing its expression,
which otherwise is overexpressed in several types of cancer (Si et al., 2007).
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The other strategy to reduce miRNA expression is to employ locked-nucleic-acid

antisense oligonucleotides or miR-masking, which requires a sequence with perfect
complementarity to the target gene such that duplexing can occur with higher affinity than
that between the target gene and its endogenous miRNA (Ebert et al., 2007). miRNAs can
also be overexpressed. For this, adenovirus-associated virus containing specific miRNAs can
be delivered to the target tissue (Kota et al., 2009).
In addition, miRNA mimics can be used to increase specific miRNA expression. These
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double-stranded RNA molecules mimic endogenous mature miRNAs (Landen et al., 2005).
Thus, there are many strategies that can be used to inhibit or overexpress miRNA of interest.
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Several of these strategies are in pipeline for neurodegenerative diseases (Meng et al., 2013)
but it will be interesting to see if these methods/strategies are useful in other complex
disorders such as psychiatric illnesses.
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Conclusion and Future Perspectives

By influencing a large number of target genes and regulating gene circuitry, miRNAs
may have major implications in disease pathophysiology. As mentioned in Table 1, miRNAs
affect many processes that are involved in MDD pathophysiology, including neural plasticity,
c.
neurogenesis, and stress response. In addition, direct studies in human postmortem brain as
well as peripheral tissues further support these ideas.
MDD is a complex disorder and heterogeneity is inherently linked to this disease
In
manifestation. Thus, before making any conclusive determinations, various clinical sub-
phenotypes and confounding variables need to be carefully considered. For example, it will
be interesting to examine whether changes in miRNAs are similar or dissimilar in
melancholic vs. non-melancholic depressed patients. Also interesting will be to examine
whether altered expression of miRNAs in depressed patients during adulthood are associated
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with childhood abuse. As indicated in animal model system, maternal separation leads to
changes in several miRNAs. Whether these miRNAs play a role in development of MDD in
later life is not known. It is noteworthy that recently, we have shown that enoxacin, a
compound that stabilizes TRBP-Dicer complex prevented learned helpless behavior in rats
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(Smalheiser et al., 2014), suggesting that alterations in miRNAs may have phenotypic
consequences.
There are many other avenues that need proper attention. For example, it will be
worthwhile examining how miRNAs that are involved in MDD are dysregulated and if SNPs
or CNVs play any role in such regulation. Also, an integrated view of miRNA network(s) and
is
the pathways that are affected by these miRNAs need to be evaluated. This is important since
not only individual miRNAs but combination of miRNAs provide more powerful regulatory
mechanism of gene regulation. A set of miRNAs that are significantly affected in MDD and
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the corresponding set of mRNAs that are affected in the same samples will help resolve this
issue.
As has been discussed earlier, presence of miRNAs biogenesis machinery in the synapse
may regulate gene expression locally. Since MDD is associated with altered synaptic
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plasticity, it will be interesting to examine whether miRNAs are synthesized at the synapse in
an activity-dependent manner and whether these miRNAs regulate synaptic proteins involved
in MDD pathogenesis.
A variety of enzymes are responsible for processing miRNAs. These include drosha,
dicer and cofactors DGCR8, TRBP, and PACT. Several of these proteins have been shown to
be modified post-transnationally in a dynamic manner. For example, altering the relative
a
expression of eIF2c may change the efficiency of translational arrest produced by a given
miRNA. Recently, it has been shown that dicer is activated by proteolytic cleavage under
conditions of elevated calcium levels (Smallheiser et al., 2008; Lugli, 2005) and eIF2C
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undergoes reversible phosphorylation within cells, which is required for its translocation to
processing bodies (Zeng et al., 2008). The phosphorylation of eIF2C appears to be due to
activation of ERK1/2 (Zeng et al., 2008). Since we have shown abnormalities in calcium-
sensing proteins and ERK1/2 signaling in the brain of MDD subjects (Dwivedi 2001, 2011,
2006, 2009), it will be worthwhile examining whether dicer cleavage patterns or eIF2C
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phosphorylation are altered in the MDD subjects.

Table 1. miRNAS, their known targets, and functional relevance
miRNA Target Known Effect/ References

Clinical Relevance
miR-124 Sox 9 Neuronal maturation, Lagos-Quintana et al.
neurogenesis 2002; Silber et al.
c.
2008; Cheng et al. in
2009; Chen et al.,
2009
miR-137 Ezh2 Neurogenesis, proliferation Szulwach et al. 2010
In
miR-26a Microtubule- Neuronal differentiation, Kye et al. 2007
associated protein 2 Neurogenesis
miR-134 Limk1 Regulates dendritic spine Schratt et al. 2006
development
miR-138 APT1 Regulates hippocampal Siegal et al. 2009
dendritic spine development
miR-132 P250GAP, MeCP2, Induces neurite outgrowth, Vo et al. 2005;
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CREB, SERT1, regulates dendritic Magill et al. 2010;
NR2A, NR2B, morphology of cortical and Fukada et al. 2005;
GLUR1 hippocampal neurons, Jugloff et al. 2005;
neuronal maturation, Klien et al. 2007;
he
synapse Formation Mellios et al. 2011
miR-16 SERT Neuronal maturation Baudry et al. 2010;
Hansen and Obrietan,
2013
miR-96, miR-141, miR-182, miR- Creb1 Downregulated in Non- Smalheiser et al.
183, miR-183, miR-198, miR- Learned Helplessness rats 2011
200a, miR-200a,
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miR-200b, miR-200b*,
miR-200c, miR-429
miR-142-5p, miR-33a, VEGFA, BCL2, Significantly downregulated Smalheiser et al.
miR-137, miR-489, miR-148b, DNMT3B, MYCN, in postmortem brain of 2013, 2014
bl
miR-101, miR-324-5p, Various transcription depressed human patients
miR-301a, miR-146a, factors and signaling who committed suicide,
miR-335, miR-494, miR-20b, proteins possibly due to decreased
miR-376a, miR-190, miR-155, transcription
miR-660, miR-552, miR-453,
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miR-130a, miR-27a, miR-497,

miR-10a, miR-20a,
miR-142-3p
miR-124, miR-139-5p, SAT1, SMOX Regulate polyamine gene Lopez et al. 2013
miR-195, miR-198, miR-320c, expression in frontal cortex
miR-33b, miR-34a, miR-34c-5p, of human suicide patients
miR-497, miR-873
miR-107, miR-133a, miR-148a, Altered expression in Belzeaux et al. 2012
a
miR-200c, miR-381, miR-425-3p, peripheral blood

miR-494, mononuclear cells of severe
miR-517b, miR-579, miR-589, MDD patients
miR-636, miR-652, miR-941, and
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miR-1243
let-7d, let-7e, miR-26a, miR-26b, Altered expression after 12 Bocchio-Chiavetto et
miR-34c-5p, miR-103, miR-128, weeks of escitalopram al. 2013
miR-132, miR-183, miR-192, treatment in MDD subjects,
miR-335, miR-494 and miR-22 neural plasticity, stress
response
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The presence of miRNAs in peripheral tissues, particularly, in blood cells provide

promising approach to use miRNAs as potential biomarkers for both diagnosis and treatment
response. However, there are several issues that need consideration for the use of circulating
miRNAs as biomarkers. For example, the source of miRNAs in blood cells is not clear at the
present time. In this regard, profiling exosomal miRNAs derived from brain may prove useful
exosomal miRNAs can be useful in detecting miRNAs. Finally, there is a possibility that
changes in circulating miRNAs may not be directly related to the changes in brain. While this
c.
complicates the study of circulating miRNAs, it still holds promise, since it is likely that
miRNA changes may reflect systemic alterations that accompany the disease process.
In
Acknowledgments
The research was supported by grants from National Institute of Mental Health
(R01MH082802; R21MH081099; R21MH091509; 1R01MH101890; R01MH100616),
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American Foundation for Suicide Prevention, and University of Illinois at Chicago Clinical
and Translational Sciences National supported by Center for Advancing Translational
Sciences, National Institutes of Health (Grant # UL1TR000050) to Dr. Dwivedi.
he
References
Ameres S. L., Zamore P. D. (2013). Diversifying microRNA sequence and function. Nat. Rev.
Mol. Cell Biol. 14, 475-488.
is
Azuma-Mukai A., Oguri H., Mituyama T., Qian Z.R., Asai K., Siomi H., Siomi M.C. (2008).
Characterization of endogenous human Argonautes and their miRNA partners in RNA
silencing. Proc. Natl. Acad. Sci. USA. 105:7964-7969.
bl
Bartel D. (2004). MicroRNA: genomics, biogenesis, mechanism and function. Cell. 23:281-
297.
Baudry A., Mouillet-Richard S., Schneider B., Launay J.M., Kellermann O. (2010). miR-16
Pu
targets the serotonin transporter: a new facet for adaptive responses to antidepressants.
Science. 329:1537-15341.
Belzeaux R., Bergon A., Jeanjean V., Loriod B., Formisano-Tréziny C., Verrier L. (2012).
Responder and nonresponder patients exhibit different peripheral transcriptional
signatures during major depressive episode. Transl. Psychiatry. 2e185.
Benito E., Barco A., (2010). CREB‘s control of intrinsic and synaptic plasticity: implications
for CREB-dependent memory models. Trends Neurosci. 33:230-40.
a
Benmansour S., Owens W.A., Cecchi M., Morilak D.A., Frazer A. (2002). Serotonin
clearance in vivo is altered to a greater extent by antidepressant-induced downregulation
of the serotonin transporter than by acute blockade of this transporter. J. Neurosci.
ov
22:6766-6772.
Bocchio-Chiavetto L., Maffioletti E., Bettinsoli P., Giovannini C., Bignotti S., Tardito D.
(2013). Blood microRNA changes in depressed patients during antidepressant treatment.
Eur. Neuropsychopharmacol. 23:602–611.
N
Borchert G. M., Lanier W., Davidson, B. L. (2006). RNA polymerase III transcribes human
microRNAs. Nature Struct. Mol. Biol. 13:1097-101.
Brodersen P., Voinnet O. (2009). Revisiting the principles of microRNA target recognition
and mode of action. Nat. Rev. Mol. Cell Biol. 10:141-148.
Caputo V., Sinibaldi L., Fiorentino A. (2011). Brain derived neurotrophic factor (BDNF)
expression is regulated by microRNAs miR-26a and miR-26b allele-specific binding.
PLoSOne 6:e28656.
c.
Cheloufi S., Dos Santos C.O., Chong M.M., Hannon G.J. (2010). A dicer-independent
miRNA biogenesis pathway that requires Ago catalysis. Nature. 465:584-589.
Chendrimada T. P., Finn K.J., Ji X., Baillat D., Gregory R.I., Liebhaber S.A., Pasquinelli A.
In
E., Shiekhattar R. (2007). MicroRNA silencing through RISC recruitment of eIF6.
Nature. 447:823-828.
Chendrimada T.P., Gregory R.I., Kumaraswamy E., Norman J., Cooch N., Nishikura K.,
Shiekhattar R. (2005). TRBP recruits the Dicer complex to Ago2 for microRNA
processing and gene silencing. Nature. 436:740–744.
rs
Cheng L. C, Pastrana E., Tavazoie M., Doetsch F. (2009). miR-124 regulates adult
neurogenesis in the subventricular zone stem cell niche. Nat Neurosci. 12:399-408.
Choi J. K., Yu U., Yoo O.J., Kim S. (2005). Differential coexpression analysis using
microarray data and its application to human cancer. Bioinformatics. 21:4348-4355.
he
Dowlatshahi D., MacQueen G.M., Wang J.F., Young L.T. (1998). Increased temporal cortex
CREB concentrations and antidepressant treatment in major depression. Lancet.
352:1754-1755.
Dwivedi Y., Mondal A.C., Rizavi H.S., Faludi G., Palkovits M., Sarosi A., Conley R.R.,
Pandey G.N. (2006a). Differential and brain region-specific regulation of Rap-1 and Epac
is
in depressed suicide victims. Arch Gen Psychiatry. 63:639-648.
Dwivedi Y., Rizavi H.S., Conley R.R., Pandey G.N. (2006b). ERK MAP kinase signaling in
post-mortem brain of suicide subjects: differential regulation of upstream Raf kinases
bl
Raf-1 and B-Raf. Mol Psychiatry. 11:86-98.
Dwivedi Y., Rizavi H. S., Conley R. R., Roberts R. C., Tamminga C.A., Pandey G.N. (2003).
Altered gene expression of brain-derived neurotrophic factor and receptor tyrosine kinase
B in postmortem brain of suicide subjects. Arch. Gen. Psychiatry. 60:804-815.
Pu
Dwivedi Y., Rizavi H. S., Roberts R.C., Conley R.C., Tamminga C.A., Pandey G.N. (2001).
Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of
depressed suicide subjects. J. Neurochem. 77(3):916-28.
Dwivedi Y., Rizavi H. S., Teppen T., Sasaki N., Chen H., Zhang H., Roberts R.C., Conley
R.R., Pandey G. N. (2007). Aberrant extracellular signal-regulated kinase (ERK) 5
signaling in hippocampus of suicide subjects. Neuropsychopharmacology. 32:2338-2350.
Dwivedi Y., Rizavi H. S., Zhang H., Roberts R.C., Conley R.R., Pandey G.N. (2009).
a
Aberrant extracellular signal-regulated kinase (ERK)1/2 signalling in suicide brain: role

of ERK kinase 1 (MEK1). Int. J. Neuropsychopharmacol. 12:1337-1354.
ov
Dwivedi Y. (2011). Evidence demonstrating role of microRNAs in the etiopathology of major

depression. J. Chem. Neuroanat. 42:142-156.
Ebert M.S., Neilson J.R., Sharp P.A. (2007). MicroRNA sponges: competitive inhibitors of
small RNAs in mammalian cells. Nat. Methods. 2007;4:721-726.
Ernst C., Chen E.S., Turecki G. (2009). Histone methylation and decreased expression of
N
TrkB.T1 in orbital frontal cortex of suicide completers. Mol. Psychiatry 14:830-832.

Fava M., Davidson K. G. (1996). Definition and epidemiology of treatment-resistant
depression. Psychiatr Clin. North Am. 19:179-200.
Fiori L. M., Gross J. A., Turecki G. (2012). Effects of histone modifications on increased
expression of polyamine biosynthetic genes insuicide. Int. J. Neuropsychopharmacol.
15:1161-1166.
Friedman J. M., Jones P A. (2009). MicroRNAs: critical mediators of differentiation,
development and disease. Swiss Med Wkly 139:466–472.
c.
Fukuda T., Yamashita Y., Nagamitsu S., Miyamoto K., Jin J.J., Ohmori I., Ohtsuka Y.,
Kuwajima K., Endo S., Iwai T., Yamagata H., Tabara Y., Miki T., Matsuishi T., Kondo I.
(2005). Methyl-CpG binding protein 2 gene (MECP2) variations in Japanese patients
In
with Rett syndrome: pathological mutations and polymorphisms. Brain Dev. 27:211-217.
Gourley S. L., Wu F. J., Taylor J. R. (2008). Corticosterone regulates pERK1/2 map kinase in
a chronic depression model. Ann. N Y Acad. Sci. 1148:509-514.
Gregory R. I., Yan K. P., Amuthan G., Chendrimada T., Doratotaj B., Cooch N., Shiekhattar
R., (2004). The Microprocessor complex mediates the genesis of microRNAs. Nature.
rs
432, 235-240.
Han J., Lee Y., Yeom K.H., Nam J.W., Heo I., Rhee J.K., Sohn S.Y., Cho Y., Zhang B.T.,
Kim V.N. (2006). Molecular basis for the recognition of primary microRNAs by the
Drosha–DGCR8 complex. Cell. 125:887-901.
he
Hansen K. F., Obrietan K. (2013). MicroRNA as therapeutic targets for treatment of
depression. Neuropsychiatr Dis Treat. 9:1011-21.
He Y., Zhou Y., Xi Q., Cui H., Luo T., Song H., Nie X., Wang L., Ying B. (2012). Genetic
variations in microRNA processing genes are associated with susceptibility in depression.
DNA Cell Biol 31:1499-506.
is
Impey S., McCorkle S. R., Cha-Molstad H., Dwyer J. M., Yochum G. S., Boss J. M.,
McWeeney S., Dunn J. J., Mandel G., Goodman R. H. (2004). Defining the CREB
regulon: a genome wide analysis of transcription factor regulatory regions. Cell.
bl
199:1041-1054.
Jugloff D. G., Jung B. P., Purushotham D., Logan R., Eubanks J. H. (2005). Increased
dendritic complexity and axonal length in cultured mouse cortical neurons
overexpressing methyl-CpG-binding protein MeCP2. Neurobiol Dis. 19:18-27.
Pu
Kawashima H., Numakawa T., Kumamaru E., Adachi N., Mizuno H., Ninomiya M., Kunugi
H., Hashido K. (2010). Glucocorticoid attenuates brain-derived neurotrophic factor-
dependent upregulation of glutamate receptors via the suppression of microRNA-132
expression. Neuroscience. 165:1301-1311.
Kessler R. C., McGonagle K. A., Zhao S. (1994). Lifetime and 12-month prevalence of DSM-
III-R psychiatric disorders in the United States. Results from the National Comorbidity
Survey. Arch. Gen. Psychiatry. 51:8-19.
a
Kosik K. S. (2006). The neuronal microRNA system. Nat. Rev. Neurosci. 7:911-920.
Kota J., Chivukula R. R., O'donnell K. A., Wentzel E.A., Montgomery C.L., Hwang H. W.,
ov
Chang T.C., Vivekanandan P., Torbenson M., Clark K.R., Mendell J.R, Mendell J.T.
(2009). Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver
cancer model. Cell. 2009;137:1005-1017.
Kozomara A., Griffiths-Jones S. (2014). miRBase: annotating high confidence microRNAs
using deep sequencing data. Nucleic Acids Res (2014 Database issue), D68-73.
N
Kye M., Liu T., Levy S. F., Xu N. L., Groves B.B., Bonneau, R., Lao L., Kosik, K.S. (2007).
Somatodendritic microRNAs identified by laser capture and multiplex RT-PCR. RNA.
13: 1224-1234.
Lagos-Quintana M., Rauhut R., Yalcin A., Meyer J., Lendeckel W., Tuschl T. (2002).
Identification of tissue-specific microRNAs from mouse. Curr. Biol 12:735-9.
Landen C.N. Jr., Chavez-Reyes A., Bucana C., Schmandt R., Deavers M.T., Lopez-Berestein
G., Sood A.K. (2005). Therapeutic EphA2 gene targeting in vivo using neutral liposomal
small interfering RNA delivery. Cancer Res. 2005;65:6910–6918.
c.
Launay J.M., Mouillet-Richard S., Baudry A., Pietri M., and Kellermann O, (2011). Raphe-
mediated signals control the hippocampal response to SRI antidepressants via miR-16.
Translational Psychiatry. 1, e56.
In
Lee Y., Hur I., Park S.Y., Kim Y. K., Suh M. R., Kim V. N. (2006). The role of PACT in the
RNA silencing pathway. EMBO J. 25:522–532
Lee Y., Kim M., Han J., Yeom K.H., Lee S., Baek S.H., Kim V.N. (2004). MicroRNA genes
are transcribed by RNA polymerase II. EMBO J. 13, 23:4051-4060.
Leistedt S.J., Linkowski P. (2013). Brain, networks, depression, and more. Eur.
rs
Neuropsychopharmacol. 23:55-62.
Leuschner P.F., Busch C.J., Kane, C., Hübel, K., Dekker, F., Hedberg, C., Rengarajan B.,
Kawashima H., Numakawa T., Kumamaru E., Adachi N., Mizuno H., Ninomiya M.,
Kunugi H., Hashido, K. (2010). Glucocorticoid attenuates brain-derived neurotrophic
he
factor dependent upregulation of glutamate receptors via the suppression of microRNA-
132 expression. Neuroscience. 165:1301-1311.
Lopez J. P., Fiori L. M., Gross J. A., Labonte B., Yerko V., Mechawar N., Turecki G. (2014).
Regulatory role of miRNAs in polyamine gene expression in the prefrontal cortex of
depressedsuicide completers. Int. J. Neuropsychopharmacol. 17:23-32.
is
Lugli G., Larson J., Martone M. E., Jones Y., Smalheiser N.R. (2005). Dicer and eIF2c are
enriched at postsynaptic densities in adult mouse brain and are modified by neuronal
activity in a calpain-dependent manner. J. Neurochem. 94:896-905.
bl
Lugli G., Torvik V. I., Larson J., Smalheiser N. R. (2008). Expression of microRNAs and
their precursors in synaptic fractions of adult mouse forebrain. J. Neurochem. 106:650-
661.
Lund E., Guttinger S., Calado A., Dahlberg J. E., Kutay U. (2004). Nuclear export of
Pu
microRNA precursors. Science. 303:95-98.

Maffioletti E, Tardito D, Gennarelli M, Bocchio-Chiavetto L. (2014). Micro spies from the
brain to the periphery: new clues from studies on microRNAs in neuropsychiatric
disorders. Front Cell Neurosci. 11;8:75.
Magill S. T., Cambronne X. A., Luikart B. W., Lioy D. T., Leighton B. H., Westbreek G. L.,
Mandel, G., Goodman, R.H. (2010). microRNA-132 regulates dendritic growth and
arborization of newborn neurons in the adult hippocampus. Proc. Natl. Acad. Sci. USA
a
106:20382-20387.
Mathonnet G., Fabian M. R., Svitkin Y. V., Parsyan A., Huck L., Murata T., Biffo S.,
ov
Merrick W. C., Darzynkiewicz E., Pillai R. S., Filipowicz W., Duchaine T.F., Sonenberg
N. (2007). MicroRNA inhibition of translation initiation in vitro by targeting the cap-
binding complex eIF4F. Science. 317:1764-1767.
Maussion G., Yang J., Yerko V., Barker P., Mechawar N., Ernst C., Turecki G. (2012).
Regulation of a truncated form of tropomyosin-related kinase B (TrkB) by Hsa-miR-185*
N
in frontal cortex of suicide completers. PLoS One. 7:e39301.

Meerson A., Cacheaux L., Goosens K. A., Sapolsky R. M., Soreq H., Kaufer D. (2010).
Changes in brain MicroRNAs contribute to cholinergic stress reactions. J. Mol. Neurosci.
40:47-55.
Mellios N., Sugihara H., Castro J. (2011). miR-132, an experience-dependent microRNA, is
essential for visual cortex plasticity. Nat Neurosci. 14:1240-1242.
c.
Meng F., Dai E., Yu X., Zhang Y., Chen X., Liu X., Wang S., Wang L., Jiang W. (2013).
Constructing and characterizing a bioactive small molecule and microRNA association
network for Alzheimer's disease. J R Soc Interface. 2013 Dec 18:;11(92):20131057.
In
Michalak P. (2006). RNA world - the dark matter of evolutionary genomics. J. Evol Biol.
19:1768-1774.
Millan M. J. (2011). MicroRNA in the regulation and expression of serotonergic transmission
in the brain and other tissues. Curr. Opin. Pharmacol. 11:11-22.
Miska E. A., Alvarez-Saavedra E., Townsend M., Yoshii A., Sestan N., Rakic P. Constantine-
rs
Paton M, Horvitz HR. (2004). Microarray analysis of microRNA expression in the
developing mammalian brain. Genome Biol. 5:R68.
Mo W. J., Fu X. P., Han X. T., Yang G. Y., Zhang J. G., Guo F. H., Huang Y., Mao Y.M., Li
Y., Xie Y. (2009). A stochastic model for identifying differential gene pair co-expression
he
patterns in prostate cancer progression. BMC Genomics.10:340.
Moya P. R., Wendland J.R., Salemme J., Fried R. L., Murphy D.L. (2012). miR-15a and miR-
16 regulate serotonin transporter expression in human placental and rat brain raphe cells.
Int. J. Neuropsychopharmacol. 16:621-629.
Nudelman A. S., DiRocco D. P., Lambert T. J., Garelick M. G., Le J., Nathanson N.M.,
is
Storm, D.R. (2011). Neuronal Activity Rapidly Induces Transcription of the CREB-
Regulated microRNA-132, in vivo. Hippocampus. 20:492-498.
O'Connor R. M., Grenham S., Dinan T. G., Cryan J. F. (2013). microRNAs as novel
bl
antidepressant targets: converging effects of ketamine and electroconvulsive shock
therapy in the rat hippocampus. Int. J. Neuropsychopharmacol. 16:1885-1892.
Ota K. T., Duman R. S. (2013). Environmental and pharmacological modulations of cellular
plasticity: role in the pathophysiology and treatment of depression. Neurobiol. Dis.
Pu
57:28-37.
Oved K., Morag A., Pasmanik-Chor M., Oron-Karni V., Shomron N., Rehavi M., Stingl J.C.,
Gurwitz D. (2012). Genome-wide miRNA expression profiling of human lymphoblastoid
cell lines identifies tentative SSRI antidepressant response biomarkers.
Pharmacogenomics. 13:1129-1139.
Pandey G. N., Dwivedi Y., Pandey S. C., Conley R. R., Roberts R. C., Tamminga C. A.
(1997). Protein kinase C in the postmortem brain of teenage suicide victims. Neurosci
a
Lett. 228:111-1114.
Pandey G. N., Ren X., Dwivedi Y., Pavuluri M.N. (2008). Decreased protein kinase C (PKC)
ov
in platelets of pediatric bipolar patients: effect of treatment with mood stabilizing drugs.
J. Psychiatr. Res. 42:106-16.
Pijnenborg R., Dixon G., Robertson W. B., Brosens I. (1980). Trophoblastic invasion of
human decidua from 8 to 18 weeks of pregnancy. Placenta. 1:3-19.
Qi X., Lin W., Li J., Li H., Wang W., Wang D., Sun M. (2008). Fluoxetine increases the
N
activity of the ERK-CREB signal system and alleviates the depressive like behavior in
rats exposed to chronic forced swim stress. Neurobiol Dis. 31:278-285.
Raabe F. J., Spengler D. (2013). Epigenetic Risk Factors in PTSD and Depression. Front
Psychiatry. 4:80.
Rinaldi A., Vincenti S., De Vito F., Bozzoni I., Oliverio A., Presutti C., Fragapane P., Mele
A. (2010). Stress induces region specific alterations in microRNAs expression in mice.
Behav Brain Res. 208:265-269.
c.
Ryan K. M., O'Donovan S. M., McLoughlin D. M. (2013). Electroconvulsive stimulation
alters levels of BDNF-associated microRNAs. Neurosci Lett. 549:125-129.
Schratt G. M., Tuebing F., Nigh E. A., Kane C. G., Sabatini M. E., Kiebler M., Greenberg
In
M.E. (2006). A brain-specific microRNA regulates dendritic spine development. Nature.
439, 283-289.
Sempere L. F., Freemantle S., Pitha-Rowe I., Moss E., Dmitrovsky E., Ambros V. (2004).
Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed
microRNAs with possible roles in murine and human neuronal differentiation. Genome
rs
Biol. 5, R13.
Serafini G., Pompili M., Innamorati M., Giordano G., Montebovi F., Sher L., Dwivedi Y.,
Girardi P. (2012). The role of microRNAs in synaptic plasticity, major affective disorders
and suicidal behavior. Neurosci Res. 73:179-190.
he
Si M. L., Zhu S., Wu H., Lu Z., Wu F., Mo Y.Y. (2007). MiR-21-mediated tumor growth.
Oncogene. 2007, 26:2799-803.
Siegel G., Obernosterer G., Fiore R., Oehmen M., Bicker S., Christensen M., Khudayberdiev
S., Drepper C., Waldmann H., Kauppinen S., Greenberg M.E., Draguhn A., Rehmsmeier
M., Martinex J., Scratt G.M. (2009). A functional screen implicates microRNA-138-
is
dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine
morphogenesis. Nat. Cell Biol. 11:705-716.
Silber J., Lim D., Petritsch C., Persson A. I., Maunakea A. K., Yu M., Vandenberg S. R.,
bl
Ginzinger D. G., James C.D., Costello J. F., Bergers G., Weiss W. A., Alvarez-Buylla A.
Hodgson, J.G. (2008). miR-124 and miR-137 inhibit proliferation of glioblastoma
multiforme cells and induce differentiation of brain tumor stem cells. BMC Med 6:14.
Silva A. J., Kogan J. H., Frankland P. W., Kida S. (1998). BREB and memory. Annu. Rev.
Pu
Neurosci 21:127-48.
Smalheiser N. R. (2008). Synaptic enrichment of microRNAs in adult mouse forebrain is
related to structural features of their precursors. Biol Direct. 3:44.
Smalheiser N. R., Lugli G., Rizavi H.S., Torvik V.I., Turecki G., Dwivedi Y. (2012).
MicroRNA expression is down-regulated and reorganized in prefrontal cortex of
depressed suicide subjects. PLoS One. 7(3):e33201
Smalheiser N. R., Lugli G., Rizavi H.S., Zhang H., Torvik V.I., Pandey G.N., Davis J.M.,
a
Dwivedi Y. (2011). MicroRNA expression in rat brain exposed to repeated inescapable

shock: differential alterations in learned helplessness vs. non-learned helplessness. Int. J.
ov
Neuropsychopharmacol. 14:1315-25.
Stäehler C.F, Keller A., Leidinger P., Backes C., Chandran A., Wischhusen J., Meder B.,
Meese E. (2012). Whole miRNome-wide differential co-expression of microRNAs.
Genomics Proteomics Bioinformatics. 10:285-294.
Szulwach K. E., Li X., Smrt R. D., Li Y., Luo Y., Lin L., Santistevan N.J., Li W., Zhao X.,
N
Jin P. (2010). Cross talk between microRNA and epigenetic regulation in adult
neurogenesis. J. Cell Biol. 189:127-141.
Todorovic C., Sherrin T., Pitts M., Hippel C., Rayner M., Spiess J. (2009). Suppression of the
MEK/ERK signaling pathway reverses depression-like behaviors of CRF2-deficient
mice. Neuropsychopharmacology. 34:1416-26.
Vo N., Klein M. E., Varlamova O., Keller D.M., Yamamoto T., Goodman R.H., Impey S.
(2005). A cAMP-response element binding protein-induced microRNA regulates
c.
neuronal morphogenesis. Proc. Natl. Acad. Sci. USA. 102:16426-16431.
Vreugdenhil E., Verissimo C. S., Mariman R., Kamphorst J.T., Barbosa J.S., Zweers T.,
Champagne D.L., Schouten T., Meijer O.C., de Kloet E.R., Fitzsimons C.P. (2009).
In
MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: implications for
glucocorticoid responsiveness in the brain. Endocrinology. 150:2220-2228.
Wang J., Cheng C. M., Zhou. J. (2004). Estradiol alters transcription factor gene expression in
primate prefrontal cortex. J. Neurosci. Res. 76:306-331.
Wayman G.A. Davare M., Ando H., Fortin D., Varlamova O., Cheng H.M., Marks D.,
rs
Obrietan K., Soderling T.R., Goodman R.H., Impey S. (2008). An activity-regulated
microRNA controls dendritic plasticity by down-regulating p250GAP. Proc. Natl. Acad.
Sci. USA. 105:9093-9098.
Westholm J. O., Ladewig E., Okamura K., Robine N., Lai E. C. (2012). Common and distinct
he
patterns of terminal modifications to mirtrons and canonical microRNAs. RNA. 18:177-
192.
Wu J., Xie X. (2006). Comparative sequence analysis reveals an intricate network among
REST, CREB and miRNA in mediating neuronal gene expression. Genome Biol. 7:R85.
Wu L., Fan J., Belasco J.G. (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc.
is
Natl. Acad. Sci. USA. 103:4034-4039.
Xu J., Li C. X., Li Y. S., Lv J. Y., Ma Y., Shao T. T., Xu L.D., Wang Y.Y., Du L., Zhang
Y.P., Jiang W., Li C.Q., Xiao Y., Li X. (2011). MiRNA-miRNA synergistic network:
bl
construction via co-regulating functional modules and disease miRNA topological
features. Nucleic Acids Res. 39:825-836.
Yang J. S., Lai E. C. (2010). Dicer-independent, Ago2-mediated microRNA biogenesis in
vertebrates. Cell Cycle. 15, 9:4455-60.
Pu
Yuan P., Zhou R., Wang Y., Li X., Li J., Chen G., Guitart X., Manji H. K. (2010). Altered
levels of extracellular signal-regulated kinase signaling proteins in postmortem frontal
cortex of individuals with mood disorders and schizophrenia. J. Affect Disord. 124:164-
169.
Zeng Y., Sankala H., Zhang X., Graves P. R. (2008). Phosphorylation of Argonaute 2 at
serine-387 facilitates its localization to processing bodies. Biochem J. 413:429-436.
Zhang H., Kolb F. A., Brondani V., Billy E., Filipowicz W. (2002). Human Dicer
a
preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J.
21:5875–5885.
ov
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Chapter 9
In
Preclinical and Clinical Aspects
of a Combination Therapy with
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Antagonists to N-Methyl-D-Aspartate
Receptor to Enhance Fluoxetine’s
he
Antidepressant Effects
Gislaine Z. Réus1,2, Helena M. Abelaira2, Fabricia Petronilho1,3

is
and João Quevedo1,2
1
Center for Experimental Models in Psychiatry,
Department of Psychiatry and Behavioral Sciences, Medical School,
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The University of Texas Health Science Center at Houston, Houston, TX, US
2
Laboratório de Neurociências, Programa de Pós-GraduaçãoemCiências da Saúde,
UnidadeAcadêmica de Ciências da Saúde,
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Universidade do ExtremoSulCatarinense, Criciúma, SC, Brazil

3
Programa de Pós-GraduaçãoemCiências da Saúde,
Universidade do Sul de Santa Catarina, Tubarão, SC, Brazil
Abstract
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Mood disorders are a major public health problem and are associated with a
considerable burden of disease, suicides, physical comorbidities, high economic costs,
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and a poor quality of life. Although drugs used for the treatment of mood disorders show
a clinical response in good a percentage of patients, around 30-40% of them do not
respond to any pharmacological drugs. Generally, to improve therapy, combinations of
agents belonging to different pharmacological groups, or a combination of an
antidepressant drug with a substance which can increase its effect are already being used
in the clinical environment. Fluoxetine (FLX), which is a selective serotonin reuptake
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inhibitor (SSRIs) has been used in combination with other drugs. In fact, a past study has

Corresponding Author address: Email: gislainezilli@hotmail.com.
168 Gislaine Z. Réus, Helena M. Abelaira, Fabricia Petronilho et al.
shown that the combination of FLX plus the antipsychotic olanzapine (OLZ) used on
subjects with treatment-resistant depression was associated with significantly greater and
faster improvement than was possible with either drug when used alone. In addition,
some agents can potentiate the efficacy of antidepressants, such as the antagonists of
glutamate receptors N-methyl-D-aspartate (NMDA), mainly, ketamine, which enhances
the effects of other antidepressant drugs in both molecular and behavioural responses.
c.
Thus, this chapter highlights the basic and clinical studies regarding the combined effects
of SSRIs when used with other drugs which have effects on the behavioural response,
and on the signalling pathways related with mood disorders.
In
Keywords: fluoxetine; NMDA receptor antagonist; combination therapy; depression
Introduction
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Major depression is a serious public health problem and one of the most common
psychiatric disorders, with a lifetime prevalence of 17% in the United States. It is estimated
that 121 million people are affected worldwide (Kessler et al., 2005; Sattler and Rothstein,
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2007). Almost 1 million lives are lost yearly due to suicide, which translates to 3,000 suicide
deaths every day (World Health Organization, 2012). Moreover, patients suffering from
severe depression have high rates of morbidity with profound economic and social
consequences (Nemeroff and Owens 2002).
The antidepressant medications which are currently used work directly on the
is
monoamine neurotransmitter systems, using targets such as serotonin, norepinephrine and
dopamine to enhance the concentration of these neurotransmitters in the synapse. Although
the treatment of depression is generally safe and effective, it is far from ideal, for example,
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standard antidepressants usually require approximately one month or more for their
antidepressant effects to manifest (Berton and Nestler, 2006). Additionally, around 36% of
patients with depression are treatment-resistant (Fava and Davidson, 1996). It is important to
note that the higher risk of suicide and other deliberate acts of self-harm during the first
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month of treatment is not uncommon, and when it does occur, it has been postulated to be due
to a mismatch in symptom improvement; that is, their physical energy improves first, while
resolution of depressive mood and negative thoughts is more gradual (Conwell and Heisel,
2012). The classes of antidepressants include the tricyclic agents (i.e., imipramine), the
selective serotonin reuptake inhibitors (SSRIs) (i.e., fluoxetine (FLX)), the selective serotonin
and noradrenaline reuptake inhibitors (i.e., venlafaxine), the selective noradrenaline reuptake
inhibitors (i.e., reboxetine), the dual inhibitors of noradrenaline and dopamine reuptake
a
(bupropion), and the monoamine oxidase inhibitors (i.e., tranilcipromine) (Susman, 2003;
Elhwuegi, 2004). For depressive patients who fail to respond to classic antidepressants
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treatments, several strategies have been proposed, such as: (a) increasing the dose of the
antidepressant, (b) substituting to a new class of antidepressant, (c) augmentation therapy, and
(d) combining two antidepressants or combining antidepressants with different
pharmacological profiles (combination pharmacotherapy) (Fredman et al., 2000; Mischoulon
et al., 2000; Carvalho et al., 2007). The use of a combination of two antidepressants from
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different classes with corresponding mechanisms of action in order to avoid the loss of partial
response to the first medication may increase the risk of drug interactions and new side
effects. However, augmenting antidepressant medication with another agent may enhance its
Preclinical and Clinical Aspects of a Combination Therapy … 169
antidepressant efficacy and thus it is possible to prevent transition from treatment with SSRI
to another medication (Maoz, 2007). The Food and Drug Administration (FDA)-approved
treatment options, for example, the quetiapine and olanzapine (OLZ)/FLX combination, are
permitted for the treatment of bipolar depression. Interestingly, individuals with treatment-
resistant depression, whom received FLX alone, OLZ alone, or a combination of both showed
c.
that the combination was associated with significantly greater and faster improvement than
was possible with either drug alone (Shelton et al., 2001). Moreover, pre-clinical studies have
shown that other agents, which act in neurotransmitter systems separatefrom the
In
monoaminergic system, may enhance the effects of FLX. In fact, FLX when administrated
alone was inactive; however, when FLX was combined with the uncompetitive N-methyl-D-
aspartate (NMDA) receptor antagonists, amantadine, memantine and neramexane, it showed
positive effects in theanimal model of depression (Rogóz et al., 2002). So, the combination of
traditional antidepressant drugs and other agents may produce enhanced antidepressant
rs
properties, and this is very important for antidepressant-resistant patients. Therefore, this
chapter highlights the basic and clinical studies concerningthe effects of combining FLX with
other therapeutic targets on the behavioural and neurochemical pathways related with mood
disorders.
he
Neurotransmitter Dysfunction in
Major Depression
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Research over the second half of the 20th century was strongly influenced by the
discovery that agents which alter monoamine metabolism, particularly that of serotonin (5-
hydroxytryptamine, 5-HT), norepinephrine (NE) and dopamine (DA) relieved depressive
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symptoms. The monoamine deficiency hypothesis posits that depressive symptoms arise from
insufficient levels of monoamine neurotransmitters (Table 1) (Delgado, 2006). This
hypothesis grew out of observations that antidepressant therapies raise neurotransmission tone
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depending on one or more of these neurotransmitters (Hamon and Blier, 2013). In this way,
the first aminergic hypothesis by Schildraut (1965), Bunney and Davis (1965) was called the
catecholamine hypothesis, which proposed that depression is associated with deficitsin
catecholamines, especially in NE. Later came the serotonergic hypothesis by Van Praag and
Korf (1971), providing a big boost to the development of the class of antidepressants called
serotonin reuptake inhibitors (SSRIs); and also the dopaminergic hypothesis by Willner et al.
(1990), relating the involvement of DA in the phenomena of brain reward, suggesting that this
a
neurotransmitter is involved with anhedonia, which is one of the main symptoms of patients
with depression. In support of this hypothesis, researches have noted alterations of NE and
5HT receptor density in the cortical and limbic formations of depressed patients (Delgado,
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2000; Drevets et al., 2007). In a positron emission tomography (PET) imaging study
comparing dopamine type-1 receptor (D1) binding potential in depressed patients vs. healthy
subjects, it was found that there was a significantreduction in D1 potential binding in the
depressed group, negatively correlating this reduction in D1 with illness duration and
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anhedonic rates (Cannon et al., 2009). Moreover, Maletic and Raison (2009) showed that
mutations in genes that code for 5 HTT, catechol-O-methyltransferase (COMT), monoamine
oxidase (MAO) and monoamine receptors have a perpetual vulnerability towards depression
(Table 1). Chiuccariello et al. (2014) have reported elevated levels of MAO-A in the brains of
depressed patients. Still, since MAO-A is a primary monoamine-lowering enzyme in the
nerve cells; its excessive activity may contribute to a disturbance of monoamine signalling in
major depressive disorder (MDD) (Meyer et al., 2006).
Despite the relevance of the monoamine hypothesis in depression, there is some
c.
resistance to its full acceptance, especially due to the fact that all antidepressant medications
instantly increase the levels of monoamines within the synaptic clefts, but their clinical effect
only occurs a few weeks later (Trivedi et al., 2006). In this way, it was necessary to establish
In
a new hypothesis to explain this latency in response, and to take into account the effects of
chronic adaptive receptors in the administration of antidepressants. Ventulani and Sulser
(1975) reported that chronic treatment with effective antidepressants shared a desensitization
of the β-adrenergic receptor coupled to a cyclic adenosine monophosphate (cAMP); and after
that, a subsequent number of preclinical studies put emphasis on what happens at the sub-
rs
cellular level after an acute increase in monoaminergic neurotransmission.
Monoamines are not the only neurotransmitters involved in mood disorders, and even
their action could be modulated by other neurotransmitters (Maletic and Raison, 2009).
Multiple lines of evidence implicate aberrant glutamate (Glu) and gamma-aminobutyric acid
he
(GABA) transmission in the etiopathogenesis of major depression. As the principal excitatory
neurotransmitter in the circuitry linking limbic and cortical areas, Glu is virtually ubiquitous
in the brain (Maletic and Raison, 2009). The glutamatergic projections regulate the function
of the monoaminergic neurons activity, including norepinephrine in the locus ceruleus,
serotonin in the nuclei raphe and dopaminergic neurons in the substantianigra and ventral
is
tegmental area (Paul and Skolnick, 2003; Kugaya and Sanacora, 2005). In addition,
glutamatergic neurotransmission relies on several classes of receptors, such as ionotropic
receptors that include N-methyl-D-aspartate (NMDA-R), alpha-amino-3-hydroxyl-5 methyl-
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4-isoxazole-propionate (AMPA-R) and kainate receptors; and metabotropic receptors, such as
mGluR1-mGluR8, which are regulators of glutamatergic excitability in the postsynaptic
membrane (Citri and Malenka, 2008). Several studies have reported that activation of
synaptic NMDA-R (for increased neuronal activity) increases the activity of MAP-kinase and
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cAMP response element-binding protein (CREB) by promoting the synthesis of the brain
derived neurotrophic factor (BDNF) (Sem and Sanacora, 2008). Binding of BDNF to
tyrosine-kinase-B (TrkB) receptors facilitates synaptic delivery of AMPA-R, thereby
mediating neural plasticity and long-term potentiation (LTP), which are key mechanisms for
translating experience into enduring modification of synaptic transmission (Carvalho et al.,
2008). On the other hand, Glu binding to extra-synaptic NMDA-R has an opposite
effect;these receptors are activated when Glu levels are high, thus suppressing BDNF
a
synthesis. In this way, when there is an excess of glutamatergic stimulation, an excess of

calcium enzymatic activity is also triggered, leading to cytoskeletal degradation of dendrites,
ov
free radical production and consequent neuronal death, caused by neuronal excitotoxicity
(Zandio et al., 2002).
In recent years, several studies have shown that glutamatergic excitation and NMDA
receptor signalling play a central role in the pathophysiology of depression (Ghasemi et al.,
2014). In this context, evidence from post-mortem studies has shown an increase in cortical
N
glutamate levels in patients with depression (Hashimoto et al., 2007). Auer et al. (2000)
demonstrated a significantly higher level of Glu/GABA in the occipital cortex of depressed
individuals compared to controls (Table 1). Furthermore, Sanacora et al. (2008) also observed
an increase in cortical glutamate in patients with depression, coupled with a decrease in

GABA levels and a change in the AMPA and NMDA receptors in certain brain areas. Several
studies have reported that major depression is associated with reduced plasma GABA levels
(Kalueff et al., 2007), as well as decreased GABA neurons in the lateral orbital prefrontal
cortex (Rajkowska et al., 2007). However, in open label studies using lamotrigine and riluzole
c.
(both inhibitors of glutamate release), as well as in a controlled randomized trial of ketamine
(an NMDA antagonist) undertaken in treatment resistant-depressed patients, preliminary
evidence of efficacy of glutamatergic modulators in the treatment of major depression is
In
provided (Zarate et al., 2006). In addition, several preclinical and clinical studies showed that
antidepressants and electroconvulsive therapy (ECT) improve GABA deficits (Krystal et al.,
2002; Herman et al., 2003).
Recent pharmacological and genetic findings indicate that the endocannabinoid system
may be involved in the pathophysiology of depression (Hill and Gorzalka, 2005) This is
rs
supported by several pieces of evidence showing that endocannabinoids and CB1 receptors
are widely distributed in brain areas that are often related to affective disorders (Devane et al.,
1988), and the density of endocannabinoids and CB1 receptor binding sitesare decreased in a
number of brain regions in the chronic unpredictable stress (CUS) model of depression (Hill
he
et al., 2008). In addition, the expression of endocannabinoids and CB1 receptorsare regulated
by antidepressant drugs (Ballmaier et al., 2007), and the administration of inhibitors of
anandamide uptake or metabolism, as well as CB1 receptor agonists, induces antidepressant-
like effects in different animal models (Adamczyk et al., 2008). Also, the blockade of the
CB1 cannabinoid receptor (endogenous) by rimonabant, which is approved for treatment of
is
obesity and nicotine addiction, has been associated with development of depressive symptoms
(Le Foll et al., 2009). However, an inconsistency remains in pre-clinical research: both
activation, as well as the blockage of the CB1 receptor exerted antidepressant effects in the
bl
forced swimming test and in the learned helplessness test (Serra and Fratta, 2007), suggesting
that more studies are needed to elucidate the true role of the endocannabinoid system in the
pathophysiology of depression.
Stress causes the release of endogenous opioids (dynorphin) in the nucleus accumbens,
Pu
which in turn, causes anhedonia (Kasper and Hamon, 2009). In this way, several reports have
shown that the activation of the opioid system is implicated in the mechanisms underlying the
effect of antidepressants (Devoize et al., 1984; Tejedor-Real et al., 1995). Moreover, the
opioid system is also implicated in the mechanisms of action of compounds, such as
adenosine and agmatine, which exert antidepressant-like effects in the forced swimming test
(Kaster et al., 2007). Furthermore, preclinical studies indicated that antidepressant-like effects
in the forced swimming test are produced by the administration of μ- and δ-opioid receptor
a
agonists (Broom et al., 2002; Fichna et al., 2007), and clinical studies have shown that opioid
compounds such as β-endorphin (Darko et al., 1992) and buprenorphine (Bodkin et al., 1995)
ov
present antidepressant effects. In addition, ECT, an effective alternative used to treat

depression, increases the plasma level of β–endorphin (Ghadirian et al., 1988); and in an
autoradiographic study, it was demonstrated that chronic antidepressant treatments increased
μ- or δ-opioid receptor binding site density in various brain regions (Vipoux et al., 2001).
Research over the last couple of years has provided extensive evidence that abnormal
N
monoamine neuronal function is an important underlying pathology in depression. However,

new theories have revealed that there is not only one system of neurotransmitters involved in
depression. Additionally, these studies have shown that currently available antidepressant
agents have effects on multiple neurotransmitter systems that account for their efficacy and
their side effects. By understanding the synergy among neurotransmitter systems across
different neural circuits, we can go on to develop synergic therapies that are more effective in
treating depressive symptoms and to help to minimize side effects.
c.
Table 1. Neurotransmitter and targets involved with depression
Neurotransmitter/Target Effect
↓ Levels and gene dysfunction.
In
Serotonin
Dopamine/Noradrenaline ↓ Levels.
MAO/COMT Gene mutation
GABA/Glutamate ↑ Levels and dysfunction.
NMDA receptor Antidepressant modulation
CB1 receptor Antidepressant modulation
rs
Limitations of Fluoxetine Monotherapy
he
Fluoxetine is the first of group of antidepressant agents known as selective serotonin (5-
HT) reuptake inhibitors, which is thought to be relevant to their antidepressant action
(Tatsumi et al., 1997). It was first used more than ten years ago, and soon after its
introduction it became the most prescribed agent used to treat depression in many countries.
is
Psychiatrists have described the discovery of fluoxetine as a giant step forward in the
pharmacotherapy of depression (Stokes, 1998), and it has also became a culturally
fashionable treatment, acquiring popularity in the lay news and media, with sociologists
describing it as a ‘socio-psychopharmaceutical‘ phenomenon (Slingsby, 2002). The
bl
phenomenal success of fluoxetine monotherapy raised some concerns because results from
randomized clinical trials did not clearly indicate substantial benefits (Cipriani et al., 2005).
A substantial body of evidence indicates that fluoxetine monotherapy poses potential
Pu
limitations in the treatment of depression related to its pharmacological activity, and for all
practical purposes, the reuptake pump blockade does not seem be overcome by increases in
medication dosage alone (Cusack et al., 1994; Tatsumi et al., 1997; Rogóz, 2013). The
pharmacological limitations of fluoxetine include its inability to act as a pharmacological
agonist and consequently control monoamine functioning in the central nervous system,
because it cannot directly activate postsynaptic receptors and is dependent almost entirely on
intact presynaptic serotonergic functioning (Bobo and Shelton, 2009). Other important points
a
are that fluoxetine does not directly enhance noradrenergic or dopaminergic

neurotransmission, presenting a negligible effect on norepinephrine and dopamine reuptake,
ov
and also that fluoxetine and other selective serotonin reuptake inhibitors may not be able to
fully optimize central monoaminergic functioning as stand-alone therapies (DiGiovanni et al.,
1999; DiMatteo et al., 1998; DiMatteo et al., 1999). The problem of antidepressant-resistant
depression has been the subject of a number of thorough studies, with no apparent therapeutic
advances. Based on clinical evidence, atypical antipsychotics may be successful adjunctive
N
medical agents for patients who fail to respond to pharmacological monotherapy with
antidepressants (Shelton and Papakostas, 2008; Wright et al., 2013). Atypical antipsychotics
(e.g., olanzapine, risperidone and clozapine) have similarly high affinities for a number of
receptor subtypes (Richelson and Souder, 2000). These drugs potently bind to more 5-HT2A
serotonin receptors than to dopamine D2 receptor, and they also bind to α1-adrenergic, α2-
adrenergic and histamine H1 receptors (Kikuchi et al., 1995; Richelson and Souder, 2000;
Jordan et al., 2002). It may be that the augmentation resulting from olanzapine plus fluoxetine
is mediated by either 5-HT2C or a1-adrenergic receptors. In addition, in combination with
c.
fluoxetine, the 5-HT2C antagonistic property of olanzapine could facilitate a persistent and
robust effect on extracellular levels of dopamine and noradrenaline in the rat prefrontal cortex
(Millan et al., 1998). It is also possible that receptors other than 5-HT2C or α1- adrenergic
In
receptors are involved in the augmentation, as olanzapine has such a promiscuous receptor
binding profile. Different trials were performed to evaluate the efficacy of olanzapine plus
fluoxetine compared with fluoxetine monotherapy in patients with treatment-resistant
depression (Shelton et al., 2001; Thase et al., 2007). Two of those trials demonstrated a
significant benefit in using olanzapine plus fluoxetine when compared with fluoxetine only
rs
for the primary outcome of the mean Montgomery-Asberg Depression Rating Scale, with the
total score change as the endpoint (Shelton et al., 2001; Thase et al., 2007). Significantly
better remission and response rates for olanzapine plus fluoxetine therapy were observed in
only one trial (Thase et al., 2007). The amelioration of treatment-resistant depression may
he
require an increase in two or more of the monoamines, and thus, a combination therapy may
have an additive and more powerful therapeutic effect compared to therapy involving an
increase of only one neurotransmitter alone.
is
Augmentation of FLX Response with Classic Drugs: Focus on the
Monoaminergic System
bl
Normally, SSRIs and other newer antidepressant drugs with a greater safety margin
constitute first-line medications for moderate-to-severe depression (Trivedi et al., 2006).
However, nonresponse to medication requires a treatment change, and one of the alternatives
is switching to an antidepressant from a different pharmacologic class to minimize
Pu
polypharmacy and reduce the risk of adverse drug interactions and side effects (Maoz, 2007).
In this line for example, the OLZ-FLX combination is FDA-approved for the acute treatment
of bipolar depression (McIntyre et al., 2013). Moreover, in a controlled study with treatment-
resistant depression it was shown that the combination of FLX and OLZ was associated with
significantly greater and faster improvements than was possible with either drug alone
(Shelton and Papakostas, 2008) It is believed that the antipsychotics have a unique
mechanism of action in depression, acting as 5-HT2A antagonists to facilitate noradrenergic
a
neurotransmission via complex cascade effects possibly mediated by GABA interneurons

(Santana et al., 2004). Nevertheless, there are other studies relating to the molecular effects of
ov
FLX plus OLZ treatment. In fact, it was discovered that treatment with FLX and OLZ alone
or in combination increased the Akt quinase, CREB, BDNF, and Bcl-2 (anti-apoptotic
protein) levels in the prefrontal cortex, hippocampus and striatum of rats, and the combination
of FLX and OLZ at high doses was associated with a greater increase in the levels of Akt in
the prefrontal cortex (Réus et al., 2012). It was also associated with an increase in
N
neurotrophin-3 (NT-3) in the prefrontal cortex (Agostinho et al., 2011a), suggesting that
treatment with FLX and OLZ alone or in combination exerts neuroprotective effects, and that
intracellular survival pathways could be involved in the therapeutic effects of combining
antipsychotic and antidepressant drugs in mood disorders (Table 2). The effects of combining
OLZ and FLX have been related to energy metabolism (Agostinho et al., 2009; 2011b;
2011c). In a study evaluating the effects of FLX plus OLZ on the monoaminergic system,
Koch et al. (2002) demonstrated that the OLZ/FLX combination increased catecholamines
(DA and NE) in the prefrontal cortex, and 5-HT concentrations in the hypothalamus to a
c.
significantly greater extent than either drug alone.
Experimental studies have also evaluated the effects of FLX with other drugs. For
example, it was related that the concomitant administration of FLU and risperidone (an
In
atypical antipsychotic drug) in rodents produced antidepressive-like effects in the forced
swimming test, which is one of the tests most commonly used by researchers to investigate
new antidepressant drugs (Abelaira et al., 2013), whereas the drugs were ineffective if
administered separately (Rogóz et al., 2012; Roman et al., 2013). On the other hand, the
administration of WAY 100635 (a 5-HT1A receptor antagonist) inhibited the antidepressant-
rs
like effect induced by the co-administration of fluoxetine or mirtazapine and risperidone,
suggesting that the 5-HT1A receptors may play some role in the antidepressant effects
exerted by FLX/risperidone administration. In fact, risperidone and FLX increased the
extracellular levels of cortical DA, 5-HT and NE, and co-treatment of both drugs was more
he
effective in increasing DA and 5-HT, release than the administration of each of the drugs
alone, but co-treatment did not increase the release of NE (Kamińska et al., 2013). In the
same sense,investigations were undertaken into the effects of FLX in combination with
pramipexole, a potent agonist of the D2 receptor and approved for Parkinson disease
treatment. This research showed that the co-administration of pramipexole and FLX may
is
induce a more pronounced antidepressive activity than treatment with pramipexole alone, and
that in addition to other mechanisms (Table 2), dopamine D2/3 and 5-HT1A receptors may
contribute to the antidepressant-like activity of pramipexole and fluoxetine in the forced
bl
swimming test in rats (Rogóz and Skuza, 2006).
The effects of methylphenidate, which is a psychostimulant that is widely used to treat
the symptoms of attention-deficit hyperactivity disorder (ADHD), have alsobeen examined in
combination with FLX (Table 2).
Pu
Table 2. Mechanism involved with synergistic effects between monoaminergic agents

and fluoxetine from animal studies
Agent Action
Olanzapine ↑ Survival pathway signaling and monoaminergic
neurotransmission. Modulates energy metabolism.
a
Improves depressive symptoms.

Risperidone ↑ Antidepressant-like behavior and DA and 5-HT levels.
ov
Pramipexole ↑ Antidepressant-like behavior.

Methylphenidate ↑ Neuropeptide and corticosterone gene regulation
Modafinil ↑ 5-HT levels.
The combination is used to accelerate treatment (Lavretsky et al., 2003), or to treat sexual
N
dysfunction related to SSRIs (Csoka et al., 2008). The studies related that FLX potentiates
methylphenidate-induced gene regulation in the striatum (Steiner, 2010; Van Waes et al.,
2012) and expression of substance P and dynorphin, markers for direct pathway neurons,
suggesting that these changes in gene regulation with the addition of FLX may enhance the
addiction liability of methylphenidate (Van Waes et al., 2012). Modanil, a psychostimulant
that affects dopaminergic neurotransmission in the nucleus accumbens (Duteil et al., 1990),
was also studied in combination with FLX. In fact, modafinil at a very low dose (3 mg/kg),
which by itself is ineffective, enhanced the fluoxetine (5 mg/kg)-induced increases of 5-HT
c.
levels in the dorsal raphe-cortical system (Table 2) (Ferraro et al., 2005). Based on pre and
clinically evaluated evidence, monoaminergic modulators may act as successful adjunctive
agents, which fail to respond to pharmacological monotherapy with FLX. However, more
In
studies are necessary to clarify the molecular mechanism by which the antidepressant effects
of FLX are potentiated mainly to dopaminergic drugs.
Augmentation of FLX Response with Other Agents: Focus

on the Glutamatergic System
rs
In clinical practice, it is common to switch to a different drug that targets
dopamine,norepinephrine, or a combination of serotonin,norepinephrine and dopamine after
he
the failure of an SSRI (Maoz, 2007). Hence, in order to improve the therapy, a combination of
antidepressants and an agent which can enhance its effects have been used in clinical practice
(Rogóz et al., 2007). Indeed, there are several clinical studies evaluating the efficacy of new
targetsin depression-resistant patients (Duncan and Zarate, 2013; Niciu et al., 2014;
Villaseñor et al., 2014). In this sense, much attention has been devoted to the glutamatergic
is
system and to NMDA receptor antagonists in particular. In fact, patients with depression
presented significant increasesin serum levels of glutamate, compared with healthy controls
(Kim et al., 1982; Mitani et al., 2006), and several basic and clinical studies have
bl
demonstrated that NMDA receptor antagonists, such as ketamine, memantine, amantadine
and others, display antidepressant effects (Berman et al., 2000; Ferguson and Shingleton,
2007; Garcia et al., 2008a, 2008b, 2009; Réus et al., 2014; Roman et al., 2009). In addition,
some studies have shown that classic antidepressantalsoact in the glutamatergic system, for
Pu
example, chronic treatment with antidepressant drugs, such as FLX, desipramine and
reboxetine significantly reduced the depolarization-evoked release of glutamate (Bonanno et
al., 2005), and a series of other antidepressants regulate metabotropic receptors (Musazzi et
al., 2013). Poleszakt et al. (2011) related that the antidepressant-like effects of imipramine,
fluoxetine and reboxetine were abolished by D-serine, a full agonist of glycine/NMDA
receptors, suggesting that glycine/NMDA receptor antagonists enhance the antidepressant-
like action of serotonin. In preclinical studies, much evidence has pointed to the synergic
a
effect using FLX in combination with glutamatergic modulator drugs (Table 3). Rogoz et al.
(2002) demonstrated that the co-administration of amantadine, memantine or neramexane and
ov
FLX at doses ineffective alone, enhanced antidepressant-like activity in rats subjected to the
forced swimming test. It is important to note that selective inhibitors of 5-HT reuptake do not
usually show antidepressant-like activity in the forced swimming test in rats (Porsolt et al.,
1978). Co-treatment with FLX and amantadine over 14 days induced a substantial increase in
BDNF gene expression in the cerebral cortex, and inhibits the behavioural syndrome induced
N
by 5-HT1A and 5-HT2 receptor agonists, compared to treatment with either drug given alone
(Table 3) (Rogóz et al., 2008). Additionally, Rogóz and Skuza (2009) used the same protocols
to show that co-treatment enhanced the effects evoked by the psychostimulants amphetamine
or quinpirole (a dopamine D2/3 agonist), when compared to treatment with either drug alone,
and that repeated co-treatment (over 7 days) with FLX and amantadine potently inhibited
more 5-HT1A neurotransmission in behavioural tests than FLX alone (but not 5-HT2). The
underlying mechanisms by which NMDA antagonists potentiate FLX antidepressant effects
remain unclear, but these effects may be involved, at least in part, to alterations in
c.
monoaminergic transmission or effects on the synthesis of monoaminergic neurotransmitters.
In fact, a previous in vivo study showed that when the antagonists of the NMDA receptor,
amantadine and budipinewere given acutely with the antidepressants reboxetine, paroxetine
In
or clomipramine, it resulted in elevated extracellular 5-HT concentrations in the frontal
cortices of freely moving rats. In addition, repeated co-treatment with amantadine or budipine
and reboxetine, paroxetine or clomipramine facilitated and potentiated these effects. On the
other hand, neither the acute nor chronic administration (7, 14 and 21 days) of amantadine or
budipine significantly changed extracellular 5-HT concentrations in the frontal cortices of
rs
freely moving rats (Owen and Whitton, 2005). FLX combined with LY379268, an mGlu2/3
agonist, showed a strong synergism in enhancing cell proliferation and inhibiting cAMP
formation (Matrisciano et al., 2008). These studies support the idea that the combination of
FLX with glutamatergic agents may be helpful in the treatment of depression. In addition, the
he
SSRI doses could be reduced and thereby reducing the side effects induced by such drugs.
Clinical and experimental studies indicate that stress and depression are associated with
the up-regulation of the immune system, including increased production of pro-inflammatory
cytokines. Increased levels of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6,
IL-8, IL-12, interferon (IFN-γ) and tumour necrosis factor-α (TNF- α) have already been
is
related in patients with depression (Schiepers et al., 2005; Raison et al., 2006; Mossner et al.,
2007). Interestingly, Roman et al. (2009) demonstrated that the co-administration of
amantadine and FLX decreased cellular adherence, a property of pro-inflammatory
bl
macrophages. As macrophages and their products (pro-inflammatory cytokines) are involved
with depressive states (Raison et al., 2006; Miller et al., 2009), it has been suggested that the
synergistic effects induced by amantadine plus FLX in reducing the macrophages activity
might be an important mechanism of successful antidepressant treatment. Indeed, an in vitro
Pu
study with human blood demonstrated that treatment with amantadine in combination with
FLX had a stronger immunomodulatory effect, reducing IFN-γ and TNF-α more than
amantadine alone (Kubera et al., 2000). Furthermore, co-treatment with amantadine and FLX
normalized the stress-induced immunoendocrine changes (Rogóz et al., 2009), suggesting that
the combined therapeutic effectiveness of antidepressants plus new agents may be associated
in not only the central nervous system, but also in the immune and endocrine systems closely
related to it. Still, FLX treatment synergized the antidepressant-like actions of intra-nucleus
a
accumbens infusions of minocycline, a tetracycline antibiotic drug, when administered to

male Wistar rats (Molina-Hernández et al., 2008).
ov
Some studies have shown that minocycline acts on both the monoaminergic and
glutamatergic systems. In fact, it decreases glutamate release and transmission and attenuates
decreases in 5-HT, DA, 5-HTT and DAT associated with psychotomimetic substance
administration. Beyond this, minocycline attenuates a decrease in the levels of dopamine,
norepinephrine and serotonin associated with 3-nitropropionic acid treatment (to review see:
N
Soczynska et al., 2012). Thus, these studies may provide some insights into how to optimally
use combinations of the amine-based antidepressant drugs with glutamatergic and/or neuro-
immune modulators, and these strategies may be of particular importance in the case of drug-
resistant patients.
Table 3. Mechanism involved with synergistic effects between glutamatergic agents

and fluoxetine
c.
Agent Action
D-serine ↓ Antidepressant-like behavior.
↑ Antidepressant-like behavior and BDNF levels.
In
Amantadine
↓ Inflammation.
Memantine ↑ Antidepressant-like behavior.
Neramexane ↑ Antidepressant-like behavior.
LY379268 ↑ Cell proliferation.
Minocycline ↑ Antidepressant-like behavior.
rs
Conclusion
he
As major depression is a heterogeneous disorder and a significant number of patients do
not respond to any single antidepressant therapy, a respectable alternative is use augmentation
or combinations of antidepressants with different pharmacological profiles. FLX has been
used in experimental and clinical studies in combination with other agents, and the results
is
have been satisfactory. Interestingly, these agents modulate different sides of the
serotoninergic system, as dopaminergic, glutamatergic and immune, which are pointed as
having important roles in the pathophysiology of depression. The FLX augmentation strategy
bl
is able to improve neurotransmitter transmission, which in turn may regulate intracellular
modulation and gene expression. In this sense, the understanding of the brain circuits
involved in the effects of FLX combined with other drugs may be of particular importance in
the case of drug-resistant patients, and could suggest a method of obtaining significant
Pu
antidepressive actions whilst limiting side effects.
References
Abelaira, H.M., Réus, G.Z., Quevedo, J., (2013). Animal models as tools to study the
a
pathophysiology of depression. Rev. Bras. Psiquiatr. 35:112-20.

Adamczyk, P., Golda, A., McCreary, A.C., Filip, M., Przegalinski, E., (2008). Activation of
endocannabinoid transmission induces antidepressant-like effects in rats. J. Physiol.
ov
Pharmacol.59-217-228.
Agostinho, F.R., Réus, G.Z., Stringari, R.B., Ribeiro, K.F., Pfaffenseller, B., Stertz, L.,
Panizzutti, B.S., Kapczinski, F., Quevedo, J., (2011a). Olanzapine plus fluoxetine
treatment increases Nt-3 protein levels in the rat prefrontal cortex. Neurosci. Lett. 497:99-
103.
N
Agostinho, F.R., Réus, G.Z., Stringari, R.B., Ribeiro, K.F., Ferreira, G.K., Jeremias, I.C.,
Scaini, G., Rezin, G.T., Streck, E.L., Quevedo, J., (2011b). Olanzapine plus fluoxetine
treatment alters mitochondrial respiratory chain activity in the rat brain. Acta
Neuropsychiatr.23:282-91.
Agostinho, F.R., Réus, G.Z., Stringari, R.B., Ribeiro, K.F., Ferraro, A.K., Benedet, J., Rochi,
N., Scaini, G., Streck, E.L., Quevedo, J., (2011c). Treatment with olanzapine, fluoxetine
and olanzapine/fluoxetine alters citrate synthase activity in rat brain. Neurosci.
c.
Lett.487:278-81.
Agostinho, F.R., Scaini, G., Ferreira, G.K., Jeremias, I.C., Réus, G.Z., Rezin, G.T., Castro,
A.A., Zugno, A.I., Quevedo, J., Streck, E.L., (2009). Effects of olanzapine, fluoxetine
In
and olanzapine/fluoxetine on creatine kinase activity in rat brain. Brain Res. Bull. 80:337-
40.
Auer, D.P., Pütz, B., Kraft, E., Lipinski, B., Schill, J., Holsboer, F., (2000). Reduced
glutamate in the anterior cingulate cortex in depression: an in vivo proton magnetic
resonance spectroscopy study. Biol. Psychiatry 47, 305–313.
rs
Ballmaier, M., Bortolato, M., Rizzetti, C., Zoli, M., Gessa, G., Heinz, A., Spano, P., (2007).
Cannabinoid receptor antagonists counteract sensorimotor gating deficits in the
phencyclidine model of psychosis. Neuropsychopharmacology 32, 2098-2107.
Berman, R.M., Cappiello, A., Anand, A., Oren, D.A., Heninger, G.R., Charney, D.S., Krystal,
he
J.H., (2000). Antidepressant effects of ketamine in depressed patients. Biol. Psychiatry
47: 351-4.
Berton, O., Nestler, E.J., (2006). New approaches to antidepressant drug discovery: beyond
monoamines. Nat. Rev. Neurosci. 7: 137-51.
Bobo, W.V., Shelton, R.C., (2009) Olanzapine and fluoxetine combination therapy for
is
treatment-resistant depression: review of efficacy, safety, and study design issues.
Neuropsychiatr. Dis. Treat. 5:369-383.
Bodkin, J.A., Zornberg, G.L., Lukas, S.E., Cole, J.O., (1995). Buprenorphin treatment of
bl
refractory depression. J. Clin. Psychopharmacol.15, 49–57.
Bonanno, G., Giambelli, R., Raiteri, L., Tiraboschi, E., Zappettini, S., Musazzi, L., Raiteri,
M., Racagni, G., Popoli, M., (2005). Chronic antidepressants reduce depolarization
evoked glutamate release and protein interactions favoring formation of SNARE complex
Pu
in hippocampus. J. Neurosci. 25:3270–9.

Broom, D.C., Jutkiewicz, E.M., Folk, J.E., Traynor, J.R., Rice, K.C., Woods, J.H., (2002).
Nonpeptidic delta-opioid receptor agonists reduce immobility in the forced swim assay in
rats. Neuropsychopharmacology 26, 744–755.
Bunney, W.E. Jr., Davis, J.M., (1965). Norepinephrine in depressive reactions.A review.
Arch. Gen. Psychiatry 13, 483-494.
Cannon, D.M., Klaver, J.M., Peck S.A., Rallis- Voak, D., Erickson, K., Drevets, W.C.,
a
(2009). Dopamine Type-1 Receptor Binding in Major Depressive Disorder Assessed

Using Positron Emission Tomography and [(11)C]NNC- 112.
ov

Carvalho, A.F., Cavalcante, J.L., Castelo, M.S., Lima, M.C., (2007). Augmentation strategies
for treatment-resistant depression: a literature review. J. Clin. Pharm. Ther. 32:415-28.
Carvalho, A.L., Caldeira, M.V., Santos S.D., Duarte C.B., (2008). Role of the brain-derived
neurotrophic factor at glutamatergic synapses. Br. J. Pharmacol. 153, 310-324.
N
Chiuccariello, L., Houle, S., Miler, L., Cooke, R.G., Rusjan, P.M., Rajkowska, G., Levitan,
R.D., Kish, S.J., Kolla, N.J., Ou, X., Wilson, A.A., Meyer, J.H., (2014). Elevated
monoamine oxidase a binding during major depressive episodes is associated with greater
severity and reversed neurovegetative symptoms. Neuropsychopharmacology 39, 973-

980.
Cipriani, A., Brambilla, P., Furukawa, T., Geddes, J., Gregis, M., Hotopf, M., Malvini, L.,
Barbui, C., (2005) Fluoxetine versus other types of pharmacotherapy for depression.
Cochrane Database Syst. Rev. 19:CD004185.
c.
Citri, A., Malenka, R.C., (2008). Synaptic plasticity: multiple forms, functions, and
mechanisms. Neuropsychopharmacology 33, 18-41.
Conwell, Y., Heisel, M.J., (2012). The elderly, in The American Psychiatric Publishing
In
Textbook of Suicide Assessment and Management.Edited by Simon RI, Hales RE. Second
Edition. Arlington, VA, American Psychiatric Publishing 367-388.
Csoka, A., Bahrick, A., Mehtonen, O.P., (2008). Persistent sexual dysfunction after
discontinuation of selective serotonin reuptake inhibitors. J. Sex. Med. 5:227–33.
Cusack, B., Nelson, A., Richelson, E., (1994) Binding of antidepressants to human brain
rs
receptors: focus on newer generation compounds. Psychopharmacology (Berl) 114:559-
565.
Darko, D.F., Rich, S.C., Gilin, J.C., Golshan, S., (1992). Association of endorphin with
specific clinical symptoms of depression. Am. J. Psychiatry 149, 1162–1167.
he
Delgado, P.L., (2000). Depression: the case for a monoamine deficiency. J. Clin. Psychiatry
6, 7-11.
Delgado, P.L., (2006). Depression: the case for a monoamine deficiency. J. Clin. Psychiatry
61, 7-11.
Devane, W.A., Dysarz, F.A., Johnson, M.R., Melvin, L.S., Howlett, A.C., (1988).
is
Determination and characterization of a cannabinoid receptor in rat brain. Mol.
Pharmacol. 34, 605-613.
Devoize, J.L., Rigal, F., Eschalier, A., Trolese, J.F., Renoux, M., (1984). Influence of
bl
naloxone on antidepressant drug effects in the forced swimming test in mice.
Psychopharmacology 84,71–75.
DiGiovanni, G., DeDeurwaerdere, P., DiMascio, M., DiMatteo, V., Esposito, E., Spampinato,
U., (1999). Selective blockade of serotonin-2C/2B receptors enhances mesolimbic and
Pu
mesostriatal dopaminergic function: a combined in vivo electrophysiological and

microdialysis study. Neuroscience 91 (2):587–597.
DiMatteo, V., DiGiovanni, G., DiMascio, M., Esposito, E., (1998). Selective blockade of
serotonin2C/2B receptors enhances dopamine release in the rat nucleus accumbens.
Neuropharmacology 37: 265–272.
DiMatteo, V., DiGiovanni, G., DiMascio, M., Esposito, E., (1999). SB 242084, a selective
serotonin2C receptor antagonist, increases dopaminergic transmission in the mesolimbic
a
system. Neuropharmacology 38: 1195–1205.

Drevets, W.C., Thase, M.E., Moses-Kolko, E.L., Price, J., Frank, E., Kupfer, D.J., Mathis, C.,
ov
(2007). Serotonin-1A receptor imaging in recurrent depression: replication and literature

review. Nucl. Med. Biol. 34, 865–877.
Duncan, W.C. Jr., Zarate, C.A. Jr., (2013). Ketamine, sleep, and depression: current status
and new questions. Curr. Psychiatry Rep. 15:394.
Duteil E., (1990). Central alpha 1-adrenergic stimulation in relation to the behaviour
N
stimulating effect of modafinil; studies with experimental animals. Eur. J. Pharmacol.

180:49-58.
Elhwuegi, A.S., (2004). Central monoamines and their role in major depression. Prog.
Neuropsychopharmacol. Biol. Psychiatry 28: 435–51.
Fava, M., Davidson, K.G., (1996). Definition and epidemiology of treatment-resistant
depression. Psychiatr. Clin. North Am. 19:179-200.
Ferguson, J.M., Shingleton, R.N., (2007). An open-label, flexible-dose study of memantine in
c.
major depressive disorder. Clin. Neuropharmacol. 30: 136-44.
Ferraro, L., Fuxe, K., Agnati, L., Tanganelli, S., Tomasini, M.C., Antonelli, T., (2005).
Modafinil enhances the increase of extracellular serotonin levels induced by the
In
antidepressant drugs fluoxetineand imipramine: a dual probe microdialysis study in
awake rat. Synapse. 55:230-41.
Fichna, J., Janecka, A., Piestrzeniewicz, M., Costentin, J., do Rego, J.C., (2007).
Antidepressantlike effect of endomorphin-1 and endomorphin-2 in mice.
Neuropsychopharmacology 32, 813–821.
rs
Fredman, S.J., Fava, M., Kienke, A.S., White, C.N., Nierenberg, A.A., Rosenbaum, J.F.,
(2000). Partial response, non response, and relapse with selective serotonin reuptake
inhibitors in major depression: a survey of current ‗‗next-step‘‘ practices. J. Clin.
Psychiatry 61: 403–8.
he
Garcia, L.B., Comim, C.M., Valvassori, S.S., Réus, G.Z., Andreazza, A.C., Stertz, L., Fries,
G.R., Gavioli, E.C., Kapczinski, F., Quevedo, J., (2008a). Chronic administration of
ketamine elicits antidepressant-like effects in rats without affecting hippocampal brain-
derived neurotrophic factor protein levels. Basic Clin. Pharmacol. Toxicol, 103: 502–06.
Garcia, L.B., Comim, C.M., Valvassori, S.S., Réus, G.Z., Barbosa, L.M., Andreazza, A.C.,
is
Stertz, L., Fries, G.R., Gavioli, E.C., Kapczinski, F., Quevedo, J., (2008b). Acute
administration of ketamine induces antidepressant-like effects in the forced swimming
test and increases BDNF levels in the rat hippocampus. Prog. Neuropsychopharmacol.
bl
Biol. Psychiatry 32: 140–144.
Garcia, L.S., Comim, C.M., Valvassori, S.S., Réus, G.Z., Stertz, L., Kapczinski, F., Gavioli,
E.C., Quevedo, J., (2009). Ketamine treatment reverses behavioral and physiological
alterations induced by chronic mild stress in rats. Prog. Neuropsychopharmacol. Biol.
Pu
Psychiatry 33: 450-55.

Ghadirian, A.M., Gianoulakis, C.H., Nair, N.P., (1988). The effect of electroconvulsive
therapy on endorphins in depression. Biol. Psychiatry 23, 459.
Ghasemi, M., Kazemi, M.H., Yoosefi, A., Ghasemi, A., Paragomi, P., Amini, H., Afzali,
M.H., (2014). Rapid antidepressant effects of repeated doses of ketamine compared with
electroconvulsive therapy in hospitalized patients with major depressive disorder.
Psychiatry Res. 215, 355-361.
a
Hamon, M., Blier, P., (2013). Monoamine neurocircuitry in depression and strategies for new
treatments. Prog. Neuropsychopharmacol. Biol. Psychiatry 45, 54-63.
ov
Hashimoto, K., Sawa, A., Iyo, M., (2007). Increased levels of glutamate in brains from
patients with mood disorders. Biol. Psychiatry 62, 1310–1316.
Herman, J.P., Renda, A., Bodie, B., (2003). Norepinephrinegamma- aminobutyric acid
(GABA) interaction in limbic stress circuits: effects of reboxetine on GABAergic
neurons. Biol. Psychiatry 53, 166-174.
N
Hill, M.N., Gorzalka, B.B., (2005). Is there a role for the endocannabinoid system in the
etiology and treatment of melancholic depression? Behav. Pharmacol. 16, 333-352.
Hill, M.N., Ho, W.S., Hillard, C.J., Gorzalka, B.B., (2008). Differential effects of the
antidepressants tranylcypromine and fluoxetine on limbic cannabinoid receptor binding
and endocannabinoid contents. J. Neural. Transm. 115, 1673-1679.
Jordan, S., Koprivica, V., Chen, R., Tottori, K., Kikuchi, T., Altar, A., (2002) The
antipsychotic aripiprazole is a potent, partial agonist at the human 5-HT1A receptor. Eur.
c.
J. Pharmacol. 441, 137–140.
Kalueff, A.V., Nutt D.J., (2007). Role of GABA in anxiety and depression. Depress. Anxiety
24, 495-517.
Kamińska, K., Gołembiowska, K., Rogóż, Z., (2013). Effect of risperidone on the fluoxetine-
In
induced changes in extracellular dopamine, serotonin and noradrenaline in the rat frontal
cortex. Pharmacol. Rep. 65:1144-51.
Kasper, S., Hamon, M., (2009). Beyond the monoaminergic hypothesis: agomelatine, a new
antidepressant with an innovative mechanism of action. World J. Biol. Psychiatry 10,
rs
117-126.
Kaster, M.P., Budni, J., Santos, A.R.S., Rodrigues, A.L.S., (2007). Pharmacological evidence
for the involvement of the opioid system in the antidepressant-like effect of adenosine in
the mouse forced swimming test. Eur. J. Pharmacol. 576, 91–98.
he
Kessler, R.C., Chiu, W.T., Demler, O., Merikangas, K.R., Walters, E.E., (2005). Prevalence,
severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity
Survey Replication. Arch. Gen. Psychiatry 62: 617-27.
Kikuchi, T., Tottori, K., Uwahodo, Y., Hirose, T., Miwa, T., Oshiro, Y., Morita, S., (1995). 7-
(4-[4-(2,3-Dichlorophenyl)-piperazinyl]butyloxy)-3,4-dihydro 2(1H)quinolinone (OPC-
is
14597), a new putative antipsychotic drug with both presynaptic dopamine autoreceptor
agonistic activity and postsynaptic D2 receptor antagonistic activity. J. Pharmacol. Exp.
Ther. 274:329–336.
bl
Kim, J.S., Schmid-Burgk, W., Claus, D., Kornhuber, H.H., (1982). Increased serum
glutamate in depressed patients. Arch. Psychiatr. Nervenkr. 23: 299–304.
Koch, S., Perry, K.W., Nelson, D.L., Conway, R.G., Threlkeld, P.G., Bymaster, F.P., (2002)
R-fluoxetine increases extracellular DA, NE, as well as 5-HT in rat prefrontal cortex and
Pu
hypothalamus: an in vivo microdialysis and receptor binding study.

Neuropsychopharmacology 27:949-59.
Krystal, J.H., Sanacora, G., Blumberg, H., Anand, A., Charney, D.S., Marek, G., Epperson,
C.N., Goddard A., Mason G.F., (2002). Glutamate and GABA systems as targets for
novel antidepressant and mood-stabilizing treatments. Mol. Psychiatry 7, 71-80.
Kubera, M., Maes, M., Budziszewska, B., Basta-Kaim, A., Leśkiewicz, M., Grygier, B.,
Rogóz, Z., Lasoń, W., (2009). Inhibitory effects of amantadine on the production of
a
proinflammatory cytokines by stimulated in vitro human blood. Pharmacol. Rep.

61:1105-12.
ov
Kugaya, A., Sanacora, G., (2005). Beyond monoamines: glutamatergic function in mood
disorders. CNS Spectr. 10, 808-819.
Lavretsky, H., Kim, M.D., Kumar, A., Reynolds, C. F., (2003). Combined treatment with
methylphenidate and citalopram for accelerated response in the elderly: an open trial. J.
Clin. Psychiatry 64:1410–4.
N
Le Foll, B., Gorelick, D.A., Goldberg, S.R., (2009). The future of endocannabinoid-oriented
clinical research after CB1 antagonists. Psychopharmacology (Berl) 205:171-174.
Maletic, V., Raison, C.L., (2009). Neurobiology of depression, fibromyalgia and neuropathic
pain. Front. Biosci. 14, 5291-5338.
Maoz, H., (2007). Failure of first SSRI for depression--what is the next step? Isr. J.
Psychiatry Relat. Sci. 44:327-9.
Matrisciano, F., Zusso, M., Panaccione, I., Turriziani, B., Caruso, A., Iacovelli, L., Noviello,
c.
L., Togna, G., Melchiorri, D., Debetto, P., Tatarelli, R., Battaglia, G., Nicoletti, F., Giusti,
P., Girardi, P., (2008). Synergism between fluoxetine and the mGlu2/3 receptor agonist,
LY379268, in an in vitro model for antidepressantdrug-induced neurogenesis.
In
Neuropharmacology 54:428-37.
McIntyre, R.S., Cha, D.S., Kim, R.D., Mansur, R.B., (2013). polar depression. CNS Spectr.
1:4-20.
Meyer, J.H., Ginovart, N., Boovariwala, A., Sagrati, S., Hussey, D., Garcia, A., Young, T.,
Praschak-Rieder N., Wilson A.A., Houle, S., (2006). Elevated monoamine oxidase a
rs
levels in the brain: an explanation for the monoamine imbalance of major depression.
Arch. Gen. Psychiatry 63, 1209-1216.
Millan, M.J., Dekeyne, A., Gobert, A., (1998). Serotonin (5-HT)2C receptors tonically inhibit
dopamine (DA) and noradrenaline (NA), but not 5-HT, release in the frontal cortex in
he
vivo. Neuropharmacology 37: 953–955.
Miller, A.H., Maletic, V., Raison, C.L., (2009). Inflammation and its discontents: the role of
cytokines in the pathophysiology of major depression. Biol. Psychiatry 65:732–41.
Mischoulon, D., Nierenberg, A.A., Kizilbash, L.,Rosenbaum, J.F., Fava, M., (2000).
Strategies for managing depression refractory to selective serotonin reuptake inhibitor
is
treatment: a survey of clinicians. Can. J. Psychiatry 45:476–81.
Mitani, H., Shirayama, Y., Yamada, T., Maeda, K., Ashby, C.R. Jr., Kawahara, R., (2006).
Correlation between plasma levels of glutamate, alanine and serine with severity of
bl
depression. Prog. Neuropsychopharmacol. Biol. Psychiatry 30:1155-8.
Molina-Hernández, M., Téllez-Alcántara, N.P., Pérez-García, J., Olivera-Lopez, J.I.
Jaramillo-Jaimes, M.T., (2008). Desipramine or glutamate antagonists synergized the
antidepressant-like actions of intra-nucleus accumbens infusions of minocycline in male
Pu
Wistar rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 32:1660–6.

Mossner, R., Mikova, O., Koutsilieri, E., Saoud, M., Ehlis, A.C., Müller, N., Fallgatter, A.J.,
Riederer, P., (2007). Consensus paper of the WFSBP Task Force on Biological Markers:
biological markers in depression. World. J. Biol. Psychiatry 8: 141–74.
Musazzi, L., Treccani, G., Mallei, A., Popoli, M., (2013). The action of antidepressants on the
glutamate system: regulation of glutamate release and glutamate receptors. Biol.
Psychiatry 73: 1180-8.
a
Nemeroff, C.B., Owens, M.J., (2002). Treatment of mood disorders. Nat. Neurosci, 5: 1068-
70.
ov
Niciu, M.J., Henter, I.D., Luckenbaugh, D.A., Zarate, C.A. Jr., Charney, D.S., (2014).
Glutamate receptor antagonists as fast-acting therapeutic alternatives for the treatment of
depression: and other compounds. Annu. Rev. Pharmacol. Toxicol. 54:119-39.
Owen, J.C.E., Whitton, P.S., (2005). Effect of amantadine and budipine on antidepressant
drug evoked changes in extracellular 5-HT in the frontal cortex of freely moving rats. Br.
N
J. Pharmacol. 145: 587–92.

Paul, I.A., Skolnick, P., (2003). Glutamate and depression: clinical and preclinical studies.
Ann. N. Y. Acad. Sci. 1003, 250-272.
Poleszak, E., Wlaź, P., Szewczyk, B., Wlaź, A., Kasperek, R., Wróbel, A., Nowak, G.,
(2011).A complex interaction between glycine/NMDA receptors and
serotonergic/noradrenergic antidepressants in theforced swim test in mice. J. Neural
Transm. 118:1535-46.
Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., (1978). Behavioral despair in rats, a new
c.
model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47: 379–91.
Raison, C.L., Capuron, L., Miller, A,H., (2006). Cytokines sing the blues: Inflammation and
the pathogenesis of major depression. Trends Immunol. 27: 24–31.
In
Rajkowska, G., O'Dwyer, G., Teleki, Z., Stockmeier, C.A., Miguel-Hidalgo J.J., (2007).
GABAergic neurons immunoreactive for calcium binding proteins are reduced in the
prefrontal cortex in major depression. Neuropsychopharmacology 32, 471-482.
Réus, G.Z., Stringari, R.B., Kirsch, T.R., Fries, G.R., Kapczinski, F., Roesler, R., Quevedo,
J., (2010). Neurochemical and behavioural effects of acute and chronic memantine
rs
administration in rats: Further support for NMDA as a new pharmacological target for the
treatment of depression? Brain Res. Bull. 81: 585–9.
Réus, G.Z., Abelaira, H.M., Agostinho, F.R., Ribeiro, K.F., Vitto, M.F., Luciano, T.F.,
Souza,C.T., Quevedo, J., (2012). The administration of olanzapine and fluoxetine has
he
synergistic effects on intracellular survival athways in the rat brain. J. Psychiatr. Res.
46:1029-35.
Richelson, E., Souder, T. (2000). Binding of antipsychotic drugs to human brain receptors
focus on newer generation compounds. Life Sci. 68, 29–39.
Rogóż, Z., (2013) Combined treatment with atypical antipsychotics and antidepressants in
is
treatment-resistant depression: preclinical and clinical efficacy. Pharmacol. Rep.
65:1535-1544.
Rogóż, Z., Kabziński, M., Sadaj, W., Rachwalska, P., Gądek-Michalska, A., (2012). Effect of
bl
co-treatment with fluoxetine or mirtazapine and risperidone on the active behaviorsand
plasma corticosterone concentration in rats subjected to the forced swim test. Pharmacol.
Rep. 64:1391-9.
Rogóz, Z., Skuza, G., (2009). Effect of repeated co-treatment with fluoxetine and amantadine
Pu
on the behavioral reactivity of the centraldopamine and serotonin system in rats.

Pharmacol Rep. 61: 924-9.
Rogóz, Z., Kubera, M., Rogóz, K., Basta-Kaim, A., Budziszewska, B., (2009). Effect of
coadministration of fluoxetine and amantadine on immunoendocrine parameters in rats
subjected to a forced swimming test. Pharmacol. Rep. 61:1050-60.
Rogóz, Z., Skuza, G., Legutko, B., (2008). Repeated co-treatment with fluoxetine and
amantadine induces brain-derived neurotrophic factor gene expression in rats.
a
Pharmacol. Rep. 60: 817–26.

Rogóz, Z., Skuza, G., Daniel, W.A., Wójcikowski, J., Dudek, D., Wróbel, A., (2007).
ov
Amantadine as an additive treatment in patients suffering from drug-resistant unipolar

depression. Pharmacol. Rep. 59:778-84.
Rogóz, Z., Skuza, G., (2006). Mechanism of synergistic action following rats. Pharmacol.
Rep. 58:493-500.
Rogóz, Z., Skuza, G., Maj, J., Danysz, W., (2002). Synergistic effect of uncompetitive
N
NMDA receptor antagonists and antidepressant drugs in the forced swimming test in rats.
Neuropharmacology 42: 1024-30.
Roman, A., Kuśmierczyk, J., Klimek, E., Rogóż, Z., Nalepa, I., (2012). Effects of
coadministration of fluoxetine and risperidone on properties of peritoeal and pleural
macrophages in rats subjected to the forced swimming test. Pharmacol. Rep. 64:1368-80.
Roman, A., Rogóz, Z., Kubera, M., Nawrat, D., Nalepa, I., (2009). Concomitant
administration of fluoxetine and amantadine modulates the activity of peritoneal
c.
macrophages ofrats subjected to a forced swimming test. Pharmacol. Rep. 61: 1069-77.
Sanacora, G., Zarate, C.A., Krystal, J.H., Manji, H.K., (2008). Targeting the glutamatergic
system to develop novel, improved therapeutics for mood disorders. Nature Rev. 7, 426-
In
437.
Santana, N., Bortolozzi, A., Serrats, J., Mengod, G., Artigas, F., (2004). Expression of
serotonin1A and serotonin2A receptors in pyramidal and GABAergicneurons of the rat
prefrontal cortex. Cereb. Cortex. 14:1100-9.
Sattler, R., Rothstein, J.D. (2007). Targeting an old mechanism in a new disease – protection
rs
of glutamatergic dysfunction in depression. Biol. Psychiatry 61: 137-8.
Schiepers, O.J., Wichers, M.C., Maes, M., (2005). Cytokines and major depression. Prog.
Neuropsychopharmacol. Biol. Psychiatry 29: 201–17.
Schildkraut, J.J., (1965). The catecholamine hypothesis of affective disorders: a review of
he
supporting evidence. J. Neuropsychiatry. Clin. Neurosci. 7, 524-533.
Sem, S., Sanacora, G., (2008). Major depression: emerging therapeutics. MT. Sinai. J. Med.
75, 204-225.
Serra, G., Fratta, W., (2007). A possible role for the endocannabinoid system in the
neurobiology of depression. Clin. Pract. Epidemiol. Ment. Health. 3:25.
is
Shelton, R.C., Papakostas, G.I., (2008) Augmentation of antidepressants with atypical
antypsychotics for treatment-resistant major depressive disorder. Acta. Psychiatr. Scand.
117: 253–259.
bl
Shelton, R.C.,Tollefson, G.D.,Tohen,M., Stahl, S., Gannon, K.S.,Acobs, T.G.,Buras, F.P.,
Bymaster, W.R., Zhang, W.,Spencer, K.A.,Feldman, P.D.,Meltzer, H.Y., (2001).A novel
augmentation strategy for treating resistant major depression. Am. J. Psychiatry 158:
131–4.
Pu
Slingsby, B.T., (2002) The Prozac boom and its placebogenic counterpart- a culturally
fashioned phenomenon. Med. Sci. Monit. 8:383–393.
Soczynska, J.K., Mansur, R.B., Brietzke, E., Swardfager, W., Kennedy, S.H.,
Woldeyohannes, H.O., Powell, A.M., Manierka, M.S., McIntyre, R.S., (2012). Novel
therapeutic targets in depression: minocycline as a candidate treatment. Behav. Brain Res.
235:302-17.
Steiner, H., (2010). Psychostimulant-induced gene regulation in corticostriatal circuits, in
a
Handbook of Basal Ganglia Structure and Function (Steiner H. and Tseng K. Y., eds)
501–525. Academic Press/Elsevier, London.
ov
Stokes, P. E., (1998) Ten years of fluoxetine. Depress. Anxiety 8:1–4.

Susman, N., (2003). SNRIs versus SSRIs: mechanisms of action in treating depression and
painful physical symptoms primary care companion. J. Clin. Psychiatry 5:19–26.
Tatsumi, M., Groshan, K., Blakely, R.D., Richelson, E., (1997). Pharmacological profile of
antidepressants and related compounds at human monoamine transporters. Eur. J.
N
Pharmacol. 340:249–258.
Tejedor-Real, P., Mico, J.A., Maldonado, R., Roques, B.P., Gibert-Rahola, J., (1995).
Implication of endogenous opioid system in the learned helplessness model of
depression. Pharmacol. Biochem. Behav. 52, 145–152.
Thase, M.E., Corya, S.A., Osuntokun, O., Case, M., Henley, D.B., Sanger, T.M., Watson,
S.B., Dubé, S., (2007). A randomized, double-blind comparison olanzapine/fluoxetine
c.
combination, olanzapine, and fluoxetine in treatment-resistant major depressive disorder.
J. Clin. Psychiatry 68:224–236.
Trivedi, M.H., Hollander, E., Nutt, D., Blier, P., (2008). Clinical evidence and potential
In
neurobiological underpinnings of unresolved symptoms of depression. J. Clin. Psychiatry
69, 246-258.
Trivedi, M.H., Rush, A.J., Wisniewski, S.R., Nierenberg, A.A.,Warden, D., Ritz, L.,
Norquist, G., Howland, R.H., Lebowitz, B., McGrath, P.J., Shores-Wilson, K., Biggs,
M.,Balasubramani, G.K., Fava, M., (2006). Evaluation of outcomes with citalopram for
rs
depression using measurement based care in STAR*D: Implications for clinical practice.
Am. J. Psychiatry 163:5–7.
van Praag, H.M., Korf, J., (1971). A pilot study of some kinetic aspects of the metabolism of
5-hydroxytryptamine in depressive patients. Biol. Psychiatry 3, 105-112.
he
Van Waes, V., Carr, B., Beverley, J.A., Steiner, H., (2012). Fluoxetine potentiation of
methylphenidate-induced neuropeptide expression in the striatum occurs selectively in
direct pathway (striatonigral) neurons. J. Neurochem. 122:1054-64.
Vetulani, J., Sulser, F., (1975). Action of various antidepressant treatments reduces reactivity
of noradrenergic cyclic AMP-generating system in limbic forebrain. Nature 257, 495-
is
506.
Villaseñor, A., Ramamoorthy, A., Silva dos Santos, M., Lorenzo, M.P., Laje, G., Zarate, C.
Jr., Barbas, C., Wainer, I.W., (2014). A pilot study of plasma metabolomicpatterns from
bl
patients treated with ketamine for bipolar depression: evidence for a response-related
difference in mitochondrial networks. Br. J. Pharmacol. 171:2230-42.
Vipoux, C., Carpentier, C., Leroux-Nicollet, I., Naudon, L., Costentin, J., (2002). Differential
effects of chronic antidepressant treatments on - and -opioid receptors in rat brain. Eur. J.
Pu
Pharmacol. 443 (2002) 85–93.

Willner, P., Sampson, D., Phillips, G., Muscat, R., (1990). A matching law analysis of the
effects of dopamine receptor antagonists. Psychopharmacology (Berl). 101, 560-567.
World Health Organization, World suicide prevention day 2012. http://www.who.int
/mediacentre/events/annual/world_suicide_prevention_day/en/ Accessed 04.30.2014
Wright, B.M., Eiland III, E.H., Lorenz, R., (2013) Augmentation with atypical antipsychotics
for depression: A review of evidence-based support from the medical literature.
a
Pharmacotherapy. 33:344–359.
Zandio, M., Ferrín, M., Cuesta, M.J., (2002). Neurobiology of depression. An. Sist. Sanit.
ov
Navar. 3, 43-62.
Zarate, C.A. Jr., Singh, J.B., Carlson, P.J., Brutsche, N.E., Ameli, R., Luckenbaugh, D.A.,
Charney, D.S., Manji, H.K., (2006). A randomized trial of an N-methyl-Daspartate
antagonist in treatment-resistant major depression. Arch. Gen. Psychiatry 63, 856-864.
N
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rs
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Chapter 10
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Fluoxetine and Glutamate Release
and Transmission
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Laura Musazzi*, Paolo Tornese, Giulia Treccani
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and Maurizio Popoli
Laboratory of Neuropsychopharmacology and Functional Neurogenomics - Dipartimento
di Scienze Farmacologiche e Biomolecolari and CEND,
Università degli Studi di Milano, Milano, Italy
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Abstract
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Dysfunction of the glutamate system has been associated with the pathophysiology
of psychiatric disorders, including mood and anxiety disorders. Changes in levels and
clearance of glutamate and its metabolites were found in cortical/limbic areas of
depressed patients, while neuroimaging and histopathological studies showed
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morphological and functional alterations in the same brain areas. Preclinical studies on
stress-based animal models of mood and anxiety disorders showed that stress potently
affects glutamate synaptic transmission and plasticity and induces consistent dendritic
remodeling and synaptic spines reduction in corresponding brain areas.
Interestingly, chronic fluoxetine, as well as other traditional antidepressants, not only
have been shown to modulate the glutamate system in basal conditions, but are also able
to prevent the enhancement of glutamate release induced by acute stress and partly
a
reverse the maladaptive changes in synapses and circuitry caused by exposure to chronic
stress. Moreover, the chronic treatment with fluoxetine induces changes in the
expression, regulation and function of glutamate receptors, reducing the activity of
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NMDA receptors, potentiating AMPA receptors, and modulating metabotropic glutamate

receptors. These findings suggest that glutamate transmission may be a relevant target for
the therapeutic action of antidepressants in general, and fluoxetine in particular.
Understanding the action of traditional drugs on glutamate transmission could be of
great help in developing new drugs directly targeted at the glutamate synapse.
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*
Corresponding Author: Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di
Milano, Via Balzaretti 9 - 20133 Milano (Italy). Email: laura.musazzi@unimi.it.
188 Laura Musazzi, Paolo Tornese, Giulia Treccani et al.
Keywords: glutamate transmission, glutamate release, glutamate receptor, dendrite

morphology
Introduction
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Major depression is a widespread debilitating psychiatric disorder characterized by
decreased mood, pleasure, motivation and reward. Since available treatments are efficacious
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only in about 60% of patients and require 2 to 6 weeks to exert therapeutic efficacy, there is a
great need for improved understanding of both the pathophysiology of depression and the
neural mechanisms involved in the action of antidepressants (Rush et al., 2003; Kessler et al.,
2003).
The most recent, and widely accepted hypothesis of depression, the so called
‗neuroplasticity hypothesis‘, claims that concomitant changes in intracellular signaling,
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neurotrophic mechanisms, neurogenesis, synaptic function and plasticity and remodelling of
neuronal cells/circuitry are involved in pathophysiology and treatment of mood disorders
(Duman and Aghajanian, 2012; Sanacora et al., 2012). These ‗neuroplastic‘ changes are
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hypothesized to lead to disruption of homeostatic mechanisms, resulting in destabilization
and loss of synaptic connections in emotional/cognitive circuitry. The hypothesis is supported
by brain-imaging studies on patients with mood and anxiety disorders showing consistent
evidence of volume and connectivity reductions in cortical and limbic brain regions, such as
prefrontal cortex and hippocampus (Gorman and Docherty, 2010; Korgaonkar et al., 2014;
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Price and Drevets, 2010) (see over). Interestingly, most of the connections between and
within these brain areas are glutamatergic, suggesting that the maladaptive changes described
in depressed subjects mainly affect glutamate neurotransmission.
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In the following sections we will briefly summarize the evidence from clinical and
preclinical studies reporting changes in glutamate metabolism and glutamatergic circuitry in
mood disorders.
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Clinical Evidence Showing Dysfunction in the

Glutamate System in Mood Disorders
Changes in Glutamate Level and Metabolism
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Growing compelling evidence has shown that neuropsychiatric disorders are associated
with dysregulation of neurotransmission. Abnormalities in both inhibitory and excitatory
transmission were found in patients with mood and anxiety disorders. In particular, changes
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in glutamate levels have been found in plasma, cerebrospinal fluid and brain tissue.
Different studies showed increased glutamate levels, and a trend for decrease in plasma
glutamine/glutamate ratio, in the plasma of depressed patients compared with healthy controls
(Altamura et al., 1993; Mauri et al., 1998; Mitani et al., 2006; Küçükibrahimoğlu et al., 2009).
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Moreover, although a study in neurosurgical frontal cortex samples did not find any
significant difference in glutamate concentrations (Francis et al., 1989), recent postmortem
studies showed that glutamate levels were significantly increased in frontal cortex and
Fluoxetine and Glutamate Release and Transmission 189
dorsolateral prefrontal cortex from depressed and bipolar patients, respectively (Hashimoto et
al., 2007; Lan et al., 2009).
In vivo proton magnetic resonance spectroscopy (1H-MRS) was also used to measure
glutamate and glutamine levels in cerebrospinal fluid and brain of depressed patients. In these
studies, higher glutamine levels were found in the cerebrospinal fluid of acutely depressed
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unmedicated patients compared with control subjects (Levine et al., 2000). 1H-MRS studies
reported alterations in Glx, a combined measure of glutamate, glutamine and GABA also in
brain areas of subjects with mood and anxiety disorders (Yüksel C and Öngür, 2010; Kondo
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et al., 2011). A large number of studies have provided evidence of a reduction in glutamate
metabolite levels in the frontal cortex and cingulate regions of depressed subjects in the midst
of a current depressive episode (Auer et al., 2000; Michael et al., 2003a, 2003b; Hasler et al.,
2007). In contrast, glutamate metabolite measures in the occipital and parietal/occipital
regions have been found to be elevated in medication-free major depression patients
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(Sanacora et al., 2004; Bhagwagar et al., 2007).
Together, these studies confirm that abnormalities in glutamate/glutamine/GABA cycling
is likely to be associated with the pathophysiology of mood and anxiety disorders (Krystal et
al., 2002; Sanacora et al., 2012).
Morphological Changes
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A large number of clinical neuroimaging studies on depressed patients have consistently
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shown regional volumetric changes in brain areas where glutamate neurons and synapses
predominate, such as hippocampus, cortical regions (including prefrontal cortex) and
amygdala.
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Interestingly, in hippocampus, the most extensively studied area, volumetric reduction
was found to be associated with repeated depressive episodes (Frodl et al., 2006; MacQueen
et al., 2006), suggesting correlation between extent of volumetric reduction and duration of
illness. Significant volumetric reduction has been found also for cortical areas, while
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volumetric enlargement, at least in the early course of illness, has been found for amygdala,
suggesting that morphological changes in this stress-related area are partly different and
dependent on the phase of illness (Lorenzetti et al., 2009).
Together with volumetric changes, significant reductions in glial cell number and density,
and neuronal atrophy were measured in patients. Indeed, in line with the dysregulation of
neurotransmitters metabolism observed in depressed subjects, it has been shown that the
expression of glial excitatory amino acid transporters, and glial cell number and/or density,
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are reduced in brain regions of patients with major depression (Choudary et al., 2005;
Rajkowska et al., 1999). Moreover, smaller size of neuronal bodies was also reported in
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prefrontal cortex and hippocampus (Rajkowsa et al., 1999; Drevets, 2000; Koolschijn et al.,
2009) and decreased expression of synapse-related genes and loss of synapses have been
described in prefrontal cortex of patients with mood disorders (Kang et al., 2012). This
suggests that synaptic dysfunction is involved in the pathophysiology of neuropsychiatric
disorders and could contribute to the volumetric changes observed in patients with mood and
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anxiety disorders.
Structural Changes Induced by Antidepressants

in Animal Models of Mood Disorders
Although the reasons for volumetric reductions of brain areas in depressed subjects have
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not yet been fully understood, it has been proposed that atrophy and remodeling of dendrites
is a major factor (Gorman and Docherty, 2010; McEwen, 2010; Musazzi et al., 2011). The
evidence for this hypothesis mainly comes from preclinical stress-based models of mood and
anxiety disorders (as a review, see Musazzi et al., 2011; Sanacora et al., 2012; Musazzi et al.,
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2013) (Figure 1). Indeed, rodent studies have shown that different forms of behavioral stress –
a major predisposing or triggering factor for mood and anxiety disorders in humans- have
powerful effects on the structure and morphology of limbic and cortical areas, inducing
atrophy, dendritic simplification, reduction of synaptic spines and global volumetric
reductions resembling those observed in patients with major depression (Radley et al., 2004;
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Michelsen et al., 2007; Radley et al., 2008; Silva-Gómez et al., 2003; Cerqueira et al., 2007).
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Figure 1. The role of glutamate release and transmission in the maladaptive neuroplasticity linked to
pathophysiology of depression. A. Neuroimaging studies have consistently shown volumetric reduction
of cortical and limbic areas in the brain of patients with mood and anxiety disorders. This has been
attributed to several factors, including dendritic atrophy and remodeling, loss of glial cells and
reduction of neurogenesis (in hippocampus). B. Preclinical studies in rodents have shown that stress,
through the action of glucocorticoids, may induce abnormal enhancement of glutamate release and
excitatory transmission in select brain areas, including amygdala, hippocampus, and prefrontal cortex.
a
Chronic stress induces atrophy and remodeling of dendritic arbor in hippocampus and prefrontal cortex,
with loss of dendritic spines and synapses. In turn, dendritic remodeling is envisaged as a major causal
factor for volumetric changes, as observed with magnetic resonance imaging in patients. The
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structural/functional changes induced by stress protocols in rodents are reversible with cessation of
stress, and are prevented or reversed by chronic antidepressant treatments and voluntary physical
exercise. HPA hypothalamic-pituitary-adrenal, CORT corticosterone. (Adapted from Musazzi et al.
2013).
On the other hand, the therapeutic effect of antidepressant drugs, involving slow adaptive
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changes in post-receptor signaling cascades and downstream mechanisms, in turn induces

increase of plasticity and dendritic spine remodeling and hippocampal neurogenesis (Castren
et al., 2007; Krishnan and Nestler, 2008; McClung and Nestler, 2008; Pittenger and Duman
2008; Racagni and Popoli 2008). Indeed, it was suggested that rapid synaptic plasticity,
especially the remodeling of dendritic spines and spine synapses of pyramidal neurons, may
play an important role in the neurobiology of depression and effects of antidepressant therapy
(Ampuero et al., 2010; Chen et al., 2008; Chen et al., 2010; Hajszan et al., 2005). Chronic
c.
SSRIs (including escitalopram, fluoxetine, paroxetine and sertraline) were shown to induce
growth of mushroom-type dendritic spines and a concomitant decrease of thin and stubby-like
spines, suggesting a stabilization of synaptic connections (Ampuero et al., 2010; Seo et al.,
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2014). Moreover, several studies have reported that imipramine and fluoxetine increase the
number of hippocampal asymmetric (i.e., excitatory) synapses in rats, suggesting the
establishment of new synaptic connections and/or the remodeling or transformation of
existing synapses (Chen et al., 2008; Chen et al., 2010; Hajszan et al., 2005).
Interestingly, pharmacological antidepressant treatments have been also shown to reverse
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the dendritic remodeling observed in the hippocampus and prefrontal cortex of animal models
of depression with a number of studies reporting that different classes of antidepressants can
rescue the morphological changes induced by stress. Tianeptine prevented and reversed the
actions of stress and glucocorticoids on dendritic remodeling in an animal model of chronic
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stress (McEwen et al., 2002). More recently, chronic fluoxetine was found to rescue the
inhibition of adult neurogenesis in the dentate gyrus and the suppression of cytogenesis in the
medial prefrontal cortex induced by chronic social stress (Czéh et al., 2007). The tricyclic
antidepressant desipramine reversed both the hippocampal spine synapse loss and the escape
deficit in the learned helplessness model of depression (Hajszan et al. 2009). Chronic
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imipramine treatment reversed the suppression of neurogenesis and synaptogenesis in the
hippocampus of the Flinders Sensitive Line rats, and this was found to be related with
behavioral improvement in the forced swim test (Chen et al., 2010). Moreover, both
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imipramine and fluoxetine have been shown to reverse the reduction in dendritic arborization
and spine density induced by chronic mild stress (Bessa et al., 2009). More recently, a single
injection of the fast acting antidepressant ketamine was reported to rapidly and completely
rescue the deficits in spine density and function induced by chronic unpredictable stress (Li et
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al., 2011).
Quantitative and qualitative changes in the morphology of synapses and circuitry
correspond to marked changes in synaptic transmission, which may be involved in the
pathophysiology of mood and anxiety disorders (Sanacora et al., 2012; Musazzi et al., 2013).
Modulation of Glutamate Transmission

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and Release by Fluoxetine

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As outlined above, the reduction of dendritic arbor and spine number is considered a
possible mechanism accounting for the volumetric changes registered in the hippocampus and
prefrontal cortex of subjects with mood and anxiety disorders. Although the mechanism of
dendritic remodeling and its significance as to pathological changes are still largely unknown,
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a major role is attributed to glucocorticoid hormones that are elevated by stressors in both
rodents and humans (Gorman and Docherty, 2010; McEwen, 2010; Musazzi et al., 2011). It is
also important to notice that a large proportion of depressed patients show a typical
dysregulation of the hypothalamic–pituitary–adrenal axis, suggesting that the alteration of the

stress response might be a hallmark of the pathophysiology (Bledsoe et al., 2011; Gillespie
and Nemeroff, 2005; Pariante and Lightman, 2008). It has been proposed that a major effect
of the increase in glucocorticoid levels in cortical and limbic areas is an enhancement of
glutamate release, which in turn is considered to be responsible for the induction of dendrites
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retraction (McEwen, 2005; Gorman and Docherty, 2010; Pittenger and Duman, 2008; Popoli
et al., 2012; Sanacora et al., 2012). Indeed, it has been argued that, similar to some
neurodegenerative disorders, the enhancement of glutamate release induced by stress might
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cause a form of excitotoxicity and, as a consequence, dendrites retraction and atrophy.
However, retraction of dendrites and spine density reduction might also be an adaptive
protective strategy to reduce the excitatory input in the face of increased glutamate release
(Gorman and Docherty, 2010; McEwen, 2010).
Since traditional monoaminergic antidepressants, particularly fluoxetine, have been
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repeatedly shown to interfere with the function of the glutamate system (Figure 2), in the
following sections of this chapter we analyze in detail the action of fluoxetine on the
glutamate system, in particular glutamate uptake and metabolism, the different classes of
glutamate receptors and the release of glutamate.
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Figure 2. Sites of action (targets) of chronic SSRIs in the glutamate synapse. Several sites/mechanisms
of regulation of the glutamate synapse have been shown to be modulated by chronic SSRIs: 1
presynaptic release of glutamate; 2 postsynaptic ionotropic receptors for glutamate (N-methyl-D-
aspartate receptors (NMDARs) and α-amino-3-hydroxy-methyl-4-isoxazole propionic acid receptors
(AMPARs); 3 metabotropic glutamate receptors (mGluR); and 4 glial-specific glutamate transporters.
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See text for details. Ca2+ calcium ions, EAAT excitatory amino-acid transporter, Gln glutamine, Na+
sodium ions, SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor, vGluT
vesicular glutamate transporter, Glu glutamate. (Adapted from Popoli et al. 2012).
Fluoxetine Modulates Glial Cells Function
A number of studies have suggested that glial function could be a target of

antidepressants (reviewed in Sanacora and Banasr 2013). In particular, fluoxetine was shown
to both promote gliogenesis and to rescue the ultrastructural astrocyte alterations induced by
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chronic stress in prelimbic cortex (Czeh et al., 2006; Kusakawa et al., 2010; Sun et al., 2012).
More directly related to glutamate uptake, treatment with fluoxetine also significantly induced
the expression of the glutamate transporter 1 in hippocampus and cortex of rats (Zink et al.,
2011). The changes in astroglial morphology and function in animal mood disorders and the
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effects of antidepressant treatments further support the notion that astroglial changes might
contribute to the pathophysiology of affective disorders as well as to the cellular actions of
antidepressants (Sanacora and Banasr 2013).
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SSRIs, Including Fluoxetine, Regulate Glutamate Receptors
Glutamate released in the synaptic cleft can bind ionotropic postsynaptic glutamate
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receptors, including α–amino-3-hydroxy-methyl-4-isoxazole propionic acid (AMPA)
receptors (mediating fast excitatory synaptic transmission), N-methyl-D-aspartate (NMDA)
receptors, generating a slower and longer-lasting response) and kainate receptors, or
metabotropic glutamate (mGlu) receptors, localized at both pre- and postsynaptic sites. A
number of studies have consistently shown that chronic antidepressants regulate glutamate
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receptors expression and function.
In early studies, antidepressants from different classes, including SSRIs, were shown to
reduce radioligand binding to rat cortical NMDA receptors (Skolnick et al., 1996). In
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particular, this effect of citalopram was shown to develop slowly, persist for some time after
cessation of treatment, and was found to be dose dependent. It was also demonstrated that
citalopram altered the levels of transcripts encoding NMDA receptor subunits in selected
mouse brain areas (Boyer et al., 1998). More recently, it was shown that early-life stress
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increased the expression of the GluN1 subunit of the NMDA receptor in hippocampus of the
Flinders Sensitive Line rat model of depression and chronic treatment with escitalopram was
able to normalize these alterations (Ryan et al., 2009).
SSRIs also exert a positive regulation of AMPA receptors expression and
phosphorylation. Chronic paroxetine treatment increased the interaction of AMPA receptor
subunits with different neuronal proteins that regulate the insertion and stability of AMPA
receptors at hippocampal membranes (Martínez-Turrillas et al., 2005). Chronic
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antidepressants, including fluoxetine, produced site-selective and area-specific effects on the

expression pattern and editing levels of AMPA and kainate glutamate receptors (Barbon et
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al., 2006). Specifically, fluoxetine treatment selectively increased AMPA subunits GluA4
transcription levels in prefrontal and frontal cortex, and variously affected GluA2, GluA5 and
GluA6 in the hippocampus. Moreover, fluoxetine slightly increased GluA2 R/G site editing in
prefrontal and frontal cortex, suggesting altered resensitization kinetics and a consequent
enhancement of the response to glutamate. Chronic fluoxetine was also reported to increase
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GluA1 phosphorylation at Ser845 (associated with increased GluA1 insertion) and at Ser-831
(associated with potentiation of AMPA currents), suggesting possible alterations in synaptic
plasticity (Svenningsson et al., 2002).
Together these data suggested that antidepressants induce an increased ratio of AMPA to
NMDA receptor mediated transmission and this shift in glutamate receptor activity was
proposed to be an important factor in antidepressant efficacy (Du et al., 2004; Bobula and
Hess, 2008; Andreasen et al., 2013; Wolak et al., 2013).
Chronic SSRIs also induce specific changes in the expression and function of individual
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mGlu receptor subtypes. mGlu7 (but not mGlu4) expression levels were decreased by
citalopram, but not imipramine, both in rat hippocampus and prefrontal cortex (Wieronska et
al., 2007). In a recent work, fluoxetine treatment (10 mg/kg for 21 days) was found to exert
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different effects in hippocampus of control rats and rats subjected to early life stress. mGlu7
and mGlu8 were up-regulated by fluoxetine in control rats but not in stressed rats, while the
expression of mGlu4 was unaffected by fluoxetine in control rats, but the drug restored
mGlu4 levels reduced by stress (O‘ Connor et al., 2012). Since mGlu4 are mainly presynaptic
and function as autoreceptors to inhibit glutamate release, fluoxetine would lead to an
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increase in the negative feedback limiting further glutamate release, therefore potentially
reducing excitation and leading to an antidepressant effect.
Recently, different antidepressant drugs, including escitalopram, were reported to
significantly increase mGlu5 postsynaptic excitatory receptor levels and binding of the
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mGlu5 radioligand [3H]MPEP in the cerebral cortex and in the hippocampus, suggesting an
adaptive upregulation of mGlu5 receptors induced by antidepressant drugs (Nowak et al.,
2014).
The modulation of glutamate receptors induced by antidepressants was demonstrated to
be also accompanied by alteration in excitability and long term potentiation and depression
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(respectively, LTP and LTD). In the following section, studies reporting changes in glutamate
neurotransmission induced by treatment with SSRIs will be discussed.
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Chronic Fluoxetine Reduces Glutamate Release and Transmission in
Cortical and Limbic Areas
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A number of studies have tested the hypothesis that chronic administration of

antidepressants changes excitatory aminoacid overflow. In particular, it was shown that the
treatment with different classes of antidepressants, including SSRIs, reduces the presynaptic
release of glutamate.
The application of fluoxetine in vitro for 10 min to Percoll-purified cerebrocortical
synaptosomes was shown to dose dependently (0.5–10 μM) inhibit 4-aminopyridine-evoked
glutamate release (Wang et al., 2003). This in vitro effect of fluoxetine was shown to be
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mediated by inhibition of P/Q-type calcium channels and not by a direct effect on the
exocytotic machinery, as shown by the lack of effect of either ionomycine or hypertonic
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sucrose.
In a different work, endogenous glutamate and GABA release were measured in Percoll-
purified hippocampal synaptosomes from rats chronically treated with various
antidepressants, including fluoxetine (Bonanno et al., 2005). Basal and depolarization-evoked
glutamate release was measured twenty-four hours after the last drug administrations, by
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using the technique of isolated synaptosomes in superfusion. This method is performed by

applying a constant top-down superfusion with physiological medium to a monolayer of
purified synaptosomes (Popoli et al. 2012; Bonanno et al. 2005). With this technique,
endogenous neurotransmitters released are immediately removed by the superfusion medium

before they can be taken up by transporters, or activate autoreceptors and heteroreceptors
present on synaptic terminals. The study showed no changes in basal glutamate or GABA
release after chronic treatment. However, depolarization evoked overflow of glutamate was
significantly reduced by chronic antidepressants. This reduction of presynaptic glutamate
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release was found to be accompanied by changes in select protein–protein interactions
regulating the assembly of the presynaptic SNARE protein complex (formed by
synaptobrevin-2, syntaxin-1 and SNAP-25) (Sudhof and Rothman, 2009). In particular, the
phosphorylation levels of α-calcium/calmodulin-dependent protein kinase II (αCaM kinase II)
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at the activation site was markedly decreased in synaptic membranes (Barbiero et al., 2007).
This change, in turn, reduced the binding of syntaxin-1 to αCaM kinase II and increased its
binding to Munc-18, decreasing availability of syntaxin 1 for the SNARE complex, thereby
reducing depolarization-evoked release of neurotransmitter (Agid et al., 2007; Bonanno et al.,
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2005).
It was also reported that fluoxetine inhibited NMDA induced currents in cortical cell
cultures with a voltage-independent mechanism, through a selective block of NMDA
receptors composed by GluN1/GluN2B subunits (Szasz et al., 2007; Kiss et al., 2012; Vizi et
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al., 2013). Since several studies have provided evidence that synaptic or extrasynaptic
GluN2A-containing receptors mediate cell survival signaling, whereas the activation of
GluN2B-containing receptors results in increased neuronal apoptosis (Vizi et al., 2010), the
inhibition of GluN1/GluN2B receptors induced by fluoxetine supported the hypothesis that
neuroplasticity may be an important target for the therapy of mood disorders. In a more recent
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study, long term fluoxetine was found to increase excitability at hippocampal Shaffer
collateral CA1 synapses, but together with an increase in spine density, impaired both LTP
and LTD (Rubio et al., 2013). The authors argued that the increased bioavailability of
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serotonin consequent to chronic fluoxetine, activating hetero-receptors on glutamatergic CA1
pyramidal neurons, might induce a tonic inhibition of neurotransmission. This, in turn, could
trigger an adaptive homeostatic response, which finally leads to an enhancement of basal
neurotransmission and dendritic spine remodeling.
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On the other hand, it was reported that 3 weeks of chronic unpredictable stress led to a
decrease in the strength of excitatory synaptic transmission in the distal apical dendrites of
hippocampal CA1 cells, an effect limited to downregulation of AMPA glutamate receptors,
without affecting NMDA receptors at the same synapses (Kallarackal et al., 2013). Chronic
fluoxetine administration in stressed animals exerts a normalizing action on AMPA receptors
mediated excitation, confirming the hypothesis that antidepressants exert long-term control of
excitatory synaptic transmission.
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A recent study on slices of human prefrontal cortex from tissues removed to gain access
for the surgical treatment of deep brain tumors, examined the effects of fluoxetine and
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physiological concentrations of serotonin on single cell-initiated polysynaptic postsynaptic

potentials (Komlósi et al., 2012). Single spikes were elicited in single pyramidal cells while
looking for polysynaptic excitatory and/or inhibitory postsynaptic potentials in
simultaneously recorded neurons. Fluoxetine and serotonin suppressed the frequency of
single-spike-triggered polysynaptic events. Since release of serotonin by activation of
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serotonergic fibers can be excluded because co-release of serotonin from glutamatergic or

GABAergic terminals has not been described, this effect of fluoxetine were mainly dependent
on the activation of heteroreceptors on glutamatergic neurons (for a discussion, see Pittaluga
et al., 2007). Indeed, the analysis of multiple parameters of monosynaptic excitatory

connections indicate a presynaptic mechanism of action of serotonin at glutamatergic
synapses possibly mediated by 5HT-1A and 2A receptors. Interestingly, in the same study,
GABAergic outputs were found to be not modulated by fluoxetine, suggesting that one of the
modes of action of antidepressants is a reduction of activity-dependent glutamate release, a
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functional change that could improve the signal to noise ratio in glutamate transmission, and
exert a protective action when the synapse is overactivated by the action of stress-related
mechanisms.
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Chronic Antidepressants Normalize Changes in Synaptic Transmission
Induced by Behavioral Stress
Acute stress paradigms and glucocorticoid administration in rodents were consistently
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reported to induce enhancement of glutamate release in select limbic and cortical areas (for
recent reviews see Musazzi et al., 2011; Popoli et al., 2012). As previously described, the
stress induced enhancement of glutamate release and transmission was proposed to be a
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causal factor in the induction of synaptic and dendritic remodeling. Since antidepressants are
able to modulate glutamate release, this effect could be part of the therapeutic action of these
drugs (Sanacora et al., 2011; Musazzi et al., 2013). A number of studies have explored the
effect of antidepressants on the stress-induced increase of glutamate release and transmission.
The effect on glutamate extracellular levels of a single administration of tianeptine or
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fluoxetine, prior to acute 1 h restraint stress, was investigated by microdialysis (Reznikov et
al., 2007). As expected, the acute stressor increased glutamate efflux in the basolateral and
central amygdala. However, the effects of the two drugs on the stress response were quite
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different: while acute tianeptine completely abolished the stress-induced increase of
extracellular glutamate in the basolateral amygdala, fluoxetine increased basal extracellular
glutamate before stress application and potentiated the increase of glutamate levels induced
by stress in both areas. The effect of fluoxetine provided a potential mechanism for the well-
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known anxiogenic properties of selective serotonin-reuptake inhibitors, which are often

observed in the initial phase of clinical treatment.
The effects of chronic treatment with different antidepressants on the stress induced
enhancement of glutamate release in prefrontal and frontal cortex were measured in a more
recent work by using purified synaptosomes in superfusion and patch-clamp
electrophysiology (Musazzi et al., 2010). Rats were treated for 2 weeks with four
antidepressants including fluoxetine, and then subjected to acute foot shock (FS)-stress, in
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order to assess the response to stress in drug treated animals. All drugs completely abolished
the stress-induced enhancement of glutamate release from prefrontal and frontal cortex
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synaptosomes. Patch-clamp recordings in acute slices of medial prefrontal cortex showed that
chronic treatment with desipramine (used as reference drug) normalized the increase of
postsynaptic excitatory current amplitude, as well as the marked reduction of paired pulse
facilitation and its calcium dependence observed in stressed rats. With regard to the
mechanism of this effect of antidepressants, it was found that the different drugs used were
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not able to block the stress-induced rise of corticosterone level, suggesting the drug action is
downstream of this mechanism.
Recently, acute FS-stress was also shown to induce in prefrontal cortex a marked increase
in the size of the readily releasable pool of glutamate vesicles, in the number of docked
vesicles, and a large sprouting of excitatory synapses (Treccani et al., 2014a; Nava et al.,
submitted). These changes were partially blocked by desipramine pretreatment, suggesting
that antidepressants may have a preventive effect on vesicle docking and small synapse
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sprouting which reflects, in turn, on reduced stress induced overexcitation and neurotoxicity.
Finally, it was recently shown that chronic fluoxetine reversed the reduction in
depolarization-evoked glutamate release and in the expression of synaptic vesicle associated
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proteins induced by prenatal restraint stress in the ventral hippocampus, a brain region
involved in the maladaptive programming caused by early life stress (Marrocco et al., 2014).
Conclusion
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Compelling evidence strongly suggests that long-term changes in brain areas and circuits
mediating complex cognitive and emotional behaviors represent the biological underpinnings
of mood and anxiety disorders. As the vast majority of neurons and synapses in these brain
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areas and circuits are glutamatergic, it is now recognized that the glutamatergic system is a
primary mediator of psychiatric pathology and, potentially, also a final common pathway for
the therapeutic action of antidepressant medications.
Starting from the effects of chronic traditional antidepressants on the glutamate system,
several lines of preclinical and clinical research are investigating the putative therapeutic
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action of molecules directly targeted on the different sites of the glutamate synapse
regulation, looking for antidepressants with faster therapeutic onset rate and a reduction in the
side effect profile. Indeed, extensive cellular and molecular investigation has recently shown
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that all major sites of regulation of the glutamate synapse, including presynaptic release
machinery, postsynaptic ionotropic receptors, pre- and postsynaptic metabotropic receptors,
glutamate reuptake and glutamate/glutamine recycling mechanism, are modulated by
traditional antidepressants and thus may represent potential targets for new pharmacological
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approaches.
In this context, we hypothesize that the molecular mechanisms regulating the presynaptic
release of glutamate could be a possible target for new compounds with antidepressant and
anxiolytic action. Indeed, chronic antidepressants (including fluoxetine) completely prevent
the stress-induced enhancement of glutamate release in prefrontal and frontal cortex,
suggesting that the stabilization of glutamate release and transmission is a relevant part of
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their therapeutic action (Musazzi et al., 2013). This drug action may protect from buildup of
dangerous concentrations of synaptic glutamate and contribute to preventing or reversing the
dendritic remodeling and synaptic disconnection which is thought to be a major factor in
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stress-related neuropsychiatric disorders (Duman and Aghajanian, 2012; Sanacora et al.,

2012; Musazzi et al., 2013). The mechanism of this protective action of antidepressants
against stress effects on neurotransmission is not clearly understood at present. We have
recently shown that corticosterone released upon acute stress binds receptors located at or
near presynaptic terminals, and rapidly, by non genomic action, increases the trafficking of
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synaptic vesicles into the pool ready for release (ready releasable pool, RRP) (Treccani et al.
2014). The RRP increase primes the terminals for the enhancement of glutamate release, but
the synaptic non-genomic action of corticosterone is not sufficient to increase glutamate

release. We speculate that slower, likely genomic effects are required to promote the
enhancement of glutamate release and transmission induced by acute stress (Popoli et al.
2012; Treccani et al. 2014).
Chronic antidepressants were shown to prevent the enhancement of glutamate release in
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prefrontal and frontal cortex, but not the rise of corticosterone levels (Musazzi et al., 2010).
However, desipramine was shown to partly or completely prevent the increase in the number
of docked glutamatergic vesicles available for release, and the increase of small excitatory
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synapses induced by acute stress (Treccani et al., 2014; Nava et al., in press). Therefore, the
action of antidepressants seems to be downstream of the rise of corticosterone but upstream of
the delayed, genomically mediated effects required for the enhancement of glutamate release
induced by stress (Figure 3). Further research is under way to dissect this mechanism, which
may serve for the identification of new drug targets.
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Figure 3. Synaptic effects of acute stress and antidepressants. Acute stress enhances glutamate release
in prefrontal and frontal cortex (Musazzi et al. 2010). Corticosterone, released upon acute stress,
increases the readily releasable pool of glutamate vesicles by rapid non genomic synaptic action.
However, corticosterone is necessary but not sufficient for the stress-induced enhancement of glutamate
release in cortical areas (Treccani et al. 2014a). Delayed, perhaps genomic, effects of corticosterone are
a
necessary for completion of corticosterone action and enhancement of glutamate release and
transmission (Treccani et al., 2014b; Yuen et al. 2011). Previous chronic antidepressant treatments
block the stress-induced enhancement of glutamate release. The mechanism of this drug action is not
ov
clear yet (see text for details). SNARE N-ethylmaleimide-sensitive fusion protein attachment protein
receptor
References
N
Agid, Y., Buzsáki, G., Diamond, D.M., Frackowiak, R., Giedd, J., Girault, J.A., Grace, A.,
Lambert, J.J., Manji, H., Mayberg, H., Popoli, M., Prochiantz, A., Richter-Levin, G.,
Somogyi, P., Spedding, M., Svenningsson, P., Weinberger, D., (2007). How can drug
discovery for psychiatric disorders be improved? Nat. Rev. Drug Discov. 6, 189-201.
Altamura, C.A., Mauri, M.C., Ferrara, A., Moro, A.R., D'Andrea, G., Zamberlan, F., (1993).
Plasma and platelet excitatory amino acids in psychiatric disorders. Am J Psychiatry. 150,
1731-3.
c.
Ampuero, E., Rubio, F.J., Falcon, R., Sandoval, M., Diaz-Veliz, G., Gonzalez, R.E., Earle,
N., Dagnino-Subiabre, A., Aboitiz, F., Orrego, F., Wyneken, U., (2010). Chronic
fluoxetine treatment induces structural plasticity and selective changes in glutamate
In
receptor subunits in the rat cerebral cortex. Neuroscience 169, 98-108.
Andreasen, J.T., Gynther, M., Rygaard, A., Bøgelund, T., Nielsen, S.D., Clausen, R.P.,
Mogensen, J., Pickering, D.S., (2013). Does increasing the ratio of AMPA-to-NMDA
receptor mediated neurotransmission engender antidepressant action? Studies in the
mouse forced swim and tail suspension tests. Neurosci. Lett. 546, 6-10.
rs
Auer, D.P., Pütz, B., Kraft, E., Lipinski, B., Schill, J., Holsboer, F., (2000). Reduced
glutamate in the anterior cingulate cortex in depression: an in vivo proton magnetic
resonance spectroscopy study. Biol. Psychiatry 47, 305-13.
Barbiero, V.S., Giambelli, R., Musazzi, L., Tiraboschi, E., Tardito, D., Perez, J., Drago, F.,
he
Racagni, G., Popoli, M., (2007). Chronic antidepressants induce redistribution and
differential activation of alphaCaM Kinase II between presynaptic compartments.
Neuropsychopharmacol. 32, 2511–9.
Barbon, A., Popoli, M., La Via, L., Moraschi, S., Vallini, I., Tardito, D., Tiraboschi, E.,
Musazzi, L., Giambelli, R., Gennarelli, M., Racagni, G., Barlati, S., (2006). Regulation of
is
editing and expression of glutamate alpha-amino-propionic-acid (AMPA)/kainate
receptors by antidepressant drugs. Biol. Psychiatry 59, 713-20.
Bessa, J.M., Ferreira, D., Melo, I., Marques, F., Cerqueira, J.J., Palha, J.A., Almeida, O.F.,
bl
Sousa, N., (2009). The mood-improving actions of antidepressants do not depend on
neurogenesis but are associated with neuronal remodeling. Mol. Psychiatry 14, 764-73.
Bhagwagar, Z., Wylezinska, M., Jezzard, P., Evans, J., Ashworth, F., Sule, A., Matthews,
P.M., Cowen, P.J., (2007). Reduction in occipital cortex gamma-aminobutyric acid
Pu
concentrations in medication-free recovered unipolar depressed and bipolar subjects.

Bledsoe, A.C., Oliver, K.M., Scholl, J.L., Forster, G.L., (2011). Anxiety states induced by
post-weaning social isolation are mediated by CRF receptors in the dorsal raphe nucleus.
Brain Res. Bull. 85, 117-22
Bobula, B., Hess, G., (2008). Antidepressant treatments-induced modifications of
glutamatergic transmission in rat frontal cortex. Pharmacol. Rep. 60, 865-71.
a
Bonanno, G., Giambelli, R., Raiteri, L., Tiraboschi, E., Zappettini, S., Musazzi, L., Raiteri,
M., Racagni, G., Popoli, M., (2005). Chronic antidepressants reduce depolarization-
ov
evoked glutamate release and protein interactions favoring formation of SNARE complex
in hippocampus. J. Neurosci. 25, 3270-9.
Boyer, P.A., Skolnick, P., Fossom, L.H., (1998). Chronic administration of imipramine and
citalopram alters the expression of NMDA receptor subunit mRNAs in mouse brain. A
quantitative in situ hybridization study. J. Mol. Neurosci. 10, 219-33.
N
Castrén, E., Võikar, V., Rantamäki, T., (2007). Role of neurotrophic factors in depression.
Curr. Opin. Pharmacol. 7, 18-21.
Cerqueira, J.J., Mailliet, F., Almeida, O.F., Jay, T.M., Sousa, N., (2007). The prefrontal
cortex as a key target of the maladaptive response to stress. J. Neurosci. 27, 2781-7.
Chen, F., Madsen, T.M.,Wegener, G., Nyengaard, J.R., (2008). Changes in rat hippocampal
CA1 synapses following imipramine treatment. Hippocampus 18, 631–9.
Chen, F., Madsen, T.M., Wegener, G., Nyengaard, J.R., (2010). Imipramine treatment
c.
increases the number of hippocampal synapses and neurons in a genetic animal model of
depression. Hippocampus 20, 1376-84.
Choudary, P.V., Molnar, M., Evans, S.J., Tomita, H., Li, J.Z., Vawter, M.P., Myers, R.M.,
In
Bunney Jr, W.E., Akil, H., Watson, S.J, Jones, E.G., (2005). Altered cortical
glutamatergic and GABAergic signal transmission with glial involvement in depression.
Proc. Natl. Acad. Sci. USA 102, 15653-8.
Czéh, B., Simon, M., Schmelting, B., Hiemke, C., Fuchs, E., (2006). Astroglial plasticity in
the hippocampus is affected by chronic psychosocial stress and concomitant fluoxetine
rs
treatment. Neuropsychopharmacol. 31, 1616-26.
Czéh, B., Müller-Keuker, J.I., Rygula, R., Abumaria, N., Hiemke, C., Domenici, E,, Fuchs,
E., (2007). Chronic social stress inhibits cell proliferation in the adult medial prefrontal
cortex: hemispheric asymmetry and reversal by fluoxetine treatment.
he
Drevets, W.C., (2000). Functional anatomical abnormalities in limbic and prefrontal cortical
structures in major depression. Prog. Brain Res. 126, 413–31.
Du, J., Gray, N.A., Falke, C.A., Chen, W., Yuan, P., Szabo, S.T., Einat, H., Manji, H.K.,
(2004). Modulation of synaptic plasticity by antimanic agents: the role of AMPA
is
glutamate receptor subunit 1 synaptic expression. J. Neurosci. 24, 6578-89.
Duman, R.S., Aghajanian, G.K., (2012). Synaptic dysfunction in depression: potential
therapeutic targets. Science 338, 68–72.
bl
Francis, P.T., Poynton, A., Lowe, S.L., Najlerahim, A., Bridges, P.K., Bartlett, J.R., Procter,
A.W., Bruton, C.J., Bowen, D.M., (1989). Brain amino acid concentrations and Ca2+-
dependent release in intractable depression assessed antemortem. Brain Res. 494, 315-24.
Frodl, T., Schaub, A., Banac, S., Charypar, M., Jäger, M., Kümmler, P., Bottlender, R.,
Pu
Zetzsche, T., Born, C., Leinsinger, G., Reiser, M., Möller, H.J., Meisenzahl, E.M.,
(2006). Reduced hippocampal volume correlates with executive dysfunctioning in major
depression. J. Psychiatry Neurosci. 31, 316–23.
Gillespie, C.F., Nemeroff, C.B., (2005). Hypercortisolemia and depression. Psychosom. Med.
67, S26–8.
Gorman, J,M,, Docherty, J,P., (2010). A hypothesized role for dendritic remodeling in the
etiology of mood and anxiety disorders. J. Neuropsychiatry Clin. Neurosci. 22, 256–64.
a
Hajszan, T., Dow, A., Warner-Schmidt, J.L., Szigeti-Buck, K., Sallam, N.L., Parducz, A.,
Leranth, C., Duman, R.S., (2009). Remodeling of hippocampal spine synapses in the rat
ov
learned helplessness model of depression. Biol. Psychiatry. 65, 392-400.

Hajszan, T., MacLusky, N.J., Leranth, C., (2005). Short-term treatment with the
antidepressant fluoxetine triggers pyramidal dendritic spine synapse formation in rat
hippocampus. Eur. J. Neurosci. 21, 1299-303.
Hashimoto, K., Sawa, A., Iyo, M., (2007). Increased levels of glutamate in brains from
N
patients with mood disorders. Biol. Psychiatry 62, 1310-6.

Hasler, G., van der Veen, J.W., Tumonis, T., Meyers, N., Shen, J., Drevets, W.C., (2007).
Reduced prefrontal glutamate/glutamine and gamma-aminobutyric acid levels in major
depression determined using proton magnetic resonance spectroscopy. Arch. Gen.

Kallarackal, A.J., Kvarta, M.D., Cammarata, E., Jaberi, L., Cai, X., Bailey, A.M., Thompson,
S.M., (2013). Chronic stress induces a selective decrease in AMPA receptor-mediated
synaptic excitation at hippocampal temporoammonic-CA1 synapses. J. Neurosci. 33,
c.
15669-74.
Kang, H.J., Voleti, B., Hajszan, T., Rajkowska, G., Stockmeier, C.A,, Licznerski, P., Lepack,
A., Majik, M.S., Jeong, L.S., Banasr, M., Son, H., Duman, R.S., (2012). Decreased
In
expression of synapse-related genes and loss of synapses in major depressive disorder.
Nat. Med. 18,1413-7.
Kessler, R.C., Berglund, P., Demler, O., Jin, R., Koretz, D., Merikangas, K.R., Rush, A.J.,
Walters, E.E., Wang, P.S., (2003). The epidemiology of major depressive disorder:
Results from the National Comorbidity Survey Replication (NCS-R). JAMA 289, 3095–
rs
105.
Kiss, J.P., Szasz, B.K., Fodor, L., Mike, A., Lenkey, N., Kurkó, D., Nagy, J., Vizi, E.S.,
(2012). GluN2B-containing NMDA receptors as possible targets for the neuroprotective
and antidepressant effects of fluoxetine. Neurochem. Int. 60, 170-6.
he
Komlósi, G., Molnár, G., Rózsa, M., Oláh, S., Barzó, P., Tamás, G., (2012). Fluoxetine
(prozac) and serotonin act on excitatory synaptic transmission to suppress single layer 2/3
pyramidal neuron-triggered cell assemblies in the human prefrontal cortex. J. Neurosci.
32, 16369-78.
Kondo, D.G., Hellem, T.L., Sung, Y.H., Kim, N., Jeong, E.K., Delmastro K.K., Shi, X.,
is
Renshaw, P.F., (2011). Review: magnetic resonance spectroscopy studies of pediatric
major depressive disorder. Depress. Res. Treat. 2011, 650450.
Koolschijn, P.C., van Haren, N.E., Lensvelt-Mulders, G.J., Hulshoff Pol, H.E., Kahn, R.S.,
bl
(2009). Brain volume abnormalities in major depressive disorder: a metaanalysis of
magnetic resonance imaging studies. Hum. Brain Mapp. 30, 3719-35.
Korgaonkar, M.S., Fornito, A., Williams, L.M., Grieve, S.M., (2014). Abnormal Structural
Networks Characterize Major Depressive Disorder: A Connectome Analysis. Biol.
Pu

Krishnan, V., Nestler, E.J., (2008). The molecular neurobiology of depression. Nature 455,
894-902.
Krystal, J.H., Sanacora, G., Blumberg, H., Anand, A., Charney, D.S., Marek, G., Epperson,
C.N., Goddard, A., Mason, G.F., (2002). Glutamate and GABA systems as targets for
novel antidepressant and mood-stabilizing treatments. Mol. Psychiatry 7, S71-80.
Küçükibrahimoğlu, E., Saygin, M.Z., Calişkan, M., Kaplan, O.K., Unsal, C., Gören, M.Z.
a
(2009). The change in plasma GABA, glutamine and glutamate levels in fluoxetine or S-
citalopram-treated female patients with major depression. Eur. J. Clin. Pharmacol. 65,
ov
571-7.
Kusakawa, S., Nakamura, K., Miyamoto, Y., Sanbe, A., Torii, T., Yamauchi, J., Tanoue, A.,
(2010). Fluoxetine promotes gliogenesis during neural differentiation in mouse
embryonic stem cells. J. Neurosci. Res. 88, 3479-87.
Lan, M.J., McLoughlin, G.A., Griffin, J.L., Tsang, T.M., Huang, J.T., Yuan, P., Manji, H.,
N
Holmes, E., Bahn, S., (2009). Metabonomic analysis identifies molecular changes
associated with the pathophysiology and drug treatment of bipolar disorder. Mol.
Levine, J., Panchalingam, K., Rapoport, A., Gershon, S., McClure, R.J., Pettegrew, J.W.,
(2000). Increased cerebrospinal fluid glutamine levels in depressed patients. Biol.
Li, N., Liu, R.J., Dwyer, J.M., Banasr, M., Lee, B., Son, H., Li, X.Y., Aghajanian, G.,
Duman, R.S., (2011). Glutamate N-methyl-D-aspartate receptor antagonists rapidly
c.
reverse behavioral and synaptic deficits caused by chronic stress exposure. Biol.
Lorenzetti, V., Allen, N.B., Fornito, A., Yücel, M., (2009). Structural brain abnormalities in
In
major depressive disorder: a selective review of recent MRI studies. J. Affect. Disord.
117, 1–17.
MacQueen, G.M., Campbell, S., McEwen, B.S., Macdonald, K., Amano, S., Joffe, R.T.,
Nahmias, C., Young, L.T., (2003). Course of illness, hippocampal function, and
hippocampal volume in major depression. Proc. Natl. Acad. Sci. USA. 100, 1387–92.
rs
Marrocco, J., Reynaert, M.L., Gatta, E., Gabriel, C., Mocaër, E., Di Prisco, S., Merega, E.,
Pittaluga, A., Nicoletti, F., Maccari, S., Morley-Fletcher, S., Mairesse, J., (2014). The
effects of antidepressant treatment in prenatally stressed rats support the glutamatergic
hypothesis of stress-related disorders. J. Neurosci. 34, 2015-24.
he
Martínez-Turrillas, R., Del Río, J., Frechilla, D., (2005). Sequential changes in BDNF mRNA
expression and synaptic levels of AMPA receptor subunits in rat hippocampus after
chronic antidepressant treatment. Neuropharmacol. 49, 1178-88.
Mauri, M.C., Ferrara, A, Boscati, L., Bravin, S, Zamberlan, F., Alecci, M., Invernizzi, G.,
(1998). Plasma and platelet amino acid concentrations in patients affected by major
is
depression and under fluvoxamine treatment. Neuropsychobiology 37, 124-9.
McClung, C.A., Nestler, E.J., (2008). Neuroplasticity mediated by altered gene expression.
bl
McEwen, B.S., Magarinos, A.M., Reagan, L.P., (2002). Structural plasticity and tianeptine:
Cellular and molecular targets. Eur. Psychiatry 17, 318–30.
McEwen, B.S., (2005). Glucocorticoids, depression, and mood disorders: structural
remodeling in the brain. Metabolism 54, 20-3.
Pu
McEwen, B.S., (2010). Stress, sex, and neural adaptation to a changing environment:
mechanisms of neuronal remodeling. Ann. N. Y. Acad. Sci. 1204, E38-59.
Michael, N., Erfurth, A., Ohrmann, P., Arolt, V., Heindel, W., Pfleiderer, B., (2003a).
Neurotrophic effects of electroconvulsive therapy: a proton magnetic resonance study of
the left amygdalar region in patients with treatment-resistant depression.
Michael, N., Erfurth, A., Ohrmann, P., Arolt, V., Heindel, W., Pfleiderer, B., (2003b).
a
Metabolic changes within the left dorsolateral prefrontal cortex occurring with
electroconvulsive therapy in patients with treatment resistant unipolar depression.
ov
Psychol. Med. 33, 1277-84.

Michelsen, K.A., van den Hove, D.L., Schmitz, C., Segers, O., Prickaerts, J., Steinbusch,
H.W. (2007). Prenatal stress and subsequent exposure to chronic mild stress influence
dendritic spine density and morphology in the rat medial prefrontal cortex. BMC
Neurosci. 8, 107.
N
Mitani, H., Shirayama, Y., Yamada, T., Maeda, K., Ashby Jr, C.R., Kawahara, R., (2006).
Correlation between plasma levels of glutamate, alanine and serine with severity of
depression. Prog. Neuropsychopharmacol. Biol. Psychiatry 30, 1155-8.
Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito, D., Barbiero, V.S.,
Bonifacino, T., Mallei, A., Baldelli, P., Racagni, G., Raiteri, M., Benfenati, F., Bonanno,
G., Popoli, M., (2010). Acute stress increases depolarization-evoked glutamate release in
the rat prefrontal/frontal cortex: the dampening action of antidepressants. PLoS One 5,
e8566.
c.
Musazzi, L., Racagni, G., Popoli, M., (2011). Stress, glucocorticoids and glutamate release:
effects of antidepressant drugs. Neurochem. Int. 59, 138-49.
Musazzi, L., Treccani, G., Mallei, A., Popoli, M., (2013). The action of antidepressants on the
In
glutamate system: regulation of glutamate release and glutamate receptors. Biol.
Nava, N., Treccani, G., Liebenberg, N., Chen, F., Popoli, M., Wegener, G., Nyengaard, J.R.,
(in press). Chronic desipramine prevents acute stress-induced reorganization of medial
prefrontal cortex architecture by blocking glutamate vesicle accumulation and excitatory
rs
synapse increase. Int. J. Neuropsychopharmacol in press.
Nowak, G., Pomierny-Chamioło, L., Siwek, A., Niedzielska, E., Pomierny, B., Pałucha-
Poniewiera, A., Pilc, A., (2014). Prolonged administration of antidepressant drugs leads
to increased binding of [(3)H]MPEP to mGlu5 receptors. Neuropharmacol. 84C, 46-51.
he
O' Connor, R.M., Pusceddu, M.M., Dinan, T.G., Cryan, J.F., (2013). Impact of early-life
stress, on group III mGlu receptor levels in the rat hippocampus: effects of ketamine,
electroconvulsive shock therapy and fluoxetine treatment. Neuropharmacol. 66, 236-41.
Pariante, C.M., Lightman, S.L., (2008). The HPA axis in major depression: classical theories
and new developments. Trends Neurosci. 31, 464–8.
is
Pittaluga, A., Raiteri, L., Longordo, F., Luccini, E., Barbiero, V.S., Racagni, G., Popoli, M.,
Raiteri, M., (2007). Antidepressant treatments and function of glutamate ionotropic
receptors mediating amine release in hippocampus. Neuropharmacol. 53, 27-36.
bl
Pittenger, C., Duman, R.S., (2008). Stress, depression, and neuroplasticity: a convergence of
mechanisms. Neuropsychopharmacol. 33, 88-109.
Popoli, M., Yan, Z., McEwen, B.S., Sanacora, G., (2012). The stressed synapse: the impact of
stress and glucocorticoids on glutamate transmission. Nat. Rev. Neurosci. 13, 22–37.
Pu
Price, J.L., Drevets, W.C., (2010). Neurocircuitry of mood disorders.

Neuropsychopharmacol. 35, 192–216.
Racagni, G., Popoli, M., (2008). Cellular and molecular mechanisms in the long-term action
of antidepressants. Dialogues Clin. Neurosci. 10, 385-400.
Radley, J.J., Sisti, H.M., Hao, J., Rocher, A.B., McCall, T., Hof, P.R., McEwen, B.S.,
Morrison JH. Chronic behavioral stress induces apical dendritic reorganization in
pyramidal neurons of the medial prefrontal cortex. Neuroscience. 2004;125:1-6.
a
Radley, J.J., Rocher, A.B., Rodriguez, A., Ehlenberger, D.B., Dammann, M., McEwen, B.S.,
Morrison, J.H., Wearne, S.L., Hof, P.R., (2008). Repeated stress alters dendritic spine
ov
morphology in the rat medial prefrontal cortex. J. Comp. Neurol. 507, 1141-50.
Rajkowska, G., Miguel-Hidalgo, J.J., Wei, J., Dilley, G., Pittman, S.D., Meltzer, H.Y.,
Overholser, J.C., Roth, B.L., Stockmeier, C.A., (1999). Morphometric evidence for
neuronal and glial prefrontal cell pathology in major depression. Biol. Psychiatry 45,
1085-98.
N
Reznikov, L.R., Grillo, C.A., Piroli, G.G., Pasumarthi, R.K., Reagan, L.P., Fadel, J., (2007).
Acute stress-mediated increases in extracellular glutamate levels in the rat amygdala:
differential effects of antidepressant treatment. Eur. J. Neurosci. 25, 3109-14.
Rubio, F.J., Ampuero, E., Sandoval, R., Toledo, J., Pancetti, F., Wyneken, U., (2013). Long-
term fluoxetine treatment induces input-specific LTP and LTD impairment and structural
plasticity in the CA1 hippocampal subfield. Front. Cell. Neurosci. 7, 66.
Rush, A.J., Trivedi, M., Fava, M., (2003). Depression, IV: STAR*D treatment trial for
depression. Am. J. Psychiatry 160, 237.
c.
Ryan, B., Musazzi, L., Mallei, A., Tardito, D., Gruber, S.H., El Khoury, A., Anwyl, R.,
Racagni, G., Mathé, A.A., Rowan, M.J., Popoli, M., (2009). Remodeling by early-life
stress of NMDA receptor-dependent synaptic plasticity in a gene-environment rat model
In
of depression. Int. J. Neuropsychopharmacol. 12, 553-9.
Sanacora, G., Gueorguieva, R., Epperson, C.N., Wu, Y.T., Appel, M., Rothman, D.L.,
Krystal, J.H., Mason, G.F., (2004). Subtype-specific alterations of gamma-aminobutyric
acid and glutamate in patients with major depression. Arch. Gen. Psychiatry 61, 705-13.
Sanacora, G., Treccani, G., Popoli, M., (2012). Towards a glutamate hypothesis of
rs
depression: an emerging frontier of neuropsychopharmacology for mood disorders.
Neuropharmacol. 62, 63–77.
Sanacora, G., Banasr, M., (2013). From pathophysiology to novel antidepressant drugs: glial
contributions to the pathology and treatment of mood disorders. Biol. Psychiatry 73,
he
1172-9.
Seo, M.K., Lee, C.H., Cho, H.Y., Lee, J.G., Lee, B.J., Kim, J.E., Seol, W., Kim, Y.H., Park,
S.W., (2014). Effects of antidepressant drugs on synaptic protein levels and dendritic
outgrowth in hippocampal neuronal cultures. Neuropharmacol. 79, 222-33.
Silva-Gómez, A.B., Rojas, D., Juárez, I., Flores, G., (2003). Decreased dendritic spine density
is
on prefrontal cortical and hippocampal pyramidal neurons in postweaning social isolation
rats. Brain Res. 983, 128-36.
Skolnick, P., Layer, R.T., Popik, P., Nowak, G., Paul, I.A., Trullas, R., (1996). Adaptation of
bl
N-methyl-D-aspartate (NMDA) receptors following antidepressant treatment:
implications for the pharmacotherapy of depression. Pharmacopsychiatry 29, 23-6.
Sudhof, T.C., (2009). Rothman JE. Membrane fusion: grappling with SNARE and SM
proteins. Science 323, 474–7.
Pu
Sun, J.D., Liu, Y., Yuan, Y.H., Li, J., Chen, N.H., (2012). Gap junction dysfunction in the
prefrontal cortex induces depressive-like behaviors in rats. Neuropsychopharmacol. 37,
1305-20.
Svenningsson, P., Tzavara, E.T., Witkin, J.M., Fienberg, A.A., Nomikos, G.G., Greengard,
P., (2002). Involvement of striatal and extrastriatal DARPP-32 in biochemical and
behavioral effects of fluoxetine (Prozac). Proc. Natl. Acad. Sci. USA 99, 3182-7.
Szasz, B.K., Mike, A., Karoly, R., Gerevich, Z., Illes, P., Vizi, E.S., Kiss, J.P., (2007). Direct
a
inhibitory effect of fluoxetine on N-methyl-D-aspartate receptors in the central nervous

system. Biol. Psychiatry. 62, 1303-9.
ov
Treccani, G., Musazzi, L., Perego, C., Milanese, M., Nava, N., Bonifacino, T., Lamanna, J.,
Malgaroli, A., Drago, F., Racagni, G., Nyengaard, J.R., Wegener, G., Bonanno, G.,
Popoli, M.. (2014a). Stress and corticosterone increase the readily releasable pool of
glutamate vesicles in synaptic terminals of prefrontal and frontal cortex. Molecul.
N
Treccani, G., Musazzi, L., Perego, C., Milanese, M., Nava, N., Bonifacino, T., Lamanna, J.,
Malgaroli, A., Drago, F., Racagni, G., Nyengaard, J.R., Wegener, G., Bonanno, G.,
Popoli, M., (2014b). Acute stress rapidly increases the readily releasable pool of
glutamate vesicles in prefrontal and frontal cortex through non-genomic action of

corticosterone. Mol. Psychiatry 19, 401.
Vizi, E.S., Fekete, A., Karoly, R., Mike, A., (2010). Non-synaptic receptors and transporters
involved in brain functions and targets of drug treatment. Br. J. Pharmacol. 160, 785-
809.
c.
Vizi, E.S., Kisfali, M., Lőrincz, T., (2013). Role of nonsynaptic GluN2B-containing NMDA
receptors in excitotoxicity: evidence that fluoxetine selectively inhibits these receptors
and may have neuroprotective effects. Brain Res. Bull. 93, 32-8.
In
Wang, S.J., Su, C.F., Kuo, Y.H., (2003). Fluoxetine depresses glutamate exocytosis in the rat
cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels.
Synapse 48, 170-7.
Wieronska, J.M., Kłak, K., Pałucha, A., Branski, P., Pilc, A., (2007). Citalopram influences
mGlu7, but not mGlu4 receptors expression in the rat brain hippocampus and cortex.
rs
Brain Res. 1184, 88–95.
Wolak, M., Siwek, A., Szewczyk, B., Poleszak, E., Pilc, A., Popik, P., Nowak, G., (2013).
Involvement of NMDA and AMPA receptors in the antidepressant-like activity of
antidepressant drugs in the forced swim test. Pharmacol. Rep. 65, 991-7.
he
Yuen, E.Y., Liu, W., Karatsoreos, I.N., Ren, Y., Feng, J., McEwen, B.S., Yan, Z., (2011).
Mechanisms for acute stress-induced enhancement of glutamatergic transmission and
working memory. Mol. Psychiatry 16, 156-70.
Yüksel, C., Öngür, D., (2010). Magnetic resonance spectroscopy studies of glutamate-related
abnormalities in mood disorders. Biol. Psychiatry. 68, 785-94.
is
Zink, M., Rapp, S., Donev, R., Gebicke-Haerter, P.J., Thome, J., (2011). Fluoxetine treatment
induces EAAT2 expression in rat brain. J. Neural. Transm. 118, 849-55.
bl
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Chapter 11
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Fluoxetine and Other SSRI
Antidepressants Potentiate Addiction-
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Related Gene Regulation by
Psychostimulant Medications
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Vincent Van Waes and Heinz Steiner
Department of Cellular and Molecular Pharmacology,
The Chicago Medical School, Rosalind Franklin University of Medicine and Science,
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North Chicago, IL, US
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Abstract
Psychostimulants such as methylphenidate and antidepressants such as fluoxetine are
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widely used in the treatment of various mental disorders or as cognitive enhancers. These
medications are often also combined, for example, to treat co-morbid disorders or to
accelerate antidepressant therapy. There is a considerable body of evidence from
preclinical studies indicating that individually these psychotropic medications can have
detrimental effects on brain and behavior, especially when given during the sensitive
period of brain development. However, very few studies have investigated possible
interactions between these drugs. This is surprising given that their combined
neurochemical effects (enhanced dopamine and serotonin neurotransmission) are similar
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to effects of illicit drugs such as cocaine and amphetamine. This chapter reviews recent
studies on the molecular effects of such combination treatments in the forebrain in
adolescent rats. Our results show that combined methylphenidate plus fluoxetine
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treatment produces potentiated changes in gene regulation in corticostriatal circuits,

effects that mimic those of cocaine exposure. These neuronal changes are present across
many functional domains of the striatum, including distinct parts of the nucleus
accumbens, but they are most robust in the lateral sensorimotor striatum. These findings
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
Present address: EA481 Laboratory of Integrative and Clinical Neuroscience, University of Franche-Comté,
Besançon, France.

Corresponding Author address: E-mail: Heinz.Steiner@rosalindfranklin.edu.
208 Vincent Van Waes and Heinz Steiner
suggest the potential for an enhanced addiction liability and other behavioral
consequences for such combination treatments.
Keywords: methylphenidate, fluoxetine, psychostimulant, gene regulation, striatum
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Introduction
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Psychotropic medications are increasingly used in children and adolescents, for example,
in the treatment of attention deficit/hyperactivity disorder (ADHD) and major depressive
disorder (MDD). These medications include selective serotonin reuptake inhibitor (SSRI)
antidepressants such as fluoxetine (FLX; Prozac), which is approved for treatment of
pediatric MDD (Iversen, 2006). Other often used drugs in pediatric populations are
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psychostimulants such as methylphenidate (MP; Ritalin), an effective agent for the
management of ADHD (Castle et al., 2007; Kollins, 2008). ADHD is diagnosed in up to 7%
of school-age children in the United States (Kollins, 2008), and a recent study estimated that
in 2008 approximately 3 million children between 4 and 17 years of age in the US alone were
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treated with psychostimulant medications for ADHD (Swanson et al., 2011).
In addition to the use of individual drugs, co-exposure to different psychotropic
medications is also prevalent. For example, MP plus SSRI combinations are indicated in the
treatment of co-morbid ADHD and MDD (Gammon and Brown, 1993; Rushton and
Whitmire, 2001; Safer et al., 2003), which occurs with up to 40% prevalence in pediatric
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ADHD (Waxmonsky, 2003; Spencer, 2006). These combination treatments can be effective
in patients who do not adequately respond to individual drugs, and the combination appears to
have no additional overt side effects (e.g., Gammon and Brown, 1993). MP+SSRI
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concomitant treatments are also used as augmentation therapy in MDD (e.g., Nelson, 2007;
Ishii et al., 2008; Ravindran et al., 2008), as acceleration treatment with SSRIs (e.g.,
Lavretsky et al., 2003), or to treat sexual dysfunction (e.g., Csoka et al., 2008).
Co-exposure to these drug classes also occurs in patients on antidepressants who use MP
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recreationally or as a cognitive enhancer (Kollins, 2008; Swanson and Volkow, 2008; Wilens
et al., 2008). This nonmedical use of MP can produce a particularly uncontrolled form of
potentially high-level exposure, as MP may be snorted or injected (e.g., Teter et al., 2006; see
Steiner and Van Waes, 2013). According to surveys, up to 10-20% or more of college
students use MP to improve concentration, stay awake to study, or party (White et al., 2006;
Kollins, 2008; Wilens et al., 2008). The 2011 National Survey on Drug Use and Health
(NSDUH) reports that approximately 1 million persons age 12 or older in the US admitted
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current nonmedical use of prescription psychostimulants (SAMHSA, 2012). The rate of co-
exposure in patients on SSRIs who also use MP recreationally or as a cognitive enhancer is
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unknown.
Use of psychotropic drugs during development and maturation of the brain is of concern.
A number of studies in animal models show that these drugs can induce maladaptive
neurobehavioral changes suggestive of an increased risk for drug addiction and other
neuropsychiatric disorders later in life (for reviews, see Carlezon and Konradi, 2004;
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Andersen, 2005; Carrey and Wilkinson, 2011; Marco et al., 2011). However, despite
prevalent MP and FLX use, the neurobiological consequences of their combined use during
development are unknown (Bhatara et al., 2004; Spencer, 2006). This is particularly striking
Fluoxetine and Other SSRI Antidepressants Potentiate Addiction … 209
because together MP and FLX may have pharmacodynamic properties similar to cocaine
(Steiner and Van Waes, 2013). MP and cocaine both inhibit the dopamine transporter
(Volkow et al., 2002). Differences between these two drugs may be related to their
differential action on the serotonin transporter, which is inhibited by cocaine, but not by MP.
MP has minimal affinity for the serotonin transporter and does not produce serotonin
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overflow (e.g., Pan et al., 1994; Kuczenski and Segal, 1997; Segal and Kuczenski, 1999; Han
and Gu, 2006; see Yano and Steiner, 2007). Therefore, combined use of MP and SSRIs may
induce emergent (―cocaine-like‖) effects by simultaneously inhibiting the reuptake of both
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dopamine and serotonin.
A role for serotonin in the molecular effects of psychostimulants such as cocaine is well
established (see Steiner and Van Waes, 2013). Dopamine is critical for gene regulation
induced by cocaine (Steiner and Van Waes, 2013), as it is for gene regulation by MP (Yano et
al., 2006; Alburges et al., 2011). However, it has been demonstrated that serotonin contributes
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to these cocaine effects. For example, attenuation of the serotonin neurotransmission by
transmitter depletion (Bhat and Baraban, 1993) or receptor antagonism (Lucas et al., 1997;
Castanon et al., 2000) reduces immediate-early gene (IEG) induction by cocaine in the
striatum. Adding serotonin (SSRI) to dopamine action (MP) may thus produce more
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―cocaine-like‖ molecular changes than MP alone. Indeed, a series of studies shows that
treatment with SSRIs such as FLX in conjunction with MP potentiates MP-induced gene
expression, including that of IEGs and neuropeptides, in the striatum (Steiner and Van Waes,
2013). These findings are summarized in the following sections.
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Methylphenidate-Induced Gene Regulation in the
Adolescent Striatum: Potentiation by Fluoxetine
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There is consensus that changes in gene regulation are critical for psychostimulant
addiction (Renthal and Nestler, 2008) and that excessive activation of the dopamine
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neurotransmission is key for these molecular changes (see Steiner and Van Waes, 2013).
Because MP acts as a dopamine reuptake inhibitor and such drugs are known to produce
various molecular changes in the striatum, several labs have investigated effects of MP on
striatal gene regulation and have compared these with the effects of psychostimulants such as
cocaine (Steiner and Van Waes, 2013). Early microarray studies in adolescent rats reported
that acute and repeated treatment even with a dose as low as 2 mg/kg (i.p.) of MP (which is
considered in the upper range or above of clinically relevant doses) altered the expression of
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hundreds of genes in the striatum (Adriani et al., 2006a; Adriani et al., 2006b). Further studies
confirmed that, similar to other psychostimulants, MP affects genes that encode transcription
factors (IEGs), neurotransmitter receptors, neuropeptides, postsynaptic density proteins, and
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other signaling- or plasticity-related molecules (Yano and Steiner, 2007; Carrey and
Wilkinson, 2011; Marco et al., 2011). Some of these molecular changes persist well past the
termination of the drug treatment, into the adulthood of the animals (Adriani et al., 2006a;
Adriani et al., 2006b; see also Chase et al., 2007; Warren et al., 2011).
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Other comparisons indicated, however, that some genes, including those encoding the
opioid peptides dynorphin and enkephalin and the IEG Homer1a, are less affected by MP
than by drugs such as cocaine (Yano and Steiner, 2005b; Cotterly et al., 2007; see Steiner and
Van Waes, 2013). It is known that these opioid peptide genes (e.g., Morris et al., 1988;
Walker et al., 1996; Horner et al., 2005), among other genes (Keefe and Horner, 2010), are
also regulated by serotonin. Therefore, given the lack of serotonin effects for MP (see above),
MP‘s reduced impact on such addiction-related gene regulation might not be surprising. This
would also be consistent with a lower addiction liability for MP compared with other
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psychostimulants (Svetlov et al., 2007). On the other hand, these findings raised the question
whether increasing serotonin action (by SSRIs) in conjunction with MP treatment would
increase MP-induced gene regulation and addiction liability. We thus investigated whether
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SSRIs modified MP effects on gene regulation in the striatum.
These studies were performed in adolescent (postnatal days 35-39) male rats. Gene
expression was measured by in situ hybridization histochemistry in 23 striatal sectors on three
rostrocaudal levels (Figure 1), which cover all major functional domains of the striatum (18
sectors represent the caudate-putamen, 5 sectors the nucleus accumbens). These sectors are
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mostly defined by their principal cortical inputs and thus allow assignment of molecular
changes to specific corticostriatal circuits (Willuhn et al., 2003; Yano and Steiner, 2005a).
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Figure 1. Schematic illustration of the 23 striatal sectors in which drug effects on gene regulation were
assessed. Gene expression was measured in coronal sections from three rostrocaudal levels [―rostral,‖ at
approximately +1.6 mm rostral to bregma (Paxinos and Watson, 1998); ―middle,‖ +0.4; ―caudal,‖ -0.8].
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These striatal sectors are defined mostly by their predominant cortical inputs (see Willuhn et al., 2003;
Yano and Steiner, 2005a). These inputs are represented by arrows for the sectors on the middle level.
Sensorimotor (dark grey), ―associative‖ (medium grey) and limbic sectors (light grey) are indicated.
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Cortical areas: CG, cingulate; I, insular; LO, lateral orbital; M2, medial agranular; M1, primary motor;
P, piriform; PL, prelimbic; SS, somatosensory.
Potentiation of Methylphenidate Effects on Immediate-Early Genes

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We first assessed the expression of IEGs (Zif268, c-Fos). IEGs are useful markers for cell
activation by neuronal activity and drug treatments due to their rapid and transient induction
(Sharp et al., 1993; Chaudhuri, 1997). However, these IEGs are also of interest because they
encode transcription factors (molecules that regulate the activity of other genes) and thus
directly participate in neuroplasticity (e.g., Knapska and Kaczmarek, 2004).
Our results demonstrate that acute treatment with the SSRI FLX (5 mg/kg, i.p.) in
conjunction with MP (5 mg/kg) robustly potentiates MP-induced expression of Zif268 and c-
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Fos throughout most of the striatum (Steiner et al., 2010; Van Waes et al., 2010; Figure 2).
FLX (5 mg/kg) by itself had no effect. Our regional analysis shows that this potentiation (i.e.,
the difference between MP and MP+FLX effects) of IEG induction is most pronounced in the
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dorsal/lateral (sensorimotor) striatum (Figure 2). However, significant but smaller
potentiation of gene regulation is also seen in select subregions of the nucleus accumbens
(medial core, lateral shell; Figure 2) (Van Waes et al., 2010). Lower doses of FLX (2.5
mg/kg; unpublished results) or MP (2 mg/kg; Van Waes et al., 2010) produced weaker
potentiation. We also assessed effects of another SSRI. Citalopram (5 mg/kg) produced
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potentiation of MP-induced IEG expression comparable to FLX (5 mg/kg) (Van Waes et al.,
2010), thus generalizing this effect to another often used SSRI.
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Figure 2. Fluoxetine potentiates acute Zif268 induction by methylphenidate in the striatum (Van Waes
et al., 2010). (A) Illustrations of film autoradiograms depict Zif268 expression in coronal sections from
the middle striatum in rats that received an injection of vehicle (V), methylphenidate (MP, 5 mg/kg),
fluoxetine (FLX, 5 mg/kg) or MP+FLX and were killed 40 min after drug administration. (B) Maps
show the distribution of the increases (vs. V) in Zif268 expression in the rostral, middle and caudal
striatum after MP, FLX or MP+FLX administration. Potentiation (POT) denotes the difference between
MP+FLX and MP treatment. The data are expressed relative to the maximal increase in Zif268
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expression (% of max.). Sectors with a significant difference [vs. vehicle (V) controls or MP+FLX vs.
MP (POT)] (p<0.05) are coded as indicated. Sectors without significant difference are in white. (C)
Mean density values (mean±SEM) measured in the whole striatum on the middle level are given for
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these groups. FLX potentiated Zif268 induction by MP, but had no effect by itself. This FLX
potentiation was most robust in the lateral striatum (see Van Waes et al., 2010). ***p<0.001, vs. V or
FLX; ###p<0.001, MP+FLX vs. MP (potentiation).
Given that in clinical treatments these psychotropic medications are administered

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chronically, the molecular consequences of repeated treatment with MP and FLX are relevant
for long-term neurobehavioral effects. Repeated psychostimulant treatments cause various
molecular changes (neuroadaptations), including upregulation of expression for some genes
and repression for others (McClung and Nestler, 2003; Yuferov et al., 2005; Heiman et al.,
2008). Most often, the exact consequences for cellular function of such alterations in gene
expression are unknown. However, the long-term functional integrity of neurons depends on
balanced regulation of gene expression, as cellular components have limited half-lives and
must be replenished. It is assumed that disruption of such homeostatic regulation by
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psychostimulants results in deficient neuronal function that contributes to behavioral
manifestations of psychostimulant addiction (e.g., Hyman and Nestler, 1996; Nestler, 2001).
One of the best-established neuroadaptations produced by repeated cocaine/amphetamine
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treatment is blunting (repression) of IEG inducibility; that is, a psychostimulant challenge
given after repeated psychostimulant pretreatment will typically produce attenuated (blunted)
IEG induction compared with acute induction (see Steiner and Van Waes, 2013). This effect
is the result of epigenetic modifications (Renthal et al., 2008) or other neuronal changes
produced by the repeated drug treatment (Steiner and Van Waes, 2013).
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Such gene blunting is a robust marker for altered gene regulation after repeated drug
treatments. Several labs showed that repeated MP treatment produces IEG blunting in the
striatum (e.g., Brandon and Steiner, 2003; Chase et al., 2003; Chase et al., 2005; Cotterly et
al., 2007). It was thus of interest to investigate whether this IEG blunting is modified by
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coadministration of FLX together with MP.
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Figure 3. Fluoxetine potentiates repeated methylphenidate-induced changes in Zif268 induction in the

striatum (Van Waes et al., 2014). (A) Illustrations of film autoradiograms depict Zif268 expression in
the middle striatum in rats that received 5 daily injections of vehicle (V), methylphenidate (MP, 5
mg/kg), fluoxetine (FLX, 5 mg/kg) or MP+FLX, followed on day 6 by a vehicle (/V) injection or a
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cocaine challenge (/C, 25 mg/kg). (B) Maps depict the distribution of blunting [i.e., the decrease vs.
acute cocaine, V/C] for Zif268 induction in the rostral, middle and caudal striatum in MP/C, FLX/C, or
MP+FLX/C groups. The differences between MP+FLX/C and MP/C treatments are shown on the right
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(POT). The data are expressed relative to the maximal decrease in Zif268 expression observed (% of
max.). Sectors with a significant decrease [vs. acute cocaine controls (V/C) or MP+FLX/C vs. MP/C
(POT)] (p<0.05) are coded as indicated. Sectors without significant difference are in white. (C) Mean
density values (mean±SEM) for Zif268 expression in the whole middle striatum are shown for these
treatment groups. Repeated MP pretreatment produced some blunting of Zif268 induction by cocaine.
Repeated FLX potentiated this MP-induced Zif268 blunting (MP+FLX), but had no effect by itself.
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***p<0.001, vs. V or FLX; ##p<0.01, MP+FLX vs. MP (potentiation).

We assessed IEG induction (Zif268, Homer1a) by a cocaine challenge (25 mg/kg) one
day or 14 days after a 5-day repeated treatment with MP (5 mg/kg) and/or FLX (5 mg/kg) in
adolescent rats. Zif268 encodes a transcription factor that is critical, for example, for
reconsolidation of drug (cocaine) memories (Lee et al., 2005; Théberge et al., 2010), and
other aspects of neuroplasticity (Knapska and Kaczmarek, 2004). Homer1a is a synaptic
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plasticity modulator (Thomas, 2002) that is also implicated in drug-induced neuroplasticity
related to addiction (for review, see Szumlinski et al., 2008). Previous studies had shown that
repeated cocaine treatment (5 days) reliably blunts the induction of both Zif268 and Homer1a
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in the striatum (Unal et al., 2009). In contrast, repeated treatment with MP alone produced
blunting of Zif268 induction (Brandon and Steiner, 2003; Cotterly et al., 2007), but had
minimal effects on Homer1a induction (Cotterly et al., 2007). Gene expression was again
mapped throughout the striatum in order to identify the functional domains affected by these
treatments. Our findings demonstrate that the 5-day repeated pretreatment with MP alone
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produced minimal IEG blunting one day later, while FLX alone had no effect (Figure 3). In
contrast, adding FLX to MP strongly potentiated blunting of Zif268 (Figure 3) and Homer1a
induction by the cocaine challenge (Van Waes et al., 2014).
Our regional analysis showed that the potentiation of blunting (i.e., the difference
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between MP and MP+FLX effects) for both Zif268 and Homer1a occurred in the same 19 (of
23) striatal sectors, with maximal potentiation in the sensorimotor striatum (Figure 4).
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Figure 4. Regional correlations for potentiation of gene regulation effects for different genes and for
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acute vs. repeated treatment. (Left) Relationship between the potentiation of blunting for Zif268
induction (by cocaine) vs. that for Homer1a induction after repeated MP+FLX treatment (Van Waes et
al., 2014). The scatterplot shows that, in the 23 striatal sectors, the difference in induction between
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MP+FLX/C and MP/C animals (potentiation) was correlated between Zif268 and Homer1a (r = 0.845).
The values are expressed as the percentages of the maximal potentiation for each gene. (Right)
Relationship between the potentiation of acute Zif268 induction (difference between MP+FLX and MP
groups; Van Waes et al., 2010) and the potentiation of Zif268 blunting (chronic; difference between
MP+FLX/C and MP/C groups; Van Waes et al., 2014), in the 23 sectors (r = 0.616). The scatterplot
shows that across the 23 sectors the magnitude of the potentiation of gene regulation was correlated
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between acute and repeated MP+FLX treatments. The open diamonds indicate sensorimotor sectors.
**p<0.01, ***p<0.001.
Overall, there was also a positive regional correlation between potentiation of acute
Zif268 induction (by MP+FLX) (Van Waes et al., 2010) vs. potentiation of blunting of Zif268
induction (by the cocaine challenge) after the repeated MP+FLX treatment (Van Waes et al.,
2014) (Figure 4). This latter correlation demonstrates that occurrence and magnitude of such
neuroadaptations after repeated MP+FLX treatment are predicted by the acute potentiation of
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gene induction, which thus serves as an acute marker for the risk for such neuroadaptations
(see Steiner and Van Waes, 2013).
Our further results show that this potentiated gene blunting by repeated MP+FLX
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treatment endures for at least 14 days after the repeated treatment, into young adulthood of
the animals (Beverley et al., 2014), similar to gene blunting by repeated cocaine treatment
(Unal et al., 2009). In summary, these findings demonstrate that SSRIs given chronically in
combination with MP produce more robust and enduring neuroadaptations in striatal gene
regulation than MP alone.
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Potentiation of Methylphenidate Effects on Neuropeptide Expression
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In other studies, we evaluated the impact of acute and repeated treatment with MP and
FLX on the expression of the neuropeptides substance P, dynorphin and enkephalin in the
striatum. These neuropeptides are selectively expressed by neurons of the direct (substance P,
dynorphin) and indirect (enkephalin) striatal output pathways (i.e., the striatonigral and
striatopallidal projection neurons, respectively; Steiner and Gerfen, 1998) and thus serve as
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cell type markers for drug actions in these neurons. These neuropeptides also function as
neuromodulators (Steiner and Gerfen, 1998) and are thought to participate in addiction
processes (Shippenberg et al., 2007). For example, there is evidence indicating that increased
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dynorphin function in striatonigral neurons after repeated psychostimulant exposure
contributes to anhedonia and depression during withdrawal (Nestler and Carlezon, 2006;
Wiley et al., 2009). Notably, increased dynorphin expression has also been found in human
cocaine addicts (Hurd and Herkenham, 1993; Frankel et al., 2008).
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We used these neuropeptides as markers to determine which striatal output pathways are
affected by MP and FLX. Previous findings showed that acute treatment with MP alone has
robust stimulatory effects on substance P and relatively minor effects on dynorphin
expression (both in striatonigral neurons), and minimal or no effects on enkephalin expression
(striatopallidal neurons) (Yano and Steiner, 2007). Adding FLX to acute MP treatment
confirmed differential effects for these neuropeptides. FLX potentiated MP-induced
expression of substance P (Figure 5) and, to some degree, dynorphin, but had no effect on
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enkephalin (Van Waes et al., 2012). These findings thus suggested some selectivity for gene
regulation in the direct pathway by the acute drug treatment.
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Drug-induced changes in neuropeptide expression are typically more pronounced (better

discernible) after repeated treatments (in part due to the long half-life of their mRNAs and
resulting accumulation with repeated treatments). This is also the case for MP (Steiner and
Van Waes, 2013). For example, repeated high-dose MP treatment [10 mg/kg, once daily, 7
days (Brandon and Steiner, 2003); 10 mg/kg, 4 injections over 6 h (Alburges et al., 2011)] did
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produce robust increases in dynorphin mRNA and peptide levels in striatonigral neurons (if
more limited compared to repeated cocaine treatment; Steiner and Van Waes, 2013). We
assessed the effects of FLX with a more moderate repeated MP treatment (5 mg/kg, once
daily, 5 days). Our results show that neither repeated treatment with MP alone, nor with FLX
(5 mg/kg) alone produced changes in dynorphin or enkephalin expression in the striatum
(Figure 6). In contrast, combined MP+FLX treatment significantly increased the expression
of dynorphin and, to a lesser extent, also enkephalin (Fig. 6) (Van Waes et al., 2015).
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Figure 5. Fluoxetine potentiates acute induction of substance P expression by methylphenidate in the
striatum (Van Waes et al., 2012). (A) Illustrations of film autoradiograms depict substance P expression
in sections from the middle striatum in rats that received an injection of vehicle (V), methylphenidate
(MP, 5 mg/kg), fluoxetine (FLX, 5 mg/kg) or MP+FLX and were killed 90 min after drug
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administration. (B) Mean density values (mean±SEM) measured in the whole striatum on the middle
level are shown for these groups. FLX potentiated substance P induction by MP, but had no effect by
itself. This FLX potentiation was most robust in the lateral striatum. **p<0.01, ***p<0.001, vs. V or
FLX; ##p<0.01, MP+FLX vs. MP (potentiation).
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Figure 6. Fluoxetine potentiation of repeated methylphenidate-induced changes in dynorphin (left) and

enkephalin expression (right) in the striatum. Mean density values (mean±SEM) are shown for rats that
received 5 daily injections of vehicle (V), MP (5 mg/kg), FLX (5 mg/kg) or MP+FLX, and were killed
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2 h later. Repeated MP+FLX treatment produced increased expression of both opioid peptides, while
neither MP nor FLX alone had a significant effect. **p<0.01, ***p<0.001 vs. V or FLX; ##p<0.01,
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p<0.001, MP+FLX vs. MP (potentiation).
These findings therefore indicate that repeated combined treatment does produce
significant molecular changes in both striatal output pathways, unlike repeated treatment with
MP alone (Brandon and Steiner, 2003), which favors the direct pathway. These effects of
repeated combined treatment are thus also more ―cocaine-like‖ than those of MP alone, as
cocaine impacts gene regulation in both pathways (Yano and Steiner, 2007). The functional
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significance of these molecular changes in striatal output pathways remains to be determined.
Discussion
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The findings reviewed here show that adolescent treatment with SSRIs in conjunction
with psychostimulants such as MP in rats produces more robust neuroadaptations in striatal
circuits than MP alone. These effects include potentiated changes in gene regulation in
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projection neurons of the striatum and the nucleus accumbens. These neuronal changes are
likely not the sole neurobiological consequences of MP+FLX co-exposure. For example,
others reported alterations in second messenger signaling in dopamine neurons of the
midbrain after repeated MP+FLX treatment in juvenile rats (Warren et al., 2011).
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Importantly, at least some of these neuronal changes endured into the adulthood of the
animals.
Behavioral Consequences of Potentiated Gene Regulation in the Striatum

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The exact behavioral consequences of these neuronal changes in striatal circuits remain to
be fully explored. An early study reported that adding FLX to acute MP treatment enhanced
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MP-induced locomotor activity in an open-field test in rats (Borycz et al., 2008). In our
studies, using slightly different MP and FLX doses, we found that FLX potentiated behavioral
stereotypies induced by MP (Van Waes et al., 2010; Beverley et al., 2014). Both hyperactivity
and behavioral stereotypies can reflect dysfunction in sensorimotor striatal circuits (Graybiel
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et al., 2000). Such motor stereotypies may be related to compulsive behavior in humans
(Graybiel et al., 2000; see Van Waes et al., 2010, for discussion).
Gene expression markers pinpoint neuronal circuits affected by drugs. In our studies,
gene regulation altered by MP+FLX was present across most functional domains of the
striatum. However, maximal MP+FLX effects occurred in the lateral sensorimotor striatum,
whereas the maximal effects of MP alone were centered more medially, involving striatal
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circuits that receive inputs from prefrontal cortical areas (Willuhn et al., 2003). With regard to
the affected corticostriatal circuits, the MP+FLX effects were thus again more ―cocaine-like‖
than those of MP alone (see Steiner and Van Waes, 2013).
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According to findings in animal models, the medial striatum subserves goal-directed

behavior, whereas the lateral, sensorimotor striatum is critical for stimulus-response (habit)
learning (Graybiel, 1995; Packard and Knowlton, 2002). Drug-induced molecular changes in
the lateral sensorimotor striatum are implicated in aberrant habit formation and compulsive
behavior in drug addiction (e.g., Berke and Hyman, 2000; Everitt and Robbins, 2005; Belin
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and Everitt, 2010), as well as in relapse to drug seeking after previous drug exposure (e.g.,
Vanderschuren et al., 2005; See et al., 2007) (see Steiner and Van Waes, 2013, for
discussion).
Enhanced Addiction Liability?
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As discussed, the observed MP+FLX-induced changes in gene regulation in the striatum
mimic to some degree molecular effects of psychostimulants such as cocaine, which are
considered part of the molecular basis of psychostimulant addiction (e.g., Berke and Hyman,
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2000; Nestler, 2001; Nestler, 2012). MP by itself can induce some of these molecular changes
(Steiner and Van Waes, 2013), but the addition of SSRIs to MP typically potentiates such
gene regulation and induces others. Our findings may thus suggest the potential of an
enhanced addiction liability for the combination treatment. If there is such a risk, this
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potential would likely be greater for MP use as a cognitive enhancer or recreational drug,
which often involves higher doses and routes of administration that result in faster uptake and
higher drug serum levels. Both of these are risk factors for addiction-related gene regulation
(Samaha and Robinson, 2005; see Steiner and Van Waes, 2013, for discussion).
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It is unclear from clinical studies that MP use in ADHD treatment (presumably oral use,
which produces slower bioavailability and lower serum levels) increases the risk for
subsequent substance use disorder, at least within a few years of the treatment when these
studies were conducted (Barkley et al., 2003; Wilens et al., 2003; Kollins, 2008). There are no
studies to date that assessed the risk of cognitive enhancer use.
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Preclinical studies addressed the question of an enhanced abuse/addiction liability in two
animal models of addiction, the cocaine self-administration model and the cocaine-
conditioned place preference (CPP) task in rodents. In the former, animals perform an operant
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response (lever pressing or nose poking) to obtain an intravenous infusion of cocaine. In the
latter, animals receive an injection of cocaine in a distinctive environment and are
subsequently tested (without drug) to see whether they now prefer (or avoid) the cocaine-
paired environment, which would indicate rewarding or aversive, respectively, properties for
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cocaine.
Regarding the exposure to MP alone, results from such studies are ambiguous (see
Steiner and Van Waes, 2013, for review). On the one hand, there is good evidence that
pretreatment with MP alone facilitates subsequent cocaine seeking and taking in the cocaine
self-administration model (Brandon et al., 2001; Schenk and Izenwasser, 2002; Crawford et
al., 2011), as occurs with cocaine pretreatment. This effect would suggest an increased
abuse/addiction risk (O'Connor et al., 2011).
a
On the other hand, results with cocaine place preference conditioning in the CPP task
indicate reduced rewarding (or even aversive) effects of cocaine after repeated MP
ov
pretreatment in juvenile rats (Andersen et al., 2002; Wiley et al., 2009; Warren et al., 2011).
These latter results would suggest that cocaine is now less preferred or even avoided, and
these findings have been taken as evidence for a ―protective‖ effect of MP pretreatment.
However, it remains unclear to what degree these apparently contradictory effects (i.e.,
enhanced intake vs. reduced preference) in these animal models were due to experimental
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variables such as the age at time of drug exposure (Meririnne et al., 2001), and/or dose and
length of treatment, or other effects. Moreover, it should be noted that pretreatment with
cocaine itself can also produce conditioned place aversion in the CPP test (Carlezon et al.,
2003).
Regarding the MP+SSRI co-exposure, it is presently unknown whether pretreatment with
SSRIs in conjunction with MP will modify subsequent cocaine self-administration in rats.
However, MP+FLX pretreatment has recently been shown to reverse the cocaine aversion in
c.
the CPP task. When FLX was added to MP in juvenile rats, they subsequently showed an
enhanced preference for the cocaine-paired environment, in contrast to the cocaine avoidance
expressed by the MP only-pretreated controls (Warren et al., 2011). This finding of an
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enhanced preference (sensitivity to cocaine reward) does suggest an increased abuse/addiction
risk for the drug combination (for review, see Steiner et al., 2014).
Significantly, this study raised another important issue – age-dependent effects. The
enhanced cocaine preference after MP+FLX pretreatment was more robust in adult rats that
were pretreated as juveniles than in juveniles tested shortly after their pretreatment (Warren et
rs
al., 2011). This finding is consistent with results from molecular studies on the developmental
effects of drug exposure. For example, prenatal cocaine exposure has been found to produce
aberrant molecular signaling and gene expression in the striatum, an effect that emerged only
in adolescent animals and was maximal in adults, but was not present in juveniles (Tropea et
he
al., 2008). Such late-emerging neurobehavioral deficits, if confirmed, would underscore the
need for reassessment of the long-term effects of MP (and MP+FLX) exposure at a later time
point in life, for ADHD treatment (see above) or cognitive enhancers.
Finally, while the focus of this discussion has been on behavioral changes relevant for
addiction, it should be noted that adverse effects of the combined MP+FLX treatment in
is
juvenile rats may not be limited to drug abuse/addiction (Steiner et al., 2014). The study by
Warren et al. (2011) showed that, in addition to the enhanced cocaine reward sensitivity (see
above), the MP+FLX-pretreated rats also displayed enhanced sensitivity to natural reward
bl
(sucrose) (Warren et al., 2011). Moreover, other findings were indicative of deficits in mood-
related behaviors and reactivity to stress (Warren et al., 2011).
In summary, while speculative, these findings in animal studies suggest a potentially
increased addiction liability and possibly other behavioral effects for such combination
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treatments. Clinical studies are needed to evaluate these potential risks.
Conclusion
In conclusion, the findings summarized in this chapter indicate that MP plus SSRI
a
combination treatments in adolescent rats can produce a variety of molecular changes that
mimic, in part, neuroadaptations induced by illicit drugs such as cocaine. Most gene
regulation effects of MP+SSRI treatments are potentiations of changes that can be induced by
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MP alone. Others are emergent with the combination treatment. In animal models, these
neuronal changes are associated with behavioral changes indicative of an increased risk for
substance use disorder, and abnormal reward sensitivity and mood regulation, with some of
these effects enduring into adulthood. These findings highlight the need for more in-depth
investigation of the effects of such combination therapies, especially when they are used in
N
the developing brain.

Acknowledgments
This work was supported in part by National Institute on Drug Abuse grants DA031916,
DA011261 (to H.S.).
c.
References
In
Adriani, W., Leo, D., Greco, D., Rea, M., di Porzio, U., Laviola, G., Perrone-Capano, C.,
(2006a). Methylphenidate administration to adolescent rats determines plastic changes in
reward-related behavior and striatal gene expression. Neuropsychopharmacology 31,
1946-1956.
Adriani, W., Leo, D., Guarino, M., Natoli, A., Di Consiglio, E., De Angelis, G., Traina, E.,
rs
Testai, E., Perrone-Capano, C., Laviola, G., (2006b). Short-term effects of adolescent
methylphenidate exposure on brain striatal gene expression and sexual/endocrine
parameters in male rats. Ann. N. Y. Acad. Sci. 1074, 52-73.
Alburges, M.E., Hoonakker, A.J., Horner, K.A., Fleckenstein, A.E., Hanson, G.R., (2011).
he
Methylphenidate alters basal ganglia neurotensin systems through dopaminergic
mechanisms: A comparison with cocaine treatment. J. Neurochem. 117, 470-478.
Andersen, S.L., (2005). Stimulants and the developing brain. Trends Pharmacol. Sci. 26, 237-
243.
Andersen, S.L., Arvanitogiannis, A., Pliakas, A.M., LeBlanc, C., Carlezon, W.A.J., (2002).
is
Altered responsiveness to cocaine in rats exposed to methylphenidate during
development. Nat. Neurosci. 5, 13-14.
Barkley, R.A., Fischer, M., Smallish, L., Fletcher, K., (2003). Does the treatment of attention-
bl
deficit/hyperactivity disorder with stimulants contribute to drug use/abuse? A 13-year
prospective study. Pediatrics 111, 97-109.
Belin, D., Everitt, B.J. (2010). Drug addiction: The neural and psychological basis of a
Pu
compulsive incentive habit. In: Handbook of Basal Ganglia Structure and Function. H.
Steiner and K. Y. Tseng (Eds.). London, Elsevier: 571-592.
Berke, J.D., Hyman, S.E., (2000). Addiction, dopamine, and the molecular mechanisms of
memory. Neuron 25, 515-532.
Beverley, J.A., Piekarski, C., Van Waes, V., Steiner, H., (2014). Potentiated gene regulation
by methylphenidate plus fluoxetine treatment: Long-term gene blunting (Zif268,
Homer1a) and behavioral correlates. Basal Ganglia 4, 109-116.
a
Bhat, R.V., Baraban, J.M., (1993). Activation of transcription factor genes in striatum by
cocaine: role of both serotonin and dopamine systems. J. Pharmacol. Exp. Ther. 267,
496-505.
ov
Bhatara, V., Feil, M., Hoagwood, K., Vitiello, B., Zima, B., (2004). National trends in
concomitant psychotropic medication with stimulants in pediatric visits: practice versus
knowledge. J. Atten. Disord. 7, 217-226.
Borycz, J., Zapata, A., Quiroz, C., Volkow, N.D., Ferré, S., (2008). 5-HT(1B) receptor-
N
mediated serotoninergic modulation of methylphenidate-induced locomotor activation in

rats. Neuropsychopharmacology 33, 619-626.
Brandon, C.L., Marinelli, M., Baker, L.K., White, F.J., (2001). Enhanced reactivity and
vulnerability to cocaine following methylphenidate treatment in adolescent rats.
Brandon, C.L., Steiner, H., (2003). Repeated methylphenidate treatment in adolescent rats
alters gene regulation in the striatum. Eur. J. Neurosci. 18, 1584-1592.
c.
Carlezon, W.A., Jr., Konradi, C., (2004). Understanding the neurobiological consequences of
early exposure to psychotropic drugs: linking behavior with molecules.
Neuropharmacology 47, 47-60.
In
Carlezon, W.A., Jr., Mague, S.D., Andersen, S.L., (2003). Enduring behavioral effects of
early exposure to methylphenidate in rats. Biol. Psychiatry 54, 1330-1337.
Carrey, N., Wilkinson, M., (2011). A review of psychostimulant-induced neuroadaptation in
developing animals. Neurosci. Bull. 27, 197-214.
Castanon, N., Scearce-Levie, K., Lucas, J.J., Rocha, B., Hen, R., (2000). Modulation of the
rs
effects of cocaine by 5-HT1B receptors: a comparison of knockouts and antagonists.
Pharmacol. Biochem. Behav. 67, 559-566.
Castle, L., Aubert, R.E., Verbrugge, R.R., Khalid, M., Epstein, R.S., (2007). Trends in
medication treatment for ADHD. J. Atten. Disord. 10, 335-342.
he
Chase, T., Carrey, N., Soo, E., Wilkinson, M., (2007). Methylphenidate regulates activity
regulated cytoskeletal associated but not brain-derived neurotrophic factor gene
expression in the developing rat striatum. Neuroscience 144, 969-984.
Chase, T.D., Brown, R.E., Carrey, N., Wilkinson, M., (2003). Daily methylphenidate
administration attenuates c-fos expression in the striatum of prepubertal rats. Neuroreport
is
14, 769-772.
Chase, T.D., Carrey, N., Brown, R.E., Wilkinson, M., (2005). Methylphenidate differentially
regulates c-fos and fosB expression in the developing rat striatum. Dev. Brain Res. 157,
bl
181-191.
Chaudhuri, A., (1997). Neural activity mapping with inducible transcription factors.
Neuroreport 8, v-ix.
Cotterly, L., Beverley, J.A., Yano, M., Steiner, H., (2007). Dysregulation of gene induction in
Pu
corticostriatal circuits after repeated methylphenidate treatment in adolescent rats:

Differential effects on zif 268 and homer 1a. Eur. J. Neurosci. 25, 3617-3628.
Crawford, C.A., Baella, S.A., Farley, C.M., Herbert, M.S., Horn, L.R., Campbell, R.H.,
Zavala, A.R., (2011). Early methylphenidate exposure enhances cocaine self-
administration but not cocaine-induced conditioned place preference in young adult rats.
Csoka, A., Bahrick, A., Mehtonen, O.P., (2008). Persistent sexual dysfunction after
a
discontinuation of selective serotonin reuptake inhibitors. J. Sex. Med. 5, 227-233.

Everitt, B.J., Robbins, T.W., (2005). Neural systems of reinforcement for drug addiction:
ov
from actions to habits to compulsion. Nat. Neurosci. 8, 1481-1489.

Frankel, P.S., Alburges, M.E., Bush, L., Hanson, G.R., Kish, S.J., (2008). Striatal and ventral
pallidum dynorphin concentrations are markedly increased in human chronic cocaine
users. Neuropharmacology 55, 41-46.
Gammon, G.D., Brown, T.E., (1993). Fluoxetine and methylphenidate in combination for
N
treatment of attention deficit disorder and comorbid depressive disorder. J. Child

Adolesc. Psychopharmacol. 3, 1-10.
Graybiel, A.M., (1995). Building action repertoires: memory and learning functions of the
basal ganglia. Curr. Opin. Neurobiol. 5, 733-741.
Graybiel, A.M., Canales, J.J., Capper-Loup, C., (2000). Levodopa-induced dyskinesias and
dopamine-dependent stereotypies: a new hypothesis. Trends Neurosci. 23, S71-S77.
Han, D.D., Gu, H.H., (2006). Comparison of the monoamine transporters from human and
c.
mouse in their sensitivities to psychostimulant drugs. BMC Pharmacol. 6, 6.
Heiman, M., Schaefer, A., Gong, S., Peterson, J.D., Day, M., Ramsey, K.E., Suárez-Fariñas,
M., Schwarz, C., Stephan, D.A., Surmeier, D.J., Greengard, P., Heintz, N., (2008). A
In
translational profiling approach for the molecular characterization of CNS cell types. Cell
135, 738-748.
Horner, K.A., Adams, D.H., Hanson, G.R., Keefe, K.A., (2005). Blockade of stimulant-
induced preprodynorphin mRNA expression in the striatal matrix by serotonin depletion.
Neuroscience 131, 67-77.
rs
Hurd, Y.L., Herkenham, M., (1993). Molecular alterations in the neostriatum of human
cocaine addicts. Synapse 13, 357-369.
Hyman, S.E., Nestler, E.J., (1996). Initiation and adaptation: a paradigm for understanding
psychotropic drug action. Am. J. Psychiatry 153, 151-162.
he
Ishii, M., Tatsuzawa, Y., Yoshino, A., Nomura, S., (2008). Serotonin syndrome induced by
augmentation of SSRI with methylphenidate. Psychiatry Clin. Neurosci. 62, 246.
Iversen, L., (2006). Neurotransmitter transporters and their impact on the development of
psychopharmacology. Br. J. Pharmacol. 147 Suppl1, S82-88.
Keefe, K.A., Horner, K.A. (2010). Neurotransmitter regulation of basal ganglia gene
is
expression. In: Handbook of Basal Ganglia Structure and Function. H. Steiner and K. Y.
Tseng (Eds.). London, Elsevier: 461-490.
Knapska, E., Kaczmarek, L., (2004). A gene for neuronal plasticity in the mammalian brain:
bl
Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Prog. Neurobiol. 74, 183-211.
Kollins, S.H., (2008). ADHD, substance use disorders, and psychostimulant treatment:
current literature and treatment guidelines. J. Atten. Disord. 12, 115-125.
Kuczenski, R., Segal, D.S., (1997). Effects of methylphenidate on extracellular dopamine,
Pu
serotonin, and norepinephrine: comparison with amphetamine. J. Neurochem. 68, 2032-

2037.
Lavretsky, H., Kim, M.D., Kumar, A., Reynolds, C.F., (2003). Combined treatment with
methylphenidate and citalopram for accelerated response in the elderly: an open trial. J.
Clin. Psychiatry 64, 1410-1414.
Lee, J.L., Di Ciano, P., Thomas, K.L., Everitt, B.J., (2005). Disrupting reconsolidation of
drug memories reduces cocaine-seeking behavior. Neuron 47, 795-801.
a
Lucas, J.J., Segu, L., Hen, R., (1997). 5-Hydroxytryptamine1B receptors modulate the effect
of cocaine on c-fos expression: converging evidence using 5-hydroxytryptamine1B
ov
knockout mice and the 5-hydroxytryptamine1B/1D antagonist GR127935. Mol.

Pharmacol. 51, 755-763.
Marco, E.M., Adriani, W., Ruocco, L.A., Canese, R., Sadile, A.G., Laviola, G., (2011).
Neurobehavioral adaptations to methylphenidate: the issue of early adolescent exposure.
N
McClung, C.A., Nestler, E.J., (2003). Regulation of gene expression and cocaine reward by
CREB and DeltaFosB. Nat. Neurosci. 6, 1208-1215.
Meririnne, E., Kankaanpaa, A., Seppala, T., (2001). Rewarding properties of

methylphenidate: sensitization by prior exposure to the drug and effects of dopamine D1-
and D2-receptor antagonists. J. Pharmacol. Exp. Ther. 298, 539-550.
Morris, B.J., Reimer, S., Hollt, V., Herz, A., (1988). Regulation of striatal prodynorphin
mRNA levels by the raphe-striatal pathway. Brain Res. 464, 15-22.
c.
Nelson, J.C., (2007). Augmentation strategies in the treatment of major depressive disorder.
Recent findings and current status of augmentation strategies. CNS Spectr. 12 Suppl 22,
6-9.
In
Nestler, E.J., (2001). Molecular basis of long-term plasticity underlying addiction. Nat. Rev.
Neurosci. 2, 119-128.
Nestler, E.J., (2012). Transcriptional mechanisms of drug addiction. Clin. Psychopharmacol.
Neurosci. 10, 136-143.
Nestler, E.J., Carlezon, W.A., Jr., (2006). The mesolimbic dopamine reward circuit in
rs
depression. Biol. Psychiatry 59, 1151-1159.
O'Connor, E.C., Chapman, K., Butler, P., Mead, A.N., (2011). The predictive validity of the
rat self-administration model for abuse liability. Neurosci. Biobehav. Rev. 35, 912-938.
Packard, M.G., Knowlton, B.J., (2002). Learning and memory functions of the basal ganglia.
he
Annu. Rev. Neurosci. 25, 563-593.
Pan, D., Gatley, S.J., Dewey, S.L., Chen, R., Alexoff, D.A., Ding, Y.S., Fowler, J.S., (1994).
Binding of bromine-substituted analogs of methylphenidate to monoamine transporters.
Eur. J. Neurosci. 264, 177-182.
Paxinos, G., Watson, C. (1998). The Rat Brain in Stereotaxic Coordinates. New York,
is
Academic Press.
Ravindran, A.V., Kennedy, S.H., O'Donovan, M.C., Fallu, A., Camacho, F., Binder, C.E.,
(2008). Osmotic-release oral system methylphenidate augmentation of antidepressant
bl
monotherapy in major depressive disorder: results of a double-blind, randomized,
placebo-controlled trial. J. Clin. Psychiatry 69, 87-94.
Renthal, W., Carle, T.L., Maze, I., Covington, H.E., Truong, H.T., Alibhai, I., Kumar, A.,
Montgomery, R.L., Olson, E.N., Nestler, E.J., (2008). Delta FosB mediates epigenetic
Pu
desensitization of the c-fos gene after chronic amphetamine exposure. J. Neurosci. 28,
7344-7349.
Renthal, W., Nestler, E.J., (2008). Epigenetic mechanisms in drug addiction. Trends Mol.
Med. 14, 341-350.
Rushton, J.L., Whitmire, J.T., (2001). Pediatric stimulant and selective serotonin reuptake
inhibitor prescription trends: 1992 to 1998. Arch. Pediatr. Adolesc. Med. 155, 560-565.
Safer, D.J., Zito, J.M., DosReis, S., (2003). Concomitant psychotropic medication for youths.
a
Am. J. Psychiatry 160, 438-449.

Samaha, A.N., Robinson, T.E., (2005). Why does the rapid delivery of drugs to the brain
ov
promote addiction? Trends Pharmacol. Sci. 26, 82-87.

SAMHSA, (2012). Results from the 2011 National Survey on Drug Use and Health:
Summary of National Findings. NSDUH Series H-44, HHS Publication No. (SMA) 12-
4713, http://www.samhsa.gov/data/NSDUH/2011SummNatFindDetTables/Index.aspx.
Schenk, S., Izenwasser, S., (2002). Pretreatment with methylphenidate sensitizes rats to the
N
reinforcing effects of cocaine. Pharmacol. Biochem. Behav. 72, 651-657.

See, R.E., Elliott, J.C., Feltenstein, M.W., (2007). The role of dorsal vs ventral striatal
pathways in cocaine-seeking behavior after prolonged abstinence in rats.
Segal, D.S., Kuczenski, R., (1999). Escalating dose-binge treatment with methylphenidate:
role of serotonin in the emergent behavioral profile. J. Pharmacol. Exp. Ther. 291, 19-30.
c.
Sharp, F.R., Sagar, S.M., Swanson, R.A., (1993). Metabolic mapping with cellular resolution:
c-fos vs. 2-deoxyglucose. Crit. Rev. Neurobiol. 7, 205-228.
Shippenberg, T.S., Zapata, A., Chefer, V.I., (2007). Dynorphin and the pathophysiology of
In
drug addiction. Pharmacol. Ther. 116, 306-321.
Spencer, T.J., (2006). ADHD and comorbidity in childhood. J. Clin. Psychiatry 67 Suppl 8,
27-31.
Steiner, H., Gerfen, C.R., (1998). Role of dynorphin and enkephalin in the regulation of
striatal output pathways and behavior. Exp. Brain Res. 123, 60-76.
rs
Steiner, H., Van Waes, V., (2013). Addiction-related gene regulation: Risks of exposure to
cognitive enhancers vs. other psychostimulants. Prog. Neurobiol. 100, 60-80.
Steiner, H., Van Waes, V., Marinelli, M., (2010). Fluoxetine potentiates methylphenidate-
induced gene regulation in addiction-related brain regions: Concerns for use of cognitive
he
enhancers? Biol. Psychiatry 67, 592-594.
Steiner, H., Warren, B.L., Van Waes, V., Bolaños-Guzmán, C.A., (2014). Life-long
consequences of juvenile exposure to psychotropic drugs on brain and behavior. Prog.
Brain Res. 211, 13-30.
Svetlov, S.I., Kobeissy, F.H., Gold, M.S., (2007). Performance enhancing, non-prescription
is
use of Ritalin: a comparison with amphetamines and cocaine. J. Addict. Dis. 26, 1-6.
Swanson, J.M., Volkow, N.D., (2008). Increasing use of stimulants warns of potential abuse.
Nature 453, 586.
bl
Swanson, J.M., Wigal, T.L., Volkow, N.D., (2011). Contrast of medical and nonmedical use
of stimulant drugs, basis for the distinction, and risk of addiction: comment on Smith and
Farah (2011). Psychol. Bull. 137, 742-748.
Szumlinski, K.K., Ary, A.W., Lominac, K.D., (2008). Homers regulate drug-induced
Pu
neuroplasticity: implications for addiction. Biochem. Pharmacol. 75, 112-133.

Teter, C.J., McCabe, S.E., LaGrange, K., Cranford, J.A., Boyd, C.J., (2006). Illicit use of
specific prescription stimulants among college students: prevalence, motives, and routes
of administration. Pharmacotherapy 26, 1501-1510.
Théberge, F.R., Milton, A.L., Belin, D., Lee, J.L., Everitt, B.J., (2010). The basolateral
amygdala and nucleus accumbens core mediate dissociable aspects of drug memory
reconsolidation. Learn. Mem. 17, 444-453.
a
Thomas, U., (2002). Modulation of synaptic signalling complexes by Homer proteins. J.

Neurochem. 81, 407-413.
ov
Tropea, T.F., Guerriero, R.M., Willuhn, I., Unterwald, E.M., Ehrlich, M.E., Steiner, H.,
Kosofsky, B.E., (2008). Augmented D1 dopamine receptor signaling and immediate-
early gene induction in adult striatum after prenatal cocaine. Biol. Psychiatry 63, 1066-
1074.
Unal, C.T., Beverley, J.A., Willuhn, I., Steiner, H., (2009). Long-lasting dysregulation of
N
gene expression in corticostriatal circuits after repeated cocaine treatment in adult rats:
Effects on zif 268 and homer 1a. Eur. J. Neurosci. 29, 1615-1626.
Van Waes, V., Beverley, J., Marinelli, M., Steiner, H., (2010). Selective serotonin reuptake
inhibitor antidepressants potentiate methylphenidate (Ritalin)-induced gene regulation in
the adolescent striatum. Eur. J. Neurosci. 32, 435-447.
Van Waes, V., Carr, B., Beverley, J.A., Steiner, H., (2012). Fluoxetine potentiation of
methylphenidate-induced neuropeptide expression in the striatum occurs selectively in
c.
direct pathway (striatonigral) neurons. J. Neurochem. 122, 1054-1064.
Van Waes, V., Ehrlich, S., Beverley, J.A., Steiner, H., (2015). Fluoxetine potentiation of
methylphenidate-induced gene regulation in striatal output pathways: Potential role for 5-
In
HT1B receptor. Neuropharmacology 89, 77-86.
Van Waes, V., Vandrevala, M., Beverley, J., Steiner, H., (2014). Selective serotonin re-
uptake inhibitors potentiate gene blunting induced by repeated methylphenidate
treatment: Zif268 versus Homer1a. Addict. Biol. 19, 986-995.
Vanderschuren, L.J., Di Ciano, P., Everitt, B.J., (2005). Involvement of the dorsal striatum in
rs
cue-controlled cocaine seeking. J. Neurosci. 25, 8665-8670.
Volkow, N.D., Wang, G.J., Fowler, J.S., Logan, J., Franceschi, D., Maynard, L., Ding, Y.S.,
Gatley, S.J., Gifford, A., Zhu, W., Swanson, J.M., (2002). Relationship between blockade
of dopamine transporters by oral methylphenidate and the increases in extracellular
he
dopamine: therapeutic implications. Synapse 43, 181-187.
Walker, P.D., Capodilupo, J.G., Wolf, W.A., Carlock, L.R., (1996). Preprotachykinin and
preproenkephalin mRNA expression within striatal subregions in response to altered
serotonin transmission. Brain Res. 732, 25-35.
Warren, B.L., Iñiguez, S.D., Alcantara, L.F., Wright, K.N., Parise, E.M., Weakley, S.K.,
is
Bolaños-Guzmán, C.A., (2011). Juvenile administration of concomitant methylphenidate
and fluoxetine alters behavioral reactivity to reward- and mood-related stimuli and
disrupts ventral tegmental area gene expression in adulthood. J. Neurosci. 31, 10347-
bl
10358.
Waxmonsky, J., (2003). Assessment and treatment of attention deficit hyperactivity disorder
in children with comorbid psychiatric illness. Curr. Opin. Pediatr. 15, 476-482.
White, B.P., Becker-Blease, K.A., Grace-Bishop, K., (2006). Stimulant medication use,
Pu
misuse, and abuse in an undergraduate and graduate student sample. J. Am. Coll. Health
54, 261-268.
Wilens, T.E., Adler, L.A., Adams, J., Sgambati, S., Rotrosen, J., Sawtelle, R., Utzinger, L.,
Fusillo, S., (2008). Misuse and diversion of stimulants prescribed for ADHD: a
systematic review of the literature. J. Am. Acad. Child Adolesc. Psychiatry 47, 21-31.
Wilens, T.E., Faraone, S.V., Biederman, J., Gunawardene, S., (2003). Does stimulant therapy
of attention-deficit/hyperactivity disorder beget later substance abuse? A meta-analytic
a
review of the literature. Pediatrics 111, 179-185.

Wiley, M.D., Poveromo, L.B., Antapasis, J., Herrera, C.M., Bolaños Guzmán, C.A., (2009).
ov
Kappa-opioid system regulates the long-lasting behavioral adaptations induced by early-

life exposure to methylphenidate. Neuropsychopharmacology 34, 1339-1350.
Willuhn, I., Sun, W., Steiner, H., (2003). Topography of cocaine-induced gene regulation in
the rat striatum: Relationship to cortical inputs and role of behavioural context. Eur. J.
Neurosci. 17, 1053-1066.
N
Yano, M., Beverley, J.A., Steiner, H., (2006). Inhibition of methylphenidate-induced gene
expression in the striatum by local blockade of D1 dopamine receptors: Interhemispheric
effects. Neuroscience 140, 699-709.
Yano, M., Steiner, H., (2005a). Methylphenidate (Ritalin) induces Homer 1a and zif 268
expression in specific corticostriatal circuits. Neuroscience 132, 855-865.
Yano, M., Steiner, H., (2005b). Topography of methylphenidate (Ritalin)-induced gene
regulation in the striatum: differential effects on c-fos, substance P and opioid peptides.
c.
Yano, M., Steiner, H., (2007). Methylphenidate and cocaine: the same effects on gene
regulation? Trends Pharmacol. Sci. 28, 588-596.
Yuferov, V., Nielsen, D., Butelman, E., Kreek, M.J., (2005). Microarray studies of
In
psychostimulant-induced changes in gene expression. Addict. Biol. 10, 101-118.
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Chapter 12
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Role of Fluoxetine on Depression-
Related Pathophysiological
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Mechanisms
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Miroslav Adzic, Milos Mitic, Iva Lukic, Jelena Djordjevic,
and Marija B. Radojcic*
Department of Molecular Biology and Endocrinology, VINCA Institute of Nuclear
Sciences, University of Belgrade, Serbia
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Abstract
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Major depression is a very common and serious mood disorder characterized by
heterogeneous etiopathology. Treatment of the disease usually involves the use of
antidepressant medications whose basic mechanism is achieved via increasing synaptic
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levels of monoamine neurotransmitters in different brain regions affected by the disease.

Currently used antidepressant medications display limited efficacy with a pronounced
delay to onset of action, and they all provoke disturbing side effects. With the ultimate
goal of uncovering the primary postsynaptic actions that may initiate cellular
antidepressive signaling, basic and clinical researches are devoted to studying the effects
of antidepressants, particularly those most commonly used, selective serotonin reuptake
inhibitors (SSRIs), on the complex signaling network that is known to be altered in
depression. This includes the effects of antidepressants on the hypothalamic-pituitary-
a
adrenal axis (HPA) activity, production of neurotrophins and regulation of neurogenesis,

pro-inflammatory and anti-inflammatory cytokines, oxidative stress and mitochondrial
functioning. Herein, we discuss the latest published data from animal and clinical studies
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that have examined the effects of different SSRIs on depression-related

pathophysiological mechanisms. Special attention will be dedicated to gender-specific
effects of SSRIs action and to their adverse effects.
Keywords: SSRI, fluoxetine, HPA axis, glucocorticoid receptor, immune system, oxidative
N
stress, neurogenesis, mitochondria
*
Corresponding author: Email: miraz@vinca.rs.
228 Miroslav Adzic, Milos Mitic, Iva Lukic et al.
Introduction
Major depressive disorder (MDD) has a multifaceted and varied etiology, including
genetic, epigenetic, and environmental factors. Among environmental factors, stress is
considered to be a leading cause of depression in depressed patients (Schüle 2007). Many
c.
animal models of depression are based on the association of stress and depression and such
models have a solid ethological basis. These models using exposure to chronic stress have
shown good face, construct, and predictive validity, making it one of the most commonly
In
used paradigms to model depression (Vollmayr et al. 2007). Even though it is not possible to
model all symptoms of depression in rodents, a battery of endophenotypes can be examined
such as anhedonia, body weight changes, behavioral despair, hypothalamic-pituitary adrenal
(HPA) axis function, alterations in neurogenic and neural plastic processes, elevation of
inflammation and oxidative processes and mitochondrial dysfunction, as have been observed
rs
in human patients with depression (Willner 2005). Regardless of the fact that the inhibitory
actions of antidepressants on reuptake or monoamine oxidase are immediate, their clinical
efficacy requires weeks of treatment. The time delay on the onset of treatment response has
resulted in the view that chronic adaptations in brain function, rather than increases in
he
synaptic norepinephrine/serotonin, underlie the therapeutic effects of antidepressant drugs.
Thus, the focus of research on antidepressant mechanisms has switched from the immediate
effects of antidepressants to effects which develop more slowly. In particular, attention is now
focused on alterations in signal transduction systems and gene expressions in the brain cells.
is
The Effects of Fluoxetine and Other SSRIs on
the Hypothalamic-Pituitary-Adrenal Axis (HPA)
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The HPA axis is a major neuroendocrine stress response system in mammalian organisms
which aberrant regulation is closely associated with the pathophysiology of affective
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disorders such as MDD (Holsboer 2000). Depression is a complex, multifactorial disease

where a large part of the environmental and genetic risk factors seem to correlate with altered
HPA-axis activity in adulthood. Confirmation that impairment in HPA axis regulation plays a
vital role in depressive pathogenesis is supported by the fact that antidepressants restore
HPA-axis function to normal in depressed patients or animal models of depression (Nemeroff
1996). Interestingly, pathological changes in HPA axis regulation could be found at all levels
of the stress system, i.e., in the brain, the paraventricular nucleus of the hypothalamus (PVN),
a
the pituitary and the adrenals (Board et al. 1957, Tsigos and Chrousos 2002, Charmandari et
al. 2005, Melmed et al. 2011).
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In mid-fifties of the last century Board and colleagues were the first to report altered
plasma cortisol levels in depressed patients (Board et al. 1957). Over the next few decades,
science accumulated substantial evidences culminated in setting the ‗‗glucocorticoid
hypothesis‘‘ of depression, This hypothesis supposes impaired glucocorticoid signalization
and disturbed HPA axis activity as the main etiological factors in depression, and that this
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state may be normalized by antidepressant therapy (Nemeroff 1996, Holsboer et al. 1996).
Despite the very complex nature of MDD, neuroendocrine symptoms are observed in
about 60% of depressed patients; namely hyper- or hypocortisolemia are associated with a
Role of Fluoxetine on Depression-Related Pathophysiological Mechanisms 229
dysregulated HPA axis function (Ising et al. 2007). In the brain, post-mortem studies on
severely depressed suicide victims revealed high levels of corticotropin-releasing hormone
(CRH), concomitant with increased levels of CRH-expressing neurons, and a decreased
amount of CRH receptors (CRHR) (Nemeroff et al. 1998, Austin et al. 2003, Merali et al.
2004, Merali et al. 2006). Also, CRH levels were increased in the cerebrospinal fluid (CSF)
c.
of MDD patients (Nemeroff et al. 1984, Banki et al. 1989). Furthermore, a number of arginine
vasopressin (AVP) -containing PVN neurons was increased in the brain of depressed subjects
(Merali et al. 2006, Meynen et al. 2006), suggesting an overstimulation of the pituitary by
In
these neuropeptides. At the pituitary level, increased expression of adrenocorticotropic
hormone (ACTH) and its precursor, proopiomelanocortin (POMC), have been reported
(Charlton et al. 1988). Consistently, increased numbers of ACTH-secretory episodes from
corticotropes and subsequent exaggerated adrenal CORT secretion have been observed in
depressed patients (Sher 2004). On the adrenal level, the increased stimulation by ACTH
rs
resulted in hyperplasia as well as hypertrophy of adrenal cells, which was associated with an
increased release of glucocorticoids from the adrenal cortex (Amsterdam et al. 1987, Szigethy
et al. 1994, Plotsky et al. 1998). Many of these clinical observations were also confirmed in
animal models of depression. Namely, stressed rats exhibited increased CRH synthesis,
he
increased levels of ACTH and corticosterone, which were followed by changes in behavior
such as sleeping difficulties, low appetite, anxiety, cognition problems, depressive-like
behavior – symptoms that are present in MDD patients (Plotsky et al. 1993, Ladd et al. 2000,
Heim et al. 2004).
The increase in CSF levels of CRH and AVP in depressed patients can be reduced by
is
following successful chronic treatment with fluoxetine (De Bellis et al. 1993). However,
research looking at the effects of fluoxetine on CRH and AVP mRNA has shown conflicting
results, with some studies reporting a decrease (Brady et al. 1992, Baker et al. 1996) and
bl
others no change (Torpy et al. 1997). Also, no effect was observed on basal ACTH and
cortisol levels when fluoxetine was administered acutely to normal volunteers (Torpy et al.
1997). Moreover, the ACTH and cortisol responses to naloxone, an opioid antagonist which
raises plasma ACTH predominantly through CRH, were not significantly altered by
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fluoxetine (Torpy et al. 1997). This clinical data are supported by animal data. Namely, in rats
fluoxetine increased the levels of both AVP and CRH in hypothalamic-hypophyseal portal
blood, and peripheral levels of AVP and ACTH, as well as in vitro (Gibbs et al. 1983, De
Bellis et al. 1993,). Also, when tricyclic antidepressants (TCAs) are administered chronically
to normal volunteers, they do not alter basal levels of cortisol and ACTH but there is evidence
for a blunting of responsiveness to ovine CRH and AVP (Michelson et al. 1997). The TCAs
may also reduce hypothalamic CRH mRNA levels in rodent models (Barden 1999). As stated,
a
AVP plays an important role in ACTH regulation, particularly in response to stress (Barden
1999). There is evidence in both human (Milsom et al. 1985, Rittmaster et al. 1987) and
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equine (Holmes et al. 1984) studies that magnocellular-derived AVP may stimulate ACTH
secretion. Data from rat studies indicate that neurons derived from the magnocellular region
of the PVN pass through the median eminence and release vasopressin, which may act
directly on the anterior pituitary (Holmes et al. 1984).
The effects of clinically active antidepressant drugs on CRH and CRHR1 has been
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extensively investigated (Mansbach et al. 1997, Arborelius et al. 1999, Yamano et al. 2000,
Gutman et al. 2003, Overstreet et al. 2004, Hodgson et al. 2007), and were further supported
with antidepressant-like effects of CRHR1 antagonists in different animal models of
depression (Stout et al. 2002, Kim et al. 2006, Santibanez et al. 2006, Raone et al. 2007,
Lowry et al. 2009, Bonne et al. 2010).
Although SSRIs are the first-line pharmacological treatment for depression, the findings
of SSRI activity on this signaling pathway are contradictory. For instance, while one study
showed that preventive treatment with fluoxetine does not exert any effect on PVN CRH
c.
overexpression (De Bellis et al. 1993), others found that fluoxetine reverses long-term
changes found in CRH and CRHR1 mRNA expression in the brains followed with
normalization of depressive-like behavior (Macedo et al. 2012). Also, it was found that the
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other SSRIs, sertraline and paroxetine, alter CRH levels. Sertraline interferes with CRH
effects in the locus coeruleus (Valentino et al. 1990), but in vitro experiments sertraline does
not modify CRH signaling (Magalhaes et al. 2010). Paroxetine normalizes pituitary CRHR
binding, reducess CRH mRNA expression in the PVN and amygdala and reduces CSF CRH
concentrations in stressed rats (Nemeroff et al. 2004). In normally reared adult rats,
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paroxetine had no effect on CRH mRNA expression. All these results seem to support the
idea that SSRI antidepressants can exhibit their effects through reducing the CRH‘s effects.
It should be noted that increased HPA axis activity is related to symptoms of melancholic
depression, while atypical depression or depression combined with posttraumatic stress
he
disorder (PTSD) was related with hypofunction of HPA axis (Gould et al. 1996, Oquendo et
al. 2003). Moreover, an impaired activity of the HPA axis is thought to be related, at least in
part, to altered glucocorticoid-mediated feedback inhibition by endogenous glucocorticoids
and especially of impaired function of the mineralocorticoid (MR) and the glucocorticoid
receptors (GR), which were reported in depressed patients (De Kloet et al. 1998, Holsboer
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2000, Pariante and Miller 2001, Young et al. 2003). The MR has a high affinity for
endogenous glucocorticoids, whereas the GR a lower affinity for endogenous glucocorticoids
and high affinity for dexamethasone, synthetic glucocorticoid (Pariante and Miller 2001).
bl
Therefore, in the stress response when glucocorticoids are high, such as in depression, general
opinion is that the role of the GR is of significantly higher relevance (Pariante and Miller
2001).
One of the supporting arguments that impaired glucocorticoid-mediated feedback
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inhibition could be crucial in the etiology of MDD comes from a number of studies showing
non-suppression of cortisol secretion following dexamethasone administration
(dexamethasone suppression test, DST) (Nemeroff 1996, Pariante and Miller 2001). Also, a
lack of adequate negative glucocorticoid feedback in patients with depression and psychosis
can be the logical explanation for the increase in the size and number of pituitary cells that
produce ACTH, leading to an increased pituitary volume, as well as the adrenals in these
patients (Pariante et al. 2004, Garner et al. 2005, Pariante et al. 2005,).
a
In agreement with the ‗‗glucocorticoid hypothesis‘‘ of depression, which supposes

impaired glucocorticoid signalization and overall impaired HPA axis activity as the main
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etiological factors in depression, there si a notion that antidepressant therapy counteracts

these disturbances (Holsboer et al. 1996, Nemeroff 1996,). Indeed, a lot of evidences,
including animal, clinical and in vitro studies, show that antidepressants, including SSRIs,
affects HPA axis activity, although the exact mechanisms by which they exert these effects
still remains to be elucidated.
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Although a significant effort has been made in assessing the impaired GR function and
cellular processes that influence the GR gene expression in patients with MDD, clinical
studies assessing this issue are rather scarce. It is very important to evaluate how changes in
GR activation and its modification are connected with depressive symptoms, and how
alterations of receptor activity could be used as a biomarker of a vulnerability of depression,
depressive status, and subtypes of depression (Maric and Adzic 2013). On the other hand,
question arises as to how the GR related cellular dynamics are modulated by antidepressants,
among others SSRI, and could they be used to predict drug treatment response in MDD
c.
patients.
The majority of studies were addressed to analyze the number of receptors capable of
binding the hormone, protein receptor levels, changes in expression of GR gene and in
In
chaperones important for the GR function (Pariante et al. 2006). Newer research, however,
are more focused on the changes in expression of genes regulated by GR, as well as in
epigenetic regulation and posttranslational modifications of GR and subcelluar distribution
(nucleo-cytoplasmic shuttling) which altogether can alter GR functioning and regulation of
HPA axis that might be crucial in pathogenesis of depression (Pariante et al. 2006).
rs
Most of these studies analyzed the GR function/expression in vitro or in vivo in
peripheral cells, such as lymphocytes, whole blood cells, fibroblasts, because of their
availability. The studies that analyzed the GR in whole lymphocyte cell lyzates in depressed
subjects, using ligand binding assays, did not found differences or found decreased GR
he
binding in depressed compared to healthy subjects (Calfa et al. 2003, Pariante et al. 2006). In
contrast, studies that analyzed the GR only in cytosolic compartment found reduced number
of GR capable of binding the ligand in MDD patients versus controls (Pariante et al. 2006,
Frodl et al. 2012). Discrepancies between results obtained in these studies lies in the binding
techniques that were used to assess GR. However, the absence of changes in GR from whole
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cell lysates combined with the reduced cytosolic GR levels suggests that the GR changes seen
in depression could rather be the consequence of disturbed nuclear trafficking of the GR or
inadequate association of the GR with its chaperon protein complex in depression (Pariante et
bl
al. 2006). Furthermore, studies that examined the GR gene expression from whole blood cells
found either no changes (Frodl et al. 2012) or decreased iRNA for GR in depressed patients
(Matsubara et al. 2006, Cattaneo et al. 2013,). Results obtained from periphery were
additionally supported by post-mortem human brain studies, which found reduced GR
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expression in hippocampus and prefrontal cortex not only in depressed patients but also in
patients with other psychiatric disorders, such as schizophrenia and bipolar disorder (Lopez et
al. 1998, Cotter and Pariante 2002, Webster et al. 2002). Data from clinical studies have been
additionally supported by findings from animal model of depression which found reduced
levels of GR levels in the forebrain of mice that coincided with depressive like behavior
(Boyle et al. 2005, Schmidt et al. 2007, Chourbaji et al. 2008). Taken together these studies
directly confirmed the idea that GR function is impaired in depression either because of
a
decreased GR function or decreased expression in the brain (Pariante and Miller 2001,
Pariante et al. 2006). Yet, we must emphasize that there are cases that reported that GR
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function could be intact in depression in some tissues. For example, it is reported that
depressed patients show increased levels of visceral fats and signs of osteoporosis, and that
these functions are mediated by increased GR signalization, which suggests that in depression
there is ‗‘localized glucocorticoid resistance‘‘ (Pariante and Miller 2001, Gold and Chrousos
2002).
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As stated above, another confirmation of the involvement of impaired GR function in

pathogenesis of depression comes from studies showing antidepressant effects on GR
function. Even though studies performed in depressed patients, animals, and cellular models
propose several mechanisms through which antidepressants exert their effects on GR, the
precise mechanism of their action is still unclear. Majority of these studies are consistent with
‗‗glucocorticoid hypothesis‘‘ of depression and report that short-term as well as long-term
treatment with antidepressants can increase GR expression and number of GR, and promote
the GR nuclear translocation. This is in turn associated with enhanced GR-mediated negative
c.
feedback by endogenous glucocorticoids, and thus with restoring of HPA axis dysfunction
(Peiffer et al. 1991, Pepin et al. 1992, Rossby et al. 1995, Lopez et al. 1998, Pariante and
Miller, 2001, Boyle et al. 2005, Pariante et al. 2006).
In
Reports how SSRIs affect GR are rather diverse. Data concerning the effect of SSRI
drugs on GR distribution/level and GR mRNA in key brain regions mainly come from rodent
models of depression, particularly from rats. Most of these studies reported that chronic
treatment with fluoxetine (Brady et al. 1992, Rossby et al. 1995, Frechilla et al. 1998,
Pariante and Miller, 2001, Mitic et al. 2013), citalopram and zimelidine (Seckl and Fink 1992,
rs
Budziszewska et al. 1994), have no effect on GR mRNA level. However, it should be
mentioned that there are also studies that reported diverse effects from previous studies. They
showed that chronic fluoxetine treatment can increase the GR expression in vitro in primary
hippocampal culture as well as in vivo (Yau et al. 2001, Lai et al. 2003,Yau et al. 2004), and
he
also decrease GR gene expression in the prefrontal cortex, amygdala, locus coeruleus and
dorsal raphé nucleus (Heydendael et al. 2010). For comparison, chronic treatment with TCAs
(imipramine, amitriptyline, desipramine) and serotonin–norepinephrine reuptake inhibitors
SNRIs (reboxetine, venlafaxine), electroconvulsive shock (a non-pharmacological therapy of
depression) and lithium increased GR expression in the rat hippocampus (Peiffer et al. 1991,
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Pepin et al. 1992, Seckl and Fink 1992, Przegalinski and Budziszewska 1993, Budziszewska
et al. 1994). Explanation for discrepancies in different effects between various classes of
antidepressants on GR expression could be found in different durations of treatments,
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different experimental designs, and analyzed brain regions.
Beside the effect of fluoxetine on GR expression, it is important to mention that there are
reports that fluoxetine also increased the MR expression in the rat hippocampus (Pariante and
Miller, 2001) which was further supported with citolopram suggesting that MR has also
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important place in antidepressant action (Pariante and Miller, 2001). Furthermore, group by
Lopez found that desipramine decreased corticosterone levels and up-regulated 5 HT1A and
MR expression which were previously decreased by chronic stress (Lopez et al. 1998). These
data are consistent with results from post-mortem brain studies, by the same group, that found
reduced 5 HT1A and MR receptors in hippocampus of suicide victims (Lopez et al. 1998).
Also, it should be noted that antidepressant drugs do not increase MR protein (Seckl and Fink
1992, Przegalinski and Budziszewska 1993, Budziszewska et al. 1994) which suggests that
a
antidepressants increase not only the biosynthesis of MR, but also their degradation and
turnover.
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Likewise GR mRNA, reports of antidepressants effects on the GR protein levels in brain

are also contradictory; as some studies found an increase in GR protein levels (Pipien et al.
1992, Hery et al. 2000, Pariante and Miller 2001, Lai et al. 2003), whereas others found a
decrease (Pariante et al. 1997, Hery et al. 2000, Pariante et al. 2003). Regarding fluoxetine
effect on GR level, few studies were conducted and they reported that chronic fluoxetine
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decreased GR protein levels in hippocampus of stressed rats which was followed with
normalization of depressive-like behavior (Szymańska et al. 2009, Mitic et al. 2013).
Although the precise mechanisms of how fluoxetine induce changes in GR levels and
distribution are still unknown, newer research propose several explanations including
activation of receptor by ligand, its phosphorylation which is essential for translocation of
receptor from the cytoplasm to the nucleus, as well as interaction with protein chaperone
complex and membrane steroid transporters. Indeed, research from our group found that
fluoxetine decreased nuclear GR translocation in hippocampus of stressed rats (Mitic et al.
c.
2013) which were further supported. Likewise, paroxetine, not only reduced the nuclear GR
levels but also reversed previously induced GR translocation by verapamil (Funato et al.
2006). However studies using TCA antidepressants, such as imipramine, clomipramine, and
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desipramine, showed enhanced nuclear GR translocation (Pariante et al. 1997, Heiske et al.
2003, Funato et al. 2006). From these data it is possible that nuclear translocation of the GR
may represent a molecular mechanism by which antidepressants normalize HPA axis
disturbances in depressed patients (Pariante and Miller 2001).
Since antidepressants have been shown to act on gene transcription (Schwaninger et al.
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1995), recently their effect on GR-mediated gene transcription was evaluated. GR is a
hormone-activated transcription factor which bind to glucocorticoid responsive element
(GRE) on DNA, yet some of its effects on gene transcription are due to interactions with
other transcription factors (CREB, AP-1, NFκB). Studies by Pariante and others have shown
he
that antidepressants facilitate glucocorticoid dependent GR-mediated gene transcription
(Pariante et al. 1997, Heiske et al. 2003, Pariante et al. 2003). Fluoxetine is also shown to
inhibit the cortisol-induced gene transcription in concentration and time-dependent manner
(Budziszewska et al. 2000). However, it is important to mention that some studies reported
that antidepressants inhibit GR-mediated gene transcription (Pariante et al. 2001). These
is
differences may underlie different second-messenger signaling mechanisms of
antidepressants in different cell types and different experimental conditions (Anacker et al.
2011). Antidepressants that are able to affect the GR-induced gene transcription can act on
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different processes connected with GR action, hormone binding, dissociation of the GR
complex from other cytosol proteins, cytosol-nuclear distribution of GR, its phosphorylation,
binding to DNA and modulation of the transcription complex (Budziszewska et al. 2000).
Research by Pariante and others suggested another mechanism which could be crucial for
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fluoxetine action in MD treatment (Pariante et al. 2001, Pariante et al. 2003, Anacker et al.
2011). Namely, they showed that antidepressants, among them and fluoxetine exert its effect
on GR function through interaction with membrane steroid transporters, such as the multiple
drug resistance p-glycoprotein (MDR PGP) (Rosby et al. 1995, Weiss et al. 2003, Anacker et
al. 2011). MDR PGP actively expels cortisol and DEX from cytoplasm, but not the
corticosterone. Using both mouse fibroblasts and rat cortical neurons, they showed that
fluoxetine blocks MDR MGP and therefore increases intracellular glucocorticoid levels (Hery
a
et al. 2000, Pariante et al. 2001) and thus indirectly enhances GR function. These findings
were further supported in vivo in MDR PGP knockout mice, where desipramine reduced
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ability to decrease plasma glucocorticoid levels (Yau et al. 2007). Also Carvalho and others
reported that different antidepressants, among them and sertraline modulated GR function in
human whole blood (Carvalho et al. 2010). Therefore, by inhibiting membrane steroid
transporters antidepressants can increase the access of cortisol to the brain, and hence
normalize the negative feedback on the HPA axis (Pariante et al. 2006, Anacker et al. 2011).
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Interestingly, this effect appears to be independent of the chemical class of the antidepressant
(Anacker et al. 2011).
Finally, altered post-translational modifications of the GR in depression, such as

phosphorylation may explain partial impairment of GR function, rather than a global
dysfunction of the receptor. Differential phosphorylation of the GR in depression and/upon
antidepressant treatment, may result in altered co-factor binding and hence changes in
subsequent expression of GR-regulated gens (Anacker et al. 2011). Such changes may explain
c.
how a partial impairment of the GR function, or rather an alteration in GR-dependent gene
transcription (Blind et al. 2008, Chen et al. 2008), may lead to decreased HPA axis feedback
inhibition and cause HPA axis hyperactivity.
In
Phosphorylation of the GR is accomplished through the activation of several cellular
kinases (Chrousos et al. 2005) which among others include cyclin-dependent kinases (CDK)
and mitogen activated protein kinases (MAPK), c-jun N-terminal kinases (JNK) and p38,
targeting the GR at specific amino-acid residues (Krstic et al. 1997, Rogatsky et al. 1997,
Ismaili and Garabedian 2004). Work from our lab reported altered phosphorylation of GR and
rs
altered GR-mediated gene transcription in stressed rat brain (Adzic et al. 2009). Furthermore
these results were complemented with finding that fluoxetine not only modulates GR
phosphorylation in hippocampus of chronically stressed rats but also changes GR
phosphorylation which is parallel with changes in upstream kinase activity (CDK5 and
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MAPK) and normalization of depressive-like behavior in a gender-specific manner (Mitic et
al. 2013, Mitic et al. 2014). Also, chronic treatment with SNRI duloxetine normalized GR
phosphorylation status in prefrontal cortex of stressed rats (Guidotti et al. 2013). Although,
Guidotti et al. did not measure concentration of endogenous glucocorticoids, or upstream
kinase signalization responsible for phosphorylation of GR, their results along with ours
is
suggest that antidepressants could modulate GR function through phosphorylation. These
results from animal studies that potentiate the importance of GR phosphorylation status in
pathogenesis of stress related disorder were further supported with our clinical reports that
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found altered GR phosphorylation status in lymphocytes of MDD (Simic et al. 2013a, Simic
et al. 2013b).
Taken the highly interrelated nature of the serotonergic and HPA system, it is
conceivable that SSRI therapy improves dysfunction in HPA axis activity found in depression
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at different levels, from the CRF to glucocorticoids, from the CNS to the periphery.
Furthermore, we can highlight the role of GR function as one of the main point in the
mechanism of antidepressant improvement on dysregulation in HPA axis activity.
The Effects of Fluoxetine and SSRIs

on Immune System
a
In recent years there has been increasing evidences that chronic low grade inflammation
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plays an important role in pathogenesis of depression, and together with dysfunctional

endocrine and neurotransmitter systems provide a network of changes that underline
depressive disorder and which may ultimately lead to neurodegeneration and
neuroprogression in a chronic course of the disorder. Patients with major depression,
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otherwise medically healthy, have been repeatedly observed to have elevated levels of
inflammatory markers such as interleukin (IL) -6, IL-1, tumor necrosis factor alpha (TNF-α),
chemokines, C-reactive protein (CRP) etc. (eg. Owen et al. 2001, Danner et al. 2003, Hestad
et al. 2003, Ford and Erlinger 2004, Alesci et al. 2005, Liu et al. 2012). Smith (1991) was one
of the first who hypothesise that IL-1 and TNF-α, released by macrophages, could produce
principal symptoms of depression, which formed the bases of the macrophage theory of
depression (Smith 1991). The credibility of this theory was further supported by the
observations that administration of pro-inflammatory cytokine interferon alpha (INF-α) or IL-
c.
2 to treat hepatitis C or some types of cancers could lead to symptoms of major depression
(depressed mood, fatigue, anhedonia, anorexia, sleep disturbances, decreased libido) in a
substantial minority of subjects (Bonaccorso et al. 2002, Capuron et al. 2002, Pavlovic et al.
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2011). Likewise, many different medical disorders that are associated with induction of
inflammatory and oxidative and nitrosative stress (IO&NS) pathways show high comorbidity
with depression, for example, chronic obstructive pulmonary disease (COPD), HIV infection,
cardiovascular disorder, rheumatoid arthritis, multiple sclerosis, Parkinson disease, etc. (Maes
et al. 2011). Finally, in laboratory animals, systemic administration of endotoxin
rs
lipopolysaccharide (LPS) produces symptoms similar to those observed in clinical depression,
such as anhedonia, anorexia, weight loss, anxiety, reduction of locomotor activity and
exploration, sleepiness, together known as ―sickness behavior‖ (Kent et al. 1992).
Interestingly, inflammatory changes associated with depression, or induced by pro-
he
inflammatory cytokines, could largely be attenuated by effective antidepressant treatment
including SSRIs.
There is, however, a question how inflammation could be initiated in depressed patients
that are otherwise presumably medically healthy? The explanation could be as follows. There
has been an increasing recognition that psychological stress, through activation of the
is
sympathetic nerve system and the HPA axis, can stimulate the inflammatory response at the
periphery and in the brain (Maes et al. 1998, Bierhaus et al. 2003, Johnson et al. 2005).
Catecholamines acting through adrenoceptors on the macrophages/monocytes at the periphery
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or on the microglia in the brain (microglia are resident macrophages of the brain) activate
inflammatory signaling pathways, in which NFkB plays a crucial role. This leads to increase
in cell mediated immunity even in the absence of an antigen challenge. Additionally, in the
conditions of chronic stress, immunosuppressive effect of glucocorticoids may be attenuated
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due to glucocorticoid resistance (discussed earlier in this chapter). Also, chronic stress leads
to mitochondria damage and release of their molecules (that are of bacterial origin) could
activate immune and inflammatory pathways as well (discussed later in this chapter).
Activated macrophages produce pro-inflammatory cytokines, such as IL-12, IL-6, IL-1β,
TNF-α. IL-12 induces differentiation of Th1 lymphocyte subtype, which mainly secrets pro-
inflammatory cytokines INF-γ, TNF-α, IL-2, and further stimulates macrophages to produce
other mediators of the cellular immunity, such as chemokines, nitric oxide (NO), reactive
a
oxygen species (ROS), prostaglandins. On the other hand, INF-γ and IL-12 inhibit the activity
of Th2 lymphocytes, which mainly secret anti-inflammatory cytokines (IL-10, IL-13) and
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inhibit Th1 activity and macrophage activation (Mosmann and Sad 1996). The same changes
are believed to happen in the brain, by inducing an inflammatory response of microglia
directly in the brain and/or by peripherally produced cytokines acting in the brain. Indeed,
there are several ways that peripheral cytokines can affect the brain: by activation of
peripheral afferent nerves (e.g., the vagal nerve); by cytokine passage through leaky regions
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in blood-brain-barrier; by saturable cytokine transporters at the blood–brain barrier; and

pathways involving IL-1 receptors located on perivascular macrophages and endothelial cells
of brain venules (Dantzer et al. 2009).
Table 1. The effects of SSRIs on immune system function in animal models
Species Sex Model Antidepressant Effects of SSRIs Reference

Rat M Olfactory Fluvoxamine Reversion of anxiety-like Song and
bulbectomized (10 mg/kg, 20 behavior; stimulation of Leonard
c.
model of days), sertraline lymphocyte proliferation 1994.
depression (10 mg/kg, 20 suppressed by olfactory
days) bulbectomy
Rat M Single LPS- Paroxetine (7.5 Desipramine normalized Shen et al.
In
injecttion mg/kg, 3 LPS-induced behavioral 1999.
weeks), changes, while venlafaxine
desipramine and paroxetine did not
(7.5 mg/kg, 3
weeks),
venlafaxine (10
rs
mg/kg, 3
weeks)
Mice M Naïve Fluoxetine and Complex immuno- Kubera et
citalopram (10 regulatory effects, al. 2000.
he
mg/kg, 1, 2 and depending on the type of
4 weeks) administrated SSRI and
duration of the treatment.
Rat M Single LPS- Fluoxetine (10 Normalization of LPS- Yirmiya et
injection mg/kg, 5 induced anorexia and body al. 2001.
is
weeks) weight loss, without
affecting TNF-α and IL-1β
mRNA levels in spleen
Rat M Chronic INF- Paroxetine (10 Normalization of INFα- Myiant et
bl
α injection mg/kg, 2 induced behavioral al. 2007.
weeks) changes, IL-10 and IL-1β
levels in the
hypothalamus, TNFα, IL-
Pu
1β, IL-10 and INFγ/IL-10

ratio in cultured blood
cells
Mice M LPS-induced Fluoxetine (20 Pre-treatment with both Roumestan
septic shock mg/kg, single drugs suppressed LPS- et al. 2007.
injection), induced rise of TNFα and
desipramine (20 increased survival
mg/kg, single
a
injection)
Mice M Chronic Fluoxetine (15 Restoration of T cell Frick et al.
ov
restrained mg/kg, 3 proliferation and IL-2, 2009.

stress, 3 weeks) INF- -α
weeks production decreased by
chronic stress
Mice M Collagen- Fluoxetine (10 Higher dose of fluoxetine Sacre et al.
induced or 25 mg/kg, 7 halted further increase in 2010.
N
arthritis (CIA) days), severity of the disease,

model more effectively than
citalopram; both SSRI
Species Sex Model Antidepressant Effects of SSRIs Reference

citalopram (25 inhibited TLR-induced
mg/kg, 7 days) TNF- α production in
murine bone marrow–
derived macrophages
c.
Mice M Single LPS- SSRIs Attenuation of LPS- Ohgi et al.
injection (fluoxetine and induced serum elevation of 2013.
paroxetine), TNF-α and decrease of IL-
SNRI 10 in dose-dependent
In
(venlafaxine manner; SSRI shortened
and duloxetine) immobility time in the tail
(3, 10, or 30 suspension test, and
mg/kg, single paroxetine simultaneously
injection) decreased TNF-α mRNA
levels in prefrontal cortex
rs
Once in the brain, cytokines could affect brain functioning in numerous ways that are
relevant for pathogenesis of depression. Pro-inflammatory cytokines have profound effects on
the metabolism of brain serotonin, dopamine and noradrenalin. For example, much attention
he
has been focused on indolamine 2,3, dioxygenase (IDO) that degrades tryptophan (the
precursor of serotonin) converting it to kynurenine and which activity was observed to be
enhanced by IFN-γ, TNF-α, IL-1β and IL-2 (Heyes et al. 1992, Babcock et al. 2000, Curier et
al. 2000, Daubener et al. 2000). It is believed that tryptophan depletion by IDO contribute to
reduced serotonin availability in cytokine-induced depression (Capuron et al. 2002, Capuron
is
et al. 2003). The kynurenine is further metabolized into other products whereby some of them
appear to be neurotoxic leading to neurodegeneration (Perkins et al. 1982, Schwartz et al.
1983) (discussed later in this chapter). It is also of note that pro-inflammatory cytokines such
bl
as IL-1β and TNF-α are potent stimulators of CRH secretion and HPA axis activity
(Berkenbosch et al. 1987, Ericsson et al. 1994, Van der Meer et al. 1996).
Since it was hypothesized that cytokines may play causative role in pathogenesis of
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depression, it was also postulated that antidepressants could counteract their effects. Indeed, a
lot of evidences, including animal, clinical and in vitro studies, support the assumption that
antidepressants, including SSRIs, affect immune system functions, although the exact
mechanisms by which they exert these effects still remains to be elucidated.
Regarding the animal models that investigated the effects of SSRIs on inflammation-
induced sickness behavior, evidence support the beneficial effects of these drugs on
behavioral changes, although the results were not always consistent (Table 1). Paroxetine pre-
a
treatment (10 mg/kg, for 2 weeks) prevented anxiety-like behavior in INF-α-challenged male
rats in the open-field test which was accompanied by the normalization of IL-10 and IL-1β
levels in the hypothalamus, while the alterations of these cytokines in the hippocampus and
ov
prefrontal cortex were not observed (Myint et al. 2007). The same treatment reduced
concentration of TNF-α, IL-1β and INF-γ/IL-10 ratio and increased IL-10 in unstimulated or
mitogen stimulated cultured blood cells (Myint et al. 2007). Likewise, fluoxetine (30 mg/kg)
and paroxetine (10 mg/kg) pre-treatment diminished LPS-induced depressive-like behavior in
N
mice (assessed by immobility time in the tail suspension test), and these paroxetine effects
were accompanied by suppression of TNF-α expression in prefrontal cortex (Ohgi et al.
2013). However, chronic pre-treatment with fluoxetine (10 mg/kg, for 5 weeks) although
managed to attenuate anorexia and body weight loss evoked by LPS injection, did not affect
TNF-α and IL-1β mRNA levels in the rat spleen (Yirmia et al. 2001). On the other hand,
chronic paroxetine pre-treatment (7.5 mg/kg, three weeks) failed to normalise LPS-elicited
behavioral changes and LPS-induced alterations in levels of TNF-α and IL-10 in rats, while
TCA desipramine prevented nearly all observed changes initiated by LPS (Shen et al. 2001).
c.
Anti-inflammatory effects of SSRIs were revealed in some other animal models that
include immune activation. For example, in a mouse model of endotoxic shock induced by a
lethal dose of LPS, acute pre-treatment with fluoxetine (20 mg/kg), but not curative post-
In
treatment, was sufficient to suppress LPS-induced rise of TNF-α and to increase survival rate
(Roumestan et al. 2007). Advantageous fluoxetine effects were also demonstrated in murine
model of rheumatoid arthritis there the antidepressant halted further increase in clinical and
histopathologic severity of the disease, at least partly by inhibiting cytokine production
induced by Toll-like receptor ligands in murine bone marrow–derived macrophages (Sacre et
al. 2010). However, fluoxetine‘s marked clinical benefits were achieved about its safe
rs
therapeutic dosages (25 mg/kg) (Sacre et al. 2010).
Table 2. The effects of SSRIs on cytokine levels in blood of depressed patients
Population
assessed
Depressed
patients
Gender
M and F Fluoxetine
he
Antidepressant
Effect on
cytokine levels
IL-6 ↓
Reference
Sluzewska et al.
1995
is
Depressed M and F Fluoxetine, IL-12 ↓ Kim et al. 2002
patients paroxetine,
nefazodone
Depressed M and F SSRI TNF-α ↓ Tuglu et al.
bl
patients 2003
Depressed M and F Paroxetine TNF-α , sTNF- Hinze-Selch et
patients αRI al. 2005
Depressed M and F SSRI IL-6 ↓ Basterzi et al.
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patients 2005
Depressed M and F Sertraline and IL-1β ↓, IL-6 ↓, Leo et al. 2006
patients citalopram TNF-α ↓
Depressed M and F Fluoxetine, IL-12 ↓ Lee and Kim
patients paroxetine, velafaxine 2006
Depressed M and F Fluoxetine mRNA: IFN-γ ↓, Tsao et al. 2006
patients TNF-α , IL-1β ,
a
IL-6
Depressed M and F Sertraline IL-12 ↓, IL-2 ↓, Sutcigil et al.
patients TNF-α ↓, IL-4 ↑ 2007
ov
Depressed M and F SSRI IL-6 , TNF-α O‘Brien et al.

patients, SSRI- 2007
resistant
Depressed M and F Excitalopram TNF-α Eller et al. 2008
patients
N
Depressed M and F SSRI IFN-γ ↑, IL-1β ↑, Hernandez et al.

patients IL-2 ↓, IL-4 ↓, IL- 2008
10 ↓, IL-13 ↓
Population Effect on
Gender Antidepressant Reference
assessed cytokine levels
Depressed M and F Fluoxetine IL-1β ↓, TNF-α Song et al. 2009
patients INF-γ , IL-10
c.
Depressed M and F Fluoxetine and IL-1β , IL-6 Jazayeri et al.
patients eicosapentaenoic acid 2010
Depressed M and F All AD All AD: IL-1β ↓, Hannestad et al.
patients TNF-α , IL-6 2011.
In
(meta-analysis) SSRI: IL-6 ↓
Immunostimulatory effects of SSRIs in experimental animals have also been described.

For example, chronic administration of fluoxetine and citalopram (10 mg/kg) in mice, have
complex immunoregulatory effects, depending on the type of administrated SSRI, or duration
rs
of the treatment (Kubera et al. 2000). Both antidepressants reduced the metabolic activity of
splenocytes after 4 weeks of administration, which was accompanied by the enhanced
secretion of anti-inflammatory IL-10. On the other hand, citalopram administration for 1, 2 or
4 weeks stimulated the proliferative activity of splenocytes and suppressed their production of
he
anti-inflammatory cytokine IL-4, while enhancing the secretion of IL-6. Similarly, fluoxetine
treatment for 1 and 2 weeks, but not 4 weeks, stimulated the proliferative activity of
splenocytes, whereas after 4 weeks it suppressed their ability to secrete IL-4 and enhanced the
secretion of IL-6 (Kubera et al. 2000). Also, chronic administration of sertraline and
fluvoxamine (10 mg/kg, 20 days) enhanced the proliferative activity of T lymphocytes from
is
blood in the olfactory bulbectomized rats (Song et al. 1994). Likewise, fluoxetine co-
treatment (15 mg/kg, 3 weeks) of rats subjected to chronic restraint stress enhanced T cell
proliferation to mitogen stimulation, reduced by chronic stress (Frick et al. 2009). Therefore,
bl
it seems that immunoregulatory effects of SSRIs vary depending on different molecular
pathways activated under certain experimetal conditions.
In depressed patients, the modulatory role of antidepressants on the immune system is not
so clear as well (Table 2). Still, the most consistent finding is that SSRIs reduce elevated
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levels of pro-inflammatory cytokine IL-6 in depressed patients, after successful

antidepressant treatment (Sluzewska et al. 1995, Basterzi et al. 2005, Leo et al. 2006, O'Brien
et al. 2007, Hannestad et al. 2011). Data concerning blood TNFα are more conflicting – while
in some studies response to SSRI therapy was associated with significant reduction of this
cytokine (Tuglu et al. 2003, Leo et al. 2006, Sutcigil et al. 2007), and SSRI-resistant patients
exhibited still high production of TNF-α (Basterzi et al. 2005), in larger studies there was no
a
difference in TNF-α between responders and non-responders to SSRIs (Eller et al. 2008,
Hannestad et al. 2011). Regarding the effects of SSRIs on IL-1β levels, studies are not
consistent as well – whereas some researchers indeed found a reduction of IL-1β levels upon
ov
SSRI treatment (Leo et al. 2006, Song et al. 2009), the others did not replicate that (Tsao et al.
2006, Hernandez et al. 2008, Jazayeri et al. 2010). There are also evidences that SSRIs
decrease pro-inflammatory cytokines IL-2 and IL-12 and increase anti-inflammatory cytokine
IL-4 levels upon treatment (Kim et al. 2001, Lee et al. 2006, Sutcigil et al. 2007). However, in
recent rigorous and long term clinical study, SSRIs increased Th1 pro-inflammatory
N
cytokines IL-1β, IL-2 and IFN-γ and decreased Th2 anti-inflammatory cytokines IL-4, IL-10
and IL-13, after 52 weeks treatment in depressed patients (Hernandez et al. 2008). Besides
this, SSRIs are also effective in treatment of depressive symptoms induced by INF-γ therapy
in patients with cancer or hepatitis C (Capuron et al. 2003, McNutt et al. 2012). Taken
together, clinical studies suggest that despite of immunoregulatory effects of SSRIs, the
improvement of depressive symptoms is not always associated with normalization of
immune-inflammatory pathways. In addition, conflicting results regarding the SSRI effects on
c.
the pro/anti-inflammatory markers in depressed patients may be related to the heterogeneity
of the disorder itself and that the specific patterns of cytokine alterations presumed to be
associated with specific sets of symptoms are affected in different ways by SSRI treatment.
In
Pronounced effects of SSRIs were observed in vitro as well (Table 3), demonstrating that
these drugs can act directly on immune cells, and also providing us with mechanisms how
they could achieve their immunoregulatory effects, independently of their actions in the
central nervous system. Indeed, immune cells express functional receptors for serotonin, as
well as 5-HT transporters.
rs
Table 3. In vitro studies of SSRI effects
Cell culture models Antidepressant Effects of SSRIs Reference
he
Mitogen-stimulated Citalopram, TCA Inhibition of pro- Xia et al.
T lymphocytes and inflammatory cytokine 1996.
monocytes production from T-
lymphocytes (IL-2, IFN-γ)
and monocytes (IL-6, TNF-α,
IL-1β); elevation of
is
intracellular cAMP
concentrations
Human synovial Fluoxetine, Inhibition of NO and Yalom et
Human cells
bl
cells stimulated by amitriptyline PGE2release, in dose al. 1999.
cytokines/LPS dependent manner
Mitogen-stimulated Fluoxetine, All drugs significantly Kubera et
whole blood from imipramne, reduced the IFN-γ/IL-10 al. 2001.
Pu
from healthy venlafaxine, l-5- ratio, without differences

controls and hydroxytryptophan between subject groups
treatment-resistant (5-HTP)
depressed patients
A549 human lung Fluoxetine, Both antidepressants Roumestan
epithelial cells desipramine repressed TNF-α induced et al. 2007
NF-κB activity and TPA-
induced AP-1 activity
a
Murine BV2 Fluoxetine Increased mRNA levels for Ha et al.

microglia cells IL-6, TNF-a and inducible 2006
NO synthase (iNOS), as well
ov Microglial cells
as NO; increased DNA

binding activity of NFkB and
phosphorylation of p38 and
ERK1/2 MAPKs
IFN- γ -stimulated Fluvoxamine, Inhibiton of microglial Hashioka et
N
murine 6-3 imipramine, production of IL-6 and NO; al. 2007.

microglial cells reboxetine cAMP and PKA inhibitors
reversed these effects
Cell culture models Antidepressant Effects of SSRIs Reference

LPS-stimulated rat Fluoxetine Inhibition of TNF-α, iNOS Lim et al.
primary microglial and IL-1β gene expressions 2009.
cells and NF-κB activity
IFN-γ-stimulated Paroxetine, Inhibition of TNF-α and NO Horikawa
c.
murine 6-3 sertraline production; reduction of et al. 2010.
microglia cells influx of calcium ions; JAK
inhibitor prevented the
effects of SSRI
In
LPS-stimulated Fluoxetine Inhibition of TNF-a, IL-6 and Liu et al.
murine BV2 NO production; inhibition of 2011.
microglia cells IkB-a degradation,
phosphorylation and nuclear
translocation of NF-kB, and
phosphorylation of p38
rs
LPS-stimulated SSRIs, SNRI SSRI inhibited TNF-α and Tynan et al.
murine BV2 (venlafaxine) NO production; PKA 2012.
microglia cells inhibitor prevented effects of
SSRI
he
The in vitro studies on lymphocytes from depressed and healthy subjects, mainly support
immunosuppressive effects of SSRI medications. For example, in vitro incubation of human
monocytes and T lymphocytes with citalopram, as well as TCAs, inhibited pro-inflammatory
cytokine production (IL-6, TNF-α, IL-1β, IL-2, INF-γ) which was at least partly mediated by
is
elevation of intracellular cAMP concentrations (Xia et al. 1996). Further, several
antidepressants, including fluoxetine, decreased production of pro-inflammatory IFN-γ, while
increasing the levels of anti-inflammatory IL-10, therefore, significantly reduced the IFN-
bl
γ/IL-10 ratio (Kubera et al. 2001). Fluoxetine and citalopram treatment also inhibit TNF and
IL-6 production in human synovial membrane cultures from patients with rheumatoid arthritis
and this inhibition was independent of serotonin increase (Sacre et al. 2010). Moreover,
fluoxetine was demonstrated to attenuate the production of NO and prostaglandin E2 induced
Pu
by IL-1α and TNFα in cultured human synovial cells in a dose dependent manner (Yaron et
al. 1999).
Actually, fluoxetine effects on lymphocyte function can be dual, depending on the degree
of their activation. Upon stimulation by optimal mitogen concentrations, fluoxetine exerted
inhibitory effects on in vitro lymphocyte proliferation which involved calcium-mediated
protein kinase C (PKC) degradation and cAMP formation; on the other hand, at submitogenic
a
concentrations, fluoxetine stimulated lymphocyte reactivity involving increased PKC

activation without affecting cAMP concentrations (Edgar et al. 1999, Frick et al. 2008).
Therefore, fluoxetine is able to act directly on T cell proliferation, independently of its central
ov
effects on serotonergic neurons (and their subsequent effects on the periphery). Furthermore,
it seems that these actions can be achieved by two mechanisms: one dependent on SSRI
actions on serotonin transporters on lymphocytes, and the other, independent of them
(Pellegrino et al. 2001, Pellegrino et al. 2002, Frick et al. 2008). Also, fluoxetine reduced
basal lymphocyte proliferation in MDD patients more efficiently compared to controls,
N
indicating that depression is associated with activation of T lymphocytes (Fazzino et al.

2008).
As it was previously mentioned, in the brain, microglia are major cell type that
participates in inflammation, both as a source and a target of cytokines. Indeed, in vitro
studies showed that SSRIs affect the microglial production of inflammatory mediators as
well. Pre-treatment with the fluvoxamine reduced microbial production of pro-inflammatory
mediators such as IL-6 and NO, and those effects were, at least partially, mediated by the
c.
cAMP-dependent potein kinase A (PKA) pathway (Hashioka et al. 2012). It was proposed
that SSRI anti-inflammatory effects could be mediated through an increase in extracellular 5-
HT due to inhibition of its reuptake by 5-HT transporters and activation of 5-HT receptors (on
In
microglial cells) which are coupled to G-proteins. Through G-protein activation of adenylate
cyclase, increased cAMP production activates PKA, which could lead to NF-κB inhibition
and decreased expression of pro-inflammatory mediators (Roumestan et al. 2007, Lim et al.
2009, Liu et al. 2011, Tynan et al. 2012). However, the recent experimental evidence offers
another way of how SSRIs can reduce the release of cytokines and exert their beneficial
rs
effects, independently of their effects on the 5-HT transporter on microglial cells. Paroxetine
and sertraline inhibited the generation of TNF-α and NO by inhibiting the influx of calcium
ions in IFN-γ-stimulated 6-3 microglial cell (Horikawa et al. 2010). Namely, activation of
microglia leads to increased calcium influx that triggers the PKC, p38, ERK1/2, JAK-STAT
he
signaling pathways which ultimately induces release of pro-inflammatory molecules (Pariante
et al. 2001, Young et al. 2003). By interfering with the influx of calcium ions, SSRIs may
inhibit above mentioned cellular pathways resulting in NF-κB inhibition and reduced
production of inflammatory mediators (Horikawa et al. 2010, Liu et al. 2011), although the
precise molecular targets of SSRIs in this signalling pathway are still required to be
is
determined. However, in contrast to these studies, fluoxetine treatment of BV2 microglia cells
resulted in significant increase in mRNAs of IL-6 and TNF-a as well as NO which was
accompanied by the increased DNA binding activity of NF-κB and increased phosphorylation
bl
of p38 and ERK1/2 MAPKs (Ha et al. 2006). These results suggest that fluoxetine can exert
immunostimulatory effects in immunologically unprovoked microglia.
The mechanisms that underlie the effects of SSRIs on cytokine production and actions,
besides their direct effects on immune cells, may also include SSRI-induced changes in
Pu
serotonin neurotransmission, which itself was shown to affect actions of cytokines in the
brain (Deleplanque et al. 1995, Pellegrino et al. 2002). Additionally, normalization of the
glucocorticoid receptor functions and HPA axis negative feedback by SSRI leading to re-
establishment of glucocorticoid anti-inflammatory effects might also modulate immune
system function (Linthorst et al. 1999, Bartolomucci et al. 2003). Vice versa, the restoration
of cytokine production and actions by SSRIs could contribute the improvement of GR
sensitivity (discussed earlier in this chapter). Namely, pro-inflammatory cytokines may
a
inhibit GR functioning through stimulation of MAPK signalling and subsequent GR

phosphorylation (Pace and Miller 2009). In this context, alterations in GR phosphorylation
ov
upon fluoxetine treatment observed in our laboratory, in the hippocampus of chronically

isolated rats (discussed earlier in this chapter) could be, at least partly, mediated by fluoxetine
effects on immune mediators as well.
Considering the adverse effects of SSRI administration, it was proposed that these drugs
increase risk of cancer development and exacerbate cancer progression (Moorman et al. 2003,
N
Kubera et al. 2009, Cosgrove et al. 2011, Kubera et al. 2011), what is of note being that
SSRIs are widely prescribed for the treatment of depressive symptoms in cancer patients.
However, opposing results are accumulating from epidemiological data and animal models as
well, suggesting inhibitory effects of SSRIs on cancer prognosis (Abdul et al. 1995, Frick et
al. 2008, Kannen et al. 2011). It is presumed that SSRIs could affect anti-tumor immunity
through modulation of the balance between Th1 and Th2 cytokines. Having in mind that
immunosuppressory as well as immunostimulatory actions of SSRIs are demonstrated, their
relevance in malignant outcomes indeed seems to be complex and need to be further studied.
c.
In conclusion, SSRIs can exert immunosuppressory, but also immunostimulatory effects,
depending on immune cell activation, and through these actions they could also contribute to
improvement of depressive symptoms.
In
The Effects of Fluoxetine and SSRIs on
Antioxidant Pathways
rs
As mentioned above, numerous studies have confirmed that mood disorders are
characterized by the activation of immune and inflammatory systems (Sluzewska et al. 1996,
Leonard 2001), both on the periphery and in the central nervous system, and that activation of
these systems favors the production of reactive oxygen species by various mechanisms
he
(Fialkow et al. 2007). The major source of ROS is also the leakage of superoxide anion (O2
•−
) from mitochondria during oxidative phosphorylation as well as from the O2 •−generating
enzymes such as xanthine oxidase (XO), NADPH oxidases, and cytochromes P450
(CytP450). Additionally, some nitrogen species can be potentially dangerous for the cell, such
as peroxynitrite (ONOO–), which is formed in a rapid reaction between O2 •− and nitric
is
oxide (NO) (Halliwell 2007). Oxidative stress is defined as a condition arising from a
disproportion between the generation of reactive oxygen species (e.g., superoxide anion,
hydrogen peroxide and hydroxyl radicals), and the activity of antioxidant defense systems.
bl
The main enzymatic antioxidant defenses include superoxide dismutase (SOD), catalase
(CAT), and glutathione peroxidase (GPx). SOD enzymes are highly efficient in the catalytic
dismutation of O2 • − and generation of H2O2 which, in turn, can be removed by two types
Pu
of enzymes—the catalases and peroxidases. Importantly, the activity of GPx is closely

dependent on glutathione reductase (GLR), a glutathione tripeptide (GSH), and others
cofactors (Kohen and Nyska 2002). Cells appear capable of handling low doses of oxidant
stress/reactive metabolites, while higher doses overwhelm their productive capacity and lead
to damage or cell death. The brain is particularly vulnerable to oxidative injury because of its
high rate of oxidative metabolic activity, intense production of reactive oxygen metabolites,
high content of polyunsaturated fatty acids, and relatively low antioxidant capacity (Evans
a
1993).
Alterations in oxidative biology are increasingly being recognized as a critical route of
damage toward the pathophysiology of neurodegenerative disorders (Halliwell 2006, Ng et al.
ov
2008). Beside activation of immune and inflammatory response systems, another mechanism
which is a source of ROS in the course of depression is a deficiency of monoamines, which
act as important free radical scavengers (Huether and Schuff-Werner 1996). On the other
hand, increased metabolism of monoamines is another pathway of ROS generation in
N
depression. Increased glutaminergic transmission and excitotoxic levels of glutamate, favor

enhanced production of ROS, which cause damage to cellular structures and lead to
disturbances in the normal function of the CNS (Dong et al. 2009). The aforementioned
processes lead to excessive production of ROS, damage of molecules such as lipids, and
consequently to the loss of cell membrane fluidity and abnormalities of monoaminergic
receptor function. The mechanism of neurogenesis inhibition has not been fully elucidated
yet, nevertheless, ROS as well as nitric oxide (NO) released by immune cells demonstrates a
negative effect on neurogenesis. Therefore, antioxidant defense is a very important aspect in
c.
the studies of stress-induced psychiatric disorders and their treatment.
Antidepressants are widely prescribed to treat these conditions. Recently, a new concept
of antidepressant action has been suggested, based on growing evidence demonstrating
In
antioxidant effects of antidepressants in the treatment of major depressive disorder (Behr et
al. 2012). Administrations of different antidepressants have been shown to influence gene
expression and activity of the important antioxidant enzymes such as blood SODs (Li et al.
2000, Kolla et al. 2005). There are increasing evidences showing that treatment with
fluoxetine can reverse and prevent psychological stress induced oxidative damage, due to
rs
elevations in SOD and other components of the antioxidant system (Zafir and Banu 2007,
Zafir et al. 2009) suggesting that alterations of antioxidant parameters in the blood could
serve as valuable biomarkers to follow the effects of chronic stress and/or pharmacological
treatment of stress induced psychiatric disorders (Galecki et al. 2009). The study of da Silva
he
and coworkers (2014) showed a significant reduction of lipid peroxidation in the
hippocampus (57%) of fluoxetine treated rat neonates, with increased activity of catalase and
glutathione-S-transferase (80% and 85% respectively). However, no changes in the
antioxidant enzyme activities were detected in the hypothalamus. The results of Zafir et al.
(2009) evidenced a significant recovery in the activities of SOD, CAT, GST, GLR, and GSH
is
levels in the rat brain by fluoxetine after restraint stress. Also, it prevented lipid and protein
oxidative damage induced by stress. Novio et al. (2011) showed that even acute treatment
with fluoxetine can partially reverse intracellular redox status in peripheral blood cells of
bl
male mice exposed to restraint stress, through normalization of SOD and CAT activity and
GSH content. In another study, acute fluoxetine treatment reduced GPx activity in the
hippocampus, whereas chronic treatment increased GSH in both hippocampus and prefrontal
cortex of female mice (Lobato et al. 2010). However, our results did not reveal any changes
Pu
in SOD, CAT or GPx status in the erythrocytes of male rats submitted to either chronic
psychosocial isolation or to fluoxetine treatment (Adzic et al. 2011). This could be due to the
low dose of fluoxetine or short period of treatment. In contrast, fluoxetine markedly reduced
GLR activity and its protein level, indicating that in spite of numerous beneficial effects, it
may compromise both hemoglobin function and oxygen transport.
Increased serum SOD and MDA levels have been found in depressive patients. Plasmatic
vitamin C levels were reduced in MDD patients compared to age- and sex-matched healthy
a
volunteers. 4 weeks of treatment with fluoxetine (20 mg/day) managed to reverse this stress
markers (SOD, vitamin C, lipid peroxidation), and the antioxidant effects were persistent
ov
even after 12 weeks of treatment (Khanzode et al. 2003). Bilici et al. (2001) also reported
increased oxidative stress in major depressive patients, indexed by higher antioxidant enzyme
activities (erythrocyte SOD, GPx, and plasmatic GLR) and MDA levels (erythrocyte and
plasmatic). They investigated the effects of four different SSRIs drugs for 12 weeks
(fluoxetine 20 mg/day, sertraline 50 mg/day, fluvoxamine 100 mg/day and citalopram 20
N
mg/day), demonstrating that antioxidant enzyme activities (plasmatic GPx) and MDA levels
(plasma and erythrocyte) were restored to control levels. Plasmatic GLR and erythrocyte
SOD were also significantly decreased in MDD patients after 12-week antidepressant
treatment. Galecki et al. (2009) tested a group of 50 MDD patients who were in remission
from their first episode of depression after 3 months of treatment with 20mg of fluoxetine,
before and after remission. Before treatment, MDD patients displayed increased erythrocyte
SOD and CAT activities, increased MDA levels, and decreased plasmatic total antioxidant
status (TAS) level. After three months of fluoxetine treatment, MDA levels returned to the
c.
control level. Decreased serum SOD and increased XO were found in 20 individuals with
MDD (Herken et al. 2007). Although increased XO levels indicate increased free radical
production, no difference was observed in serum total nitrite levels (a marker of nitrosative
In
stress) between control and MDD patients before treatment. Also, no significant relationship
was found between the duration of illness and SOD, XO activities, or nitrite levels in this
cohort. Treatment with citalopram (20mg/day), fluoxetine (20mg/day), fluvoxamine (150
mg/day), or sertraline (50mg/day) for 8 weeks increased SOD activity whereas decreased XO
levels suggesting that normalization of these enzymes was associated with symptomatic
rs
improvement. Cumurcu et al. (2009) investigated whether 3 different total antioxidant
parameters total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress
index (OSI) were associated with MDD and evaluated the impact of antidepressant treatment
on these oxidative/antioxidant parameters in a cohort of 57 MDD patients. TOS and OSI were
he
higher and TAC level was lower in the MDD group compared with controls. Furthermore, the
authors found a positive correlation between the severity of the disease and serum TOS and
OSI. These further suggest that recovery from a major depressive episode may be associated
with normalization of antioxidant potential induced by antidepressants. More recently, a 24-
week follow-up study evaluated the effects of long-term antidepressant treatment
is
(venlafaxine, milnacipran, paroxetine, escitalopram, sertraline, citalopram, fluoxetine,
tianeptine and andmoclobemide) on oxidative/ antioxidant status in a cohort of 50 MDD
subjects (Kotan et al. 2011). Plasmatic MDA, serum oxidized LDL (OxLDL) levels, and
bl
erythrocyte SOD activity was increased in MDD patients before treatment, and MDA levels
were positively correlated with the severity of MDD. After 24-weeks of treatment, MDA and
SOD levels decreased. However, TAC was also decreased after 24-week treatment with
antidepressants, indicating that the oxidative stress observed in depressed patients was partly
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improved during 24 weeks of antidepressant treatment. Patients on venlafaxine were also

compared with patients on SSRIs in the aspect of oxidative stress parameters in the follow-up
period, but no significant differences were found. Sarandol et al. (2007) found that short-term
antidepressant treatment of MDD (6 weeks) did not alter oxidative/antioxidant systems in a
cohort of 96 patients. In this study, MDD patients had increased plasmatic MDA levels and
increased susceptibility of red blood cells (RBCs) to oxidation. Also, SOD activity was
increased in patients with MDD, and there was a positive correlation between the severity of
a
depressive symptoms and SOD activity. After 6 weeks of treatment with venlafaxine,
sertraline, or reboxetine, these oxidative parameters were not altered. Although it is very
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important to recognize sexual dimorphisms in the susceptibility to psychiatric disorders and

their treatment, the studies regarding the association of gender and oxidative stress markers
upon fluoxetine treatment are scarce. Wiener et al. (2014) tried to determine whether any
gender-related difference exists concerning oxidative stress parameters in a population of 231
subjects, and if these changes might be related to gender-associated differences in major
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depressive disorder (MDD) or bipolar disorder (BD) vulnerability. After sample stratification
by gender, no association was found between oxidative stress parameters and clinical
diagnosis of MDD and BD for women or men. Although women seemed more susceptible to
oxidative stress than male, these gender-based differences did not seem to provide a
biochemical basis for the epidemiologic differences in mood disorder susceptibility between
sexes.
Although the brain is the main target of fluoxetine actions, the central organ for its
activation is the liver, where it undergoes an extensive metabolic conversion, leading to the
c.
formation of the active metabolite norfluoxetine among multiple other metabolites (Altamura
et al. 1994).
Due to inhibition of its own metabolism, elimination of fluoxetine and norfluoxetine from
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the body is extremely slow. When fluoxetine is administered intraperitoneally, the drug
rapidly reaches high concentrations in the liver. Fluoxetine and norfluoxetine were found to
exert potentially toxic multiple effects on energy metabolism in rat liver mitochondria (Souza
et al. 1994). This seems to be a consequence of the solubilization of the drug and/or its
metabolites in the inner mitochondrial membrane. Its administration in mice has been
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reported to cause changes such as steatosis (fatty change) and hepatocyte enlargement
(Bendele et al. 1992).
However, the molecular basis of fluoxetine-induced hepatotoxicity (Friedenberg and
Rothstein 1996, Johnston and Wheeler 1997, Cia et al. 1999) is not well understood as yet.
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There is also scarce information on the influence of fluoxetine on antioxidant enzymes. Some
reports suggest that fluoxetine restores the antioxidant capacity in rat liver, as well as in the
brain (Bilici et al. 2001, Zafir and Banu 2007). The antioxidant effect of fluoxetine (Kirkov et
al. 2010) might also be related to the fact that this drug increases the level of serotonin, which
has been reported to display antioxidant effects (Huether et al. 1997).
is
The results of our study revealed that administration of fluoxetine in both chronically-
isolated and control animals resulted in downregulation of SOD activity and upregulation of
GPx activity, while glutathione reductase activity and total antioxidant status were elevated
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only in stressed animals (Djordjevic et al. 2011). Our results suggested that fluoxetine
interfered with stress-induced pathways of oxidative defense in the liver. In addition, in both
experimental groups, fluoxetine induced several hallmarks of apoptosis in the liver, including
a decrease in Bcl-2 expression and increased DNA fragmentation. However, apoptotic
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alterations were more pronounced in stressed animals, suggesting that stress related oxidative
damage could have primed apoptotic effects of fluoxetine. Zlatkovic et al. (2014) reported
increased serum ALT and AST activity in chronically-isolated and control animals treated
with fluoxetine.
Also, due to increased carbonyl content, MDA, GST activity and decreased GSH levels
in drug-treated controls/chronically-isolated animals, they suggested a link between drugs and
hepatic oxidative stress. Inkielewicz-Stepniak (2011) also reported increased ALT, AST, GST
a
in the serum and TBARS, carbonyl groups and uric acid content in the liver of male rates
treated with fluoxetine for one month (8 and 24 mg/kg).
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To conclude, although fluoxetine treatments generate acceptable outcomes in mood and

anxiety disorders, and to some extent they restore antioxidative status, they may result in
hepatic toxicity and undesirable side effects.
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The Effects of Fluoxetine and Other SSRIs on

the Production of Neurotrophins and Regulation
of Neurogenesis
c.
Among biological theories postulated about MDD, an impairment of neuroplasticity has
been suggested. Neuroplasticity refers to nervous system changes that occur in response to
experience (Berlucchi and Buchtel 2009). It is believed that all areas of the brain are plastic
even after childhood (Rakic 2002). It is of particular importance that environmental changes
In
could alter behavior and cognition by modifying connections between existing neurons and
via neurogenesis in the hippocampus and other parts of the brain, like the cerebellum
(Bonfanti and Ponti 2008, Bonfanti et al. 2008, Ponti et al. 2008). Several neurotoxic agents
such as chronic stress, excessive concentrations of glutamate, biogenic amines and
glucocorticoids may affect the morphology of some neural cells, such as hippocampal CA3
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pyramidal neurons and pyramidal cells of prefrontal cortex. According to the theory of
neuroplasticity, experience actually changes not only the brain's physical structure but also
functional organization. Thus, the changes in neuroplasticity process are of great importance
he
in the etiology of depression and have an important role during antidepressant treatment. The
process of neural plasticity encompasses an array of mechanisms, from the birth, survival,
migration, and integration of new neurons to neurite outgrowth, synaptogenesis, and the
modulation of mature synapses.
Neural stem cells are found across a variety of species (Eriksson et al. 1998) and the
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production of new neurons is limited to two regions: the subventricular zone, which lines the
lateral ventricles and sends new neurons to the olfactory bulb via the rostral migratory stream,
and the subgranular zone of the hippocampus. Neurogenesis is a process which requires the
proliferation, survival, and differentiation of newly born cells into neurons. It is as a dynamic
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process that includes development of dendritic spines and synaptic adaptations which are
essential for normal functioning. Aberrant neural production, connectivity, or transmission is
documented under disease states, such as Alzheimer‘s disease, schizophrenia, or depression
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(Varea et al. 2012).

Moreover, brain imaging studies revealed modification of synaptic and structural
plasticity in different regions of the brain, including the frontal cortex and hippocampus in
MDD patients. At the pathophysiological level, it was proposed that these alterations could
prevent the brain from making appropriate adaptive responses to environmental stimuli.
Studies in rodents and nonhuman primates demonstrate that exposure to stress can cause not
only the alteration in neuronal processes, but also could affect the number of neurons. For
a
example, repeated stress cause atrophy of CA3 pyramidal neurons in the hippocampus,
including a decrease in the number and length of apical dendrites. In addition, exposure to
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stress has been shown to decrease the proliferation of cells in the dentate gyrus of the
hippocampus. Each aspect of neural plasticity can independently and additively cause or
contribute to the disease state.
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Table 4. The effect of antidepressant treatment on neurogenesis and the functional

importance of neurogenesis in antidepressant efficacy estimated on animal models of
depression
Species Sex Model Antidepressant Method of ablation Summary of findings Reference
c.
Mouse M and F Chronic Fluoxetine X-ray Neurogenesis-dependent (NSF) and - David et al. 2009
corticosterone independent (OFT, FST) aspects of
administration antidepressant efficacy
Rat M Chronic mild Imipramine; MAM Neurogenesis-dependent (NSF) and - Bessa et al. 2009
stress fluoxetine independent (SC, FST) aspects of
antidepressant efficacy. Significant
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alterations in neural plasticity associated with
antidepressant efficacy
Mouse M Chronic Imipramine; Neurogenesis-dependent (NSF, CS, ST) and - Surget et al. 2008
unpredictable fluoxetine independent (A) aspects of antidepressant
stress efficacy
Mouse M — Fluoxetine X-ray Neurogenesis-independent effects of Holick et al. 2008
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antidepressant efficacy
Rat F Chronic mild Imipramine; X-ray Neurogenesis-dependent (NSF) and - Airan et al. 2007
stress fluoxetine independent (OFT) aspects of antidepressant
efficacy
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Mouse M Chronic mild Fluoxetine — Decreased cell proliferation in the DG, while Alonso et al. 2004
stress chronic fluoxetine blocked this effect
Mouse M — Imipramine; X-ray Neurogenesis-dependent (NSF) Santarelli et al.

fluoxetine antidepressant efficacy 2003
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NSF: novelty suppressed feeding; FST: forced swim test; SC: sucrose consumption; OFT: open field
test; CS: coat state; ST: splash test; A: actimeter; MAM: methylazoxymethanol acetate (cytostatic
agent)
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Table 5. The effect of antidepressant treatment on neurogenesis and the functional
importance of neurogenesis in antidepressant efficacy estimated in human studies of
depression
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Subjects Sex Population assessed Antidepressant Effect on neurogenesis Reference
Humans M and F Depressed patients SSRIs; TCAs Depression is associated with a Boldrini et al.2013
postmortem decreased number of granule
neurons, correlated with reduced
DG volume. SSRI and TCA
treatment increase granule neuron
number and DG volume
a
Humans M and F Depressed patients SSRIs; TCAs Both antidepressant classes increase Boldrini et al.2012
postmortem cell proliferation over untreated
depressed patients and controls;
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NPCs associated with angiogenesis
Humans M and F Depressed patients SSRIs; TCAs Both antidepressant classes increase Boldrini et al.2009
postmortem cell proliferation over untreated
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depressed patients and controls

Considering this, it is of fundamental importance to elucidate the neurological

underpinnings of these disease states at the cellular level, in order to understand the etiology
and to elucidate the effect of currently used antidepressants on molecular mechanisms related
to the neuroplasticty processes and neurogenesis. All these facts have attracted the attention
of neuroscientists to studythe process of neuroplastiticy and neurogenesis both in animal
c.
models of depression and in MDD patients (Table 4 and 5).
The most studied molecular mechanisms associated with neuroplastic processes are
related to neutrofines. Neurotrophins are neuron survival factors and have a critical role in the
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establishment and maintenance of neural circuits during development and during adulthood
(Jiang and Salton 2013, Oliveira et al. 2013). They belong to a family of 4 distinct secreted
growth factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF;
neurotrophin-3, NT-3; and neurotrophin-4, NT-4) which upon binding to membrane receptors
activate an intracellular cascade which promotes survival and trophic effects. Neurotrophins
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bind with high affinity to specific members of the tyrosine kinase receptor family (Trk
receptors; NGF binds to TrkA, BDNF and NT-4 bind to TrkB and, NT-3 binds not only to
TrkC but also to the other Trk receptors with low affinity). In addition, all the neurotrophins
bind with low affinity to p75 (NTR), which is responsible for storing and transporting
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neurotrophins, and promotes neuronal cell death during development. Preclinical studies
indicating that expression of BDNF were deregulated in hippocampal dentate gyrus of rats
subjected to chronic stress (Smith et al. 1995) indicating potential involvement of BDNF in
depression. Data accumulated in the last decade indicates that this neurotrophin is a central
target in the pathogenesis of depression and suicidal behavior (Dwivedi 2009). Expressions of
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BDNF, BDNF-regulated genes, and the TrkB are decreased in postmortem brain samples
from depressed humans (Tripp et al. 2012) and in lymphocytes of depressed patients (Pandey
et al. 2012). Consistent with these findings, serum levels of BDNF also decrease in MDD
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patients (Karege et al. 2002). In addition, BDNF and NT3 produced an antidepressant effect
on behavioral models of depression (Shirayama et al. 2002) which are abolished in mice
deficient in TrkB receptor (Saarelainen et al. 2003). Together, these findings support a causal
implication of BDNF in the genesis of MDD.
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BDNF is created by proteolytic cleavage of a larger precursor protein termed proBDNF.

ProBDNF is able to bind the low-affinity receptor p75 NTR exerting an opposite effect to that
of BDNF/TrkB signaling (Woo et al. 2005). It has been found that the serum levels and the
expression of both proBDNF and p75 NTR in circulating lymphocytes are up-regulated in
MDD. According to this, it has been proposed that not only the expression of BDNF and
TrkB but also the ratio between BDNF-TrkB and proBDNF-p75 NTR is dysregulated in
MDD (Zhou et al. 2013). Evidences for a role of the p75 NTR receptor in depression is also
a
confirmed by genetic, because the mission Ser205Leu polymorphism of this gene appears to
deliver a protective effect against the development of MDD in women. Several lines of
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evidence demonstrate that some modern antidepressants may reverse neuroplasticity and
neurogenesis modifications induced by chronic stress (Pittenger and Duman 2008). In animal
models, the antidepressants may reverse and remodel many of the stress-induced
neurohistological changes. It is possible to speculate that by reversing the neurohistological
effects of stress in animal models, the antidepressants may attenuate depression in human
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subjects. There is evidence that treatment with modern antidepressants significantly improves
both hippocampal shrinkage (Dranovsky and Hen 2006, Fuchs 2009) and functions (e.g.,
cognitive functions) (Yan et al. 2011). Interestingly, both tianeptine and fluoxetine, a
common SSRI antidepressant medication, may reverse the inhibition of long-term

potentiation not only in the hippocampus but also in prefrontal cortex (Rocher et al. 2004). In
addition, tianeptine may inhibit the stress-induced reduced number of hippocampal astrocytes
reversing morphological modifications induced by stress (Czéh et al. 2001). Clinical and
basic researches demonstrate that chronic antidepressant treatment increases the rate of
c.
neuroplasticity processes in the adult hippocampus. In particular, antidepressants increase the
expression of several molecules, related to neuroplasticity, in particular BDNF and its
receptor TrkB. Moreover, antidepressants up-regulate cAMP and the neurotrophin signaling
In
pathways involved in plasticity and survival. In vitro and in vivo data provide direct evidence
that the transcription factor, cAMP response element-binding protein (CREB) and BDNF are
key mediators of the therapeutic response to antidepressants. In particular, antidepressants, by
activating cellular signaling cascades that culminate in the activation of CREB (cyclic-AMP
response element binding protein), function to enhance levels of BDNF and other growth
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factors like VEGF (vascular endothelial growth factor), which promote the proliferation and
differentiation of hippocampal progenitors and alter monoaminergic synaptic transmission
(Krishnan and Nestler 2008, Pittenger and Duman 2008, Balu and Lucki 2009). Another
mechanism underlying neuroplasticity dysfunctions include both N-Methyl-D-aspartate
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(NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate
receptors. The activation of glutamate receptors induced morphological changes such as
dendritic length and branching, spine density and volumes of several brain regions,
specifically in the hippocampal dentate gyrus. Glutamate, in certain concentrations and
presumably under the influence of elevated glucocorticoids levels, mediates a structural
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remodelling of neurons leading to reversible modifications such as reduced neurogenesis,
neuronal shrinkage and decreased growth. It was shown that the inhibition of glutamate
release by NMDA receptors prevents this remodeling. Of particular interest is also the
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evidence suggesting that suicide in depressed subjects is associated with genes encoding
ionotropic glutamate receptors. Some antidepressants may reverse reduced neurogenesis,
neuronal shrinkage and decreased growth, modifying glutamate impairment in the anterior
cingulated of depressed individuals (Michael et al. 2003). Particularly, it was reported that
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that tianeptine prevented the retraction of apical dendrites of hippocampal CA3 pyramidal
neurons and the increased granule cell proliferation (Malberg et al. 2000). The precise
mechanism responsible for this includes decreasing of glutamate efflux in the basolateral
nucleus of the amygdala, the effect which seems not to be induced by the administration of
fluoxetine. Moreover, it was suggested that antidepressants may induce a phosphorilation of
AMPA receptors in two main sites of the subunit GluR1: Ser831 which is phosphorilated by
protein kinase C or CaMK-II determining elevations in hippocampal currents (Barria et al.
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1997) and Ser845 which appears crucial for protein kinase A amplification of peak current by
GluR1 receptors (Roche et al. 1996). Imipramine and fluoxetine act increasing the
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phosphorylation at Ser845 on the subunit GluR1 (Svenningsson et al. 2002, Du et al. 2007),
while tianeptine may reverse stress-induced changes in glutamate receptors expression
(Reagan et al. 2004), impairment of neurogenesis (Czéh et al. 2001) and reduced stress-
induced apoptosis in the hippocampus and temporal cortex (Lucassen et al. 2004).
The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin
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superfamily of cell adhesion molecules with an important role in synaptic function, synaptic
plasticity, and remodeling of neural circuits (Dalva et al. 2007). Through homo and
heterophilic interactions, NCAM plays an important role in cell migration, neurite outgrowth
and targeting, axonal branching, synaptogenesis, and synaptic plasticity (Bonfanti 2006,
Bonfanti and Theodosis 2009, Senkov et al. 2012). Neural plasticity mediated through the
NCAM protein is facilitated through posttranslational modifications. The most important is
glycosylation with polysialic acid (PSA) (Finne et al. 1983). Polysialic acid is a linear
homopolymer of α 2,8-linked sialic acid, which bears a negative charge, acting at NCAM-
c.
NCAM interactions and therefore interfere with cell adhesion (Gascon et al. 2007). In the
adult brain PSA-NCAM is expressed on the cell surface of newly generated daughter cells, on
neurites during outgrowth and path finding, and at the synapse of mature neurons (Bonfanti
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2006) and it is essential to neural remodelling and synaptic plasticity (Muller et al. 1996;
Burgess et al. 2008). Selective cleavage of PSA in the adult brain inhibits activity-induced
synaptic plasticity, induction of long-term potentiation and long-term depression, and alters
the normal migration and integration of newly generated neurons within the hippocampus.
PSA-NCAM is therefore of particular interestsince it mediates plasticity at multiple levels,
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from neural proliferation, integration, differentiation, neuritic outgrowth, synaptogenesis, and
modulation of mature synapses. Alterations in PSA-NCAM expression following stress have
been observed in brain regions associated with depression including the amygdala,
hippocampus and prefrontal cortex (Gilabert-Juan et al. 2012a). Similarly, PSA-NCAM
he
expression is reduced in the amygdala of patients with major depressive disorder (Maheu et
al. 2013); however no change is seen in the PFC (Gilabert-Juan et al. 2012b). Conversely,
chronic antidepressant treatment, including fluoxetine, modulates PSA-NCAM expression
throughout the limbic system in animal models of depression (Sairanen et al. 2007, Varea et
al. 2007a, Varea et al. 2007b, Homberg et al. 2011, Djordjevic et al. 2012b). Moreover,
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antidepressant treatment reduces PSA-NCAM expression in the dorsal raphe nucleus
(Homberg et al. 2011), implicating that PSA-NCAM in antidepressant-induced plasticity is
related to serotonergic neurotransmission. Overall, these findings suggest that PSA-NCAM
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may play a fundamental role in mediating broad effects of antidepressant treatment across
multiple forms of neural plasticity.
Overall, the antidepressants may reverse the stress-induced loss of neuronal cells by
reducing the retraction of hippocampal neurons (neuroplasticity) or increasing cell survival
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and functions (neurogenesis). Also, the antidepressants may reverse the structural and
functional consequences of stress in a site-specific manner in both the hippocampus and
prefrontal cortex, but not in the amygdala. This presumably explains why, although most of
depressive clinical manifestations may be reversed with the administration of antidepressants.
However, future longitudinal studies, including larger samples of subjects should deeply
investigate the potential of antidepressants, as long-term modulators of neuroplasticity and
neurogenesis mechanisms, allowing a more detailed understanding of the pathophysiology of
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major depression.
Since, women are twice as likely as men to develop depression (Gutiérrez-Lobos et al.
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2002), it is very important to understand the effects of antidepressant action from the
prospective of gender. Namely, sex differences are seen in antidepressant efficacy as men
have a better response to TCAs, while women have a better response to SSRIs (Kornstein et
al. 2000). These indicate that gonadal hormones are involved in the etiology of the disease as
well as inadequate medical treatment of MDD. Regarding neuroplastic processes, it is well
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established that gonadal hormones modulate adult neurogenesis, BDNF levels, and PSA-
NCAM expression in the hippocampus. Both, testosterone and its metabolite
dihydrotestosterone (DHT) enhance hippocampal neurogenesis by improving cell survival via
an androgen-dependent mechanism within the dentate gyrus (Spritzer and Galea 2007).
Likewise, estrogens are able to increase adult neurogenesis via proliferation or survival
depending on duration of treatment (Galea et al. 2006). In addition, testosterone and DHT
interact with BDNF and modulate synaptic plasticity, dendritic morphology, and
neurogenesis (MacLusky et al. 2006, Hatanaka et al. 2009) while estradiol regulates
c.
hippocampal BDNF (Franklin TB and Perrot-Sinal TS, 2006) through an estrogen-sensitive
response element on the BDNF gene (Sohrabji et al. 1995). On the other hand, BDNF
mediates estradiol-induced alterations in hippocampal dendritic spine density (Murphy et al.
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1998). Thus, it is of great importance the knowledge that gonadal hormones may play a role
in facilitating antidepressant efficacy through the modulation of neural plasticity. Recently,
we provided evidence that even though fluoxetine normalized depressive-like behavior in
both genders, the antidepressant elevated BDNF expression only in females, probably through
normalization of GR transcriptional activity (Mitic et al. 2013). Nevertheless, there are
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comparatively few data about the effects of antidepressants on neurotrophins and
neurogenesis in females. To date, a small number of studies were done on females indicated
that a high dose of fluoxetine (20–25 mg/kg) increased neurogenesis in the hippocampus of
female mice (Engesser-Cesar et al. 2007) and rats (Airan et al. 2007). The greatest increase in
he
proliferation in male hippocampus was found at a dose that decreased cell survival in the
frontal cortex and caused no other alterations in other areas. Females were most responsive in
the hippocampus to a higher dose, which also increased cell proliferation in the cerebellum
and the amygdala, suggesting that plasticity in multiple regions of the brain may be involved
in antidepressant efficacy in females (Hodes et al. 2010). These gender-related differences
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could be related to different pharmacokinetics of fluoxetine in males and females (Hodes et
al. 2010). Indeed, female mice more efficiently metabolized fluoxetine than males and
produced lower levels of fluoxetine, higher levels of its major metabolite norfluoxetine, and
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maintained a higher metabolite/parent ratio in both plasma and brain (Hodes et al. 2010).
Likewise, one human study documented that even though males and females did not differ in
plasma concentrations of fluoxetine, women received significantly higher levels of
norfluoxetine and exhibited slower clearance of norfluoxetine. This finding reflects gender-
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specific differences in the activity of cytochrome P450 enzymes, which are involved in the
metabolism of fluoxetine (Anderson 2005). In particular, this increased metabolism of
fluoxetine in human females may be related to increased enzymatic activity of CYP3A4.
Thus, pharmacokinetic differences in fluoxetine metabolism as well as gonadal hormones
may lead to gender differences in the effects of fluoxetine on neurogenesis.
Even though neuroplasticity and neurogenesis theory of depression are thought to be a
core pathophysiological feature of MDD, this theory also has significant limitations. One that
a
has received attention recently is that alterations of neuroplasticity in some brain regions
produce a pro-depressive effect. Namely, the expression of CREB in the nucleus accumbens
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increases behavioral despair and helplessness while CREB inhibition has an antidepressant
effect (Pliakas et al. 2001, Newton et al. 2002). Similarly, increased expression of BDNF in
the mesolimbic dopamine system produces a pro-depressive effect in the forced swim test
(Eisch et al. 2003). These studies indicate that neuroplasticity in the mesolimbic dopamine
circuit can produce effects that are opposite to the antidepressant effects observed in the
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hippocampus. In addition, chronic stress and glucocorticoid treatment increase dendritic

branching and synaptic connectivity in the amygdala, perhaps paralleling the increased size of
the amygdala reported in some neuroimaging studies of depressed patients. It appears
probable that these plastic events contribute to the evolution of a depressive state, rather than
contributing to antidepressant response. This suggestion raises the interesting possibility that
novel therapeutic strategies targeted at the enhancement of neuroplasticity may have
unintended pro-depressant effects on the nucleus accumbens or other structures outside the
hippocampus.
c.
Taken together, all these data indicate a convergence of neuroplasticity/neurogenesis
processes, effects of antidepressants, and the consequences of chronic stress on the set of
molecular and cellular mechanisms suggesting a deep connection between these three
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phenomena. However, further explorations and better understanding of these relationships is
likely to point the way toward a deeper understanding of appropriate treatments for MDD.
The Effects of Fluoxetine on
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Mitochondrial Functioning
Depression is commonly associated with frontal hypometabolic activity accompanied by
hypermetabolism in certain limbic regions. Functional imaging studies have demonstrated
he
regional blood flow and metabolic abnormalities in depressed patients. It has been reported a
decrease in metabolic activity in the prefrontal cortex in individuals with major depression,
particularly in the dorsolateral and medial areas (Mayberg 1994, Mayberg 1997). Regions of
decreased metabolism in the dorsal anterior cingulate as well as in paralimbic regions such as
anterior insula, inferior orbital, and inferior temporal cortex, have also been identified
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(Newton et al. 2002). In addition, the significant correlation between the normalization of
previous elevated activity in the anterior cingulate and the reduction in depressive symptoms
in response to sertraline treatment was reported (Buchsbaum et al. 1997). Furthermore,
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increased metabolic activity in left prefrontal, inferior parietal, dorsal anterior and posterior
cingulate areas were also associated with remission during fluoxetine treatment. All these
findings have led to the development of an additional theory of depression called ‗‗the
mitochondrial dysfunction hypothesis‘‘, suggesting that impaired functions in mitochondria
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could be also associated with psychiatric conditions such as bipolar disorder (Konradi et al.
2004), major depression and a spectrum of other affective disorders (Gardner and Boles
2011). According to this theory, depression is considered as a disease of mitochondrial energy
metabolism, which could be responsible for an abnormal brain energy metabolism found in
the frontal and temporal lobes of depressed patients. This is further supported by the fact that
the brain is a tissue with high energy demands containing a large number of mitochondria and
a
therefore is more susceptible to a reduction of the aerobic metabolism (Boekema and Braun
2007). It has been suggested that mitochondrial disease results from a malfunction in
biochemical cascade and the damage to the mitochondrial electron transport chain (MRC)
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which could be an important factor in the pathogenesis of a range of neuropsychiatric

disorders, such as BD, depression and schizophrenia (Prabakaran et al. 2004, Fattal et al.
2006). Research data indicated marked alterations regarding mitochondrial processes
following chronic stress, including, respiratory chain dysfunction, decreased ATP production,
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mitochondrial structural abnormalities and apoptosis (Alesci et al. 2006). The cytochrome
(cyt) c oxidase as terminal respiratory enzyme in MRC and as a key point during ATP
synthesis serves as an endogenous metabolic marker for neuronal functional activity (Wong-
Riley, 1989). In an animal model of depression, detrimental effects of chronic stress on

mitochondrial COX 1 and COX 3 subunits of cyt c oxidase in the rat prefrontal cortex and
hippocampus was found (Adzic et al. 2009).
Besides well-known effects of fluoxetine action on inhibition of 5-HT reuptake in the
central nervous system, it also affects electron transport and F1Fo-ATPase activity by
c.
inhibiting oxidative phosphorylation in isolated rat brain and liver mitochondria (Souza et al.
1994, Curti et al. 1999). These effects were mediated by the drug interferes with the physical
state of the lipid bilayer in the mitochondria. This was not surprising since it was found that
In
after fluoxetine administration, 40% of the drug and its metabolite were found to be localized
in brain mitochondria. In vitro research, suggests that a different class of antidepressant
mostly inhibits enzymes of MRC even though the direct interaction of antidepressants and
enzymes of MRC have not been detected (Hroudova and Fisar 2010, Abdel-Razaq et al.
2011). However, the literature data regarding effects of particular antidepressant on the
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mitochondrial function in an animal model of depression are very scarce. Recently, we
documented not only the effects of fluoxetine on the mitochondrial biogenesis but also we
elucidate that fluoxetine could act on the mitochondrial process via GR in a gender-specific
manner (Adzic et al. 2013). In particular, while fluoxetine did not affect COXs gene
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expression and cyt c oxidase activity in the prefrontal cortex of both genders, it exhibited sex-
specific effects on mitochondrial COXs expression and cyt c oxidase activity in the
hippocampus. Namely, in females, fluoxetine increased COXs expression and cyt c oxidase
activity and mitochondrial pGR232 levels while diminishment of mitochondrial COXs gene
expressions, found in males upon fluoxetine treatment, was not parallel with the changes in
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cyt c oxidase activity and, on the other hand, it was not related to accumulation of
mitochondrial GR and its pGR232 isoform. Such findings suggested that the action of the GR
in male hippocampal mitochondria could be diminished by additional mechanism also
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influencing COXs gene expression, perhaps estrogen receptors (Chen et al. 2005). In addition,
concomitant elevation of BDNF expression and cyt c oxidase activity upon fluoxetine
treatment in female hippocampus confirmed a previous study in which a positive correlation
between BDNF and cyt c oxidase activity was found (Aguiar et al. 2007). Since BDNF is
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closely linked to brain energy metabolism (Vaynman et al. 2006) and has been shown to
impact mitochondrial activity (El Idrissi and Trenkner 1999), we suggest that normalization
of female behavior upon fluoxetine treatment could be accomplished through hippocampal
BDNF-cyt c oxidase system, i.e., by synergistic actions of the nuclear (Mitic et al. 2013) and
mitochondrial pGR232 isoform. Nevertheless, these results for the first time indicate that sex-
and tissue-specific modulation of cyt c oxidase activity by fluoxetine could be related to
mitochondrial GR and its phosphoisoforms. Moreover, fluoxetine-induced increase in cyt c
a
oxidase activity in female hippocampus indicated that antidepressant treatment could also
potentiate the oxidative metabolism, which could be responsible for better outcomes in
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women following antidepressant therapy (Yang et al. 2011), particularly in the treatments
with SSRI.
In general, mitochondrial dysfunction contributes to neurodegeneration either by
apoptosis or through the generation of reactive oxygen species (ROS). Moreover, stressed
mitochondria can also induce inflammatory processes. Oxidized mtDNA and other damage-
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associated factors released from impaired mitochondria act as pro-inflammatory molecules

that engage immune and inflammatory processes (Escames et al. 2012). Oxidized mtDNA
activates pro-inflammatory pathways both intracellular (Shimada et al. 2012) and
systemically (Zhang et al. 2010). Mitochondrial stress and the resultant oxidative stress can,
therefore, lead to systemic inflammation via a direct mechanism involving the release of pro-
inflammatory molecules that activate gene expression characteristic of inflammation. There
have been discussions about oxidative stress and inflammation contributing to the
pathophysiology of mood disorders and the effects of antidepressants on these processes in
c.
separate parts of this chapter.
As already mentioned, mitochondria, besides an important role in cellular energy
production, also have a crucial role in determining cell fate through the control of apoptosis,
In
the process that has been proposed as a cellular mechanism contributing to the structural
alterations observed in stress-related mood disorders in both humans and animal models
(McKernana et al. 2009). The apoptotic process is programmed and critically controlled by
the balance and dimerization between proapoptotic (Bax) and antiapoptotic (Bcl-2) proteins
in the outer mitochondrial membrane (Lindsten et al. 2005). In response to stress, these
rs
proteins regulate the release of cytochromec, which activates caspases, the main executors of
apoptosis. An increase in the ratio of proapoptotic vs. antiapoptotic proteins is associated with
greater vulnerability to apoptotic activation and cell death. It was shown that restraint stress
caused a decrease in the Bcl-2 level in the hippocampus (Luo et al. 2004) and that chronic
he
social isolation is related to proapoptotic signaling illustrated by the relocation of
mitochondrial Bcl-2 protein to its soluble cytoplasmic form (Djordjevic et al. 2009). In
addition, a significant increase in caspase-3 positive neurons was found following chronic
unpredictable stress (Bachis et al. 2008) and maternal separation was found to increase cell
death in the dentate gyrus of rats (Lee et al. 2001). The antidepressant treatment, on the other
is
hand, has been hypothesized to mediate protective processes and inhibit apoptosis to
counteract the structural impairments and disruption in molecular mechanisms governing
neural plasticity (Duman 2004). Nevertheless, the effect of antidepressants on apoptosis
bl
appears to be complex and depends presumably on cell and brain structure type. For example,
while fluoxetine fails to prevent stress-induced initiation of apoptosis in the prefrontal cortex
(Djordjevic et al. 2012a), it successfully prevents chronic stress-induced apoptosis in male
hippocampus (Djordjevic et al. 2012a, Djordjevic et al. 2012b).
Pu
Taken together, it seems that mitochondria via controlling major cellular processes, such
as energy production, cell death and the generation of reactive oxygen species, in parallel
with structural neural changes achieved via neuroplastic and neurogenic processes, present an
important target of antidepressant action. However, further study of mitochondrial
dysfunction in psychiatric disorder is expected to be useful for the development of cellular
disease markers and new psychotropics.
a
Conclusion
ov
Treatments with SSRIs, particularly with fluoxetine, can block or even reverse the
deleterious effects of stress and depression, in part via regulation of the intracellular pathways
discussed in this chapter, notably elements of HPA axis, neuroplastic processes and
mechanisms of neurogenesis. To some extent SSRI, could also improve anti-oxidative status
N
and inflammatory processes, while their effects on mitochondrial functions are rather related
to achieving new steady state. The ability, in the first place, of fluoxetine to reverse BDNF
and mitochondrial biogenesis in stressed-females, further demonstrate the significance of

gender in etiology and the treatment of depression. Even though the complex signaling
pathways that underlie the pathophysiology of depression have been elucidated, many
questions remain unanswered regarding disease etiology, the role of specific signaling
molecules, and the efficacy and safety of either already used or novel antidepressants that
c.
directly target these systems.
References
In
Abdel-Razaq, W., Kendall, D. A. & Bates, T. E. (2011). The Effects of Antidepressants on
Mitochondrial Function in a Model Cell System and Isolated Mitochondria. Neurochem.
Res., 36, 327-338.
rs
Abdul, M., Logothetis, C. J. & Hoosein, N. M. (1995). Growth-inhibitory effects of serotonin
uptake inhibitors on human prostate carcinoma cell lines. J Urol., 154, 247-250.
Adzic, M., Djordjevic, A., Demonacos, C., Krstic-Demonacos M. & Radojcic, M. B. (2009).
The role of phosphorylated glucocorticoid receptor in mitochondrial functions and
he
apoptotic signalling in brain tissue of stressed Wistar rats. Int. J. Biochem. Cell. Bio., l41,
2181-2188.
Adzic, M., Djordjevic, J., Djordjevic, A., Niciforovic, A., Demonacos, C., Radojcic, M. &
Krstic-Demonacos M. (2009). Acute or chronic stress induce cell compartment-specific
phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar
is
rat brain. J. Endocrinol., 202, 87-97.
Adzic, M., Djordjevic, J., Mitic, M., Simic, I., Rackov, G., Djordjevic, A., Elakovic, I., Matic,
G. & Radojcic, M. (2011). Fluoxetine decreases glutathione reductase in erythrocytes of
bl
chronically isolated Wistar rats. Acta Chim. Slov., 58, 785-791.
Adzic, M., Lukic, I., Mitic, M., Djordjevic, J., Elakovic, I., Djordjevic, A., Krstic-
Demonacos, M., Matic, G. & Radojcic, M. (2013). Brain region- and sex-specific
Pu
modulation of mitochondrial glucocorticoid receptor phosphorylation in fluoxetine

treated stressed rats: effects on energy metabolism. Psychoneuroendocrinology, 38, 2914-
2924.
Aguiar, A. S., Jr. Tuon, T. & Pinho, C. A. (2007). MitochondrialI V complex and brain
neurothrophic derived factor responses of mice brain cortex after downhill training.
Neurosci. Lett. 426, 171-174.
Airan, R. D., Meltzer, L. A., Roy, M., Gong, Y., Chen, H. & Deisseroth, K. (2007). High-
a
speed imaging reveals neurophysiological links to behavior in an animal model of

depression. Science, 317, 819-823.
Alesci, S., Manoli, I., Michopoulos, V. J., Brouwers, F. M., Le, H., Gold, P. W., Blackman,
ov
M. R., Rennert, O. M., Su, Y. A. & Chrousos, G. P. (2006). Development of a human

mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle
cells: implications for pharmaco-and mitogenomics. Pharmacogenomics, 6, 333-342.
Alesci, S., Martinez, P. E., Kelkar, S., Ilias, I., Ronsaville, D. S., Listwak, S. J., Ayala, A. R.,
N
Licinio, J., Gold, H. K., Kling, M. A., Chrousos, G. P. & Gold, P. W. (2005). Major
depression is associated with significant diurnal elevations in plasma interleukin-6 levels,
a shift of its circadian rhythm, and loss of physiological complexity in its secretion:
clinical implications. J. Clin. Endocrinol. Metab., 90, 2522-2530.
Alonso, R., Griebel, G., Pavone, G., Stemmelin, J., Le Fur, G. & Soubrié, P. (2004).
Blockade of CRF1 or V1b receptors reverses stress-induced suppression of neurogenesis
in a mouse model of depression. Mol. Psychiatry, 9, 278–286.
c.
Altamura, A. C., Moro, A. R. & Percudani, M. (1994). Clinical pharmacokinetics of
fluoxetine. Clin. Pharmacokinet. 26, 201-214.
Amsterdam, J. D., Marinelli, D. L., Arger, P. & Winokur, A. (1987). Assessment of adrenal
In
gland volume by computed tomography in depressed patients and healthy volunteers: a
pilot study. Psychiatry Res., 21, 189-197.
Anacker, C., Zunszain, P. A., Carvalho, L. A. & Pariante, C. M. (2011). The glucocorticoid
receptor: pivot of depression and of antidepressant treatment?
Psychoneuroendocrinology, 36, 415-425.
rs
Anderson, G. D. (1999). Sex and racial differences in pharmacological response: where is the
evidence? Pharmacogenetics, pharmacokinetics, and pharmacodynamics. J. Womens
Health, 14, 19-29.
Arborelius, L., Owens, M. J., Plotsky, P. M. & Nemeroff, C. B. (1999). The role of
he
corticotropin-releasing factor in depression and anxiety disorders. J Endocrinol., 160, 1-
12.
Austin, M. C., Janosky, J. E. & Murphy, H. A. (2003). Increased corticotropin-releasing
hormone immunoreactivity in monoamine-containing pontine nuclei of depressed suicide
men. Mol. Psychiatry, 8, 324-332.
is
Babcock, T. A. & Carlin, J. M. (2000). Transcriptional activation of indoleamine dioxygenase
by interleukin 1 and tumor necrosis factor alpha in interferon-treated epithelial cells.
Cytokine, 12, 588-594.
bl
Bachis, A., Cruz, M. I., Nosheny, R. L. & Mocchetti, I. (2008). Chronic unpredictable stress
promotes neuronal apoptosis in the cerebral cortex. Neurosci. Lett., 442, 104–108.
Baker, R. A., Herkenham, M. & Brady, L. S. (1996). Effects of long-term treatment with
antidepressant drugs on proopiomelanocortin and neuropeptide mRNA expression in the
Pu
hypothalamic arcuate nucleus of rats. J. Neuroendocrinol., 8, 337-343.

Balu, D. T. & Lucki, I. (2009). Adult hippocampal neurogenesis: regulation, functional
implications, and contribution to disease pathology. Neurosci. Biobehav. Rev., 33, 232-
252.
Banki, C. M., Bissette, G., Arato, M., O'Connor, L. & Nemeroff, C. B. (1989). CSF
corticotropin-releasing factor-like immunoreactivity in depression and schizophrenia.
Am. J. Psychiatry, 144, 873-877.
a
Barden, N. (1999). Regulation of corticosteroid receptor gene expression in depression and

antidepressant action. J. Psychiatry Neurosci., 24, 25-39.
ov
Barria, A., Muller, D., Derkach, V., Griffith, L. C., Soderling, T.R., (1997). Regulatory
phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term
potentiation. Science, 276, 2042-2045.
Bartolomucci, A., Palanza, P., Parmigiani, S., Pederzani, T., Merlot, E., Neveu, P.J., Dantzer,
R., (2003). Chronic psychosocial stress down-regulates central cytokines mRNA. Brain
N
Res. Bull. 62, 173-178.

Basterzi, A.D., Aydemir, C., Kisa, C., Aksaray, S., Tuzer, V., Yazici, K., Göka, E., (2005).
IL-6 levels decrease with SSRI treatment in patients with major depression. Hum.
Psychopharm. Clin. 20, 473-476.
Behr, G.A., Moreira, J.C., Frey, B.N., (2012). Preclinical and Clinical Evidence of
Antioxidant Effects of Antidepressant Agents: Implications for the Pathophysiology of
c.
Major Depressive Disorder. Oxid. Med. Cell Longev. 2012, 609421.
Bendele, R.A., Adams, E.R., Hoffman, W.P., Gries, C.L., Morton, D.M., (1992).
Carcinogenicity studies of fluoxetine hydrochloride in rats and mice. Cancer Res. 52,
In
6931-6935.
Berkenbosch F, van Oers J, del Rey A, Tilders, F., Besedovsky, H., (1987). Corticotropin-
releasing factor-producing neurons in the rat activated by interleukin-1. Science, 238,
524-526.
Berlucchi, G. & Buchtel, H. A. (2009). Neuronal plasticity: historial roots and evolution of
rs
meaning. Xp. Brain Res., 192, 307-319.
Bessa, J. M., Ferreira, D., Melo, I., Marques, F., Cerqueira, J. J., Palha, J. A., Almeida, O. F.
& Sousa, N. (2009). The mood-improving actions of antidepressants do not depend on
neurogenesis but are associated with neuronal remodeling. Mol. Psychiatry, 14, 764-773.
he
Bierhaus, A., Wolf, J., Andrassy, M., Rohleder, N., Humpert, P. M., Petrov, D., Ferstl, R.,
von Eynatten, M., Wendt, T., Rudofsky, G., Joswig, M., Morcos, M., Schwaninger, M.,
McEwen, B., Kirschbaum, C. & Nawroth, P. P. (2003). A mechanism converting
psychosocial stress into mononuclear cell activation. Proc. Natl. Acad. Sci. U S A., 100,
1920-1925.
is
Bilici, M., Efe, H., Köroğlu, M. A., Uydu, H. A., Bekaroğlu, M. & Değer, O. (2001).
Antioxidative enzyme activities and lipid peroxidation in major depression: alterations by
antidepressant treatments. J. Affect. Disord., 64, 43-51.
bl
Blind, R. D. & Garabedian, M. J. (2008). Differential recruitment of glucocorticoid receptor
phospho-isoforms to glucocorticoid-induced genes. J. Steroid. Biochem. Mol. Biol., 109,
150-157.
Board, F., Wadeson, R. & Persky, H. (1957). Depressive affect and endocrine functions;
Pu
blood levels of adrenal cortex and thyroid hormones in patients suffering from depressive
reactions. AMA. Arch. Neurol. Psychiatry, 78, 612-620.
Boekema, E. J. & Braun, H. P. (2007). Supramolecular structure of the mitochondrial
oxidative phosphorylation system. J. Bio. Chem., 282,1-4.
Boldrini, M., Hen, R., Underwood, M. D., Rosoklija, G. B., Dwork, A. J., Mann, J. J. &
Arango, V. (2012). Hippocampal angiogenesis and progenitor cell proliferation are
increased with antidepressant use in major depression. Biol. Psychiatry, 72, 562-571.
a
Boldrini, M., Santiago, A. N., Hen, R., Dwork, A. J., Rosoklija, G. B., Tamir, H., Arango, V.
& John Mann, J. (2013). Hippocampal granule neuron number and dentate gyrus volume
ov
in antidepressant-treated and untreated major depression. Neuropsychopharmacology, 38,

1068-1077.
Boldrini, M., Underwood, M. D., Hen, R., Rosoklija, G. B., Dwork, A. J., John Mann, J. &
Arango, V. (2009). Antidepressants increase neural progenitor cells in the human
hippocampus. Neuropsychopharmacology, 34, 2376-2389.
N
Bonaccorso, S., Marino, V., Biondi, M., Grimaldi, F., Ippoliti, F. & Maes, M. (2002).
Depression induced by treatment with interferon-alpha in patients affected by hepatitis C
virus. J. Affect. Disord., 72, 237-241.
Bonfanti, L., Aimar, P., Ponti, G. & Canalia, N. (2008). Immuno-electromicroscopic

approach for the study of neural stem cell niches. Vet. Res. Commun., 32, 107-110.
Bonfanti, L. & Ponti, G. (2008). Adult mammalian neurogenesis and the New Zealand white
rabbit. Vet. J., 175, 310-331.
Bonfanti, L. & Theodosis, D. T. (2009). Polysialic acid and activity-dependent synapse
c.
remodeling. Cell Adhe and Migration, 3, 43–50.
Bonfanti, L. (2006). PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog.
Neurob., 80, 129-164.
In
Bonne, O., Gill, J. M., Luckenbaugh, D. A., Collins, C., Owens, M. J., Alesci, S., Neumeister,
A., Yuan, P., Kinkead, B., Manji, H. K., Charney, D. S. & Vythilingam, M. (2010).
Corticotropin-releasing factor, interleukin-6, brain-derived neurotrophic factor, insulin-
like growth factor-1, and substance P in the cerebrospinal fluid of civilians with
posttraumatic stress disorder before and after treatment with paroxetine. J. Clin.
rs
Psychiatry, 72, 1124–1128.
Boyle, M. P., Brewer, J. A. & Funatsu, M. (2005). Acquired deficit of forebrain
glucocorticoid receptor produces depression-like changes in adrenal axis regulation and
behavior. Proc. Natl. Acad. Sci., 102, 473-478.
he
Brady, L. S., Gold, P. W., Herkenham, M., Lynn, A. B., Whitfield H. J., Jr. (1992). The
antidepressants fluoxetine, idazoxan and phnelzine alter corticotropin- releasing hormone
and tyrosine hydroxylase mRNA levels in rat brain: therapeutic implications. Brain Res.,
572, 117-125.
Buchsbaum, M. S., Wu, J., Siegel, B. V., Hackett, E., Trenary, M., Abel, L. & Reynolds, C.,
is
(1997). Effect of sertraline on regional metabolic rate in patients with affective disorder.
Biol. Psychiatry, 41, 15-22.
Budziszewska, B., Jaworska-Feil, L., Kajta, M. & Lasoń, W. (1994). Antidepressant drugs
bl
inhibit glucocorticoid receptor-mediated gene transcription - a possible mechanism. Br. J.
Pharmacol., 130, 1385-1393.
Budziszewska, B., Siwanowicz, J. & Przegaliński, E. (1994). The effect of chronic treatment
with antidepressant drugs on the corticosteroid receptor levels in the rat hippocampus.
Pu
Pol. J. Pharmacol., 46, 147-152.

Burgess, A., Wainwright, S. R., Shihabuddin, L. S., Rutishauser, U., Seki, T. & Aubert, I.,
(2008). Polysialic acid regulates the clustering, migration, and neuronal differentiation of
progenitor cells in the adult hippocampus. Develop. Neurob., 68, 1580-1590.
Cai, Q., Benson, M. A., Talbot, T. J., Devadas, G., Swanson, H. J., Olson, J. L. & Kirchner, J.
P. (1999). Acute hepatitis due to fluoxetine therapy. Mayo Clin. Proc., 74, 692-694.
Calfa, G., Kademian, S., Ceschin, D., Vega, G., Rabinovich G. A. & Volosin, M. (2003).
a
Characterization and functional significance of glucocorticoid receptors in patients with

major depression: modulation by antidepressant treatment. Psychoneuroendocrinology,
ov
28, 687-701.
Capuron, L., Neurauter, G., Musselman, D. L., Lawson, D. H., Nemeroff, C. B., Fuchs, D. &
Miller, A. H. (2003). Interferon-alpha-induced changes in tryptophan metabolism.
relationship to depression and paroxetine treatment. Biol. Psychiatry, 54, 906-914.
Capuron, L., Ravaud, A., Neveu, P. J., Miller, A. H., Maes, M. & Dantzer, R. (2002).
N
Association between decreased serum tryptophan concentrations and depressive

symptoms in cancer patients undergoing cytokine therapy. Mol. Psychiatry, 7, 468-473.
Carvalho, L. A., Garner, B. A., Dew, T., Fazakerley, H. & Pariante, C. M. (2010).
Antidepressants, but not antipsychotics, modulate GR function in human whole blood: an
insight into molecular mechanisms. Eur. Neuropsychopharmacol., 20, 379-387.
Cattaneo, A., Gennarelli, M. & Pariante, C., (2013). Candidate Genes Expression Profile
Associated with Antidepressants Response in the GENDEP Study: Differentiating
c.
between Baseline ‗Predictors' and Longitudinal ‗Targets‘. Neuropsychopharmacol., 38,
3765-3778.
Charlton, B. G., Ferrier, I. N., Leake, A., Edwardson, J. A., Eccleston, D., Crowcombe, K.,
In
McLean, J., Jackson, S. & Lowry, P. (1988). A multiple time-point study of N-terminal
proopiomelanocortin in depression using a two-site recognition immunoradiometric
assay. Clin. Endocrinol., 28, 165-172.
Charmandari, E., Tsigos, C. & Chrousos, G. P. (2005). Endocrinology of the stress response.
Annu. Rev. Physiol., 67, 259-284.
rs
Chen, J. Q., Yager, J. D. & Russo, J. (2005). Regulation of mitochondrial respiratory chain
structure and function by estrogens/estrogen receptors and potential physiological/
pathophysiological implications. Biochim. Biophys. Acta., 1746, 1-17.
Chen, W., Dang, T., Blind, R. D., Wang, Z., Cavasotto, C. N., Hittelman, A. B., Rogatsky, I.,
he
Logan, S. K. & Garabedian, M. J. (2008). Glucocorticoid receptor phosphorylation
differentially affects target gene expression. Mol. Endocrinol., 22, 1754-1766.
Chourbaji, S., Vogt, M. A. & Gass, P. (2008). Mice that under- or overexpress glucocorticoid
receptors as models for depression or posttraumatic stress disorder. Prog. Brain Res.,
167, 65-77.
is
Chrousos, G. P. & Kino, T. (2005). Intracellular glucocorticoid signaling: a formerly simple
system turns stochastic. Sci. STKE. pe48.
Coogan, P. F., Strom, B. L. & Rosenberg, L. (2009). Antidepressant use and colorectal cancer
bl
risk. Pharmacoepidemiol. Drug Saf., 18, 1111-1114.
Cosgrove, L., Shi, L., Creasey, D. E., McKivergan, M. A., Myers J. A. & Huybrechts, K. F.
(2011). Antidepressants and breast and ovarian cancer risk: a review of the literature and
researchers' financial associations with industry. PloS ONE, 6, e18210.
Pu
Cotter, D. & Pariante, C. M. (2002). Stress on the progress of the developmental hypothesis
of schizophrenia? Br. J. Psychiatry, 181, 363-365.
Cumurcu, B. E., Ozyurt, H., Etikan, I., Demir, S. & Karlidag, R. (2009). Total antioxidant
capacity and total oxidant status in patients with major depression: impact of
antidepressant treatment. Psychiatry Clin. Neurosci., 63,639-645.
Currier, A. R., Ziegler, M. H., Riley, M. M., Babcock, T. A., Telbis, V. P. & Carlin, J. M.
(2000). Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced
a
antichlamydial indoleamine dioxygenase activity independently. J. Interferon Cytokine

Res., 20, 369-376.
ov
Curti, C., Mingatto, F. E., Polizello, A. C., Galastri, L. O., Uyemura, S. A. & Santos A. C.
(1999). Fluoxetine interacts with the lipid bilayer of the inner membrane in isolated rat
brain mitochondria, inhibiting electron transport and F1F0-ATPase activity. Mol. Cell
Biochem., 199, 103-109.
Czéh, B., Michaelis, T., Watanabe, T., Frahm, J., de Biurrun, G., van Kampen, M.,
N
Bartolomucci, A. & Fuchs, E. (2001). Stress-induced changes in cerebral metabolites,

hippocampal volume, and cell proliferation are prevented by antidepressant treatment
with tianeptine. Proc. Natl. Acad. Sci. USA., 98, 12796-12801.
da Silva, A. I., Monteiro Galindo, L. C., Nascimento, L., Moura Freitas, C., Manhaes-de-
Castro, R., Lagranha, C. J. (2014). Lopes de Souza S. Fluoxetine treatment of rat
neonates significantly reduces oxidative stress in the hippocampus and in behavioral
indicators of anxiety later in postnatal life. Can. J. Physiol. Pharmacol., 92, 330-337.
Dalva, M. B., McClelland, A. C. & Kayser, M. S. (2007). Cell adhesion molecules: signalling
c.
functions at the synapse. Nat. Rev. Neurosci., 8, 206–220.
Danner, M., Kasl, S. V., Abramson, J. L. & Vaccarino, V. (2003). Association between
depression and elevated C-reactive protein. Psychosom. Med., 65, 347-356.
In
Dantzer, R. (2009). Cytokine, sickness behavior, and depression. Immunol. Allergy Clin.
North Am., 29, 247-264.
Daubener, W. & MacKenzie, C. R. (1999). IFN-gamma activated indoleamine 2,3-
dioxygenase activity in human cells is an antiparasitic and an antibacterial effector
mechanism. Adv. Exp. Med. Biol., 467, 517-524.
rs
David, D. J., Samuels, B. A., Rainer, Q., Wang, J. W., Marsteller, D., Mendez, I., Drew, M.,
Craig, D. A., Guiard, B. P. & Guilloux, J. P. (2009). Neurogenesis-dependent and-
independent effects of fluoxetine in an animal model of anxiety/depression. Neuron, 62,
479-493.
he
De Bellis, A., Bizzarro, A., Rossi, R., Paglionico, V. A., Criscuolo, T., Lombardi, G. &
Bellastella, A. (1993). Remission of subclinical adrenocortical failure in subjects with
adrenal autoantibodies. J. Clin. Endocrinol. Metab., 76, 1002-1007.
de Kloet, E. R., Vreugdenhil, E., Oitzl, M. S. & Joels, M. (1998). Brain Corticosteroid
Receptor Balance in Health and Disease. Endocr. Rev., 19, 269-301.
is
Deleplanque, B. & Neveu, P. (1995). Immunological effects of neuropsychotropic substances.
In: M. Guenounou, editor. Forum on immunomodulators. Paris, 287-302.
Djordjevic, A., Adzic, M., Djordjevic, J. & Radojcic, M. B. (2009). Chronic social isolation is
bl
related to both upregulation of plasticity genes and initiation of proapoptotic signaling in
Wistar rat hippocampus. J. Neural. Transm., 116, 1579-1589.
Djordjevic, A., Djordjevic, J., Elakovic, I., Adzic, M., Matic, G. & Radojcic, M. B. (2012a).
Effects of fluoxetine on plasticity and apoptosis evoked by chronic stress in rat prefrontal
Pu
cortex. Eur. J. Pharmacology., 693, 37-44.

Djordjevic, A., Djordjevic, J., Elakovic, I., Adzic, M., Matic, G. & Radojcic, M. B. (2012b).
Fluoxetine affects hippocampal plasticity, apoptosis and depressive-like behavior of
chronically isolated rats. Prog. Neuropsychopharmacol. Biol. Psychiatry, 36, 92-100.
Djordjevic, J., Djordjevic, A., Adzic, M., Elaković, I., Matić, G. & Radojcic, M. B. (2011).
Fluoxetine affects antioxidant system and promotes apoptotic signaling in Wistar rat
liver. Eur. J. Pharmacol., 659, 61-6.
a
Dong, X. X., Wang, Y. & Qin, Z. H. (2009). Molecular mechanisms of excitotoxicity and
their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol. Sin., 30,
ov
379-387.
Dranovsky, A. & Hen, R. (2006). Hippocampal neurogenesis: regulation by stress and
antidepressants. Biol. Psychiatry, 59, 1136-1143.
Du, J., Suzuki, K., Wei, Y., Wang, Y., Blumenthal, R., Chen, Z., Falke, C., Zarate, C. A. &
Manji, H. K. (2006). The anticonvulsants lamotrigine, riluzole, and valproate
N
differentially regulate AMPA receptor membrane localization: relationship to clinical

effects in mood disorders. Neuropsychopharmacology, 32, 793-802.
Duman, R. S. (2004). A case of neuronal life and death? Biol. Psychiatry., 56, 140–145.
Dwivedi, Y. (2009). Brain-derived neurotrophic factor: role in depression and suicide.

Neuropsychiatr. Dis. Treat., 5, 433–449.
Ayelli Edgar, V., Sterin-Borda, L., Cremaschi, G. A. & Genaro, A. M. (1999). Role of protein
kinase C and cAMP in fluoxetine effects on human T-cell proliferation. Eur. J.
Pharmacol., 372, 65-73.
c.
Eisch, A. J., Bolaños, C. A., de Wit, J., Simonak, R. D., Pudiak, C. M., Barrot, M.,
Verhaagen, J. & Nestler, E. J. (2003). Brain-derived neurotrophic factor in the ventral
midbrain-nucleus accumbens pathway: a role in depression. Biol. Psych., 54, 994-1005.
In
El Idrissi, A. & Trenkner, E. (1999). Growth factors and taurine protect against excitotoxicity
by stabilizing calcium homeostasis and energy metabolism. J. Neurosci., 19, 9459-9468.
Eller, T., Vasar, V., Shlik, J. & Maron, E. (2008). Pro-inflammatory cytokines and treatment
response to escitaloprsam in major depressive disorder. Prog. Neuropsychopharmacol.
Biol. Psychiatry, 32, 445-450.
rs
Engesser-Cesar, C., Anderson, A. J. & Cotman, C. W. (2007). Wheel running and fluoxetine
antidepressant treatment have differential effects in the hippocampus and the spinal cord.
Neuroscience, 144, 1033–1044.
Ericsson, A., Kovacs, K. J. & Sawchenko, P. E. (1994). A functional anatomical analysis of
he
central pathways subserving the effects of interleukin-1 on stress-related neuroendocrine
neurons. J. Neurosci., 14, 897-913.
Eriksson, P. S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.-M., Nordborg, C., Peterson, D.
A. & Gage, F. H. (1998). Neurogenesis in the adult human hippocampus. Nat. Med., 4,
1313-1317.
is
Escames, G., López, L. C., García, J. A., García-Corzo, L., Ortiz, F. & Acua-Castroviejo, D.
(2012). Mitochondrial DNA and inflammatory diseases. Hum. Genet., 131, 161-173.
Evans, P. H. (1993). Free radicals in brain metabolism and pathology. Br. Med. Bull., 49,
bl
577-87.
Fattal, O., Budur, K., Vaughan, A. J. & Franco, K. (2006). Review of the literature on major
mental disorders in adult patients with mitochondrial diseases. Psychosomatics, 47, 1-7.
Fazzino, F., Montes, C., Urbina, M., Carreira, I. & Lima, L., (2008). Serotonin transporter is
Pu
differentially localized in subpopulations of lymphocytes of major depression patients.

Effect of fluoxetine on proliferation. J. Neuroimmunol., 196, 173-180.
Fialkow, L., Wang, Y. & Downey, G. P. (2007). Reactive oxygen and nitrogen species as
signaling molecules regulating neutrophil function. Free Radic. Biol. Med., 42, 153-164.
Finne, J., Finne, U., Deagostini-Bazin, H. & Goridis, C. (1983). Occurrence of α2-8 linked
polysialosyl units in a neural cell adhesion molecule. Biochem. and Biophys. Res. Comm.,
112, 482-487.
a
Ford, D. E. & Erlinger, T. P. (2004). Depression and C-reactive protein in US adults: data
from the Third National Health and Nutrition Examination Survey. Arch. Intern. Med.,
ov
164, 1010-1014.
Franklin, T. B. & Perrot-Sinal, T. S. (2006). Sex and ovarian steroids modulate brain-derived
neurotrophic factor (BDNF) protein levels in rat hippocampus under stressful and non-
stressful conditions. Psychoneuroendocrinology, 31, 38-48.
Frechilla, D., Otano, A. & Del Río, J. (1998). Effect of chronic antidepressant treatment on
N
transcription factor binding activity in rat hippocampus and frontal cortex. Prog.
Neuropsychopharmacol. Biol. Psychiatry, 22, 787-802.
Frick, L. R., Palumbo, M. a. L., Zappia, M. a. P., Brocco, M. A., Cremaschi, G. A. & Genaro,
A. M. a. (2008). Inhibitory effect of fluoxetine on lymphoma growth through the
modulation of antitumor T-cell response by serotonin-dependent and independent
mechanisms. Biochem. Pharm., 75, 1817-1826.
Frick, L. R., Rapanelli, M., Cremaschi, G. A. & Genaro, A. M. (2009). Fluoxetine directly
c.
counteracts the adverse effects of chronic stress on T cell immunity by compensatory and
specific mechanisms. Brain Behav. Immun., 23, 36-40.
Friedenberg, F. K. & Rothstein, K. D. (1996). Hepatitis secondary to fluoxetine treatment.
In
Am. J. Psychiatry, 153, 580.
Frodl, T. & O'Keane, V. (2012). How does the brain deal with cumulative stress? A review
with focus on developmental stress, HPA axis function and hippocampal structure in
humans. Neurobiol. Dis., 52, 24-37.
Fuchs, E. (2009). Neuroplasticity: a new approach to the pathophysiology of depression.
rs
Neuroplasticity: new biochemical mechanisms. London: Springer 1-12.
Funato, H., Kobayashi, A. & Watanabe, Y. (2006). Differential effects of antidepressants on
dexamethasone-induced nuclear translocation and expression of glucocorticoid receptor.
Brain Res., 1117, 125-134.
he
Galea, L. A., Spritzer, M. D., Barker, J. M. & Pawluski, J. L. (2006). Gonadal hormone
modulation of hippocampal neurogenesis in the adult. Hippocampus, 16, 225-232.
Galecki, P., Szemraj, J., Bienkiewicz, M., Zboralski, K. & Galecka, E. (2009). Oxidative
stress parameters after combined fluoxetine and acetylsalicylic acid therapy in depressive
patients. Hum. Psychopharmacol., 24, 277-286.
is
Gardner, A. & Boles, R. G. (2011). Beyond the serotonin hypothesis: mitochondria,
inflammation and neurodegeneration in major depression and affective spectrum
disorders. Prog. Neuropsychopharmacol. Biol. Psychiatry, 35, 730-743.
bl
Garner, B., Pariante, C. M. & Wood, S. J. (2005). Pituitary volume predicts future transition
to psychosis in individuals at ultra-high risk of developing psychosis. Biol. Psychiatry,
58, 417-423.
Gascon, E., Vutskits, L., Kiss, J. Z. Polysialic acid-neural cell adhesion molecule in brain
Pu
plasticity: from synapses to integration of new neurons. Brain Res. Rev., 56, 101–118.
Gibbs, D. M. & Vale, W. (1983). Effect of the serotonin reuptake inhibitor fluoxetine on
corticotropin-releasing factor and vasopressin secretion into hypophysial portal blood.
Brain Res., 280, 176-9.
Gilabert-Juan, J., Castillo-Gomez, E., Guirado, R., Molto, M. D. & Nacher, J. (2012a).
Chronic stress alters inhibitory networks in the medial prefrontal cortex of adult mice.
Brain Struct. Func., 218, 1591-1605.
a
Gilabert-Juan, J., Varea, E., Guirado, R., Blasco-Ibanez, J. M., Crespo, C. & Nacher, J.
(2012b). Alterations in the expression of PSA-NCAM and synaptic proteins in the
ov
dorsolateral prefrontal cortex of psychiatric disorder patients. Neurosc. Lett., 530, 97-102.
Gold, P. W. & Chrousos, G. P. (2002). Organization of the stress system and its dysregulation
in melancholic and atypical depression: high vs low CRH/NE states. Mol. Psychiatry 7,
254-275.
Gould, M. S., Fisher, P., Parides, M., Flory, M. & Shaffer, D. (1996). Psychosocial risk
N
factors of child and adolescent completed suicide. Arch. Gen. Psychiatry, 53, 1155-1162.
Guidotti, G., Calabrese, F., Anacker, C., Racagni, G., Pariante, C. M. & Riva, M. A. (2013).
Glucocorticoid receptor and FKBP5 expression is altered following exposure to chronic
stress: modulation by antidepressant treatment. Neuropsychopharmacology, 38, 616-627.
Gutiérrez-Lobos, K., Scherer, M., Anderer, P. & Katschnig, H. (2002). The influence of age
on the female/male ratio of treated incidence rates in depression. BMC psychiatry, 2, 3.
c.
Gutman, D. A. & Nemeroff, C. B. (2003). Persistent central nervous system effects of an
adverse early environment: clinical and preclinical studies. Physiol. Behav., 79, 471-478.
Ha, E., Jung, K. H., Choe, B. K., Bae, J. H., Shin, D. H., Yim, S.-V. & Baik, H. H. (2006).
In
Fluoxetine increases the nitric oxide production via nuclear factor kappa B-mediated
pathway in BV2 murine microglial cells. Neurosci. Lett., 397, 185-189.
Halliwell, B. (2007). Biochemistry of oxidative stress. Biochem. Soc. Trans., 35, 1147-1150.
Halliwell, B. (2006). Oxidative stress and neurodegeneration: where are we now? J.
Neurochem., 97, 1634-1658.
rs
Hannestad, J., DellaGioia, N. & Bloch, M. (2011). The effect of antidepressant medication
treatment on serum levels of inflammatory cytokines: a meta-analysis.
Neuropsychopharmacology, 36, 2452-2459.
Hashioka, S., Klegeris, A., Monji, A., Kato, T., Sawada, M., McGeer, P. L. & Kanba, S.
he
(2007). Antidepressants inhibit interferon-gamma-induced microglial production of IL-6
and nitric oxide. Exp. Neurol., 206, 33-42.
Hatanaka, Y., Mukai, H., Mitsuhashi, K., Hojo, Y., Murakami, G., Komatsuzaki, Y., Sato, R.
& Kawato, S. (2009). Androgen rapidly increases dendritic thorns of CA3 neurons in
male rat hippocampus. Biochemical and Biophysical Research Comm., 381, 728-732.
is
Heim, C., Plotsky, P. M. & Nemeroff, C. B. (2004). Importance of Studying the
Contributions of Early Adverse Experience to Neurobiological Findings in Depression.
Neuropsychopharmacology, 29, 641–648.
bl
Heiske, A., Jesberg, J., Krieg, J. C. & Vedder, H. (2003). Differential effects of
antidepressants on glucocorticoid receptors in human primary blood cells and human
monocytic U-937 cells. Neuropsychopharmacology, 28, 807-817.
Herken, H., Gurel, A., Selek, S., Armutcu, F., Ozen, M. E., Bulut, M., Kap, O., Yumru, M.,
Pu
Savas, H. A. & Akyol, O. (2007). Adenosine deaminase, nitric oxide, superoxide

dismutase, and xanthine oxidase in patients with major depression: impact of
antidepressant treatment. Arch. Med. Res., 38, 247-252.
Hernandez, M. a. E., Mendieta, D., Martinez-Fong, D., Lora, F., Moreno, J., Estrada, I.,
Bojalil, R. & Pavon, L. (2008). Variations in circulating cytokine levels during 52 week
course of treatment with SSRI for major depressive disorder. Eur.
Neuropsychopharmacol., 18, 917-924.
a
Hery, M., Semont, A., Fache, M. P., Faudon, M. & Hery, F., (2000). The effects of serotonin
on glucocorticoid receptor binding in rat raphe nuclei and hippocampal cells in culture. J.
ov
Neurochem., 74, 406-413.

Hestad, K. A., Tonseth, S., Stoen, C. D., Ueland, T. & Aukrust, P. (2003). Raised plasma
levels of tumor necrosis factor [alpha] in patients with depression: normalization during
electroconvulsive therapy. J. ECT, 19, 183-188.
Heydendael, W. & Jacobson, L. (2010). Widespread hypothalamic-pituitary-adrenocortical
N
axis-relevant and mood-relevant effects of chronic fluoxetine treatment on glucocorticoid

receptor gene expression in mice. Eur. J. Neurosci., 31, 892-902.
Heyes, M. P., Saito, K. & Markey, S. P. (1992). Human macrophages convert L-tryptophan
into the neurotoxin quinolinic acid. Biochem. J., 283, 633-635.
Hinze-Selch, D., Schuld, A., Kraus, T., Kühn, M., Uhr, M., Haack, M. & Pollmächer, T.
(2000). Effects of antidepressants on weight and on the plasma levels of leptin, TNF-α
and soluble TNF receptors: a longitudinal study in patients treated with amitriptyline or
c.
paroxetine. Neuropsychopharmacology, 23, 13-19.
Hodes, G. E., Hill-Smith, T. E., Suckow, R. F., Cooper, T. B. & Lucki, I. (2010). Sex-
Specific Effects of Chronic Fluoxetine Treatment on Neuroplasticity and
In
Pharmacokinetics in Mice. J. Pharmacol. Exp. Ther., 332, 266-273.
Hodgson, R. A., Higgins, G. A., Guthrie, D. H., Lu, S. X., Pond, A. J., Mullins, D. E., Guzzi,
M. F., Parker, E. M. & Varty, G. B. (2007). Comparison of the V1b antagonist,
SSR149415, and the CRF1 antagonist, CP-154, 526, in rodent models of anxiety and
depression. Pharmacol. Biochem. Behav. 86, 431-440.
rs
Holick, K. A., Lee, D. C., Hen, R. & Dulawa, S. C. (2008). Behavioral effects of chronic
fluoxetine in BALB/cJ mice do not require adult hippocampal neurogenesis or the
serotonin 1A receptor. Neuropsychopharmacology, 33, 406-417.
Holmes, M. C., Antoni, F. A. & Szentendrei, T. (1984). Pituitary receptors for corticotropin-
he
releasing factor: no effect of vasopressin on binding or activation of adenylatecyclas.
Neuroendocrinology, 39, 162-169.
Holsboer, F. & Barden, N. (1996). Antidepressants and hypothalamic-pituitary-adrenocortical
regulation. Endocr. Rev., 17, 187-205.
Holsboer, F. (2000). The corticosteroid receptor hypothesis of depression.
is
Homberg, J. R., Olivier, J. D. A., Blom, T., Arentsen, T., van Brunschot, C., Schipper, P.,
Korte-Bouws, G., van Luijtelaar, G. & Reneman, L. (2011). Fluoxetine exerts age-
bl
dependent effects on behavior and amygdala neuroplasticity in the rat. PLoS ONE, 6,
e16646.
Horikawa, H., Kato, T. A., Mizoguchi, Y., Monji, A., Seki, Y., Ohkuri, T., Gotoh, L.,
Yonaha, M., Ueda, T., Hashioka, S. & Kanba, S. (2010). Inhibitory effects of SSRIs on
Pu
IFN-γ induced microglial activation through the regulation of intracellular calcium. Prog.
Hroudova, J. & Fisar, Z. (2010). Activities of respiratory chain complexes and citrate
synthase influenced by pharmacologically different antidepressants and mood stabilizers.
Neuro. Endocrinol. Lett., 31, 336-342.
Huether, G. & Schuff-Werner, P. (1996). Platelet serotonin acts as a locally releasable
antioxidant. Adv. Exp. Med. Biol., 398, 299-306.
a
Inkielewicz-Stepniak, I. (2011). Impact of fluoxetine on liver damage in rats. Pharmacol.

Rep., 63, 441-447.
ov
Ising, M., Horstmann, S., Kloiber, S., Lucae, S., Binder, E. B., Kern, N., Künzel, H. E.,
Pfennig, A., Uhr, M. & Holsboer, F. (2007). Combined dexamethasone/corticotropin
releasing hormone test predicts treatment response in major depression - a potential
biomarker? Biol. Psychiatry, 62, 47-54.
Ismaili, N. & Garabedian, M. J. (2004). Modulation of glucocorticoid receptor function via
N
phosphorylation. Ann. N. Y. Acad. Sci., 1024, 86-101.

Jazayeri, S., Keshavarz, S.A., Tehrani-Doost, M., Djalali, M., Hosseini, M., Amini, H.,
Chamari, M. & Djazayery, A. (2010). Effects of eicosapentaenoic acid and fluoxetine on
plasma cortisol, serum interleukin-1beta and interleukin-6 concentrations in patients with

major depressive disorder. Psychiatry Res., 178, 112-115.
Jiang, C. & Salton, S. R. (2013). The role of neurotrophins in majord epressive disorder.
Transl. Neurosci., 4, 46-58.
Johnson, J. D., Campisi, J., Sharkey, C. M., Kennedy, S. L., Nickerson, M., Greenwood, B.
c.
N. & Fleshner, M. (2005). Catecholamines mediate stress-induced increases in peripheral
and central inflammatory cytokines. Neuroscience, 135, 1295–1307.
Johnston, D. E. & Wheeler, D. E. (1997). Chronic hepatitis related to use of fluoxetine. Am. J.
In
Gastroenterol., 92, 1225-1226.
Kannen, V., Marini, T., Turatti, A., Carvalho, M. C., Brandão, M. L., Jabor, V. A., Bonato, P.
S., Ferreira, F. R., Zanette, D. L., Wilson, S. A. Jr. & Garcia, S. B. (2011). Fluoxetine
induces preventive and complex effects against colon cancer development in epithelial
and stromal areas in rats. Toxicol. Lett., 204, 134-140.
rs
Karege, F., Perret, G., Bondolfi, G., Schwald, M., Bertschy, G. & Aubry, J. M. (2002).
Psy. Res., 109, 143-148.
Kent, S., Bluthé, R. M., Kelley, K. W. & Dantzer, R. (1992). Sickness behavior as a new
he
target for drug development. Trends Pharmacol. Sci., 13, 24-28.
Khanzode, S.D., Dakhale, G.N., Khanzode, S.S., Saoji, A., Palasodkar, R., (2003). Oxidative
damage and major depression: the potential antioxidant action of selective serotonin-re-
uptake inhibitors. Redox Rep. 8, 365-370.
Kim, S.J., Park, S.H., Choi, S.H., Moon, B.H., Lee, K.J., Kang, S.W., Lee, M.S., Choi, S.H.,
is
Chun, B.G., Shin, K.H., (2006). Effects of repeated tianeptine treatment on CRF mRNA
expression in non-stressed and chronic mild stress-exposed rats. Neuropharmacology 50,
824–833.
bl
Kim, Y., Suh, I., Kim, H., Han, C., Lim, C., Choi, S., Licinio, J., (2001). The plasma levels of
interleukin-12 in schizophrenia, major depression, and bipolar mania: effects of
psychotropic drugs. Mol. psychiatry 7, 1107-1114.
Kirkova, M., Tzvetanova, E., Vircheva, S., Zamfirova, R., Grygier, B., Kubera, M., (2010).
Pu
Antioxidant activity of fluoxetine: studies in mice melanoma model. Cell Biochem.

Funct. 28, 497-502.
Kohen, R., Nyska, A., (2002). Oxidation of biological systems: oxidative stress phenomena,
antioxidants, redox reactions, and methods for their quantification. Toxicol. Pathol. 30,
620-650.
Kolla, N., Wei, Z., Richardson, J.S., Li, X.M., (2005). Amitriptyline and fluoxetine protect
PC12 cells from cell death induced by hydrogen peroxide. J. Psychiatry Neurosci. 30,
a
196-201.
Konradi, C., Eaton, M., MacDonald, M.L., Walsh, J., Benes, F.M., Heckers, S., (2004).
ov
Molecular evidence for mitochondrial dysfunction in bipolar disorder. Arch. Gen.

Kornstein, S.G., Schatzberg, A.F., Thase, M.E., Yonkers, K.A., McCullough, J.P., Keitner,
G.I., Gelenberg, A.J., Davis, S.M., Harrison, W.M., Keller, M.B., (2000). Gender
differences in treatment response to sertraline versus imipramine in chronic depression.
N
Am. J. Psychiatry 157, 1445-1452.

Kotan, V.O., Sarandol, E., Kirhan, E., Ozkaya, G., Kirli, S., (2011). Effects of long-term
antidepressant treatment on oxidative status in major depressive disorder: a 24-week
follow-up study. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 1284-1290.
Krishnan, V., Nestler, E.J., (2008). The molecular neurobiology of depression. Nature 455,
894-902.
c.
Krstic, M.D., Rogatsky, I., Yamamoto, K.R., Garabedian, M.J., (1997). Mitogen-activated
and cyclin-dependent protein kinases selectively and differentially modulate
transcriptional enhancement by the glucocorticoid receptor. Mol. Cell Biol. 17, 3947-
In
3954.
Kubera, M., Grygier, B., Arteta, B., Urbańska, K., Basta-Kaim, A., Budziszewska, B.,
Leśkiewicz, M., Kołaczkowska, E., Maes, M., Szczepanik, M., Majewska, M., Lasoń,
W., (2009). Age-dependent stimulatory effect of desipramine and fluoxetine pretreatment
on metastasis formation by B16F10 melanoma in male C57BL/6 mice. Pharmacol. Rep.
rs
61, 1113-1126.
Kubera, M., Grygier, B., Wrona, D., Rogóż, Z., Roman, A., Basta-Kaim, A., Budziszewska,
B., Leskiewicz, M., Jantas, D., Nowak, W., Maes, M., Lason, W., (2011). Stimulatory
effect of antidepressant drug pretreatment on progression of B16F10 melanoma in high-
he
active male and female C57BL/6J mice. J. Neuroimmunol. 240-241, 34-44.
Kubera, M., Lin, A.H., Kenis, G., Bosmans, E., van Bockstaele, D., Maes, M., (2001). Anti-
inflammatory effects of antidepressants through suppression of the interferon-
γ/interleukin-10 production ratio. J. Clin. Psychopharmacol. 21, 199-206.
Kubera, M., Simbirtsev, A., Mathison, R., Maes, M., (2000). Effects of repeated fluoxetine
is
and citalopram administration on cytokine release in C57BL/6 mice. Psychiatry Res. 96,
255-266.
Ladd, C.O., Huot, R.L., Thrivikraman, K.V., Nemeroff, C.B., Meaney, M.J., Plotsky, P.M.,
bl
(2000). Long-term behavioral and neuroendocrine adaptations to adverse early
experience. Prog. Brain Res. 122, 81-103.
Lai, M., McCormick, J.A., Chapman, K.E., Kelly, P.A., Seckl, J.R., Yau, J.L., (2003).
Differential regulation of corticosteroid receptors by monoamine neurotransmitters and
Pu
antidepressant drugs in primary hippocampal culture. Neuroscience 118, 975-984.

Lee, H.J., Kim, J.W., Yim, S.V., Kim, M.J., Kim, S.A., Kim, Y.J., Kim, C.J., Chung, J.H.,
(2001). Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of
maternally separated rats. Mol. Psychiatry 6,725-728.
Lee, K.M., Kim, Y.K., (2006). The role of IL-12 and TGF-β1 in the pathophysiology of
major depressive disorder. Int. Immunopharmacol. 6, 1298-1304.
Leo, R., Di Lorenzo, G., Tesauro, M., Razzini, C., Forleo, G.B., Chiricolo, G., Cola, C.,
a
Zanasi, M., Troisi, A., Siracusano, A., (2006). Association between enhanced soluble
CD40 ligand and proinflammatory and prothrombotic states in major depressive disorder:
ov
pilot observations on the effects of selective serotonin reuptake inhibitor therapy. J. Clin.
Leonard, B.E., (2001). The immune system, depression and the action of antidepressants.
Prog. Neuropsychopharmacol. Biol. Psychiatry 25,767-780.
Li, X.M., Chlan-Fourney, J., Juorio, A.V., Bennett, V.L., Shrikhande, S., Bowen, R.C.,
N
(2000). Antidepressants upregulate messenger RNA levels of the neuroprotective enzyme

superoxide dismutase (SOD1). J. Psychiatry Neurosci. 25, 43-47.
Lim, C.M., Kim, S.W., Park, J.Y., Kim, C., Yoon, S.H., Lee, J.K., (2009). Fluoxetine affords
robust neuroprotection in the postischemic brain via its anti-inflammatory effect. J.
Neurosci. Res., 87, 1037-1045.
Lindsten, T., Zong, W. X. & Thompson, C. B. (2005). Defining the role of the Bcl-2 family
of proteins in the nervous system. Neuroscientist., 11, 10-15.
c.
Linthorst, A. C. & Reul, J. M. (1999). Inflammation and brain function under basal conditions
and during long-term elevation of brain corticotropin-releasing hormone levels. In
Bantzer R, Wollmann EE, Yimiya R, editors. Cytokines, Stress, and Depression. New
In
York: Kluwer/Plen; p. 129-52.
Liu, D., Wang, Z., Liu, S., Wang, F., Zhao, S. & Hao, A., (2011). Anti-inflammatory effects
of fluoxetine in lipopolysaccharide (LPS)-stimulated microglial cells.
Neuropharmacology, 61, 592-599.
Liu, Y., Ho, R. C. & Mak, A. (2012). Interleukin (IL)-6, tumour necrosis factor alpha (TNF-
rs
alpha) and soluble interleukin-2 receptors (sIL-2R) are elevated in patients with major
depressive disorder: a meta-analysis and meta-regression. J. Affect. Disord., 139, 230-
239.
Lobato, K. R., Cardoso, C. C., Binfaré, R. W., Budni, J., Wagner, C. L., Brocardo, P. S., de
he
Souza, L. F., Brocardo. C., Flesch. S., Freitas, A. E., Dafré, A. L. & Rodrigues, A. L.
(2010). α-Tocopherol administration produces an antidepressant-like effect in predictive
animal models of depression. Behav. Brain Res., 209, 249-259.
Lopez, J. F., Chalmers, D. T., Little, K. Y. & Watson, S. J. (1998). Regulation of serotonin
1A, glucocorticoid and mineralocorticoid receptor in rat and human hippocampus:
is
Implications for the neurobiology of depression. Biol. Psychiatry, 43, 547-573.
Lowry, C. A., Hale, M. W., Burke, K. A., Renner, K. J. & Moore, F. L. (2009). Fluoxetine
potentiates the effects of corticotropin-releasing factor on locomotor activity and
bl
serotonergic systems in the rough skin newt. Horm. Behav., 56, 177-184.
Lucassen, P. J., Fuchs, E. & Czéh, B. (2004). Antidepressant treatment with tianeptine
reduces apoptosis in the hippocampal dentate gyrus and temporal cortex. Biol.
Psychiatry, 55, 789-796.
Pu
Luo, C., Xu, H. & Li, X. M. (2004). Post-stress changes in BDNF and Bcl-2
immunoreactivities in hippocampal neurons: effect of chronic administration of
olanzapine. Brain Res., 1025, 194–202.
Macedo, G. V. F., Cladouchos, M. L. & Sifonios, L. (2012). Effects of fluoxetine on CRF and
CRF1 expression in rats exposed to the learned helplessness paradigm.
Psychopharmacology (Berl), 225, 647-659.
MacLusky, N. J., Hajszan, T., Johansen, J. A., Jordan, C. L. & Leranth, C. (2006). Androgen
a
effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats with
defective androgen receptors. Endocrinology, 147, 2392–2398.
ov
Maes, M., Kubera, M., Obuchowiczwa, E., Goehler, L. & Brzeszcz, J. (2011). Depression's
multiple comorbidities explained by (neuro)inflammatory and oxidative & nitrosative
stress pathways. Neuro. Endocrinol. Lett., 32, 7-24.
Maes, M., Song, C., Lin, A., De Jongh, R., Van Gastel, A., Kenis, G., Bosmans, E., De
Meester, I., Benoy, I., Neels, H., Demedts, P., Janca, A., Scharpé, S. & Smith, R. S.
N
(1998). The effects of psychological stress on humans: increased production of pro-

inflammatory cytokines and Th1-like response in stress-induced anxiety. Cytokine, 10,
313-318.
Magalhaes, A. C., Holmes, K. D. & Dale, L. B. (2010). CRF receptor 1 regulates anxiety
behavior via sensitization of 5-HT2 receptor signaling. Nat. Neurosci., 13, 622-629.
Maheu, M. E., Davoli, M. A., Turecki, G. & Mechawar, N. (2013). Amygdalar expression of
proteins associated with neuroplasticity in major depression and suicide. J. Psych. Res.,
47, 384-390.
c.
Malberg, J. E., Eisch, A. J., Nestler, E. J. & Duman, R. S. (2000). Chronic antidepressant
treatment increases neurogenesis in adult rat hippocampus. J. Neurosci., 20, 9104-9110.
Mansbach, R. S., Brooks, E. N. & Chen, Y. L. (1997). Antidepressant-like effects of CP-
In
154,526, a selective CRF1 receptor antagonist. Eur. J. Pharmacol., 323, 21-26.
Maric, N. & Adzic, M. (2013). Pharmacological modulation of HPA axis in depression – new
avenues for potential therapeutic benefits. Psychiatria Danubina, 25, 299-305.
Matsubara, T., Funato, H., Kobayashi, A., Nobumoto, M. & Watanabe, Y. (2006). Reduced
Glucocorticoid Receptor alpha Expression in Mood Disorder Patients and First-Degree
rs
Relatives. Biol. Psychiatry., 59, 689-695.
Mayberg, H. S. (1997). Limbic-cortical dysregulation: a proposed model of depression. J.
Neuropsychiatry Clin. Neurosci., 9, 471-481.
Mayberg, H. S. (1994). Frontal lobe dysfunction in secondary depression. J. Neuropsychiatry
he
Clin. Neuroscience, 6, 428-442.
McKernana, D. P., Dinana, T. G. & Cryan, J. F. (2009). ‖Killing the Blues‖: a role for cellular
suicide (apoptosis) in depression and the antidepressant response? Prog. Neurobiol., 88,
246–263.
McNutt, M. D., Liu, S., Manatunga, A., Royster, E. B., Raison, C. L., Woolwine, B. J.,
is
Demetrashvili, M. F., Miller, A. H. & Musselman, D. L. (2012). Neurobehavioral effects
of interferon-alpha in patients with hepatitis-C: symptom dimensions and responsiveness
to paroxetine. Neuropsychopharmacology, 37, 1444-1454.
bl
Melmed, S., Polonsky, K. S., Larsen, P. R. & Kronenberg, H. M. (2011). Williams Textbook
of Endocrinology 12th Edition. Elsevier Saunders. Philadelphia 978-1-4377-0324-5.
Merali, Z., Du, L., Hrdina, P., Palkovits, M., Faludi, G., Poulter, M. O. & Anisman, H.
(2004). Dysregulation in the suicide brain: mRNA expression of corticotropin-releasing
Pu
hormone receptors and GABA(A) receptor subunits in frontal cortical brain region. J.
Neurosci., 24, 1478-1485.
Merali, Z., Kent, P., Du, L, Hrdina, P., Palkovits, M., Faludi, G., Poulter, M. O., Bedard, T. &
Anisman, H. (2006). Corticotropin-releasing hormone, arginine vasopressin, gastrin-
releasing peptide, and neuromedin B alterations in stress-relevant brain regions of
suicides and control subjects. Biol. Psychiatry, 59, 594-602.
Meynen, G., Unmehopa, U. A., van Heerikhuize, J. J., Hofman, M. A., Swaab, D. F. &
a
Hoogendijk, W. J. G. (2006). Increased arginine vasopressin mRNA expression in the

human hypothalamus in depression: A preliminary report. Biol. Psychiatry, 60, 892-895.
ov
Michael, N., Gösling, M., Reutemann, M., Kersting, A., Heindel, W., Arolt, V. & Pfleiderer,
B. (2003). Metabolic changes after repetitive transcranial magnetic stimulation (rTMS) of
the left prefrontal cortex: a sham-controlled proton magnetic resonance spectroscopy (1H
MRS) study of healthy brain. Eur. J. Neurosci., 17, 2462-2468.
Michelson, D., Galliven, E., Hill, L., Demitrack, M., Chrousos, G. & Gold, P. (1997).
N
Chronic imipramine is associated with diminished hypothalamic-pituitary-adrenal axis

responsivity in healthy humans. J. Clin. Endocrinol. Metab., 82, 2601-2606.
Milsom, S. R., Conaglen, J. V, Donald, R. A., Espiner, E. A., Nicholls, M. G. & Livesey, J.
H. (1985). Augmentation of the response to CRF in man: relative contributions of
endogenous angiotensin and vasopressin. Clinical Endocrinology, 22, 623-628.
Mitic, M., Lukic, I., Bozovic, N., Djordjevic, J. & Adzic, M. (2014). Fluoxetine Signature on
Hippocampal MAPK Signalling in Sex-Dependent Manner. J. Mol. Neurosci., DOI
c.
10.1007/s12031-014-0328-1.
Mitic, M., Simic, I., Djordjevic, J., Radojcic, M. B. & Adzic, M. (2013). Gender-specific
effects of fluoxetine on hippocampal glucocorticoid receptor phosphorylation and
In
behavior in chronically stressed rats. Neuropharmacology, 70, 100-111.
Moorman, P. G., Grubber, J. M., Millikan, R. C. & Newman, B. (2013). Antidepressant
medications and their association with invasive breast cancer and carcinoma in situ of the
breast. Epidemiology, 14, 307-314.
Mosman, T. R. & Sad, S. (1996). The expanding universe of T-cell subsets: Th1, Th2 and
rs
more. Immunol. Today, 17, 138-146.
Muller, D., Wang, C., Skibo, G., Toni, N., Cremer, H., Calaora, V., Rougon, G. & Kiss, J. Z.
(1996). PSA-NCAM is required for activity-induced synaptic plasticity. Neuron, 17, 413-
422.
he
Murphy, D. D., Cole, N. B. & Segal, M. (1998). Brain-derived neurotrophic factor mediates
estradiol-induced dendritic spine formation in hippocampal neurons. PNAS, 95, 11412-
11417.
Myint, A. M., O'Mahony, S., Kubera, M., Kim, Y. K., Kenny, C., Kaim-Basta, A.,
Steinbusch, H. W. & Leonard, B. E. (2007). Role of paroxetine in interferon-alpha-
is
induced immune and behavioural changes in male Wistar rats. J. Psychopharmacol., 21,
843-850.
Nemeroff, C. B., Owens, M. J., Bissette, G., Andorn, A. C. & Stanley, M. (1998). Reduced
bl
corticotropin releasing factor binding sites in the frontal cortex of suicide victims. Arch.
Gen. Psychiatry, 45, 577-579.
Nemeroff, C. B. & Owens, M. J. (2004). Antidepressant actions on brain noradrenergic
neurons. Pharmacologic differences among the SSRIs: focus on monoamine transporters
Pu
and the HPA axis. CNS Spectr., 6, 23-31.

Nemeroff, C. B., Widerlov, E., Bissette, G., Walleus, H., Karlsson, I., Eklund, K., Kilts, C.
D., Loosen, P. T. & Vale, W. (1984). Elevated concentrations of CSF corticotropin-
releasing factor-like immunoreactivity in depressed patients. Science, 226, 1342-4134.
Nemeroff, C. B. (1996). The corticotropin-releasing factor (CRF) hypothesis of depression:
new findings and new directions. Mol. Psychiatry, 1, 336-342.
Newton, S. S., Thome, J., Wallace, T. L., Shirayama, Y., Schlesinger, L., Sakai, N., Chen, J.,
a
Neve, R., Nestler, E. J. & Duman, R. S. (2002). Inhibition of cAMP response element-
binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like
ov
effect. J. Neuroscience, 22, 10883-10890.

Ng, F., Berk, M., Dean, O., Bush, AI., (2008). Oxidative stress in psychiatric disorders:
evidence base and therapeutic implications. Int. J. Neuropsychopharmacol., 11, 851-876.
Novio, S., Nunez, M. J., Amigo, G. & Freire-Garabal, M. (2011). Effects of fluoxetine on the
oxidative status of peripheral blood leukocytes of restraint-stressed mice. Basic Clin.
N
Pharmacol. Toxicol., 109, 365-371.

O'Brien, S. M., Scully, P., Fitzgerald, P., Scott, L. V., Dinan T. G. (2007). Plasma cytokine
profiles in depressed patients who fail to respond to selective serotonin reuptake inhibitor
therapy. J. Psychiatry Res., 41, 326-331.
Ohgi, Y., Futamura, T., Kikuchi, T. & Hashimoto, K. (2013). Effects of antidepressants on
alternations in serum cytokines and depressive-like behavior in mice after
c.
lipopolysaccharide administration. Pharmacol. Biochem. Behav., 103, 853-859.
Oliveira, S. L., Pillat, M. M., Cheffer, A., Lameu, C., Schwindt, T. T. & Ulrich, H. (2013).
Functions of neurotrophins and growth factors in neurogenesis and brain repair. J. Int.
In
Soc. Adv. Cyt., 83, 76-89.
Oquendo, M. A., Echavarria, G., Galfalvy, H. C., Grunebaum, M. F., Burke, A., Barrera, A.,
Cooper, T. B., Malone, K. M. & John Mann, J. (2003). Lower cortisol levels in depressed
patients with comorbid post-traumatic stress disorder. Neuropsychopharmacoogyl., 28,
591-598.
rs
Overstreet, D. H. & Griebel, G. (2004). Antidepressant-like effects of CRF1 receptor
antagonist SSR125543 in an animal model of depression. Eur. J. Pharmacol., 497, 49-53.
Owen, B. M., Eccleston, D., Ferrier, I. N., Young, A. H. (2001). Raised levels of plasma
interleukin-1beta in major and postviral depression. Acta Psychiatr. Scand., 103, 226-
he
228.
Pace, T. W. & Miller, A. H. (2009). Cytokines and glucocorticoid receptor signaling. Ann. N.
Y. Acad Sci., 1179, 86-105.
Pandey, G. N., Dwivedi, Y., Rizavi, H. S., Ren, X., Zhang, H. & Pavuluri, M. N. (2012).
Brain-derived neurotrophic factor gene and protein expression in pediatric and adult
is
depressed subjects. Prog. Neuropsychopharmacol. Biol. Psychiatry, 34, 645-651.
Pariante, C. M., Vassilopoulou, K., Velakoulis, D., Phillips, L., Soulsby, B., Wood, S. J.,
Brewer, W., Smith, D. J., Dazzan, P., Yung, A. R., Zervas, I. M., Christodoulou, G. N.,
bl
Murray, R., McGorry, P. D. & Pantelis, C. (2004). Pituitary volume in psychosis. Br. J.
Pariante, C. M., Dazzan, P., Danese, A., Morgan, K. D., Brudaglio, F., Morgan, C., Fearon,
P., Orr, K., Hutchinson, G., Pantelis, C., Velakoulis, D., Jones, P. B., Leff, J. & Murray,
Pu
R. M. (2005). Increased pituitary volume in antipsychotic-free and antipsychotic-treated

patients of the AEsop first-onset psychosis study. Neuropsychopharmacol., 30, 1923-
1931.
Pariante, C. M., Kim, R. B., Makoff, A. & Kerwin, R. W. (2003). Antidepressant fluoxetine
enhances glucocorticoid receptor function in vitro by modulating membrane steroid
transporters. Br. J. Pharmacol., 139, 1111-1118.
Pariante, C. M., Makoff, A., Lovestone, S., Feroli, S., Heyden, A., Miller, A. H. & Kerwin, R.
a
W. (2001). Antidepressants enhance glucocorticoid receptor function in vitro by

modulating the membrane steroid transporters. Br. J. Pharmacol., 134, 1335-1343.
ov
Pariante, C. M. & Miller, A. H. (2001). Glucocorticoid receptors in major depression:

relevance to pathophysiology and treatment. Biol. Psychiatry., 49, 391-404.
Pariante, C. M., Pearce, B. D., Pisell, T. L., Owens, M. J. & Miller, A. H. (1997). Steroid-
independent translocation of the glucocorticoid receptor by the antidepressant
desipramine. Mol. Pharmacol., 52, 571-581.
N
Pariante, C. M. (2006). The glucocorticoid receptor: part of the solution or part of the
problem? J. Psychopharmacol., 20, 79-84.
Pavlovic, Z., Delic, D., Maric, N. P., Vukovic, O. & Jasovic-Gasic, M. (2011). Depressive
symptoms in patients with hepatitis C treated with pegylated interferon alpha therapy: a
24-week prospective study. Psychiatr. Danub., 23, 370-377.
Peiffer, A., Veilleux, S. & Barden, N. (1991). Antidepressant and other centrally acting drugs
regulate glucocorticoid receptor messenger RNA levels in rat brain. PNEC, 16, 505-515.
c.
Pellegrino, T. C. & Bayer, B. M. (2001). Specific serotonin reuptake inhibitor-induced
decreases in lymphocyte activity require endogenous serotonin release.
Neuroimmunomodulation, 8, 179-187.
In
Pellegrino, T. C. & Bayer, B. M. (2002). Role of central 5-HT (2) receptors in fluoxetine-
induced decreases in T lymphocyte activity. Brain. Behav. Immun., 16, 87-103.
Pepin, M. C., Govindan, M. V. & Barden, N. (1992). Increased glucocorticoid receptor gene
promoter activity after antidepressant treatment. Mol. Pharmacol., 41, 1016-1022.
Perkins, M. N. & Stone, T. W. (1982). An iontophoretic investigation of the actions of
rs
convulsant kynurenines and their interaction with the endogenous excitant quinolinic
acid. Brain. Res., 247, 184-187.
Pittenger, C. & Duman, R. S. (2008). Stress, depression, and neuroplasticity: a convergence
of mechanisms. Neuropsychopharmacology, 33, 88-109.
he
Pliakas, A. M., Carlson, R. R., Neve, R. L., Konradi, C., Nestler, E. J. & Carlezon, W. A., Jr.
(2001). Altered responsiveness to cocaine and increase immobility in the forced swim
test associated with elevate cAMP response element-binding protein expression in
nucleus accumbens. J. Neurosci., 21, 7397-7403. Carlezon, W.A. Jr,.
Plotsky, P. M. & Meaney, M. J. (1993). Early, postnatal experience alters hypothalamic
is
corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-
induced release in adult rats. Brain Res. Mol., 18, 195-200.
Plotsky, P. M., Owens, M. J. & Nemeroff, C. B. (1998). Psychoneuroendocrinology of
bl
depression. Hypothalamic-pituitary-adrenal axis. Psychiatr. Clin. North. Am., 21, 293-
307.
Ponti, G., Peretto, P. & Bonfanti, L. (2008). Genesis of neuronal and glial progenitors in the
cerebellar cortex of peripuberal and adult rabbits. PLoS One, 3, e2366.
Pu
Prabakaran, S., Swatton, J. E., Ryan, M. M., Huffaker, S. J., Huang, J. T., Griffin, J. L.,
Wayland, M., Freeman, T., Dudbridge, F., Lilley, K. S., Karp, N. A., Hester, S., Tkachev,
D., Mimmack, M. L., Yolken, R. H., Webster, M. J., Torrey, E. F. & Bahn, S. (1993).
Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism
and oxidative stress. Mol. Psychiatry, 9, 684–687.
Przegalinski, E. & Budziszewska, B. (1993). The effect of long-term treatment with
antidepressants drugs on the hippocampal mineralocorticoid and glucocorticoid receptors
a
in rats. Neurosci. Lett., 161, 215–218.

Rakic, P. (2002). Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat.
ov
Rev. Neuosci, 3, 65-71.

Raone, A., Cassanelli, A., Scheggi, S., Rauggi, R., Danielli, B. & De Montis, M. G. (2007).
Hypothalamus–pituitary–adrenal modifications consequent to chronic stress exposure in
an experimental model of depression in rats. Neuroscience, 146, 1734-1742.
Reagan, L. P., Rosell, D. R., Wood, G. E., Spedding, M., Muñoz, C., Rothstein, J. &
N
McEwen, B. S. (2004). Chronic restraint stress up-regulates GLT-1 mRNA and protein
expression in the rat hippocampus: reversal by tianeptine. Proc. Natl. Acad. Sci. USA.,
101, 2179-2184.
Rittmaster, R. S., Cutler, G. B., Jr. Gold, P. W., Brandon, D. D., Tomai, T., Loriaux, D. L. &
Chrousos, G. P. (1987). The relationship of saline-induced changes in vasopressin
secretion to basal and corticotropin-releasing hormone-stimulated adrenocorticotropin
and cortisol secretion in man. J. Clin. Endocrinol. Metab., 642, 371-376.
Roche, K. W., O‘Brien, R. J., Mammen, A. L., Bernhardt, J. & Huganir, R. L. (1996).
c.
Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit.
Neuron, 16, 1179-1188.
Rocher, C., Spedding, M., Munoz, C. & Jay, T. M. (2004). Acute stress-induced changes in
In
hippocampal/prefrontal circuits in rats: effects of antidepressants. Cereb. Cortex., 14,
224-229.
Rogatsky, I., Logan, S. K. & Garabedian, M. J. (1998). Antagonism of glucocorticoid
receptor transcriptional activation by the c-Jun N-terminal kinase. Proc. Natl. Acad. Sci.,
95, 2050-2055.
rs
Rossby, S. P., Nalepa, I., Huang, M., Perrin, C., Burt, A. M., Schmidt, D. E., Gillespie, D. D.
& Sulser, F. (1995). Norepinephrine-independent regulation of GRII mRNA in vivo by a
tricyclic antidepressant. Brain Res., 687, 79-82.
Roumestan, C., Michel, A., Bichon, F., Portet, K., Detoc, M., Henriquet, C., Jaffuel, D. &
he
Mathieu, M. (2007). Anti-inflammatory properties of desipramine and fluoxetine. Respir.
Res., 8, 35.
Saarelainen, T., Hendolin, P., Lucas, G., Koponen, E., Sairanen, M., MacDonald, E.,
Agerman, K., Haapasalo, A., Nawa, H., Aloyz, R., Ernfors, P. & Castrén, E. (2003).
Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is
is
required for antidepressant-induced behavioral effects. J. Neurosci., 23, 349-357.
Sacre, S., Medghalchi, M., Gregory, B., Brennan, F. & Williams, R. (2010). Fluoxetine and
citalopram exhibit potent antiinflammatory activity in human and murine models of
bl
rheumatoid arthritis and inhibit toll-like receptors. Arthritis Rheum., 62, 683-693.
Sairanen, M., O'Leary, O. F., Knuuttila, J. E. & Castrén, E. (2007). Chronic antidepressant
treatment selectively increases expression of plasticity-related proteins in the
hippocampus and medial prefrontal cortex of the rat. Neuroscience, 144, 368-374.
Pu
J., Duman, R., Arancio, O., Belzung, C. & Hen, R. (2003). Requirement of hippocampal
neurogenesis for the behavioral effects of antidepressants. Science, 301, 805-809.
Santibanez, M., Gysling, K. & Forray, M. I. (2006). Desipramine prevents the sustained
increase in corticotropin-releasing hormone-like immunoreactivity induced by repeated
immobilization stress in the rat central extended amygdala. J. Neurosci. Res., 84, 1270–
1281.
a
Sarandol, A., Sarandol, E., Eker, S. S., Erdinc, S., Vatansever, E. & Kirli, S. (2007). Major
depressive disorder is accompanied with oxidative stress: short-term antidepressant
ov
treatment does not alter oxidative-antioxidative systems. Hum. Psychopharmacol., 22,

67-73.
Schmidt, H. D. & Duman, R. S. (2007). The role of neurotrophic factors in adult hippocampal
neurogenesis, antidepressant treatments and animal models of depressive-like behavior.
Behav. Pharmacol., 18, 391-418.
N
Schüle, C. (2007). Neuroendocrinological mechanisms of actions of antidepressant drugs. J.

Neuroendocrinol., 19, 213-226.
Schwaninger, M., Schöfl, C. & Blume, R., (1995). Inhibition by antidepressant drugs of
cyclic AMP response element-binding protein/cyclic AMP response element-directed
gene transcription. Mol. Pharmacol., 47, 1112-1118.
Schwarcz, R., Whetsell, W. O. Jr. & Mangano, R. M. (1983). Quinolinic acid: an endogenous
metabolite that produces axon-sparing lesions in rat brain. Science, 219, 316-318.
c.
Seckl, J. R. & Fink, G. (1992). Antidepressants increase glucocorticoid and mineralocorticoid
receptor mRNA expression in rat hippocampus in vivo. Neuroendocrinology, 55, 621-
666.
In
Senkov, O., Tikhobrazova, O. & Dityatev, A. (2012). PSA-NCAM: synaptic functions
mediated by its interactions with proteoglycans and glutamate receptors. The Int. J.
Bioch. & C. Biol., 44, 591-595.
Shen, Y., Connor, T. J., Nolan, Y., Kelly, J. P. & Leonard, B. E. (1999). Differential effect of
chronic antidepressant treatments on lipopolysaccharide-induced depressive-like
rs
behavioural symptoms in the rat. Life Sci., 65, 1773-1786.
Sher, L., (2004). Daily hassles, cortisol, and the pathogenesis of depression. Med.
Hypotheses, 62, 198-202.
Shimada, K., Crother, T. R., Karlin, J., Dagvadorj, J., Chiba, N., Chen, S., Ramanujan, V. K.,
he
Wolf, A. J., Vergnes, L., Ojcius, D. M., Rentsendorj, A., Vargas, M., Guerrero, C.,
Wang, Y., Fitzgerald, K. A., Underhill, D. M., Town, T. & Arditi, M. (2012). Oxidized
mitochondrial DNA activates the NLRP3 inflammasome duringa poptosis. Immunity, 36,
401-414.
Shirayama, Y., Chen, A. C. H. & Nakagawa, S. (2002). Brain-derived neurotrophic factor
is
produces antidepressant effects in behavioral models of depression. J. Neurosci., 22,
3251–3261.
Simic, I., Adzic, M., Maric, N., Savic, D., Djordjevic, J., Mihaljevic, M., Mitic, M., Pavlovic,
bl
Z., Soldatovic, I., Krstic-Demonacos, M., Jasovic-Gasic, M. & Radojcic, M. (2013a). A
preliminary evaluation of leukocyte phospho-glucocorticoid receptor as a potential
biomarker of depressogenic vulnerability in healthy adults. Psychiatry Res., 209, 658-
664.
Pu
Simic, I., Maric, N. P., Mitic, M., Soldatovic, I., Pavlovic, Z., Mihaljevic, M., Andric, S.,
Radojcic, M. B. & Adzic, M. (2013b). Phosphorylation of leukocyte glucocorticoid
receptor in patients with current episode of major depressive disorder. Prog.
Sluzewska, A., Rybakowski, J., Bosmans, E., Sobieska, M., Berghmans, R., Maes, M. &
Wiktorowicz, K. (1996). Indicators of immune activation in major depression. Psychiatr.
Res. 64, 161-167.
a
Sluzewska, A., Rybakowski, J., Laciak, M., Mackiewicz, A., Sobieska, M. & Wiktorowicz,
K. (1995). Interleukin 6 Serum Levels in Depressed Patients before and after Treatment
ov
with Fluoxetine. Ann. N. Y. Acad. Sci., 762, 474-476.

Smith, M.A., Makino, S., Kvetnansky, R. & Post, R. M. (1995). Stress, and glucocorticoids
affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in
the hippocampus. J. Neurosci., 15, 1768-1777.
Smith, R. S. (1995). The macrophage theory of depression. Med. Hypotheses., 35, 298-306.
N
Sohrabji, F., Miranda, R. C. G. & Toran-Allerand, C. D. (1995). Identification of a putative

estrogen response element in the gene encoding brain-derived neurotrophic factor. PNAS,
92, 110-114.
Song, C., Halbreich, U., Han C., Leonard, B. E. & Luo, H. (2009). Imbalance between pro-
and anti-inflammatory cytokines, and between Th1 and Th2 cytokines in depressed
patients: the effect of electroacupuncture or fluoxetine treatment. Pharmacopsychiatry,
42, 182-188.
Song, C., Leonard, B., (1994). Serotonin reuptake inhibitors reverse the impairments in
c.
behaviour, neurotransmitter and immune functions in the olfactory bulbectomized rat.
Hum. Psychopharmacol. Clin. Exp., 9, 135-146.
Souza, M. E., Polizello, A. C., Uyemura, S. A., Castro-Silva, O. & Curti, C. (1994). Effect of
In
fluoxetine on rat liver mitochondria. Biochem. Pharmacol., 48, 535-541.
Souza, M. E., Polizello, A. C. M., Uyemura, S. A., Castro-Silva, O. & Curti, C. (1994). Effect
of fluoxetine on rat liver mitochondria. Biochem. Pharm., 8, 535-541.
Spritzer, M. D. & Galea, L. A. M. (2007). Testosterone and dihydrotestosterone, but not
estradiol, enhance survival of new hippocampal neurons in adult male rats. Develop.
rs
Neurob., 67, 1321-1333.
Stout, S. C., Owens, M. J. & Nemeroff, C. B. (2002). Regulation of corticotropin-releasing
factor neuronal systems and hypothalamic–pituitary–adrenal axis activity by stress and
chronic antidepressant treatment. J. Pharmacol. Exp. Ther., 300, 1085-1092.
he
Surget, A., Saxe, M., Leman, S., Ibarguen-Vargas, Y., Chalon, S., Griebel, G., Hen, R.,
Belzung, C., (2008). Drug-dependent requirement of hippocampal neurogenesis in a
model of depression and of antidepressant reversal. Biol. Psychiatry., 64, 293-301.
Sutcigil, L., Oktenli, C., Musabak, U., Bozkurt, A., Cansever, A., Uzun, O., Sanisoglu, S. Y.,
Yesilova, Z., Ozmenler, N., Ozsahin, A. & Sengul, A. (2007). Pro-and anti-inflammatory
is
cytokine balance in major depression: effect of sertraline therapy. Clin. Dev. Immunol.,
2007, p. 76396.
Svenningsson, P., Tzavara, E. T., Witkin, J. M., Fienberg, A. A., Nomikos, G. G. &
bl
Greengard, P. (1994). Involvement of striatal and extrastriatal DARPP-32 in biochemical
and behavioral effects of fluoxetine (Prozac). Proc. Natl. Acad. Sci. USA, 99, 3182-3187.
Szigethy, E., Conwell, Y., Forbes, N. T., Cox, C. & Caine, E. D. (1994). Adrenal weight and
morphology in victims of completed suicide. Biol. Psychiatry, 36, 374-380.
Pu
Szymańska, M., Budziszewska, B., Jaworska-Feil, L., Basta-Kaim, Kubera, M., Leśkiewicz,
Regulska, M. & Lason, W. (2009). The effect of antidepressant drugs on the HPA axis
activity, glucocorticoid receptor level and FKBP51 concentration in prenatally stressed
rats. PNEC, 34, 822-832.
Torpy, D. J., Grice, J. E., Hockings, G. I., Walters, M. M., Crosbie, G. V. & Jackson, R.V.
(1997). Diurnal effects of fluoxetine and naloxone on the human hypothalamic-pituitary-
adrenal axis. Clin. Exp. Pharmacol. Physiol., 24, 421-423.
a
Tripp, A., Oh, H., Guilloux, J. P., Martinowich, K., Lewis, D. A. & Sybille, E. (2012). Brain-
derived neurotrophic factor signaling and subgenual anterior cingulate cortex dysfunction
ov
in major depressive disorder. Am. J. of Psy., 169, 1194-1202.

Tsao, C. W., Lin, Y. S., Chen, C. C., Bai, C. H. & Wu, S. R. (2006). Cytokines and serotonin
transporter in patients with major depression. Prog. Neuropsychopharmacol. Biol.
Tsigos, C. & Chrousos, G. P. (2002). Hypothalamic-pituitary-adrenal axis, neuroendocrine
N
factors and stress. J. Psychosom. Res., 53, 865-871.

Tuglu, C., Kara, S. H., Caliyurt, O., Vardar, E. & Abay, E. (2003). Increased serum tumor
necrosis factor-alpha levels and treatment response in major depressive disorder.
Psychopharmacology, 170, 429-433.
Tynan, R.J., Weidenhofer, J., Hinwood, M., Cairns, M. J., Day, T. A. & Walker, F. R. (2012).
A comparative examination of the anti-inflammatory effects of SSRI and SNRI
c.
antidepressants on LPS stimulated microglia. Brain Behav. Immun., 26, 469-479.
Valentino, R. J., Curtis, A. L., Parris, D. G. & Wehby, R. G. (1990). Antidepressant actions
on brain noradrenergic neurons. J. Pharmacol. Exp. Ther., 253, 833-840.
In
Van der Meer, M. J., Sweep, C. G., Rijnkels, C. E., Pesman, G. J., Tilders, F. J.,
Kloppenborg, P. W. & Hermus, A. R. (1996). Acute stimulation of the hypothalamic-
pituitary-adrenal axis by IL-1 beta, TNF alpha and IL-6: a dose response study. J.
Endocrinol. Invest., 19, 175-82.
Varea, E., Blasco-Ibáñez, J. M., Gómez-Climent, M. Á., Castillo-Gómez, E., Crespo, C.,
rs
Martínez -Guijarro, F. J. & Nácher, J. (2006). Chronic fluoxetine treatment increases the
expression of PSA-NCAM in the medial prefrontal cortex. Neuropsychopharmacology,
32, 803-812.
Varea, E., Castillo-Gomez, E., Gomez-Climent, M., Blasco-Ibanez, J., Crespo, C., Martinez-
he
Guijarro, F. & Nacher, J. (2007). Chronic antidepressant treatment induces contrasting
patterns of synaptophysin and PSA-NCAM expression in different regions of the adult rat
telencephalon. European Neuropsychopharmacology, 17, 546-557.
Varea, E., Guirado, R., Gilabert-Juan, J., Martí, U., Castillo-Gomez, E., Blasco- Ibanez, J. M.,
Crespo, C. & Nacher, J. (2012). Expression of PSA-NCAM and synaptic proteins in the
is
amygdala of psychiatric disorder patients. J. Psychiatr. Res., 46, 189-197.
Vaynman, S., Ying, Z., Wu, A. & Gomez-Pinilla, F. (2006). Coupling energy metabolism
with a mechanism to support brain-derived neurotrophic factor-mediated synaptic
bl
plasticity. Neuroscience, 139, 1221-1234.
Vollmayr, B., Mahlstedt, M.M., Henn, F.A., (2007). Neurogenesis and depression: what
animal models tell us about the link. Eur. Arch. Psychiatry. Clin. Neurosci., 257, 300-
303.
Pu
Webster, M., Knable, M., O'Grady, J., Orthmann, J. & Weickert, C. S. (2002). Regional
specificity of brain glucocorticoid receptor mRNA alterations in subjects with
schizophrenia and mood disorders. Mol. Psychiatry, 7, 985-994.
Weiss, J., Dormann, S. M., Martin-Facklam, M., Kerpen, C. J., Ketabi-Kiyanvash, N.,
Haefeli, W. E. (2003). Inhibition of P-glycoprotein by newer antidepressants. J.
Pharmacol. Exp. Ther., 305, 197-204.
Wiener, C., Rassier, G., Kaster, M., Jansen, K., Pinheiro, R., Klamt, F., Magalhães, P.,
a
Kapczinski, F., Ghisleni, G. & da Silva, R. (2014). Gender-based differences in oxidative

stress parameters do not underlie the differences in mood disorders susceptibility between
ov
sexes. Eur. Psychiatry, 29, 58-63.

Willner, P. (2005). Chronic mild stress (CMS) revisited: consistency and behavioural-
neurobiological concordance in the effects of CMS. Neuropsychobiology, 52, 90-110.
Wong-Riley, M. T. (1989). Cytochrome oxidase: an endogenous metabolic marker for
neuronal activity. Trends Neurosci., 12, 94-101.
N
Woo, N. H., Teng, H. K., Siao, C. J., Chiaruttini, C., Pang, P. T., Milner, T. A., Hempstead,
B. L. & Lu, B. (2005). Activation of p75NTR by proBDNF facilitates hippocampal long-
term depression. Nat. Neurosci., 8, 1069-1077.
Xia, Z., Depierre, J. W. & Nassberger, L. (1996). Tricyclic antidepressants inhibit IL-6, IL-1β
and TNF-α release in human blood monocytes and IL-2 and interferon-γ in T cells.
Immunopharmacology, 34, 27-37.
Yamano, M., Yuki, H., Yasuda, S. & Miyata, K. (2000). Corticotropin-releasing hormone
receptors mediate consensus interferon-alpha YM643-induced depression-like behavior
c.
in mice. The J. Pharmacol. Exp. Ther., 292, 181-187.
Yan, H. C., Cao, X., Gao, T. M. & Zhu, X. H. (2011). Promoting adult hippocampal
neurogenesis: a novel strategy for antidepressant drug screening. Curr. Med. Chem., 18,
In
4359-4367.
Yang, S. J., Kim, S. Y., Stewart, R., Kim, J. M., Shin, I. S., Jung, S. W., Lee, M. S., Jeong, S.
H. & Jun, T. Y. (2011). Gender differences in 12-week antidepressant treatment
outcomes for a naturalistic secondary care cohort: The CRESCEND study. Psychiatry
Res., 189, 82-90.
rs
Yaron, I., Shirazi, I., Judovich, R., Levartovsky, D., Caspi, D. & Yaron, M. (1999).
Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid
production in human synovial cells and synovial tissue cultures. Arthritis Rheum., 42,
2561-2568.
he
Yau, J. L., Noble, J., Chapman, K. E. & Seckl, J. R. (2004). Differential regulation of variant
glucocorticoid receptor mRNAs in the rat hippocampus by the antidepressant fluoxetine.
Brain Res. Mol. Brain Res., 129, 189-192.
Yau, J. L., Noble, J., Hibberd, C. & Seckl, J. R. (2001). Short-term administration of
fluoxetine and venlafaxine decreases corticosteroid receptor mRNA expression in the rat
is
hippocampus. Neurosci. Lett., 306, 161-164.
Yau, J. L., Noble, J. & Thomas, S. (2007). The antidepressant desipramine requires the
ABCB1 (Mdr1)-type p-glycoprotein to upregulate the glucocorticoid receptor in mice.
bl
Yirmiya, R., Pollak, Y., Barak, O., Avitsur, R., Ovadia, H., Bette, M., Weihe, E. &
Weidenfeld, J. (2001). Effects of antidepressant drugs on the behavioral and
physiological responses to lipopolysaccharide (LPS) in rodents.
Pu

Young, E. A., Lopez, J. F., Murphy-Weinberg, V., Watson, S. J. & Akil, H. (2003).
Mineralocorticoid receptor function in major depression. Arch. Gen. Psychiatry, 60, 24-
28.
Zafir, A., Ara, A. & Banu, N., (2009). In vivo antioxidant status: a putative target of
antidepressant action. Prog. Neuropsychopharmacol. Biol. Psychiatry 33, 220-228.
Zafir, A. & Banu, N., (2007). Antioxidant potential of fluoxetine in comparison to Curcuma
a
longa in restraint-stressed rats. Eur. J. Pharmacol., 572, 23-31.

Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Itagaki, K. &
ov
Hauser, C. J. (2010). Circulating mitochondrial DAMPs cause inflammatory responses to

injury. Nature, 464, 104-107.
N
Zhou, L., Xiong, J., Lim, Y., Ruan, Y., Huang, C., Zhu, Y., Zhong, J. H., Xiao, Z. & Zhou, X.
F. (2013). Upregulation of blood proBDNF and its receptors in major depression. J.
Affect. Dis., 150, 776-784.
Zlatkovic, J., Todorovic, N., Tomanovic, N., Boskovic, M., Djordjevic, S., Lazarevic-Pasti,
T., Bernardi, R. E., Djurdjevic, A. & Filipovic, D. (2014). Chronic administration of
c.
fluoxetine or clozapine induces oxidative stress in rat liver: A histopathological study.
Eur. J. Pharm. Sci., 59 C, 20-30.
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Chapter 13
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Clinical Implications of Fluvoxamine
and Fluoxetine with Sigma-1 Receptor
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Chaperone Activity in the Treatment of
Neuropsychiatric Disorders
1
2 he
Yakup Albayrak1 and Kenji Hashimoto2,*
Namık Kemal University, Medical Faculty, Department of Psychiatry, Tekirdag, Turkey
Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health,
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Chiba, Japan
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Abstract
Selective serotonin reuptake inhibitors (SSRIs) are used as therapeutic drugs for a
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number of neuropsychiatric diseases throughout the world. Although all SSRIs act by
blockading serotonin transporters, leading to elevated serotonin levels in the central
nervous system (CNS), it is well known that their pharmacology is quite heterogeneous.
Endoplasmic reticulum protein sigma-1 receptors represent a unique chaperone activity,
and they exert a potent influence on a number of neurotransmitter systems. Accumulating
evidence suggests that sigma-1 receptors play a role in the pathophysiology of
neuropsychiatric and cardiovasular diseases, as well as in the mechanisms of some
SSRIs. Among the SSRIs, the order of affinity for sigma-1 receptors is as follows:
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fluvoxamine > sertraline > fluoxetine > escitalopram > citalopram >> paroxetine. In cell
culture systems, some SSRIs (e.g., fluvoxamine, fluoxetine and escitalopram) potentiate
nerve-growth factor (NGF)-induced neurite outgrowth in PC12 cells, and these effects
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can be antagonized by the selective sigma-1 receptor antagonist, NE-100. Furthermore,

fluvoxamine, but not paroxetine or sertraline, improved phencyclidine-induced cognitive
impairment in mice, and again, this effect could be antagonized by NE-100. Several
clinical studies have found that fluvoxamine may offer beneficial effects in patients with
neuropsychiatric disorders. In this chapter, the authors will discuss the role of sigma-1
receptors in the mechanistic action of some SSRIs and new clinical implications for
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SSRIs with potent sigma-1 receptor chaperone activity.
*
Corresponding author: 1-8-1 Inohana, Chiba 260-8670, Japan, E-mail: hashimoto@faculty.chiba-u.jp.
280 Yakup Albayrak and Kenji Hashimoto
Keywords: cognition; neurite outgrowth; neuroprotection; neuroplasticity; selective serotonin

reuptake inhibitors (SSRIs); sigma-1 receptor chaperone
Introduction
c.
Sigma receptors were identified by Martin and coworkers in 1976, and were initially
considered a subclass (sigma opioid receptor) of opioid receptors (Martin et al., 1976).
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Intense research on these receptors eventually led to their reclassification from opioid
receptors, to a distinct category of sigma receptors. More confusion ensued when sigma
receptors were mistaken for the phencyclidine (PCP) binding site on N-methyl-D-aspartate
(NMDA) receptors. Fortunately, over the last fifteen years, research has produced enhanced
knowledge of the structure and function of sigma receptors providing a better understanding
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in this field (Maurice and Su, 2009; Hayashi and Su, 2004; Fishback et al., 2010). Sigma
receptors have recently been confirmed as a non-opioid, endoplasmic reticulum (ER) protein,
with two subtypes, namely sigma-1 and sigma-2 receptors.
Sigma-1 receptors comprise 223 amino acids with two potential trans-membrane
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domains. They confer effects on various cellular fuctions, such as Ca2+ signaling, ion channel
firing, protein kinase translocation or activation, cellular redox, neurotransmitter release,
inflammation, cellular differentiation, neuronal survival and synaptogenesis (Marrazzo et al.,
2005; Maurice and Su, 2009; Hayashi and Su, 2004; Fishback et al., 2010; Yao et al., 2010;
Hayashi et al., 2011). Hayashi and Su (2007) demonstrated that the C-terminus of the sigma-1
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receptor possess a molecular chaperone activity that stabilizes ER proteins, thus regulating
their degradation (Hayashi and Su, 2007; Hayashi et al., 2011). Both preclinical and clinical
studies have recorded effects for sigma receptors in cardiovascular diseases (CVD), immune
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reactions, pain, cancer proliferation, liver function and various neuropsychiatric conditions
(Hayashi and Su, 2004; Hashimoto and Ishiwata, 2006; Hayashi et al., 2011; Hashimoto,
2013).
Recent research has presented beneficial clinical effects for SSRI mediated sigma-1
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receptor agonism, in a number of neuropsychiatric diseases. Although to date there are a

limited numbers of clinical studies using SSRI agonism at sigma-1 receptors to treat
psychiatric and neurological disorders, the results of fluvoxamine, an SSRI with potent
sigma-1 receptor chaperone activity seem promising. In this chapter, we will discuss the
clinical implications of SSRIs with sigma-1 receptor chaperone activity, in neuropsychiatric
and other diseases.
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Interaction of SSRIs with Sigma-1 Receptors

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SSRIs are widely used in the treatment of major depressive disorder (MDD), as well as
other psychiatric illnesses, such as anxiety and eating disorders. While all SSRIs elevate
serotonin levels in the central nervous system (CNS) by blockade of serotonin transporters, it
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is well known that their pharmacology is very heterogeneous (Goodnick and Goldstein, 1998;
Stahl, 1998; Nemeroff and Owens, 2004). SSRIs are reported to have moderate to high
affinity for sigma-1, but not sigma-2 receptors (Narita et al., 1996; Ishima et al., 2014)
Clinical Implications of Fluvoxamine and Fluoxetine with Sigma-1 Receptor … 281
(Table 1). The order of potency for SSRIs at sigma-1 receptor chaperones is as follows:
fluvoxamine (Ki = 17.0 nM) > sertraline (Ki = 31.6 nM) > fluoxetine (Ki = 191.2 nM) >
escitalopram (Ki = 288.3 nM) > citalopram (Ki = 403.8 nM) >> paroxetine (Ki = 2,041 nM)
(Ishima et al., 2014). In contrast, serotonin and norepinephrine reuptake inhibitors (SNRIs)
have no affinity at sigma-1 receptors (Ishima et al., 2014). A study using positron emission
c.
tomography showed that orally administered fluvoxamine bound to sigma-1 receptors in the
intact human brain (Ishikawa et al., 2007). This suggests that sigma-1 receptors play a role in
the mechanistic action of SSRIs, such as fluvoxamine, fluoxetine and escitalopram
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(Hashimoto, 2009a; 2013; Ishikawa and Hashimoto, 2010; Ishima et al., 2014).
Cognition and Sigma-1 Receptors
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Cognitive deficits are seen in several psychiatric disorders such as major depressive
disorder (MDD) and schizophrenia. The severity of depression in patients with MDD is
associated with the extent of cognitive, psychomotor and memory impairment (Cohen et al.,
1982; Porter et al., 2003; Hashimoto, 2009b; Hindmarch and Hashimoto, 2010; Yoshida et
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al., 2012).
Sigma-1 receptors can affect a number of neurotransmitter systems (Hayashi and Su,
2004; Hashimoto and Ishiwata, 2006; Hayashi et al., 2011; Hashimoto, 2013). SSRIs such as
fluvoxamine, fluoxetine and escitalopram can potentiate nerve-grouwth factor (NGF)-induced
neurite outgrowth in PC12 cells, via sigma-1 receptor activation, whereas sertraline does not
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(Takebayashi et al., 2002; Nishimura et al., 2008; Ishima et al., 2014). Furthermore, this
fluvoxamine potentiation of NGF-induced neurite outgrowth is significantly antagonized by
sertraline, indicating that sertraline may act as an antagonist at sigma-1 receptors (Ishima et
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al., 2014) (Table 1). The available evidence suggests that activation or up-regulation of
sigma-1 receptors may promote neuronal differentiation and neuroprotection (Hayashi and
Su, 2004; Hashimoto and Ishiwata, 2006; Hayashi et al., 2011). Moreover, fluvoxamine, but
not sertraline or paroxetine, improve phencyclidine (PCP)-induced cognitive deficits in mice,
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while the selective sigma-1 receptor antagonist, NE-100 significantly antagonizes this effect
(Hashimoto et al., 2007; Ishima et al., 2008). Cumulative reports, propose the possibility that
sigma-1 receptor agonists, typified by fluvoxamine promote improved memory and learning
processes (Debonnel and deMontigny, 1996; Takebayashi et al., 2002; Guitart et al., 2004;
Volz and Stoll, 2004; Stall, 2005; Hashimoto et al., 2007; Ishima et al., 2009; Niitsu et al.,
2012b).
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A randomized, double-blind study showed that fluvoxamine significantly increased

performance in the digit symbol substitution test (Perez and Ashford, 1990). In patients with
MDD, a four week treatment regimen with fluvoxamine was associated with significant
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symptomatic remission, with higher total Wechsler IQ scores. Also, a lower incidence of
cognitive impairment was reported in patients who responded to fluvoxamine treatment
(Mandelli et al., 2006). Two case reports showed that fluvoxamine improved cognitive
impairment in patients with schizophrenia (Iyo et al., 2008; Niitsu et al., 2010). A subsequent
randomized, double-blind, placebo-controlled study showed no major benefit for fluvoxamine
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in alleviating cognitive impairment in schizophrenics. However, secondary analysis found

improved executive function with fluvoxamine treatment (Niitsu et al., 2012a). These
findings highlight the need for further large scale studies to confirm the effects of SSRIs with
sigma-1 receptor activity on cognitive impairment.
Psychotic Depression and Fluvoxamine
c.
MDD is the most prevalent of all the psychiatric disorders. It is estimated that
aproximately 17 percent of the American population suffers from depressive disorder at some
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point of their lives (Kessler et al., 2003; 2005). Furthermore, MDD is considered the single
most burdensome disease in the world, in terms of total disability among people in midlife,
according to a World Health Organization (WHO) Global Burden of Disease Study (Murray
and Lopez, 1996). MDD is often associated with poor physical health, such as fibromyalgia,
higher rates of cardiac problems and higher rates of smoking (Nicholson et al., 2006; Wulsin
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and Singal, 2003). Due to its debilitating nature, depression is strongly linked to increased
levels of economic loss. The symptamotology commomly consists of depressed mood,
anhedonia, poor apetite, weight loss, sleep disturbances, feelings of guilt, agitation or
psychomotor retardation, fatigue or loss of energy, diminished ability to concentrate and
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recurrent thoughts of death. Suicidal behaviour is not uncommon in patients with severe
depression (Kessler et al., 2003; 2005). In 1999, the number of suicides in the USA was
reported at 30,000, with suicide being the the third leading cause of death between the ages of
15 and 24 years (Charney and Manji, 2004).
Psychotic depression is a clinical subtype of MDD and is characterized by psychosis
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accompanied by relatively severe depressive symptoms, which include psychomotor
impairment, morbid cognition, suicidal ideation and neuropsychological impairment. A
number of clinical studies have demonstrated the efficacy of combined therapy using an
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antidepressant (e.g., tricyclic antidepressant or SSRI) and an atypical antipsychotic or
electroconvulsive therapy (ECT), in treating psychotic depression. In some instances, both the
treating clinician and patient try to avoid using antipsychotics due to the risk of
extrapyramidal side effects (EPS) induced by this class of drugs (Hamoda abd Osser, 2008).
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Interestingly, monotherapy using fluvoxamine is effective against both the psychotic and
depressive symptoms of this disorder (Gatti et al., 1996; Zanardi et al., 2000), whereas
paroxetine has a lesser effect (Zanardi et al., 1997). It seems likely that both serotonin
reuptake inhibition and sigma-1 receptor activation are utilitized in the action of fluvoxamine,
since paroxetine exerts a lesser effect in psychotic depression (Zanardi et al., 1997). Furuse
and Hashimoto (2009) reported five patients with psychotic depression who were treated
a
effectively with fluvoxamine monotherapy. Kishimoto et al. (2010) reported a case of

relapsed psychotic depression after a switch from fluvoxamine to sertraline treatment. In this
case, the patient was restarted on fluvoxamine at 150 mg/day and psychotic depression
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symptoms gradually resolved after a week of treatment.

These combined findings led to the proposal that sigma-1 receptors are functional in the
efficacy of fluvoxamine when used in psychotic depression (Hayashi and Su, 2005; Stahl,
2005; Hashimoto, 2009a; Ishikawa and Hashimoto, 2010). However, they do not confim
involvement of sigma-1 receptor agonism as an active mechanism of fluvoxamine. To
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achieve this, randomized double-blind studies of fluvoxamine and sertraline (a sigma-1

receptor antagonist) in patients with psychotic depression may be helpful.
Table 1. Pharmacology of SSRIs at sigma-1 receptors in rat brains
Drugs Ki (nM) Pharmacology

Fluvoxamine 17 agonist
Sertraline 31.6 antagonist
c.
Fluoxetine 191.2 agonist
Escitalopram 288.3 agonist
Citalopram 403.8 agonist
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Paroxetine 2,041 —
From Ishima et al., 2014.
Delirium and Fluvoxamine
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Delirium is a clinical condition characterized by an acute disturbance in consciousness
and cognition, particulary in attention. Delirium often includes perceptual disturbances,
abnormal psychomotor activity, and sleep cycle impairment. It is a common occurrence
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among inpatients and is sometimes difficult to recognise. Unfortunately, it also complicates
patients‘ prognoses and is linked to a high mortality rate. The pathophysiology of delirium is
still not clearly understood. Data suggests that drug toxicity, inflammation and acute stress
responses all contribute to the disruption of neurotransmitters, such as acetylcholine,
glutamate, γ-aminobutyric acid, dopamine, serotonin and norepinephrine, and consequently,
is
to the development of delirium (Girard and Pandharipande, 2008; Fong et al., 2009).
Besides the non-pharmacological management of delirium, both typical and atypical
antipsychotics are used in its treatment. In fact, antipsychotics are the most frequently used
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drugs to treat this syndrome. However, patients treated with antipsychotic drugs need closer
monitoring for a variety of adverse events, including hypotension, dystonia, extrapyramidal
effects, laryngeal spasms, malignant hyperthermia, glucose and lipid dysregulation and
anticholinergic effects such as dry mouth, constipation and urinary retention (Girard and
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Pandharipande, 2008; Maldonado, 2008; Gunther et al., 2008; Fong et al., 2009). The United
States Food and Drug Administration (FDA) report has cautioned against the use of atypical
antipsychotics in elderly patients with dementia, as they are associated with a higher
incidence of mortality. Benzodiazepines are also used in the management of delirium to
sedate agitated patients. When the agitation is associated with sedative-hypnotic withdrawal,
benzodiazepines are the treatment of choice (Solai, 2009).
a
There is limited data on the association between SSRIs and delirium. What is available
appears to suggest that a combination of SSRIs with antipsychotic drug(s) and concomitant
benztropine may increase the risk of delirium in patients. Byerly et al. (1996) reported on a
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patient who developed delirium after receiving a combination of sertraline, haloperidol and
benztropine. Similarly, Armstrong et al. (1997) reported on a case who developed delirium
after being administered benztropine and paroxetine concomitantly. More recently, Delić and
Pregelj (2013) reported on a case who exhibited delirium three days after citalopram (20 mg
i.v.) treatment. As well as SSRI induced delirium, there have been three patients who
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developed the disorder during SNRI treatment; one patient was treated with duloxetine
(Suzuki et al., 2012) and the other two, with venlafaxine (Howe and Ravasia, 2003;
Alexander and Nillsen, 2011).
In contrast, there are case reports which show beneficial effects for fluvoxamine in
treating delirium. Furuse and Hashimoto (2010a; 2010b; 2010c) reported effective use of
fluvoxamine in treating delirium in patients with Alzheimer‘s disease (Furuse and Hashimoto,
c.
2010a), patients in intensive care units (Furuse and Hashimoto, 2010b), and elderly patients
with postoperative delirium (Furuse and Hashimoto, 2010c). The Delirium Rating Scale in
these elderly patients decreased after treatment with fluvoxamine (50-150 mg). These case
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reports suggest that fluvoxamine shows good potential for clinical use in treating this disorder
in elderly cohorts, although appropriate clinical studies will be needed to confirm its efficacy.
At present, it is unclear whether the actions of fluvoxamine against delirium are mediated by
sigma-1 receptors. Evidence points towards sigma-1 receptor agonism, since sigma-1
receptors regulate a number of neurotransmission pathways, including those in cognition
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(Hashimoto, 2009a; Hayashi et al., 2011). These reports suggest that fluvoxamine may be a
potential agent for the treatment of delirium in older adults, with minimal side effects
(Hashimoto and Furuse, 2012). Further testing is needed to verify this conclusion.
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Akathisia and Fluvoxamine
The term of akathisia, derived from the Greek language, means ‗inability to sit‘. This
clinical condition is characterized by inner restlesness and an inability to sit or remain
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motionless (Owens, 1999). It was introduced into literature by Hascovec in 1901. Since the
first use of neuroleptics in schizophrenia treatment, the emergence of akathisia has garnered
increased attention (Mohr and Volavka, 2002). Akathisia is widely accepted as an
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extrapyramidal side effect (EPS), seen during treatment with neuroleptics. Its development
can adversely affect patients‘ adherence to medication, and as a consequence, have a negative
impact on long-term treatment outcomes in schizophrenia (Kane, 2001; Kane et al., 2009;
2010). Furthermore, as a side effect, it is serious enough in some cases to cause suicidal
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behaviour. Although akathisia is considered an EPS, induced by typical antipsychotics, it is

not uncommon in atypical antipsychotic treatment. Due to its close association with drug
treatment, efforts to unravel the pathophysiology of akathisia have focussed on
neurotransmitter systems, particularly the dopaminergic system (Sachdev, 2005).
There are several treatment options for akathisia. Besides prevention, β-adrenergic
blockers, benzodiazepines and anticholinergics are commonly used in the management of
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akathisia (Kumar and Sachdev, 2009). There are three reports showing potential positive
results using fluvoxamine in patients with akathisia. Furuse and Hashimoto (2010d; 2010e)
reported that fluvoxamine alleviated akathisia in schizophrenic patients previously treated
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with aripiprazole or blonanserin. Recently, we reported on a patient with tardive akathisia

who was improved with fluvoxamine over a short time period (Albayrak and Hashimoto,
2013). From these case reports, it is unclear whether sigma-1 receptor agonism is relevant to
the anti-akathitic action of fluvoxamine. A number of neurotransmitter systems are thought to
be involved in the complex pathophysiology of akathisia (Kane et al., 2009). Considering the
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importance of sigma-1 receptors in the regulation of neurotransmitter systems (Hayashi and

Su, 2004; Hashimoto and Ishiwata, 2006; Hashimoto, 2009a; Ishikawa and Hashimoto, 2010;
Hayashi et al., 2011), it is likely that indirect modulation of some of these systems by sigma-1
receptor agonists may facilitate the action of this drug, although further detailed studies will
of course be necessary.
c.
Prevention of Psychosis by Fluvoxamine
Schizophrenia is a chronic, disabling psychiatric disorder, affecting roughly one percent
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of the world‘s population. The symptoms of schizophrenia include positive and negative
symptoms, cognitive impairment and deterioration in social and occupational functioning
(Tandon et al., 2009). In the past decade, there has been increasing interest in the potential
benefit of early pharmacological and psychological intervention in this disease. Patients with
schizophrenia show non-psychotic and non-specific prodromal symptoms (such as depression
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and cognitive impairment) for several years preceding the onset of frank psychosis. Although
several studies have demonstrated that therapy with atypical antipsychotic drugs in prodromal
symptom sufferers may reduce the risk of subsequent transition to schizophrenia, the use of
antipsychotic drugs is hampered by their sometimes severe adverse effects (de Koning et al.,
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2009; Liu and Demjaha, 2013).
A recent meta-analysis showed that adolescents and young adults at high-risk for
developing psychosis, showed cognitive impairment before the onset of psychosis (Fusar-Poli
et al., 2012). Given that we already know sigma-1 receptors modulate cognitive function
(Niitsu et al., 2012b), Hashimoto (2009c) was the first to put forward the hypothesis that
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fluvoxamine might reduce the risk of subsequent transition to psychosis, based on sigma-1
receptor mediated neuroprotective or neurotrophic activity. Two years after this hypothesis,
Takodoro et al. (2011) presented a case report showing that fluvoxamine could prevent the
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onset of psychosis in a high-risk subject. Futher clinical trials are required to verify this
hypothesis.
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Cardiovascular Disease and Fluvoxamine

Sigma-1 receptors are not confined to the brain. They are also widely distributed in
peripheral organs, such as lung, liver, adrenal glands, testis, kidney and heart (Su et al.,
1988a; 1988b; Hashimoto and Ishiwata, 2006; Collier et al., 2007; Hashimoto, 2013). In the
heart, sigma-1 receptors are present in cardiomyocytes from neonatal rats (Ela et al., 1994)
a
and on the membranes of cardiac myocytes from adult rats (Novakova et al., 1995).
Moreover, sigma-1 receptors show greater expression in whole-cell extracts from the left
(LV) and right heart ventricles (RV), compared with brain samples (Bhuiyan et al., 2011;
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Bhuiyan and Fukunaga, 2011). It is highly plausible that cardiovascular function is affected
by complex pathways, initiated by the binding of cardiac sigma-1 receptors to agonist ligands
(Bhuiyan and Fukunaga, 2011). Studies have found that patients with progressive LV
hypertrophy show reduced expression of sigma-1 receptors in LV tissue and a significant
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negative correlation between sigma-1 receptor expression in the LV and heart failure
(Bhuiyan et al., 2011; Bhuiyan and Fukunaga, 2011). Itoh et al. (2012) reported that
decreased levels of sigma-1 receptors in the brain are crucial to the link between heart failure
and depression. Mice with impaired cardiac function induced by aortic banding and high salt
intake, exhibited reduced levels of sigma-1 receptors in the brain and depression-like
behavior. Intracerebroventricular (ICV) infusion of the sigma-1 receptor agonist, PRE084,
increased brain sigma-1 receptor expression, lowered sympathetic activity resulting in
improved cardiac function, and improved depression-like behavior in this model (Itoh et al.,
c.
2012). In contrast, ICV infusion of the sigma-1 receptor antagonist, BD1063, increased
sympathetic activity and decreased cardiac function in sham mice. Conversely, oral
administration of fluvoxamine attenuated sympathetic hyperactivation, in addition to
In
improving depression-like behavior in mice with aortic banding (Itoh et al., 2012). These
findings suggest that sigma-1 receptors are integral to the pathophysiology of cardiac
hypertrophy and heart failure and therefore, receptor agonists, such as fluvoxamine represent
potential therapeutic drugs for these diseases (Bhuiyan and Fukunaga, 2011; Hashimoto,
2013).
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There is a strong association between cardiovascular disease (CVD) and depression
(Baune et al., 2012; Nemeroff and Goldschmidt-Clermont, 2012). Available data suggested
that treatment of depression by SSRIs was potentially safe for patients with CVD (Taylor et
al., 2005). However, a recent review reported only minimal benefit for SSRIs on depression
he
outcomes in patients with CVD (Baumeister et al., 2011). Preclinical studies show that
fluvoxamine offers positive cardioprotective effects via sigma-1 receptor activation
(Tagashira et al., 2010; Bhuiyan et al., 2010; Bhuiyan et al., 2011). Despite the promising
reports of preclinical efficacy for fluvoxamine in CVD, there are no clinical studies
evaluating these effects. Taken together, it would be of great interest to examine the effects of
is
fluvoxamine in depressed patients with CVD (Hashimoto, 2013; Bhuiyan et al., 2013).
Hyperkinetic Movement Disorders

bl
It has been proposed that sigma receptor function is needed for the brain mechanisms
involved in movement (Walker et al., 1994). Several case studies show beneficial effects for
Pu
fluvoxamine in several diseases, mediated by possible sigma-1 receptor activation. We

reported on two patients with schizophrenia who suffered from tardive dyskinesia and who
achieved a gradual lessening of symptoms with fluvoxamine (Albayrak and Hashimoto,
2013). In addition, we reported five cases with post-psychotic depressive disorder and
schizophrenia, where depression and tardive dyskinesia improved with fluvoxamine
(Albayrak and Hashimoto, 2012). Similarly, we also reported a case who developed tardive
a
dyskinesia during duloxetine treatment, which resolved after switching to fluvoxamine

(Albayrak and Ekinci, 2012). Other reports found improvements in chorea and hemiballism in
patients with Huntington‘s disease and depression, respectively (Caykoylu et al., 2011;
ov
Albayrak and Ekinci, 2012). While these results highlight the benefit of fluvoxamine therapy
as a safe alternative treatment in hyperkinetic movement disorders, more clinical trials are
needed to confirm this effect.
N
Conclusion
There are a number of preclinical and clinical findings that emphasize the beneficial
effects of fluvoxamine treatment in various neuropsychiatric disorders. Given that
fluvoxamine is an SSRI with potent sigma-1 receptor chaperone activity, we conclude that
c.
these receptors mediate its action. This conclusion will no doubt be tested in future detailed
studies examining the action of fluvoxamine on various neuropsychiatric and CVD.
In
Acknowledgment
The authors would like to thank our collaborators who worked on some papers described
in this chapter, and who are listed as the co-authors of our papers in the reference list. This
rs
study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas of the
Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.H.).
he
References
Albayrak, Y. & Ekinci, O. (2012). Duloxetine-associated tardive dyskinesia resolved with
fluvoxamine: a case report. J. Clin. Psychopharmacol. 32, 723-724.
is
Albayrak, Y. & Hashimoto, K. (2012). Beneficial effects of the sigma-1 agonist fluvoxamine
for tardive dyskinesia in patients with post-psychotic depressive disorder of
schizophrenia: report of 5 cases. Prim. Care Companion CNS 14, 12br0140.
Albayrak, Y. & Hashimoto, K. (2013). Beneficial effects of sigma-1 agonist fluvoxamine for
bl
tardive dyskinesia and tardive akathisia in patients with schizophrenia: report of three
cases. Psychiatry Investig. 10, 417-420.
Albayrak, Y., Uğurlu, G. K., Uğurlu, M. & Cayköylü, A. (2012). Beneficial effects of
Pu
fluvoxamine for chorea in a patient with Huntington's disease: a case report. Prim. Care
Companion CNS 14, 12l01369.
Alexander, J. & Nillsen, A. (2011). Venlafaxine-induced delirium. Aust. N. J. Psychiatry, 45,
606.
Armstrong, S. C. & Schweitzer, S. M. (1997). Delirium associated with paroxetine and
benztropine combination. Am. J. Psychiatry, 154, 581-582.
Baumeister, H., Hutter, N. & Bengel, J. (2011). Psychological and pharmacological
a
interventions for depression in patients with coronary artery disease. Cochrane Data.
Syst. Rev. 9, CD008012.
ov
Baune, B. T., Stuart, M., Gilmour, A., Wersching, H., heindel, W., Arolt, V. & Berger, K.
(2012). The relationship between subtypes of depression and cardiovascular disease: a
systematic review of biological models. Transl. Psychiatry, 2, e92.
Bhuiyan, M. S. & Fukunaga, K. (2011). Targeting sigma-1 receptor signaling by endogenous
ligands for cardioprotection. Expert Opin. Ther. Targets, 15, 145-155.
N
Bhuiyan, M. S., Tagashira, H. & Fukunaga, K. (2011). Sigma-1 receptor stimulation with
fluvoxamine activates Akt-eNOS signaling in the thoracic aorta of ovariectomized rats
with abdominal aortic banding. Eur. J. Pharmacol. 650, 621-628.
Bhuiyan, M. S., Tagashira, H. & Fukunaga, K. (2013). Crucial interactions between selective
serotonin uptake inhibitors and sigma-1 receptor in heart failure. J. Pharmacol. Sci. 121,
177-184.
Bhuiyan, M. S., Tagashira, H., Shioda, N. & Fukunaga, K. (2010). Targeting sigma-1
receptor with fluvoxamine ameliorates pressure-overload-induced hypertrophy and
c.
dysfunctions. Expert Opin. Ther. Targets, 14, 1009-1022.
Byerly, M. J., Christensen, R. C. & Evans, D. (1996). Delirium associated with a combination
of sertraline, haloperidol, and benztropine. Am. J. Psychiatry, 153, 965-966.
Cayköylü, A., Albayrak, Y., Uğurlu, G.K., Ekinci, O. (2011). Beneficial effects of
In
fluvoxamine for hemiballism in a patient with depressive disorder: a case report. Acta.
Neurol. Belg. 111, 62-65.
Charney, D. S. & Manji, H. K. (2004). Life stress, genes, and depression: multiple pathways
lead to increased risk and new opportunities for intervention. Sci. STKE. 2004, re5.
rs
Cohen, R. M., Weingartner, H., Smallberg, S. A., Pickar, D. & Murphy, D. L. (1982). Effort
and cognition in depression. Arch. Gen. Psychiatry, 39, 593-597.
Collier, T. L., Waterhouse, R. N. & Kassiou, M. (2007). Imaging sigma receptors:
applications in drug development. Curr. Pharm. Des. 13, 51-72.
he
de Koning, M. B., Bloemen, O. J. N., van Amelsvoort, T. A. M., Becker, H. E., Nieman, D.
H., van der Gaag, M. & Linszen, D. H. (2009). Early intervention in patients at ultra high
risk of psychosis: benefits and risks. Acta. Psychiatr. Scand. 119, 426-442.
Debonnel, G. & de Montigny, C. (1996). Modulation of NMDA and dopaminergic
neurotransmissions by sigma ligands: possible implications for the treatment of
is
psychiatric disorders. Life Sci., 58, 721-734.
Delić, M. & Pregelj, P. (2013). Delirium during i. v. citalopram treatment: a case report.
Pharmacopsychiatry, 46, 37-38.
bl
Ela, C., Barg, J., Vogel, Z., Hasin, Y. & Eilam, Y. (1994). Sigma receptor ligands modulate
contractility, Ca2+ influx and beating rate in cultured cardiac myocytes. J. Pharmacol.
Exp. Ther. 269, 1300-1309.
Fishback, J. A., Robson, M. J., Xu, Y. T. & Matsumoto, R. R. (2010). Sigma receptors:
Pu
potential targets for a new class of antidepressant drug. Pharmacol. Ther. 127, 271-282.
Fong, T. G., Tulebaev, S. R. & Inouye, S. K. (2009). Delirium in elderly adults: diagnosis,
prevention and treatment. Nat. Rev. Neurol. 5, 210-220.
Furuse, T. & Hashimoto, K. (2009). Fluvoxamine monotherapy for psychotic depression: the
potential role of sigma-1 receptors. Ann. Gen. Psychiatry, 8, 26.
Furuse, T. & Hashimoto, K. (2010a). Sigma-1 receptor agonist fluvoxamine for delirium in
patients with Alzheimer's disease. Ann. Gen. Psychiatry, 9, 6.
a
Furuse, T. & Hashimoto, K. (2010b). Sigma-1 receptor agonist fluvoxamine for delirium in
intensive care units: report of five cases. Ann. Gen. Psychiatry, 9, 18.
ov
Furuse, T. & Hashimoto, K. (2010c). Sigma-1 receptor agonist fluvoxamine for postoperative
delirium in older adults: report of three cases. Ann. Gen. Psychiatry, 9, 28.
Furuse, T. & Hashimoto, K. (2010d). Fluvoxamine for aripiprazole-associated akathisia in
patients with schizophrenia: a potential role of sigma-1 receptors. Ann. Gen. Psychiatry,
9, 11
N
Furuse, T. & Hashimoto, K. (2010e). Fluvoxamine for blonanserin-associated akathisia in

patients with schizophrenia: report of five cases. Ann. Gen. Psychiatry, 9, 17.
Fusar-Poli, P., Deste, G., Smieskova, R., Barlati, S., Yung, A. R., Howes, O., Stieglitz, R. D.,
Vita, A., McGuire, P. & Borgwardt, S. (2012). Cognitive functioning in prodromal
psychosis: a meta-analysis. Arch. Gen. Psychiatry, 69, 562-571.
Gatti, F., Bellini, L., Gasperini, M., Perez, J., Zanardi, R. & Smeraldi, E. (1996).
Fluvoxamine alone in the treatment of delusional depression. Am. J. Psychiatry, 153,
c.
414-416.
Girard, T. D., Pandharipande, P. P. & Ely, E. W. (2008). Delirium in the intensive care unit.
Crit. Care, 12(Suppl 3), S3.
In
Goodnick, P. J. & Goldstein, B. J. (1998). Selective serotonin reuptake inhibitors in affective
disorders-I: basic pharmacology. J. Psychopharmacol. 12(3 Suppl B), S5-S20.
Guitart, X., Codony, X. & Monroy, X. (2004). Sigma receptors: biology and therapeutic
potential. Psychopharmacology, 174, 301-319.
Gunther, M. L., Morandi, A. & Ely, E. W. (2008). Pathophysiology of delirium in the
rs
intensive care unit. Crit. Care Clin. 24, 45-65.
Hamoda, H. & Osser, D. N. (2008). The psychopharmacology algorithm project at the
Harvard South Shore Program: an update on psychotic depression. Harv. Rev. Psychiatry,
16, 235-247.
he
Hashimoto, K. (2009a). Sigma-1 receptors and selective serotonin reuptake inhibitors: clinical
implications of their relationship. Cent. Nerv. Syst. Agents Med. Chem. 9, 197-204.
Hashimoto, K. (2009b). Emerging role of glutamate in the pathophysiology of major
depressive disorder. Brain Res. Rev. 61, 105-123.
Hashimoto, K. (2009c). Can the sigma-1 receptor agonist fluvoxamine prevent
is
schizophrenia? CNS Drug Targets, 8, 470-474.
Hashimoto, K. (2013). Sigma-1 receptor chaperone and brain-derived neurotrophic factor:
emerging links between cardiovascular disease and depression. Prog. Neurobiol. 100, 15-
bl
29.
Hashimoto, K., Fujita, Y. & Iyo, M. (2007). Phencyclidine-induced cognitive deficits in mice
are improved by subsequent subchronic administration of fluvoxamine: role of sigma-1
receptors. Neuropsychopharmacology, 32, 514-521.
Pu
Hashimoto, K. & Furuse, T. (2012). Sigma-1 receptor agonist fluvoxamine for delirium in
older adults. Int. J. Geriatr. Psychiatry, 27, 981-983.
Hashimoto, K. & Ishiwata, K. (2006). Sigma receptor ligands: possible application as
therapeutic drugs and as radiopharmaceuticals. Curr. Pharm. Des. 12, 3857-3876.
Hayashi, T. & Su, T. P. (2004). Sigma-1 receptor ligands: potential in the treatment of
neuropsychiatric disorders. CNS Drugs, 18, 269-284.
Hayashi, T. & Su, T. P. (2005). Understanding the role of sigma-1 receptors in psychotic
a
depression. Psychiatric Times, 22, 54-63.

Hayashi, T. & Su, T. P. (2007). Sigma-1 receptor chaperones Cell, 131, 596-610.
ov
Hayashi, T., Tsai, S. Y., Mori, T., Fujimoto, M. & Su, T. P. (2011). Targeting ligand Expert
Opin. Ther. Targets, 15, 557-577.
Hindmarch, I. & Hashimoto, K. (2010). Cognition and depression: the effects of fluvoxamine,
a sigma-1 receptor agonist, reconsidered. Hum. Psychopharmacol. 25, 193-200.
Howe, C. & Ravasia, S. (2003). Venlafaxine-induced delirium. Can. J. Psychiatry, 48, 129.
N
Ishiguro, H., Ohtsuki, T., Toru, M., Itokawa, M., Aoki, J., Shibuya, H., Kurumaji, A., Okubo,
Y., Iwawaki, A., Ota, K., Shimizu, H., Hamaguchi, H. & Arinami, T. (2001). Association
between polymorphisms in the type 1 sigma receptor gene and schizophrenia. J.

Psychiatry Neurosci. 26, 203-220.
Ishikawa, M. & Hashimoto, K. (2010). The role of sigma-1 receptors in the pathophysiology
of neuropsychiatric diseases. J. Receptor Ligand Channel Res. 3, 25-36.
Ishikawa, M., Ishiwata, K., Ishii, K., Kimura, Y., Sakata, M., Naganawa, M., Oda, K.,
c.
Miyatake, R., Fujisaki, M., Shimizu, E., Shirayama, Y., Iyo, M. & Hashimoto, K. (2007).
High occupancy of sigma-1 receptors Biol. Psychiatry, 62, 878-883.
Ishima, T., Fujita, Y. & Hashimoto, K. (2014). Interaction of new antidepressants Eur. J.
In
Pharmacol. 727, 167-173.
Ishima, T., Fujita, Y., Kohno, M., Kunitachi, S., Horio, M., Takatsu, Y., Minase, T.,
Tanibuchi, Y., Hagiwara, H., Iyo, M. & Hashimoto, K. (2009). Improvement of
phencyclidine-induced cognitive deficits in mice by subsequent subchronic
administration of fluvoxamine, but not sertraline. Open. Clin. Chem. J. 2, 7-11.
rs
Itoh, K., Hirooka, Y., Matsukawa, R., Nakano, M. & Sunagawa, K. (2012). Decreased brain
sigma-1 receptor contributes to the relationship between heart failure and depression.
Cardiovas. Res. 93, 33-40.
Iyo, M., Shirayama, Y., Watanabe, H., Fujisaki, M., Miyatake, R., Fukami, G., Shiina, A.,
he
Nakazato, M., Shiraishi, T., Ookami, T. & Hashimoto, K. (2008). Fluvoxamine as a
sigma-1 receptor agonist improved cognitive impairments in a patient with schizophrenia.
Prog. Neuropsychopharmacol. Biol. Psychiatry, 32, 1072-1073.
Kane, J. H. (2001). Extrapyramidal side effects are unacceptable. Eur.
Neuropsychopharmacol. 11(Suppl 4), S397-S403.
is
Kane, J. M., Barnes, T. R., Correll, C. U., Sachs, G., Buckley, P., Eudicone, J., McQuade, R.,
Van Tran, Q., Pikalov, A. & Assunção-Talbott, S. (2010). Evaluation of akathisia in
patients with schizophrenia, schizoaffective disorder, or bipolar I disorder: a post hoc
bl
analysis of pooled data from short- and long-term aripiprazole trials. J.
Psychopharmacol. 24, 1019-1029.
Kane, J. M., Fleischhacker, W. W., Hansen, L., Perlis, R., Pikalov, A. 3rd & Assunção-
Talbott, S. (2009). Akathisia: an updated review focusing on second-generation
Pu
antipsychotics. J. Clin. Psychiatry, 70, 627-643.

Kessler, R. C., Berglund, P., Demler, O., Jin, R., Koretz, D., Merikangas, K. R., Rush, A. J.,
Walters, E. E. & Wang, P. S. (2003). The epidemiology of major depressive disorder:
results from the National Comorbidity Survey Replication (NCS-R). JAMA, 289, 3095-
3105.
Kessler, R. C., Berglund, P., Demler, O., Jin, R., Merikangas, K. R. & Walters, E. E. (2005).
Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National
a
Comorbidity Survey Replication. Arch. Gen. Psychiatry, 62, 593-602.

Kishimoto, A., Todani, A., Miura, J., Kitagaki, T. & Hashimoto, K. (2010). The opposite
ov
effects of fluvoxamine and sertraline in the treatment of psychotic major depression: a

case report. Ann. Gen. Psychiatry, 9, 23.
Kumar, R. & Sachdev, P. S. (2009). Akathisia and second-generation antipsychotic Akathisia
and second-generation antipsychotic drugs Curr. Opin. Psychiatry, 22, 293-299.
Liu, C. C. & Demjaha, A. (2013). Antipsychotic interventions in prodromal psychosis: safety
N
issues. CNS Drugs, 27, 197-205.

Maldonado, J. R. (2008). Delirium in the acute care setting: characteristics, diagnosis and
treatment. Crit. Care Clin. 24, 657-722.
Mandelli, L., Serretti, A., Colombo, C., Florita, M., Santoro, A., Rossini, D., Zanardi, R. &
Smeraldi, E. (2006). Improvement of cognitive functioning in mood disorder patients
with depressive symptomatic recovery during treatment: an exploratory analysis.
Psychiatry Clin. Neurosci. 60, 598-604.
Marrazzo, A., Caraci, F., Salinaro, E. T., Su, T. P., Copani, A. & Ronsisvalle, G. (2005).
c.
Neuroprotective effects of sigma-1 receptor agonists against β-amyloid-induced toxicity.
Neuroreport, 16, 1223-1226.
Martin, W., Eades, C. E., Thompson, J. A. & Huppler, R. E. (1976). The effects of morphine-
In
and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal
dog. J. Pharmacol. Exp. Ther.197, 517-532.
Maurice, T. & Su, T. P. (2009). The pharmacology of sigma-1 receptors. Pharmacol. Ther.
124, 195-206.
Mohr, P. & Volavka, J. (2002). Ladislav Haskovec and akathisia: 100th anniversary. Br. J.
rs
Murray, C. J. L. & Lopez, A. D. (Eds.). (1996). The global burden of disease: A
comprehensive assessment of mortality and disability from diseases, injuries, and risk
factors in 1990 and projected to 2020. Cambridge, MA: Harvard University Press.
he
Narita, N., Hashimoto, K., Tomitaka, S. & Minabe, Y. (1996). Interactions of selective
serotonin reuptake inhibitor Eur. J. Pharmacol. 307, 117-119.
Nemeroff, C. B. & Goldschmidt-Clermont, P. J. (2012). Heartache and heartbreak – the link
between depression and cardiovascular disease. Nat. Rev. Cardiol. 9, 526-539.
Nemeroff, C. B. & Owens, M. J. (2004). Pharmacologic differences among the SSRIs: focus
is
on monoamine transporters and the HPA axis. CNS Spectr. 9(6 Suppl 4), 23-31.
Nicholson, A., Kuper, H. & Hemingway, H. (2006). Depression as an aetiologic and
prognostic factor in coronary heart disease: a meta-analysis of 6,362 events among 146,
bl
538 participants in 54 observational studies. Eur. Heart J. 27, 2763-2774.
Nishimura, T., Ishima, T., Iyo, M. & Hashimoto, K. (2008). Potentiation of nerve growth
factor-induced neurite outgrowth by fluvoxamine: role of sigma-1 receptors, IP3 receptors
and cellular signaling pathways. PLoS ONE, 3, e2558.
Pu
Niitsu, T., Fujisaki, M., Shiina, A., Yoshida, T., Hasegawa, T., Kanahara, N., Hashimoto, T.,
Shiraishi, T., Fukami, G., Nakazato, M., Shirayama, Y., Hashimoto, K. & Iyo, M.
(2012a). A randomized, double-blind, placebo-controlled trial of fluvoxamine in patients
with schizophrenia: a preliminary study. J. Clin Psychopharmacol. 32, 593-601.
Niitsu, T., Iyo, M. & Hashimoto, K. (2012b). Sigma-1 receptor agonists as therapeutic drugs
for cognitive impairment in neuropsychiatric diseases. Curr. Pharm. Des. 18, 875-883.
Niitsu, T., Shirayama, Y., Fujisaki, M., Hashimoto, K. & Iyo, M. (2010). Fluvoxamine
a
improved negative symptoms and cognitive impairments in a patient with schizophrenia.

Prog. Neuropsychopharmacol. Biol. Psychiatry, 34, 1345-1346.
ov
Novakova, M., Ela, C., Barg, J., Vogel, Z., Hasin, Y. & Eilam, Y. (1995). Inotropic action of
sigma receptor ligands in isolated cardiac myocytes from adult rats. Eur. J. Pharmacol.
286, 19-30.
Owens, D. G. C. (1999). Akathisia. In: A Guide to the Extrapyramidal Side- Effects of
Antipsychotic Drugs. New York, N Y: Cambridge University Press. 130-162.
N
Perez, A. & Ashford, J. J. (1990). A double-blind, randomized comparison of fluvoxamine

with mianserin in depressive illness. Curr. Med. Res. Opin. 12, 234-241.
Porter, R. J., Gallagher, P., Thompson, J. M. & Young, A. H. (2003). Neurocognitive

impairment in drug-free patients with major depressive disorder. Br. J. Psychiatry, 182,
214-220.
Sachdev, P. S. (2005). Neuroleptic-induced movement disorders: an overview. Psychiatr.
Clin. North. Am. 28, 255-274.
c.
Solai, L. K. K. (2009). Delirium. Sadock, B.J., Kaplan, H.I., Sadock, V.A., Kaplan &
Sadock's Synopsis of Psychiatry 9th edition. Philadelphia: Wolter Kluwer/Lippincott
Williams & Wilkins.
In
Stahl, S. M. (2005). Antidepressant treatment of psychotic major depression: potential role of
the sigma receptor. CNS Spectr. 10, 319-323.
Stahl, S. M. (1998). Using secondary binding properties to select a not so selective serotonin
selective reuptake inhibitor. J. Clin. Psychiatry, 59, 642-643.
Su, T. P., London, E. D. & Jaffe, J. H. (1988a). Steroid binding at sigma receptors Science,
rs
240, 21-221.
Su, T. P., Schell, S. E., Ford. & London, E. D. (1988b). Correlation of inhibitory potencies of
putative antagonists for sigma receptors in brain and spleen. Eur. J. Pharmacol. 148, 467-
470.
he
Suzuki, Y., Saito, M. & Someya T. (2012). Delirium associated with duloxetine in a
depressed patient with Alzheimer's dementia. Psychiatry Clin. Neurosci. 66, 166.
Tadokoro, S., Kanahara, N., Kikuchi, S., Hashimoto, K. & Iyo, M. (2011). Fluvoxamine may
prevent onset of psychosis: a case report of a patient at ultra-high risk of psychotic
disorder. Ann. Gen. Psychiatry, 10, 26.
is
Tagashira, H., Bhuiyan, S., Shioda, N., Hasegawa, H., Kanai, H. & Fukunaga, K. (2010).
Sigma-1 receptor stimulation with fluvoxamine ameliorates transverse aortic constriction-
induced myocardial hypertrophy and dysfunction in mice. Am. J. Physiol. Heart Circ.
bl
Physiol. 299, H1535-H1545.
Takebayashi, M., Hayashi, T. & Su, T. P. (2002). Nerve growth factorinduced neurite
sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants. J.
Pharmacol. Exp. Ther. 303, 1227-1237.
Pu
Tandon, R., Nasrallah, H. A. & Keshavan, M. S. (2009). Schizophrenia, "just the facts" 4.
Clinical features and conceptualization. Schizophr. Res. 110, 1-23.
Taylor, C. B., Youngblood, M. E., Catellier, D., Veith, R. C., Carney, R. M., Burg, M. M.,
Kaufmann, P. G., Shuster, J., Mellman, T., Blumenthal, J. A., Krishnan, R., Jaffe, A. S. &
ENRICHD Investigators, (2005). Effects of antidepressant medication on morbidity and
mortality in depressed patients after myocardial infarction. Arch. Gen. Psychiatry, 62,
792-798.
a
Volz, H. P. & Stoll, K. D. (2004). Clinical trials with sigma ligands. Pharmacopsychiatry, 37
(Suppl 3), S214-S220.
ov
Walker, J. M., Martin, W. J., Hohmann, A. G., Hemstreet, M. K., Roth, J. S., Leitner, M. L.,
Weiser, S. D., Patrick, S. L., Patrick, R. L. & Matsumoto, R. R. (1994). Role of sigma
receptors in brain mechansism of movement. In: ―Sigma Receptors‖, edited by Itzhak, Y.,
Academic Press, New York, 205-224.
Wulsin, L. R., Evans, J. C., Vasan, R. S., Murabito, J. M., Kelly-Hayes, M. & Benjamin, E. J.
N
(2005). Depressive symptoms, coronary heart disease, and overall mortality in the
Framingham Heart Study. Psychosomatic Med. 67, 697-702.
Yao, H., Yang, Y., Kim, K. J., Bethel-Brown, C., Gong, N., Funa, K., Gendelman, H. E., Su,
T. P., Wang, J. Q. & Buch, S. (2010). Molecular mechanisms involving sigma receptor-
mediated induction of MCP-1: implication for increased monocyte transmigration. Blood,
115, 4951-4962.
Yoshida, T., Ishikawa, M., Niitsu, T., Nakazato, M., Watanabe, H., Shiraishi, T., Shiina, A.,
c.
Hashimoto, T., Kanahara, N., Hasegawa, T., Enohara, M., Kimura, A., Iyo, M. &
Hashimoto, K. (2012). Decreased serum levels of mature brain-derived neurotrophic
factor (BDNF), but not its precursor proBDNF, in patients with major depressive
In
disorder. PLoS One, 7, e42676.
Zanardi, R., Franchini, L., Serretti, A., Perez, J. & Smeraldi, E. (2000). Venlafaxine versus
fluvoxamine in the treatment of delusional depression: a pilot double-blind controlled
study. J. Clin. Psychiatry, 61, 26-29.
Zanardi, R., Franchini, L., Gasperini, M., Smeraldi, E. & Perez, J. (1997). Long-term
rs
treatment of psychotic (delusional) depression with fluvoxamine: an open pilot study. Int.
Clin. Psychopharmacol. 12, 195-197.
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Chapter 14
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Fluoxetine: Pharmacological Analysis
of Depression-Like Responses
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in Zebrafish
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Julian Pittman*, Roaa Hadi and Katie Ichikawa
Department of Biological & Environmental Sciences, Troy University, Troy, AL, US
Abstract
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Due to their high throughput value, genetic tractability, physiological homology to
humans, low cost, and quick reproductive cycle, zebrafish (Danio rerio) have emerged as
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a promising new model species for studying various central nervous system disorders. In
this chapter, we discuss the current state of utilizing zebrafish to study depression, and
outline further directions of investigation, using this model organism, to understand the
pathogenesis and develop innovative therapeutic approaches for depression.
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Keywords: fluoxetine; anxiety/depression; model development; behavioral tests
Introduction
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Nearly one half-century ago several classes of medications, discovered by serendipity,

were introduced for the treatment of depression. These medications revolutionized our
approach to mood disorders and helped launch the modern era of psychiatry. However, our
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progress since those serendipitous discoveries has been rather disappointing. We still do not
understand with absolute certainty how those medications produce their desired clinical
effects. We have not introduced newer medications with fundamentally different
mechanisms-of-action. We have not identified the genetic and neurobiological mechanisms
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underlying depression, nor do we understand the mechanisms by which nongenetic factors
*
Corresponding author: Department of Biological & Environmental Sciences, Troy University, McCall Hall
Troy, Al 36083, (334) 808-6478, jtpittman@troy.edu.
296 Julian Pittman, Roaa Hadi and Katie Ichikawa
influence these disorders. We have only a rudimentary understanding of the circuits in the
brain responsible for the normal regulation of mood and affect and of those circuits that
function abnormally in mood disorders. In approaching these gaps in our knowledge, there
are three primary areas that need to be addressed: development of animal models,
identification of genetic determinants, and discovery of novel targets/biomarkers of
c.
depression. This chapter discusses how zebrafish (Danio rerio) may be utilized in the
modeling and analysis of the mechanisms of depression. Furthermore, this chapter argues that
the foundation of research into the mechanisms of depression is that behavioral paradigms, as
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they allow the quantification of functional changes in the brain induced by mutations or drugs
(fluoxetine), will facilitate the discovery of underlying mechanisms and drug targets. The
chapter also provides a detailed description of the behavioral responses and makes
recommendations for further development of these methods.
Most of the antidepressants available today inhibit the reuptake or breakdown of
rs
serotonin, noradrenaline, or both in the brain. However, elevation of the extracellular
concentration of these monoamines, which occurs rapidly, does not explain the time lag of
several weeks to months that is required before a therapeutic response is achieved (Berton and
Nestler 2006; Wong and Licinio 2004). Selective serotonin reuptake inhibitors (SSRIs) are
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the most commonly prescribed drugs for the treatment of depression. Among SSRIs,
fluoxetine is widely used to treat depression, and its biochemical and pharmacological
properties have been studied extensively in animals and humans (Wong et al. 2005).
Zebrafish are rapidly becoming a promising model organism for experimental studies of
affective disorders (Best 2008, Flint 2008, Kyzar 2012). Nevertheless, while zebrafish are
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becoming a prevalent model in the field of biological psychiatry, their application as a robust
translational model is still very much in its infancy. This species demonstrates the potential to
be an exceptional animal for investigating experimental, genetic, and pharmacological models
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of neurobehavioral disorders, such as anxiety/depression (Dooley K 2000, Sprague J 2001,
Zon L 2005, Blaser R 2006, Alsop D 2008, Flint 2008, Alsop D 2008, Mathur P 2010, Norton
W 2010). As a result of the past three decades of intensive investigation with zebrafish, this
species has become geneticists‘ favorite model organisms (Dooley K 2000). The accumulated
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genetic knowledge about, and the genetic methods specifically developed for the zebrafish
now make this species particularly attractive for several research fields. One of these fields is
behavioral neuroscience. Zebrafish models strike an optimal balance between system
complexity and practical simplicity, possessing brain anatomy, physiology, and genome very
similar to those of other vertebrates including mammals (Lele Z 1996, Dooley K 2000,
Wullimann M 2000, Moorman 2001, Shin J 2002, Ward A 2002, McGrath P 2008).
Furthermore, they are small, easy and cheap to maintain in the laboratory, and are highly
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amenable to high-throughput screening (e.g., forward genetic or drug screens). The latter is
particularly noteworthy for the purposes of unraveling the genetic, and in general the
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biological, mechanisms of complex brain functions and the disorders of these functions.
High-throughput screens may have the ability to identify a significant proportion of the
potentially large number of molecular players involved in these functions (Norton W 2010,
Blackburn J 2011). Anxiety/depression are common, serious brain disorders (Kessler R
2005). Numerous studies have examined the biological mechanisms of anxiety/depression,
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and a considerable amount of effort has been invested in the development of pharmacological
treatments (P. 1984, Willner 1990, C. 1998, Leonard 1998, Geyer M 2002, Kato T 2007, Flint
2008, Egan R 2009). For preclinical research, most of these studies have used rodents. Since a
Fluoxetine 297
large amount of data has been accumulated on rodent species, it may seem logical to think
that building upon this well-laid foundation is the only way to proceed. The abandonment of
rodent research is certainly not likely or recommended; however, utilization of another
vertebrate, zebrafish, appears to be a fruitful direction to pursue namely because they are
robust, small, reproduce quickly, possess evolutionarily conserved traits.
c.
Development of Models
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Notwithstanding our increased understanding of both the pathophysiology and treatment
of anxiety/depression, it remains highly prevalent; affecting 6.7 percent of adult Americans in
a given year (Kessler R 2005). Moreover, there are no validated, diagnostically useful
biological tests for depression that reliably predict a response to well-established and effective
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treatments for depression. Similarly, there are no biomarkers, such as expression of a gene,
that reliably change as a function of treatment response to antidepressants or psychotherapy.
While there is little doubt that numerous neurotransmitter systems are pathologically
involved in the etiology of anxiety/depression, no single neurotransmitter systems appear to
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be solely responsible. This is not surprising given the panoply of symptoms that comprise the
depressive syndrome: depressed mood, loss of interest in usual activities, inability to
experience pleasure, impaired concentration, disturbed sleep, decreased appetite, and
suicidality. A more recent conceptual approach to the biology of anxiety/depression is to
consider it a systems-level ‗spectrum‘ disorder involving several critical brain regions and
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pathways involving these regions. A comprehensive understanding of the genetic and
environmental contributions to anxiety/depression and its associated neurobiology is required
before scientifically-based rational new treatment strategies are likely to be developed.
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The current view of the etiology of anxiety/depression is based on gene-environment
interactions, with a focus on the three major monoamine systems—serotonin (5-
hydroxytryptamine, 5HT), norepinephrine (NE), and dopamine (DA). The emerging new
tools of molecular neurobiology and functional brain imaging have provided additional
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support for the involvement of these three systems. In contrast with previous views (C. 1998),
considerable evidence now supports a preeminent role for central nervous system DA circuits
(Dunlop B 2007), with many investigators suggesting that the now well-documented
suboptimal therapeutic responses to SSRIs and selective serotonin-norepinephrine reuptake
inhibitors (SNRIs) may be due, in part, to their relative lack of effect on brain DA circuits.
It is exceedingly difficult to develop an animal model that perfectly reproduces the
symptoms of anxiety/depression (Willner 1990, Weiss 1995, Leonard 1998). Animals lack
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self-consciousness, self-reflection, and consideration; moreover, hallmarks of the disorder

such as depressed mood, low self-esteem, or suicidality are hardly accessible in non-humans.
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However, anxiety/depression, as with other mental disorders, consists of endophenotypes

(Hasler 2004) that can be reproduced independently and evaluated in animals. An ideal
animal model offers an opportunity to understand molecular, genetic, and epigenetic factors
that may lead to depression. By using animal models, the underlying molecular alterations
and the causal relationship between genetic or environmental alterations and depression can
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be examined, which affords insight into the pathology of anxiety/depression. In addition,

animal models of anxiety/depression are indispensable for identifying novel therapies for
depression. For medical disorders of unclear pathophysiology or genetic etiology, such as
depression, the emerging approach to developing an animal model is to model a single

symptom or endophenotype of the disorder, rather than attempting to create a full phenotypic
recreation of anxiety/depression. For example, anxiety/depression symptomology is often
characterized by lack of activity or motivation to be active. In modeling behaviors, in general,
the behavioral measures should satisfy the criteria of being robust and reliable, having strong
c.
predictive validity to be initially used as a ―new model‖ (Geyer M 1995, Geyer M 2002).
While several groups have actively modeled anxiety in zebrafish (and many of those tests
may offer clues into the mechanisms of depression), very few studies have attempted to
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model depression per se in zebrafish. The following endophenotypes have been described in
rodents; some of the below tests can be adapted, or serve as inspiration for new tests to be
developed, using zebrafish. Anhedonia: The loss of interest is a core symptom of depression.
Anhedonia in rodents can be assessed by sucrose preference or by intracranial self-
stimulation. Behavioral despair: Behavioral despair may be assessed with tests such as the
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forced-swimming test or the tail suspension test. Changes in appetite or weight gain:
Depression is often associated with changes in appetite or weight gain, which is easily
measured in rodents. Neuroanatomy: Depressed subjects display decreased hippocampal
volume and rodents exposed to chronic stress or excess glucocorticoids exhibit similar signs
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of hippocampal loss of neurons and dendritic atrophy. Neuroendocrine disturbances:
Disturbances of the hypothalamic–pituitary–adrenal axis (HPA) are one of the most
consistent symptoms in major depression. The functionality of the HPA can be assessed by
dexamethasone suppression test. Alterations in sleep architecture: Disturbances in the
circadian rhythm and especially in the sleep architecture are often observed in depression. In
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rodents, it is accessible via electroencephalography (EEG). Anxiety-related behavior: Anxiety
is a symptom with high prevalence in depression. Therefore, animal models of depression
often display altered anxiety-related behavior.
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a
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Figure 1. General approach to model development relevant to zebrafish anxiety/depression-like

behavior.
Fluoxetine 299
Classic Behavioral Tests
There are several ways one can induce and study the effects of depression-related
behaviors. Novelty has long been known to induce anxiety responses in a variety of species
including humans. For example, the ―open field task‖ has been extensively used with rodents
c.
(Crusio E 1986, Prut L 2003) and other animals including fish (Csányi V 1988, Egan R
2009). In this task, the subject is exposed to an unfamiliar environment. The response to this
novel environment is believed to arise as a result of a compromise between opposing forces
or tendencies; exploration, which is believed to be associated with active responses, and
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anxiety, which is often associated with passive responses. Exploratory activity is considered
adaptive as it may lead to finding food, mates and escape routes, while passive anxiety-
induced responses (immobility/freezing) are argued to reduce predation risk (Crusio E 1986).
The adaptive aspect of these responses may seem speculative, but quantitative genetic
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analyses have confirmed ambidirectional selection forces underlying open field behavior.
That is, in the evolutionary past of mice/rats, individuals that performed at intermediate levels
(not too active but not too passive either) had been favored (Crusio E 1986), a finding that
extends to other vertebrates including fish (Gerlai R 1990). This could prove as an extremely
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valuable metric for depressive behavior in zebrafish and offer possible utility in identifying
new genetic lines.
It is likely that the evolutionary past of zebrafish is similar to mice/rats in that this species
too has been under ambidirectional selection with regard to novelty induced behavioral
responses. Therefore, exposing zebrafish to a novel environment is expected to induce
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moderate levels of anxiety. Importantly, behavioral experimentation almost always includes
at least some level of handling of animals by humans, which is also expected to induce
anxiety. Novelty induced anxiety responses have been analyzed in zebrafish by (Levin E
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2007), who demonstrated an initially low level of exploratory activity of zebrafish that
gradually increased with time. Levin also described a ―diving‖ response, i.e., increased
amount of time spent on the bottom of the test tank, a response that slowly habituated as the
fish adapted to their novel environment. (Egan R 2009) also reported similar findings.
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Furthermore, (Levin E 2007) showed nicotine had anxiolytic properties as this drug reduced
novelty induced fear responses.
Importantly, a decrease in serotonergic activity is associated with depression. In
experimental studies, reduced brain serotonergic activity due to social isolation has been
known for decades (Garattini S 1967). Specifically, rodents show hyperactive and aggressive
behavior during long-term social isolation, which can be blocked with anti-depressant
treatment (Garzon J 1981). These social isolation paradigms based on serotonin deficiency
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are used as experimental depression models in rodents (Leonard 1998). Similar tests may be
useful experimentally for zebrafish. However, with all behavioral tests and endpoints, one
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must exercise extreme care and ensure there is some ability to provide a dissection between
anxiety and depression (Table 1).
Pharmacological Models of Anxiety/Depression-like Responses

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Some approaches that can be employed to model depression, are currently being used in
our lab (Pittman J. 2012). One such model involves the administration of one drug followed
by the administration of another that will elicit conflicting effects. For example,
administration of psychostimulants, such as amphetamine, leading to hyperactivity, that can
then be used to test the efficacy of anti-manic treatments, such as Valproate. Furthermore,
behavioral sensitization by repeated administration of psychostimulants can also be used as a
model of bipolar disorder (Kato T 2007). Since repeated exposure to cocaine can induce a
c.
―cycling‖ in a variety of neurochemical and physiological systems (Antelman S 1998), it may
be possible to evoke bipolar-like behavior in zebrafish, for example, by using a combination
of cocaine and anti-psychotic agents.
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Another approach that can be employed to induce anxiety/depressive-like behavior is
withdrawal from an anxiolytic agent, such as ethanol; this methodology involves chronic
administration (3+weeks) of high doses (1-3%) of ethanol, and at least 7 days post-
withdrawal before behavioral symptoms are readily evident. Additionally, these depressive-
like behaviors can be reversed with fluoxetine. We have had particular success with this
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approach in our lab using some of the behavioral tests (Figure 2). In addition, quantitative
changes in immunoreactive neurons are also quite evident following this course of treatment
with ethanol, and mirrors many of the neurochemical hallmarks of clinical depression (Figure
3)(Pittman J. 2012).
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Table 1. Tests that can be used to study animal depression-related behavior
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a
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. B. C.
Figure 2. (A) Locomotion trajectories during the Novel Tank Diving Test of fish exposed to fluoxetine
(100 μg/L). Swimming path tracings generated by iPhone App (SwingReader Golf Lite™). (B) Image
frames where the corresponding images of 2 zebrafish were processed in overlay. (C) Image frames
where the corresponding images of 2 zebrafish were identified, tagged, and processed in overlay from
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(B).
Fluoxetine 301
c.
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Figure 3. Representative images of coronal sections through the telencephalon (hippocampus) showing
an example of immunopositive staining of serotonin terminals. (A) control, (B) ethanol withdrawal, (C)
ethanol, (D) fluoxetine. Images (100x).
The motivation for the continued search for improved drugs to treat depression is not
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only to improve the quality of life of those suffering from it, but also to aid in our
understanding of how depression develops, and what biological mechanisms may underlie
this disorder cluster. Another reason is that the currently available, however numerous, drugs
are often not efficacious or do not work for all patients. One way zebrafish may be beneficial
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for such research is by speeding up the discovery of the biological mechanisms responsible
for the symptoms of depression. This may be achieved using, for example, forward genetic
screens that identify mutations leading to the isolation of underlying genes. Another
completely different approach has been to search for compounds, or ―small molecules‖,
which may alter expression–like symptoms. It is thus important to consider what is known
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about the psychopharmacological properties of zebrafish in the context of depression. For
example, can one consistently detect the efficacy of ―gold standard‖ drugs for depression
using zebrafish? That is to say, does the zebrafish model have predictive validity? Predictive
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validity is an important question for the use of novel model organisms. The principal theme
with regard to the translational relevance of laboratory model organisms concerns the notion
―evolutionary homology‖, i.e., conservation of biological function across previously utilized
species (e.g., rodents), the novel laboratory species (e.g., zebrafish), and humans.
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Zebrafish have been used very infrequently in psychopharmacological analyses,

nevertheless, the few studies that have been completed suggest a possibly bright future for
drug development with the use of zebrafish. Alcohol (ethanol) is one of the best studied drugs
in zebrafish research. For example, the effect of developmental alcohol exposure has been
shown to be strain dependent (Loucks E 2004), early embryonic alcohol exposure has been
found to exert significant behavioral effects in the adult (Fernandes Y 2009), adaptation
(tolerance) after chronic alcohol exposure as well as alcohol withdrawal induced behavioral
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responses were all demonstrated (Gerlai R 1990, Gerlai R 2000, Gerlai R 2009), and
numerous changes induced by acute alcohol administration have also been revealed (Gerlai R
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2000). Importantly, alcohol has both anxiolytic (for the effects of lower doses of alcohol in
zebrafish see (Gerlai R 2000), also see (Egan R 2009) as well as anxiogenic properties (for
the effects of prolonged exposure to alcohol and during withdrawal in zebrafish see (Gerlai R
2009), also see (Egan R 2009) depending on concentration and mode or regime of its
administration. Other drugs of abuse have also been shown, to exert significant behavioral
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effects in zebrafish. For example, the rewarding properties of cocaine have been shown and
mutants with altered cocaine reinforced place preference have already been identified in
forward genetic screens (Darland T 2001). The reinforcing properties of drugs of abuse have
also been analyzed (Ninkovic J 2006). Drugs of abuse, similarly to alcohol, often have
anxiety/depression altering properties depending on concentration and dosing regimen
employed. For example, cocaine withdrawal induces anxiety and depressive responses in
zebrafish (Lopez-Patino M 2008).
Some classical anti-anxiety drugs have also been tested using zebrafish, e.g.,
c.
fluromethylhistidine exhibited an anxiolytic profile (Peitsaro N 2003), diazepam reversed
cocaine withdrawal induced anxiety, and the benzodiazepine inverse agonist FG-7142
induced anxiety in zebrafish (Lopez-Patino M 2008). Also, acute administration of caffeine,
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known to induce anxiety in humans (Childs E 2008) and rodents (El Yacoubi M 2000), also
led to increased anxiety responses; reduced frequency of visits to the upper water layer and
increased erratic movements in zebrafish (Egan R 2009).
Levels of stress hormones have also been analyzed in zebrafish (Alsop D 2008) and
numerous similarities between zebrafish and human stress responses have been revealed,
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which strengthen the translational relevance of zebrafish in depression research. For example,
the sight of a predator elevates cortisol levels in zebrafish (Barcellos G 2007) (also see Egan
et al. 2009, Cachat et al. 2010). It is important to note that cortisol, as in zebrafish, is also the
primary stress hormone of the hypothalamic-pituitary-adrenal (HPA) axis in human but not in
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rodents (using corticosterone instead). At the Society for Neuroscience meeting in San Diego
(2010), Dr. Herwig Baier (University of California, San Fransico) presented some interesting
findings related to the HPA axis (Baier 2010). Baier and his team found that disrupting the
stress response in zebrafish can generate behavioral phenotypes that resemble behaviors
characteristic of depression. His work suggests that depression could be linked to an
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individual‘s ability to cope with stress. The zebrafish displaying depression-like behaviors
carried a mutation in the glucocorticoid receptor gene, which is involved in stress
management. However, the depression-like behaviors were ameliorated when the fish were
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given fluoxetine (Prozac; a selective serotonin reuptake inhibitor (SSRI)). New therapies
might be able to play into the activity of the glucocorticoid receptor, and promoting its
activity instead of blocking it. Treatment with the widely prescribed antidepressant Prozac in
zebrafish has been shown to reduce anxiety responses, with more time spent in the top portion
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of a novel tank and also performing fewer erratic movements, also accompanied by reduced
whole-body cortisol levels (Egan R 2009), generally paralleling the responses seen in rodents
(Dulawa S 2004).
Kalueff‘s group has recently reported depression-like motor retardation in adult zebrafish
several days after an exposure to reserpine (Kyzar 2012) – a dopamine-depleting agent known
to evoke depression like responses in rodents and trigger clinical depression in humans.
However, with the use of all the above pharmacological treatments, one must exercise
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extreme care and ensure there is some ability to provide a dissection between anxiety and
depression endpoints, especially given a high degree of comorbidity of anxiety with
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depression clinically. How will this be accomplished? Through careful selection of both the
pharmacological agent used to induce depression-like symptoms, employing robust
behavioral tests (much development is needed in this area), and finally confirmation via
quantitative changes in neural circuitry involved in depression.
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Fluoxetine 303
Conclusion
A significant difficulty with using zebrafish is that the behavior of this species is not well
characterized. While there is an increasing number of behavioral studies published on
zebrafish, compared to classical laboratory study species such as the rat, mouse, or even the
c.
fruit fly, zebrafish behavioral research is very much still in its infancy (Weiss 1995). Without
robust behavioral tests, and without thorough understanding of the behavioral features of
zebrafish, it is not possible to utilize behavioral phenotyping of mutation or drug effects, and
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how these manipulations may influence brain function becomes exceedingly difficult to
investigate. Notably, the majority of zebrafish behavioral publications have appeared only
recently, demonstrating a clear upsurge of interest in this species, as behavioral
neuroscientists and behavioral geneticists have discovered novel ways to use zebrafish
models.
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It is challenging to predict how beneficial zebrafish may become in modeling and
analysis of the biological mechanisms of human depression. At this juncture, however, it
seems that the main components, necessary for such research to be successful in the future,
already exist. While only distantly related to humans, the zebrafish has already proven its
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translational relevance. However, perhaps the most important advantage of this species as a
laboratory tool may be best described with one word: numbers. Complex biological
phenomena are associated with large numbers of mechanisms. These may be discovered
using broad screens, genetic, or pharmacological tools. Zebrafish have been shown to be ideal
for large-scale screens due to several of its features, but principally to the fact that a large
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amount of these fish can be produced fast and can be maintained and now tested efficiently in
the laboratory.
Given the complexity of the mechanisms of depression, one may assume the need to
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identify a large number of molecular players, i.e., genes and their protein products and the
biochemical interactions between the proteins. It can be argued that this complexity may be
best tackled, at least initially, using large scale screens for mutations and drugs. These screens
are the key to the identification of potential targets and leads that may subsequently be
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followed up on by more targeted hypothesis driven analyses. It is important to note that I am

not advocating the screening approach as the only possible or only potentially fruitful one.
There are a large number of unknown mechanisms waiting to be discovered and their
discovery may be significantly facilitated by ―blind‖, i.e., unbiased, screening applications.
This is exactly where zebrafish have a major advantage over other, more traditional
laboratory organisms. It is imperative that additional novel behavioral endpoints and
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observational methodologies, such as automated video-tracking systems, strengthen the utility

of zebrafish for use as an animal model for depression research. Using biomolecular markers,
such as gene expression, immunohistochemistry etc., to parallel zebrafish physiology with
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behavioral data gives us another essential research direction. Lastly, expanding the area of
zebrafish research by including cross-domain modeling, for example drug withdrawal/
depression, as is currently done in my laboratory, may well lead to new translational models
using a combination of both larval and adult zebrafish.
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References
Alsop, D. V. M. (2008). "Development of the corticosteroid stress axis and receptor
expression in zebrafish. " Am J Physiol Regul Integr Comp Physiol, 294, 711-719.
Alsop, D. V. M. (2008). "The zebrafish stress axis, Molecular fallout from the teleost specific
c.
genome duplication event." Gen Comp Endocrinol, 161, 62-66.
Antelman, S. C. A., Kucinski, B., Fowler, H., Gerhon, S. & Edwards, D. (1998). "The effects
of lithium on a potential cycling model of bipolar disorder." Progress in neuro-
In
psychopharmacology and biological psychiatry. 22, 495-510.
Baier, H. (2010). "Depression-like behavior in zebrafish mutants with disruption of the
glucocorticoid receptor " Society for Neuroscience Annual Meeting Abstract 884.1.
Barcellos, G. R. F., Kreutz, C., Quevedo, M., Bolognesi da Silva, L. & Bedin, C. (2007).
"Whole-body cortisol increases after direct and visual contact with a predator in zebrafish
rs
Danio rerio." Aquaculture, 272, 774-778.
Best, J. A. W. (2008). "Zebrafish,, An in vivo model for the study of neurological diseases."
Neuropsychiatric Disease and Treatment, 4(3), 567-576.
Blackburn, J. L. S., Raimondi, A., Ignatius, M., Salthouse, C. & Langenau, D. (2011). "High-
he
throughput imaging of adult fluorescent zebrafish with an LED fluorescence
macroscope." Nat Protoc. 6, 229-241.
Blaser, R. G. R. (2006). "Behavioral phenotyping in Zebrafish, Comparison of three
behavioral quantification methods." Behav Res Methods, 38, 456-469.
C. N. (1998). "The neurobiology of depression." Sci Am, 278, 42-49.
is
Childs, E. H. C., Deckert, J., Xu, K., Badner, J. & de Wit, H. (2008). "Association between
ADORA2A and DRD2 polymorphisms and caffeine-induced anxiety."
bl
Crusio, E. A. J. (1986). "The genetic architecture of behavioral responses to novelty in mice."
Heredity, 56, 55-63.
Csányi, V. G. R. (1988). "Open-field behavior and the behavior-genetic analysis of the
paradise fish (Macropodus opercularis)." J Comp Psychol, 102, 326-336.
Pu
Darland, T. D. J. (2001). "Behavioral screening for cocaine sensitivity in mutagenized

zebrafish." Proc Natl Acad Sci, 98, 11691-11696.
Dooley, K. Z. L. (2000). "Zebrafish, a model system for the study of human disease." Curr
Opin Genet Dev, 10, 252-256.
Dooley, K. Z. L. (2000). "Zebrafish, A model system for the study of human disease." Curr
Opin Genet Dev, 10, 252-256.
a
Dulawa, S. H. K., Gundersen, B. & Hen R. (2004). "Effects of chronic fluoxetine in animal
models of anxiety and depression." Neuropsychopharmacology, 29, 1321-1330.
Dunlop, B. N. C. (2007). "The role of dopamine in the pathophysiology of depression." Arch
ov
Gen Psychiatry, 64, 327-337.

Egan, R. B. C., Hart, P., Cachat, J., Canavello, P., Elegante, M., Elkhayat, S., Bartels, B.,
Tien, A., Tien, D., Mohnot, S., Beeson, E., Glasgow, E., Amri, H., Zukowska, Z. &
Kalueff, A. (2009). "Understanding behavioral and physiological phenotypes of stress
and anxiety in zebrafish. " Behav Brain Res, 205, 38-44.
N
Fluoxetine 305
El Yacoubi, M. L. C., Parmentier, M., Costentin, J. & Vaugeois, J. (2000). "The anxiogenic-
like effect of caffeine in two experimental procedures measuring anxiety in the mouse is
not shared by selective A(2A) adenosine receptor antagonists." Psychopharmacology,
148, 153-163.
Fernandes, Y. G. R. (2009). "Long-term behavioral changes in response to early
c.
developmental exposure to ethanol in zebrafish." Clinical and Experimental Research,
33, 601-609.
Flint, J. S. S. (2008). "Animal models of psychiatric disease. " Curr Opin Genet Dev, 18, 235-
In
240.
Garattini, S. G. E. & Valzelli L. (1967). "Isolation, aggressiveness and brain 5-
hydroxytriptamine turnover." Journal of Pharmacy and Pharmacology, 19, 338-339.
Garzon, J. & d. R. J. (1981). "Hypersensitivity induced in rats by long term isolation, Further
studies on a new animal model for the detection of antidepressants." European Journal of
rs
Pharmacology, 74, 287-294.
Gerlai, R. C. D., Pereira, T., Sawashima, T. & Krishnannair, R. (2009). "Acute and chronic
alcohol dose, Population differences in behavior and neurochemistry of zebrafish."
Genes, Brain and Behavior, 8, 586-599.
he
Gerlai, R. C. V. (1990). "Genotype environment interaction and the correlation structure of
behavioral elements in paradise fish (Macropodus opercularis)." Physiol Behav, 47, 343-
356.
Gerlai, R. L. M., Guo, S. & Rosenthal, A. (2000). "Drinks like a fish, Zebra fish (Danio rerio)
as a behavior genetic model to study alcohol effects." Pharmacol. Biochem. Behav, 67,
is
773-782.
Geyer, M. M. A. (1995). "Animal models of psychiatric disorders." In, Bloom F, Kupfer D
(eds). Psychopharmacology, The Fourth Generation of Progress. Raven Press, New
bl
York., 787-798.
Geyer, M. M. A. (2002). "The role of preclinical models in the development of psychotropic
drugs." In, Davis K, Charney D, Coyle J, Nemeroff C (eds). Neuropsychopharmacology,
The Fifth Generation of Progress. Lippincott Williams & Wilkins, Philadelphia., 446-
Pu
455.
Hasler. (2004). "Discovering endophenotypes for major depression.."
Kato, T. K. M. & Kasahara, T. (2007). "Animal models of bipolar disorder." Neuroscience
and Biobehavvioral Reveiws, 31, 832-842.
Kessler, R. C. W., Demler, O. & Walters, E. (2005). "Prevalence, severity, and comorbidity
of twelve-month DSM-IV disorders in the National Comorbidity Survey Replication
a
(NCS-R)." Archives of General Psychiatry, 62(6), 617-627.

Kyzar, E. R. A., Gaikwad, S., Green, J., Collins, C., El-Ounsi, M., Davis, A., Pham, M.,
ov
Stewart, A. M., Cachat, J., Zukowska, Z. & Kalueff, A. V. (2012). "On Making Zebrafish
Sad and Anxious, Developing Novel Aquatic Models of Affective Disorders." IBNS
Abstract.
Lele, Z. K. P. (1996). "The zebrafish as a model system in developmental, toxicological and
transgenic research. " Biotechnol Adv, 14, 57-72.
N
Leonard, B. (1998). "Animal models of depression." In Antidepressant Therapy, Briley, M.,

Montgomery, S. eds, Martin Dunitz Ltd, London, 87-109.
Levin, E. B. Z. & Cerutti D. (2007). "Anxiolytic effects of nicotine in zebrafish." Physiology

and Behavior, 90, 54-58.
Lopez-Patino, M. Y. L., Cabral, H. & Zhdanova I. (2008). "Anxiogenic effects of cocaine
withdrawal in zebrafish. " Physiol Behav, 93, 160-171.
Loucks, E. C. I. M. (2004). "Strain-dependent effects of developmental ethanol exposure in
c.
zebrafish." Neurotoxicol Teratol, 26, 745-755.
Mathur, P. G. S. (2010). "Use of zebrafish as a model to understand mechanisms of addiction
and complex neurobehaviroal phenotypes." Neurobiol Dis., 40, 66-72.
In
McGrath, P. L. C. (2008). "Zebrafish, a predictive model for assessing drug-induced
toxicity." Drug Discov Today, 13, 394-401.
Moorman, S. (2001). "Development of sensory systems in zebrafish (Danio rerio)." ILAR J,
42, 292-298.
Ninkovic, J. B. C. L. (2006). "The zebrafish as a model system for assessing the reinforcing
rs
properties of drugs of abuse." Methods, 39, 262-274.
Norton, W. B. C. L. (2010). "Adult zebrafish as a model organism for behavioral genetics."
BMC Neurosci., 11, 90.
P. W. (1984). "The validity of animal models of depression." Psychopharmacology, 83(1), 1-
he
16.
Peitsaro, N. K. J., Anichtchik, O. & Panula, P. (2003). "Modulation of the histaminergic
system and behaviour by alpha-fluoromethylhistidine in zebrafish." J Neurochem, 86,
432-441.
Pittman, J. & Lott, C. (2014). Startle response memory and hippocampal changes in adult
is
zebrafish pharmacologically-induced to exhibit anxiety/depression-like behaviors.
Physiology & Behavior., 123, 174–179.
Pittman, J. & Ichicawa, K. (2013). iPhone® Applications as Versatile Video Tracking Tools
bl
to Analyze Anxiety/Depression-Related Behaviors in Zebrafish (Danio rerio). Pharm,
Biochem and Behavior. 106, 137-142.
Prut, L. B. C. (2003). "The open field as a paradigm to measure the effects of drugs on
anxiety-like behaviors, A review." European Journal of Pharmacology, 463, 3-33.
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Shin, J. F. M. (2002). "From zebrafish to human, modular medical models." Annu Rev
Genomics Hum Genet, 3, 311-340.
Sprague, J. D. E., Douglas, S. & Westerfield, M. (2001). "The zebrafish information network
(ZFIN), A resource for genetic, genomic and developmental research." Nucleic Acids
Res, 29, 87-90.
Ward, A. L. G. (2002). "The zebrafish as a model system for human disease. " Front Biosci,
7, d827-d833.
a
Weiss, J. K. C. (1995). "Animal models of depression and schizophrenia." American

Psychiatric Press, Washington, DC, 89-131.
ov
Willner, P. (1990). "Animal models of depression, An overview." Pharmacol. Ther., 45, 425-
455.
Wullimann, M. K. S. (2000). "Proliferation pattern changes in the zebrafish brain from
embryonic through early postembryonic stages." Anat Embryol, 202, 385-400.
Zon, L. P. R. (2005). "In vivo drug discovery in the zebrafish." Nat Rev Drug Discov, 4, 35-
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44.
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Chapter 15
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How Could Fluoxetine Exert
Therapeutic Effects in Stroke?
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Virginie Beray-Berthat, Michel Plotkine and Raymond Mongeau*
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Pharmacologie de la circulation cérébrale EA4475,
Université Paris Descartes, Paris, France
Abstract
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Stroke is among the leading causes of morbidity and mortality worldwide. The
current pharmacological treatment of stroke is early reperfusion and fibrinolysis of the
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ischemic brain area using the recombinant tissue plasminogen activator (rt-PA, alteplase).
Much effort has been invested into the discovery of other pharmacological therapies to
provide neuroprotection and to reduce the late neurological and psychiatric deficits
associated with stroke. Among the numerous strategies proposed, there is the
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administration of antidepressant drugs. The recent report that the selective serotonin
reuptake inhibitor (SSRI) fluoxetine provides neuroprotection and can improve motor
deficits in patients is particularly encouraging. In addition to its well-known therapeutic
effect in depression, a frequent long-term consequence of stroke, fluoxetine might exert
additional effects after cerebral ischemia independently of its antidepressant effect per se.
The present review examines various short and long-term mechanisms through which
fluoxetine and other SSRIs might exert their therapeutic effects in stroke. In addition to
the facilitation of serotonergic transmission, via long term regulations of 5-HT receptor
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functions and neuroplasticity, SSRIs could have important beneficial effects on

haemostasis, vasomotricity, trophic and inflammation factors (BDNF, cytokines,
microglia, …) that can reduce the initial consequences (infarct size, sensorimotor
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deficits) as well as the long term emotional deficits of stroke.
Keywords: antidepressant drugs; blood brain barrier; cerebral ischemia; cerebral blood flow;
haemostasis; inflammation; neurogenesis; neuroprotection; neurovascular unit; post-
stroke depression; trophic factors
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*
Corresponding author: R. Mongeau, Université Paris Descartes, EA4475, Pharmacologie de la circulation
cérébrale, Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de l‘Observatoire, 75270, Paris
Cedex 06, France; raymond.mongeau@parisdescartes.fr.
308 Virginie Beray-Berthat, Michel Plotkine and Raymond Mongeau
1. Introduction
Fluoxetine and other selective serotonin reuptake inhibitors (SSRIs) are first line
treatments for depression and anxiety. Furthermore, SSRIs are also effective treatments for
the long-term emotional deficits associated with stroke, namely post-stroke depression, and
c.
could possibly be effective also for the long term cognitive deficits associated with this
disease since, as in depression, these might be associated with chronically low extracellular 5-
HT levels (Cowen & Sherwood, 2013; Jorge et al., 2010). However, prior to cognitive and
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depressive symptoms, stroke is mostly recognized by sensory-motor deficits. Cholet et al.
FLAME multicenter study about the beneficial effect of fluoxetine for stroke recovery, in
particular for motor functions, had recently a considerable impact (Chollet et al., 2011;
Cramer, 2011). Indeed, patients that had suffered stroke of moderate severity, but with severe
arm and leg motor weakness, treated few days after with fluoxetine experienced great
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neurological benefits independently of the therapeutic effect of fluoxetine on depressive
symptoms. As indicated by functional magnetic imaging, these effects on motor performances
are reflected by activity changes at the level of the motor cortex (Pariente et al., 2001). Since
then, systematic reviews and meta-analyses have shown the efficacy of SSRIs in reducing
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dependency and disabilities associated with stroke (Mead et al., 2012), in agreement with
older studies suggesting that antidepressant drugs (ADs), and in particular those acting on the
serotonergic system, reduce the neurological disabilities occurring after stroke (Cramer, 2011;
Mead et al., 2012).
Because fluoxetine might eventually become a first line treatment for stroke neurological
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symptoms, our goal here was to review, at physiological, cellular and behavioral levels, how
SSRIs could exert therapeutic effects on the various facets of stroke.
Stroke is defined by the interruption of blood perfusion to a part of the brain. This cuts
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off the oxygen and nutrients supplies, and thereby causes damages to brain tissues. The
majority of strokes is ischemic (85% are due to the occlusion of a blood vessel by a thrombus
or an embolus), while a minority of them is hemorrhagic. Stroke is a serious neurological
disease, particularly common and disabling. It affects 22 million people worldwide each year,
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and this number is increasing because of the aging population (IST-3 collaborative group et
al., 2012). It is the second cause of death worldwide after cardiovascular diseases, being
responsible for 9.7% of deaths in 2004 and this is expected to reach 12.1% in 2030 (Mathers
et al., 2009). In addition, stroke is the leading cause of acquired disability in adults and is the
second cause of dementia after Alzheimer's disease (Acheampong & Ford, 2012). Stroke
diagnosis needs to be done as early as possible to limit its consequences and implement an
appropriate treatment. The phrase ―time is brain‖ emphasizes that nervous tissue is rapidly
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lost as a stroke progresses, since a typical patient loses 1.9 million neurons each minute stroke
is untreated (Saver, 2006). Sudden neurological symptoms rapidly progressing over a few
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minutes or a few hours may be signs of a stroke. The most common stroke symptoms include
hemiparesis that may involve face, arm and leg, hemisensory disturbances, gaze deviation,
language disorder (dysphasia) and visuospatial neglect.
Risk factors for ischemic stroke include age, the most powerful factor (the risk of stroke
doubles every decade after 55 years), gender (prevalence is higher in men), ethnic
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background, and family history. Modifiable risk factors are represented by hypertension,
which is the major risk factor, heart diseases (atrial fibrillation being the most important),
How Could Fluoxetine Exert Therapeutic Effects in Stroke? 309
smoking, diabetes and hyperlipidemia. Other possible modifiable risk factors are obesity, diet,
physical activity, alcohol consumption, contraception, and hormone replacement therapy
(Goldstein et al., 2011). Among psychological risk factors, a depressive mood may predispose
to a stroke, in particular an ischemic stroke (May et al., 2002; Ohira et al., 2013). Metabolic
changes characteristic of depressive patients may underlie this increased propensity for
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stroke. Depressed individuals very often have increased autonomic sympathetic activity that
may lead to increased platelet activity. Furthermore, a hallmark of depression is the excessive
secretion of glucocorticoids that itself promotes conventional stroke risk factors, such as
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hypertension. Elevated glucocorticoids are also known to decrease neurogenesis and trophic
factors, which as we will see in section 3.2, may be stroke recovery mechanisms.
Interestingly, depression and anxiety are associated with exaggerated serotonin-mediated
platelet activation (Zafar et al., 2010), but patients treated with SSRIs may be protected
against this risk factor, as 5-HT uptake inhibition have an inhibitory effect on platelet
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activation by decreasing 5-HT content inside platelets and thereby reducing platelet plug
formation (Geiser et al., 2011; Hergovich et al., 2000).
Beyond a risk factor, depression is also known as a main consequence of stroke. The
psychological distress reactions generated by the stroke itself may explain this late emotional
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deficit (Gainotti et al., 1997). Nevertheless, many authors insist instead on biological causes.
Depression occurs more often in the stroke population than in patients presenting similar
levels of disability from other diseases (Folstein et al., 1977). Post-stroke depression could be
the result of a brain lesion in a region regulating emotions such as the prefrontal cortex
(Terroni et al., 2011). However, whether an augmented risk of depression does exist after a
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stroke in a specific brain location rather than another remains a matter of debate (Carson et
al., 2000; Narushima et al., 2003). In any case, lesions in areas such as the prefrontal cortex
should have an important impact through their projections on monoaminergic systems. These
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systems and the neural circuits regulating mood and anxiety might suffer from a generalized
damage to the brain vasculature, as first claimed in the ―vascular hypothesis‖ of depression
(Taylor et al., 2013). In contrast to post-stroke depression, which involves an acute and focal
lesion, vascular depression would be the consequence of chronic small ischemic lesions to
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frontal and subcortical circuits regulating emotion and cognition. It is particularly fitted to
explain late-onset depressions occurring after age 50. In old depressed patients who have
experienced a brutal mood transformation, symptoms are more likely accounted by a
biological dysfunction than traits and personality. Imaging studies have revealed abnormally
of the white matter (white matter hyperintensities) in several brain areas of depressed patients,
including the prefrontal cortex (Tupler et al., 2002). These small infarcts largely
asymptomatic at first could eventually trigger a major depression, dementia or the occurrence
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of a larger ischemic stroke, itself leading to post-stroke depression. Note that fluoxetine and
other SSRIs are well-tolerated first line agents to treat late-life depression, whether or not
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associated with stroke (Solai et al., 2001). We will argue in the present review that fluoxetine
is a pleiotropic drug for stroke treatment. Putative mechanisms explaining the beneficial
effects of fluoxetine are multiple and may explain as much the therapeutic effects on motor
functions as those on mood and cognition. First, SSRIs are known to have neurotrophic
effects by acting on the production of growth factors, such as the brain derived nerve growth
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factor (BDNF) and other processes such as hippocampal neurogenesis. Migration of new
neurons to damaged brain areas have been shown to occur after stroke (Wiltrout et al., 2007).
Both neurogenesis and angiogenesis in stroke areas appear to be facilitated by SSRI
treatments (Espinera et al., 2013). Second, SSRIs may have neuroprotective effects by
reducing inflammation via an action on cytokines and by repressing microglia activation, and
third, they might be acting on vascular and haemostasis parameters immediately after stroke
(Geiser et al., 2011; Ofek et al., 2012). Finally, on the psychological sphere, the
antidepressant and anxiolytic effects of chronic SSRI may help stroke recovery by attenuating
c.
a cascade of distress related processes.
1.1. The Neurovascular Unit and the Cerebral Blood Flow
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To better understand brain damages caused by stroke, we need to consider primary events
taking place at the blood brain barrier (BBB). The BBB regulates the entry of nutrients and
drugs into the brain from circulating blood. As shown on figure 1, the BBB is composed of
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endothelial cells, a basement membrane, pericytes and astrocytic feet. All these elements are
necessary to control exchanges between the blood and the brain parenchyma. Together with
neurons and glial cells, they form a functional unit called the neurovascular unit.
Changes in the integrity of the BBB are central to the pathophysiology of cerebral
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ischemia. Ischemic stroke can disrupt the neurovascular unit at both the structural and
functional levels, which leads to abnormal leaks across the BBB. Models of cerebral ischemia
revealed how expression of both inter-endothelial junction proteins (such as occludin) and
components of the basal membrane (such as collagen IV and laminin) can be decreased.
Swelling of astrocytes leads to the detachment of their microvascular endfeet (figure 1).
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Among the mechanisms underlying post-ischemic damage of the BBB, those involving
metalloproteinases (MMPs) play a major role by degrading the extracellular matrix and
interendothelial junctions. These proteolytic enzymes expressed by endothelial cells,
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microglial cells, neurons, astrocytes and infiltrating inflammatory cells are involved in brain
remodeling after cerebral ischemia (section 1.4). Fluoxetine may inhibit ischemia-induced
cell death and cognitive impairment, by preventing BBB disruption indirectly via its action on
astrocyte / microglial activation and inflammation (Lee et al., 2014). It may also possibly act
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directly at the level of the neurovascular unit itself because expression of the 5-HT transporter
(5-HTT) mRNA has been detected in cultured immortalized rat cerebral endothelial cell lines
(Brust et al., 2000). This transporter appears to be localized on both the luminal and the
abluminal sides of brain capillaries (figure 1; Wakayama et al., 2002).
Following an ischemic insult, there is a persistent microvascular constriction that results
in part from pericyte contraction (Liu et al., 2012; figure 1). Post-stroke vasospasms and BBB
dysfunctions also involve blood vessels composed of vascular smooth muscles regulating
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cerebral blood flow through innervation from noradrenergic, GABAergic, cholinergic and
serotonergic neurons (Hawkins & Davis, 2005). Both in vitro and in vivo studies have shown
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that 5-HT act not only as a synaptic neurotransmitter in the central nervous system, but also
as a potent vasoactive agent of the brain vasculature. 5-HT nerve fibers have been observed
through the use of an antibody to tryptophan hydroxylase (the 5-HT synthesis enzyme), in
major cerebral arteries and small pial vessels. These projections appear to be either of central
origin, from the rostral raphe nucleus, or of peripheral origin, from the superior cervical
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ganglion (Bonvento et al., 1991). Because of its localization in key vascular targets, 5-HT is
thought to play a pivotal role in coupling cerebral blood flow to metabolism. It decreases
oxygen and glucose consumption, depresses electrical activity in the cortex and reduces
cerebral blood flow, mainly when the BBB is disrupted (for a review see Bonvento et al.,
1991). Heterogeneous post-synaptic receptors (5-HT1, 5-HT2, 5-HT7, …) are involved in the
vasomotor effects of 5-HT depending on the species, anatomically distinct vessels and even
different segments of the same vessel (Bonvento et al., 1991). Some of the vasoconstrictor
effects of 5-HT in small cerebral arteries are mediated by 5-HT2A receptors via voltage-
c.
dependent Ca2+ channels (Ungvari et al., 1999). Furthermore, activation of 5-HT3 receptors
on GABAergic interneurons can indirectly mediate either constriction or vasodilatation via
neuropeptide Y or NO, respectively (Perrenoud et al., 2012). Finally, the inhibitory
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somatodendritic 5-HT1A autoreceptors on 5-HT neurons of the raphe nuclei could also play a
role in increasing cerebral blood flow (Bonvento et al., 1997; Marco et al., 1999). Activation
of inhibitory autoreceptors, as occurring when extracellular 5-HT concentration is increased
by an SSRI, may thus decrease the postsynaptic receptor-mediated vasoconstrictor effect of 5-
HT.
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Figure 1. The principal elements constituting the neurovascular unit.

Endothelial cells form a continuous layer at the luminal surface of the capillary. They limit the
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permeability of the barrier via four defense levels: 1) interendothelial tight and adherens junctions that
greatly reduce the paracellular flux through various proteins such as the tight junction protein occludin,
2) few pinocytotic vesicles forming a transcellular barrier, 3) enzymatic barriers and 4) endothelial cells
expressing a large number of efflux transporters that allow efflux of toxins and drugs. Some of them
participate to a certain extent to the inactivation of several neurotransmitters, including glutamate,
GABA, noradrenaline and, in particular, 5-HT via the 5-HTT localized on both the luminal and
abluminal sides. It is noteworthy that endothelial cells also express endothelial NO synthase (eNOS)
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exerting endothelium-dependent vasodilatation, platelet aggregation inhibition and anti-oxidant effects

contrasting with the deleterious action of the inducible NOS (iNOS) triggered during inflammation. The
basal membrane is an extracellular matrix on which is based the cerebral endothelium. It is composed
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of various proteins such as the cell adhesion molecule laminin and collagen IV. Astrocytes form a link
between the endothelium and neurons. The astrocytic endfeet cover over 99% of the surface on the
basement membrane of capillaries and microvessels. Microglial cells are also found in the perivascular
space. They play a very important immunological role since they are the resident macrophages of brain
tissue. Pericytes are poorly differentiated contractile cells spreading around the capillary wall. Drowned
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in the basement membrane, pericytes cover the endothelium and provide structural support to blood
vessels. Pericytes regulate brain capillary blood flow through contraction and relaxation (the figure was
modified from Abbott et al., 2010).
1.2. Stroke and Cerebral Ischemia
Haemostasis, that prevents blood loss when vessels are damaged, is a vital defense
process. It involves activation and adhesion of platelets, and subsequently fibrin matrix and
blood clot formations. It can pathologically occur, however, in absence of bleeding, such as in
c.
stroke. The majority of strokes are ischemic, that is - due to the occlusion of a blood vessel by
a thrombus or an embolus. A « haemostatic » plug interrupts blood flow, thereby causing
cerebral ischemia and death of brain tissue downstream (infarction). In focal ischemia,
occlusion of a cerebral artery occurs after migration of an embolus of extracerebral origin or
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by the formation of a thrombus in situ. The latter emerges in reaction to atherosclerosis or
other vascular dysfunctions. The most frequently occluded cerebral artery is the middle
cerebral artery (MCA). Ischemic areas are divided into the core area, where the fall in
cerebral blood flow is severe enough to causes irreversible tissue loss (i.e., cell death), and the
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penumbra, which has residual blood flow and consists of cells initially not showing
alterations of morphological integrity, but having impaired functions. The latter cells remain
alive for a few hours and are the target of neuroprotective and reperfusion treatments.
Most of the preclinical studies on cerebral ischemia, in particular those about the effects
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of SSRIs (section 2 and 3), derive from animal models of cerebral ischemia that reproduce
focal ischemia. They consist in MCA occlusion (MCAo), for instance by a microclip or by
introducing a nylon filament to block blood flow. In other models, occlusion is carried out by
injecting blood clots or thrombin into the artery. In vitro models are performed on cell
cultures, more rarely on cultured brain slices. Different cell types can be cultured (neurons,
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astrocytes, oligodendrocytes, microglia, endothelial cells; see figure 1) alone or in co-
cultures, such as astrocytes / neurons co-cultures. Cells are exposed to deprivation of either
oxygen or glucose reproducing in vitro ischemic conditions. They can also be subjected to
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conditions that mimic some other events of cerebral ischemia: oxidative stress or glutamate-
induced excitotoxic stress. These studies revealed the important consequences of ischemia on
brain cells, as detailed in the following section.
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1.3. Cellular Consequences of Brain Ischemia: Glutamate-Induced

Excitotoxicity, Oxidative Stress and Inflammation
Following cerebral ischemia, the decrease in cerebral blood flow reduces input of energy
substrates, oxygen and glucose. Energy depletion notably disrupts the function of the Na+/K+
ATPase, which consumes under physiological conditions, 70% of the brain energy resources.
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The resulting loss of ionic gradients triggers neuronal depolarization and increases the release
of neurotransmitters, particularly the main excitatory amino acid glutamate. This leads to an
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accumulation of glutamate into the extracellular medium and excessive stimulation of its
receptors, including the NMDA (N-methyl-D-aspartate) receptor. These receptors operating
channels, widespread in the central nervous system, are permeable to Ca2+. The glutamate-
mediated increase in intracellular Ca2+ concentration stimulates proteases, lipases,
endonucleases, and neuronal NO synthase (nNOS), all contributing to the so-called
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excitotoxic cell death (Doyle et al., 2008; Turner et al., 2013).

In parallel with the huge increase in glutamate extracellular concentration, there is also a
dramatic increase in 5-HT levels associated with brain ischemia (Kanthan et al., 1996; Nagao
et al., 1995). Similarly to glutamate, 5-HT is associated with the negative consequences of
brain injuries, such as brain edema formation and BBB disruption, because these
consequences are attenuated by either 5-HT biosynthesis inhibition or 5-HT antibodies
(Sharma et al., 2007). Furthermore, following BBB disruption, increasing 5-HT levels
produces marked decreases in blood perfusion in numerous brain areas (Grome & Harper,
c.
1983). Acute 5-HT reuptake inhibition with citalopram was shown to reduce ischemia-
induced 5-HT hypermetabolism (Nagao et al., 1995).
Under physiological conditions, cells produce various free radicals, in particular through
In
their mitochondrial respiratory chain. There is normally a dynamic equilibrium between the
formation of free radicals and their disposal by endogenous enzymatic antioxidant systems
(superoxide dismutase, glutathione peroxidase) and non-enzymatic processes (glutathione,
vitamin E, vitamin C, …). After brain ischemia, mitochondrial respiratory chain dysfunction
and post-ischemic activation of various enzymes, such as iNOS and COX, leads to
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overproduction of free radicals, especially reactive oxygen species (ROS) including
superoxide anion (O2-.), hydroxyl radical (.OH) and nitric oxide (.NO). Under ischemic
conditions, depletion of the L-arginine substrate and/or the co-factor BH4, required for
coupling two functional eNOS subunits, generates excessive ROS in endothelial cells leading
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to eNOS uncoupling. In an uncoupled state, eNOS produces harmful O2-. instead of the
beneficial endothelial NO (wich exerts vasodilation and antithrombotic effects). Once
generated, O2-. activates several stress-sensitive pathways and scavenges NO to produce
ONOO-, a highly reactive pro-oxidant that elicits direct neurotoxic effects (Srivastava et al.,
2012). This ONOO- causes DNA damages that hyperactivate the poly(ADP-
is
ribose)polymerase (PARP), a DNA repair enzyme known to be deleterious when it is over
activated during cerebral ischemia (Haddad et al., 2006; Szabó & Dawson, 1998). Post-
ischemic overproduction of ROS also exceeds the capacity of endogenous antioxidant
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systems causing membrane lipid peroxidation, protein oxidation and cell death (Pradeep et
al., 2012).
Post-ischemic oxidative stress and the resulting necrotic cells trigger the production of
inflammatory cytokines. These initial events lead to the activation of microglial cells that in
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turn, produce glutamate, cytokines and chemokines in large amounts, which themselves
causes the expression of adhesion molecules by endothelial cells. This triggers the adhesion
of circulating inflammatory cells, i.e leukocytes (neutrophils, lymphocytes and monocytes) to
the endothelium and their subsequent infiltration into the brain parenchyma. Infiltrated
leukocytes and microglial cells express and release various inflammatory mediators
(cytokines, MMPs, iNOS, ...; Ronaldson & Davis, 2012). High levels of neurotoxic
mediators, such as high concentration of NO and peroxide, inflammatory cytokines and
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proteases are involved in the post-ischemic disruption of the BBB (Yenari et al., 2006). We
will see that fluoxetine could target many of these damaging processes (see section 2.3).
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1.4. Brain Remodeling after Cerebral Ischemia
Post-ischemic disruption of the BBB notably results from a rise in MMPs, proteolytic
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enzymes that have the ability to remodel the extracellular matrix. These enzymes are
necessary for inflammation and wound repair by cleaving several substrates including growth
factors, cytokines, cell surface receptors and cell adhesion molecules. Studies have
demonstrated the induction of several MMPs (MMP-2,-3,-7,-9) following cerebral ischemia

(Jin et al., 2010; Sandoval & Witt, 2008). Post-ischemic oxidative stress is implicated since
MMPs can be strongly activated by free radicals. MMPs are secreted by astrocytes,
endothelial, microglial and neuronal cells, and by neutrophils infiltrated in the damaged brain
parenchyma (Lakhan et al., 2009).
c.
MMPs can also cleave and activate proneurotrophins, leading to mature forms of growth
factors such as BDNF, regulating neuronal survival and synaptic plasticity via its tyrosine
kinase (TrkB) receptor (Hwang et al., 2005; Lee et al., 2001). BDNF levels are increased at
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and around the lesion site of an ischemic stroke, through a timely secretion from neuronal and
non-neuronal sources (endothelial cells, astrocytes and microglia; Béjot et al., 2011).
Together with growth factors secretion, stroke induces brain repair mechanisms by
stimulating new neuronal formation. Neurogenesis is triggered by neural stem cells in the
dentate subgranular zone of the hippocampus and the subventricular zone lining ventricles
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adjacent to this area. New neurons migrate within the hippocampus and other areas of the
brain such as the striatum, and this migration occurs in close apposition to blood vessels. The
vasculature thus plays an essential role in this migration since neurogenesis was shown to
occur in parallel with hypoxia-induced angiogenesis (Thored et al., 2007). Furthermore, in
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addition to growth factors (such as BDNF), various mediators of the ischaemic-hypoxic
response (erythropoietin) as well as glutamate intervene at different steps to regulate neural
progenitor proliferation, migration and survival in the post-ischemic brain (Kokaia &
Lindvall, 2003; Wiltrout et al., 2007). This cell proliferation appears important since
irradiation-induced ablation of neurogenesis exacerbates behavioral deficits after cerebral
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ischemia (Raber et al., 2004). Enhancement of serotonergic transmission by chronic
fluoxetine might also boost brain remodeling after stroke as it stimulates BDNF secretion and
neurogenesis (see section 2.4).
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2. Experimental and Clinical Stroke Therapeutic

and the Acute Effects of Fluoxetine
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2.1. Drugs Acting on Haemostasis
Thrombolysis with the recombinant tissue plasminogen activator (rt-PA, alteplase)

remains the only pharmacological treatment currently available for acute ischemic stroke.
Endogenous tPA converts inactive plasminogen to plasmin, which in turn degrades the fibrin
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matrix. Treatment with rt-PA thus aims to lyse the thrombus to allow recanalization of clotted
artery, and thereby permits tissue reperfusion. Rt-PA is the only thrombolytic agent proved
clinically effective when administered 4.30 hours after symptoms onset. However, to date,
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only 3-4% of patients suffering from an acute ischemic stroke are treated with intravenous rt-
PA. The barriers to thrombolysis are the delay in recognizing stroke symptoms at hospital
arrival and the multiple contraindications to rt-PA administration. The use of rt-PA is indeed
not without risk since it has been shown to enhance the occurrence of symptomatic
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hemorrhagic transformations via multiple factors, including inflammation (Haddad et al.,

2013). Furthermore, early reperfusion after intravenous rt-PA can effectively be established in
fewer than 40% of treated stroke patients (von Kummer & Hacke, 1992).
Interestingly, one possible mechanism through which fluoxetine exerts a beneficial action
in stroke recovery is via an action on blood coagulation and fibrinolysis. 5-HTT plays a role
in the activity of platelets: these anucleated cells are unable to synthesize 5-HT, but they
actively uptake it from the plasma via 5-HTT. Endothelium damage, caused among other by
arteriosclerosis triggers platelet activation and the content of platelet granules, including 5-
c.
HT, is released thereby enhancing the Ca2+-dependent amplification of platelet activation.
Although 5-HT per se does not activate platelets, it enhances platelet activation by factors
such as ADP, adrenaline and thrombin via 5-HT2A receptors (Li et al., 1997). Note that the
In
latter receptors are also expressed on the vascular walls to exert, concomitantly with
haemostasis, vasoconstriction (section 1.1). Interestingly, anxious and depressed patients
treated with serotonergic ADs displayed enhanced fibrinolysis; these drugs thus prevent a
basal state of enhanced blood coagulation produced by platelets hyperactivity in these patients
(Geiser et al., 2011). Furthermore, 5-HT appears to have a crucial role in the formation of a
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subpopulation of platelets, the coated-platelets dually activated by collagen and thrombin,
which may lead to a robust pro-thrombotic state, and accordingly, individuals treated with
SSRIs have a reduced proportion of these platelets (Prodan et al., 2007). By blocking uptake
and storage of 5-HT into platelets via 5-HTT, fluoxetine may prevent the platelet aggregation
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and coagulation cascade. Clinical studies that have investigated the existence of an
association between chronic SSRI treatments and stroke did not detect, however, a reduced
risk (de Abajo, 2011). Nevertheless, it is conceivable that acute fluoxetine administration
after a stroke prevents reocclusion contributing to chronic small ischemic lesions. However,
similarly to rt-PA, SSRIs might also increase the risk of brain hemorrhage, although this risk
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appears to be moderate (Hackam & Mrkobrada, 2012).
2.2. Drugs Promoting Vasodilation and Blood Reperfusion

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Recanalization with rt-PA is necessary, albeit not sufficient to allow full blood
reperfusion of brain tissue via microvessels. Reperfusion is, in fact, a better predictor of
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stroke recovery than recanalization (Soares et al., 2010). Following cerebral ischemia,
calcium not only triggers several deleterious enzymatic cascades (section 1.3), but also
induces vasomotor contraction through voltage dependent-gated channels on microvessels
smooth muscles, leading to vasospasms (Cipolla et al., 2014). Based on preclinical studies,
the vasodilation effect of calcium antagonists, combined with their neuroprotectant action via
an attenuation of intracellular Ca2+ concentrations, was hypothesized to confer therapeutic
efficacy. Unfortunately, calcium antagonists, such as nimodipine, failed to show clinical
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benefits in several trials on ischemic stroke patients (Zhang et al., 2012).

Other drugs might be more suitable to release the vasospams. Interestingly, fluoxetine
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might be such an agent because its acute administration was shown to produce vasodilation of
cerebral arterioles and to ameliorate reperfusion of the ischemic brain tissue. Furthermore, it
was shown to prevent the vasoconstrictor response triggered by either 5-HT or by a calcium
channel opener in vascular smooth muscle of small cerebral arteries (Ungvari et al., 1999).
This may occur through the release of endothelial NO since a eNOS antagonist blocked this
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vasodilation effect (Ofek et al., 2012). Apparently this is a 5-HT-independent effect because it
was not blocked by the non-selective 5-HT antagonist methysergide (Ofek et al., 2012).
Nevertheless, whether selective 5-HT3 receptor antagonists would alter the effect of
fluoxetine needs to be assessed as 5-HT3 receptors also mediate vasodilatation through NO

(Perrenoud et al., 2012). According to Ofek et al. (2012), the indirect effect of fluoxetine
involves rather an action of acetylcholine released from parasympathetic fibers and binding of
this neurotransmitter on muscarinic receptors of endothelial cells (Ofek et al., 2012). This
results into the release of calcium from intracellular stores in these cells and facilitates
c.
binding of calmodulin to eNOS that then produces NO from L-arginine. NO subsequently
diffuses to vascular smooth muscles to induce vasodilation. Fluoxetine at low doses may also
enhance noradrenaline release from cerebral perivascular sympathetic nerves and may cause,
through 2 receptors, a NO-mediated vasodilation via axo-axonal mechanism, independently
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of the endothelium (Chen et al., 2012).
2.3. Drugs Preventing Cellular Damages
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As we have seen in section 1.3, neuroinflammation is one of the most important
mechanisms leading to brain cell damages. A major event at the cellular level leading to the
inflammation cascade is oxidative stress. Therefore, preventing oxidation stress appeared, at
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least in several experimental stroke models, as one of the most promising pharmacological
target. Several anti-oxidant compounds have been tested clinically, including NXY-059,
which unfortunately was revealed ineffective in large trials on acute ischemic stroke (Diener
et al., 2008). Another important process in inflammation is immune cells adhesion and
infiltration in the infarct area. Enlimomab, developed to prevent these processes, did not show
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efficacy in a clinical trial and had even worsen stroke outcomes (Enlimomab Acute Stroke
Trial Investigators, 2001). Among the plethora of strategies researchers had hope would be
beneficial in human stroke, there is also the prevention of excitoxicity with glutamate
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antagonists, but these drugs also failed to fulfill expectancies (Muir, 2006).
Fluoxetine and other SSRIs shown to be clinically effective have, in turn, an interesting
pattern of action at some of these same cellular processes. First, fluoxetine may inhibit cell
death after stroke by blocking BBB disruption. In an experimental model of global ischemia,
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subchronic treatment with fluoxetine inhibited MMP-2 mRNA expression, reduced MMP-9
activity and attenuated the loss of laminin and occludin, substrates of MMPs (Lee et al.,
2014). As visualized with occludin antibodies, the vascular network integrity was restore after
fluoxetine treatment in ischemic animals, and this probably explains the decrease in immune
blood cell infiltration concomitantly observed (Lee et al., 2014; Lim et al., 2009). Second,
recent in vivo studies as well as studies on cultured cells indicate that fluoxetine can suppress
microglial and astrocyte activation resulting from ischemic insults (Dhami et al., 2013; Lee et
a
al., 2014; Lim et al., 2009). These therapeutic-like effects were correlated with improvements
both in terms of motor activity and memory, and in terms of infarct volume and apoptotic cell
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death (Lee et al., 2014; Lim et al., 2009). Importantly, treatment with high acute doses of
SSRIs appears particularly effective, contrary to chronic SSRI treatments that failed to induce
therapeutic effects at this level (Jolkkonen et al., 2000; Lim et al., 2009; Windle & Corbett,
2005; Zhao et al., 2005). In a co-cultured cell study, oxygen-glucose deprived cortical
neurons showed greater survival in presence of activated microglial cells when they were pre-
N
treated with fluoxetine, but not with other types of ADs such as tricyclic antidepressants,
(TCA) and monoamine oxidase inhibitors (MAOI) (Dhami et al., 2013). Neurons would be
protected under fluoxetine apparently because glutamate excitotoxicity resulting from
microglial activation is reduced (Dhami et al., 2013). The primary mechanism accounting for
the decreased glutamate release from activated microglia in presence of fluoxetine remains
unclear, although interactions between serotonergic and glutamatergic systems are known to
occur in the brain (Drago et al.,2011).
Fluoxetine not only influences immune cells infiltration and microglial activation, but
c.
also the capacity of these cells to produce proinflammatory agents. Protein and mRNA
expression of inflammatory mediators such as TNF-, IL-1, IL-6, COX-2 and iNOS and
several chemokines triggered by ischemia are markedly decreased by fluoxetine (Lee et al.,
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2014; Lim et al., 2009). This is associated with an inhibition of NF-B, a transcription factor
that controls DNA elements involved in cellular responses to cytokines (Lim et al., 2009).
Although drugs having a primary action on oxidation/inflammation, such as NXY-059
and enlimomab, failed to show clinical efficacy (Diener et al., 2008; Enlimomab Acute Stroke
Trial Investigators, 2001; Muir, 2006), it remains possible that fluoxetine‘s peculiar pattern of
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action on glutamate excitotoxicity and inflammation explain its therapeutic action in stroke
patients.
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3. Antidepressant-Like Effects of Fluoxetine in
Post-Stroke Depression
Preclinical research interest has recently shifted toward the long-term cognitive and
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emotional deficits of stroke. About one-third of patients display anxio-depressive symptoms
after stroke onset (Carota et al., 2005; Hackett et al.,2014). Post-stroke depression is a major
problem as it slows recovery, predicts poorer functional outcomes and increases the risks of
mortality, including suicide (Kronenberg et al., 2014). Depression itself, which as we have
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seen increases the risks of stroke (May et al., 2002; Ohira et al., 2001), could be associated
with BBB barrier disruption, eNO uncoupling, MMPs induction (Najjar et al., 2013; Taylor et
al., 2013; Yoshida et al., 2012) and the generation of chronic small ischemic lesions (Dieguez
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et al., 2004). It is thus becoming urgent to adequately model at the preclinical level this
stroke-depression association, especially with the prospect that drugs with antidepressant
properties, SSRIs, might become the first pharmacological treatment for both the subacute
and the chronic phases of stroke recovery (Kronenberg et al., 2014).
Behavioral models commonly used to rapidly screen for ADs such as the forced swim
test (FST) may not be adequate. Cerebral ischemia in rodents is very often associated with
motor hyperactivity resulting in decreased immobility in the FST, an antidepressant-like
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effect. In fact, the SSRI fluvoxamine and cerebral ischemia were shown to have additive
antidepressant-like effects in the FST (Deplanque et al., 2011). Modeling symptoms of
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depression such as anhedonia, i.e., the inability to experience pleasure, provides more
interesting information. It has been reported that MCAo can decrease by itself mice sucrose
consumption, and this was associated with a degeneration of mesolimbic dopaminergic
pathways involved in reward behaviors (Kronenberg et al., 2012). Long-term treatment with
the SSRI citalopram prevented anhedonia and the neurodegeneration occurring after cerebral
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ischemia (Kronenberg et al., 2012). MCAo alone might sometimes be insufficient to trigger
anhedonia and it is often combined with a protocol of chronic mild stress to produce a
depression-like state (Wang et al., 2009). Here again, at various time windows, several weeks
after MCAo, chronic SSRI treatment was shown to prevent the decrease in sucrose
consumption (Wang et al., 2009; Wang et al., 2010; Wang et al., 2010). As in patients,
cerebral ischemia sometimes produces in rodents long term anxiety and spatial cognitive
impairments that are likewise corrected by long term fluoxetine or citalopram treatments
(Kronenberg et al., 2012; Li et al., 2009).
c.
The behavioral deficits modeling post-stroke depression are associated with decreased 5-
HT levels in the frontal cortex and the hippocampus (Ji et al., 2014). Serotonergic projections
to brain areas involved in emotional and cognitive functions are most likely crucial for stroke
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recovery. Destruction of hippocampal serotonergic fibers with the toxin 5,7-
dihydroxytryptamine aggravates ischemic neuronal damages (Nakata et al., 1997). Although
it is beneficial to limit, not abolish, serotonergic mechanisms at the initial phase of a stroke
(see section 2), on the long term, the enhancement of serotonergic transmission (as explained
below) would rather favor brain remodeling and antidepressant-like behavioral effects.
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3.1. Chronic Fluoxetine Increases Post-Synaptic 5-HT1A Receptor-Mediated
Neurotransmission
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Microdialysis studies have clearly shown that 5-HTT blockade rapidly increases 5-HT
extracellular concentration following either systemic SSRI injections or local SSRI injections
into a brain area. However, when 5-HTT is blocked locally prior to a systemic SSRI injection,
the latter induces a decrease of extracellular 5-HT concentration. This is surprising but
is
explained by a decrease release of 5-HT resulting from activation of negative feedback
mechanisms involving inhibitory autoreceptors (Auerbach et al.,1995; Hervás & Artigas,
1998). Acutely, SSRI administration decreases the spontaneous activity of serotonergic
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neurons, and this can obviously not explain their long-term therapeutic effect in post-stroke
depression. Following weeks of treatment with these same SSRIs, the basal firing rate of
neurons is re-established because of somatodendritic 5-HT1A autoreceptors desensitization. A
majority of authors consensually admit that this desensitization is at the core of their
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therapeutic effect in depression (Blier & de Montigny, 1994). Some other studies have also
shown that inhibitory terminal 5-HT1B or 5-HT1D autoreceptors, regulating 5-HT release,
might also become desensitized after chronic ADs (Chaput et al., 1986; Newman et al., 2004).
Beside autoreceptors, other desensitizations occurring at post-synaptic 5-HT2A/2C receptor
subtypes might further explain the anxiolytic effect of SSRIs (Martin et al., 2014; Mongeau et
al., 2010; Weisstaub et al., 2006). Activation of brain 5-HT2C receptors during stress may
indirectly inhibit 5-HT released at post-synaptic 5-HT1A receptors and thereby limit the
a
capacity of an animal to cope with stress (Mongeau et al., 2010). Similarly to other ADs
(TCA, MAOIs), SSRIs are known to increase post-synaptic 5-HT1A neurotransmission.
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Chronic SSRIs do not change the sensitivity of post-synaptic 5-HT1A receptors, but rather
increase the amount of 5-HT released per action potential acting at these receptors (Chaput et
al., 1991).
In the anhedonia model of post-stroke depression, animals displayed reduced 5-HT1A
receptor protein and mRNA levels in the hippocampus, and these changes were reversed by a
N
citalopram treatment (Wang et al., 2009). In keeping with the view that increasing post-
synaptic 5-HT1A transmission leads to favorable outcomes in stroke, activation of 5-HT1A
receptors prevents cAMP-dependent protein kinase A phosphorylation of a NMDA receptor
subunit contributing to neuronal death in the post-ischemic brain (Salazar-Colocho et al.,

2007), and 5-HT1A receptors may have the ability to decrease the excessive release of
glutamate induced by ischemia (Semkova et al.,1998). Furthermore, differential activation of
either 5-HT2C or 5-HT1A neurotransmission increase and decrease, respectively, cellular
damages after cerebral ischemia (Torup & Diemer, 2000), while impairments of glucose
c.
uptake and hippocampal electrical activity after cerebral ischemia were both prevented by
either 5-HT1A receptor agonists or by 5-HT2A/2C receptor antagonists (Shibata et al., 1992).
In
3.2. Chronic Fluoxetine Alters Trophic Factors and Cell Proliferation
Contrary to post-stroke depression, there are few neuro-anatomical correlates of major

depression itself. Nevertheless, many clinical imaging studies indicate reduced hippocampal
rs
volume in depressed patients and this degeneration occurs as a function of stress chronicity
and depression duration (McKinnon et al., 2009). ADs, whether or not they have a primary
action on 5-HT, may induce the production of trophic factors, including BDNF. Stimulation
of TrkB by BDNF triggers intracellular cascades inducing trophic effects in the hippocampus
he
and other brain regions. The fact that tissue levels of BDNF are decreased in the brain of
suicide patients and in the serum or the plasma of depressed patients (Lee & Kim, 2010) may
explain, at least in part, hippocampal volume loss in these patients. Furthermore, there is a
strong relationship between prior serum BDNF levels and the development of post-stroke
depression (Li et al., 2014). Interestingly, the progressive fall in hippocampal BDNF levels
is
occurring in animal models of transient ischemia was prevented by either fluoxetine or
escitalopram treatment (Kim et al., 2007; Lee et al., 2011). Beyond the hippocampus, the
beneficial effect of BDNF secretion following chronic SSRI treatment extends to sensory-
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motor cortical areas (Espinera et al., 2013; Maya Vetencourt et al., 2008). Post-stroke
depression-like behaviors and the reduction in 5-HT neurotransmission are both associated
with depletion of other trophic factors, such as the fibroblast growth factor (FGF-2) known to
protect cerebral tissue from ischemia by activating anti-apoptotic proteins (Ji et al., 2014).
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Another important recovery process that could alter hippocampal morphology is

neurogenesis. It is now well-known that 1) new neurons are normally generated in this area
during the whole life, 2) a decrease in neurogenesis occurs during chronic stress, and 3) the
latter can be corrected by all class of ADs (Gould, 2007; Malberg, 2004; Paizanis et al.,
2007). Despite being a major stress, cerebral ischemia triggers hippocampal neurogenesis, but
this neurogenesis is inversely related to ischemia severity (Winkelheide et al., 2008).
Neurogenesis is a mechanism necessary to reinstate some functions after brain insults since
a
its conditional ablation attenuates recovery of motor function (Sun et al., 2012). The
depressive-like behaviors of MCAo rats submitted to chronic mild stress were shown to be
ov
associated with reductions of various ischemia-induced neurogenesis processes, including cell

proliferation, survival and neuronal differentiation (Wang et al., 2008). Chronic treatments
with either fluoxetine or citalopram were shown to increase hippocampal neurogenesis in
experimental models of stroke, and this was correlated with improvements of late emotional
and cognitive deficits (Li et al., 2009; Wang et al., 2010). Interestingly, these post-stroke
N
improvements in terms of neurogenesis and behavioral antidepressant-like effects were

accelerated by co-administration of an antagonist at the inhibitory 5-HT1A somatodendritic
autoreceptors (Wang et al., 2010). Furthermore, chronic buspirone, a partial agonist at post-
synaptic 5-HT1A receptors (and effective as fluoxetine as an anxiolytic drug) also increase
neuronal cells proliferation in the hippocampus (Campbell Burton et al., 2011; Grabiec et al.,
2009).
The effect of SSRIs on neurogenesis and BDNF may be implicated in neurovascular unit
repair in the peri-infarct area. Citalopram was shown to stimulate neuroblasts proliferation, to
c.
enhance cells migration from the hippocampus to the peri-infarct regions, to increase BDNF
protein and to improve microvessels density in the damaged cortex (Espinera et al., 2013).
Interestingly, there might be a causal relationship between angiogenesis and neurogenesis
In
stimulation by SSRIs even in treated depressed patients that had not suffered a stroke
(Boldrini et al., 2012)
Conclusion and Perspectives
rs
Clinical studies have confirmed the efficacy of ADs, mostly SSRIs, not only in post-
stroke depression but also in alleviating cognitive and motor deficits occurring after a stroke.
In view of the shortcomings in the development of new pharmacological treatments for
he
stroke, these are by any standards exciting results. Nonetheless, as argued by Cramer (2011),
many questions remain at the clinical level regarding therapeutic time windows, the prognosis
and the range of stroke deficits that can effectively be treated with fluoxetine or other SSRIs.
Furthermore, larger clinical trials should be designed based on accumulated knowledge at the
preclinical level about fluoxetine‘s mechanism of action. Yet, we do not really know how
is
SSRIs exert their therapeutic effect in stroke, although several hypotheses have been
reviewed here. Blocking 5-HT uptake and storage into platelets may reduce platelet
aggregation and the coagulation cascade leading to either less recurrent strokes or chronic
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small ischemic lesions. A second possibility is the indirect vasodilator effect of SSRI via
endothelial NO that might help reperfusion of penumbra areas. Not only would SSRI
counteract hypoperfusion, they would also prevent the post-stroke increase in BBB
permeability through an action on MMPs at the basal membrane of the neurovascular unit. In
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these two cases, by either preventing 5-HT uptake or by indirectly increasing 5-HT
autoreceptor-mediated negative feedback, acute SSRIs would reduce the increased 5-HT
metabolism generated by ischemia. Acute high doses of SSRIs were also shown to decrease
the cellular (microglial activation) and the molecular (cytokines, chemokines) actors of the
inflammation cascade and this might be explained notably by 5-HT / glutamate interactions.
One may wonder, however, how important any of these mechanisms are in explaining the
a
therapeutic effect of fluoxetine. Nevertheless, at this point, it remains reasonable to assume

that fluoxetine is a pleiotropic drug exerting action on distinct mechanisms that are
evolutionary linked to the role of 5-HT in defense to acute injuries.
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This contrasts with our traditional views about fluoxetine being an antidepressant
medication. Post-stroke depression and cognitive decline are associated with decreased 5-HT
transmission. It has long been argued that chronic ADs enhance 5-HT neurotransmission,
stimulate the secretion of neurotrophic factors and facilitate neurogenesis in brain areas such
as the hippocampus. These physiological effects have been associated with antidepressant-
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like behaviors in various animal models of anxious-depressive disorders as well as in models

of post-stroke depression. SSRIs might have a dual benefit in stroke; at high acute doses, or
after relatively short term treatments, they would reduce post-stroke 5-HT hypermetabolism,
trigger anti-thrombotic, vasodilator and anti-inflammatory mechanisms, while on the long
term, SSRIs would rather desensitize autoreceptor-mediated feedback mechanisms and
enhance 5-HT transmission, thereby favoring brain repair processes through neurotrophic
factors and cell proliferation. Clinical studies remain necessary to determine how various
c.
fluoxetine administration protocols differentially favor short and long term recovery
mechanisms in stroke patients, and to which extent any of these effects are based on either
neurovascular, neuroprotectant or antidepressant actions.
In
References
Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R. & Begley, D. J. (2010).
rs
Structure and function of the blood-brain barrier. Neurobiol. Dis. 37, 13-25.
Acheampong, P. & Ford, G. A. (2012). Pharmacokinetics of alteplase in the treatment of
ischaemic stroke. Expert Opin. Drug Metab. Toxicol. 8, 271-281.
Auerbach, S. B., Lundberg, J. F. & Hjorth, S. (1995). Differential inhibition of serotonin
he
release by 5-HT and NA reuptake blockers after systemic administration.
Neuropharmacology, 34, 89-96.
Béjot, Y., Prigent-Tessier, A., Cachia, C., Giroud, M., Mossiat, C., Bertrand, N., … Marie, C.
(2011). Time-dependent contribution of non neuronal cells to BDNF production after
ischemic stroke in rats. Neurochem. Int. 58, 102-111.
is
Blier, P. & de Montigny, C. (1994). Current advances and trends in the treatment of
depression. Trends Pharmacol. Sci. 15, 220-226.
Boldrini, M., Hen, R., Underwood, M. D., Rosoklija, G. B., Dwork, A. J., Mann, J. J. &
bl
Arango, V. (2012). Hippocampal angiogenesis and progenitor cell proliferation are
increased with antidepressant use in major depression. Biol. Psychiatry, 72, 562-571.
Bonvento, G., Borredon, J., Seylaz, J. & Lacombe, P. (1997). Cerebrovascular consequences
of altering serotonergic transmission in conscious rat. Brain Res. 767, 208-213.
Pu
Bonvento, G., MacKenzie, E. T. & Edvinsson, L. (1991). Serotonergic innervation of the

cerebral vasculature: relevance to migraine and ischaemia. Brain Res. Brain Res. Rev. 16,
257-263.
Brust, P., Friedrich, A., Krizbai, I. A., Bergmann, R., Roux, F., Ganapathy, V. & Johannsen,
B. (2000). Functional expression of the serotonin transporter in immortalized rat brain
microvessel endothelial cells. J. Neurochem. 74, 1241-1248.
a
Campbell Burton, C. A., Holmes, J., Murray, J., Gillespie, D., Lightbody, C. E., Watkins, C.
L. & Knapp, P. (2011). Interventions for treating anxiety after stroke. Cochrane
Database Syst. Rev. CD008860.
ov
Carota, A., Berney, A., Aybek, S., Iaria, G., Staub, F., Ghika-Schmid, F., … Bogousslavsky,
J. (2005). A prospective study of predictors of poststroke depression. Neurology, 64, 428-
433.
Carson, A. J., MacHale, S., Allen, K., Lawrie, S. M., Dennis, M., House, A. & Sharpe, M.
(2000). Depression after stroke and lesion location: a systematic review. Lancet, 356,
N
122-126.
Chaput, Y., Blier, P. & de Montigny, C. (1986). In vivo electrophysiological evidence for the
regulatory role of autoreceptors on serotonergic terminals. J. Neurosci. 6, 2796-2801.
Chaput, Y., de Montigny, C. & Blier, P. (1991). Presynaptic and postsynaptic modifications
of the serotonin system by long-term administration of antidepressant treatments. An in
vivo electrophysiologic study in the rat. Neuropsychopharmacol. 5, 219-229.
c.
Chen, M.-F., Huang, Y.-C., Long, C., Yang, H.-I., Lee, H.-C., Chen, P.-Y., … Lee, T. J.-F.,
(2012). Bimodal effects of fluoxetine on cerebral nitrergic neurogenic vasodilation in
porcine large cerebral arteries. Neuropharmacology, 62, 1651-1658.
Chollet, F., Tardy, J., Albucher, J.-F., Thalamas, C., Berard, E., Lamy, C., … Loubinoux, I.
In
(2011). Fluoxetine for motor recovery after acute ischaemic stroke (FLAME): a
randomised placebo-controlled trial. Lancet Neurol. 10, 123-130.
Cipolla, M. J., Chan, S.-L., Sweet, J., Tavares, M. J., Gokina, N. & Brayden, J. E. (2014).
Postischemic reperfusion causes smooth muscle calcium sensitization and
rs
vasoconstriction of parenchymal arterioles. Stroke 45, 2425-2430.
Cowen, P. & Sherwood, A. C. (2013). The role of serotonin in cognitive function: evidence
from recent studies and implications for understanding depression. J. Psychopharmacol.
27, 575-583.
he
Cramer, S. C. (2011). Listening to fluoxetine: a hot message from the FLAME trial of
poststroke motor recovery. Int. J. Stroke, 6, 315-316.
De Abajo, F. J. (2011). Effects of selective serotonin reuptake inhibitors on platelet function:
mechanisms, clinical outcomes and implications for use in elderly patients. Drugs Aging
28, 345-367.
is
Deplanque, D., Venna, V. R. & Bordet, R. (2011). Brain ischemia changes the long term
response to antidepressant drugs in mice. Behav. Brain Res. 219, 367-372.
Dhami, K. S., Churchward, M. A., Baker, G. B. & Todd, K. G. (2013). Fluoxetine and
bl
citalopram decrease microglial release of glutamate and D-serine to promote cortical
neuronal viability following ischemic insult. Mol. Cell. Neurosci. 56, 365‑374.
Dieguez, S., Staub, F., Bruggimann, L. & Bogousslavsky, J. (2004). Is poststroke depression
a vascular depression? J. Neurol. Sci. 226, 53-58.
Pu
Diener, H. C., Lees, K. R., Lyden, P., Grotta, J., Davalos, A., Davis, S. M., … SAINT I and II
Investigators., (2008). NXY-059 for the treatment of acute stroke: pooled analysis of the
SAINT I and II Trials. Stroke 39, 1751-1758.
Doyle, K. P., Simon, R. P. & Stenzel-Poore, M. P. (2008). Mechanisms of ischemic brain
damage. Neuropharmacology, 55, 310-318.
Drago, A., Crisafulli, C., Sidoti, A. & Serretti, A. (2011). The molecular interaction between
the glutamatergic, noradrenergic, dopaminergic and serotoninergic systems informs a
a
detailed genetic perspective on depressive phenotypes. Prog. Neurobiol. 94, 418-460.

Enlimomab Acute Stroke Trial Investigators (2001). Use of anti-ICAM-1 therapy in ischemic
ov
stroke: results of the Enlimomab Acute Stroke Trial. Neurology, 57, 1428-1434.
Espinera, A. R., Ogle, M. E., Gu, X. & Wei, L. (2013). Citalopram enhances neurovascular
regeneration and sensorimotor functional recovery after ischemic stroke in mice.
Folstein, M. F., Maiberger, R. & McHugh, P. R. (1977). Mood disorder as a specific
N
complication of stroke. J. Neurol. Neurosurg. Psychiatry, 40, 1018-1020.

Gainotti, G., Azzoni, A., Gasparini, F., Marra, C. & Razzano, C. (1997). Relation of lesion
location to verbal and nonverbal mood measures in stroke patients. Stroke 28, 2145-2149.
Geiser, F., Conrad, R., Imbierowicz, K., Meier, C., Liedtke, R., Klingmüller, D., …
Harbrecht, U. (2011). Coagulation activation and fibrinolysis impairment are reduced in
patients with anxiety and depression when medicated with serotonergic antidepressants.
c.
Psychiatry Clin. Neurosci. 65, 518-525.
Goldstein, L. B., Bushnell, C. D., Adams, R. J., Appel, L. J., Braun, L. T., Chaturvedi, S., …
Council on Peripheral Vascular Disease, and Interdisciplinary Council on Quality of Care
In
and Outcomes Research. (2011). Guidelines for the primary prevention of stroke: a
guideline for healthcare professionals from the American Heart Association/American
Stroke Association. Stroke 42, 517-584.
Gould, E. (2007). How widespread is adult neurogenesis in mammals? Nat. Rev. Neurosci. 8,
481‑488.
rs
Grabiec, M., Turlejski, K. & Djavadian, R. L. (2009). The partial 5-HT1A receptor agonist
buspirone enhances neurogenesis in the opossum (Monodelphis domestica). Eur.
Grome, J. J. & Harper, A. M. (1983). The effects of serotonin on local cerebral blood flow. J.
he
Cereb. Blood Flow Metab. 3, 71-77.
Hackam, D. G. & Mrkobrada, M. (2012). Selective serotonin reuptake inhibitors and brain
hemorrhage: a meta-analysis. Neurology, 79, 1862-1865.
Hackett, M. L., Köhler, S., O‘Brien, J. T. & Mead, G. E. (2014). Neuropsychiatric outcomes
of stroke. Lancet Neurol. 13, 525-534.
is
Haddad, M., Beray-Berthat, V., Coqueran, B., Plotkine, M., Marchand-Leroux, C. &
Margaill, I. (2013). Combined therapy with PJ34, a poly(ADP-ribose)polymerase
inhibitor, reduces tissue plasminogen activator-induced hemorrhagic transformations in
bl
cerebral ischemia in mice. Fundam. Clin. Pharmacol. 27, 393-401.
Haddad, M., Rhinn, H., Bloquel, C., Coqueran, B., Szabó, C., Plotkine, M., … Margaill, I.,
(2006). Anti-inflammatory effects of PJ34, a poly(ADP-ribose) polymerase inhibitor, in
transient focal cerebral ischemia in mice. Br. J. Pharmacol. 149, 23-30.
Pu
Hawkins, B. T. & Davis, T. P. (2005). The blood-brain barrier/neurovascular unit in health

and disease. Pharmacol. Rev. 57, 173-185.
Hergovich, N., Aigner, M., Eichler, H. G., Entlicher, J., Drucker, C. & Jilma, B. (2000).
Paroxetine decreases platelet serotonin storage and platelet function in human beings.
Clin. Pharmacol. Ther. 68, 435-442.
Hervás, I., & Artigas, F., (1998). Effect of fluoxetine on extracellular 5-hydroxytryptamine in
rat brain. Role of 5-HT autoreceptors. Eur. J. Pharmacol. 358, 9-18.
a
Hwang, J. J., Park, M.-H., Choi, S. Y. & Koh, J. Y. (2005). Activation of the Trk signaling
pathway by extracellular zinc. Role of metalloproteinases. J. Biol. Chem. 280, 11995-
ov
12001.
IST-3 collaborative group, Sandercock, P., Wardlaw, J. M., Lindley, R. I., Dennis, M.,
Cohen, G., … Arauz, A. (2012). The benefits and harms of intravenous thrombolysis
with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the
third international stroke trial [IST-3]): a randomised controlled trial. Lancet, 379, 2352-
N
2363.
Jin, R., Yang, G. & Li, G. (2010). Molecular insights and therapeutic targets for blood-brain
barrier disruption in ischemic stroke: critical role of matrix metalloproteinases and tissue-
type plasminogen activator. Neurobiol. Dis. 38, 376-385.
Ji, X.-W., Wu, C. L., Wang, X. C., Liu, J., Bi, J. Z. & Wang, D.-Y, (2014). Monoamine
neurotransmitters and fibroblast growth factor-2 in the brains of rats with post-stroke
c.
depression. Exp. Ther. Med. 8, 159-164.
Jolkkonen, J., Puurunen, K., Rantakömi, S., Sirviö, J., Haapalinna, A. & Sivenius, J. (2000).
Effects-of fluoxetine on sensorimotor and spatial learning deficits following focal
In
cerebral ischemia in rats. Restor. Neurol. Neurosci. 17, 211-216.
Jorge, R. E., Acion, L., Moser, D., Adams, H. P. & Robinson, R. G. (2010). Escitalopram and
enhancement of cognitive recovery following stroke. Arch. Gen. Psychiatry, 67, 187-196.
Kanthan, R., Shuaib, A., Griebel, R., el-Alazounni, H., Miyashita, H. & Kalra, J. (1996).
Evaluation of monoaminergic neurotransmitters in the acute focal ischemic human brain
rs
model by intracerebral in vivo microdialysis. Neurochem. Res. 21, 563-566.
Kim, D. H., Li, H., Yoo, K.-Y., Lee, B.-H., Hwang, I. K. & Won, M. H. (2007). Effects of
fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the
gerbil hippocampal CA1 region induced by transient ischemia. Exp. Neurol. 204, 748-
he
758.
Kokaia, Z. & Lindvall, O. (2003). Neurogenesis after ischaemic brain insults. Curr. Opin.
Neurobiol. 13, 127‑132.
Kronenberg, G., Balkaya, M., Prinz, V., Gertz, K., Ji, S., Kirste, I., … Endres, M. (2012).
Exofocal dopaminergic degeneration as antidepressant target in mouse model of
is
poststroke depression. Biol. Psychiatry, 72, 273-281.
Kronenberg, G., Gertz, K., Heinz, A. & Endres, M. (2014). Of mice and men: modelling post-
stroke depression experimentally. Br. J. Pharmacol. 171, 4673-89.
bl
Lakhan, S. E., Kirchgessner, A. & Hofer, M. (2009). Inflammatory mechanisms in ischemic
stroke: therapeutic approaches. J. Transl. Med. 7, 97.
Lee, B. H. & Kim, Y. K. (2010). The roles of BDNF in the pathophysiology of major
depression and in antidepressant treatment. Psychiatry Investig. 7, 231-235.
Pu
Lee, C. H., Park, J. H., Yoo, K.-Y., Choi, J. H., Hwang, I. K., Ryu, P. D., … Won, M. H.,
(2011). Pre- and post-treatments with escitalopram protect against experimental ischemic
neuronal damage via regulation of BDNF expression and oxidative stress. Exp. Neurol.
229, 450-459.
Lee, J. Y., Lee, H. E., Kang, S. R., Choi, H. Y., Ryu, J. H. & Yune, T. Y. (2014). Fluoxetine
inhibits transient global ischemia-induced hippocampal neuronal death and memory
impairment by preventing blood-brain barrier disruption. Neuropharmacology, 79, 161-
a
171.
Lee, R., Kermani, P., Teng, K. K. & Hempstead, B. L. (2001). Regulation of cell survival by
ov
secreted proneurotrophins. Science, 294, 1945-1948.

Li, J., Zhao, Y. D., Zeng, J.-W., Chen, X. Y., Wang, R. D. & Cheng, S. Y. (2014). Serum
brain-derived neurotrophic factor levels in post-stroke depression. J. Affect. Disord. 168,
373-379.
Lim, C. M., Kim, S. W., Park, J. Y., Kim, C., Yoon, S. H. & Lee, J.-K. (2009). Fluoxetine
N
affords robust neuroprotection in the postischemic brain via its anti-inflammatory effect.
J. Neurosci. Res. 87, 1037-1045.
Li, N., Wallén, N. H., Ladjevardi, M. & Hjemdahl, P. (1997). Effects of serotonin on platelet
activation in whole blood. Blood Coagul. Fibrinolysis, 8, 517-523.
Liu, S., Agalliu, D., Yu, C. & Fisher, M. (2012). The role of pericytes in blood-brain barrier
function and stroke. Curr. Pharm. Des. 18, 3653-3662.
Li, W. L., Cai, H. H., Wang, B., Chen, L., Zhou, Q. G., Luo, C.-X., … Zhu, D. Y. (2009).
c.
Chronic fluoxetine treatment improves ischemia-induced spatial cognitive deficits
through increasing hippocampal neurogenesis after stroke. J. Neurosci. Res. 87, 112-122.
Malberg, J. E. (2004). Implications of adult hippocampal neurogenesis in antidepressant
In
action. J. Psychiatry Neurosci. 29, 196-205.
Marco, E. J., Moreno, M. J. & de Pablo, A. L. (1999). Local treatments of dorsal raphe
nucleus induce changes in serotonergic activity in rat major cerebral arteries. Stroke 30,
1695-1701.
Martin, C. B. P., Hamon, M., Lanfumey, L. & Mongeau, R. (2014). Controversies on the role
rs
of 5-HT(2C) receptors in the mechanisms of action of antidepressant drugs. Neurosci.
Biobehav. Rev. 42, 208-223.
Mathers, C. D., Boerma, T. & Ma Fat, D. (2009). Global and regional causes of death. Br.
Med. Bull. 92, 7-32.
he
Maya Vetencourt, J. F., Sale, A., Viegi, A., Baroncelli, L., De Pasquale, R., O‘Leary, O. F.,
… Maffei, L. (2008). The antidepressant fluoxetine restores plasticity in the adult visual
cortex. Science, 320, 385-388.
May, M., McCarron, P., Stansfeld, S., Ben-Shlomo, Y., Gallacher, J., Yarnell, J., … Ebrahim,
S. (2002). Does psychological distress predict the risk of ischemic stroke and transient
is
ischemic attack? The Caerphilly Study. Stroke 33, 7-12.
McKinnon, M. C., Yucel, K., Nazarov, A. & MacQueen, G. M. (2009). A meta-analysis
examining clinical predictors of hippocampal volume in patients with major depressive
bl
disorder. J. Psychiatry Neurosci. 34, 41-54.
Mead, G. E., Hsieh, C.-F., Lee, R., Kutlubaev, M. A., Claxton, A., Hankey, G. J. & Hackett,
M. L. (2012). Selective serotonin reuptake inhibitors (SSRIs) for stroke recovery.
Cochrane Database Syst. Rev. 11, CD009286.
Pu
Mongeau, R., Martin, C. B. P., Chevarin, C., Maldonado, R., Hamon, M., Robledo, P. &
Lanfumey, L. (2010). 5-HT2C receptor activation prevents stress-induced enhancement
of brain 5-HT turnover and extracellular levels in the mouse brain: modulation by chronic
paroxetine treatment. J. Neurochem. 115, 438-449.
Muir, K. W. (2006). Glutamate-based therapeutic approaches: clinical trials with NMDA
antagonists. Curr. Opin. Pharmacol. 6, 53-60.
Nagao, T., Ibayashi, S., Sadoshima, S., Izumi, J. & Fujishima, M. (1995). Citalopram, a
a
serotonin reuptake inhibitor, and brain ischemia in SHR. Brain Res. Bull., 38, 49-52.
Najjar, S., Pearlman, D. M., Devinsky, O., Najjar, A. & Zagzag, D. (2013). Neurovascular
ov
unit dysfunction with blood-brain barrier hyperpermeability contributes to major

depressive disorder: a review of clinical and experimental evidence. J.
Neuroinflammation, 10, 142.
Nakata, N., Suda, H., Izumi, J., Tanaka, Y., Ikeda, Y., Kato, H., … Kogure, K. (1997). Role
of hippocampal serotonergic neurons in ischemic neuronal death. Behav. Brain Res. 83,
N
217‑220.
Narushima, K., Kosier, J. T. & Robinson, R. G. (2003). A reappraisal of poststroke

depression, intra- and inter-hemispheric lesion location using meta-analysis. J.
Neuropsychiatry Clin. Neurosci. 15, 422-430.
Newman, M. E., Shalom, G., Ran, A., Gur, E. & Van de Kar, L. D. (2004). Chronic
fluoxetine-induced desensitization of 5-HT1A and 5-HT1B autoreceptors: regional
c.
differences and effects of WAY-100635. Eur. J. Pharmacol. 486, 25-30.
Ofek, K., Schoknecht, K., Melamed-Book, N., Heinemann, U., Friedman, A. & Soreq, H.
(2012). Fluoxetine induces vasodilatation of cerebral arterioles by co-modulating
In
NO/muscarinic signalling. J. Cell. Mol. Med. 16, 2736-2744.
Ohira, K., Takeuchi, R., Shoji, H. & Miyakawa, T. (2013). Fluoxetine-induced cortical adult
neurogenesis. Neuropsychopharmacol. 38, 909-920.
Ohira, T., Iso, H., Satoh, S., Sankai, T., Tanigawa, T., Ogawa, Y., … Shimamoto, T. (2001).
Prospective study of depressive symptoms and risk of stroke among japanese. Stroke 32,
rs
903-908.
Paizanis, E., Kelaï, S., Renoir, T., Hamon, M. & Lanfumey, L. (2007). Life-long hippocampal
neurogenesis: environmental, pharmacological and neurochemical modulations.
Neurochem. Res. 32, 1762-1771.
he
Pariente, J., Loubinoux, I., Carel, C., Albucher, J. F., Leger, A., Manelfe, C., … Chollet, F.
(2001). Fluoxetine modulates motor performance and cerebral activation of patients
recovering from stroke. Ann. Neurol. 50, 718-729.
Perrenoud, Q., Rossier, J., Férézou, I., Geoffroy, H., Gallopin, T., Vitalis, T. & Rancillac, A.
(2012). Activation of cortical 5-HT(3) receptor-expressing interneurons induces NO
is
mediated vasodilatations and NPY mediated vasoconstrictions. Front. Neural Circuits, 6,
50.
Pradeep, H., Diya, J. B., Shashikumar, S. & Rajanikant, G. K. (2012). Oxidative stress--
bl
assassin behind the ischemic stroke. Folia Neuropathol. 50, 219-230.
Prodan, C. I., Joseph, P. M., Vincent, A. S. & Dale, G. L. (2007). Coated-platelet levels are
influenced by smoking, aspirin, and selective serotonin reuptake inhibitors. J. Thromb.
Haemost. 5, 2149-2151.
Pu
Raber, J., Fan, Y., Matsumori, Y., Liu, Z., Weinstein, P. R., Fike, J. R. & Liu, J. (2004).
Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Ann.
Neurol. 55, 381-389.
Ronaldson, P. T. & Davis, T. P. (2012). Blood-brain barrier integrity and glial support:
mechanisms that can be targeted for novel therapeutic approaches in stroke. Curr. Pharm.
Des. 18, 3624-3644.
Salazar-Colocho, P., Del Río, J. & Frechilla, D. (2007). Serotonin 5-HT1A receptor activation
a
prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia. J.

Physiol. Biochem. 63, 203-211.
ov
Sandoval, K. E. & Witt, K. A. (2008). Blood-brain barrier tight junction permeability and
ischemic stroke. Neurobiol. Dis. 32, 200-219.
Saver, J. L. (2006). Time is brain--quantified. Stroke 37, 263-266.
Semkova, I., Wolz, P. & Krieglstein, J. (1998). Neuroprotective effect of 5-HT1A receptor
agonist, Bay X 3702, demonstrated in vitro and in vivo. Eur. J. Pharmacol. 359, 251-260.
N
Sharma, H. S., Patnaik, R., Patnaik, S., Mohanty, S., Sharma, A. & Vannemreddy, P. (2007).
Antibodies to serotonin attenuate closed head injury induced blood brain barrier
disruption and brain pathology. Ann. N. Y. Acad. Sci. 1122, 295-312.
Shibata, S., Kagami-Ishi, Y., Tominaga, K., Kodama, K., Ueki, S. & Watanabe, S. (1992).
Ischemia-induced impairment of 2-deoxyglucose uptake and CA1 field potentials in rat
hippocampal slices: protection by 5-HT1A receptor agonists and 5-HT2 receptor
antagonists. Eur. J. Pharmacol. 229, 21-29.
Soares, B. P., Tong, E., Hom, J., Cheng, S.-C., Bredno, J., Boussel, L., … Wintermark, M.
c.
(2010). Reperfusion is a more accurate predictor of follow-up infarct volume than
recanalization: a proof of concept using CT in acute ischemic stroke patients. Stroke 41,
34‑40.
In
Solai, L. K., Mulsant, B. H. & Pollock, B. G. (2001). Selective serotonin reuptake inhibitors
for late-life depression: a comparative review. Drugs Aging, 18, 355-368.
Srivastava, K., Bath, P. M. W. & Bayraktutan, U. (2012). Current therapeutic strategies to
mitigate the eNOS dysfunction in ischaemic stroke. Cell. Mol. Neurobiol. 32, 319-336.
Sun, F., Wang, X., Mao, X., Xie, L. & Jin, K. (2012). Ablation of neurogenesis attenuates
rs
recovery of motor function after focal cerebral ischemia in middle-aged mice. PloS One,
7, e46326.
Szabó, C. & Dawson, V. L. (1998). Role of poly(ADP-ribose) synthetase in inflammation and
ischaemia-reperfusion. Trends Pharmacol. Sci. 19, 287-298.
he
Taylor, W. D., Aizenstein, H. J. & Alexopoulos, G. S. (2013). The vascular depression
hypothesis: mechanisms linking vascular disease with depression. Mol. Psychiatry, 18,
963-974.
Terroni, L., Amaro, E., Iosifescu, D. V., Tinone, G., Sato, J. R., Leite, C. C., … Fráguas, R.
(2011). Stroke lesion in cortical neural circuits and post-stroke incidence of major
is
depressive episode: a 4-month prospective study. World J. Biol. Psychiatry, 12, 539-548.
Thored, P., Wood, J., Arvidsson, A., Cammenga, J., Kokaia, Z. & Lindvall, O. (2007). Long-
term neuroblast migration along blood vessels in an area with transient angiogenesis and
bl
increased vascularization after stroke. Stroke 38, 3032-3039.
Torup, L. & Diemer, N. H. (2000). Is the 5-HT2C receptor a therapeutic target in cerebral
ischaemia? Pharmacol. Toxicol. 87, 74-78.
Tupler, L. A., Krishnan, K. R. R., McDonald, W. M., Dombeck, C. B., D‘Souza, S. &
Pu
Steffens, D. C. (2002). Anatomic location and laterality of MRI signal hyperintensities in

late-life depression. J. Psychosom. Res. 53, 665-676.
Turner, R. C., Dodson, S. C., Rosen, C. L. & Huber, J. D. (2013). The science of cerebral
ischemia and the quest for neuroprotection: navigating past failure to future success. J.
Neurosurg. 118, 1072-1085.
Ungvari, Z., Pacher, P., Kecskeméti, V. & Koller, A. (1999). Fluoxetine dilates isolated small
cerebral arteries of rats and attenuates constrictions to serotonin, norepinephrine, and a
a
voltage-dependent Ca(2+) channel opener. Stroke 30, 1949-1954.

Von Kummer, R. & Hacke, W. (1992). Safety and efficacy of intravenous tissue plasminogen
ov
activator and heparin in acute middle cerebral artery stroke. Stroke. 23, 646-652.
Wakayama, K., Ohtsuki, S., Takanaga, H., Hosoya, K. & Terasaki, T. (2002). Localization of
norepinephrine and serotonin transporter in mouse brain capillary endothelial cells.
Neurosci. Res. 44, 173-180.
Wang, S. H., Zhang, Z., Guo, Y. J., Zhou, H., Teng, G. J. & Chen, B. A. (2009). Anhedonia
N
and activity deficits in rats: impact of post-stroke depression. J. Psychopharmacol. 23,

295-304.
Wang, S. H., Zhang, Z. J., Guo, Y. J., Sui, Y. X. & Sun, Y. (2010). Notch1 signaling related
hippocampal neurogenesis in adult poststroke depression rats: a valid index for an
efficient combined citalopram and WAY100635 pharmacotherapy. Behav. Pharmacol.
21, 47-57.
Wang, S., Zhang, Z., Guo, Y., Sui, Y. & Sun, Y. (2010). Involvement of serotonin
c.
neurotransmission in hippocampal neurogenesis and behavioral responses in a rat model
of post-stroke depression. Pharmacol. Biochem. Behav. 95, 129-137.
Wang, S., Zhang, Z., Guo, Y., Teng, G. & Chen, B. (2008). Hippocampal neurogenesis and
In
behavioural studies on adult ischemic rat response to chronic mild stress. Behav. Brain
Res. 189, 9-16.
Wang, S., Zhang, Z., Guo, Y., Teng, G. & Chen, B. (2009). Decreased expression of
serotonin 1A receptor in the dentate gyrus in association with chronic mild stress: a rat
model of post-stroke depression. Psychiatry Res. 170, 245-251.
Weisstaub, N. V., Zhou, M., Lira, A., Lambe, E., González-Maeso, J., Hornung, J.-P., …
rs
Gingrich, J. A. (2006). Cortical 5-HT2A receptor signaling modulates anxiety-like
behaviors in mice. Science, 313, 536-540.
Wiltrout, C., Lang, B., Yan, Y., Dempsey, R. J. & Vemuganti, R. (2007). Repairing brain
he
after stroke: a review on post-ischemic neurogenesis. Neurochem. Int., 50, 1028-1041.
Windle, V. & Corbett, D. (2005). Fluoxetine and recovery of motor function after focal
ischemia in rats. Brain Res. 1044, 25-32.
Winkelheide, U., Engelhard, K., Kaeppel, B., Winkler, J., Hutzler, P., Werner, C. & Kochs,
E. (2008). Cerebral ischemia and neurogenesis: a two-time comparison. Neurocrit. Care
is
9, 387-393.
Yenari, M. A., Xu, L., Tang, X. N., Qiao, Y. & Giffard, R. G. (2006). Microglia potentiate
damage to blood-brain barrier constituents: improvement by minocycline in vivo and in
bl
vitro. Stroke 37, 1087-1093.
Yoshida, T., Ishikawa, M., Niitsu, T., Nakazato, M., Watanabe, H., Shiraishi, T., …
Hashimoto, K. (2012). Decreased serum levels of mature brain-derived neurotrophic
factor (BDNF), but not its precursor proBDNF, in patients with major depressive
Pu
disorder. PloS One, 7, e42676.

Zafar, M. U., Paz-Yepes, M., Shimbo, D., Vilahur, G., Burg, M. M., Chaplin, W., …
Badimon, J. J. (2010). Anxiety is a better predictor of platelet reactivity in coronary
artery disease patients than depression. Eur. Heart J. 31, 1573-1582.
Zhang, J., Yang, J., Zhang, C., Jiang, X., Zhou, H. & Liu, M. (2012). Calcium antagonists for
acute ischemic stroke. Cochrane Database Syst. Rev. 5, CD001928.
Zhao, C. S., Puurunen, K., Schallert, T., Sivenius, J. & Jolkkonen, J. (2005). Behavioral and
a
histological effects of chronic antipsychotic and antidepressant drug treatment in aged

rats with focal ischemic brain injury. Behav. Brain Res. 158, 211-220.
ov
N
c.
In
Index
rs
adverse effects, 98, 218, 227, 242, 263, 285
# adverse event, 283
aetiology, 5, 7, 9
20th century, 169
affective disorder, 19, 37, 41, 72, 107, 139, 165, 171,
A 184, 193, 228, 253, 259, 289, 296
he
afferent nerve, 235
abuse, 95, 158, 217, 218, 219, 222, 223, 224, 301, aggression, 22, 26, 27, 30, 31, 39, 51, 103
306 aggressive behavior, 25, 26, 30, 31, 32, 40, 299
access, 27, 28, 36, 127, 195, 233 aggressiveness, 29, 34, 35, 141, 305
accessibility, 127 agonist, 15, 18, 40, 56, 89, 172, 174, 175, 181, 182,
accounting, 148, 191, 317 283, 285, 287, 288, 289, 290, 302, 319, 323, 326
is
acetylation, 83, 84, 125, 128, 133, 134, 140, 141, 144 akathisia, 12, 19, 284, 287, 288, 290, 291
acetylcholine, 18, 283, 316 alanine, 182, 202
acid, 40, 41, 89, 112, 114, 157, 170, 176, 180, 192, alcohol consumption, 309
bl
193, 199, 200, 204, 234, 250, 251, 259, 263, 274, alcohol withdrawal, 301
277, 283 alcoholism, 18
ACTH, 46, 47, 48, 68, 229, 230 algorithm, 289
action potential, 318 allele, 107, 109, 116, 145, 154, 161
Pu
activation state, 127 Allopregnanolone, 26, 27, 28, 29, 31, 33, 39, 41, 42
acute stress, 187, 196, 197, 198, 203, 205, 283 ALT, 246
adaptation(s), 86, 108, 135, 142, 202, 221, 224, 228, alteplase, 307, 314, 321
247, 267, 301 alters, 48, 60, 62, 67, 69, 70, 79, 97, 112, 130, 139,
adenosine, 88, 117, 118, 120, 140, 144, 150, 170, 140, 165, 166, 178, 199, 203, 219, 220, 224, 251,
171, 181, 305 263, 272
adenovirus, 157 American Heart Association, 323
ADHD, 174, 208, 217, 218, 220, 221, 223, 224 amine(s), 176, 203, 247
a
adhesion, 61, 81, 250, 261, 262, 263, 311, 312, 313, amino, 45, 56, 109, 114, 119, 170, 189, 192, 193,
316 199, 200, 202, 234, 250, 280, 312
adolescents, 208, 285 amino acid(s), 45, 109, 119, 189, 199, 200, 202, 280,
ov
ADP, 313, 315, 323, 327 312

adrenal gland(s), 257, 285 AMPA receptors, 193, 195, 205, 250
adrenaline, 315 amphetamines, 223
adrenoceptors, 235 amplitude, 196
adrenocorticotropic hormone, 229 amygdala, 29, 30, 31, 37, 39, 41, 62, 90, 108, 135,
Adult neurogenesis, 62, 97 141, 152, 189, 190, 196, 203, 223, 230, 232, 250,
N
adulthood, 46, 48, 49, 50, 55, 79, 158, 209, 214, 216, 251, 252, 265, 273, 276
218, 224, 228, 249 Amygdala, 144
adults, 114, 137, 218, 262, 274, 284, 288, 289, 308 analgesic, vii, 1
330 Index
anatomy, 296 atrial fibrillation, 308

androgen(s), 51, 54, 56, 60, 61, 68, 69, 131, 252, 268 atrophy, 34, 42, 72, 84, 108, 114, 136, 140, 189, 190,
anesthetics, 41 192, 247, 298
angiogenesis, 258, 309, 314, 320, 321, 327 attachment, 192, 198
annotation, 139, 142 autism, 44, 61, 63, 119
anorexia, 235, 236, 238 autoantibodies, 261
c.
ANOVA, 11, 12 aversion, 9, 52, 218
antagonism, 209 avoidance, 7, 19, 21, 78, 130, 218
anterior cingulate cortex, 82, 178, 199, 275 avoidance behavior, 130
antibody, 76, 310 axons, 109
In
anticholinergic, 283
anticholinergic effect, 283
B
anticonvulsant, 29, 38
bacteria, 145
Antidepressant drugs, 92, 259, 307
barriers, 311, 314
antidepressant medication, vii, 1, 2, 4, 41, 44, 45, 49,
basal forebrain, 73, 78, 90
59, 60, 86, 93, 120, 123, 168, 170, 197, 227, 250,
rs
basal ganglia, 219, 221, 222
264, 292, 320
basement membrane, 310, 311
Antidepressants, v, vi, 71, 81, 82, 87, 104, 135, 143,
BBB, 310, 313, 316, 317, 320
145, 147, 190, 196, 207, 233, 244, 256, 258, 260,
behavioral change, 218, 236, 237, 305
264, 265, 267, 271, 274
he
behavioral genetics, 306
antigen, 235
behavioral manifestations, 212
antioxidant, 243, 244, 246, 260, 261, 265, 266, 277,
behavioral models, 93, 249, 274
313
behavioral sensitization, 300
antipsychotic, 105, 155, 168, 174, 181, 183, 271,
Behavioral tests, 295
282, 283, 284, 285, 290, 328
behavior(s), 27, 31, 42, 45, 49, 51, 52, 54, 56, 59, 60,
antipsychotic drugs, 155, 183, 283, 285
is
66, 67, 70, 91, 94, 130, 131, 135, 136, 166, 197,
antisense, 95, 112, 123, 157
204, 218, 298, 299, 300, 302, 305, 306, 317, 319,
antisense oligonucleotides, 112, 157
320, 328
antisense RNA, 95, 123
Belgium, 43
antitumor, 263
bl
beneficial effect, 84, 136, 237, 242, 244, 279, 284,
anxiety, v, 1, 3, 9, 18, 20, 60, 181, 184, 199, 295,
286, 287, 307, 308, 309, 319
296, 298, 299, 306, 328
benefits, 60, 172, 238, 288, 308, 315, 323
anxiety disorder, vii, viii, 1, 3, 16, 25, 26, 27, 29, 35,
benzodiazepine, viii, 25, 29, 31, 35, 36, 40, 41, 302
38, 39, 60, 97, 187, 188, 189, 190, 191, 197, 200,
Pu
binding globulin, 48, 49, 54, 61, 67

246, 257
bioavailability, 195, 217
apoptosis, 65, 74, 78, 80, 89, 95, 110, 111, 129, 139,
biological markers, 182
141, 246, 250, 253, 254, 255, 261, 267, 268, 269
biological psychiatry, 296, 304
arginine, 47, 128, 229, 269
biological systems, 266
aripiprazole, 181, 284, 288, 290
biomarkers, 119, 140, 159, 164, 244, 296, 297
arteries, 311
biosynthesis, viii, 25, 26, 29, 31, 32, 33, 34, 35, 36,
arterioles, 315, 322, 326
37, 38, 40, 129, 232, 313
arteriosclerosis, 315
a
bipolar disorder, 93, 119, 131, 133, 141, 201, 231,

artery, 104, 312, 314, 327
245, 253, 266, 300, 304, 305
arthritis, 236
Blood brain barrier, 307
ov
aspartate, 109, 112, 116, 155, 168, 169, 170, 192,

blood flow, 253, 307, 310, 311, 312
193, 202, 204, 250, 280, 312
blood monocytes, 131, 277
assessment, 2, 19, 291
blood vessels, 310, 311, 314, 327
astrocytes, 9, 73, 80, 85, 106, 133, 153, 250, 310,
blood-brain barrier, 321, 323, 324, 325, 328
312, 314
body weight, 228, 236, 238
astrocytoma, 137
N
bone marrow, 237, 238

asymptomatic, 309
brain abnormalities, 202
atherosclerosis, 312
brain damage, 310, 322
ATP, 166, 253
Brain derived neurotrophic factor (BDNF), 121, 161
Index 331
brain functioning, 148, 237 cerebellum, 26, 29, 96, 108, 112, 247, 252
brain functions, 147, 205, 296 cerebral arteries, 310, 315, 322, 325, 327
brain structure, 62, 66, 81, 114, 255 cerebral blood flow, 310, 312, 323
brain tumor, 165, 195 Cerebral blood flow, 307
brainstem, 19, 138 cerebral cortex, 4, 81, 85, 104, 106, 175, 194, 199,
branching, 113, 250, 251, 252 257
c.
Brazil, 167 Cerebral ischemia, 307, 317, 328
breast cancer, 270 cerebrospinal fluid, 9, 22, 26, 41, 42, 188, 189, 202,
breast milk, 44, 59 229, 259
breastfeeding, 63 chemical, 2, 111, 233
In
breeding, 9, 21 chemokines, 234, 235, 313, 317, 320
bromine, 222 Chicago, 25, 160, 207
childhood, 84, 145, 158, 223, 247
C children, 45, 48, 51, 66, 67, 208, 224
cholesterol, 28
Ca2+, 75, 82, 114, 115, 119, 128, 192, 200, 205, 280,
choline, 86, 94
rs
288, 311, 312, 315
chorea, 286, 287
caffeine, 38, 302, 304
choriocarcinoma, 157
calcium, 94, 112, 144, 153, 158, 170, 183, 192, 194,
choroid, 3
195, 196, 241, 242, 262, 315, 322
chromosome, 157
he
cancer, 144, 157, 161, 240, 242, 259, 280
Chronic mild stress (CMS), 276
cancer progression, 242
chronic obstructive pulmonary disease, 235
capillary, 311, 327
Chronic treatment, 80, 95, 105, 123
carbonyl groups, 246
CIA, 236
carcinoma, 270
cingulate region, 189
cardiovascular disease, 280, 286, 287, 289, 291, 308
circadian rhythm(s), 8, 20, 257, 298
is
cardiovascular function, 285
circulation, 47, 307
cascades, 73, 74, 75, 79, 82, 93, 94, 190, 250, 315,
classes, 6, 20, 72, 80, 117, 128, 168, 170, 191, 192,
319
193, 194, 208, 232, 295
caspases, 255
cleavage, 73, 74, 89, 109, 110, 149, 158, 249, 251
bl
catalysis, 161
Clinical, vi, 26, 44, 48, 69, 106, 107, 119, 159, 160,
catecholamines, 4, 46, 169, 174
167, 176, 185, 188, 207, 218, 250, 257, 258, 270,
causal relationship, 297, 320
279, 292, 305, 314, 315, 320, 321
CBP, 84, 86, 128, 141
clinical application, viii, 66, 103, 144
Pu
cDNA, 256
clinical depression, 5, 235, 300, 302
cell biology, 145
clinical diagnosis, 245
cell body, 101
clinical symptoms, 179
cell culture, 195, 279, 312
clinical trials, 172, 285, 286, 320, 325
cell cycle, 151
clozapine, 172, 278
cell death, 49, 74, 100, 108, 111, 142, 243, 249, 255,
clustering, 259
266, 310, 312, 313, 316
clusters, 5, 113
cell differentiation, 79
CNS, 85, 98, 108, 181, 182, 221, 222, 234, 243, 270,
a
cell division, 45, 106, 127

287, 289, 290, 291, 292
cell fate, 108, 255
cocaine, 78, 86, 89, 90, 91, 92, 139, 142, 207, 209,
cell line(s), 80, 132, 137, 144, 154, 155, 164, 256,
ov
212, 213, 214, 216, 217, 218, 219, 220, 221, 222,
310
223, 224, 225, 272, 300, 301, 302, 304, 306
cell signaling, 8
coding, 73, 125, 129, 140, 148, 149
cell surface, 131, 251, 313
codon, 109, 116, 130
cellular energy, 255
coenzyme, 127
cellular immunity, 235
cognition, 45, 107, 145, 229, 247, 282, 283, 284,
N
cellular signaling pathway, 291

288, 309
central nervous system, 46, 93, 95, 98, 108, 117,
Cognition, 280, 281, 289
124, 144, 172, 176, 204, 240, 243, 254, 264, 279,
cognitive abilities, 44
280, 295, 297, 310, 312
332 Index
cognitive deficit(s), 42, 104, 131, 281, 289, 290, 308, cortical neurons, 76, 95, 162, 233, 316
319, 325 corticotropin, 47, 62, 138, 229, 257, 259, 263, 265,
cognitive function, 34, 71, 249, 285, 291, 318, 322 268, 269, 270, 272, 273, 275
cognitive impairment, 279, 281, 285, 290, 291, 310, cortisol, 46, 48, 61, 67, 228, 229, 230, 233, 266, 271,
318 273, 274, 302, 304
cognitive performance, 112 CpG sites, 126, 131
c.
cognitive process, 152 CPP, 217, 218
cognitive tasks, 44, 69 CRF, 199, 234, 266, 268, 269, 270, 272
collagen, 310, 311, 315 critical period, 54, 63
collateral, 195 CRP, 234
In
colon, 266 CSF, 26, 27, 31, 34, 229, 230, 257, 270
colon cancer, 266 culture, 85, 112, 113, 232, 240, 241, 264, 267
colorectal cancer, 260 CVD, 286, 287
combination therapy, 173, 178 cysteine, 70, 110
Combination therapy, 168 cytochrome(s), 243, 252, 253
combined effect, 168 cytokines, 107, 176, 181, 182, 227, 235, 237, 239,
rs
communication, 73 240, 242, 243, 257, 262, 264, 266, 268, 271, 275,
comorbidity, 61, 181, 223, 235, 302, 305 307, 310, 313, 317, 320
complementarity, 157 cytoplasm, 129, 149, 233
complexity, 7, 77, 94, 162, 257, 296, 303 cytosine, 126
he
compliance, 2, 4
complications, 10
D
composition, 31, 37, 40, 42, 103
damages, 308, 316, 318, 319
compounds, 1, 2, 3, 5, 10, 16, 125, 171, 179, 182,
dark matter, 164
183, 184, 197, 301, 316
deacetylation, 128
compulsive behavior, 216
is
deaths, 168, 308
computed tomography, 257
decay, 142, 149, 150
conception, 44
declarative memory, 145
conceptualization, 292
deficiency, 145, 169, 179, 243, 299
concordance, 276
bl
deficit, 26, 65, 122, 174, 191, 208, 219, 220, 224,
condensation, 127, 128
259, 309
conditioned response, 10
degradation, 129, 149, 170, 232, 241, 280
conditioning, 34, 35, 217
delirium, 283, 284, 287, 288, 289
configuration, 127, 133
Pu
Delta, 222
confounding variables, 158
dementia, 283, 292, 308, 309
connectivity, 72, 73, 84, 188, 247, 252
dendrites, 27, 30, 34, 77, 109, 113, 115, 116, 151,
consensus, 182, 209, 277
170, 190, 192, 195, 247, 250
consolidation, 107, 112, 141
dendritic arborization, 27, 28, 71, 191
consumption, 9, 248, 310, 317
dendritic spines, 113, 151, 190, 191, 247
control group, 152, 153
density values, 211, 212, 215
controversies, 104
depolarization, 112, 113, 175, 178, 194, 197, 199,
convergence, 30, 142, 203, 253, 272
a
203, 312
COPD, 235
Depression, v, vi, 1, 5, 18, 20, 44, 61, 62, 65, 69, 70,
coronary artery disease, 287, 328
72, 83, 87, 112, 113, 114, 119, 140, 147, 157,
ov
coronary heart disease, 291, 292

165, 168, 169, 173, 179, 204, 227, 228, 253, 258,
correlation, 26, 60, 113, 153, 154, 189, 214, 245,
262, 264, 268, 282, 291, 295, 298, 299, 304, 306,
253, 285, 305
309, 317, 321
correlations, 107, 153, 213
depressive symptoms, vii, 26, 31, 34, 64, 69, 169,
cortex, 26, 30, 34, 38, 46, 47, 77, 81, 82, 88, 91, 92,
171, 172, 174, 231, 240, 242, 243, 245, 253, 259,
97, 100, 101, 102, 103, 105, 108, 112, 131, 140,
N
282, 308, 317, 326

143, 153, 154, 161, 164, 173, 184,188, 189, 190,
deprivation, 22, 312
191, 193, 196, 205, 229, 237, 250, 253, 256, 258,
derivatives, 41
268, 272, 273, 308, 309, 310, 320, 325
desensitization, 17, 170, 222, 318, 326
Index 333
despair, 6, 20, 21, 22, 52, 183, 228, 252, 298 dysphoria, viii, 25, 26, 33, 35, 36
destruction, 129 dystonia, 283
detachment, 310
detectable, 151
E
detection, 6, 10, 12, 18, 88, 154, 305
eating disorders, 107, 119, 280
developing brain, 218, 219
c.
economic status, 44
Development, 17, 45, 63, 67, 256, 297, 304, 306
edema, 313
developmental change, 44
editors, 36, 268
deviation, 308
EEG, 298
dexamethasone suppression test, 230, 298
In
efflux transporters, 311
diabetes, 309
eicosapentaenoic acid, 239, 265
diamonds, 213
ejaculation, 55
diet, 14, 309
Electroconvulsive Therapy, 94
dimerization, 74, 255
electroencephalography, 298
direct action, 114
electron, 253, 254, 260
disability, 72, 91, 282, 291, 308, 309
rs
elongation, 45
diseases, 70, 103, 137, 148, 155, 262, 279, 280, 286,
embolus, 308, 312
290, 291, 309
embryonic stem cells, 201
dissociation, 75, 233
emotion, 45, 152, 309
distribution, 69, 84, 88, 122, 126, 211, 212, 231, 232,
he
emotional state, 79
233
emotional stimuli, 78, 85
diversity, 66
enantiomers, 3, 4, 23
DNA, 69, 83, 88, 90, 91, 94, 125, 126, 127, 128,
encoding, 30, 143, 193, 209, 250, 274
129, 130, 131, 133, 134, 137, 139, 141, 143, 144,
endocrine, 86, 117, 120, 176, 219, 234, 258
145, 151, 162, 233, 240, 242, 246, 262, 313, 317
endocrine system, 176
DNA damage, 313
is
endocrinology, 143
DNA methylation, 83, 88, 91, 94, 125, 126, 127,
endogenous depression, 64, 70
128, 129, 130, 131, 133, 134, 137, 139, 143, 144,
endophenotypes, 9, 228, 297, 298, 305
145
endorphins, 180
DNA repair, 313
bl
endothelial cells, 235, 310, 311, 312, 313, 314, 316,
DOI, 270
321, 327
dopamine, 62, 81, 85, 86, 92, 138, 144, 168, 169,
endothelial NO synthase, 311
172, 174, 175, 176, 179, 181, 182, 185, 207, 209,
endothelium, 311, 313, 316
216, 219, 221, 222, 223, 224, 237, 252, 283, 297,
Pu
energy, 124, 143, 168, 174, 246, 253, 254, 255, 256,
302, 304
262, 276, 282, 312
Dopamine, 37, 142, 178, 209
enlargement, 189, 246
dopaminergic, 37, 169, 170, 172, 175, 177, 179, 219,
entorhinal cortex, 118
284, 288, 317, 322, 324
environment, 10, 15, 42, 76, 79, 126, 167, 204, 217,
dorsolateral prefrontal cortex, 189, 202, 263
218, 264, 297, 299, 305
dosage, 2, 29, 172
environmental change, 247
down-regulation, 4, 40, 115, 130, 154
environmental factors, 83, 126, 127, 228
drug abuse, 218
a
environmental stimuli, 117, 247

drug action, 196, 197, 198, 214, 221
enzymatic activity, 133, 170, 252
drug addict, 51, 208, 216, 220, 222, 223
enzyme(s), 28, 36, 38, 127, 128, 133, 149, 154, 158,
ov
drug addiction, 51, 208, 216, 220, 222, 223

165, 170, 243, 244, 245, 246, 252, 253, 254, 258,
drug discovery, 5, 18, 143, 178, 199, 306
267, 310, 313
drug interaction, 168, 173
epidemiologic, 246
drug resistance, 233
epidemiology, 161, 180, 201, 290
drug targets, 198, 296
epigenetic alterations, 125, 137, 143
drug toxicity, 283
N
Epigenetic mechanisms, 126, 137, 141, 142, 145,

drug treatment, 18, 20, 86, 92, 120, 122, 201, 205,
222
209, 210, 212, 214, 231, 284, 328
epigenetic modification, 125, 129, 130, 133, 134,
D-serine, 175, 177, 322
136, 212
334 Index
epigenetics, 72, 126 fibroblast growth factor, 117, 118, 121, 122, 319,
epilepsy, 25, 33 324
epinephrine, 46 fibroblasts, 231, 233
episodic memory, 109 fibromyalgia, 182, 282
epithelial cells, 240, 257 filament, 312
epithelium, 57 financial, 260
c.
EPS, 282, 284 fish, 149, 299, 300, 302, 303, 304, 305
equilibrium, 313 fluctuations, 38
erythrocytes, 244, 256 fluorescence, 304
erythropoietin, 314 fluvoxamine, 26, 31, 42, 202, 239, 242, 244, 279,
In
escitalopram, 121, 133, 134, 137, 155, 159, 191, 193, 280, 281, 282, 284, 285, 286, 287, 288, 289, 290,
194, 245, 279, 281, 319, 324 291, 292, 293, 317
Escitalopram, 52, 62, 118, 324 Fluvoxamine, vi, 236, 240, 279, 282, 283, 284, 285,
estrogen, 51, 56, 70, 153, 252, 254, 260, 274 288, 289, 290, 291, 292
ethanol, 18, 300, 301, 305, 306 food, 12, 15, 22, 299
ethnic background, 308 Food and Drug Administration, 169, 283
rs
etiology, 26, 36, 49, 72, 120, 180, 200, 228, 230, Ford, 235, 262, 292, 308, 321
247, 249, 251, 256, 297 forebrain, 18, 45, 66, 78, 85, 105, 133, 151, 163,
eukaryotic, 148 165, 185, 207, 231, 259
evolution, 155, 253, 258 formation, 31, 45, 69, 76, 80, 88, 90, 113, 126, 130,
he
excitability, 42, 64, 95, 170, 194, 195 150, 151, 176, 178, 199, 200, 216, 241, 246, 267,
excitation, 170, 194, 195, 201 270, 309, 312, 313, 314, 315
excitatory synapses, 197, 198 foundations, 143
excitotoxicity, 100, 170, 192, 205, 261, 262, 316, France, 207, 307
317 free radicals, 313, 314
excretion, 64 frontal cortex, 29, 30, 34, 72, 81, 82, 101, 133, 140,
is
executive function, 281 152, 153, 159, 161, 163, 166, 181, 182, 188, 189,
exercise, 65, 119, 123, 299, 302 193, 196, 197, 198, 199, 203, 204, 205, 247, 252,
exocytosis, 205 262, 270, 318
exons, 73, 84, 87, 130 functional changes, 72, 133, 190, 296
bl
experimental condition, 233 functional MRI, 109
experimental design, 232 fusion, 198, 204
externalizing behavior, 51
extinction, 30, 34, 35, 39, 91
G
Pu
extracellular matrix, 310, 311, 313

GABA, 13, 25, 29, 36, 41, 101, 153, 170, 172, 173,
extracts, 18, 23, 285
180, 181, 189, 194, 201, 269, 311
extrapyramidal effects, 283
GABAA receptors, 25, 26, 27, 28, 29, 31, 37, 38, 40,
F 42
ganglion, 111, 310
false negative, 1, 5 gastrin, 269
FDA, vii, 169, 173, 283 gender differences, 77, 91, 252
a
FDA approval, vii Gender-specific, 270

FDR, 153 gene promoter, 130, 131, 145, 272
fear response, 25, 26, 30, 32, 35, 299 gene regulation, 86, 91, 148, 149, 158, 174, 184,
ov
feedback inhibition, 230, 234 207, 208, 209, 210, 211, 212, 213, 214, 216, 217,
female rat, 33, 37, 38, 65, 68, 70 218, 219, 220, 223, 224, 225
fertilization, 45 gene silencing, 139, 150, 161
fetal development, 43, 54, 66 gene targeting, 163
fetal growth, 61, 62 genes, 74, 75, 79, 84, 89, 90, 95, 111, 114, 119, 126,
N
fetus, 44, 45, 47, 69 128, 129, 136, 141, 142, 143, 149, 150, 151, 154,
fiber(s), 30, 93, 123, 141, 195, 316, 318 156, 158, 162, 163, 169, 189, 201, 209, 211, 213,
fibrin, 312, 314 219, 231, 249, 250, 258, 261, 288, 301, 303
fibrinolysis, 307, 315, 323 genetic background, 7
Index 335
genetic marker, 19 hepatitis, 235, 240, 258, 259, 266, 269, 272
genetics, 41, 63 hepatitis d, 259
genome, 148, 162, 296, 304 hepatotoxicity, 246
genomics, 160, 164 heterogeneity, 42, 158, 240
genotype, 67 HHS, 222
gestation, 44, 54, 55 Hippocampus, 41, 42, 63, 70, 87, 92, 94, 95, 164,
c.
gestational age, 44 200, 263
glia, 9, 130, 144 histochemistry, 210
glial cells, 28, 72, 82, 84, 190, 310 histone(s), 83, 125, 127, 128, 130, 131, 133, 134,
glioblastoma, 137, 165 135, 137, 139, 140, 141, 142, 143, 144, 151, 153,
In
glioblastoma multiforme, 165 162
glioma, 141 Histone acetylation, 134, 141
glucocorticoid receptor, 46, 47, 48, 62, 65, 66, 70, histone deacetylase, 83, 127, 128, 133, 143, 153
95, 114, 123, 152, 166, 227, 230, 242, 256, 257, history, 37, 44
258, 259, 260, 263, 264, 265, 267, 270, 271, 272, HIV, 235
273, 274, 275, 276, 277, 302, 304 homeostasis, 45, 262
rs
glucose, 85, 138, 283, 310, 312, 316, 319 homogeneity, 2
Glutamate receptor, 182 hormone(s), 36, 38, 44, 46, 47, 51, 62, 63, 64, 67, 70,
Glutamate release, 193 108, 131, 191, 229, 233, 251, 257, 258, 259, 263,
Glutamate transmission, 188 265, 268, 269, 277, 302, 309
he
glutamine, 188, 189, 192, 197, 200, 201, 202 hormone levels, 51, 268
glutathione, 243, 244, 246, 256, 313 HPA axis, 45, 46, 47, 48, 54, 61, 62, 63, 203, 227,
glycine, 175, 183 228, 229, 230, 231, 232, 233, 234, 235, 237, 242,
glycogen, 111 255, 263, 269, 270, 275, 291, 302
glycosylation, 251 human brain, 31, 42, 94, 105, 132, 139, 145, 179,
goal-directed behavior, 216 183, 231, 281, 324
is
grants, 23, 160, 219 human cerebral cortex, 69
granules, 315 human development, 70
gray matter, 100 human genome, 126
growth, 44, 45, 70, 72, 83, 85, 94, 107, 108, 110, human subjects, 100, 101, 249
bl
113, 115, 117, 118, 121, 137, 142, 155, 163, 191, Hunter, 70, 143
249, 250, 259, 263, 271, 279, 292, 309, 313, 314 hybridization, 88
growth arrest, 83 hydrogen, 126, 243, 266
growth factor, 70, 72, 85, 94, 115, 117, 118, 121, hydrogen bonds, 126
Pu
249, 250, 259, 271, 279, 292, 309, 313, 314 hydrogen peroxide, 243, 266
guanine, 126 hydroxyl, 170, 243, 313
guidelines, 221 hyperactivity, 8, 21, 51, 79, 174, 208, 216, 219, 224,
234, 300, 315, 317
H hyperlipidemia, 309
hypermethylation, 83, 130, 131, 133, 134
habituation, 8, 20
hyperplasia, 229
haemostasis, 307, 310, 315
hypertension, 308
a
half-life, 4, 214
hypertrophy, 229, 285, 288, 292
HDAC, 133
hypotension, 283
head injury, 326
ov
Hypothalamic-pituitary-adrenal (HPA) axis, 67

health, 67, 323
hypothalamo-pituitary-adrenal axis, 60
heart disease, 308
hypothalamus, 45, 46, 47, 48, 62, 65, 92, 108, 174,
heart failure, 285, 288, 290
181, 228, 236, 237, 244, 269
heart rate, 21, 44, 48
hypothesis, 9, 26, 27, 32, 34, 64, 71, 72, 73, 114,
helplessness, 6, 52, 165, 252
115, 117, 121, 156, 169, 170, 181, 184, 188, 190,
N
hemiparesis, 308
194, 195, 202, 204, 221, 228, 230, 232, 253, 260,
hemispheric asymmetry, 87, 200
263, 265, 270, 285, 303, 309, 327
hemoglobin, 244
hypoxia, 314
hemorrhage, 315, 323
336 Index
inflammatory disease, 262

I inflammatory mediators, 242, 313, 317
inflammatory responses, 277
ICAM, 322
inhibition, 4, 13, 17, 25, 32, 38, 42, 48, 56, 75, 80,
identification, 156, 198, 296, 303
112, 132, 137, 149, 151, 155, 163, 191, 194, 195,
IFN, 176, 237, 238, 239, 240, 241, 242, 261, 265
205, 241, 242, 244, 246, 250, 252, 254, 282, 309,
c.
IL-13, 235, 238, 239
311, 313, 317, 321
IL-8, 176
inhibitor, vii, 2, 4, 6, 18, 22, 23, 30, 37, 43, 56, 64,
ILAR, 306
67, 81, 117, 137, 138, 209, 224, 241, 263, 272,
illumination, 15
292, 323, 325
In
images, 99, 102, 300, 301
initiation, 85, 149, 163, 255, 261
immobilization, 115, 140, 273
injections, 52, 53, 58, 212, 214, 215, 318
immune activation, 238, 274
injuries, 97, 291, 313, 320
immune function, 275
injury, 88, 100, 103, 109, 111, 243, 277, 328
immune reaction, 280
insulin, 117, 259
immune system, 176, 227, 236, 237, 239, 242, 267
integration, 76, 142, 247, 251, 263
rs
immunity, 235, 243, 263
integrity, 91, 212, 310, 312, 316, 326
immunoglobulin, 250
intensive care unit, 284, 288, 289
immunoglobulin superfamily, 250
interaction effect, 11
immunohistochemistry, 303
interference, 77, 79
he
immunomodulatory, 176
interferon, 176, 235, 257, 258, 260, 264, 267, 269,
immunoreactivity, 37, 61, 86, 90, 120, 124, 257, 270,
270, 272, 277
273
interferon-γ, 267, 277
immunostimulatory, 242, 243
internalizing, 48, 51
impairments, 33, 37, 44, 55, 69, 109, 255, 275, 319
interneurons, 30, 100, 101, 104, 114, 173, 311, 326
improvements, 173, 286, 316, 319
interphase, 127
is
impulsive, 26
intervention, 66, 285, 288
impulsivity, 44, 69
intracellular calcium, 265
in situ hybridization, 199, 210
ions, 192, 241, 242
in utero, 61, 66, 135
irradiation, 314
bl
in vitro, 3, 4, 33, 34, 37, 48, 62, 76, 82, 137, 163,
ischemia, 100, 101, 102, 103, 104, 145, 307, 310,
176, 181, 182, 194, 229, 230, 231, 232, 237, 240,
312, 313, 314, 315, 316, 317, 319, 320, 322, 323,
241, 242, 271, 310, 312, 326, 328
324, 325, 326, 327, 328
in vivo, 4, 34, 37, 61, 76, 88, 117, 142, 145, 160,
islands, 79, 126
Pu
163, 164, 176, 178, 179, 181, 182, 199, 231, 232,
isolation, 26, 29, 30, 32, 39, 40, 41, 42, 79, 95, 105,
233, 250, 273, 274, 304, 310, 316, 322, 324, 326,
199, 204, 244, 255, 261, 299, 301, 305
328
isomers, 32, 33
incidence, 264, 281, 283, 327
issues, 2, 10, 160, 178, 290
indirect effect, 316
Italy, 187
individuals, 46, 141, 145, 153, 155, 157, 166, 169,
170, 245, 250, 253, 263, 299, 309, 315 J
inducible protein, 83
a
induction, 63, 81, 88, 92, 111, 112, 119, 130, 135, Japan, 97, 279, 287
151, 192, 196, 209, 210, 211, 212, 213, 214, 215, Jordan, 173, 181, 268
220, 223, 235, 251, 293, 314, 317 juveniles, 218
ov
INF, 235, 236, 237, 239, 240, 241

infancy, 63, 296, 303
K
infants, 48, 67
K+, 312
inflammasome, 274
Ketamine, 180
inflammation, 65, 228, 234, 235, 237, 242, 255, 263,
kidney, 285
N
280, 283, 307, 310, 311, 313, 314, 316, 317, 320,
kinase activity, 76, 178, 234
327
kinetics, 193
Inflammation, 177, 182, 183, 268, 307, 312
inflammatory cells, 310, 313
Index 337
major depressive disorder(MDD), 9, 20, 107, 125,

L 126, 139, 143, 147, 148, 170, 180, 184, 185, 201,
202, 208, 222, 228, 244, 245, 251, 262, 264, 266,
lactation, 62, 65, 67
267, 268, 274, 275, 276, 280, 281, 289, 290, 292,
language development, 44
293, 325, 328
L-arginine, 313, 316
majority, 5, 10, 15, 76, 197, 231, 303, 308, 312, 318
c.
latency, 15, 55, 56, 77, 170
malignant hyperthermia, 283
LCLs, 137, 155
mammal(s), 62, 97, 98, 100, 114, 126, 141, 296, 323
LDL, 245
mammalian brain, 150, 164, 221
leakage, 243
mammalian cells, 149, 150, 161
In
learned helplessness, 77, 165, 171, 185, 191, 200,
management, 45, 208, 283, 284, 302
268
mania, 103, 104, 266
learning, 45, 51, 77, 90, 91, 112, 114, 123, 135, 216,
manic, 131, 300
221, 281
manic episode, 131
LED, 304
manipulation, 56, 59, 70, 81
leptin, 265
mapping, 220, 223
rs
lesions, 100, 105, 274, 309, 315, 317, 320
Marx, 26, 39
leukocytes, 270, 313
maternal mood, 43, 44, 51, 59, 67
ligand, 37, 79, 110, 231, 233, 267, 289
matrix, 73, 108, 221, 312, 314, 324
limbic system, 125, 251
matrix metalloproteinase, 108, 324
he
lipases, 312
matter, 309
lipid dysregulation, 283
MCP, 293
lipid peroxidation, 244, 258, 313
MCP-1, 293
lipids, 244
measurement(s), 82, 119, 185
lithium, 79, 86, 139, 232, 304
Mechanism of action, 38, 105
liver, 162, 246, 254, 261, 265, 275, 278, 280, 285
median, 229, 272
is
liver cancer, 162
medical, 172, 185, 223, 235, 251, 297, 306
liver damage, 265
medication, 2, 13, 48, 49, 55, 67, 116, 118, 168, 172,
localization, 95, 113, 122, 124, 166, 261, 310
173, 189, 199, 219, 220, 222, 224, 284
locomotor, 8, 10, 16, 21, 40, 216, 219, 235, 268
medicine, 104
bl
locus, 132, 136, 144, 156, 170, 230, 232
MEK, 121, 166
long-term memory, 77, 90, 112, 113
melanoma, 266, 267
LSD, 13, 14
melatonin, 17, 21
LTD, 194, 195, 204
membranes, 27, 28, 193, 195, 285
Pu
luciferase, 154, 157

memory, 34, 39, 71, 76, 112, 114, 122, 123, 135,
Luo, 104, 162, 165, 255, 268, 275, 325
139, 140, 141, 142, 143, 160, 165, 219, 221, 222,
luteinizing hormone, 66, 69
223, 281, 306, 316, 324
lymphocytes, 231, 234, 235, 240, 241, 249, 262, 313
memory formation, 71, 112, 142
lymphoma, 132, 263
memory function, 222
lysine, 83, 127, 128, 130, 131, 135, 136, 139, 142,
memory processes, 135
151
mental development, 44
M mental disorder, 72, 131, 207, 262, 297
a
mental health, 62
machinery, 120, 127, 129, 139, 149, 151, 158, 194, mesenchymal stem cells, 142
ov
197 messenger RNA, 89, 144, 267, 272

macrophages, 176, 184, 235, 237, 238, 265, 311 meta-analysis, 61, 106, 239, 264, 268, 285, 289, 291,
magnetic resonance, 178, 189, 190, 199, 201, 202, 323, 325, 326
269 Metabolic, 202, 223, 269, 309
magnetic resonance imaging, 190, 201 metabolism, 42, 85, 138, 143, 156, 169, 171, 174,
magnetic resonance spectroscopy, 178, 189, 199, 185, 188, 189, 192, 237, 243, 246, 252, 253, 254,
N
201, 269 256, 259, 262, 272, 276, 310, 320

magnitude, 213, 214 metabolites, 3, 34, 37, 54, 56, 187, 243, 246, 260
metabolized, 237, 252
338 Index
metalloproteinase, 109 mortality, 91, 283, 291, 292, 307, 317

metastasis, 267 mortality rate, 283
methodology, 300 Moses, 44, 66, 179
methyl group(s), 126, 127, 128 motif, 109, 112, 152
methylation, 79, 83, 88, 89, 91, 94, 95, 125, 126, motivation, 188, 298, 301
127, 128, 129, 130, 131, 133, 134, 135, 137, 139, motor activity, 316
c.
140, 141, 142, 143, 144, 145, 161 movement disorders, 286, 292
methylphenidate, 174, 181, 185, 207, 208, 209, 210, MR, 46, 47, 62, 230, 232
211, 212, 214, 215, 219, 220, 221, 220, 222, 223, MRI, 202, 327
224, 225 mRNAs, 30, 70, 93, 122, 129, 140, 149, 150, 153,
In
microdialysis, 179, 180, 181, 196, 324 158, 199, 214, 242, 274, 277
microRNA, 89, 137, 138, 139, 142, 143, 154, 160, mtDNA, 254
161, 162, 163, 164, 165, 166 muscarinic receptor, 316
midbrain, 45, 77, 87, 216, 262 muscles, 316
migration, 45, 76, 104, 247, 250, 259, 312, 314, 320, mutant, 7, 20, 79, 82, 122
327 mutation(s), 83, 112, 113, 139, 162, 169, 172, 296,
rs
mineralocorticoid, 46, 47, 62, 230, 268, 272, 274 301, 302, 303
Ministry of Education, 287 myocardial infarction, 292
mitochondria, 235, 243, 246, 253, 254, 255, 256,
260, 263, 275
N
he
Mitochondria, 227, 256
Na+, 192, 312
mitochondrial DNA, 274
NAD, 143, 144
mitogen, 87, 110, 111, 112, 119, 139, 234, 237, 239,
National Academy of Sciences, 104
241
National Health and Nutrition Examination Survey,
MMP(s), 73, 310, 313, 314, 316, 317, 320
262
MMP-2, 314, 316
is
National Institute of Mental Health, 160
MMP-3, 73
National Institutes of Health, 160
MMP-9, 316
National Survey, 208, 222
moclobemide, 6, 18, 19
NCS, 201, 290, 305
Model development, 295
bl
necrosis, 110, 176, 260, 268
model system, 16, 148, 157, 158, 304, 305, 306
negative consequences, 313
modelling, 324
neocortex, 97, 104, 272
modifications, 6, 48, 83, 113, 126, 127, 130, 133,
neonates, 44, 48, 244, 261
134, 136, 137, 139, 145, 162, 166, 199, 231, 234,
Pu
nerve, 73, 88, 89, 90, 94, 107, 108, 115, 117, 122,
249, 251, 272, 322
123, 170, 205, 235, 249, 279, 281, 291, 309, 310
modules, 166
nerve growth factor, 73, 88, 89, 90, 94, 107, 108,
molecular weight, 86, 108
115, 117, 122, 123, 249, 291, 309
molecules, vii, 1, 2, 5, 7, 16, 75, 83, 110, 129, 142,
nervous system, 46, 64, 89, 98, 102, 108, 111, 121,
149, 156, 157, 197, 209, 211, 220, 235, 242, 244,
123, 247, 268, 279, 280
250, 254, 256, 261, 262, 301, 313
Netherlands, 43, 71
monoamine oxidase inhibitors, 81, 117, 168, 316
neural development, 131
Monoamines, 170
a
Neural plasticity, 140, 251

monolayer, 194
Neurite outgrowth, 280
mood disorder(s), vii, 20, 22, 46, 49, 51, 54, 59, 62,
neurobiology, 91, 139, 184, 191, 201, 267, 268, 297,
ov
65, 87, 103, 107, 114, 120, 134, 140, 144, 145,
304
166, 167, 169, 170, 174, 180, 181, 182, 184, 188,
neuroblastoma, 76, 89
189, 193, 195, 200, 202, 203, 204, 205, 227, 243,
neuroblasts, 105, 320
246, 255, 261, 276, 291, 295
neurodegeneration, 120, 234, 237, 254, 263, 264,
morbidity, 168, 292, 307
317
morphine, 78, 94, 291
N
neurodegenerative diseases, 90, 157, 261

morphogenesis, 151, 165, 166
neurodegenerative disorders, 192, 243
morphology, 34, 35, 36, 68, 70, 108, 113, 151, 159,
Neuroendocrine, v, 43, 46, 298
188, 190, 191, 193, 202, 203, 247, 252, 275, 319
Index 339
Neurogenesis, v, 62, 70, 76, 87, 97, 98, 100, 104, nuclei, 6, 17, 144, 156, 157, 170, 257, 264, 311
113, 150, 159, 227, 247, 261, 262, 272, 276, 307, nucleosome, 127
314, 319, 324 nucleotides, 126, 129, 149
neuroimaging, 140, 187, 189, 190, 252 nucleus, 18, 27, 37, 45, 47, 61, 64, 72, 78, 85, 87, 88,
neuroinflammation, 316 91, 92, 95, 127, 129, 130, 133, 134, 135, 136,
neurokinin, 20 149, 171, 175, 176, 179, 182, 199, 207, 210, 211,
c.
neuroleptics, 284 216, 223, 228, 232, 233, 250, 251, 252, 257, 262,
neurological disease, 304, 308 270, 272, 310, 325
neuronal apoptosis, 195, 257 nutrients, 308, 310
neuronal cells, 73, 82, 87, 188, 251, 314, 320, 321
In
neuronal circuits, 31, 216
O
neuronal stem cells, 150
obesity, 171, 309
neuronal systems, 45, 107, 275
occipital cortex, 170, 199
neuropathic pain, 182
occipital regions, 189
neuropeptides, 209, 214, 229
occlusion, 104, 308, 312
Neuroplasticity, 202, 247, 263, 265, 280
rs
OH, 4, 15, 18, 43, 313
neuroprotection, 34, 100, 103, 104, 268, 280, 281,
olanzapine, 20, 104, 168, 169, 172, 174, 177, 178,
307, 324, 327
183, 185, 268
neuropsychopharmacology, 31, 204
oligodendrocytes, 111, 120, 312
neuroscience, 296
he
opiates, 117
neurosecretory, 46
opioids, 171
Neurosteroidogenesis, 28
opportunities, 288
Neurosteroids, 27, 37, 38, 40, 42
organ(s), 56, 57, 246, 285
neurotoxicity, 144, 197
organism, 46, 76, 127, 137, 295, 296, 306
neurotransmission, vii, 26, 28, 29, 31, 39, 59, 66,
osteoporosis, 231
100, 114, 117, 169, 170, 172, 173, 174, 175, 176,
is
ovarian cancer, 260
188, 194, 195, 197, 199, 207, 209, 242, 251, 284,
oxidation, 245, 316, 317
318, 319, 320, 328
oxidative damage, 244, 246
neurotransmitter(s), vii, 4, 45, 49, 62, 63, 71, 72, 82,
oxidative stress, 227, 243, 244, 246, 255, 261, 263,
107, 112, 119, 168, 169, 170, 171, 172, 173, 176,
bl
264, 266, 270, 272, 273, 276, 278, 312, 313, 314,
177, 189, 195, 209, 227, 234, 267, 275, 279, 280,
316, 324, 326
281, 283, 284, 297, 310, 311, 312, 316, 324
oxygen, 243, 244, 262, 308, 310, 312, 316
neurotrophic factors, 108, 114, 115, 117, 120, 199,
273, 320 P
Pu
Neurotrophins, v, 72, 107, 108, 109, 110, 111, 112,

117, 120, 121, 247, 249 pain, 48, 49, 64, 67, 107, 280
Neurovascular unit, 307, 325 panic disorder, 3, 42
neutrophils, 313, 314 parallel, 51, 234, 254, 255, 303, 312, 314
New Zealand, 259 parenchyma, 92, 310, 313, 314
nicotine, 39, 171, 299, 305 paroxetine, 7, 42, 44, 82, 137, 145, 155, 176, 191,
nitric oxide, 235, 243, 244, 264, 277, 313 193, 230, 233, 236, 237, 238, 245, 259, 265, 269,
nitrite, 245
a
270, 279, 281, 282, 283, 287, 325

nitrogen, 243, 262 Paroxetine, 32, 133, 230, 236, 237, 242, 323
NMDA receptor antagonist, 168, 175, 183 participants, 134, 291
ov
NMDA receptors, 112, 171, 175, 183, 187, 193, 195, partition, 11
201, 205, 250 pathogenesis, 125, 132, 147, 148, 155, 158, 183,
N-methyl-D-aspartate (NMDA), 168, 169, 193, 204, 228, 231, 234, 237, 249, 253, 261, 274, 295
280 pathology, 84, 156, 171, 197, 203, 204, 257, 262,
norepinephrine, 20, 44, 46, 60, 116, 117, 168, 169, 297, 326
170, 172, 175, 176, 221, 228, 232, 281, 283, 297, pathophysiological, 108, 227, 247, 252, 260
N
327 pathophysiology, 71, 72, 73, 80, 82, 93, 130, 131,
Norfluoxetine, 23 135, 137, 142, 143, 148, 154, 158, 164, 170, 171,
Norway, 14 177, 182, 187, 188, 189, 190, 191, 192, 193, 201,
340 Index
204, 223, 228, 243, 251, 255, 256, 263, 267, 271, placental barrier, 44, 59
279, 283, 284, 286, 289, 290, 297, 304, 310, 324 plasma levels, 182, 202, 264, 265, 266
pathways, 8, 19, 45, 47, 91, 105, 107, 110, 111, 114, plasminogen, 314, 324
115, 118, 122, 129, 131, 135, 148, 150, 158, 168, Plasticity, v, 49, 67, 70, 71, 76, 79, 112, 151
169, 173, 214, 216, 223, 224, 235, 239, 240, 242, platelet aggregation, 311, 315, 320
246, 254, 255, 262, 268, 284, 285, 288, 297, 313, platelets, 164, 309, 312, 315, 320
c.
317 pleasure, 188, 297, 317
PBMC, 134 polyamine, 154, 159, 162, 163
PCP, 280, 281 polymerase, 149, 160, 163, 313, 323
PCR, 154, 162 polymorphism(s), 113, 116, 121, 123, 130, 139, 140,
In
peptide(s), 37, 209, 210, 214, 215, 225, 269 144, 154, 162, 249, 290, 304
perfusion, 308, 313 polysialic acid, 251
pericytes, 310, 311, 325 polyunsaturated fat, 243
perinatal, vii, 43, 44, 45, 46, 50, 52, 55, 56, 57, 59, polyunsaturated fatty acids, 243
61, 64, 65, 66, 67, 68, 83, 92, 143 population, 2, 57, 67, 95, 105, 148, 151, 245, 282,
peripheral blood, 130, 134, 142, 147, 154, 159, 244, 285, 309
rs
270 positive correlation, 153, 245, 254
peripheral blood mononuclear cell, 142, 154, 159 positron, 169, 281
peripheral nervous system, 63, 108 positron emission tomography, 169, 281
permeability, 311, 320, 326 postnatal exposure, 55
he
peroxynitrite, 243 postpartum depression, 37
personality, 309 Post-stroke depression, 307, 309, 317, 319, 320
PET, 169 post-transcriptional regulation, 154
pharmaceutical, 2 posttraumatic stress, 36, 39, 40, 41, 145, 230, 259,
pharmacokinetics, 19, 60, 63, 104, 252, 257 260
pharmacological treatment, 230, 244, 296, 302, 307, post-traumatic stress disorder, vii, 18, 25, 40, 136,
is
314, 317, 320 271
pharmacology, 2, 4, 41, 139, 279, 280, 289, 291 potassium, 112, 113
pharmacotherapy, vii, 125, 168, 172, 179, 204, 223, Preclinical, vi, 37, 43, 51, 52, 55, 57, 167, 187, 190,
328 217, 249, 258, 286, 317
bl
phencyclidine, 178, 279, 280, 281, 290 precursor cells, 95
phenotype(s), 77, 79, 88, 128, 131, 135, 143, 147, predictive validity, 5, 20, 222, 228, 298, 301
148, 151, 158, 302, 304, 306, 322 pregnancy, 43, 44, 59, 60, 61, 62, 63, 64, 65, 66, 67,
Philadelphia, 144, 269, 292, 305 69, 164
Pu
phosphate, 126 premature death, 148

phosphorylation, 8, 75, 76, 82, 86, 94, 96, 112, 113, premenstrual syndrome, 36
119, 120, 128, 158, 193, 195, 233, 234, 240, 241, prevention, 185, 284, 288, 316, 323
242, 243, 250, 254, 256, 257, 258, 260, 265, 270, primary visual cortex, 81
273, 318, 326 primate, 104, 166, 272
photomicrographs, 50 prodromal symptoms, 285
physical activity, 309 productive capacity, 243
physical aggression, 145 professionals, 323
a
physical exercise, 123, 190 progenitor cells, 45, 76, 85, 88, 96, 97, 98, 100, 104,
physical health, 282 105, 113, 114, 258, 259
ov
physical structure, 247 progesterone, 25, 26, 31, 32, 34, 37, 42, 70
Physiological, 27 prognosis, 243, 320
physiology, 76, 296, 303 programming, 59, 63, 197
PI3K, 112, 114, 121, 123 pro-inflammatory, 176, 227, 235, 237, 239, 240, 241,
pilot study, 17, 27, 31, 185, 257, 293 242, 254, 268
pineal gland, 45 Pro-inflammatory cytokines, 237, 262
N
pituitary gland, 47 proliferation, 20, 34, 42, 45, 49, 50, 51, 60, 65, 71,
placebo, 2, 104, 222, 281, 291, 322 72, 74, 76, 80, 87, 93, 95, 96, 98, 100, 103, 104,
placenta, 43, 62 113, 115, 117, 121, 122, 123, 141, 151, 159, 165,
Index 341
176, 177, 200, 236, 239, 241, 247, 250, 251, 252,
258, 260, 262, 267, 280, 314, 319, 320, 321
R
promoter, 67, 73, 79, 83, 84, 89, 91, 114, 119, 126,
radicals, 243, 262, 313
130, 131, 133, 134, 135, 141, 143, 145, 151
reactions, 68, 178, 258, 266, 309
prophylactic, 139
reactive oxygen, 235, 243, 254, 255, 313
prophylaxis, 106
c.
reactivity, 48, 52, 62, 65, 67, 89, 118, 183, 185, 218,
prostaglandins, 235
220, 224, 241, 328
prostate cancer, 164
reciprocal relationships, 129
prostate carcinoma, 256
recognition, 3, 143, 152, 161, 162, 235, 260
protein family, 110
In
recommendations, 60, 296
protein kinase C, 150, 164, 241, 250, 262
recovery, 48, 104, 114, 123, 145, 244, 245, 291, 308,
protein kinases, 119, 234, 267
309, 310, 315, 317, 318, 319, 321, 322, 324, 325,
protein oxidation, 313
327, 328
protein synthesis, 90, 129, 150, 151
recurrence, 154
proteins, 63, 70, 74, 75, 83, 86, 95, 108, 111, 127,
red blood cells, 245
128, 131, 143, 149, 153, 158, 159, 166, 183, 193,
rs
redistribution, 199
197, 204, 209, 223, 233, 242, 255, 263, 268, 269,
regeneration, 322
273, 276, 280, 303, 310, 311, 319
relevance, vii, 1, 29, 97, 119, 136, 159, 170, 230,
proteoglycans, 274
243, 261, 271, 301, 302, 303, 321
proteolytic enzyme, 310, 313
he
remission, 62, 136, 143, 173, 245, 253, 281
pro-thrombotic, 315
remodelling, 188, 250, 251
Prozac, vii, 23, 184, 204, 208, 275, 302
replication, 126, 179
psychiatric disorders, vii, viii, 26, 33, 34, 35, 36, 38,
repression, 83, 84, 89, 127, 128, 130, 131, 135, 136,
41, 60, 83, 84, 86, 94, 97, 103, 125, 139, 141,
138, 151, 212
145, 148, 155, 162, 168, 187, 199, 231, 244, 245,
repressor, 83, 127
270, 281, 282, 288, 305
is
RES, 166
psychiatric illness, 157, 224, 280
researchers, 33, 174, 239, 260, 316
psychiatry, 89, 264, 266, 295
residues, 29, 70, 74, 110, 112, 119, 126, 127, 128,
psychoactive drug, 157
234
psychological distress, 309, 325
bl
resilience, 71, 77, 94
psychological stress, 61, 235, 244, 268
resistance, 21, 27, 31, 76, 91, 142, 170, 231, 235
psychopathology, 20, 63, 154
resolution, 168, 223
psychopharmacology, 2, 221, 289, 304
responsiveness, 74, 89, 92, 166, 219, 229, 269, 272
psychosis, 103, 178, 230, 263, 271, 282, 285, 288,
Pu
restoration, 242
289, 290, 292
retardation, 282, 302
psychosocial stress, 87, 200, 257, 258
reticulum, 109, 279, 280
Psychostimulant, vi, 184, 207, 208
retina, 80, 90
psychostimulants, vii, 117, 175, 208, 209, 210, 212,
rheumatoid arthritis, 235, 238, 241, 273
216, 217, 223, 300
rhythm, 8, 21
psychotherapy, 297
ribose, 313, 323, 327
psychotropic drugs, vii, 27, 89, 117, 121, 208, 220,
ribosome, 150
223, 266, 305
a
righting, 32, 39
psychotropic medications, 66, 207, 208, 211
risk(s), 44, 51, 60, 62, 63, , 65, 84, 107, 154, 168,
PTSD, vii, viii, 25, 26, 27, 28, 29, 33, 34, 35, 36, 39,
173, 208, 214, 217, 218, 223, 228, 242, 260, 263,
ov
156, 165, 230

282, 283, 285, 288, 291, 292, 299, 308, 309, 314,
public health, 60, 147, 167, 168
315, 317, 325, 326
pyramidal cells, 115, 195, 247
risk factors, 84, 217, 228, 263, 291, 308
Q risperidone, 172, 174, 181, 183, 184
RNA, 77, 79, 125, 127, 129, 140, 148, 149, 155, 157,
N
quality of life, 148, 167, 301 160, 161, 162, 163, 164, 166
quantification, 266, 296, 304 rodents, 4, 5, 8, 9, 12, 16, 18, 19, 29, 31, 32, 46, 62,
quetiapine, 95, 169 69, 73, 78, 82, 132, 134, 174, 190, 191, 196, 217,
quinolinic acid, 265, 272 228, 247, 277, 296, 298, 299, 301, 302, 317
342 Index
Romania, 107 Sexual differentiation, 54

sexual dimorphism, 60, 77, 245
S sexual motivation, 55, 58, 63
sham, 99, 269, 286
safety, 3, 63, 173, 178, 256, 290
shock, 152, 155, 156, 164, 165, 196, 203, 232, 238
SAMHSA, 208, 222
shock therapy, 155, 164, 203
c.
schizophrenia, 39, 107, 119, 141, 144, 145, 166, 231,
showing, 34, 47, 55, 59, 72, 76, 80, 82, 148, 171,
247, 253, 257, 260, 266, 272, 276, 281, 284, 285,
188, 230, 231, 244, 284, 285, 301, 312
286, 287, 288, 289, 290, 291, 306
sialic acid, 251
schizophrenic patients, 284
side effects, 25, 36, 41, 103, 105, 168, 172, 173, 176,
In
science, 139, 228, 327
177, 208, 227, 246, 282, 284, 290
secrete, 109, 239
Sigma-1 receptor chaperone, 280, 289
secretion, 46, 68, 77, 83, 108, 109, 112, 113, 116,
signal transduction, 88, 89, 115, 118, 121, 153, 228
140, 229, 230, 237, 239, 257, 263, 273, 309, 314,
signaling pathway, 92, 114, 119, 128, 139, 166, 230,
319, 320
235, 242, 250, 256, 323
sedative, 29, 31, 36, 283
signalling, 19, 44, 46, 50, 91, 110, 111, 144, 161,
rs
seizure, 20, 39, 92, 122
168, 170, 223, 242, 256, 261, 326
selective serotonin reuptake inhibitor, 27, 44, 60, 61,
signals, 111, 113, 120, 142, 163
62, 63, 66, 67, 71, 80, 97, 98, 108, 117, 126, 155,
signs, 66, 231, 298, 308
167, 168, 172, 179, 180, 182, 208, 220, 222, 227,
Sinai, 184
he
267, 271, 289, 291, 302, 307, 308, 322, 326
single-nucleotide polymorphism, 109
Selective serotonin reuptake inhibitors (SSRIs), 62,
sleep disturbance, 235, 282
279, 280, 296, 325
smoking, 282, 309, 326
selectivity, 111, 214
smooth muscle, 95, 111, 310, 315, 322
self-consciousness, 297
smooth muscle cells, 95, 111
seminal vesicle, 57
SNAP, 195
is
seminiferous tubules, 57
social consequences, 168
sensitivity, 9, 15, 17, 19, 21, 22, 39, 64, 137, 218,
social interactions, 68
242, 304, 318
social isolation, 29, 41
sensitization, 222, 269, 322
social stress, 87, 191, 200
bl
sensorimotor gating, 178
social support, 44
sensory systems, 306
sodium, 112, 192
septic shock, 236
solution, 8, 271
septum, 41, 78, 92
spatial learning, 122, 324
Pu
sequencing, 142, 162

spatial memory, 93, 112
Serbia, 227
species, 5, 6, 8, 10, 15, 53, 109, 148, 235, 243, 247,
serine, 166, 182, 202
254, 255, 262, 295, 296, 299, 301, 303, 311, 313
Serotonin (5-HT), 182
spectroscopy, 205
Sertoli cells, 57
sperm, 55, 57
sertraline, 20, 44, 118, 138, 191, 230, 233, 236, 239,
spinal cord, 45, 108, 120, 262
241, 242, 244, 253, 259, 266, 275, 279, 281, 282,
spinal cord injury, 120
283, 288, 290
spine, 34, 35, 36, 49, 81, 88, 113, 151, 159, 165, 190,
a
serum, 9, 26, 31, 34, 42, 48, 49, 54, 61, 83, 85, 88,
191, 195, 200, 202, 203, 204, 250, 252, 268, 270
89, 93, 116, 120, 121, 123, 175, 181, 217, 237,
spleen, 236, 238, 292
244, 246, 249, 259, 264, 266, 271, 276, 293, 319,
ov
Sprague-Dawley rats, 11, 12, 15

328
sprouting, 80, 94, 197, 292
sex, 31, 50, 51, 54, 58, 59, 60, 68, 69, 202, 244, 251,
stability, 150, 193
254, 256
stabilization, 191, 197
sex differences, 51, 54, 59, 69, 251
stabilizers, 143, 155, 265
sex steroid, 54, 60
state(s), 4, 71, 72, 80, 83, 108, 127, 128, 134, 135,
N
sexual activity, 58, 70

140, 143, 176, 199, 228, 247, 248, 249, 253, 254,
sexual behavio(u)r, 38, 55, 56, 57, 58, 59, 65, 68, 69,
255, 263, 267, 295, 313, 315, 317
85
status epilepticus, 141
sexual development, 66
Index 343
stem cell differentiation, 151 143, 173, 183, 195, 236, 238, 247, 249, 250, 251,
stem cells, 92, 151, 165, 247, 314 275, 280, 314, 316, 319, 324
stereospecificity, 32 susceptibility, 39, 63, 126, 130, 142, 162, 245, 276
Steroid hormones, 36 sympathetic nervous system, 46
steroids, 26, 36, 37, 39, 41, 42, 66, 262 symptomology, 298
stimulant, 25, 27, 31, 39, 221, 222, 223, 224 symptoms, 5, 16, 27, 28, 34, 39, 44, 133, 134, 135,
c.
stimulation, 12, 55, 56, 60, 61, 65, 88, 117, 122, 140, 137, 138, 152, 169, 174, 179, 184, 185, 228, 230,
165, 170, 179, 229, 236, 239, 241, 242, 269, 276, 235, 240, 272, 274, 282, 285, 286, 291, 292, 297,
287, 292, 298, 312, 320 298, 300, 301, 302, 308, 309, 314, 317
stimulus, 135, 216 synapse, 49, 76, 81, 88, 112, 113, 132, 151, 158,
In
stochastic model, 164 159, 168, 187, 189, 191, 192, 196, 197, 200, 201,
storage, 65, 315, 320, 323 203, 251, 259, 261, 268
stratification, 245 synaptic clefts, 170
Stress, 3, 39, 47, 50, 60, 62, 63, 64, 65, 91, 93, 107, synaptic plasticity, 8, 71, 72, 76, 94, 107, 108, 112,
114, 115, 145, 152, 165, 171, 196, 202, 203, 204, 113, 129, 131, 135, 139, 141, 148, 155, 158, 160,
260, 268, 272, 274, 312 165, 191, 193, 200, 204, 213, 250, 252, 270, 276,
rs
stress response, 45, 46, 63, 67, 137, 147, 155, 158, 314
159, 192, 196, 228, 230, 260, 302 Synaptic plasticity, 179
stressors, 8, 51, 61, 81, 126, 191 synaptic strength, 73
striatum, 27, 29, 78, 86, 92, 104, 142, 173, 174, 185, synaptic transmission, 112, 121, 170, 187, 191, 193,
he
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 195, 201, 250
217, 218, 219, 220, 223, 224, 225, 314 synaptic vesicles, 197
stroke, vi, vii, 100, 103, 104, 105, 106, 114, 123, synaptogenesis, 45, 73, 75, 79, 84, 107, 108, 191,
307, 308, 309, 310, 312, 314, 315, 316, 317, 318, 280
319, 320, 321, 322, 323, 324, 325, 326, 327, 328 syndrome, 40, 68, 153, 162, 175, 221, 283, 297
stroke symptoms, 308, 314 synergistic effect, 174, 176, 177, 183
is
structural changes, 51, 113 synovial membrane, 241
structural modifications, 113 synovial tissue, 277
structure, 3, 40, 73, 85, 113, 114, 115, 123, 127, 128, synthesis, 26, 32, 56, 69, 112, 128, 145, 151, 170,
130, 138, 141, 145, 149, 190, 258, 260, 263, 280, 176, 229, 253, 310
bl
305
subacute, 7, 317
T
substance abuse, 142, 224
T cell, 143, 236, 239, 241, 263, 277
substance use, 217, 218, 221
Pu
T lymphocytes, 239, 240, 241

substitution, 107, 109, 116, 130, 281
tardive dyskinesia, 286, 287
substrate(s), 18, 126, 127, 128, 131, 143, 149, 312,
target, 30, 36, 41, 79, 84, 96, 108, 126, 129, 130,
313, 316
132, 134, 143, 147, 149, 150, 151, 152, 153, 154,
Subventricular zone, 105
156, 157, 158, 161, 183, 187, 193, 195, 197, 200,
sucrose, 8, 9, 78, 194, 218, 248, 298, 317
242, 246, 249, 255, 256, 260, 266, 277, 312, 313,
suicidal behavior, 84, 89, 130, 141, 154, 165, 249
316, 324, 327
suicidal ideation, 116, 282
telencephalon, 95, 276, 301
suicide, 72, 83, 87, 89, 98, 116, 120, 121, 123, 131,
a
TEM, 143
138, 140, 141, 142, 144, 150, 152, 153, 154, 159,
temperament, 66
161, 163, 164, 165, 168, 185, 229, 232, 250, 257,
temporal lobe, 253
ov
262, 263, 269, 270, 275, 282, 317, 319

terminals, 112, 195, 197, 204, 205, 301, 322
suicide completers, 140, 153, 154, 161, 163
testing, 2, 5, 9, 10, 11, 12, 140, 284
supplementation, 141
testis, 57, 89, 285
suppression, 15, 46, 102, 112, 134, 152, 162, 163,
testosterone, 54, 56, 60, 61, 66, 68, 69, 70, 251
191, 230, 237, 257, 267
TGF, 267
survival, 34, 42, 49, 50, 51, 61, 70, 71, 72, 73, 74,
N
thalamus, 92
75, 76, 79, 80, 83, 84, 85, 87, 90, 93, 95, 107,
therapeutic approaches, 20, 147, 157, 295, 324, 325,
108, 110, 111, 113, 114, 117, 119, 120, 121, 122,
326
therapeutic benefits, 269
344 Index
therapeutic effects, vii, 27, 98, 101, 103, 105, 125, tumorigenesis, 162
132, 135, 136, 137, 173, 228, 307, 308, 309, 316 Turkey, 279
therapeutic targets, 147, 157, 162, 169, 184, 200, 324 turnover, 80, 93, 153, 232, 305, 325
therapeutic use, 39 tyrosine, 73, 74, 76, 87, 108, 110, 141, 143, 153,
therapeutics, 184 161, 170, 249, 259, 314
therapy, vii, 22, 77, 90, 104, 115, 135, 137, 155, 167, tyrosine hydroxylase, 259
c.
168, 171, 173, 175, 177, 180, 191, 195, 202, 207,
208, 224, 228, 230, 232, 234, 239, 254, 259, 263,
U
264, 267, 271, 272, 275, 282, 285, 286, 309, 322,
ubiquitin, 153
323
In
underlying mechanisms, 51, 101, 103, 176, 296
thrombin, 312, 315
United States (USA), 25, 70, 104, 160, 162, 163,
thrombus, 308, 312, 314
166, 168, 200, 202, 204, 208, 260, 272, 275, 282,
thyroid, 258
283
tissue, 4, 79, 94, 100, 131, 134, 139, 145, 157, 163,
uric acid, 246
188, 253, 254, 256, 285, 307, 308, 311, 312, 314,
urinary retention, 283
315, 319, 323, 324, 327
rs
tissue plasminogen activator, 307, 314, 323, 327 V
TLR, 237
TNF, 176, 234, 235, 236, 237, 238, 239, 240, 241, validation, 256
242, 265, 268, 276, 277, 317 valine, 107, 116, 130
he
TNF-alpha, 268 variations, 127, 154, 162
TNF-α, 176, 234, 235, 236, 237, 238, 239, 240, 241, vascular wall, 315
242, 265, 277 vascularization, 327
tonic, 42, 195 vasculature, 309, 310, 314, 321
toxicity, 246, 291, 306 vasoconstriction, 315, 322
TPA, 240 vasodilation, 313, 315, 322
is
trafficking, 109, 113, 116, 130, 131, 139, 197, 231 vasodilator, 320, 321
traits, 297, 309 vasomotor, 311, 315
transcription factors, 79, 91, 127, 131, 135, 150, 153, vasopressin, 47, 229, 263, 265, 269, 270, 273
159, 209, 211, 220, 233 venlafaxine, 6, 95, 168, 232, 236, 237, 240, 241,
bl
transcripts, 87, 92, 120, 123, 129, 130, 140, 152, 193 245, 277, 284
transduction, 89, 138 ventricle, 92
transformation(s), 191, 309, 314, 323 venules, 235
transient ischemic attack, 325 Venus, 102
Pu
translation, 73, 129, 149, 150, 151, 156, 163 vertebrates, 166, 296, 299
translocation, 89, 158, 232, 233, 241, 263, 271, 280 vesicle, 197, 203
transmembrane region, 110 vessels, 310, 312
transmission, 46, 140, 164, 170, 176, 177, 179, 187, victims, 72, 89, 116, 121, 138, 161, 164, 229, 232,
188, 190, 194, 195, 196, 197, 198, 199, 200, 203, 270, 275
205, 224, 243, 247, 307, 314, 318, 320, 321 violence, 103, 104
transport, 60, 93, 123, 244, 253, 254, 260 vitamin C, 244, 313
traumatic brain injury, 145
a
vitamin E, 313
trial, 2, 9, 39, 104, 134, 171, 173, 181, 185, 204, 221, volumetric changes, 189, 190, 191
222, 291, 316, 322, 323 vulnerability, 21, 51, 169, 220, 231, 245, 255, 274
ov
tricyclic antidepressant(s), 6, 55, 66, 80, 191, 229,

273, 282, 316 W
triggers, 34, 46, 74, 75, 88, 200, 242, 312, 313, 315,
319 Washington, 39, 306
Trophic factors, 307 water, 5, 8, 12, 302
tryptophan, 19, 45, 56, 141, 237, 259, 265, 310 weight loss, 235, 282
N
tumor, 165, 234, 243, 257, 264, 276 white matter, 100, 105, 309
tumor growth, 165 withdrawal, 51, 98, 214, 283, 300, 301, 302, 306
tumor necrosis factor, 234, 257, 264, 276 Wnt signaling, 156
workers, 15, 131, 133, 134, 135, 136, 137
Index 345
World Health Organization (WHO), 168, 185, 282

Z
X Zebrafish, vi, 295, 296, 301, 303, 304, 306

zinc, 323
X-irradiation, 80
c.
Y
yang, 90, 110, 122

yin, 90, 110, 122
In
rs
he
is
bl
Pu
a
ov
N
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Published by Nova Science Publishers, Inc. † New York

Chapter 5 Fluoxetine and Its Novel Effect on Adult Neurogenesis 97

Reuptake Inhibitors 125

Chapter 8 microRNAs As Novel Players in Depression Pathogenesis

and Yogesh Dwivedi

Chapter 9 Preclinical and Clinical Aspects of a Combination

neurotransmission as well as the combination of fluoxetine with other psychotropic drugs is

and the impact of perinatal fluoxetine exposure.

demonstrating efficacy in a range of anxiety disorders. Evaluation of new molecules in

models has indicated antidepressant activity but demonstration of anxiolytic effects in

Keywords: models, depression, anxiety, behaviour, fluoxetine

widely available as an antidepressant agent. Following its introduction as an antidepressant,

Table 1. Binding of fluoxetine and its enantiomers to serotonin receptor subtypes

Serotonin Receptor Subtype

Data are expressed as Ki values nM; from Wong et al., 1991.

antidepressant administration to increase BDNF in the hippocampus (Nibuya et al., 1995).

Activity in Depression Models

Overview of Studies Reviewed

assess antidepressant-like activity. As antidepressants require chronic administration for

Activity in Anxiety Models

Other factors may also be important determinants of behavioural outcome. One

Furthermore, as with the antidepressant paradigms discussed above strain, species

TIME IN THE LIGHT AREA (SECONDS)

TIME IN INTERACTION (SECONDS)

16 = 8.38; P<0.05). There was no = 6.28; P<0.05). There was no

Figure 2. Diurnal variation of performance on anxiety tests.

(Unpublished data from the laboratory of the author).

HT2A receptors, resulting in inhibition of DA release (Hansen, 2001). Continued treatment

Animal Species /Strain Dose (mg/kg) * Outcome Reference

Light Dark Box

HSD rats 10 Anxiogenic File et al. 1999

Marble Burying Test

Wistar rats 10 Anxiogenic Kshama et al. 1990

Table 3. Effects of chronic fluoxetine in various animal tests of anxiolytic-like activity

Animal Species /Strain Dose (mg/kg)* Outcome Reference

increasing the anxiogenic-like behaviour.

hyponeophagia, has been taken as a model of anxiety and is reversed by benzodiazepines

‗anxiety‘ in animals which in model system is based on innate or conditioned fear

representation of the psychopharmacological profile of antidepressants, Prog.

accelerated desensitization of 5-HT(2C) receptor following combined treatment with

Neuropharmacol. 39, 1222–1236.

anxiety, locomotion and exploration, Psychopharmacol. 96, 528–533.

fluoxetine treatment on chronic mild stress-Induced cardiovascular changes and

in young and elderly volunteers, J. Clin. Psychopharmacol. 21, 161-166.

anxiety, Pharmacol. Biochem. Behav. 73, 703–712.

stress-induced depression-like behaviour in mice, Life. Sci. 91, 1252–1262.

Neurosci. 15, 7539 –7547.

Pharmacotherapy, J.F.Cryan and B.E.Leonard (eds), Modern Trends in

disorder: a useful new model? Psychopharmacol. 134, 365–367.

chronic anxiogenic-like response to fluoxetine in rats in the elevated plus-maze:

Rodriguez-Filho, R., Takahashi, R.N., (1999). Antinociceptive effects induced by

by chronic fluoxetine or ipsapirone treatment, Neuropharmacol. 38, 279–282.

depression: A 10-year review and evaluation. Psychopharmacol. 134, 319 –329.

animal model of depression. Neurosci. Biobehav. Rev. 16, 525–534.

have demonstrated that fluoxetine and norfluoxetine ameliorate anxiety-like behavior,