Editorial

ear Colleagues,

The nervous system is made up of specific elements (neurons and glial cells) which are organized into networks. Each of these has its own particular activity, and its functions are coordinated by various regulatory systems. Some parts of the nervous system are characterized by their plasticity, and consequently their capacity to respond in an adaptive fashion to events (both positive and negative). These events can take the form either of an attack against the nerve cells (neurotoxicity) or of protection of these cells (neuroprotection). They may result from pathologies (depression, delusional syndromes, neurodegenerative diseases) or they may be linked to the action of either therapeutic agents (antidepressant medications with a neuroprotective action, for example) or to that of toxic agents (drug abuse). For a long time it was difficult to investigate neurotoxicity and neuroprotection, as there was a lack of specifically designed technologies for the examination of the microanatomy of the nervous system. Now, new methods of detailed microimaging and experimental protocols allowing their use have allowed us to observe the results of these processes. We are now able to obtain both qualitative and quantitative information on nerve cells, and to assess their functioning. It seemed an opportune moment to invite researchers in these particular areas to share their knowledge and to provide an update on the status of their research. There are now new models available to explain disorders of the nervous system and their treatment. Consequently, this is the focus we have chosen for this issue of Dialogues in Clinical Neuroscience. We warmly thank the brilliant authors who have lent their expertise to this issue, and naturally we also particularly thank our colleagues David Rubinow and Pierre Schulz, who agreed to coordinate it.

Sincerely yours,

Jean-Paul Macher, MD

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Dialogues in Clinical Neuroscience is a quarterly publication that aims to serve as an interface between clinical neuropsychiatry and the neurosciences by providing state-of-the-art information and original insights into relevant clinical, biological, and therapeutic aspects. Each issue addresses a specific topic, and also publishes free contributions in the field of neuroscience as well as other nontopic-related material. All contributions are reviewed by members of the Editorial Board and submitted to expert consultants for peer review. Indexed in MEDLINE, Index Medicus, EMBASE, Scopus, Elsevier BIOBASE, and PASCAL/INIST-CNRS. EDITORIAL OFFICES Editor in Chief Jean-Paul MACHER, MD BP30 - F-68250 Rouffach - France Tel: + 33 3 89 49 56 60 / Fax: +33 3 89 49 56 74 Secretariat, subscriptions, and submission of manuscripts Marc-Antoine CROCQ, MD BP29 - F-68250 Rouffach - France Tel: +33 3 89 78 71 20 (direct) or +33 3 89 78 70 10 (secretariat) Fax: +33 3 89 78 72 00 / E-mail: ma.crocq@ch-rouffach.fr Annual subscription rates: Europe 150; Rest of World 170. Production Editor Catriona DONAGH, BAppSc Servier International - Medical Publishing Division 35 rue de Verdun 92284 Suresnes - France Tel: +33 1 55 72 32 79 / Fax: +33 1 55 72 58 84 E-mail: catriona.donagh@fr.netgrs.com PUBLISHER Les Laboratoires Servier 22 rue Garnier - 92578 Neuilly-sur-Seine Cedex - France E-mail: mail.dialneuro@fr.netgrs.com Copyright 2009 by Les Laboratoires Servier All rights reserved throughout the world and in all languages. No part of this publication may be reproduced, transmitted, or stored in any form or by any means either mechanical or electronic, including photocopying, recording, or through an information storage and retrieval system, without the written permission of the copyright holder. Opinions expressed do not necessarily reflect the views of the publisher, editors, or editorial board. The authors, editors, and publisher cannot be held responsible for errors or for any consequences arising from the use of information contained in this journal. ISSN 1294-8322
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Contents
Page

233 237 239 257 269 281 297 305 319 333 350

Editorial
Jean-Paul Macher

In this issue
David R. Rubinow

State of the art

Neuronal damage and protection in the pathophysiology and treatment of psychiatric illness: stress and depression Ronald S. Duman (USA)

Translational research
Chromatin regulation in drug addiction and depression William Renthal, Eric J. Nestler (USA) Neuroplasticity of excitatory and inhibitory cortical circuits in schizophrenia David A. Lewis (USA) The role of astroglia in neuroprotection Mireille Blanger, Pierre J. Magistretti (Switzerland) Estradiol: a hormone with diverse and contradictory neuroprotective actions Phyllis M. Wise, Shotaro Suzuki, Candice M. Brown (USA) Neurotoxicity of drugs of abusethe case of methylenedioxyamphetamines (MDMA, ecstasy), and stimulant amphetamines Euphrosyne Gouzoulis-Mayfrank, Joerg Daumann (Germany)

Pharmacological aspects
The impact of neuroimmune dysregulation on neuroprotection and neurotoxicity in psychiatric disordersrelation to drug treatment Norbert Mller, Aye-Mu Myint, Markus J. Schwarz (Germany) The neurotrophic and neuroprotective effects of psychotropic agents Joshua Hunsberger, Daniel R. Austin, Ioline D. Henter, Guang Chen (USA)

Brief report
Neuroplasticity in addictive disorders Charles P. OBrien (USA)

ISSUE COORDINATED BY: David R. Rubinow

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Contributors
Ronald S. Duman, PhD Euphrosyne Gouzoulis-Mayfrank, MD Author affiliations: Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA Author affiliations: Department of Psychiatry and Psychotherapy, University of Cologne, Germany

Eric J. Nestler, MD, PhD

Norbert Mller, MD, PhD, DipPsych

Author affiliations: Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA

Author affiliations: Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-Universitt Mnchen, Germany

David A. Lewis, MD

Guang Chen, MD, PhD

Author affiliations: Departments of Psychiatry and Neuroscience, University of Pittsburgh, Pennsylvania, USA

Author affiliations: Laboratory of Molecular Pathophysiology and Experimental Therapeutics, Mood and Anxiety Disorders Program, NIMH, NIH, Bethesda, Maryland, USA

Pierre J. Magistretti, MD, PhD

Charles P. OBrien, MD, PhD

Author affiliations: Centre de Neurosciences Psychiatriques, CHUV, Dpartement de Psychiatrie, Site de Cery, Lausanne, Switzerland

Author affiliations: Department of Psychiatry, University of Pennsylvania, Treatment Research Center, Philadelphia, Pennsylvania, USA

Phyllis M. Wise, PhD

Author affiliations: Departments of Physiology and Biophysics), Biology, and Obstetrics and Gynecology, University of Washington, Seattle, Washington, USA

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In this issue...
Our brains are incredibly plasticable to learn, remember, and change in the service of adaptation. As such, unlike what many of us were taught, the brain is not a collection of structurally invariant components, but rather is a dynamic organ, both structurally and functionally. And just as alterations in gene expression and synaptic connectivity and strength are critical to learning and stress adaptation, so can disturbances in brain plasticity result in behavioral disturbances and, indeed, classical psychiatric disorders. This issue of Dialogues in Clinical Neuroscience discusses neuroprotection and neurotoxicity, not as trivial academic conceits, but as processes that can now be shown to underpin a variety of psychiatric disorders and that may offer future directions for novel therapeutics. In the State of the art opening article (p 239), Ron Duman summarizes a burgeoning literature demonstrating the structural and cellular effects of stressors and depression as well as the mechanisms underlying these effects. An impressively consistent story implicates opposing effects of stress and depression versus antidepressant therapies on growth factors, particularly brain-derived neurotrophic factor, glutamate signaling, apoptosis, and inflammation. These data suggest that targeted neuroprotective mechanisms together with an understanding of genetic determinants of susceptibility will usher in a new realm of effective and individualized prevention and treatment of depression. Although the basis of heredity is found in the structure of DNA, the basis of much of the behavioral variance in psychiatric disorders is found in gene expression. In the first Translational research article, Renthal and Nestler (p 257) provide a lucid and yet sophisticated discussion of epigenesis, a remarkable process by which environmental events can be transduced into potentially long-lasting changes in chromatin structure (chromatin remodeling) with associated changes in gene transcription. Not only do epigenetic changes represent a form of cellular memory, but as well they represent compelling explanations for both the development of drug addiction and the behavioral response to stress and to antidepressant treatments. For some psychiatric disorders, environmental and genetic risks alter brain development, resulting in enduring molecular and cellular disturbances in neural circuits, with resultant emergent and characteristic symptomatology. In the second Translational research article, David Lewis (p 269) performs an anatomical and biochemical dissection of the circuitry of a brain region that mediates cognitive processes known to be disturbed in schizophrenia, the dorsolateral prefrontal cortex (DLPFC). In a technically detailed and yet lucid presentation, he describes how developmentally determined deficiencies in -aminobutyric acid (GABA) signaling and neuronal synchronization in the DLPFC may result in core cognitive and behavioral deficits in schizophrenia. In the third article in this section, Belanger and Magistretti (p 281) authoritatively trounce the antiquated yet previously prevalent view that glia are largely inert neural components, mere structural nursemaids. Quite to the contrary, astrocytes are major homeostatic defenders of neurons, exquisitely sensitive to and dynamic modulators of neuronal activity. Under pathogenic conditionsneuroinflammation, oxidative stress, excitotoxicitynot only can the neuroprotective functions of astrocytes be overwhelmed, but as well the astrocytes can paradoxically advance the deleterious processes and the onset of disorders like Alzheimers disease. In the following article, Drs Wise, Suzuki, and Brown (p 297) update our understanding of the dramatic yet highly context-dependent neuroprotective and neuroplastic effects of estradiol. Of particular clinical relevance are the observations of the anti-inflammatory actions of estradiol and the elegant demonstration that the latency following cessation of ovarian function critically determines the cellular and antiinflammatory effects of estrogen replacementlong latency, absence of beneficial effects. These findings help explain ostensible contradictions in the literature and support the optimism that estradiol and related steroids may yet be rescued as neuroprotective therapeutic agents. In general throughout this volume, neuroplastic and neurotoxic effects cannot be inferred with certainty absent knowledge of timing and context. In the fifth Translational research article, Drs Gouzoulis-Mayfrank and Daumann (p 305) turn our attention from neuroprotection to neurotoxicity in their description of the effects of drugs of abuse, namely MDMA (ecstasy) and the stimulant amphetamines. Brain morphological and neurochemical abnormalities (particularly involving serotonergic and dopaminergic systems) in animals and brain imaging abnormalities in humans consequent to stimulant abuse are comprehensively reviewed and juxtaposed with reports of behavioral abnormalities. While evidence of the neurotoxicity of these agents is substantial, the authors highlight the lack of parallelism in reported effects and consequent gaps in our knowledge that must critically be addressed.

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In this issue...
As many psychiatric disorders are systemic illnesses, it should surprise no one that they are characterized and contributed to by immune disturbances. In the first Pharmacological aspects article, Norbert Mller and colleagues (p 319) provide an extensive review of inflammatory processes as they apply to schizophrenia and depression. Mechanisms of action of psychopharmacotherapies and pathogenic contributions of neurotransmitter dysregulation are translated into descriptions of the restoration or perturbation of immune balance, respectively. Consideration of immunologic contributions to major psychiatric disorders may generate novel therapeutic interventions for these conditions. While most of the medications in our current psychiatric armamentarium antedate the turn of the century, the originally described mechanisms of action increasingly appear to be relatively primitive. In the second Pharmacological aspects article, Chen and colleagues (p 333) review the cellular effects of psychotropic drug classesantidepressants, antipsychotics, and mood stabilizerswith exquisite attention paid to cell signaling pathways and molecules that mediate the trophic and neuroprotective actions of these drugs. Compelling evidence is presented from animal and human studies for the role of these neuroplastic actions in the therapeutic efficacy of psychotropic agents, thus paving the way for broader potential future therapeutic indications. Finally, in the Brief report, Charles OBrien (p 350) eloquently and convincingly demonstrates that the phenomenology surrounding one psychiatric disorderdrug addictioncan best, if not only, be understood as representing neuroplastic changes in susceptible individuals in response to drugs that co-opt our reward/learning pathways. As such, treatments designed to restore normal synaptic plasticity in the nucleus accumbens might be predicted to lyse drug-seeking behaviors.

David R. Rubinow, MD

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State of the art

Neuronal damage and protection in the pathophysiology and treatment of psychiatric illness: stress and depression
Ronald S. Duman, PhD

The discovery that stress and depression, as well as other psychiatric illnesses, are characterized by structural alterations, and that these changes result from atrophy and loss of neurons and glia in specific limbic regions and circuits, has contributed to a fundamental change in our understanding of these illnesses. These structural changes are accompanied by dysregulation of neuroprotective and neurotrophic signaling mechanisms that are required for the maturation, growth, and survival of neurons and glia. Conversely, behavioral and therapeutic interventions can reverse these structural alterations by stimulating neuroprotective and neurotrophic pathways and by blocking the damaging, excitotoxic, and inflammatory effects of stress. Lifetime exposure to cellular and environmental stressors and interactions with genetic factors contribute to individual susceptibility or resilience. This exciting area of research holds promise and potential for further elucidating the pathophysiology of psychiatric illness and for development of novel therapeutic interventions.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:239-255.

role for damage and protection of neurons in the pathophysiology and treatment of psychiatric illness, including major depressive disorder (MDD) is based on molecular, cellular, and morphological studies in experimental animals and in human patients. Preclinical studies demonstrate that chronic stress causes alterations to the number and shape of neurons and glia in brain regions implicated in mood disorders.1-4 Advances in human brain imaging have also reported decreased volumes of limbic brain regions implicated in depression.5,6 Preclinical and postmortem studies of signal transduction pathways and target genes have extended this work at the molecular level, demonstrating dysregulation of neurotrophic factors and neuroprotective mechanisms in response to stress and in depressed patients. 1,2,7 Conversely, chronic administration of therapeutic agents blocks the effects of stress or leads to induction of neurotrophic and neuroprotective pathways. 2,8 Together, these findings have contributed to a fundamental shift in our understanding of the cause and treatment of psychiatric illnesses and the role of neurotrophic and neuroprotective mechanisms. This review will present evidence demonstrating neuronal damage, atrophy, and cell loss in response to stress and depression, and the mechanisms underlying these effects. Studies demonstrating the neuroprotective actions of therapeutic agents that counteract the effects of stress and depression will also be discussed. Related aspects of this work are the effects of environment, cellular stressors, insults, and interactions with genetic factors that increase susceptibility and thereby cause damAddress for correspondence: Department of Psychiatry, Yale University School of Medicine, 34 Park Street, 3rd floor, New Haven, CT 06508, USA (e-mail: ronald.duman@yale.edu)

Keywords: synapse, neurogenesis, antidepressant, signal transduction, gene expression Author affiliations: Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA

Copyright 2009 LLS SAS. All rights reserved

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State of the art

Selected abbreviations and acronyms
ADT AMPA BDNF IDO MDD NMDA PFC TNF antidepressant treatment -amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid brain derived neurotrophic factor indoleamine 2,3-dioxygenase major depressive disorder N-methyl-D-aspartic acid prefrontal cortex tumor necrosis factor studies have consistently reported that the volume of the hippocampus is decreased 10% to 20% in MDD patients.9-12,13,14 There is also evidence of a negative correlation with the length of illness and reversal with antidepressant treatment (ADT),15 but additional studies are needed to further examine these relationships and to determine whether the reduction is a result or a cause of depressive illness. It is also notable that hippocampal volume reductions have been reported in other stressrelated illnesses, including post-traumatic stress disorder (PTSD)16,17 and schizophrenic patients.18 The PFC is another stress-responsive brain region implicated in depression. The primary function of the PFC is cognition, working memory, and inhibitory control of brain regions that underlie fear and emotion. Brain imaging studies have reported a significant reduction in the volume of the PFC in MDD patients, which could underlie the reported hypofunction of this structure, most notably decreased cognition.9,15,19,20 Cellular alterations Different types of cellular alterations could account for the volume reductions observed in the hippocampus,
Cellular/neuronal stress and damage Environment Stress Trauma Illness Neuroprotective mechanisms Life history Reduce stress Exercise, diet Medication Genetic factors Increased resilience

age and illness (Figure 1). Conversely, life history of behavior or therapies that reduce stress and enhance neuronal survival, such as exercise, diet, medications, and interactions with genetic factors that increase resilience are neuroprotective, and reverse or block the damaging effects of stress. In addition, cellular growth and survival are intimately controlled by neuronal activity (Figure 1). This is due to the activity-dependent requirement for expression of neurotrophic factors and other survival pathways and mechanisms that control neurotransmission and neuroplasticity, as well as proliferation, growth, and survival.

Structural/cellular alterations in mood disorders

Depression, like most other major psychiatric illnesses, is widely accepted to be caused by neurochemical imbalances in regions of the brain that are known to control mood, anxiety, cognition, and fear. These regions include the hippocampus, prefrontal cortex (PFC), cingulate cortex, nucleus accumbens, and amygdala. In addition, brain imaging and postmortem studies have identified structural alterations in MDD patients that indicate reductions in dendrite arborization and complexity, and decreased numbers of neurons and glia in these brain regions, all of which could contribute to depressive symptoms (Figure 2). Together, these findings provide compelling evidence for disruption of neurotrophic factors and neuroprotective mechanisms in the pathophysiology of depression. Structural alterations One of the regions of interest in depression, as well as other disorders, is the hippocampus, a structure that contains high levels of receptors for glucocorticoids. Imaging

Genetic factors Increased susceptibility

Trophic support Excitotoxicity Inammation/cytokines Metabolic support Viral/toxic load

Neuronal & glial proliferation, growth, function, and survival

Emotional and cognitive function and health

Figure 1. Schematic demonstrating the effects of stress and neuroprotective mechanisms on the proliferation, growth, and survival of neurons and glia. Interactions with environment, genetic factors, and life history also influence these cellular processes, which then regulate emotional and cognitive health or illness. Normal, healthy activity of the brain circuits that underlie emotion and cognition also influence cell survival and function as the expression and function of neurotrophic and neuroprotective mechanisms requires neuronal activity.

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including reductions in the number, size, and proliferation of neurons and glia. There is one report that the size of neurons in the major subfields of the hippocampus is reduced,21 suggesting a reduction in neuropil that could contribute to decreased hippocampal volume in MDD patients. There were no changes in the numbers of neurons or glia reported in this study or in other qualitative studies, although more subtle synaptic changes have been reported.22
Control Stress

Studies of the PFC and cingulate cortex have been more extensive, and have shown a reduction in the size of neuronal cell bodies, suggestive of reduced dendritic arborization and complexity.23,24 In addition, the most consistent finding in studies of PFC is a decrease in the number of glia in MDD patients.23-25 Reductions of both astrocytes26 and oligodendrocytes27,28 have been reported. Given the significant role of glia in providing metabolic support for neurons as well as control of neurotransmitter activity (eg, synthesis and reuptake), it is reasonable to speculate that neuronal atrophy, damage, and hypofunction of PFC could be related to the loss of glia.

PFC pyramidal cell apical dendrites

Cellular alterations in animal models of depression

Animal models of depression have been used to further elucidate the ultastructural and molecular alterations that underlie the morphological changes observed in MDD patients. Most of these models are based on acute or chronic-stress paradigms, as stress is a critical factor in the etiology of depression. In addition, these models have been used to demonstrate that antidepressants can reverse or block the effects of stress on cellular morphology, which might contribute to the therapeutic actions of these agents. Cell morphology Early studies of cell morphology found that repeated stress causes atrophy of CA3 pyramidal neurons in the hippocampus, characterized by a decreased number and length of apical dendrites.29,30 More recent studies have shown that pyramidal neurons in the PFC undergo a similar retraction/atrophy of apical dendrites, and a reduction in spine number in response to immobilization stress (Figure 2).31 Chronic exposure to high levels of exogenous corticosterone, the rodent equivalent of cortisol, causes a similar atrophy of hippocampal and PFC neurons.32,33 In contrast to most neurological disorders, in which the structural alterations and loss of neurons is permanent, the stress-induced atrophy of hippocampal and PFC neurons is reversible. Most notably, removing animals from stress normalizes the dendritic arborization of pyramidal neurons over a period of several weeks.3,32,34 Moreover, chronic administration of certain antidepressants blocks or reverses hippocampal atrophy, even with continued stress exposure.29,30 This reversibility supports

PFC pyramidal cell spines

Hippocampal granule cell neurogenesis

Hippocampal BDNF expression

Figure 2. Influence of stress on the morphology and proliferation of neurons and neurotrophic factor expression. Exposure to immobilization stress decreases the number and length of pyramidal cell apical dendrites, and the number of spines in the prefrontal cortex (PFC). Stress also decreases the birth of new neurons and expression of brain derived neurotrophic factor (BDNF) in the adult hippocampus (HP). Images courtesy of Drs G. Aghajanian and R-J. Li.

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the notion that dendritic alterations represent a type of structural plasticity that has functional consequences. Cell proliferation In addition to dendritic atrophy, chronic stress decreases the proliferation of new cells in the adult hippocampus and PFC. The dentate gyrus of the hippocampus is one of the few regions of the brain that continues to give rise to new neurons in adulthood, in rodents as well as nonhuman primates and humans.35,36 Interestingly, the rate of neurogenesis is influenced by environmental and endocrine factors, and stress is one of the most consistent and robust negative regulators (Figure 2). The proliferation of new neurons is decreased by different types of stress, including restraint, footshock, maternal separation, predator odor, psychosocial stress, and sleep deprivation, and by administration of exogenous corticosterone.37 In the PFC the proliferation of glia is decreased by exposure to repeated stress38 or corticosterone treatment.39 Chronic stress also decreases the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus.40 In contrast, antidepressants increase the proliferation of neurons and glia in the hippocampus and/or PFC, and block or reverse the effects of stress.37,38,41,42 These effects require chronic administration (weeks), consistent with the time course for the therapeutic response to antidepressants. Different classes of antidepressant increase cell proliferation in rats, including serotonin selective transporter inhibitors, norepinephrine selective reuptake inhibitors (NSRIs), and electroconvulsive seizures (ECS),41,43,44 indicating that this is a common target of ADT. Behavioral consequences of altered cell morphology A major question is whether the cellular alterations lead to changes in behavior. This has been addressed by blockade studies (ie, focused irradiation or genetic manipulation), which demonstrate that neurogenesis is required for the actions of antidepressants in certain behavioral models,42,45,46 although there are exceptions.47,48 Ablation of glia in the PFC decreases sucrose consumption, a measure of anhedonia, indicating a requirement for glial function in this model.49 Decreased PFC dendrite arborization in response to stress is also correlated with a reduction in attention set shifting, a PFC-depenImportance of life stress/trauma: gene-environment interactions There is also evidence that exposure to traumatic or stressful life events can have a cumulative effect that increases susceptibility or vulnerability to mood disorders51 (see Figure 1). Interactions of stress and genetic factors have also been reported, most notably for lifetime stress and the serotonin (5-HT) transporter short allele polymorphism52; however, a recent meta-analysis suggests that additional studies of this polymorphism are required.53 Studies of genes that increase resilience to stress and mood disorders have also been conducted. 54 Recent studies have also reported an interaction between early life stress or trauma and neurotrophic factors (see below). dent behavior.50 These studies demonstrate a causal and/or correlative relationship between cell number and complexity with behavior.

Mechanisms underlying structural alterations and neuroprotection: gene-environment interactions

Cellular and structural alterations in response to stress, depression, and antidepressant medications could result from a number of different mechanisms that alter the proliferation, growth, survival, and function of neurons and glia. These include altered neurotrophic/growth factor support, excitotoxicity, inflammation/cytokines, metabolic/vascular support, viral, and toxic insults. The influence of these factors and insults on cell function and survival could occur rapidly after a single major event or could occur gradually over time with the accumulation of one or more insults, also referred to as allostatic load (Figure 1).55 The effects of these cellular stressors and insults are also influenced by genetic factors that can either increase susceptibility to cellular damage, or conversely decrease susceptibility and increase resilience and neuroprotection. This complex interaction of gene-environment interactions over the lifespan is thought to contribute to the heterogeneity of depression, other psychiatric illnesses, as well as treatment of these disorders. Characterization of the molecular mechanisms and genetic factors that underlie the structural alterations and that play a key role in neuroprotection will provide

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important information for the diagnosis and treatment of depression. The following sections will discuss the major molecular and cellular mechanisms underlying the actions of stress, depression, and ADT. The latter will include not only chemical antidepressants, but also other strategies that have neuroprotective actions, including exercise. As discussed above, postmortem studies demonstrate a decrease in the size, but not the number, of neurons, indicating that cell death probably does not play a major role in depression. These findings suggest that mechanisms that control maintenance of neuronal size and function, and counteract stress-induced atrophy, such as neurotrophic factors, could be critical mediators. Other important mechanisms to be discussed are glutamate excitoxicity, apoptosis, and inflammation/immune responses.

that lead to activation of one of three major intracellular signaling cascades: the microtubule associated protein kinase (MAPK), the phosphatidylinositol-3 kinase (PI3K), and the phospholipase-C- (PLC) pathways (Figure 3).8 These cascades have been linked to the neuroprotective effects of BDNF, as well as regulation of cell proliferation, differentiation, and survival.56 Stress, depression, and regulation of BDNF Smith and colleagues were the first to report that exposure to immobilization stress results in a dramatic reduction in levels of BDNF in the rodent hippocampus,57 and this effect has been reported with many other types of stress.1 Decreased expression of BDNF is observed in the major subfields of the hippocampus, including those layers where dendritic atrophy (CA3 pyramidal cell layer) and decreased neurogenesis (dentate gyrus granule cell layer) are observed in response to stress.57 Expression of BDNF in the PFC is also decreased by chronic, but not acute stress.58 Postmortem studies are consistent with the rodent work, reporting decreased levels of BDNF in the hippocampus of suicide-MDD subjects.59-61 These findings provide further support for the hypothesis that the morphological and behavioral abnormalities associated with MDD could result, in part, from decreased BDNF expression. There are several possible mechanisms that could underlie the regulation of BDNF by stress. This includes a reduction of neuronal firing, as BDNF expression is dependent on activity and Ca2+-stimulated gene transcription.62 BDNF expression is also decreased by adrenal-glucocorticoids, which are induced by stress and activation of the hypothalamo-pituitary-adrenal (HPA) axis.57 There is also evidence that downregulation of BDNF by acute stress is mediated by interleukin-1 (IL1),63 and epigenetic regulation of BDNF expression in response to chronic social defeat stress.64 Genetic studies of BDNF and interactions with stress A relationship between BDNF, morphology, and behavior is supported by genetic studies of BDNF. Most of this work has focused on a functional polymorphism, Val66Met, that decreases the processing and release of BDNF.65 The Met allele has been associated with reduced hippocampal size and decreased memory and executive function in humans.65-67 The met allele has also been asso-

Neurotrophic/growth factors
The nerve growth factor (NGF) family has been the focus of much of the work on stress and depression, and the most widely studied member of this family is brain derived neurotrophic factor (BDNF). In addition, several other growth factors, including VEGF, IGF-1, and FGF2 have also been implicated in the effects of stress, depression, and ADT. Because these factors play a critical role in the proliferation, growth, and survival of neurons and glia in the adult brain, their altered expression or function could contribute to the cellular and morphological changes in animal models of depression and in MDD patients. This section will review key evidence demonstrating dysregulation of neurotrophic/growth factors in stress and depression. Role of BDNF in stress, depression, and ADT BDNF and related family members, including NGF and neurotrophin-3 (NT-3), influence the proliferation, differentiation, and growth of neurons during development, but are also expressed in the adult brain and play a critical role in the survival and function of mature neurons.56 BDNF is expressed at relatively high levels in limbic brain structures implicated in mood disorders, including the hippocampus, PFC, and amygdala, and acts through a transmembrane tyrosine kinase receptor referred to as TrkB. Functional BDNF acts as a dimer to stimulate the intracellular tyrosine kinase domain of TrkB, resulting in autophosphorylation of the receptor and interactions with docking proteins

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Antidepressant/exercise Stress Inammation Oxidative stress Aging IL-1

5-HT/NE VEGF/IGF1/FGF2 BDNF

GPCR AC Gs

Y IRS/FRS

Ras Sos Raf

Shc Grb2 Sos PI-3K IKK

Grb2 PI-3K

cAMP
MEK Akt PKA Akt IB ERK NFB IB

Degradation
GSK-3 Bad Rsk NFB

Neuroprotection, survival, resilience, antiapoptosis, proliferation

Apoptosis

CREB

Gene expression (BDNF, Bcl-2) Nucleus

Figure 3. Regulation of neurotrophic/growth factors signaling is decreased by stress and increased by antidepressant treatment. Activation of these pathways leads to neuroprotection, survival, resilience, Antiapoptosis, and proliferation of neurons and glia in limbic brain regions. 5-HT, 5hydroxytryptamine; NE, norepinephrine; GPCR, G protein coupled receptor; AC, adenylyl cyclase; Gs, stimulatory G protein; cAMP, cyclic adenosine 3,5-monophosphate; PKA, cAMP-dependent protein kinase; CREB, cAMP response element binding protein; VEGF, vascular endothelial growth factor; IGF-1, insulin-like growth factor-1; FGF2, fibroblast growth factor-2; BDNF, brain derived neurotrophic factor; IRS, insulin receptor substrate ; FRS, fibroblast growth factor receptor substrate; Grb2, adaptor protein; Sos, guanine nucleotide exchange factor; PI-3K, phosphatidylinositol 3-kinase; Akt, protein kinase B, serine.threonine kinase; GSK-3, glycogen synthase kinase-3; Bad, proapoptotic factor; Ras, small guanosine triphosphatase; Raf, serine/threonine kinase; MEK, ERK kinase; ERK, extracellular signal regulated kinase; Rsk, ribosomal S6 kinase; Shc, Src homology domain adaptor protein; IL-1b, interleukin-1b; IKK, kappaB inhibitory protein kinase; IkB, kappaB inhibitory protein; NFkB, nuclear factor kappaB; Bcl2, antiapoptotic factor

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ciated with smaller volume of cingulate cortex, and this effect is greater in patients with bipolar disorder.68 There are also reports that patients carrying the Met allele, either young or aged, have an increased incidence of depression when exposed to stress or trauma.69-71 These latter studies highlight the importance of gene x environment interactions in complex, multifactorial illnesses such as depression. Evidence for a direct relationship between the Met allele and neuronal structure has also been reported in rodent models. In mice expressing the Met allele there is a decrease in the number and length of apical dendrites in both the hippocampus72 and PFC,73 similar to the actions of stress.73,74 Although deletion of BDNF is not sufficient to produce depressive-like behaviors, except in female mice,75-77 blockade or reduction of BDNF expression increases the susceptibility to the effects of stress. Exposure of BDNF heterozygous deletion mutant mice to stress or blockade of BDNF-TrkB signaling produces a depressive-like phenotype in the forced swim test.78 A gene-environment interaction is also supported by the genetic association studies of the BDNF Met allele discussed above.70,71 A complicating factor in understanding the functions of trophic factors is the possibility that there are opposing, region-specific effects of BDNF. This is based on studies demonstrating that BDNF is increased by stress in the mesolimbic dopamine system and has a depressive effect in the social defeat model, and conversely, that ADT decreases BDNF in this reward pathway.79 These findings demonstrate that the expression and function of BDNF, and possibly other trophic factors, is circuitdependent and that findings in one region cannot be extrapolated to others. Antidepressants increase BDNF In contrast to the effects of stress, chronic, but not acute ADT increases the expression of BDNF in the hippocampus and frontal cortex.1,80-82 Induction of BDNF is observed with different classes of chemical antidepressants as well as electroconvulsive seizures.1,80,82,83 Other agents known to have antidepressant efficacy also increase BDNF expression in the hippocampus, including -amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor potentiators, NMDA receptor antagonists, transcranial magnetic stimulation, and exercise.1 These findings indicate that increased expression of

BDNF is a common target for different therapeutic strategies. Postmortem studies also demonstrate that BDNF levels are increased in the hippocampus of patients receiving antidepressant medication at the time of death, demonstrating the clinical relevance of ADT induction of BDNF.59 These effects are thought to occur via activation of cAMP and/or Ca2+-dependent BDNF gene transcription that are activated by ADT.84-86 Neuroprotective, neurogenic, and behavioral actions of BDNF The neuroprotective effects of BDNF have been well documented, primarily in cultured cell systems, but also in vivo. This includes studies demonstrating that BDNF increases survival and has neuroprotective actions in models of hypoxia, ischemia, excitotoxicity, hypoclycemia, and inflammation87-91; for reviews see refs 92,93. As discussed above, hippocampal pyramidal cell dendrite complexity is decreased in BDNF Met allele or heterozygous deletion mutants.74 Similar effects have been observed in PFC pyramidal cells, and stress does not produce further atrophy of apical dendrites in BDNF heterozygous deletion mutants, indicating that decreased BDNF underlies the effects of stress.73 These findings indicate that a full complement of functional BDNF is required for maintenance of normal dendritic arbor in both the hippocampus and PFC. BDNF has also been shown to influence hippocampal neurogenesis. Infusions of BDNF increase hippocampal neurogenesis,94,95 and BDNF is necessary for the survival of new neurons in response to ADT.96 The BDNF receptor, TrkB is also required for antidepressant induction of hippocampal neurogenesis, as well as the behavioral actions of antidepressants.97 BDNF has also been implicated in the behavioral actions of ADT. BDNF infusions are sufficient to produce an antidepressant response in rodent behavioral models of depression,98-100 and mutant mouse studies demonstrate that BDNF is required for the behavioral actions of antidepressants.75-77 These findings are consistent with the hypothesis that induction of BDNF contributes to the neurogenic and behavioral actions of antidepressants. Other neurotrophic/growth factors There is now strong evidence demonstrating a role for several other growth factors in the actions of stress,

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depression, and ADT, including vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF-1). VEGF was originally characterized as a vascular permeability factor and an endothelial cell mitogen,101 but is also expressed in the brain in both neurons and glia, and has been shown to play a role in hippocampal neuroplasticity, memory, and neurogenesis.102,103 Chronic unpredictable stress decreases the expression of VEGF, as well as its receptor, Flk-1,102 while ADT increases VEGF expression in the granule cell layer of the hippocampus.104 Different classes of chemical antidepressants, including SSRI, NSRI, and ECS, increase VEGF expression in the hippocampus, indicating that VEGF is a common downstream target of these treatments. The opposing actions of stress and ADT on VEGF suggest a possible relationship between neurogenesis and behavior. Stress has a greater effect on newborn cells associated with endothelial cells than nonvascular associated cells.102 In addition, VEGF is sufficient to induce neurogenesis and produce antidepressant effects in behavioral models of depression, whereas inhibition of Flk-1 blocks the induction of adult neurogenesis and the behavioral effects of ADT.104 A recent postmortem study found that the expression of FGF2 and its receptors (FGFR2 and FGFR3) are reduced in the PFC and cingulate cortex of MDD patients,105 and social defeat stress decreases FGF2 in the hippocampus.106 Conversely, chronic ADT increases the expression of FGF2 in cerebral cortex and hippocampus of rodents107,108 and FGF2 infusions are sufficient to produce an antidepressant response in behavioral models.109 The role of FGF2 in the proliferative actions of ADT, on both neurons and glia, is currently being investigated. The expression of IGF-1 in the hippocampus is increased by chronic administration of two different monoamine oxidase inhibitor antidepessants.110 In addition to expression in brain, circulating IGF-1, derived primarily from the liver, is actively transported into the brain and is required for the induction of neurogenesis in response to exercise.111 Recent studies have also demonstrated that IGF-1 administration, or agents that increase IGF-1 levels, produce antidepressant-like actions in behavioral models of depression.98,112,113 Together, these findings suggest that peripheral production and/or the central actions of IGF-1 could be novel targets for the treatment of depression. Neuroprotective and neurotrophic effects of exercise Exercise is reported to increase the expression of neurotrophic/growth factors, including BDNF, VEGF, FGF2, and IGF-1.111,114-117 In addition, exercise increases neurogenesis in the adult hippocampus, an effect that is dependent on increased expression of IGF-1 and VEGF.111,114 IGF-1 has also been shown to underlie the neuroprotective effects of exercise against different types of brain insults.118 In addition to the regulation of these growth factors, exercise has also been shown to influence other neuroprotective mechanisms.119 These positive, neuroprotective actions make exercise one of the key behavioral factors for protecting, or even reversing the damage that can be caused by environmental, physical, and psychological stressors, and even the susceptibility resulting from genetic vulnerabilities (see Figure 1).

Glutamatergic excitotoxicity: stress, depression, and ADT

Excess glutamatergic excitotoxicity is one of the major mechanisms underlying neuronal damage and loss in the brain, and has been implicated in the pathophysiology of a variety of disorders, including those resulting from acute insult (eg, stroke induced ischemia or trauma) and neurodegenerative disorders (eg, amyotrophic lateral sclerosis, Huntingtons chorea, epilepsy, and Alzheimers disease.120,121 This section discusses evidence for excess glutamate in stress related mood disorders, the cellular mechanisms that contribute to glutamate excitotoxicity, and pharmacological strategies for intervention and treatment. Excess glutamate in depression and stress Abnormal glutamate levels and function have been implicated in psychiatric illnesses, including schizophrenia, anxiety, and mood disorders.122-124 Glutamatergic abnormalities have been reported in the plasma, serum, cerebrospinal fluid (CSF), and brain tissue of individuals suffering from mood disorders.123 Functional in vivo measures of glutamate content in the brain using proton magnetic resonance spectroscopy (H-MRS) show elevated glutamate levels in the occipital cortex of depressed patients, although decreases have been reported in other regions.123,125

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Preclinical studies also demonstrate a role for glutamate in the actions of stress. Microdialysis studies have shown that stress increases extracellular levels of glutamate in the PFC and hippocampus,126,127 consistent with the possibility that atrophy of CA3 neurons arises in part through increased glutamate neurotransmission.128,129 This hypothesis is supported by studies demonstrating that N-methyl-D-aspartic acid (NMDA) receptor antagonists attenuate stress-induced atrophy of CA3-pyramidal neurons.29,32,130 Stress or glucocorticoid treatment also increases the susceptibility to other types of neuronal insults, including excitotoxins and ischemia.129,131 There are several possible mechanisms that could contribute to the overactivation of glutamate in response to stress and in depression, including a decrease or loss of mechanisms for inactivation of glutamate. Glial cells are responsible for the reuptake and inactivation of glutamate from synaptic and extrasynaptic sites.123 Reductions in the number or function of glia are thought to play a role in the atrophy of limbic brain regions observed in brain imaging studies, as well as decreased neuronal cell body size in postmortem brains of depressed patients.123,132,133 Recent studies demonstrate that agents that increase glial reuptake of glutamate, such as riluzole and ceftriaxone, have antidepressant effects in rodent behavioral models and in depressed patients.132-134 Mechanisms of glutamate excitotoxicity Glutamate neurotoxicity results from excessive flux of Ca2+ via ionoptopic receptors, including AMPA, kainiate, and NMDA type receptors.120,121,123 Uncontrolled elevation of intracellular Ca2+ leads to further loss of Ca2+ buffering and homeostasis, and then to a cascade of events that contribute to cell damage and death. These include oxidative stress resulting in generation of reactive oxygen species (ROS) and nitric oxide, which results in necrotic cell death characterized by swelling, membrane damage, DNA degradation, and eventually inflammation and cell lysis.120,121,135 There are multiple sites for controlling glutamate release and activity at pre- and postsynaptic sites, as well as for buffering intracellular Ca2+ that protects against cell damage. These mechanisms are typically overcome only by severe conditions, such as those that would occur during stroke-induced ischemia, prolonged hypoxia, uncontrolled seizures or head trauma. As discussed above, most studies do not report a loss of neurons in post-

mortem tissue from depressed patients, or in animal models. However, excess glutamate is still thought to play a role in psychiatric illnesses, and this has resulted in targeting glutamatergic sites for development of therapeutic agents for mood disorders, as well as for other psychiatric, neurological, and neurodegenerative illnesses. Glutamate and neuroprotection: therapeutic targets Glutamate neurotransmission is controlled by a complex system of pre- and postsynaptic receptors, including ionotropic and metabotropic subtypes. In addition, regulation of tropic factor signaling cascades, including extracellular signal-related kinase (ERK), Akt, and cAMP response element binding (CREB) can serve as neuroprotective targets for excitoxicity. There is also evidence that chronic ADT regulates the phosphorylation, trafficking, and expression of glutamate receptors, providing further evidence that the actions of ADT involves this neurotransmitter system. These topics have been extensively covered by a number of recent reviews.121,123,136,137 A brief discussion of the major glutamatergic targets will be discussed here. One of the key targets for regulation of glutamate is glial reuptake, which is the primary mechanism for inactivation of glutamate neurotransmission. Agents that increase this process, notably riluzole and ceftriaxone, are reported to have antidepressant efficacy in rodent models and in clinical trials.123,132,138 These effects are mediated in part by increased expression of glial excitatory amino acid transporters. Riluzole also has several other interesting properties, including the ability to decrease glutamate and increase neurotrophic factor expression, making this an interesting, and potentially useful therapeutic compound. Clinical and preclinical studies are currently underway to further test the therapeutic efficacy and mechanisms underlying the actions of riluzole. Lamotragine is another compound that acts in part by decreasing glutamate release and is used for treating mood disorders, although with limited efficacy.123 Blockade of the NMDA ionotropic receptor represents another primary target for neuroprotection, although this is a complex issue as glutamate is the major excitatory neurotransmitter in the brain. However, agents that block the NMDA channel, most notably memantine and ketamine, are reported to have antidepressant actions in clinical trials and rodents.123,137 The actions of memantine

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have been more modest, with greater effects when coadministered with other antidepressants. However, reports on ketamine have been extraordinary, with several studies demonstrating a rapid and sustained antidepressant response in approximately 60% of patients tested, which have all been resistant to other chemical antidepressants.139,140 A single intravenous dose of ketamine, which produces transient and mild psychotomimetic effects, results in an antidepressant response within 6 to 12 hours, and this effect is sustained for at least 7 days. These effects are dramatic compared with all other chemical antidepressants, which require weeks or months of treatment before a therapeutic response is observed. Further studies are needed to identify safer drugs that have rapid antidepressant effects similar to ketamine. The most direct mechanism to explain the antidepressant action of ketamine is its direct inhibitory effect on NMDA receptors. In particular, the hypothesis that blockade of the extrasynaptic NR2B receptor subtype, which is activated by excess glutamate, underlies the therapeutic action of ketamine has received the most attention. This possibility is supported by a recent study demonstrating that a selective NR2B receptor inhibitor, CP-101,606, produces a rapid antidepressant response in treatment resistant MDD patients.141 Another possible mechanism to account for the rapid actions of these agents is via blockade of NMDA receptors on GABAergic inhibitory neurons, which leads to disinhibition or activation of glutamatergic transmission. The latter possibility is supported by studies in rodents demonstrating that NMDA channel blockers increase BDNF expression in limbic structures, indicating stimulation of neuronal activity,142,143 and by a recent report that the behavioral actions of ketamine are blocked by inhibition of AMPA receptor activity.144 The metabotropic glutamate receptors represent another interesting and diverse set of targets for drug development.123,136 Group I receptors, particularly mGluR1/R5 subtypes located at postsynaptic sites as well as on glia, influence both the function and release of glutamate. Drugs acting at these receptors are reported to have anxiolytic effects in rodent models. Group II receptors, mGluR2 and R3, located at presynaptic sites and on glia and regulate glutamate release, have also been targets of interest. Both agonists and antagonists of group II receptors have shown promise, with reports that mGluR2/R3 antagonists have antidepressant actions and agonists showing anxiolytic and antipsychotic effects. Most promising is a clinical report demonstrating antipsychotic efficacy of an mGluR2/3 agonist.145 Allosteric AMPA receptor potentiator (ARP) agents make up another interesting group of drugs. These agents do not directly stimulate AMPA receptors, but slow the inactivation or desensitation of the receptors. The idea of using drugs that enhance AMPA receptor function would appear to be counterintuitive given the possibility of an overactive glutamate system. However, preclinical studies of these agents, which were first developed for enhancing cognition, demonstrate positive antidepressant-like effects in rodent models of depression.123,146

Programmed cell death (apoptosis) in stress and depression

Programmed cell death is a critical mechanism for regulation of the appropriate complement of neurons during development, but apoptotic signaling pathways are also regulated in the adult brain and influence the number and function of mature cells. Apoptosis is a highly regulated signaling process, which includes the Bcl-2 family of proteins, cytochrome C, a cytosolic adaptor protein, and caspase activation, which results in energy-dependent death.135,147 The Bcl-2 family includes antiapoptotic factors (ie, Bcl-2 and Bcl-xl) that antagonize proapoptotic factors (eg, Bax and Bak). Upon activation of apoptotic pathways, Bax and Bak insert into the mitochrondrial membrane and promote the release of cytochrome C, which in turn binds to the apoptotic activator factor (Apaf1), leading to activation of capases 9 and 3. Regulation of apoptosis by depression, stress, and ADT Analsysis of postmortem tissue and rodent models has provided some evidence for apoptotic cell death and/or signaling in depression and stress.135 There is a postmortem report of low levels of apoptosis in the temporal cortex and hippocampus of depressed patients. 148 Rodent studies demonstrate that social stress increases the number of apoptotic cells in the hippocampus and temporal cortex,149 and chronic unpredictable stress increases the number of caspase 3 positive neurons in the cerebral cortex.150 Maternal separation of rats is also reported to increase cell death in the dentate gyrus of hippocampus.151

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Genetic association studies have also provided evidence for a link between apoptosis signaling and depression. Polymorphisms of the adaptor protein Apaf1 were found to be associated with major depression.152 These polymorphisms increase the activity of caspase 9 and would thereby increase the vulnerability of neurons to apoptotic cell death. There are also several studies that have examined levels of apoptotic signaling proteins in models of stress and ADT. Chronic unpredictable stress is reported to decrease levels of the antiapoptotic factors Bcl-2 and Bcl-xl, but does not influence levels of Bax.153 Administration of a high dose of adrenal-glucocorticoids reduces Bcl-2 levels, and this effect corresponds with increased sensitivity to excitotoxic damage.154 Conversely, chronic ADT increases the expression of Bcl-2 and/or Bcl-xl in limbic brain regions.153,155,156 Chronic administration of lithium or valproate also increases Bcl-2 in the hippocampus and PFC.157 The antiapoptotic actions of lithium and valproate have also been demonstrated in studies of cultured cells.158-160 Antidepressants also influence other signaling cascades that indirectly influence apoptotic processes. Most notable are the effects of stress and ADT on neurotrophic factors and related signaling cascades, including ERK and Akt, which increase cell survival in part via inhibition of apoptotic, and induction of antiapoptotic factors such as Bcl-2.135,161 Finally, it is also notable that certain members of the Bcl-2 family have also been implicated in other cellular functions, including neurotransmission, which could be involved in the actions of stress and ADT.162 Mitochondria play a primary role in the storage, processing, and release of proteins involved in apoptosis, and recent studies demonstrate a role for other aspects of mitochondria function in the pathophysiology and treatment of mood disorders.154,163

tem, elevating cytokine production, and thereby stimulate inflammatory processes.164-167 Inflammatory and immune processes can lead to multiple actions that have acute protective actions, but that also can have damaging effects on cells and tissue. This includes many of the same actions implicated in the responses to stress and depression, including activation of the HPA axis, alterations of neurotransmitter systems, decreased neurotrophic factor expression, and increased oxidative stress.167 Depending on the severity and length of the inflammatory response, these effects can result in significant actions on neuronal and glial function and cell survival or death. There are several proinflammatory cytokines of interest, including IL-1, IL-6, and tumor necrosis factor (TNF), that have been implicated in the pathophysiology and treatment of depression. Also of interest are studies of interferon-, used for the treatment of hepatitis or cancer, which results in depressive-like symptoms in a large number of patients. Here we discuss a few of the most interesting targets for treatment of depression; for a more thorough review see ref 167. TNF and depression One of the most consistently altered proinflammatory cytokines in depressed subjects is TNF. An inverse correlation between levels of TNF and treatment response has been reported.168,169 TNF immunotherapy also causes depression, indicating that this cytokine may contribute to the etiology of mood disorders and is not simply a marker for depression (for reviews see refs 168,170). Moreover, a recent large clinical trial using an antibody neutralization approach demonstrated significant antidepressant effects of TNF reduction.171 This finding is supported by preclinical studies demonstrating that TNF infusions produce a prodepressive effect,172 and that TNF receptor null mutant mice have an antidepressant phenotype in the forced swim and sucrose consumption tests.173 Taken together, the preclinical and clinical studies provide strong support for TNF receptors, particularly TNFR2, as targets for the treatment of mood disorders. IL-1, stress, and depression There is also strong evidence that the proinflammatory cytokine IL-1 plays a key role in the pathophysiology of

Inflammation/immune responses
Inflammation and immune responses are major factors contributing to the etiology and pathophysiology of many medical illnesses, including depression and other psychiatric disorders. Inflammation can be caused by other medical conditions, including infection, stroke, trauma, and abnormal or autoimmune responses. However, it is also now clear that psychological stress, such as social stress, can activate the innate immune sys-

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stress and depression, and that the IL-1 signaling is a relevant target for drug development.174,175 These findings include: i) clinical studies reporting an increase in serum levels IL-1 in MDD176-180; ii) reports that IL-1 produces stress like effects, including activation of the HPA axis, regulation of monoamines, and behavioral responses in rodent models181; iii) evidence that IL-1 contributes to conditioned fear and depressive like behavior,182 and produces anhedonia and disrupts incentive motivation in rodent models183; iv) preclinical reports that IL-1 decreases hippocampal neurogenesis and underlies the decrease observed in response to stress184; v) our report that CUS-induced anhedonia and decreased neurogenesis produced by is blocked by pharmacological inhibition or null mutation of IL-1 receptors.184 Studies are currently underway to determine if blockade of peripheral, as well as central IL-1 signaling is sufficient to block the effects of stress and produce antidepressant actions. Interferon and IDO Recent studies demonstrate that one of the key factors contributing to the depressive actions of inflammation and activation of the innate immune system is the induction of a tryptophan degradative enzyme, indoleamine 2,3-dioxygenase (IDO). Chronic inflammation and infection can lead to sustained induction of interferon, which is then responsible for the increased levels of IDO. The induction of IDO then results in diversion of tryptophan from the synthesis of serotonin to kyneurenic acid, which can be further converted to toxic metabolites, most notably quinolinic acid. Evidence for IDO in depression is supported by studies demonstrating that decreased levels of tryptophan and increased kyneurenin is associated with inflammation and depression.185 Increased IDO has also been positively correlated with depression, although a direct causal relationship has not been demonstrated. A recent study has now provided direct evidence that induction of IDO underlies the depressive behaviors caused by inflammation/activated immunologic conditions. This work was conducted using a bacterial immune activation model, Bacille Calmette-Guerin (BCM), which induces a long-lasting induction of interferon and results in depressive behaviors in animal models.185,186 The results demonstrate that BCM-mediated immobility in the forced swim test is reversed by an IDO inhibitor, 1-methyltryptophan, and in mice that are deficient in IDO.185 In addition, BCM also increases the expression of a downstream enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO) that is involved in the synthesis of quinolinic acid. These studies indicate that an IDO inhibitor, and possibly an inhibitor of 3-HAO, could have efficacy for the treatment of depression and related mood disorders.

Summary and future directions

Significant advances have been made in characterizing the neuronal and glial damage, or structural alterations, at the cellular and anatomical levels in stress-related mood disorders and other psychiatric illnesses, and in elucidating the molecular signaling pathways and mechanisms that underlie these changes. However, this work is still at a relatively early stage, and a more complete characterization of these complex alterations and signaling mechanisms will require extensive resources and time. Moreover, identification of genetic polymorphisms that impact these pathways and systems and that influence susceptibility or resilience to illness is a major area of research that will continue to develop and unfold. When combined with studies of environmental risk factors and lifetime history of stress, this work will define and describe the mechanisms underlying individual variations of illness. Together, the results of this work can be used to formulate a comprehensive approach for the prevention and treatment of psychiatric illnesses. Changes in lifestyle and behavior can reduce stress and exposure to environmental factors that influence cellular risk and damage and prevent illness. These approaches, as well as behavioral interventions that enhance the activity and function of specific neural circuits, and thereby provide protection, can also be used once a person has become ill. Development of therapeutic agents that target neuroprotective mechanisms, combined with genetic information will ultimately provide tailored approaches for highly specific and efficacious treatments for depression and other illnesses.
Acknowledgements: This work is supported by USPHS grants MH45481 and 2 P01 MH25642 and by the Connecticut Mental Health Center. We would also like to thank Mr Xiaowe Su for assistance with literature research, and Drs G. Aghajanian and R-J. Li for providing images of PFC dendrites in Figure 2.

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Dao y proteccin neuronal en la fisiopatologa y el tratamiento de la enfermedad psiquitrica: el estrs y la depresin

El haber descubierto que el estrs y la depresin, al igual que otras enfermedades psiquitricas, se caracterizan por alteraciones estructurales, y que estos cambios se deben a atrofia y prdida de neuronas y gla en regiones y circuitos lmbicos especficos ha contribuido a un cambio fundamental en la comprensin de estas enfermedades. Estos cambios estructurales se acompaan de una falta de regulacin de los mecanismos de seales neuroprotectoras y neurotrficas, los cuales son requeridos para la maduracin, crecimiento y supervivencia de las neuronas y la gla. A la inversa, las intervenciones conductuales y teraputicas pueden revertir estas alteraciones estructurales mediante la estimulacin de las vas neuroprotectoras y neurotrficas y a travs del bloqueo de los efectos dainos, excitotxicos e inflamatorios del estrs. La exposicin a lo largo de la vida a estresores celulares y ambientales, y las interacciones con factores genticos contribuyen a la susceptibilidad o a la resiliencia. Esta interesante rea de investigacin abriga potenciales esperanzas para favorecer la dilucidacin de la fisiopatologa de las enfermedades psiquitricas y el desarrollo de nuevas intervenciones teraputicas. REFERENCES
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Lsions et protection neuronales dans la physiopathologie et le traitement des maladies psychiatriques : stress et dpression
Notre comprhension du stress et de la dpression, comme dautres maladies psychiatriques, a t profondment transforme en dcouvrant que ces maladies sont caractrises par des modifications structurales rsultant de latrophie et de la perte des neurones et de la glie dans des rgions et des circuits limbiques spcifiques. Ces modifications structurales saccompagnent dune dysrgulation des mcanismes de signalisation neurotrophiques et neuroprotecteurs ncessaires la maturation, la croissance et la survie des neurones et de la glie. loppos, des interventions comportementales et thrapeutiques peuvent inverser ces modifications structurales en stimulant les voies neuroprotectrices et neurotrophiques et en bloquant les effets lsionnels, excitotoxiques et inflammatoires du stress. Lexposition durant la vie aux agents stressants cellulaires et environnementaux et les interactions avec des facteurs gntiques participent la susceptibilit individuelle ou la rsilience. Ce domaine de recherche passionnant est prometteur et devrait permettre d' expliquer plus prcisment la physiopathologie des maladies psychiatriques et de participer au dveloppement de nouveaux traitements.
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Chromatin regulation in drug addiction and depression
William Renthal, PhD; Eric J. Nestler, MD, PhD

Alterations in gene expression are implicated in the pathogenesis of several neuropsychiatric disorders, including drug addiction and depression. Increasing evidence indicates that changes in gene expression in neurons, in the context of animal models of addiction and depression, are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. This review discusses recent findings from behavioral, molecular, and bioinformatic approaches that are being used to understand the complex epigenetic regulation of gene expression in brain by drugs of abuse and by stress. These advances promise to open up new avenues for improved treatments of these disorders.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:257-268.

Keywords: chromatin remodeling; histone acetylation; histone methylation; DNA methylation; BDNF; glucocorticoid receptor; cocaine; stress Author affiliations: Medical Scientist Training Program, The University of Texas Southwestern Medical Center, Dallas, Texas, USA (William Renthal); Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA (Eric J. Nestler) Address for correspondence: Eric J. Nestler, Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA (e-mail: eric.nestler@mssm.edu) Copyright 2009 LLS SAS. All rights reserved

amily history is one of the greatest risk factors for psychiatric disorders, yet their genetic basis remains poorly understood despite substantial advances in whole genome sequencing techniques. While the search for genetic mutations continues at a rapid pace, the field is also investigating the environmental component of family history, which has remained more difficult to explain mechanistically. One hypothesis is that environmental stimuli alter gene expression patterns in certain brain regions that ultimately change neural function and behavior. Support for this hypothesis has been observed in animal models of psychiatric illness, as well as in human patients. The interactions between the environment and the genes that give rise to specific phenotypes are termed epigenetic.1 An example of this process is observed in cellular differentiation, where unique chemical signals induce totipotent stem cells to differentiate into genetically identical cell types with vastly different functions. This is due in part to the vastly different sets of genes expressed between distinct cell types (eg, neurons vs hepatocytes), despite their identical DNA templates. Mechanistic insight into this process has recently been uncovered, and involves the transduction of unique environmental signals into precise and highly stable alterations in chromatin structure that ultimately gate access of transcriptional machinery to specific gene programs, thereby providing unique gene expression profiles in response to specific environmental cues.2 Importantly, many of these chromatin remodeling mechanisms are highly stable, contributing to the maintenance of specific gene expression programs in the correct tissues throughout the life of an individual. The strong control exerted by chromatin remodeling on gene expression, and the potential stability of chromatin
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Selected abbreviations and acronyms
BDNF cAMP CREB H HAT HDAC HDM HMT brain derived neurotrophic factor cyclic adenosine monophosphate cAMP-response element binding protein histone histone acetyltransferase histone deacetylase histone demethylases histone methyltransferase mechanisms, make chromatin regulation a prime candidate for mediating aspects of the long-lasting neural plasticity that ultimately results in psychiatric syndromes. It is also interesting to note that certain neurological and psychiatric diseases are caused by rare genetic mutations in chromatin remodeling enzymes (Table I). While these mutations are rare, they directly illustrate how disruption of chromatin regulation can profoundly affect neural function and lead to complex behavioral abnormalities. Thus, epigenetic research in psychiatry is aimed
Clinical features Autosomal dominant inheritance Mental retardation Abnormal facial features, blunted growth X-linked inheritance Most common inherited form of mental retardation, signs of autistic behavior Macrocephaly, long and narrow face with large ears, macro-orchidism, hypotonia X-linked inheritance Psychomotor retardation Craniofacial and skeletal abnormalities X-linked, affecting predominantly girls Pervasive developmental disorder associated with arrested brain development, cognitive decline, and autistic-like behavior X-linked inheritance Mental retardation Hemolytic anemia, splenomegaly, facial, skeletal, and genital anomalies Autosomal recessive Mild mental retardation Marked immunodeficiency, facial anomalies Autosomal dominant Mild mental retardation, myotonia, abnormal cardiac conduction, insulin-dependent diabetes, testicular atrophy, premature balding Imprinting+mutation Mild mental retardation, endocrine abnormalities Imprinting+mutation Cortical atrophy, cerebellar dysmyelination, cognitive abnormalities

Disease Rubinstein-Taybi syndrome

Chromatin defect Heterozygous mutations in CBP

Fragile X syndrome

Hypermethylation of DNA at the FMR1 and FMR2 (Fragile X mental retardation-1,2) promoters, caused by trinucleotide repeat expansion

Coffin-Lowry syndrome

Rett syndrome

Mutation in RSK2 (ribosomal S6 kinase-2), which can interact with CREB and CBP and can phosphorylate H3 in vitro Mutations in MeCP2

Alpha-thalassemia/mental retardation syndrome, X-linked (ATR-X)

Immunodeficiencycentromeric instabilityfacial anomalies syndrome (ICF) Myotonic dystrophy

Prader-Willi syndrome Angelman syndrome

Mutations in ATRX gene, encoding the X-linked helicase-2 (XH2) a member of SWI/SNF family of proteins Defective chromatin remodeling thought to downregulate the -globin locus Mutations in Dnmt3B Hypomethylation at centromeric regions of chromosomes 1, 9, and 16 Abnormal CTG repeat expansion at the 3UTR of the DM1-Protein Kinase gene favors chromatin condensation, affecting expression of many neighboring genes Imprinting (DNA methylation) of maternal chromosomal region 15q11-13 Imprinting (DNA methylation) of paternal chromosomal region 15q11-13

Table I. Examples of diseases of chromatin remodeling. CBP, CREB binding protein; CREB, cyclic AMP-response element binding protein; DMPK, DM1 protein kinase; Dnmt3B, DNA methyltransferase 3B; FMR1, fragile X mental retardation protein 1; MeCP2, methyl-CpG-binding protein 2; RSK2, ribosomal S6 kinase 2; SWI/SNF, mating switching and sucrose non-fermenting complex; UTR, untranslated region; XH2, Xlinked helicase 2

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at identifying whether environmental stimuli induce changes in chromatin structure which ultimately contribute to transcriptional programs in neurons that cause psychiatric illness, much in the same way as environmental cues differentiate a stem cell into specific lineages. While this field is still in its infancy, great progress is being made in identifying epigenetic alterations in many neuropsychiatric syndromes, including drug addiction, depression, schizophrenia, Alzheimers disease, and Rett syndrome, among others. Focusing on drug addiction and depression, this review briefly discusses the molecular machinery underlying epigenetic mechanisms in brain, and how their dysregulation may contribute to these chronic psychiatric illnesses.

the apparent stability of some epigenetic mechanisms in vivo, all types of chromatin modifications identified to date are potentially reversible and have specific enzymes or processes which mediate the addition or removal of each mark.6 The in vivo mechanisms which maintain easily reversible histone modifications (eg, acetylation) on some genes or delete highly stable marks (eg, DNA methylation) on other genes are currently not clear. Histone acetylation Acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and DNA, which relaxes chromatin structure and improves access of transcriptional regulators to DNA (Figure 1).6 Genome-wide studies indicate that high levels of histone acetylation in gene promoter regions are generally associated with higher gene activity, while low levels of acetylation correlating with reduced gene activity. 9 Most genome-wide studies of histone acetylation have focused on acetylation of the N-terminal lysine residues in histones H3 and H4, but histone acetylation can occur on other histone proteins as well as in their globular domains. Histone acetylation is a dynamic process, controlled by specific enzymes which either add or remove the acetyl mark. There are over a dozen known histone acetyltransferases (HATs) which catalyze the addition of acetyl groups onto lysine residues of histones with varying degrees of specificity. Many HATs can also acetylate nonhistone proteins such as transcription factors (eg, p53), and some transcription factors (eg, ATF2 [activating transcription factor 2], CLOCK) even possess intrinsic HAT activity that contributes to gene activation.10,11 Histone deacetylases (HDACs), which remove acetyl groups from histones, are divided into four classes. Class I HDACs (eg, HDAC1, 2, 3, and 8) are ubiquitously expressed and likely mediate the majority of deacetylase activity within cells. Class II HDACs (eg, HDAC4, 5, 7, 9, 10) are only expressed in specific tissues such as heart and brain and are much larger enzymes that also contain an N-terminal regulatory domain that enables them to be shuttled in and out of the nucleus in a neural activitydependent manner.12 While Class II HDACs can deacetylate histones, they are much less efficient enzymes than Class I HDACs, and may also deacetylate other cellular substrates.13,14 There is currently one Class IV HDAC, HDAC11, and it has characteristics of both

Epigenetic mechanisms
Chromatin is the complex of DNA, histones, and associated nonhistone proteins in the cell nucleus. DNA wraps around histone octamers made up of two copies of histone H2A, H2B, H3, and H4,3 which then supercoil to form a highly condensed structure (Figure 1). Initially, it was thought that this elaborate chromatin structure only functioned to condense meters of DNA into the microscopic cell nucleus, but it is now known to participate directly in gene regulation. Because DNA is tightly associated with histones and often embedded deep within chromatin supercoils,4,5 cellular mechanisms exist to modify and remodel chromatin structure to allow for the coordinated expression of specific transcriptional programs and the silencing of others.6 Such modifications typically occur on N-terminal histone tails and include acetylation, phosphorylation, methylation, or several other covalent modifications of histones, methylation of DNA, and many others, with each modification either directly altering histone-DNA interactions or serving as a mark that recruits specific proteins to positively or negatively regulate the underlying genes activity. Ultimately, dozens of potential modifications that occur at many distinct histone residues summate to determine the final transcriptional output of a given gene.7 As mentioned earlier, genetic mutations in many of these chromatin remodeling enzymes are associated with severe neurological and psychiatric disorders (see Table I). An especially important aspect of certain chromatin modifications is their apparent stability, as is seen with genetic imprinting or X-inactivation, where DNA methylation contributes to lifelong gene silencing.8 However, despite

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A
Histone tail

DNA H2B H4 H3 H2A

Histone

A M P

Acetylation Methylation Phosphorylation Permissive

B Active
Histones A Histone Transcription factor tail + A A P

M A A

Co-Act DNA

A Basal transcription complex

Inactive P Rep M A M

P Rep M

M Repressed M M Rep Rep M M A ? M M Rep M M

Rep M M M Demethylation HDM Methylation (activating) HMT HDM

Demethylation HMT Methylation (repressing)

M
H3

M
K9

M M
S1 0

K4

A P Acetylation (activating) HAT Deacetylation HDAC

4 K1
A

8 K1
A

K23
A

K27

S28

K36

K79
Histone tail

Phosphorylation (activating) PK Dephosphorylation PP

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Class I and Class II enzymes.15 Class III HDACs (also referred to as sirtuins) are mechanistically distinct from the other HDACs, and have been implicated in the regulation of lifespan and metabolism.16 The individual functions of each HDAC remain an active topic of investigation. Histone phosphorylation Histone phosphorylation is generally associated with transcriptional activation; it can be observed on the promoters of immediate early genes such as c-fos when they are induced after cyclic adenosine monophosphate (cAMP) stimulation or glutamate treatment in cultured striatal neurons.17,18 One of the best-characterized histone phosphorylation sites is serine 10 on histone H3 (H3S10). This modification stabilizes the HAT, GCN5, on associated gene promoters while antagonizing the repressive modificationmethylation of lysine 9 on histone H3 (H3K9) and its subsequent recruitment of HP1 (heterochromatin protein 1, see below). 6 Since phosphorylation at H3S10 recruits a HAT, the neighboring lysine residue at H3K9 is often acetylated in concert with phosphorylation, a process called phosphoacetylation that further potentiates gene activation. There are several nuclear protein kinases and protein phosphatases known to regulate histone phosphorylation.6 The mitogen-activated protein kinase, MSK1, and the dopamine and cyclic-AMP regulated protein phosphatase inhibitor, DARRP-32, are elegant examples shown to regulate H3S10 phosphorylation in the adult brain in response to cocaine exposure.19,20 Furthermore, genetic disruption of the histone-modifying ability of MSK1 or DARRP-32 in vivo has dramatic effects on behavioral responses to cocaine. Thus, histone phospho-

rylation likely plays an important role in the regulation of brain function. Histone methylation Histone methylation generates unique docking sites that recruit transcriptional regulators to specific gene loci. Histone methylation occurs on lysine residues in mono-, di-, or trimethylated states, enabling each state to recruit unique coregulators and exert distinct effects on transcriptional activity.6 Additionally, methylation of different histone lysine residues can exert opposite effects on transcription. In gene promoter regions for example, trimethylation of H3K4 is highly associated with gene activation, whereas trimethylation of H3K9 or H3K27 is repressive.5 The repression caused by trimethylation of H3K9 is mediated in part via the recruitment of corepressors, such as HP1, as stated earlier. However, even this is an oversimplification, as methylated H3K9 is often found in the coding region downstream of a gene promoter and may be involved in transcriptional elongation.6,21 Thus, histone methylation provides each cell with exquisite control over an individual genes activity through numerous combinatorial possibilities. Histone methyltransferases (HMTs) add methyl groups to specific lysine residues of histones, and histone demethylases (HDMs) remove them (Figure 1). Like HATs and HDACs, HMTs and HDMs also have activity towards nonhistone proteins.6 HMTs and HDMs not only discriminate between various histone lysine residues, but each enzyme is also unique in its ability to catalyze mono-, di-, or trimethylation or demethylation at that site.6 For example, the HMT, KMT1C (G9a), is specific for histone H3K9 but only adds 1 or 2 methyl groups, with the distinct HMT, KMT1A (SUV39H1),

Figure 1. Chromatin remodeling. A. Picture of a nucleosome showing a DNA strand wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. The amino (N) termini of the histones face outward from the nucleosome complex. B. Chromatin can be conceptualized as existing in two primary structural states: as active, or open, euchromatin (top left) in which histone acetylation (A) is associated with opening the nucleosome to allow binding of the basal transcriptional complex and other activators of transcription; or as inactive, or condensed, heterochromatin where all gene activity is permanently silenced (bottom left). In reality, chromatin exists in a continuum of functional states in between (eg, active; permissive (top right); repressed (bottom right); and inactive). Enrichment of histone modifications such as acetylation and methylation (M) at histone N-terminal tails and related binding of transcription factors and coactivators (CoAct) or repressors (Rep) to chromatin modulates the transcriptional state of the nucleosome. Recent evidence suggests that inactivated chromatin may in some cases be subject to reactivation in adult nerve cells, although this remains uncertain. C. Summary of common covalent modifications of H3, which include acetylation, methylation, and phosphorylation (P) at several amino acid residues. H3 phosphoacetylation commonly involves phosphorylation of S10 and acetylation of K14. Acetylation of lysine residues is catalysed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs); lysine methylation (which can be either activating or repressing) is catalyzed by histone methyltransferases (HMTs) and reversed by histone demethylases (HDMs); and phosphorylation is catalysed by protein kinases (PK) and reversed by protein phosphatases (PP), which have not yet been identified with certainty. K, lysine residue; S, serine residue.
From ref 8: Tsankova N, Renthal W, Kumar A, Nestler EJ. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci. 2007;8:355-367.

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catalyzing trimethylation of this site. Similarly, the HDM, KDM3A (JHDM2a), can demethylate 1 or 2 methyl groups on H3K9, requiring a distinct demethylase, eg, KDM4D (JMJD2D) to fully demethylate the trimethylated state. Thus, large complexes of enzymes are required to move between the unmethylated and fully trimethylated states. Proper balance of histone methylation has already been strongly implicated in normal brain function, as the HDM, KMT5C (SMCX), controls dendritic spine density and is mutated in patients with mental retardation.22,23 DNA methylation DNA methylation refers to the enzymatic methylation of cytosine bases, a fundamental cellular process required for development, tissue-specific gene expression, X-inactivation, and genetic imprinting, to name a few examples.24 DNA methylation is thought to repress gene expression by interfering with the binding of transcription factors to their target sequences or by initiating the recruitment of corepressors. For example, the cAMP-response element (CRE) contains a cytosineguanine dinucleotide in the middle of its consensus sequence, which, when methylated, prevents the transcription factor CRE-binding protein (CREB) from binding.25 Thus, for genes at which CREB is necessary to initiate transcription, methylation at this site is repressive. Methylated DNA can also recruit methyl-binding domain-containing proteins, such as MeCP2, which can then recruit and stabilize transcriptional corepressors such as HDACs on specific gene promoters. Mutations in MeCP2 cause the autistic spectrum disorder, Rett syndrome, illustrating the importance of DNA methylation in normal brain development.26 While there is a strong correlation between methylated DNA and repressed gene activity, recent studies of MeCP2 indicate it may also serve to activate gene activity under some circumstances,27 suggesting that the context in which DNA methylation occurs is an important factor in its ultimate effect on transcription. There are three known enzymes which catalyze DNA cytosine methylation: DNMT1, DNMT3a, and DNMT3b. DMNT2 was recently shown to methylate RNA rather than DNA.28 Together, these enzymes establish and maintain the unique methylation patterns that exist within each cell type. While the regulation of these enzymes in brain remains unclear, pharmacological inhibition of DNA methylation in the brain in vivo results in rapid demethylation of specific gene targets and severe deficits in learning and memory.29 The mechanism by which this occurs, however, remains unclear because, unlike other chromatin modifications, the existence of DNA demethylases remains controversial.30 Nevertheless, regulation of DNA methylation by environmental stimuli remains an attractive mediator of long-lasting changes in transcription in adult neurons.

Epigenetic mechanisms in drug addiction

Drug addiction is a chronic relapsing disorder where motivation to seek and take drugs of abuse becomes compulsive and pathological.31 The process by which repeated drug experimentation transitions into a chronically addicted state is the focus of intense research, as clues into these mechanisms may help better manage or perhaps fully treat addicted patients. Another avenue of intense research focuses on the mechanisms driving drug relapse, which occurs even after long periods of drug abstinence and is a major clinical challenge for successful treatment. The exciting new possibility that druginduced alterations in chromatin structure may contribute to long-lasting behavioral changes provides a new avenue for novel therapeutics that improve drug rehabilitation. The first studies to implicate changes in chromatin structure in responses to drugs of abuse found that acute administration of cocaine rapidly increased histone H4 acetylation on the immediate early genes c-fos and fosB in striatum,32 two genes known to play a critical role in cocaine-related behaviors.33 The histone acetyltransferase CBP appears to be required for the drug-induced acetylation of the fosB promoter, and probably many other, yet to be identified genes as well.34 Interestingly, despite several control gene promoters where acute cocaine does not affect histone acetylation, an acute cocaine dose increases total levels of histone H4 acetylation, and histone H3 phosphoacetylation in striatum, as measured by Western blotting.19,32 These global increases in histone acetylation, which are also observed in response to environmental enrichment and tests of learning and memory,35,36 may be accounted for by high levels of acetylation on specific subsets of genes. This is likely, as global increases in histone K9 methylation, a repressive histone modification, are also observed after cocaine exposure37 and appear to occur on unique subsets of genes.38

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The promoters of certain genes induced by chronic cocaine exposure are hyperacetylated for days to weeks after the last drug exposure (Figure 2). For example, the expression of cdk5 (cyclin-dependent kinase 5), bdnf (brain derived neurotrophic factor),32 npy (neuropeptide Y),39 and sirt1 and sirt2 (two subtypes of sirtuins), among many other genes,38 were found to be upregulated after chronic cocaine administration and their gene promoters hyperacetylated, while egr-1 (early growth response 1) was found to be downregulated and hypoacetylated

after cocaine withdrawal.39 Moreover, altered expression of each of these genes has been shown to contribute to the addiction behavioral phenotype. These findings suggest a role of histone acetylation in the maintenance of gene expression involved in drug addiction, including drug withdrawal and relapse. Cocaine-induced alterations in chromatin structure in the nucleus accumbens (NAc), the ventral portion of striatum heavily implicated as a brain reward region, have been shown to regulate behavioral responses to
Cocaine/amphetamine

Cytoplasm

HDMs? DNMT? CaMK P HDAC 5

cAMP

MAPK/ERK

PKA

RSK/MSK1

CaMK

Nucleus

HMT

DARPP32

PP1
+ HDAC 5 + P CREB CBP + HDAC 1 FosB P CREB CBP P H3S10 +

Cdk5, BDNF, NPY

NK1R

FosB

c-F os

Figure 2. Regulation of chromatin structure by drugs of abuse. Drug-induced signaling events are depicted for psychostimulants such as cocaine and amphetamine. These drugs increase cAMP levels in striatum, which activates protein kinase A (PKA) and leads to phosphorylation of its targets. This includes the cAMP response element binding protein (CREB), the phosphorylation of which induces its association with the histone acetyltransferase, CREB binding protein (CBP) to acetylate histones and facilitate gene activation. This is known to occur on many genes including fosB and c-fos in response to psychostimulant exposure. FosB is also upregulated by chronic psychostimulant treatments, and is known to activate certain genes (eg, cdk5) and repress others (eg, c-fos) where it recruits HDAC1 as a corepressor. This repression of c-fos also involves increased repressive histone methylation, which is thought to occur via the induction of specific histone methyltransferases (HMTs). In addition, cocaine regulates the HMT, KMT1C/G9a, which alters histone H3 methylation on K9. It is not yet known how cocaine regulates histone demethylases (HDM) or DNA methyltransferases (DNMTs). Cocaine also activates the mitogen activated protein kinase (MAPK) cascade, which through MSK1 can phosphorylate CREB and histone H3 at serine 10. Cocaine promotes H3 phosphorylation via a distinct pathway, whereby PKA activates protein phosphatase 2A, leading to the dephosphorylation of serine 97 of DARPP32. This causes DARPP32 to accumulate in the nucleus and inhibit protein phosphatase-1 (PP1) which normally dephosphorylates H3. Chronic exposure to psychostimulants increases glutamatergic stignaling from the prefrontal cortex to the NAc. Glutamatergic signaling elevates Ca2+ levels in NAc postsynaptic elements where it activates CaMK (calcium/calmodulin protein kinases) signaling, which, in addition to phosphorylating CREB, also phosphorylates HDAC5. This results in nuclear export of HDAC5 and increased histone acetylation on its target genes (eg, NK1R [NK1 or substance P receptor]).
From ref 8: Tsankova N, Renthal W, Kumar A, Nestler EJ. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci. 2007;8:355-367.

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drugs of abuse. Pharmacological inhibition of HDACs in the NAc, which increases histone acetylation in this brain region, significantly potentiates the locomotor-activating and rewarding responses to cocaine. 32,40,41 Conversely, reducing histone acetylation by overexpressing certain HDACs, or knockdown of the HAT, CBP, results in less sensitivity to cocaine.32,34,40 Two reports have extended these findings in rat models of cocaine self-administration, where animals are trained to press levers to receive the drug. Interestingly, delivery of the HDAC inhibitor, sodium butyrate, potentiates drug-taking42 while delivery of the HDAC inhibitor, trichostatin A, attenuates it.43 The explanation for these different observations is unclear, but it may involve experimental differences with the self-administration paradigm or the HDAC inhibitor used. Cocaine alters histone acetylation through many enzymes in the NAc, but one particular HDAC, HDAC5, responds uniquely to chronic cocaine administration, raising the interesting possibility that this HDAC is involved in the behavioral transitions which occur between acute and chronic cocaine exposure (eg, drug experimentation to compulsive drug use). Chronic cocaine administration increases the phosphorylation of HDAC5 and shuttles it out of the nucleus, permitting hyperacetylation of histones at target genes for HDAC5 (Figure 2).40 This phosphorylation reaction may be mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), since ex vivo inhibition of CaMKII reduces the activity-induced phosphorylation of HDAC5. Consistent with its regulation by cocaine, mice deficient for HDAC5 display normal rewarding responses to initial cocaine exposures, but become hypersensitive when treated with a chronic course of cocaine.40 Thus, pharmacological and genetic manipulations that increase histone acetylation appear to potentiate behavioral responses to cocaine and suggest that altered histone acetylation may contribute to establishment of an addicted state. Histone H3 phosphorylation and phosphoacetylation also appear to play key roles in drug-regulated behaviors. Global levels of histone H3 phosphorylation at serine 10 are induced by acute cocaine in striatum, a process which requires the kinase MSK1.19,32 The function of MSK1 is behaviorally important, as mice lacking this kinase have attenuated locomotor responses to cocaine. Cocaine-induced inhibition of protein phosphatase-1 also plays an important role in H3 phosphorylation in striatum (Figure 2). Dopamine D1 receptor activation alters the phosphorylation of dopamine-regulated and cyclic-AMP-regulated phosphoprotein of 32kD (DARPP-32) at particular serine residues; the protein then accumulates in the nucleus to inhibit protein phosphatase-1 from dephosphorylating histone H3.20 The simultaneous activation of an H3 kinase and inhibition of an H3 phosphatase results in the robust increase in H3 phosphorylation after acute cocaine exposure. The genes at which histone phosphorylation is occurring in response to cocaine remain poorly defined with an exception of c-fos, where dramatic histone phosphorylation occurs in conjunction with acetylation (phosphoacetylation). As mentioned earlier, global histone methylation of H3K9 is also regulated by cocaine and, in turn, alters behavioral responses to the drug. For example, inhibition of a particular H3K9 histone methyltransferase, KMT1C (G9a), whose expression is regulated in the NAc by chronic cocaine administration, potentiates behavioral responses to the drug. 37 These findings are consistent with histone acetylation findings, since inhibition of H3K9 methylation would also be expected to enhance gene activity. Together, these data suggest that, in general, increases in gene expression potentiate behavioral sensitivity to drugs of abuse. As well, advances are being made in identifying the individual gene promoters where chronic cocaine induces alterations in H3K9 methylation and thereby regulates gene expression in the NAc.37 Overall, these findings implicate changes in histone acetylation, phosphorylation, and methylation in mediating expression changes in specific sets of genes that are crucial for controlling behavioral responses to drugs of abuse.

Epigenetic mechanisms in depression

Depression is a chronic disorder characterized by many debilitating symptoms including dysphoria, anhedonia, sleep disturbances, and weight changes. Most people diagnosed with depression are prescribed some type of antidepressant medication, of which selective serotonin reuptake inhibitors (SSRIs) or mixed serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most common. Unfortunately, less than 50% of patients exhibit a complete response to SSRIs, SNRIs, or related antidepressants, thus leaving a substantial portion of depressed

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patients with a chronic syndrome for which few effective clinical alternatives are available. Psychiatric research is thus focused on identifying new mechanisms that are involved in the pathogenesis and maintenance of depression, which may serve as novel targets for more effective therapeutics. One of the most challenging obstacles for depression research has been the development of an animal model that accurately recapitulates human depression. While no model can effectively model all aspects of human depression (eg, suicide), some of the major symptoms such as anhedonia and sleep and weight disturbances, and their reversal by antidepressant treatment, can be studied in rodents. The pathogenesis of depressed-like states is typically modeled in rodents by chronic exposure to stress.44 One such model, chronic social defeat stress, involves the repeated exposure of an experimental mouse to a series of aggressive mice over 10 days. Each day the stress begins as a brief physical encounter (typically 5 to 10 minutes) followed by a full day of sensory contact (eg, smell, sight) as the mice are separated by a screen. After 10 days of social defeat, the experimental mice develop a chronic syndrome (lasting more than a month) that is characterized by anhedonia, anxiety-like symptoms, weight loss, and loss of interest in social interaction. Importantly, SSRIs or SNRIs reverse most of these behavioral end points, making chronic social defeat stress an attractive model in which to study the molecular adaptations associated with a depressed-like state and those involved with antidepressant action.45,46 Brain derived neurotrophic factor (BDNF) plays a critical role in the development of the social defeat phenotype and its reversal by antidepressant treatment. It was observed that BDNF in the hippocampus is downregulated for at least 1 month after chronic social defeat stress, and that chronic antidepressant treatment reversed this downregulation.46 A mechanism for this long-lasting regulation of gene expression was identified as methylation of H3K27, a repressive histone modification, that remains hypermethylated on the bdnf promoter within hippocampus for at least a month after defeat stress. While chronic antidepressant treatment of mice exposed to chronic social defeat ameliorates many of the behavioral deficits and restores bdnf mRNA to normal levels, H3K27 remains hypermethylated. The maintenance of H3K27 methylation even after chronic antidepressant treatment suggests that BDNF expression might revert to a repressed state if drug adminis-

tration were stopped. This novel epigenetic mechanism, which was proposed as a form of molecular scar, may describe a potential mechanism by which the symptoms of depressed patients reappear after cessation of antidepressant treatment, however, this remains speculative and further research is needed. The recovery of bdnf expression after antidepressant treatment is likely mediated by the antidepressantinduced increase in histone H3K4 methylation and H3 polyacetylation in hippocampus, which are associated with gene activation.46 Interestingly, tranylcypromine, which inhibits monoamine oxidases and is used as an antidepressant, is actually a much stronger inhibitor of the histone H3K4 demethylase KMT1A (formerly, LSD1) than it is of either monamine oxidase A or B.47 Thus, it will be interesting to determine whether any of the antidepressant properties of tranylcypromine derive from its blockade of KMT1A and the subsequent facilitation of H3K4 methylation. Arguing against this interpretation is the knowledge that several structurally unrelated monoamine oxidase inhibitors, which have not been shown to inhibit histone demethylases, are still effective antidepressants. The increase in H3 acetylation by antidepressant treatment suggested that HDAC inhibitors may also have antidepressant-like effects. Indeed, in both the chronic social defeat model and in the forced swim test, HDAC inhibitors demonstrated antidepressant-like prosperities.46,48 This was especially apparent when an HDAC inhibitor was administered in addition to an SSRI, fluoxetine. While these inhibitors target numerous HDACs, one specific isoform, HDAC5, stood out because it was oppositely regulated by stress and antidepressant treatment.46 Indeed, overexpression of HDAC5 in the hippocampus blocks the behavioral effects of chronic antidepressant treatment, suggesting that increased histone acetylation on the bdnf promoter is a key mechanism to overcome the repressive effects of H3K27 methylation. Another intriguing aspect of chronic social defeat stress is that the severity of the depression-like phenotype varies within a cohort of inbred (ie, virtually genetically identical) mice. It was observed that mice susceptible to defeat stress show significantly higher firing rates of dopaminergic neurons in the ventral tegmental area (VTA) after stress exposure compared with resilient mice. These resilient mice had normal VTA firing rates because of a stress-induced upregulation of potassium channels in this brain region. Why do certain mice

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upregulate protective potassium channels in the VTA while others fail to do this and become depressed? Perhaps an epigenetic mechanism is involved in altering the promoters of certain potassium channels to ultimately determine if the gene will be induced in response to chronic stress. If so, which life experiences trigger these chromatin remodeling events? These are important questions that may shed fundamentally new light onto an extraordinarily complex syndrome, and provide new avenues for the development of more effective antidepressants. Another important epigenetic mechanism that may contribute to long-lasting changes in neural function and behavior is DNA methylation. Early insight into the role of DNA methylation in behavior followed from studies of maternal care that clearly demonstrate an experiencedependent rather than genetic basis for how rats treat their offspring. Rats that receive poor maternal care as pups grow up to become poor mothers to their pups. In addition to becoming poor mothers, these rats also develop long-lasting heightened anxiety and stress responses. Meaney and colleagues identified a region of the glucocorticoid receptor (GR) gene, which was hypermethylated throughout adulthood in rats who received poor maternal care. Treatment with an HDAC inhibitor not only reduced DNA methylation on the GR receptor gene but also improved anxiety and stress responses in these rats.49 More recently, these studies have been translated from rats into humans by studying the hippocampus of patients who committed suicide with or without a history of child abuse. In patients with a history of child abuse, it was observed that DNA methylation on the GR gene promoter was significantly higher, while GR mRNA expression was significantly lower than patients with no history of child abuse.50 While these data do not demonstrate causation, they are among the best evidence to date implicating epigenetic mechanisms in anxiety and stress and suggest that DNA methylation at the GR gene promoter (and probably other genes) in both rats and humans may contribute to this phenomenon. REFERENCES
1. Bird A. Perceptions of epigenetics. Nature. 2007;447:396-398. 2. Surani MA, Hayashi K, Hajkova P. Genetic and epigenetic regulators of pluripotency. Cell. 2007;128:747-762. 3. Luger K, Richmond TJ. The histone tails of the nucleosome. Curr Opin Genet Dev. 1998;8:140-146. 4. Felsenfeld G, Groudine M. Controlling the double helix. Nature. 2003;421:448-453.

Taken together, these studies demonstrate that chromatin structure is an important substrate for long-lasting changes in behavioral responses to stress and antidepressant treatments. While the precise signaling mechanisms by which environmental stresses converge on chromatin are still under investigation (eg, Figure 2), these early studies suggest the exciting possibility that pharmacological manipulation of chromatin remodeling pathways could be a novel approach to new antidepressant development.

Concluding remarks
Chromatin structure is emerging as a key substrate in the pathogenesis and maintenance of chronic psychiatric illnesses. This is important because novel therapeutics could target chromatin remodeling enzymes or chromatin itself to ultimately block or even reverse, for example, a chronically addicted or depressed state. Ultimately though, the key function of chromatin remodeling is to alter the transcription or the transcriptional potential of genes which eventually affect neural function, so any study of chromatin regulation is, in theory, inexorably linked with the study of the underlying gene activity. While extremely exciting, epigenetic research in psychiatry is still in its infancy, and far more research is needed to identify both the dysregulated genes and chromatin modifications responsible for individual psychiatric diseases. Fortunately, new advances in high-throughput sequencing are enabling such characterization of chromatin regulation and gene expression, genome-wide, at an incredible rate and resolution. Armed with these and other new research tools, epigenetic research in psychiatry is progressing at a spectacular speed, and may soon prove to be a major avenue for novel therapeutics.
Acknowledgments: Preparation of this review was supported by grants from NIDA and NIMH, and the Medical Scientist Training Program at UT Southwestern Medical Center. Parts of this review were based on ref 8 and ref 51 with permission. The authors declare no conflicts of interest. 5. Li B, Carey M, Workman JL. The role of chromatin during transcription. Cell. 2007;128:707-719. 6. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693-705. 7. Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000;403:41-45. 8. Tsankova N, Renthal W, Kumar A, Nestler EJ. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci. 2007;8:355-367. 9. Pokholok DK, Harbison CT, Levine S, et al. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell. 2005;122:517-527.

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Regulacin de la cromatina en la adiccin a drogas y en la depresin

Las alteraciones en la expresin gnica estn implicadas en la patognesis de varios trastornos neuropsiquitricos, incluyendo la adiccin a drogas y la depresin. Existe una evidencia creciente que indica que los cambios en la expresin gnica en las neuronas, en el contexto de modelos animales de adiccin y depresin, estn mediados en parte por mecanismos epigenticos que alteran la estructura de la cromatina de genes promotores especficos. Esta revisin discute hallazgos recientes que provienen de aproximaciones conductuales, moleculares y bioinformticas los cuales estn siendo utilizados para comprender la compleja regulacin epigentica de la expresin gnica en el cerebro por las drogas de abuso y por el estrs. Estos avances prometen establecer nuevos caminos para mejores tratamientos de estos trastornos.

Rgulation de la chromatine dans la toxicomanie et la dpression

Des modifications de lexpression gnique sont impliques dans la pathogense de plusieurs maladies neuropsychiatriques, y compris la toxicomanie et la dpression. Des modles animaux de ces pathologies montrent de plus en plus que des variations de lexpression des gnes dans les neurones sont transmises en partie par des mcanismes pigntiques qui changent la structure de la chromatine sur les promoteurs spcifiques des gnes. Cet article analyse les rsultats rcents des approches comportementales, molculaires et bioinformatiques utilises pour comprendre la rgulation pigntique complexe de lexpression gnique dans le cerveau par labus de substances et par le stress. Ces avances ouvrent de nouvelles voies pour mieux traiter ces maladies.

10. Doi M, Hirayama J, Sassone-Corsi P. Circadian regulator CLOCK is a histone acetyltransferase. Cell. 2006;125:497-508. 11. Kawasaki H, Schiltz L, Chiu R, et al. ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. Nature. 2000;405:195-200. 12. Chawla S, Vanhoutte P, Arnold FJ, Huang CL, Bading H. Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5. J Neurochem. 2003;85:151-159. 13. Fischle W, Dequiedt F, Hendzel MJ, et al. Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR. Mol Cell. 2002;9:45-57. 14. Lahm A, Paolini C, Pallaoro M, et al. Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases. Proc Natl Acad Sci U S A 2007;104:17335-17340. 15. Yang XJ, Seto E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell. Biol 2008;9:206-218. 16. Haigis MC, Guarente LP. Mammalian sirtuins--emerging roles in physiology, aging, and calorie restriction. Genes Dev. 2006;20:2913-2921. 17. Brami-Cherrier K, Lavaur J, Pages C, Arthur JS, Caboche J. Glutamate induces histone H3 phosphorylation but not acetylation in striatal neurons: role of mitogen- and stress-activated kinase-1. J Neurochem. 2007;101:697-708. 18. Li J, Guo Y, Schroeder FA, et al. Dopamine D2-like antagonists induce chromatin remodeling in striatal neurons through cyclic AMP-protein kinase A and NMDA receptor signaling. J Neurochem. 2004;90:1117-1131. 19. Brami-Cherrier K, Valjent E, Herve D, et al. Parsing molecular and behavioral effects of cocaine in mitogen- and stress-activated protein kinase-1deficient mice. J Neurosci. 2005;25:11444-11454. 20. Stipanovich A, Valjent E, Matamales M, et al. A phosphatase cascade by which rewarding stimuli control nucleosomal response. Nature. 2008;453:879884. 21. Vakoc CR, Mandat SA, Olenchock BA, Blobel GA. Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin. Mol Cell. 2005;19:381-391. 22. Iwase S, Lan F, Bayliss P, et al. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell. 2007;128:1077-1088.

23. Jensen LR, Amende M, Gurok U, et al. Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation. Am J Hum Genet. 2005;76:227-236. 24. Suzuki MM, Bird A. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet. 2008;9:465-476. 25. Zhang X, Odom DT, Koo SH, et al. Genome-wide analysis of cAMPresponse element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc Natl Acad Sci U S A. 2005;102:44594464. 26. Ramocki MB, Zoghbi HY. Failure of neuronal homeostasis results in common neuropsychiatric phenotypes. Nature. 2008;455:912-918. 27. Chahrour M, Jung SY, Shaw C, et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science. 2008;320:12241229. 28. Goll MG, Kirpekar F, Maggert KA, et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science. 2006;311:395-398. 29. Miller CA, Sweatt JD. Covalent modification of DNA regulates memory formation. Neuron. 2007;53:857-869. 30. Ooi SK, Bestor TH. The colorful history of active DNA demethylation. Cell. 2008;133:1145-1148. 31. Hyman S, Malenka R, Nestler E. Neural mechanisms of addiction: the role of reward-related learning and memory. Annu Rev Neurosci. 2006;29:565-598. 32. Kumar A, Choi KH, Renthal W, et al. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron. 2005;48:303-314. 33. Nestler EJ. Review. Transcriptional mechanisms of addiction: role of DeltaFosB. Philos Trans R Soc Lond B Biol Sci. 2008;363:3245-3255. 34. Levine AA, Guan Z, Barco A, et al. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum. Proc Natl Acad Sci U S A. 2005;102:19186-19191. 35. Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai LH. Recovery of learning and memory is associated with chromatin remodelling. Nature. 2007;447:178-182. 36. Levenson JM, O'Riordan KJ, Brown KD, et al. Regulation of histone acetylation during memory formation in the hippocampus. J Biol Chem. 2004;279:40545-40559.

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37. Maze I, Covington HE, 3rd, Laplant Q, et al. Histone methylation in the nucleus accumbens controls behavioral responses to cocaine. Soc Neurosci Abs. In press. 38. Renthal W, Kumar A, Xiao G, et al. Genome wide analysis of chromatin regulation by cocaine reveals a novel role for sirtuins. Neuron. 2009;62:335348. 39. Freeman WM, Patel KM, Brucklacher RM, et al. Persistent alterations in mesolimbic gene expression with abstinence from cocaine self-administration. Neuropsychopharmacology. 2008;33:1807-1817. 40. Renthal W, Maze I, Krishnan V, et al. Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli. Neuron. 2007;56:517-529. 41. Schroeder FA, Penta KL, Matevossian A, et al. Drug-induced activation of dopamine D(1) receptor signaling and inhibition of class I/II histone deacetylase induce chromatin remodeling in reward circuitry and modulate cocaine-related behaviors. Neuropsychopharmacology. 2008;33:2981-2992. 42. Sun J, Wang L, Jiang B, et al. The effects of sodium butyrate, an inhibitor of histone deacetylase, on the cocaine- and sucrose-maintained self-administration in rats. Neurosci Lett. 2008;441:72-76. 43. Romieu P, Host L, Gobaille S, et al. Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats. J Neurosci. 2008;28:9342-9348. 44. Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature. 2008;455:894-902. 45. Berton O, McClung CA, Dileone RJ, et al. Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science. 2006;311:864868. 46. Tsankova N, Berton O, Renthal W, et al. Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. Nat Neurosci. 2006;9:519-525. 47. Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R. Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol. 2006;13:563-567. 48. Schroeder FA, Lin CL, Crusio WE, Akbarian S. Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse. Biol Psychiatry. 2007;62:55-64. 49. Weaver IC, Cervoni N, Champagne FA, et al. Epigenetic programming by maternal behavior. Nat Neurosci. 2004;7:847-854. 50. McGowan PO, Sasaki A, D'Alessio AC, et al. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. Nat Neurosci. 2009;12:342-348. 51. Renthal W, Nestler EJ. Epigenetic mechanisms in drug addiction. Trends Mol Med. 2008;14:341-350.

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Neuroplasticity of excitatory and inhibitory cortical circuits in schizophrenia
David A. Lewis, MD

Schizophrenia is a neurodevelopmental disorder characterized by deficits in cognitive processes mediated by the circuitry of the dorsolateral prefrontal cortex (DLPFC). These deficits are associated with a range of alterations in DLPFC circuitry, some of which reflect the pathology of the illness and others of which reflect the neuroplasticity of the brain in response to the underlying disease process. This article reviews disturbances in excitatory and inhibitory components of DLPFC circuitry from the perspective of developmental neuroplasticity and discusses their implications for the identification of novel therapeutic targets.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:269-280.

Keywords: GABA; glutamate; prefrontal cortex; working memory Author affiliations: Departments of Psychiatry and Neuroscience, University of Pittsburgh, Pennsylvania, USA Address for correspondence: David A. Lewis, MD, Department of Psychiatry, University of Pittsburgh, 3811 OHara Street, W1651 BST, Pittsburgh, PA 15213, USA (e-mail: lewisda@upmc.edu) Copyright 2009 LLS SAS. All rights reserved

europlasticity can be broadly considered to be the capacity of the brain to change the molecular and structural features that dictate its functions in response to a disease process (or other factors) that disrupts those functions.1 For a disorder such as schizophrenia, the disease process appears to result from a complex interplay of an unknown number of genetic liabilities and environmental risk factors that unleash pathogenetic mechanisms which produce a pathological entity, a conserved set of molecular and cellular disturbances in specific neural circuits. These pathological changes so alter the normal function of the affected circuits that the resulting pathophysiology gives rise to the emergent properties recognized as the clinical features of the illness.2 One approach to dissecting this disease process involves focusing on a well-defined clinical component of the illness. For example, deficits in cognitive abilities are thought to be the core features of schizophrenia because they occur with high frequency in individuals with schizophrenia, are relatively stable over the course of the illness, are independent of the psychotic symptoms of the disorder, are present in a milder form in individuals at genetic risk who do not become clinically ill,3 and are the best predictor of long-term functional outcome.4 Of the domains of cognition affected in schizophrenia, disturbances in working memory, the ability to transiently maintain and manipulate a limited amount of information in order to guide thought or behavior, are accompanied by altered activation of the dorsolateral prefrontal cortex (DLPFC, Figure 1 A, B). The altered activation of the DLPFC under such conditions might be specific to the disease process of schizophrenia because these disturbances are present in medication-nave individuals with schizophrenia, but not in subjects with other psychotic disorders or major depression.5,6
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Selected abbreviations and acronyms
CCK DLPFC GABA GAD GAT NMDA PV cholecystokinin dorsolateral prefrontal cortex -aminobutyric acid glutamic acid decarboxylase GABA membrane transporter N-methyl-D-aspartic acid parvalbumin tional studies are required, existing data suggests that many of the alterations described below are probably also present in other cortical regions that are dysfunctional in schizophrenia.7

Neuroplasticity of excitatory cortical connections in schizophrenia

Excitatory connections in the DLPFC are altered in schizophrenia The disease process of schizophrenia appears to involve deficient glutamate-mediated excitatory neurotransmission through the N-methyl-D-aspartic acid (NMDA) receptor.8,9 NMDA receptor antagonists such as phencyclidine (PCP) or ketamine increase both positive and

This review examines alterations in components of excitatory and inhibitory neurotransmission in DLPFC circuitry that might contribute to the impairments in working memory in schizophrenia. Each mediator is considered from the perspective of which alterations reflect the disease process and which might be neuroplastic responses of the affected circuits. Although addi-

A
Dorsolateral

B
1 2

Apical dendrites 3 V e n t r o m e d i a l Orbital

4 5 Basilar dendrites

White matter

Contralateral ipsilateral

Striatum or brain stem

Thalamus

Figure 1. A) Photograph of an unstained coronal block, containing the prefrontal cortex, cut immediately anterior to the corpus callosum through the left hemisphere of a postmortem human brain. This block also includes the adjacent anterior cingulate gyrus (ACG) of the limbic lobe. The portion of the dorsolateral prefrontal cortex (DLFPC) delineated by the small rectangle is shown at higher magnification in panel B. B) Nissl-stained section showing the typical appearance of six layers or lamina, numbered from the pial surface of the cortex to the underlying white matter, based on the size and packing density of neurons. C) Schematic representation of neurons across cortical layers. Pyramidal neurons (red) represent about 75% of cortical neurons and typically have triangularly-shaped cell bodies, a single apical dendrite directed towards the pial surface, and an array of basilar dendrites. Depending on their laminar location, the axons of pyramidal neurons preferentially provide excitatory projections to different brain regions. Axons that project to the DLPFC from other brain regions also tend to innervate different subsets of cortical layers. For example, axonal projections (green) from the thalamus terminate in layers deep 3 and 4. The remaining ~25% of DLPFC neurons are local circuit or interneurons (blue). These neurons use the inhibitory neurotransmitter GABA, and have axons that arborize locally and innervate other neurons in the same area of the prefrontal cortex.
Reproduced from ref 1: Lewis DA, Gonzalez-Burgos G. Neuroplasticity of neocortical circuits in schizophrenia. Neuropsychopharmacology. 2008;33:141-165. Copyright Nature Publishing Group 2008

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negative symptoms in patients with schizophrenia, and the administration of subanesthetic doses of ketamine to healthy individuals produces thought disorder and other features similar to those seen in schizophrenia.10 In addition, systemic administration of NMDA receptor antagonists disrupts working memory in rats,11 and application of an NMDA receptor antagonist to the DLPFC impairs working memory performance in monkeys. 12 However, although postmortem studies have reported alterations in measures of glutamate receptor binding, transcription, and subunit protein expression in several brain regions in subjects with schizophrenia,13 such findings for mRNA and protein levels of NMDA receptor subunits in the DLPFC have been limited in magnitude and not always replicated, suggesting that other components of NMDA receptor signaling might be affected in the illness.14 Anatomical studies do support the presence of inputspecific alterations of excitatory connections in the DLPFC in schizophrenia. In the DLPFC, pyramidal neurons (Figure 1C) are the principal source of glutamate neurotransmission, as well as the targets of the majority of glutamate-containing axon terminals. Although the number of these neurons does not appear to be altered in schizophrenia,15,16 neuronal density in the DLPFC has been reported to be increased in schizophrenia. 17 Increased cell packing density has been interpreted as evidence of a reduction in the amount of cortical neuropil, the axon terminals, dendritic spines, and glial processes that occupy the space between neurons. 18 Consistent with this interpretation, synaptophysin protein, a marker of axon terminals, has been reported to be decreased in the DLPFC of subjects with schizophrenia.19-21 Furthermore, gene expression profiling studies have found reduced tissue levels of gene transcripts that encode proteins involved in the presynaptic regulation of neurotransmission.22 Dendritic spines are the principal targets of excitatory synapses to pyramidal neurons. Although most dendritic spines present are stable in number during adulthood,23 they are subject to a number of neuroplastic changes, such as a loss of their presynaptic excitatory input. In schizophrenia, dendritic spine density in pyramidal neurons has been reported to be lower in the DLPFC 24,25; understanding the nature of these neuroplastic responses requires knowledge of the specific circuits that are affected and the developmental mechanisms that might underlie these changes.

Reduced excitatory connections in schizophrenia are specific to a subset of pyramidal neurons Pyramidal neurons can be divided into subgroups based on the brain region targeted by their principal axonal projection and the sources of their excitatory inputs; both of these characteristics are associated with the location of pyramidal cell bodies in different layers of the cortex (Figure 1C). For example, many pyramidal cells in layers 2 to 3 send axonal projections to other cortical regions, pyramidal neurons in layer 5 tend to project to the striatum and other subcortical structures, and pyramidal neurons in layer 6 furnish projections primarily to the thalamus.26 Studies of basilar dendritic spine density on Golgi-impregnated pyramidal neurons in each cortical layer of the DLPFC in the same cohort of subjects found a significant effect of diagnosis on spine density only for pyramidal neurons in deep layer 3 (Figure 2).25,27 The functional integrity of the pyramidal neurons with lower dendritic spine densities may be reflected in changes in their somal volume. For example, shifts in somal size may indicate disturbances in neuronal connectivity, given that somal size has been shown to be correlated with measures of a neuron's dendritic tree28 and axonal arbor.29 Indeed, the mean cross-sectional somal area of the Golgi-impregnated, deep layer 3 pyramidal neurons was 9% smaller in the subjects with schizophrenia relative to normal control subjects.25 Consistent with this observation, the mean somal volume of Nisslstained pyramidal neurons in DLPFC deep layer 3 was also 9% smaller in a different cohort of subjects with schizophrenia.30 Similarly, in another study, the mean somal size of all layer 3 neurons in DLPFC area 9 was smaller in subjects with schizophrenia, and was accompanied by a decrease in the density of the largest neurons in deep layer 3, without a change in somal volume in layer 5.31 Furthermore, in both primary and association auditory cortices, somal volumes of deep layer 3, but not of layer 5, pyramidal neurons were smaller in schizophrenia.32,33 Together, these findings suggest that in schizophrenia: i) basilar dendritic spine density is lower and somal volume is smaller in deep layer 3 pyramidal neurons; ii) these alterations are specific to or at least most prominent in deep layer 3; iii) this pattern of alterations is not restricted to the DLPFC; and iv) these differences reflect the underlying disease process and not confounding factors.

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A
Dendritic spine

D
0.6 Deep layer 3

F2,37=6.01, P=.006 0.5 Mean spine density (spines/m)

0.4

0.3

0.2

E
Layer Superficial 3 Deep 3 Layer 5 Layer 6 Change in Schizophrenia -13% -23% +3% +12% 0.1 P value NS .003 NS NS P=.003 23% 0 Control Schizophrenia Subject group Psychiatric P=.08 16%

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The contribution of developmental plasticity to dendritic spine alterations in schizophrenia Dendritic spine density on DLPFC layer 3 pyramidal neurons undergoes a substantial decline during adolescence in primates.34 Consistent with the findings that dendritic spines are the main site of excitatory synaptic input onto pyramidal cells and that all mature dendritic spines contain an excitatory synapse, 35 the number of excitatory synapses declines in a similar age-related fashion in both monkey and human DLPFC.36,37 In humans, this synaptic pruning is thought to underlie the decrease in cortical gray matter thickness that occurs during adolescence.38,39 Interestingly, the late developmental refinements in excitatory connectivity are more marked in layer 3 than in the deeper cortical layers,36 suggesting that they may be associated with the apparent laminaspecific alterations in spine density in schizophrenia. The observation of alterations in the expression of certain synaptic proteins in schizophrenia suggested the possibility that the exuberant synapses present before adolescence somehow compensated for a dysfunction in excitatory transmission in individuals with schizophrenia.40 Alternatively, such alterations in synaptic protein expression might disturb the mechanisms of adolescence-related synapse elimination leading, for instance, to excessive synapse pruning and decreased spine number in the illness.41,42 The potential contribution of excitatory synapse pruning during adolescence to disease-related changes in DLPFC function depends, in part, on the functional properties of the synapses that are pruned. During early brain development, pruned synapses are functionally immature. Immature glutamate synapses are relatively weak and their maturation involves an activity-dependent increase in strength. Such activity-dependent strengthening might underlie synapse stabilization, and thus mark for elimination the immature synapses that are not strengthened.43,44 However, recent findings in the developing monkey DLPFC indicate that the excitatory

inputs to layer 3 pyramidal neurons mature functionally during the age range when they are present in high density and before synaptic pruning begins.45 Thus, these data suggest that the substantial remodeling of excitatory connectivity of the primate DLPFC during adolescence primarily involves the elimination of mature synapses, and that some other factor, such as the neuronal source of input, somehow tags mature synapses for pruning.46 Thus, the presence of functionally mature synapses prior to adolescence supports the hypothesis that the excess in excitatory synapse number prior to adolescence might be able to compensate for a molecular based dysfunction of these synapses in individuals with schizophrenia, and thereby forestall the appearance of the clinical features of the illness until synapse number falls below some critical threshold.40

Neuroplasticity of inhibitory cortical connections in schizophrenia

Prefrontal inhibitory neurotransmission is altered in schizophrenia Studies from multiple laboratories have consistently found lower levels of the mRNA for the 67 kilodalton isoform of glutamic acid decarboxylase (GAD67), the principal synthesizing enzyme for -aminobutyric acid (GABA), in the DLPFC of subjects with schizophrenia.16,22,47-52 At the cellular level, the expression of GAD67 mRNA was not detectable in ~25% to 35% of GABA neurons in layers 15 of the DLPFC, but the remaining GABA neurons exhibited normal levels of GAD67 mRNA.16,47 Similarly, expression of the mRNA for the GABA membrane transporter (GAT1), a protein responsible for reuptake of released GABA into nerve terminals, was decreased in a similar minority of GABA neurons.53 These findings suggest that both the synthesis and reuptake of GABA are lower in a subset of DLPFC neurons in schizophrenia. Subclasses of cortical GABA neurons can be distinguished on the basis of a number of molecular, electro-

Figure 2. Pyramidal neuron dendritic spines in the human DLPFC. A) Schematic diagram illustrating the dendritic tree and dendritic spines on a prototypic pyramidal neuron. B) Electron micrograph showing a dendrite (D) with two spines (S). Each spine receives an asymmetric (presumably excitatory) synapse from an axon terminal (at). C) Golgi-impregnated basilar dendrites and spines on deep layer 3 pyramidal neurons from a normal comparison (top) and two subjects with schizophrenia (bottom). Note the reduced density of spines in the subjects with schizophrenia in these extreme examples. D) Scatter plot demonstrating the lower density of spines on the basilar dendrites of deep layer 3 pyramidal neurons in the DLPFC of subjects with schizophrenia relative to both normal and psychiatrically-ill comparison subjects. E) Laminar-specificity of the spine density differences in the same subjects.
Reproduced from ref 1: Lewis DA, Gonzalez-Burgos G. Neuroplasticity of neocortical circuits in schizophrenia. Neuropsychopharmacology. 2008;33:141-165. Copyright Nature Publishing Group 2008

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physiological, and anatomical properties. For example, the affected GABA neurons in schizophrenia include the subclass that contain the calcium-binding protein, parvalbumin (PV), which comprise ~25% of GABA neurons in the primate DLPFC. PV-containing neurons include fast-spiking chandelier and basket neurons that principally target the axon initial segments and cell body/proximal dendrites, respectively, of pyramidal neurons54,55 (Figure 3). In individuals with schizophrenia the expression level of PV mRNA is reduced, although the number of PV neurons appears to be unchanged56; in addition, approximately half of PV mRNA-containing neurons lack detectable levels of GAD67 mRNA.57 In contrast, the ~50% of GABA neurons that express the calcium binding protein calretinin appear to be unaffected.57 In the DLPFC of subjects with schizophrenia, GAT1 immunoreactivity is selectively reduced in the characteristic axon terminals (cartridges) of PV-containing chandelier neurons. 58 In the postsynaptic targets of these axon cartridges, the axon initial segments of pyramidal neurons, immunoreactivity for the GABAA receptor 2 subunit (which is present in most GABAA receptors in this location 59) is markedly increased in schizophrenia.60 Several lines of evidence suggest that the reductions in presynaptic GABA markers (GAT1 and PV) and increased postsynaptic GABA A receptors are compensatory responses to a deficit in GABA release from chandelier neurons. For example, PV is a slow calcium buffer that does not affect the amplitude, but accelerates the decay, of Ca2+ transients in GABA nerve terminals. 61,62 Thus, PV decreases the residual Ca2+ levels that normally accumulate in nerve terminals and facilitate GABA release during repetitive firing.61 Studies in PV-deficient mice have demonstrated that a decrease in PV increases residual Ca2+ and favors synaptic facilitation. 61,63 Furthermore, the enhanced facilitation of GABA release from fast-spiking neurons with reductions in PV is associated with increased power of gamma oscillations 63 (which is, as explained below, deficient in schizophrenia). Similarly, the blockade of GABA reuptake via GAT1 prolongs the duration of inhibitory postsynaptic currents (IPSCs) when synapses located close to each other are activated synchronously64; the resulting prolongation of IPSCs increases the probability of IPSC summation, enhances the total efficacy of IPSC trains, and thereby augments GABA signaling. The upregulation of the postsynaptic GABA A receptors that contain 2 subunits would be expected to increase the efficacy of the GABA that is released from chandelier neurons. Thus, the combined reduction of PV and GAT1 proteins in chandelier cell axon cartridges, and the upregulation of postsynaptic GABAA receptors, appear to represent neuroplastic responses that might act synergistically to increase the efficacy of GABA neurotransmission at pyramidal neuron axon initial segments during the types of repetitive neuronal activity associated with working memory. However, the persistence of cognitive impairments in individuals with schizophrenia suggests that these neuroplastic changes in GABA neurotransmission from chandelier neurons are insufficient as compensatory responses. Alternatively, it is possible that compensation at chandelier cell synapses is not effective because additional interneuron subclasses are also functionally deficient in schizophrenia.65 Consistent with this interpretation, other findings indicate that alterations in PVcontaining GABA neurons cannot account for all of the observed findings in postmortem studies of schizophrenia. For example, the levels of GAD67 and GAT1 mRNAs are reduced to comparable degrees in layers 2-5,47,53 even though the density of PV neurons is much greater in layers 3 and 4 than in layers 2 and 5.66 In addition, PV mRNA expression was reduced in layers 3 and 4, but not in layers 2 and 5, in subjects with schizophrenia.57 Indeed, other studies have found lower tissue concentrations of the mRNAs for the neuropeptides somatostatin (SST) and cholecystokinin (CCK) in the DLPFC of subjects with schizophrenia (Figure 3).51 In the cortex, SST is expressed by GABA neurons located in layers 2 and 5 that do not express PV or CR.67 CCK is also heavily expressed in GABA neurons that do not contain either PV or SST located principally in layers 2-3 of the primate prefrontal cortex. 68 Interestingly, the axon terminals of CCK-containing large basket neurons, which target selectively pyramidal neuron cell bodies, contain type I cannabinoid receptors (CB1R),69 and the mRNA and protein levels of CB1R are also lower in schizophrenia. 70 Because activation of the CB1R suppresses GABA release from the terminals of CCK neurons, the downregulation of this receptor may represent a compensatory response to reduce the ability of endogenous cannabinoids to decrease GABA release from CCK/CB1R-containing axon terminals.70

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Figure 3. Schematic summary of putative alterations in DLPFC circuitry in schizophrenia. Pyramidal neurons (light blue) in deep layer 3 have smaller somal size, shorter basilar dendrites, lower dendritic spine density, and a reduced axonal arbor in schizophrenia. Altered GABA neurotransmission by PV-containing neurons (green) is indicated by expression deficits in several gene products as well as by lower levels of GAT1 protein in the terminals of chandelier neurons and upregulated GABAA receptor 2 subunits at their synaptic targets, the axon initial segments of pyramidal neurons (enlarged square). Expression of the neuropeptide somatostatin (SST) is decreased in GABA neurons (dark blue) that target the distal dendrites of pyramidal neurons. Decreased cholecystokinin (CCK) and cannabinoid receptor 1 (CB1) mRNA levels, and lower CB1 protein in axon terminals, suggest altered regulation of GABA neurotransmission in a subset of basket neurons (purple) that target the cell body and proximal dendrites of pyramidal neurons. Gene expression does not seem to be altered in calretinin (CR)-containing GABA neurons (red) that primarily target other GABA neurons (gray). Putative alterations in thalamic and dopamine (DA) cell bodies and their projections to the DLPFC are also shown. Some studies indicate that the number and/or gene expression in oligodendrocytes is also altered. Not all of the circuitry alterations shown here have been sufficiently replicated or demonstrated to be specific to the disease process of schizophrenia to be considered established facts; solid arrows indicate abnormalities supported by convergent and/or replicated observations.
Reproduced from ref 2: Lewis DA, Sweet RA. Schizophrenia from a neural circuitry perspective: advancing toward rational pharmacological therapies. J Clin Invest. 2009;119:706-716. Copyright American Society for Clinical Investigation 2009

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Altered GABA neurotransmission in PV-containing neurons impairs prefrontal network synchrony in schizophrenia Reduced GABA signaling from PV-containing GABA neurons to the perisomatic region of pyramidal neurons in the DLPFC might contribute to the pathophysiology of working memory dysfunction via the following mechanisms. First, the activity of DLPFC GABA neurons is essential for normal working memory function in monkeys.71,72 Second, PV-positive GABA neurons and pyramidal neurons share common sources (eg, thalamic afferents) of excitatory input.55 The resulting feed-forward, disynaptic inhibition creates a time window during which the number of excitatory inputs required to evoke pyramidal neuron firing must occur.73 Third, both chandelier and basket neurons target multiple pyramidal neurons,74 enabling them to use this timing mechanism to synchronize the activity of local populations of pyramidal neurons.75 Fourth, networks of PV-positive GABA neurons, formed by both chemical and electrical synapses, give rise to oscillatory activity in the gamma band range, the synchronized firing of a neuronal population at 30 to 80 Hz.76,77 Interestingly, gamma band oscillations in the human DLPFC increase in proportion to working memory load,78 and in subjects with schizophrenia, prefrontal gamma band oscillations are reduced bilaterally during a working memory task. 79 Thus, a deficit in the synchronization of pyramidal cell firing, resulting from impaired regulation of pyramidal cell networks by PV-positive GABA neurons, may contribute to reduced levels of induced gamma band oscillations, and consequently to impairments in cognitive tasks that involve working memory in subjects with schizophrenia.65 Interestingly, CCK/CB1R- and PV-containing cells provide convergent sources of perisomatic inhibition to pyramidal neurons that play specific roles in shaping network activity, including complementary roles in regulating gamma band oscillations.80 Thus, alterations in CCKcontaining basket cells could also contribute to impaired gamma oscillations in schizophrenia. The contribution of developmental plasticity to GABA neuron alterations in schizophrenia In the monkey prefrontal cortex DLPFC, the density of symmetric, presumably GABA, synapses rises rapidly during the third trimester of gestation and perinatal period until stable, adult levels are achieved at 3 months postnatal.36 In contrast, pre- and postsynaptic markers of the functional properties of chandelier axon inputs to the axon initial segment (AIS) of pyramidal neurons exhibit a very protracted maturation. Presynaptically, immunoreactivities for the calcium-binding protein PV and GAT1 in chandelier axon cartridges are not detectable or low at birth, rise (albeit with different developmental time courses) to peak levels early in postnatal development that are sustained until ~15 months of age, and then rapidly decline during adolescence until stable adult levels are achieved.34,81,82 Since chandelier cartridges are readily visualized with Golgi staining across postnatal development,83 these changes in PV and GAT1 immunoreactivity are likely to reflect shifts in the concentration of these proteins rather than changes in the presence of, or in the density of, axon terminals within chandelier axon cartridges.82 Postsynaptically, GABAA receptors containing 2 subunits predominate in pyramidal neuron AIS especially in cortical layers 24.84 The density of pyramidal neuron AIS immunoreactive for 2 subunits is high at birth, then significantly declines during adolescence before achieving stable adult levels.82 These findings indicate that both pre- and postsynaptic markers of GABA neurotransmission undergo significant changes during postnatal development, suggesting that the capacity to synchronize pyramidal neuron output in the prefrontal cortex (PFC) might be in substantial flux until adulthood. The significance of these changes depends, at least in part, on how GABA synapses are stabilized at the AIS while the functional properties of GABA neurotransmission at this location are changing during postnatal development. For example, pyramidal neuron AIS contain specific proteins that regulate synapse structure and receptor clustering: i) Ankyrin-G, an adaptor molecule that links various membrane proteins to the cytoskeleton, is localized to AIS,85 and interactions between ankyrin-G and the cell adhesion molecule, neurofascin, are required for the formation and stabilization of GABA synapses at AIS86; ii) IV spectrin, which is localized to the AIS of pyramidal neurons through its direct interaction with ankryin-G,87 is a critical component in the organization and stabilization of membrane proteins at the AIS88; iii) The scaffolding protein gephyrin facilitates the preferential accumulation of gephyrin-GABAA receptor clusters, especially those containing 2 subunits.89 In monkey DLPFC, ankyrin-G-

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and IV spectrin-labeled AIS decline in density during the first 6 months postnatal, but then remain stable, whereas the density of gephyrin-labeled AIS is stable through early postnatal development and then then markedly declines during adolescence.90 Thus, molecular determinants of the structural features that define GABA inputs to pyramidal neuron AIS in monkey PFC undergo distinct developmental trajectories, with different types of changes occurring during the perinatal period and adolescence (Figure 4). This complex and protracted postnatal maturation of the inputs from PV-containing GABA neurons in the primate DLPFC provides a number of opportunities for even subtle disturbances to have their effects amplified as they alter the trajectories of the developmental events that follow. For example, the marked developmental changes in the axon terminals of PV-containing chandelier neurons, and their postsynaptic receptors, during the perinatal period and adolescence, raises the possibility that the alterations in schizophrenia of these markers
3m 100 15 m 42 m Ankyrin-G AIS IV spectrin AIS Gephyrin AIS GABAA 2 AIS GAT1 cartridges PV cartridges Adolescence

reflect a disturbance in these patterns of development. These temporal correlations may explain how a range of environmental factors (eg, labor-delivery complications, urban place of rearing, and marijuana use during adolescence) are all associated with increased risk for the appearance of schizophrenia later in life. Although speculative, the current literature raises the possibility that the GABA-related disturbances in schizophrenia represent an arrest of normal developmental trajectories. For example, recent studies indicate that mRNA expression for the 1 subunit of the GABAA receptor in the DLPFC increases across postnatal development, whereas the expression of the 2 subunit declines.91 Thus, the elevated 2 subunit expression60 and the decreased 1 mRNA levels92 in schizophrenia might reflect a developmental dysregulation of GABAA receptor subunit expression, where the changes in subunit expression with age fail to undergo their full course. Under this scenario, the alterations of DLPFC circuitry in schizophrenia may render it unable to support higher levels of working memory load, rendering the impaired performance in schizophrenia analogous to the immature levels of working memory function seen in children.93,94

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Relative density (%)

Neuroplastic responses as targets for treatment

The findings reviewed above indicate that working memory and related cognitive impairments in schizophrenia are likely the result of a complex set of alterations in prefrontal excitatory and inhibitory circuitry. Some of these alterations appear to be deleterious causes or consequences of disturbances in the functional architecture of the DLPFC and interconnected brain regions, whereas others may be best explained as compensatory responses. In each case, they reflect the morphological and molecular neuroplasticity of DLPFC circuitry in a disease state. Understanding whether the disease-related change in a given molecule is a consequence or compensation in the disease process has important implications both for the nature of activity of the drugs designed against that target and for the potential therapeutic value of the target. For example, is the neuroplastic capacity of cortical circuitry sufficiently limited that pharmacological augmentation of a compensatory response is feasible? The results of a recent proofof-concept clinical trial suggest that this may be the case. For example, the idea that GABAA receptors contain-

60

40

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0 .01 .1 1 10 100 1000

Log age (mo)

Figure 4. Schematic summary of the trajectories of pyramidal neuron axon initial segments and chandelier neuron axon cartridges labeled with different markers across postnatal development in area 46 of monkey PFC. Lines for each marker represent the percent maximal value achieved plotted against age in months after birth on a log scale. Arrowheads demarcate the indicated ages in months, and the white area indicates the approximate age range corresponding to adolescence in this species. GAT1 indicates GABA membrane transporter 1; PV indicates parvalbumin.
Reproduced from ref 90: Cruz DA, Lovallo EM, Stockton S, Rasband M, Lewis DA. Postnatal development of synaptic structure proteins in pyramidal neuron axon initial segments in monkey prefrontal cortex. J Comp Neurol. 2009;514:353-367. Copyright Wiley-Liss 2009

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ing 2 subunits are upregulated in pyramidal neurons due to a deficit in GABA input from chandelier neurons led to the use of a novel, positive allosteric modulator of this receptor subtype that improved both working memory function and prefrontal gamma band oscillations in a small randomized controlled trial of subjects with schizophrenia.95 Given the marked developmental changes that occur in each of these systems during adolescence, this type of pharmacological intervention may have particular value as a treatment strategy for highrisk adolescents in the prodromal phase of the illness. However, the effectiveness and safety of such interventions requires a fuller understanding of the maturation of these neural circuits, of the functional consequences of these circuitry changes and of the vulnerability of these developmental processes to pharmacological agents.
Acknowledgments: Cited work conducted by the author was supported by NIH grants MH045156, MH051234 and MH043784. Disclosure/conflict of interest: David A. Lewis currently receives investigator-initiated research support from the BMS Foundation, Bristol-Myers Squibb, Curridium Ltd and Pfizer, and in 2007-2009 served as a consultant in the areas of target identification and validation and new compound development to AstraZeneca, Bristol-Myers Squibb, Hoffman-Roche, Lilly, Merck, and Neurogen.

Neuroplasticidad de los circuitos corticales excitatorios e inhibitorios en la esquizofrenia

La esquizofrenia es un trastorno del neurodesarrollo que se caracteriza por dficit en los procesos cognitivos, los que estn mediados por los circuitos de la corteza prefrontal dorsolateral (CPFDL). Estos dficit estn asociados con una amplia gama de alteraciones en los circuitos de la CPFDL; algunos de ellos reflejan la patologa de la enfermedad y otros cmo la neuroplasticidad cerebral responde al proceso patolgico subyacente. En este artculo se revisan las alteraciones en los componentes excitatorios e inhibitorios de los circuitos de la CPFDL desde la perspectiva de la neuroplasticidad del desarrollo y se discuten sus implicancias en la identificacin de nuevas terapias.

Neuroplasticit des circuits corticaux excitateurs et inhibiteurs dans la schizophrnie

La schizophrnie est une maladie dorigine neurodveloppementale caractrise par un dficit des processus cognitifs transmis par les circuits du cortex dorsolatral prfrontal (CPFDL). Ces dficits sassocient un ensemble daltrations des circuits du CPFDL, dont certains refltent la physiopathologie du trouble et dont dautres sont la consquence de la neuroplasticit crbrale en rponse au processus pathologique sous-jacent. Cet article propose une revue des troubles des composants excitateurs et inhibiteurs des circuits du CPFDL selon une perspective de neuroplasticit dveloppementale, et traite de leurs implications pour lidentification de nouvelles cibles thrapeutiques.

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Protracted developmental trajectories of GABAA receptor alpha 1 and alpha 2 subunit expression in primate prefrontal cortex. Biol Psychiatry. 2009;65:1015-1023. 92. Hashimoto T, Arion D, Unger T, et al. Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia. Mol Psychiatry. 2008;13:147-161. 93. Crone EA, Wendelken C, Donohue S, van Leijenhorst L, Bunge SA. Neurocognitive development of the ability to manipulate information in working memory. Proc Natl Acad Sci U S A. 2006;103:9315-9320. 94. Luna B, Garver KE, Urban TA, Lazar NA, Sweeney JA. Maturation of cognitive processes from late childhood to adulthood. Child Devel. 2004;75:1357-1372. 95. Lewis DA, Cho RY, Carter CS, et al. Subunit-selective modulation of GABA type A receptor neurotransmission and cognition in schizophrenia. Am J Psychiatry. 2008;165:1585-1593.

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The role of astroglia in neuroprotection
Mireille Blanger, PhD; Pierre J. Magistretti, MD, PhD

Astrocytes are the main neural cell type responsible for the maintenance of brain homeostasis. They form highly organized anatomical domains that are interconnected into extensive networks. These features, along with the expression of a wide array of receptors, transporters, and ion channels, ideally position them to sense and dynamically modulate neuronal activity. Astrocytes cooperate with neurons on several levels, including neurotransmitter trafficking and recycling, ion homeostasis, energy metabolism, and defense against oxidative stress. The critical dependence of neurons upon their constant support confers astrocytes with intrinsic neuroprotective properties which are discussed here. Conversely, pathogenic stimuli may disturb astrocytic function, thus compromising neuronal functionality and viability. Using neuroinflammation, Alzheimers disease, and hepatic encephalopathy as examples, we discuss how astrocytic defense mechanisms may be overwhelmed in pathological conditions, contributing to disease progression.
2009, LLS SAS

n the last two decades, intense research efforts aiming to provide a better understanding of astroglial cell function have revealed a number of previously unsuspected roles for these neural cells, which were long considered as relatively passive structural elements of the brain. It has now become quite clear that a plethora of cooperative metabolic processes and interdependencies exist between astrocytes and neurons. As a result of the growing appreciation of the role of astrocytes in both the normal and diseased brain, the traditional neuroncentric conception of the central nervous system (CNS) has been increasingly challenged. Astrocytes are territorial cells: they extend several processes with little overlap between adjacent cells, forming highly organized anatomical domains1-3 which are interconnected into functional syncytia via abundant gap junctions.4 These astrocytic processes closely ensheath synapses and express a wide range of receptors for neurotransmitters, cytokines, and growth factors, as well as various transporters and ion channels.5-11 In addition, astrocytes project specialized astrocytic endfeet which are in close contact with intraparenchymal blood vessels, almost entirely covering their surface.12,13 Together, these cytoarchitectural and phenotypical features ideally position astrocytes to fulfill a pivotal role in brain homeostasis, allowing them not only to sense their surroundings but also to respond toand consequently
Author affiliations: Laboratory of Neuroenergetics and Cellular Dynamics, Brain Mind Institute, Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland (Mireille Blanger); Centre de Neurosciences Psychiatriques, CHUV, Dpartement de Psychiatrie, Site de Cery, Lausanne, Switzerland (Pierre J. Magistretti) Address for correspondence: Prof Pierre J. Magistretti, EPFL SV BMI LNDC, SV 2511 (Btiment SV), Station 19, 1015 Lausanne, Switzerland (e-mail: pierre.magistretti@epfl.ch)

Dialogues Clin Neurosci. 2009;11:281-295.

Keywords: astrocyte; astrocyte-neuron interaction; brain homeostasis; neuroinflammation; Alzheimers disease; hepatic encephalopathy

Copyright 2009 LLS SAS. All rights reserved

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Selected abbreviations and acronyms
A AD GSH MCT ROS amyloid-beta Alzheimers disease glutathione monocarboxylate transporter reactive oxygen species strophic consequences for neurons. In the present review we discuss the intrinsically protective role of astrocytes in the normal brain, and examine how these defense mechanisms may be overwhelmed in pathological conditions, contributing to disease progression.

modulatechanges in their microenvironment. Indeed, astrocytes can respond to neurotransmitters with transient increases in their intracellular Ca2+ levels, which can travel through the astrocytic syncytium in a wavelike fashion.14,15 These Ca2+ signals can trigger the release of neuroactive molecules from astrocytes (or gliotransmitters), such as glutamate, D-serine, or adenosine triphosphate (ATP) which in turn modulate synaptic activity and neuronal excitability (see ref 16 for review). This process, for which the term gliotransmission has been coined, marks the emergence of an exciting new notion that information processing may not be a unique feature of neurons. Remarkably, the phylogenetic evolution of the brain correlates with a steady increase of the astrocyte-toneuron ratiogoing from about 1/6 in nematodes to 1/3 in rodents, and reaching up to 1.65 astrocytes per neuron in the human cortex.3,17 Importantly, more than simply outnumbering their rodent counterparts, human astrocytes are also strikingly more complex, both morphologically and functionally. In comparison, human neocortical astrocytes are 2.5 times larger, extend 10 times more processes, and display unique microanatomical features (Figure 1).2 In addition, they generate more robust intracellular Ca2+ responses to neurotransmitter receptor agonists and display a 4-fold increase in Ca 2+ wave velocity.2 In light of these evolution-driven modifications, it is tempting to hypothesize that the astrocytic contribution to the overall neural network complexity may in part provide the fine tuning necessary to take information processing to a higher level of competence, such as that seen in humans. At the very least, the evolutionary pressure exerted on astrocytes highlights the importance of this glial cell type in sustaining normal brain function as the brain itself becomes more complex. A continuously growing body of evidence demonstrates that astrocytes are essential sentinels and dynamic modulators of neuronal function. Considering the strong metabolic cooperation that exists between these two cell types, it is not surprising that alterations in astrocytic function have been shown to have potentially cata-

Astrocytes in the normal brain: maintenance of extracellular homeostasis

Despite the fact that the brain has a very high metabolic rate, neurons are by nature particularly sensitive to minute changes in their microenvironment. In this context, neuronal function and viability would rapidly be compromised without effective mechanisms for the supply of metabolic substrates andequally as important for the removal of waste products. In this respect, astrocytes play an essential role through a number of cellular processes; some of the most important are outlined in the following section.

Figure 1. Human astrocytes are more complex then their rodent counterparts. Typical human (A) and mouse (B) protoplasmic astrocytes are shown at the same scale for comparison. Based on glial fibrillary acidic protein (GFAP) immunostaining, human protoplasmic astrocytes are 2.5-fold larger and project 10 times more main processes than mouse astrocytes. (GFAP, white. Scale bar, 20 M).
Adapted from ref 2: Oberheim NA, Takano T, Han X, et al. Uniquely hominid features of adult human astrocytes. J Neurosci. 2009;29:32763287. Copyright Society for Neuroscience 2009

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Glutamate uptake and recycling Astrocytic processes surrounding synaptic elements express transporters for a variety of neurotransmitters and neuromodulators including glutamate, -aminobutyric acid (GABA), glycine, and histamine.5-8 These transporters participate in the rapid removal of neurotransmitters released into the synaptic cleft, which is essential for the termination of synaptic transmission and maintenance of neuronal excitability. In the specific case of glutamate, its uptake by astrocytes is also crucial in protecting neurons against glutamate-induced excitotoxicity. Indeed, although glutamate is the primary excitatory neurotransmitter in the brain, overstimulation of glutamate receptors is highly toxic to neurons (reviewed in detail by Sattler and Tymianski).18 While basal extracellular glutamate levels are maintained in the low micromolar range, they increase dramatically during glutamatergic neurotransmission, reaching up to 1 mM for a few milliseconds in the synaptic cleft.19 This concentration of glutamate would cause extensive neuronal injury in the absence of highly efficient mechanisms for its removal at the synapse. This is primarily achieved by the astrocyte-specific sodium-dependent high-affinity glutamate transporters GLT-1 and GLAST (corresponding to human EAAT2 and EAAT1, respectively) and to a lesser extent by the neuronal glutamate transporters EAAC1 (human EAAT3) and EAAT4.7 A number of in vitro and in vivo studies demonstrate the primary importance of astrocytic glutamate uptake in preventing glutamate-induced excitotoxicity.20-23 A good example is provided by the phenotypical changes displayed by knockout mice for the various glutamate transporters. Indeed, knockout mice for GLT-1, considered the main astrocytic glutamate transporter, suffer lethal spontaneous seizures and selective hippocampal neuronal degeneration,24 whereas knockout mice for the neuronal EAAC1 display no apparent neurodegeneration. 25 Interestingly, beta-lactam antibiotics have been shown to upregulate the expression of GLT-1 and to prevent neuronal loss both in vitro and in vivo in models involving excitotoxicity.26 This suggests that modulation of the glutamate uptake capacity of astrocytes may be achievable in vivo with classical pharmacological tools, thus representing a promising therapeutic target for pathologies involving excitotoxicity. Astrocytes also play a central role in the transfer of glutamate back to neurons following its uptake at the

synapse. Failure to do so would result in the rapid depletion of the glutamate pool in presynaptic neurons and subsequent disruption of excitatory neurotransmission. This transfer is achieved by the well-described glutamate-glutamine cycle (Figure 2, pink box).27,28 In short, glutamate is converted to glutamine by the astrocytespecific enzyme glutamine synthetase (GS).29 Glutamine is then transferred to neurons in a process most likely involving the amino acid transport systems N, L, and ASC in astrocytes and system A in neurons.27 Glutamine is then converted back to glutamate via deamination by phosphate-activated glutaminase which is enriched in the neuronal compartment. The ammonia produced in the process is thought to be shuttled back to astrocytes following its incorporation into leucine and/or alanine.27 It is important to note that glutamate can be metabolized in a number of different pathways in astrocytes and neurons, including oxidation in the tricarboxylic acid (TCA) cycle.28 Astrocytes are responsible for the replenishment of brain glutamate, as they are the only neural cell type expressing pyruvate carboxylase, a key enzyme in the main anaplerotic pathway in the brain, effectively allowing them to synthesize glutamate from glucose.30,31 This represents another level of cooperation between astrocytes and neurons. K+ buffering Apart from the release of neurotransmitters which have to be rapidly removed from the synaptic cleft, neuronal activity and the resulting propagation of action potentials causes substantial local increases of extracellular potassium ions (K+) in the restricted extracellular space. Without tight regulatory mechanisms, this could dramatically alter the neuronal membrane potential, leading to neuronal hyperexcitability and seriously compromising CNS function.32 Such a scenario is prevented by the buffering of extracellular K+ by glial cells33,34 (Figure 2, orange box). Indeed, astrocytes have a strongly negative resting potential and express a number of potassium channels, resulting in a high membrane permeability to K+.35 These features, in conjunction with the action of the Na+/K+ ATPase, enable astrocytes to accumulate the excess extracellular K+,36 which can then travel in the astrocytic syncitium through gap junctions down its concentration gradient.34,35 This allows for the spatial dispersion of K+ from areas of high concentration to areas of lower concentration where it can be extruded either

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Neurons Astrocytes Capillary

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Figure 2. Simplified representation of the main roles of astrocytes in brain homeostasis. Pink box: glutamate-glutamine cycle. Astrocytic excitatory amino acid transporters (EAATs) are responsible for the uptake of a large fraction of glutamate at the synapse. Glutamate is converted into glutamine by glutamine synthetase (GS) and shuttled back to neurons for glutamate resynthesis. Blue boxes: Lactate shuttle. Glutamate uptake by astrocytes is accompanied by Na+ entry which is counteracted by the action of the Na+/K+ ATPase. The resulting increase in ADP/ATP ratio triggers anaerobic glucose utilization in astrocytes and glucose uptake from the circulation through the glucose transporter GLUT1. The lactate produced is shuttled to neurons through monocarboxylate transporters (mainly MCT-1 in astrocytes and MCT-2 in neurons), where it can be used as an energy substrate after its conversion to pyruvate. Yellow box: pH buffering. Abundant carbonic anhydrase (CA) in astrocytes converts CO2 into H+ and HCO3-. Two HCO3- are transported into the extracellular space along with one Na+ via the Na+-HCO3- cotransporter (NBC), thereby increasing the extracellular buffering power. Protons left in the glial compartment may drive the transport of lactate outside of astrocytes and into neurons through MCTs. Excess H+ in neurons is extruded via sodium-hydrogen exchange (NHE). Orange box: K+ buffering. Astrocytes buffer excess K+ released into the extracellular space as a result of neuronal activity. Potassium ions travel through the astrocytic syncitium down their concentration gradient and are released in sites of lower concentration. Green box: Glutathione metabolism. Astrocytes release glutathione (GSH) in the extracellular space where it is cleaved by the astrocytic ectoenzyme -glutamyl transpeptidase (GT). The resulting CysGly serves as a precursor for neuronal GSH synthesis. X represents an acceptor for the -glutamyl moiety in the reaction catalyzed by GT.

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into the extracellular space or the circulation, thus maintaining the overall extracellular K+ concentration within the physiological range. In addition to spatial buffering, other mechanisms such as the transient storage of K + ions appear to contribute to the potassium-buffering capacity of astrocytes.32 Supply of energy substrates Although the brain represents only 2% of the body weight, it is responsible for the consumption of an estimated 25% of all glucose in the body.37 This disproportionate energy need compared with other organs can be largely explained by the energetic cost of maintaining the steep ion gradients necessary for the transmission of action potentials.38 For this reason, neurons in particular have very high energy requirements, and are therefore highly dependent upon a tight regulation of energy substrate supply in order to sustain their normal function and cellular integrity. As mentioned previously, the morphological features of astrocytes ideally position them to sense neuronal activity at the synapse and respond with the appropriate metabolic supply via their astrocytic endfeet which almost entirely enwrap the intracerebral blood vessels (Figure 3). In line with this, an increasing body of evidence suggests that astrocytes play a key role in the spatiotemporal coupling between neuronal activity and cerebral blood flow (known as functional hyperemia) in a process that involves transient neurotransmitterinduced increases of [Ca2+]i in astrocytes, the subsequent propagation of Ca2+ waves through the astrocytic syncitium and the release of vasoactive substances (such as arachidonic acid metabolites or ATP) by astrocytic endfeet.13 Importantly, the role of astrocytes in functional hyperemia does not preclude a concerted contribution of neurons via the release of vasoactive substances such as neurotransmitters, nitric oxide, H+, and K+ to name a few.39 Although neurons can import glucose directly from the extracellular space, astrocytes have been proposed to play an instrumental role in coupling neuronal activity and brain glucose uptake through a mechanism referred to as the astrocyteneuron lactate shuttle (ANLS) (Figure 2, blue boxes).40,41 In brief, according to the ANLS, glutamate uptake into astrocytes following synaptic release causes a stimulation of anaerobic glycolysis and glucose uptake from the circulation via

GLUT1, a glucose transporter expressed specifically by glial and capillary endothelial cells in the brain. 42 Lactate produced by astrocytes as an end result of glycolysis is released into the extracellular space and taken up by neurons via monocarboxylate transporters (MCTs) expressed on astrocytes and neurons.42 Once into neurons, lactate can be used as an energy substrate via its conversion to pyruvate by the action of lactate

Figure 3. Astrocytic endfeet in humans. (A) Protoplasmic astrocytes project specialized processes towards the intraparenchymal vasculature (part of a blood vessel is highlighted in the yellow box) (glial fibrillary acidic protein (GFAP), white; nuclei (4',6diamidino-2-phenylindole - DAPI), blue. Scale bar, 20 M). (B) Astrocytic endfeet are in close contact with blood vessels and almost entirely cover their surface (GFAP, white. Scale bar, 20 M).
Adapted from ref 2: Oberheim NA, Takano T, Han X, et al. Uniquely hominid features of adult human astrocytes. J Neurosci. 2009;29:32763287. Copyright Society for Neuroscience 2009

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dehydrogenase and subsequent oxidation in the mitochondrial TCA cycle. The existence of a lactate shuttle between astrocytes and neurons is supported by a number of experimental studies (reviewed in ref 41). For instance, in an elegant study by Rouach and colleagues,43 it was recently demonstrated that 2-NBDG (a fluorescent glucose analogue) injected into a single astrocyte in hippocampal slices traffics through the astrocytic network as a function of neuronal activity. The diffusion of 2-NBGD across the astrocytic syncitium was indeed reduced when spontaneous neuronal activity was inhibited with tetrodotoxin, whereas increasing neuronal activity by means of epileptiform bursts or stimulation of the Schaffer collaterals resulted in the trafficking of 2-NBDG to a larger number of astrocytes.43 They next went on to show that during glucose deprivation which resulted in a 50% depression of synaptic transmission in hippocampal slices, glucose delivery into a single astrocyte and its subsequent (and necessary) diffusion through the astrocytic syncitium could rescue neuronal activity. This effect was mimicked by lactate but was abolished in the presence of the MCT inhibitor cyano-4-hydroxycinnamic acid (4-CIN), demonstrating that glucose present in the astrocytic network is metabolized to lactate, transported out of astrocytes, and used by neurons to sustain their activity.43 Interestingly, lactate has also been shown to preserve neuronal function in experimental models of excitotoxicity,44 posthypoxic recovery,45,46 cerebral ischemia,47 and energy deprivation,48 highlighting the importance of astrocyte-derived lactate for neuronal function and viability. Another key feature of astrocytes is their capacity to store glucose in the form of glycogen. Indeed, in the CNS glycogen is almost exclusively present in astrocytes and virtually constitutes the only energy reserve. 37,49 Interestingly, it has recently been demonstrated that neurons also possess the enzymatic machinery to synthesize glycogen, but that it normally is tightly suppressed.50 Failure to do so results in neuronal apoptosis, suggesting that intracellular glycogen is actually toxic to neurons.50 In astrocytes, glycogen can be rapidly mobilized in response to neuronal activity. 51,52 The glycosyl units resulting from glycogen breakdown are fed into the glycolytic pathway of astrocytes, and released into the extracellular space in the form of lactate which can be used to face the transiently elevated energy requirements associated with neuronal activation.49,52-54 Storage of energy in the form of glycogen is also essential for the preservation of neuronal viability in situations where glucose becomes scarce. For example, it has been demonstrated that brain glycogen levels are increased following mild hypoxic preconditioning in vivo, resulting in significant protection from brain damage as a result of subsequent cerebral hypoxic-ischemic injury.55 Beyond lactate, it is of interest to note that astrocytes may also transfer other energy substrates to neurons. Indeed, evidence suggests that in certain conditions, astrocytes may be able to metabolize fatty acids or leucine to produce ketone bodies which are know to be readily used by neurons as an energy substrate.56-58 It has been suggested that this pathway may also serve a neuroprotective purpose by scavenging nonesterified phospholipids which can lead to the production of proapoptotic sphingolipids.58,59 pH buffering Another instrumental function of astrocytes in supporting proper neuronal function is their contribution to pH regulation of the brain microenvironment (Figure 2, yellow box).60-62 Several neuronal processes are strongly affected by relatively small shifts in pH, including energy metabolism, membrane conductance, neuronal excitability, synaptic transmission, and gap junction communication.60,62 The main feature of glial cells, endowing them with a high pH buffering capacity, is their enriched expression of carbonic anhydrase (CA) which converts CO2 into H+ and HCO3-effectively allowing them to act as a CO2 sink. Indeed, CA is preferentially expressed in astrocytes and oligodendrocytes,63,64 although low activity levels are also observed in neurons and in the extracellular space.62 A coupling mechanism which integrates synaptic transmission, pH regulation, and energy supply between neurons and glia has been proposed by J. W. Deiter.61,65 According to this model, during periods of high neuronal activity, the CO2 produced by elevated (mostly neuronal) oxidative metabolism diffuses into glial cells and is converted to H+ and HCO3- by the action of glial CA. Two HCO3- can then be transported into the extracellular space along with one Na + via the Na+- HCO3- cotransporter (NBC), thereby increasing the extracellular buffering power. The protons left in the glial compartment could be used to drive the transport of lactate outside of astrocytes through MCT-1 and -4 and its subsequent transport by MCT-2 into neurons, since MCTs exploit proton gradients for the transport of

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lactate.41,61 As previously discussed, according to the ANLS hypothesis, this lactate can then be used as an energy substrate by neurons.40,41 Alternatively, protons released into the extracellular space may also be reconverted to CO2 and water by the action of extracellular CA at the expense of one HCO3-.61 This model suggests that pH buffering taking place in glial cells during neuronal activation may also act cooperatively to: i) contribute, via the Na+- HCO3- cotransporter, to the extrusion against its concentration gradient of the excess intracellular Na+ resulting from glutamate uptake in astrocytes, thereby alleviating the metabolic burden on the glial Na+/K+ ATPase; and ii) drive the efflux of lactate which is produced in response to glutamate uptake in astrocytes, thus providing an energy substrate for the neuronal TCA cycle.61,65 Defense against oxidative stress Oxidative stress occurs as a result of an imbalance between the production of reactive oxygen species (ROS) and antioxidant processes. It is known to be involved in a number of neuropathological conditions, including neurodegenerative diseases, traumatic brain injury, and stroke,66 suggesting that the CNS is particularly vulnerable to oxidative injury. This can be explained by the brains high rate of oxidative energy metabolism (which inevitably generates ROS), combined with a relatively low intrinsic antioxidant capacity.67 Compared with neurons, astrocytes display a much more effective artillery against ROS. Accordingly, cooperative astrocyte-neuron defense mechanisms against oxidative stress seem to be essential for neuronal viability.68 This is supported by a number of studies demonstrating that when cultured in the presence of astrocytes, neurons show increased resistance to toxic doses of nitric oxide, 69,70 hydrogen peroxide,71-73 superoxide anion combined with nitric oxide,69,74 or iron.69,74 This neuroprotective capacity of astrocytes may derive from the fact that they possess significantly higher levels of a variety of antioxidant molecules (including glutathione, ascorbate, and vitamin E) and display greater activities for ROS-detoxifying enzymes (including glutathione S-transferase, glutathione peroxidase, and catalase).68,72,75-78 In addition, it appears that astrocytes may also play an active role in preventing the generation of free radicals by redox active metals, as they participate in metal sequestration in the brain.79 This is

achieved in part through their high expression levels of metallothioneins and ceruloplasmin, which are involved in metal binding and iron trafficking, respectively.80-82 Glutathione (GSH) is the most important antioxidant molecule found in the brain.83 This thiol compound can act as an electron donor, and thus fulfills its antioxidant role either by directly reacting with ROS or by acting as a substrate for glutathione S-transferase or glutathione peroxidase. Both neurons and astrocytes can synthesize the GSH tripeptide (L-glutamyl-Lcysteinylglycine) by the sequential action of glutamate cysteine ligase and glutathione synthetase. However, neurons are highly dependent on astrocytes for their own GSH synthesis, as illustrated by the fact that GSH levels are higher in neurons when they are cultured in the presence of astrocytes.84 Astrocytes release GSH in the extracellular space, where it is cleaved by the astrocytic ectoenzyme -glutamyl transpeptidase (GT) to produce CysGly, which can then be taken up by neurons directly or after undergoing further cleavage by extracellular neuronal aminopeptidase N to form glycine and cysteine.83 This shuttling of GSH between astrocytes and neurons is essential in providing precursors for neuronal GSH synthesis (Figure 2, green box). This is especially true for cysteine, the rate-limiting substrate for GSH synthesis, since neurons, unlike astrocytes, cannot use the cysteine-oxidation product cystine as a precursor.83 The importance of this cooperative process for neuronal defense against oxidative stress is evidenced by the reduced ability of GSHdepleted astrocytes to protect neurons against oxidative injury. 85,86 Conversely, increasing the capacity to synthesize GSH specifically in astrocytes by increasing their capacity to uptake cystine significantly enhances the neuroprotective effect of astrocytes against oxidative stress.87 The recycling of ascorbate is another example of cooperation between astrocytes and neurons for antioxidant defense. Ascorbate can directly scavenge ROS, and is also an important cofactor for the recycling of oxidized vitamin E and GSH.68 Astrocytes are responsible for the uptake of the oxidation product of ascorbate, dehydroascorbic acid, from the extracellular space and its recycling back to ascorbic acid. The latter can then either be used intracellularly in astrocytes, or released into the extracellular space to be utilized by neurons for their own antioxidant defense.68

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Astrocytes in the diseased brain: a fine balance
Considering the extensive functional cooperativity that exists between neurons and astrocytes, one can expect that alterations of astrocytic pathways in response to pathological stimuli will result in (or at least contribute to) neuronal dysfunction. Interestingly, several neurological diseases share common pathogenic processes, such as oxidative stress, excitotoxicity, metabolic failure, or inflammationmany of which are known to be counteracted by the function of astrocytes in the normal brain (see previous sections). This may reflect a common underlying phenomenon by which disease progression is associated with chronic and/or escalating harmful stimuli that eventually exhaust the neuroprotective mechanisms of astrocytes. Even worse, deleterious pathways may then be turned on in astrocytes, directly contributing to the pathogenic process. A role of astrocytes has been described in a number of brain pathologies, and a complete review is beyond the scope of this article (see refs 88-90). Instead, we focus on three pathological processes that well illustrate the dual role of astrocytes in neuroprotection and neurotoxicity, namely neuroinflammation, Alzheimers disease, and hepatic encephalopathy. Neuroinflammation The brain can mount an immune response as a result of various insults such as infection, injury, cellular debris, or abnormal protein aggregates. In most cases, it constitutes a beneficial process aiming to protect the brain from potentially deleterious threats. In some situations, however, the insult may persist and/or the inflammatory process may get out of control. Chronic neuroinflammation sets in as a result, and may negatively affect neuronal function and viability, thus contributing to disease progression. Neuroinflammation has indeed been implicated in several neuropathologies including Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, multiple sclerosis, and stroke.91 While microglial cells are generally considered the main resident immune cells of the brain, it is important to note that astrocytes are immunocompetent cells as well, and that they act as important regulators of brain inflammation. Like microglia, astrocytes can become activated a process known as astrogliosis, which is characterized by altered gene expression, hypertrophy, and proliferation.92 Activated astrocytes can release a wide array of immune mediators such as cytokines, chemokines, and growth factors, that may exert either neuroprotective or neurotoxic effects.93 Additionally, activated astrocytes can release potentially deleterious ROS and form a glial scar which may impede axon regeneration and neurite outgrowth.94 This has led to considerable debate as to whether activation of astrocytes is beneficial or detrimental to neighbouring neurons. The most likely answer is that it is neither exclusively one nor the other, and that the overall consequences of an immune activation of astrocytes is the result of a complex interplay between pro- and anti-inflammatoryas well as neurotoxic and neurotrophicprocesses. Cytokines, for instance, are major effectors in this fine balance as they exert a dual role, potentially sustaining or suppressing neuroinflammation (hence their traditional labeling as pro- or anti-inflammatory). In this regard, dissecting out the exact neuroprotective and neurotoxic contributions of astrocytes in neuroinflammatory processes has proven to be extremely challenging because they are capable of releasing such an extensive repertoire of cytokines in response to various stimuli (some examples include interleukin (IL)-1, TNF, IL6, IL-10, IL-15, INF, and TGF).93 Adding another level of complexity, astrocytes express several cytokine receptors and can therefore also be a target of cytokine signaling through autocrine or paracrine mechanisms.11 While cytokines are categorized as proinflammatory or anti-inflammatory, understanding their exact individual effect is far more complex, as many of them interact with each other (either antagonistically or synergistically) and may additionally have pleiotropic effects.11,95 As a result, cytokines can potentially mediate both neuroprotective and neurotoxic processes at once. For example, ample evidence indicates that IL-1 may exacerbate neuronal injury both in vivo and in vitro.96-99 In contrast, IL-1 has also been implicated in neuroprotective processes such as remyelination,100 blood-brain barrier repair,101 ischemic tolerance,102 and neurotrophic factor production.103-106 Importantly, astrocytes can themselves respond to IL-1 by releasing a number of potentially neuroprotective trophic factors such as nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor GDNF, and fibroblast growth factor (FGF)-2.11,107-109 Taken together, studies such as those mentioned above provide important information about the multiple

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effects of individual cytokines. However, they also have major limitations, in that they can only take into account a few pro- and anti-inflammatory pathways at a time. As such, they may only reflect a small fraction of an infinitely more intricate process in which astrocytes take part. For this reason, the use of genetically manipulated animal models specifically preventing the proliferation of reactive astrocytes or the activation of their core inflammatory pathways, has provided important new insight into their overall role in response to brain injury. For instance, it has been demonstrated that the selective attenuation of astrocytic proinflammatory processes, through genetic inactivation of the transcription factor NF-B specifically in this cell type, affords substantial neuroprotection following spinal cord injury.110 By contrast, using a transgenic mouse model in which dividing reactive astrocytes were selectively ablated, Sofroniew and colleagues have demonstrated that following various types of brain injury, reactive astrocytes play an essential role in temporally and spatially restricting neuroinflammation, as well as in promoting blood-brain barrier repair, limiting brain edema, and preserving neuronal viability.94,111-113 Consistent with a role of astrocytes in containing neuroinflammation, it is interesting to note that astrocytes appear to participate in the suppression of microglial activation through negative feedback loops. Activated microglial cells release high levels of proinflammatory cytokines and toxic ROS which may negatively impact neuronal survival.114 Several in vitro studies have demonstrated that astrocyte-conditioned medium or the presence of astrocytes attenuates microglial activation in response to various proinflammatory stimuli.115-117 The exact nature of the astrocyte-derived factors involved has not been fully elucidated, but transforming growth factor (TFG) is thought to contribute to this process.115 This may in part explain the neuroprotective effect of TGF in experimental models of excitotoxicity or ischemia.118-120 To summarize, if inflammatory activation of astrocytes unquestionably has consequences for neuronal function and viability, it must be emphasized that the overall effect is dependent on the fine balance between a number of factors including the type, duration, and severity of the insult, the complex interplay between the various cytokines released by astrocytes and surrounding cells, and the receptors for cytokines and growth factors expressed by these neighboring cells.

Alzheimers disease Alzheimers disease (AD), the most prevalent neurodegenerative disorder, is characterized by the progressive decline of cognitive functions including memory and mental processing, and by disturbances in behavior and personality.121 Typical histopathological features of the AD brain are amyloid- (A) plaques which may contain dystrophic neurites, intracellular neurofibrillary tangles, vascular amyloidosis, neuronal and synaptic loss, and reactive gliosis. Though the exact pathophysiological mechanisms leading to synaptic loss and the resulting cognitive decline have not been fully elucidated, a central role of A peptides in concert with neuroinflammation is generally accepted. 122 Alois Alzheimer himself in 1910 suggested that glial cells may participate in the pathogenesis of dementia123; however, their exact role is still a matter of debate, as available evidence can argue both for neuroprotective or neurotoxic effects. Reactive astrocytes, like microglia, are observed in close association with A plaques in the brains of AD patients,124,125 and both cell types have been shown to be capable of internalizing and degrading A peptides.126-129 This is thought to be a neuroprotective mechanism by contributing to the clearance of A from the extracellular space, thus avoiding the accumulation of toxic extracellular A. Several observations support an active role of astrocytes in A clearance. For example, astrocytes surrounding plaques in autopsy material from the brain of AD patients contain intracellular A deposits.128,130 In addition, when exogenous astrocytes were transplanted into the brain of A plaque-bearing transgenic mice, they migrated towards A deposits and internalized Apositive material.129 Similarly in ex vivo studies, binding, internalization, and degradation of A could be observed when cultured astrocytes were seeded on top of plaque-bearing sections prepared either from the brains of AD patients or transgenic mice models of AD.127,129 The physiological importance of A clearance by glial cells in vivo is evidenced by the increased A accumulation and premature death observed in a transgenic mouse model of AD when microglial activation was impaired.131 Interestingly, glial cell activation and astrocytic accumulation of A can be observed even preceding plaque formation,128,132 suggesting that astrocyte cells attempt to scavenge A early in the progression of the disease, which likely reflects an effort to limit its extracellular deposition.

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Although their contribution to the clearance of A deposits is thought to be protective, there is also evidence to suggest that microglia and astrocytes contribute to the progression of AD. One obvious explanation is that the physiological functions of astrocytes may be directly affected by A. For instance, in a elegant study using fluorescence imaging microscopy in live mice bearing AD-like pathology, intracellular Ca2+ signaling was reported to be abnormally increased in astrocytes, sometimes propagating as intracellular calcium waves.133 These Ca2+ transients were only observed after the mice developed senile plaques and were uncoupled from neuronal activity, suggesting that A interacts directly with the astrocytic network.133 The involvement of glial cells in the pathogenesis of AD is supported by several in vitro studies demonstrating that their interaction with A impairs neuronal viability or worsens the neurotoxic effect of A.134-138 Upon their activation by A, astrocytes and microglia can release a number of inflammatory mediators which may be toxic for surrounding neurons. Examples include proinflammatory cytokines such as IL-1 and IL-6, and reactive oxygen and nitrogen species (RN/ROS) such as NO and O2-.132,139-143 Proinflammatory cytokines have been shown to exacerbate the microglial response to A and to enhance its neurotoxic effects.144-146 Moreover, it appears that proinflammatory cytokines can also increase the expression of the amyloid precursor protein and its processing through amyloidogenic pathways.147-149 A accumulation may therefore establish a vicious circle whereby neuronal stress and glial activation initiates an inflammatory response, which in turn promotes the synthesis and accumulation of more A, thus perpetuating glial cell activation. This may in part explain why age is the most important risk factor for developing AD since increased neuroinflammation is associated with normal aging.150 This enhancement of the basal inflammatory state, together with the gradual accumulation of A which is also seen in the normal aging brain, may provide the trigger necessary for this vicious circle to set in. Because of their central role in neuroinflammation (see previous section), glial cells may provide a valuable therapeutic target for the treatment of AD. This is supported by studies testing newly identified antiinflammatory molecules which selectively suppress proinflammatory cytokines production in glia, resulting in a significant attenuation of synaptic dysfunction and neurodegeneration and in behavioral improvements in experimental models of AD.151,152 Besides proinflammatory cytokines, RN/ROS produced by activated astrocytes and microglia may contribute to disease progression by inducing oxidative stress, a hallmark of AD.142,153 Astrocytes have been proposed to take part in this process. For example, A causes intracellular Ca2+ transients and stimulates the production of ROS by NADPH oxidase in astrocytes but not in neurons.154-156 In mixed cultures, these effects were accompanied by decreases in GSH levels in both astrocytes and neurons, resulting in neuronal cell death.154-156 Conversely, in the presence of microglia, astrocytes may provide significant protection through the negative regulation of microglial reactivity following exposure to A.137,157 However, this must be interpreted with caution since, as previously discussed, increased microglial phagocytosis associated with their activated state may be neuroprotective. In line with this, microglial phagocytosis was shown to be markedly suppressed in the presence of astrocytes, which resulted in increased persistence of senile plaques when presented to microglia in vitro.158 In summary, the apparently conflicting roles of astrocytes in the progression of AD may be explained by the coexistence of potentially protective and deleterious pathways in activated astrocytes. As the disease progresses, the overwhelming combined effect of A accumulation, neuroinflammation, and oxidative stress may tip the scales away from the neuroprotective functions of astrocytes and towards the activation of deleterious pathways. Hepatic encephalopathy Hepatic encephalopathy (HE), a neuropsychiatric syndrome occurring as a result of chronic or acute liver failure, is one of the first identified neurological disorders involving astroglial dysfunction as its primary cause. In its acute form, the symptoms of HE can progress rapidly from altered mental status to stupor and coma, and may cause death within days. The most important cause of mortality in acute liver failure is brain herniation, which occurs as a result of cytotoxic swelling of astrocytes, leading to intracranial hypertension.159 Although HE is a multifactorial disorder, ammonia is thought to play a central role in its pathogenesis.159 Ammonia rapidly accumulates in the blood as a result of acute liver failure and can readily cross the blood-brain barrier. Because the brain does not possess an effective urea cycle, it relies

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almost exclusively on glutamine synthesis for the detoxification of ammonia.159 As mentioned before, this is accomplished by the enzyme glutamine synthetase (GS) which is exclusively localized in astrocytes.29 Ammonia detoxification is an essential homeostatic function of astrocytes, as excess hyperammonemia has profound effects on various brain functions.159 However, the astrocytic accumulation of osmotically active glutamine as a result of ammonia detoxification is thought to contribute at least in part to the swelling of astrocytes in hyperammonemic conditions. This is supported by the demonstration that inhibition of GS with methionine sulfoxide prevents brain edema in experimental hyperammonemia.160 Alternatively, glutamine may also induce astrocytic swelling via other mechanisms, including oxidative and nitrosative stress.161 Interestingly, glutamine efflux from asctrocytes through the system N transporter appears to be negatively regulated by elevated extracellular glutamine in hyperammonemic conditions.162 Such a mechanism may contribute to trap glutamine in astrocytes and promote swelling. In contrast with its acute form, chronic hepatic encephalopathy, which is associated with more modest increases in brain ammonia, does not result in overt cerebral edema,163 suggesting the existence of compensatory mechanisms taking place in astrocytes in order to prevent excessive swelling. This is thought to be accomplished by the release of osmolytes such as taurine and myo-inositol by astrocytes in response to glutamine accumulation. However, it appears that when osmolyte pools are depleted as a result of excessive hyperammonemia, for REFERENCES
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example during acute liver failure, this protective mechanism is exhausted and astrocytes swell as a result. This, together with an impaired capacity of astrocytes to fulfill their role in ammonia detoxification, seriously compromises brain function in acute liver failure.

Conclusion
Astrocytes are known to be the most important neural cell type for the maintenance of brain homeostasis. It is safe to assume that, as technology advances in the years to come, we will continue to uncover the multiple facets of astroglia. It has already become quite clear however that it is unrealistic to approach brain function and dysfunction from a uniquely neuronal standpoint. Because of their involvement in such a wide range of homeostatic functions, any brain insult is likely to have an impact on astrocytes. Their capacity to adapt to these changes weighs heavily in the fine balance between neuroprotection and neurotoxicity as illustrated by the three neuropathological conditions discussed above. In this context, understanding astrocytic function is key to providing a better grasp of brain function in general and how it may go awry. This may lead to the identification of better suited therapeutic targets, as they should take into account the multiple interactions and interdependencies between neural cell types.
Acknowledgements: The authors wish to thank Drs Igor Allaman and Nicolas Aznavour for their help with the manuscript. Work in PJM's laboratory is supported by the Swiss National Science Foundation (grant no. 3100AO-108336/1 to PJM). MB was supported by the Fonds de la Recherche en Sant du Qubec (FRSQ).

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El papel de la astrogla en la neuroproteccin
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Rle de lastroglie dans la neuroprotection

Les astrocytes sont le principal type de cellules neuronales responsables de lentretien de lhomostasie crbrale. Ils sinterconnectent en rseaux tendus, formant des rgions anatomiques trs organises. Cette organisation qui saccompagne de toute une srie de rcepteurs, transporteurs et canaux ioniques, les met en position idale pour pressentir et moduler de faon dynamique lactivit neuronale. Les astrocytes cooprent avec les neurones diffrents niveaux, dont le recyclage et la circulation des neurotransmetteurs, lhomostasie ionique, la neuronergtique et la dfense contre le stress oxydant. Les neurones sont trs dpendants du soutien constant des astrocytes, ce qui donne ces derniers des proprits neuroprotectrices que nous analysons dans cet article. loppos, lorsque des stimuli pathognes troublent la fonction astrocytaire, la fonctionnalit et la viabilit des neurones sont compromises. En prenant pour exemples la neuro-inflammation, la maladie dAlzheimer et lencphalopathie hpatique, nous montrerons comment les mcanismes de dfense astrocytaires peuvent tre dbords en situation pathologique, participant ainsi la progression de la maladie.

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Selkoe DJ. Alzheimer disease: mechanistic understanding predicts novel therapies. Ann Intern Med. 2004;140:627-638. 123. Rodriguez JJ, Olabarria M, Chvatal A, Verkhratsky A. Astroglia in dementia and Alzheimer's disease. Cell Death Differ. 2009;16:378-385. 124. Shao Y, Gearing M, Mirra SS. Astrocyte-apolipoprotein E associations in senile plaques in Alzheimer disease and vascular lesions: a regional immunohistochemical study. J Neuropathol Exp Neurol. 1997;56:376-381. 125. Schwab C, McGeer PL. Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders. J Alzheimers Dis. 2008;13:359-369. 126. Frautschy SA, Cole GM, Baird A. Phagocytosis and deposition of vascular beta-amyloid in rat brains injected with Alzheimer beta-amyloid. Am J Pathol. 1992;140:1389-1399. 127. Wyss-Coray T, Loike JD, Brionne TC, et al. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med. 2003;9:453-457. 128. Nagele RG, D'Andrea MR, Lee H, Venkataraman V, Wang HY. Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains. Brain Res. 2003;971:197-209. 129. Pihlaja R, Koistinaho J, Malm T, Sikkila H, Vainio S, Koistinaho M. Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease. Glia. 2008;56:154-163. 130. Kurt MA, Davies DC, Kidd M. beta-Amyloid immunoreactivity in astrocytes in Alzheimer's disease brain biopsies: an electron microscope study. Exp Neurol. 1999;158:221-228. 131. El Khoury J, Toft M, Hickman SE, et al. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med. 2007;13:432-438. 132. Heneka MT, Sastre M, Dumitrescu-Ozimek L, et al. Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice. J Neuroinflammation. 2005;2:22.

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133. Kuchibhotla KV, Lattarulo CR, Hyman BT, Bacskai BJ. Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science. 2009;323:1211-1215. 134. Malchiodi-Albedi F, Domenici MR, Paradisi S, Bernardo A, Ajmone-Cat MA, Minghetti L. Astrocytes contribute to neuronal impairment in beta A toxicity increasing apoptosis in rat hippocampal neurons. Glia. 2001;34:6872. 135. Domenici MR, Paradisi S, Sacchetti B, et al. The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures. J Physiol Paris. 2002;96:313-316. 136. Paradisi S, Sacchetti B, Balduzzi M, Gaudi S, Malchiodi-Albedi F. Astrocyte modulation of in vitro beta-amyloid neurotoxicity. Glia. 2004;46:252-260. 137. von Bernhardi R, Eugenin J. Microglial reactivity to beta-amyloid is modulated by astrocytes and proinflammatory factors. Brain Res. 2004;1025:186-193. 138. Saez ET, Pehar M, Vargas MR, Barbeito L, Maccioni RB. Production of nerve growth factor by beta-amyloid-stimulated astrocytes induces p75NTRdependent tau hyperphosphorylation in cultured hippocampal neurons. J Neurosci Res. 2006;84:1098-1106. 139. Sheng JG, Mrak RE, Griffin WS. Neuritic plaque evolution in Alzheimer's disease is accompanied by transition of activated microglia from primed to enlarged to phagocytic forms. Acta Neuropathol. 1997;94:1-5. 140. Akama KT, Albanese C, Pestell RG, Van Eldik LJ. Amyloid beta-peptide stimulates nitric oxide production in astrocytes through an NFkappaB-dependent mechanism. Proc Natl Acad Sci U S A. 1998;95:57955800. 141. Bianca VD, Dusi S, Bianchini E, Dal P, I, Rossi F. beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease. J Biol Chem. 1999;274:15493-15499. 142. Luth HJ, Munch G, Arendt T. Aberrant expression of NOS isoforms in Alzheimer's disease is structurally related to nitrotyrosine formation. Brain Res. 2002;953:135-143. 143. Farfara D, Lifshitz V, Frenkel D. Neuroprotective and neurotoxic properties of glial cells in the pathogenesis of Alzheimer's disease. J Cell Mol Med. 2008;12:762-780. 144. Craft JM, Watterson DM, Hirsch E, Van Eldik LJ. Interleukin 1 receptor antagonist knockout mice show enhanced microglial activation and neuronal damage induced by intracerebroventricular infusion of human betaamyloid. J Neuroinflammation. 2005;2:15. 145. Bate C, Kempster S, Last V, Williams A. Interferon-gamma increases neuronal death in response to amyloid-beta1-42. J Neuroinflammation. 2006;3:7. 146. Ramirez G, Rey S, von Bernhardi R. Proinflammatory stimuli are needed for induction of microglial cell-mediated AbetaPP_{244-C} and Abeta-neurotoxicity in hippocampal cultures. J Alzheimers Dis. 2008;15:45-59. 147. Goldgaber D, Harris HW, Hla T, et al. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells. Proc Natl Acad Sci U S A. 1989;86:7606-7610.

148. Forloni G, Demicheli F, Giorgi S, Bendotti C, Angeretti N. Expression of amyloid precursor protein mRNAs in endothelial, neuronal and glial cells: modulation by interleukin-1. Brain Res Mol Brain Res. 1992;16:128-134. 149. Blasko I, Veerhuis R, Stampfer-Kountchev M, Saurwein-Teissl M, Eikelenboom P, Grubeck-Loebenstein B. Costimulatory effects of interferongamma and interleukin-1beta or tumor necrosis factor alpha on the synthesis of Abeta1-40 and Abeta1-42 by human astrocytes. Neurobiol Dis. 2000;7:682-689. 150. Sparkman NL, Johnson RW. Neuroinflammation associated with aging sensitizes the brain to the effects of infection or stress. Neuroimmunomodulation. 2008;15:323-330. 151. Craft JM, Watterson DM, Frautschy SA, Van Eldik LJ. Aminopyridazines inhibit beta-amyloid-induced glial activation and neuronal damage in vivo. Neurobiol Aging. 2004;25:1283-1292. 152. Ralay RH, Craft JM, Hu W, Guo L, et al. Glia as a therapeutic target: selective suppression of human amyloid-beta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration. J Neurosci. 2006;26:662-670. 153. Pratico D. Evidence of oxidative stress in Alzheimer's disease brain and antioxidant therapy: lights and shadows. Ann N Y Acad Sci. 2008;1147:70-78. 154. Abramov AY, Canevari L, Duchen MR. Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. J Neurosci. 2003;23:5088-5095. 155. Abramov AY, Canevari L, Duchen MR. Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture. Biochim Biophys Acta. 2004;1742:81-87. 156. Abramov AY, Canevari L, Duchen MR. Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J Neurosci. 2004;24:565-575. 157. Smits HA, van Beelen AJ, de Vos NM, et al. Activation of human macrophages by amyloid-beta is attenuated by astrocytes. J Immunol. 2001;166:6869-6876. 158. DeWitt DA, Perry G, Cohen M, Doller C, Silver J. Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer's disease. Exp Neurol. 1998;149:329-340. 159. Felipo V, Butterworth RF. Neurobiology of ammonia. Prog Neurobiol. 2002;67:259-79. 160. Takahashi H, Koehler RC, Brusilow SW, Traystman RJ. Inhibition of brain glutamine accumulation prevents cerebral edema in hyperammonemic rats. Am J Physiol. 1991;261:H825-H829. 161. Norenberg MD, Jayakumar AR, Rama Rao KV, Panickar KS. New concepts in the mechanism of ammonia-induced astrocyte swelling. Metab Brain Dis. 2007;22:219-234. 162. Kanamori K, Ross BD. Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain. J Neurochem. 2005;94:74-85. 163. Haussinger D, Schliess F. Pathogenetic mechanisms of hepatic encephalopathy. Gut. 2008;57:1156-1165.

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Estradiol: a hormone with diverse and contradictory neuroprotective actions
Phyllis M. Wise, PhD, Shotaro Suzuki, PhD, Candice M. Brown, PhD

n 2002 we contributed an article to Dialogues in Clinical Neuroscience which discussed the neuroprotective actions of estrogens.1 In this review we build on the understanding we had at that point, and will discuss the importance of the accumulating data that point to the complexities of estrogen action. Clearly, many factors, including the type of estrogen used, the dose and route of administration, and the age and previous hormonal and health status of the women being treated, must be taken into consideration when designing clinical studies and when interpreting results. Women usually undergo the menopausal transition when they are about 51 years of age. This dramatic physiological change, which may be abrupt or may occur over a period The concept that estrogens exert important neuroprotective actions has gained considerable attention during the past decade. Numerous studies have provided a deep understanding of the seemingly contradictory actions of estrogens. We realize more than ever that the effects of estrogens (with and without simultaneous or sequential progestins) are diverse and sometimes opposite, depending on the use of different estrogenic and progestinic compounds, on different delivery routes, on different concentrations, on treatment sequence, and on the age and health status of the women who receive hormone therapy. During the past few years, we have gained an increasing appreciation of the impact of estrogens on the immune system and on inflammation. In addition, we have learned that estrogens cannot only protect against cell death, but can also stimulate the birth of new neurons. Here we posit the concept that estrogens modulation of the immune status may be the basic mechanism that underlies its ability to protect against neurodegeneration and its powerful neuroregenerative actions. We hope that this update will encourage even richer dialogues between basic and clinical scientists to ensure that future clinical studies fully consider the information that can be derived from basic science studies. Only then will we have a better understanding of the impact of hormones on the menopausal and postmenopausal period in a womans life.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:297-303.

Keywords: estradiol; stroke; estrogen therapy; menopause; brain injury; cell death; neuroprotection; neurogenesis; inflammation; immune system; animal model Author affiliations: Departments of Physiology and Biophysics (Phyllis M. Wise, Shotaro Suzuki, Candice M. Brown), Biology (Phyllis M. Wise), and Obstetrics and Gynecology (Phyllis M. Wise),University of Washington, Seattle, Washington, USA Copyright 2009 LLS SAS. All rights reserved

Address for correspondence: Dr Phyllis M. Wise, University of Washington, 301 Gerberding Hall, Box 351237, Seattle, WA 98195-1237, USA (E-mail: pmwise@u.washington.edu)

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of a few years, is marked by a decline in ovarian hormone secretion and cessation of reproductive fertility. The postmenopausal period is often associated with vaginal dryness, urinary symptoms, osteoporosis, and a panel of neurological manifestations such as hot flushes, and greater instances of sleep disturbance, emotional instability, cerebrovascular stroke, and Alzheimer's disease. Over the past century the average life expectancy in the United States has increased to over 80 years, while the age of menopause has remained fixed at age 51. Thus, today, more than ever before, a greater number of women and a larger proportion of women are destined to spend over three decades of their lives in a hypoestrogenic state. This makes it imperative that we understand the intricacies of estrogen action, when it is protective, and when it increases risk. affects 500 000 Americans each year.3 Every year approximately 40 000 more women than men are affected by stroke.4 Initially, this gender difference was thought to be explained by a combination of both the longer life expectancy of women and the protective roles of estrogen, since the incidence of stroke increases after menopause and the risk continues to rise with age. 5 However, this interpretation has been questioned since recent clinical trials including the Women's Health initiative (WHI) reported negative impact of estrogen therapy (ET)4,6-8 and some studies in animal models also suggest that estrogens are not universally protective and can be deleterious under some circumstances.9 In an attempt to reconcile these seemingly contradictory data, our lab has used animal models to explore the mechanisms of estrogens neuroprotective and neuroregenerative actions.

Estrogens and progestins

Estradiol and progesterone belong to a family of steroid hormones with complex actions. Estradiol-17 (E2), the predominant and most biologically active estrogen, is an 18 carbon (C-18) steroid with an aromatic A-ring. It is synthesized mainly by the ovary; however, other organs and tissues, including adipose tissue, the brain (neurons, astrocytes, and microglia), cells of the immune system, and bone, are thought to produce it as well. Progesterone is a C-21 steroid hormone, which is not only an active hormone in and of itself, but is also a precursor to estrogens. In addition to the estrogens and progestins produced in human tissues, a wide variety of estrogenic and progestogenic compounds are synthesized in other species or are pharmacologically manufactured through pathways that have been developed by researchers and have been used widely by the pharmaceutical industry. When used in hormone therapy for postmenopausal women, the actions of these different compounds are diverse, complex, and sometimes contradictory. Their effects depend upon the concentration, whether they are given simultaneously or sequentially, the route of delivery, and on the age and health status of the women who receive hormone therapy. Turgeon et al2 have provided a detailed review of our current understanding of estrogens, progestogens, their related compounds, agonists, and antagonists.

Estrogens and stroke: use of animal models to decipher mechanisms of action

Even the best, well-designed clinical studies cannot benefit from the experimental advantages of many basic science studies, since studies performed with experimental animal models allow replication with adequate numbers of animals, controls with equivalent genetic backgrounds and previous exposure to similar environments, wellcontrolled environments during the entire study, and lack of selection or recall bias. Thus, investigators have developed several animal models to investigate the pathophysiology and potential treatments for stroke. Since most cerebrovascular strokes (>70%) in aging human populations are ischemic, and not hemorrhagic, we adopted an animal model that reproduces ischemic infarcts. We have utilized permanent middle cerebral artery occlusion (MCAO) as a model of permanent occlusion of the middle cerebral artery, which vascularizes the cerebral cortex, the striatum, and the hippocampus, to examine the effects of estrogen in neurodegeneration. Blockade of this artery at its base results in about a 50% decrease in blood flow and causes severe metabolic impairment in a core region, called the ischemic core and many neurons in these regions die by necrosis within hours following injury. In contrast, regions that surround the ischemic core, the ischemic penumbra, undergo more moderate metabolic impairment and are potentially salvageable by effective therapeutic agents. We have previously demonstrated that E2

Estrogens and stroke

Stroke is the third leading cause of death for middleaged and older women and a major health problem that

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exerts powerful neuroprotective action in ischemic penumbra where E2 protects neurons from delayed programmed cell death or apoptosis.10-12 Studies in the early 1990s suggested that estradiol is a neuroprotective factor that profoundly attenuates the degree of ischemic brain injury: i) female gerbils demonstrate less neuronal pathology than males after ischemia induced by unilateral carotid artery occlusion; ii) female rats sustain over 50% less infarction than males and ovariectomized female rats following ischemia induced by transient occlusion of the middle cerebral artery (MCA); iii) both ovariectomized females and castrated males that are treated with estradiol suffer less MCAO-induced injury than vehicle-treated gonadecetomized controls.1 Our work has contributed significantly to the understanding of the neuroprotective actions of physiological levels of E2. We have shown that low, physiological levels of E2, exert profound neuroprotective actions after MCAO.10-13 We have also demonstrated that E2 effectively reduces the infarct volume in middle-aged animals, suggesting that a constellation of factors responsible for mediating E2s protective actions is preserved during the initial stages of aging.14 Further, we have found that E2 must be administered prior to the onset of injury, since acute administration of E2 at the time of injury does not reduce the extent of infarction.15 Collectively, the results of these studies suggest that postmenopausal women who are estrogenreplaced may suffer a decreased degree of brain injury following a stroke, compared with their hypoestrogenic counterparts. However, we must be careful in extrapolating from rodents to humans until the appropriate clinical studies are performed.

the ability of E2 to exert protective action against ischemic injury.18 Taken together, the injured brain seems to provide signals conveying the need for the reappearance of ER, which may mediate the ability of E2 to protect against neuronal apoptosis and possibly reinitiate differentiation of the injured brain.

Anti-inflammatory actions of estrogens

More recently we have begun to appreciate the importance of inflammation in neurodegeneration and the role of E2 acting as an anti-inflammatory factor. Many neurological disorders such as Alzheimers disease, Parkinsons disease, multiple sclerosis, and cerebrovascular stroke include inflammation processes in the underlying mechanisms of the disorder.19 In vivo and in vitro models of brain injury and neurodegenerative diseases have provided substantial evidence that physiological levels of E2 suppress inflammation through ER and ER (reviewed in refs 19-22). These studies demonstrate that estrogens act not only on neurons and astrocytes, but also on microglia, the resident macrophages of the brain (Figure 1). These studies also highlight the tremendous importance of understanding the crosstalk between the nervous, endocrine, and immune systems to fully appreciate the protective role of E2 during neurological diseases and injury.19,23 The inflammatory response associated with stroke is complex, but an accumulating body of evidence clearly shows that estrogens may directly or indirectly regulate three components of the inflammatory response: i) microglial activation; ii) activation of the enzyme; inducible nitric oxide synthase (iNOS); and iii) the activation of cytokines/chemokines. These components of inflammation may interact with each other and are not mutually exclusive. Microglia become activated in response to injury, proliferate, migrate to the site of injury, and change in both morphology and cell surface markers. E2 suppresses microglial activation, and this response is regulated by both estrogen receptors. Microglia, peripheral infiltrating macrophages, and astrocytes are the primary source of the iNOS enzyme during stroke. Activation of iNOS during stroke produces high, concentrated levels of nitric oxide that promote neuronal cell death. Many studies have shown that E2 suppresses iNOS in animal models of neuroinflammation, stroke, and Alzheimers disease, and this response is also regulated by both estrogen receptors.19,24,25

Estrogen receptor mediates estrogens neuroprotective actions

To date, two forms of nuclear estrogen receptor (ER) have been cloned. Since the discovery of the second form of ER (ER) in 1996, researchers have investigated the similarities and differences in the distribution and actions of these two forms of ER. Recently, we found that ischemic injury upregulates ER expression in the cortex of ovariectomized animals without influencing ER expression.16 Consequently, we hypothesized that the dramatic re-expression of ER after stroke injury mediates E2s profound neuroprotection against ischemia.17,18 Using ER knockout mice, we found that the presence of this receptor subtype is a prerequisite for

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Cytokines are secreted proteins that appear to play a critical role in the pathophysiology of human cerebral ischemia. There is a positive correlation between high levels of proinflammatory cytokines in serum or cerebrospinal fluid greater stroke severity. These cytokines include: interleukin 6 (IL-6), IL-1, tumor necrosis factor alpha (TNF-), and macrophage chemoattractant protein1 (MCP-1). Conversely, increased levels of antiinflammatory cytokines (eg, IL-10) correlate with diminished stroke severity and an improved outcome. Cytokines in the brain perform pleiotropic functions in inflammation and are synthesized primarily by microglia and astrocytes, but also can be produced by neurons.26,27 In several different brain injury paradigms, subcutaneous E2 generally suppresses proinflammatory cytokines, and enhances the production anti-inflammatory cytokines.16,27 Studies from our laboratory have shown that immediate treatment with E2 following MCAO suppresses brain levels of the proinflammatory cytokines IL-6 and MCP1.16

Estradiol and neurogenesis

One of the most remarkable discoveries in modern neuroscience is that the adult brain continues to generate new neurons under both normal and neurodegenerative conditions. We have explored whether E2 stimulates generation of newborn neurons after stroke. We have found that low, physiological levels of E2 increase the number of newborn neurons (Figure 2). Interestingly, both ER and ER play essential functional roles, and the presence of both receptor forms is the prerequisite for E2 to enhance neurogenesis. Although precise roles for each ER form are yet to be determined, our study clearly demonstrates that the presence of both receptors is important in expansion of neuronal populations in the subventricular zone after ischemic injury.28 So far, we have not been able to determine whether these newborn neurons actually differentiate into mature neurons at this time point, whether they migrate to the site of injury, and whether they undergo synapse formation with neighboring neurons. These future studies will allow us to determine whether this elevated level of neurogenesis is critical to the recovery of function.

Importance of timing of estrogen therapy

Recent studies describing the seemingly contradictory actions of estrogens in stroke injury have led us to believe that the timing of estrogen therapy relative to the time of the menopause may be an important factor to consider. It is important to remember that in the WHI, the mean age of the subjects was 63 years, and thus, the majority of subjects were 12 years past the perimenopausal transition prior to the initiation of any horA B
neurons 250 200 150 100 50 0 Oil E2
#

Figure 1. Overview of the brain cell types and neuromodulators influenced by estrogens. The ability of estrogens to exert trophic and protective actions depends upon their ability to alter the birth and death of neurons, synaptogenesis, and neuritogenesis. Estradiol influences neurons, astrocytes, and microglia through altering the expression of a broad profile of neurotransmitters and neuropeptides and their receptors, pro- and anti-inflammatory agents, and factors which influence, birth, survival, growth, and maturation of neurons.

Figure 2. Estradiol influences the number of newborn neurons. Panel A shows confocal micrographs of newborn neurons dual-labeled with bromodeoxyuridine and doublecortin in vehicle and estradiol-treated mice following stroke injury. Panel B shows the mean of groups of 4 to 6 animals in each experimental group and shows that the differences are statistically significant.

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mone treatment.5,29 We tested the hypothesis that an extended period of hypoestrogenicity both prevents E2 from protecting the brain against ischemia, and simultaneously suppresses its anti-inflammatory actions. We found that E2 exerts profound neuroprotective action when administered immediately upon ovariectomy, but not when administered after 10 weeks of hypoestrogenicity. This dichotomous action is due to differential actions that estradiol has when administered immediately versus when treatment is initiated after a delay (Figure 3). Consistently, E2 treatment given immediately at the time of ovariectomy attenuated central and peripheral
Estrogen therapy Early Brain infarction ER expression Inflammation Overall effect Beneficial Detrimental Delayed

production of proinflammatory cytokines after ischemic stroke. In contrast, E2 did not suppress production of proinflammatory molecules when it was administered 10 weeks postovariectomy.16 These results demonstrate that a prolonged period of hypoestrogenicity disrupts both neuroprotective and anti-inflammatory actions of E2. Our findings may help to explain the results of the WHI that reported no beneficial effect of ET against stroke because the majority of the subjects initiated ET after an extended period of hypoestrogenicity.

Summary
We have summarized recent studies that have increased our understanding of the complex actions of estrogens on the brain. These basic science and clinical studies give us a new appreciation of the breadth of estrogen actions in the adult brain to maintain function after injury or during disease. Much more work is necessary before we fully understand the many ways through which estrogens exert beneficial actions, but it is clear that estradiol protects the brain from injury and enhances neurogenesis by acting to both enhance survival of neurons and stimulate the birth of new neurons, respectively. Estradiols anti-inflammatory actions may underpin both the protective and reparative effects. We hope that our growing knowledge of the pleiotropic actions of this hormone will lead to preventative and restorative therapies for neurodegenerative conditions, which will, in turn improve the lives of our aging population.
Acknowledgements: This work was supported by the NIH: AG02224 (PMW) and NRSA AG27614 (CMB).

Figure 3. Estradiol protects the brain only if treatment is initiated immediately after hypoestrogenicity is induced. Estradiol decreases the size of the infarct, induces estrogen receptor (ER) and suppresses inflammation only if it is administered immediately after ovariectomy. We have used ovariectomy to mimic the menopause. These findings strongly suggest that, if estrogen therapy (ET) is initiated after several years of postmenopause, as was the case in the Womens' Health Initiative, that ET will not be effective in protecting the brain against neurodegeneration.

REFERENCES
1. Dubal DB, Wise PM. Estrogen and neuroprotection: from clinical observations to molecular mechanisms. Dialogues Clin Neurosci. 2002;4:149-161. 2. Turgeon JL, Carr MC, Maki PM, Mendelsohn ME, Wise PM. Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies. Endocr Rev. 2006;27:575-605. 3. Paganini-Hill A. Estrogen replacement therapy and stroke. Prog Cardiovasc Dis. 1995;38:223-242. 4. Bushnell CD. Hormone replacement therapy and stroke: the current state of knowledge and directions for future research. Semin Neurol. 2006;26:123-130. 5. Mitka M. Studies explore stroke's gender gap. JAMA. 2006;295:17551756.

6. Nordell VL, Scarborough MM, Buchanan AK, Sohrabji F. Differential effects of estrogen in the injured forebrain of young adult and reproductive senescent animals. Neurobiol Aging. 2003;24:733-743. 7. Sohrabji F, Bake S. Age-related changes in neuroprotection: is estrogen pro-inflammatory for the reproductive senescent brain? Endocrine. 2006;29:191-197. 8. Viscoli CM, Brass LM, Kernan WN, Sarrel PM, Suissa S, Horwitz RI. A clinical trial of estrogen-replacement therapy after ischemic stroke. N Engl J Med. 2001;345:1243-1249. 9. Bushnell CD, Hurn P, Colton C, et al. Advancing the study of stroke in women: summary and recommendations for future research from an NINDS-sponsored multidisciplinary working group. Stroke. 2006;37:23872399. 10. Prewitt AK, Wilson ME. Changes in estrogen receptor-alpha mRNA in the mouse cortex during development. Brain Res. 2007;1134:62-69.

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Estradiol: una hormona con diversas y contradictorias acciones neuroprotectoras
Durante la ltima dcada el concepto de que los estrgenos ejercen importantes acciones neuroprotectoras ha ganado considerable atencin. Hay numerosos estudios que han proporcionado una comprensin profunda de las acciones aparentemente contradictorias de los estrgenos. Actualmente se comprende ms que nunca que los efectos de los estrgenos (con y sin progestinas simultneas o en secuencia) son diversos y algunas veces opuestos. Estos efectos dependen del uso de diferentes compuestos estrognicos y progestnicos, de las variadas vas de administracin, de las diversas concentraciones, de la secuencia de tratamiento y de la edad y estado de salud de la mujer que recibe la terapia hormonal. Durante los ltimos aos, se ha alcanzado una mayor comprensin acerca del impacto de los estrgenos en el sistema inmune y en la inflamacin. Adems, se sabe que los estrgenos no slo pueden proteger contra la muerte celular, sino que tambin pueden estimular el nacimiento de nuevas neuronas. Se propone que el concepto de la modulacin que tienen los estrgenos sobre el sistema inmune puede ser el mecanismo bsico que subyace a su capacidad de proteccin contra la neurodegeneracin y sus poderosas acciones neurorregenerativas. Se espera que esta actualizacin fomente los enriquecedores dilogos entre los cientistas bsicos y los clnicos para asegurar que los futuros estudios clnicos consideren muy bien la informacin que pueda derivarse de estudios de ciencia bsica. Slo entonces se tendr una mejor comprensin del impacto de las hormonas en el perodo menopusico y postmenopusico en la vida de la mujer.

stradiol : une hormone aux actions neuroprotectrices diverses et contradictoires

Ces 10 dernires annes, lide dune action neuroprotectrice importante des strognes a retenu particulirement lattention. Les actions apparemment contradictoires de ces hormones sont nettement mieux comprises grce aux nombreuses tudes cliniques. Nous ralisons plus que jamais que leurs effets (avec ou sans progestatif associ de faon simultane ou squentielle) sont varis et parfois opposs, dpendant de lutilisation des diffrents composs strogniques ou progestatifs, des diffrents modes dadministration et concentrations, de la chronologie des traitements, et enfin de lge et de ltat de sant des femmes qui reoivent le traitement hormonal. Ces dernires annes, lapprciation de limpact des strognes sur le systme immunitaire et linflammation sest considrablement tendue. Nous avons appris non seulement quils protgeaient les cellules de lapoptose, mais quils stimulaient galement la production de nouveaux neurones. Nous postulons dans cet article que la modulation strognique de ltat immunitaire pourrait tre le mcanisme de base qui sous-tend sa capacit protectrice contre la neurodgnration et sa puissante activit neurorgnratrice. Nous esprons que cette mise jour encouragera un dialogue plus riche entre des scientifiques cliniciens et fondamentalistes pour sassurer que les tudes cliniques futures prendront compltement en compte linformation provenant de la recherche fondamentale. Cest seulement alors que nous comprendrons limpact des hormones sur la priode de mnopause et de post-mnopause dans la vie dune femme.

11. Solum DT, Handa RJ. Localization of estrogen receptor alpha (ER[alpha]) in pyramidal neurons of the developing rat hippocampus. Dev Brain Res. 2001;128:165-175. 12. Shughrue P, Stumpf W, Maclusky N, Zielinski N, Hochberg R. Developmental changes in estrogen receptors in mouse cerebral cortex between birth and postweaning: studied by autoradiography with 11 beta-methoxy-16 alpha-[125I]iodoestradiol. Endocrinology. 1990;126:11121124. 13. Wilson ME, Liu Y, Wise PM. Estradiol enhances Akt activation in cortical explant cultures following neuronal injury. Mol Brain Res. 2002;102:4854.

14. Bryant D, Shedahl L, Marriott L, Shapiro R, Dorsa D. Multiple pathways transmit neuroprotective effects of gonadal steroids. Endocrine. 2006;29:199-207. 15. Dubal DB, Kashon M, Pettigrew L, et al. Estradiol protects against ischemic injury. J Cereb Blood Flow Metab. 1998;18:1253-1258. 16. Suzuki S, Brown CM, Dela Cruz CD, Yang E, Bridwell DA, Wise PM. Timing of estrogen therapy after ovariectomy dictates the efficacy of its neuroprotective and antiinflammatory actions. Proc Natl Acad Sci U S A. 2007;104:6013-6018. 17. Dubal DB, Shughrue PJ, Wilson ME, Merchenthaler I, Wise PM. Estradiol modulates bcl-2 in cerebral ischemia: a potential role for estrogen receptors. J Neurosci. 1999;19:6385-6393.

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Neurotoxicity of drugs of abusethe case of methylenedioxyamphetamines (MDMA, ecstasy), and amphetamines
Euphrosyne Gouzoulis-Mayfrank, MD; Joerg Daumann, PhD

mphetamines and ring substituted methylenedioxyamphetamines are the most commonly used illicit drugs after cannabis. Amphetamines are psychostimulants, and methylenedioxyamphetamines are entactogenspsychoactive drugs with emotional and social effects. Both drug groups are derivatives of -phenethylamine and they share chemical and pharmacological similarities (Figure 1). 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is the most popular entactogen, and methamphetamine (METH, speed) is the most popular stimulant. In Germany about 5% of young adults have used these drugs at least once.1 However, this percentage is 5 to 10 times higher among people who regularly attend parties and raves, and seems to be generally higher in other countries such as the UK and USA.2-5 Ecstasy (MDMA, 3,4-methylendioxymethamphetamine) and the stimulants methamphetamine (METH, speed) and amphetamine are popular drugs among young people, particularly in the dance scene. When given in high doses both MDMA and the stimulant amphetamines are clearly neurotoxic in laboratory animals. MDMA causes selective and persistent lesions of central serotonergic nerve terminals, whereas amphetamines damage both the serotonergic and dopaminergic systems. In recent years, the question of ecstasy-induced neurotoxicity and possible functional sequelae has been addressed in several studies in drug users. Despite large methodological problems, the bulk of evidence suggests residual alterations of serotonergic transmission in MDMA users, although at least partial recovery may occur after long-term abstinence. However, functional sequelae may persist even after longer periods of abstinence. To date, the most consistent findings associate subtle cognitive impairments with ecstasy use, particularly with memory. In contrast, studies on possible long-term neurotoxic effects of stimulant use have been relatively scarce. Preliminary evidence suggests that alterations of the dopaminergic system may persist even after years of abstinence from METH, and may be associated with deficits in motor and cognitive performance. In this paper, we will review the literature focusing on human studies.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:305-317.

Keywords: ecstasy; MDMA; methamphetamine; stimulant; amphetamine; neurotoxicity; serotonin; dopamine; memory Author affiliations: Department of Psychiatry and Psychotherapy, University of Cologne, Germany (Euphrosyne Gouzoulis-Mayfrank, Joerg Daumman); LVR Clinics of Cologne, Cologne, Germany (Joerg Daumman) Copyright 2009 LLS SAS. All rights reserved

Address for correspondence: Professor Euphrosyne Gouzoulis-Mayfrank, MD, Department of Psychiatry and Psychotherapy, University of Cologne, Kerpener Strasse 62, D-50924 Cologne, Germany (e-mail: e.gouzoulis@uni-koeln.de)

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Selected abbreviations and acronyms
5-HT 5-HIAA DA MDMA METH SERT serotonin 5-hydroxyindoleacetic acid dopamine methylenedioxymethamphetamine (ecstasy) methamphetamine serotonin transporter nous monoamines from presynaptic terminals. The main mechanism of amphetamines is the enhanced release of dopamine (DA), particularly in the striatal system, and norepinephrine (NE). MDMA binds most strongly to the serotonin (5-HT) transporter (SERT) and induces rapid and powerful release of both 5-HT and DA. Depending on the dose and route of administration, effects of stimulants may last from 3 to about 8 hours. They include increased drive, hypervigilance, pressure of ideas and speech, euphoria, and expansive behavior, but sometimes dysphoric mood, agitation, and aggression may occur. The psychological effects of MDMA last about 3 to 5 hours, and are more complex: they include relaxation, feelings of happiness, empathy, and closeness to other people, along with stimulant-like effects, alterations of perception, and other mild hallucinogenic effects.7 The addictive potential of amphetamines is generally lower than that of cocaine or heroin, but it becomes high when the drugs are used intravenously. MDMA is considerably less addictive, and is mostly used as a recreational drug during weekends; however, a minority of about 15% to 20% of users develop a more frequent or compulsive use pattern, and they may ingest 10 or even more pills per occasion.8 Beside the issue of addiction, there is a range of acute and subacute complications including drug-induced psychoses, seizures, myocardial infarction, or stroke resulting from hypertension and/or hemorrhage, hyperpyrexia with rhabdomyolysis, disseminated intravascular coagulation (DIC) and organ failure, toxic hepatitis, and many others. Moreover,

Both ecstasy and amphetamines are easy to manufacture in underground laboratories. Ecstasy is almost always sold as tablets or pills with various imprinted logos (Figure 2). The pills typically contain 70 to 120 mg of MDMA, although the concentration may sometimes be higher or lower. Occasionally ecstasy tablets will contain similarly acting analogues (3,4-methylenedioxy-Nethylamphetamine [MDE], 3,4-methylenedioxyamphetamine [MDA], or 3,4-methylenedioxy-alpha-ethylN-methylphenethylamine [MBDB], Figure 1) or amphetamines, and more rarely they may also contain substances from different classes. Amphetamines are mostly sold as powder which can be inhaled, smoked, ingested, or injected, although intranasal use (snorting) is now particularly common. The acute pharmacology of MDMA and amphetamines has been widely studied.6 Among other actions, both drug groups bind to presynaptic monoamine transporters, and act as inhibitors on these sites and releasers of the endoge6 5 4 3 1 2

H NH2

-Phenethylamine

Ecstasy
R2 N O O H R1

Stimulants
CH 3 N H R R 1 = CH 3; R 2 = CH 3: MDMA R 1 = H; R2 = CH: MDA R 1 = C 2H5; R 2 = CH 3: MDE R 1 = CH 3; R 2 = C 2H5: MBDB

R = H: amphetamine R = CH 3 : methamphetamine

Figure 1. Chemical structures of amphetamines and ring-substituted methylenedioxyamphetamines (ecstasy). MDMA, methylenedioxymethamphetamine; MDE, 3,4-methylenedioxy-Nethylamphetamine; MDA, 3,4-methylenedioxyamphetamine; MBDB, 3,4-methylenedioxy-alpha-ethyl-N-methylphenethylamine

Figure 2. Ecstasy pills from the illicit market.

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amphetamines and MDMA have been shown to be neurotoxic in animal studies, particularly when given at high and repeated doses. This neurotoxic potential of the drugs may be relevant for humans. In the following sections we review the evidence for neurotoxicity in animal studies and in human populations.

Animal studies
Brain morphology and neurochemistry Several studies in different laboratories and with different species demonstrate long-term alterations in brain 5-HT systems following high and repeated doses of MDMA. In studies with primates, even single doses of MDMA were found to elicit some degree of serotonergic depletion lasting over a few weeks.4 However, the lowest MDMA dose which was shown to produce longterm neurotoxic effects that persisted over months and years has been 5 mg/kg given parenterally twice daily over 4 days, ie, 40 mg/kg overall in 4 days.9-11 The alterations include depletion of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA), reduced [3H]paroxetine binding, reflecting reduced density of SERT, and reduced serotonergic axonal density in several brain regions.6-12 All but one species tested so far, including nonhuman primates, have confirmed the pattern of selective neurotoxicity for serotonergic axons, with the sole exception of mice, which exhibit neurotoxic alterations of serotonergic and dopaminergic axons.4 The rate of recovery was shown to be region-dependent. This probably corresponds to the very different distances that must be covered in the process of reinnervation. Axons need to be regrown from their origin in the serotonergic cell bodies in the raphe nuclei of the brain stem to the different terminal areas of the brain. In rats, full recovery was shown in most studies and most brain regions after 1 year, but some individual studies reported only partial recovery in the hippocampus and some cortical areas and hyperinnervation in the hypothalamus. In nonhuman primates, sensitivity to the neurotoxic effects of MDMA was shown to be more pronounced than in rodents, resulting in higher rates of 5-HT depletion with smaller doses of MDMA and persisting hypoinnervation patterns in most neocortical regions and the hippocampus in the range of 20% to 40% lower SERT binding depending on the brain region examined) for as long as 7 years post-treatment.9-11

Similarly to MDMA, stimulant amphetamines, particularly METH, have also been shown to be neurotoxic in rodent and nonhuman primate studies.6,13 Typical neurotoxic METH regimens are 5 to 10 mg/kg given parenterally 4 to 10 times within 1 to 4 days. Stimulant-related neurotoxicity is not restricted to the serotonergic system. High and/or repeated doses of METH induce widespread degeneration of presynaptic serotonergic axon terminals and degeneration of dopaminergic terminals, which is most prominent in the striatum. The consequences are 5-HT and dopamine (DA) depletion, and lower 5-HT and DA transporter densities (SERT and DAT) in brain tissue, with the effects being more pronounced on the striatal DA system.6,13-15 A recent study in vervet monkeys used an escalating-dose METH exposure which models a common human abuse pattern, and demonstrated persistent changes in the presynaptic striatal DA system 3 weeks after abstinence (20% lower striatal DA content, 35% lower DAT binding).16 However, METH toxicity to DA and 5-HT terminals had been previously shown to be considerably more long-lasting, and can persist for up to 4 years after drug administration in nonhuman primates.17 The mechanism of neurotoxicity resulting from amphetamines and MDMA is not entirely understood. However, data from animal studies strongly suggest that the formation of free radicals is a key factor, that hyperthermia enhances the formation of free radicals, and that both hyperthermia and high ambient temperatures enhance the neurotoxic effects of the drugs.4-6 Functional consequences of neurotoxic drug regimens Generally, the long-term functional abnormalities seen in laboratory animals after neurotoxic MDMA regimens have been only subtle.18-20 This may correspond to a complex role of the neuromodulator 5-HT in fine tuning and stabilizing neural transmission in cerebral networks.21-22 Broadly speaking, 5-HT appears to play important roles in several functional systems such as cognition, stimulus processing, psychological well-being, sleep control, and vegetative and neuroendocrine functions, without it being critical for the essential functioning of any of these domains. Nevertheless, some studies which used specialized behavioral test methods and pharmacological challenges reported subtle functional disturbances such as increased anxiety and poor memory performance in MDMA-treated rodents and monkeys.19-20,23-30 However, other studies reported normal

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or back-to-normal performance within 2 to 3 weeks following MDMA treatment,31-33 and studies which used behavioral tests for the assessment of anxiety and risktaking behavior yielded conflicting results.27-28,34-35 These data strongly suggest that if ecstasy users are indeed suffering neurotoxic damage to their serotonergic system, the functional consequences may be subtle. Similarly, neurotoxic METH regimens which are sufficient to produce neurotoxicity were shown to induce only moderate, if any, alterations in behaviour of laboratory animals. These moderate effects may be best explained by the fact that METH-induced degeneration of DA and 5-HT axon terminals is incomplete and that long-term reductions in monoamine concentration levels and transporter densities are in the range of 20% to 45%.16,36 Indeed, higher reductions in the range of 80% to 95% may be required to produce gross abnormalities such as parkinsonian-like motor deficits. Accordingly, reduction of spontaneous locomotor activity was reported only 3 days after a neurotoxic METH regimen, but not after 1, 2, and 4 weeks in rodents. 33 However, using more subtle motor tests, persisting deficits in active avoidance performance (24% increase in response latency) and balance beam performance (2to 3-fold increase in footfalls) were demonstrated. 36 In mice, an impairment of consolidation of learned place preference was reported after neurotoxic METH doses.37 Rats treated with a neurotoxic regimen of METH were impaired on a radial maze sequential learning task when tested after 3 weeks,38 and on a novelty preference object recognition (OR) task when tested after 1 week and 4 weeks. 39-40 Interestingly, a recent study reported than an escalating dose regimen which appears to mimic a common human pattern of escalating drug intake attenuates the neurotoxic effects and the OR deficits after METH treatment.41 Similarly, in nonhuman primates progressive increases in METH doses in an escalating dose regimen induced abnormal behavior and decreases in social behavior on injection days with aggression decreasing throughout the study; however, after 3 weeks of abstinence no differences in baseline vs post-METH behaviors were observed.16 These recent studies suggest that many METH users may not present with functional abnormalities despite residual dopaminergic toxicity; however, the extent of toxic damage and functional sequelae may well be more severe in heavy users with binge use behavior.

Are the animal data relevant for humans?

The key question is whether illicit drug users may suffer similar neurotoxic brain lesions as experimental animals. Over the last 10 to 15 years this question has received particular attention for MDMA,42 while studies with amphetamine users very been relatively scarce. Two reasons may account for the relatively lower interest in amphetaminerelated neurotoxicity in humans: first, the neurotoxic doses in experimental animals are much higher than the typical human recreational doses of 20 to 40 mg of AMPH or METH, and second, amphetamines have been used therapeutically for the treatment of attention deficit-hyperactivity disorder (ADHD) and narcolepsy for decades without clear evidence of long-term adverse effects.43 Hence, the interest in possible long-term sequelae of neurotoxic drug use has focused highly on MDMA. Compared with a neurotoxic MDMA regimen in primates (5 mg/kg twice daily over 4 days sc or ip the typical dose of a recreational MDMA weekend user (1 to 2 pills of 75 to 125 mg MDMA or analogue every 1 to 4 weeks) is still considerably lower.44 However, according to some formulae for interspecies scaling, the recreational MDMA doses might well approach doses commonly given to animals in experimental studies. 4 Moreover, some heavy users take MDMA more frequently than just at weekends; they ingest up to 10 or even more pills in one night and they typically use MDMA over years, which may increase the risk for long-term cumulative neurotoxic effects. Although these heavy users are a minority, given the widespread use of MDMA, their absolute number is large. Interestingly, a study which looked at the effects of self-administration of MDMA in primates over a period as long as 18 months showed 5-HT depletions in the order of 25% to 50% lower (5-HT concentration depending on the region examined, in various cortical and subcortical regions.45 These decrements in 5-HT content did not reach statistical significance, possibly due to the small sample in this study (n=3). Nevertheless, if the results are confirmed by further studies, they are clearly alarming.45 Furthermore, the widespread parallel use of different neurotoxic substances such as MDMA, METH, and alcohol may act synergistically and enhance the neurotoxic effects of the single drugs. Finally, neurotoxicity may be enhanced by the typical conditions associated with MDMA and METH use such as hot, overcrowded surroundings and long periods of dancing, leading to fur-

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ther increases in body temperature.46 In conclusion, it is possible that the animal data demonstrating MDMA and METH-induced neurotoxicity are indeed relevant for humans, and that club drug users may be exposing themselves to the risk of neurotoxic brain damage.

Studies in ecstasy users

Brain morphology and global brain function In principle, it is rather unlikely that neurotoxic damage confined to the serotonergic system will be visible in routine brain imaging procedures in terms of loss of brain volume or atrophy, or that it will manifest itself as an alteration of global cerebral activity in positron emission tomography and singlephoton emission tomography (PET and SPECT). However, serotonin is more than a neurotransmitter or neuromodulator in neuronal tissues; it also exerts powerful vasoconstrictive actions on small brain vessels,47 has neurotrophic effects on brain tissue not confined to the period of brain maturation,48 and has been shown to stimulate neurogenesis in the hippocampus throughout adulthood.49 Routine structural magnetic resonance imaging (MRI), perfusion and diffusion MRI, SPECT with 133Xe, and 99m Tc-hexamethylpropylene amine oxime (HMPAO) and H215O PET were generally found to be normal in ecstasy users.50-53 However, one study reported an association between longer periods of MDMA use and decreased global brain volume,50 and another study54 demonstrated reduced grey matter density in several cortical regions. In addition, studies with MR spectroscopy reported higher concentration of the glia marker myoinositole with heavier use of MDMA,55 dose-dependent reductions of N-acetylaspartate (NAA) levels (NAA:creatine and NAA:choline ratios) in the frontal cortex of MDMA users,56 and a tendency towards lower NAA:creatine ratios in the hippocampus of MDMA users compared with controls.57 These findings could be related to neurotoxic damage and glial proliferation, indicating a repair mechanism. In addition, another small pilot study reported a high diffusion coefficient (ADC) and high regional cerebral blood volume (rCBV) in the globus pallidus, a brain area that is particularly rich in 5-HT. This finding could be related to vasodilatation due to low serotonergic tone following degeneration of serotonergic axons.52 Recently, a large study with 71 ecstasy polydrug users reported alterations in the thalamus asso-

ciated specifically with MDMA use: decreased fractional anisotropy (FA) in diffusion tensor imaging (DTI) was suggestive of axonal loss; whereas increased regional cerebral blood volume (rCBV) in perfusion weighted imaging (PWI) may have been caused by 5-HT depletion.58 In the same study no effects of ecstasy use on apparent diffusion coefficients and brain metabolites (MR spectroscopy) were detected. Finally, an ambitious and methodologically sound prospective study examined a large number of young subjects who socialized in the drug scene, but had not yet used amphetamines or ecstasy (The Netherlands XTC Toxicity [NeXT] study).59 After a mean period of 17 months' follow-up, neuroimaging was repeated in 59 incident ecstasy users and 56 matched persistent ecstasy-naives using multiple NMR techniques and SPECT for measurement of SERT availability. Although the novice MDMA users reported only very sporadic and low-dose use of MDMA in the follow-up period (mean 6.0, median 2.0 tablets), the MRI examinations showed decreases in rCBV in the globus pallidus and putamen (PWI), decreases in FA (indicator of axonal integrity) in the thalamus and frontoparietal white matter (DTI) and increases of FA in globus pallidus, and increase of apparent diffusion coefficient in the thalamus. Although relatively subtle, these findings are alarming, because they are in line with sustained effects of ecstasy on brain microvasculature, white matter maturation, and possibly axonal damage, even after very low dosages of ecstasy.59 Central serotonergic parameters Reduced 5-HT concentration would be the expected outcome of widespread neurotoxic damage of serotonergic axon terminals in the brain tissue of MDMA users. As the 5-HT concentration cannot be measured in vivo in human brains, we may use the concentration of both 5-HT and its main metabolite, 5-HIAA, in cerebrospinal fluid (CSF) as a proxy for the concentration in the brain. An early study on a small number of ecstasy users reported normal levels of 5-HIAA in the CSF.60 Since then several studies with larger samples showed reduced concentrations of 5-HIAA in cerebrospinal fluid of ecstasy users compared with control groups.61-64 However, only one study reported a correlation between the 5HIAA concentration and the extent of earlier ecstasy use.62

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PET and SPECT using suitable ligands make the in-vivo examination of brain tissue receptors and/ or binding sites feasible. An early PET study in 14 ecstasy users and the SERT ligand [11C] (+)McN5652 demonstrated a dose-dependent reduction in its binding, both globally and in most cortical and subcortical brain regions examined.65 A further study in 10 ecstasy users also demonstrated reduced cortical SERT availability using SPECT and the SERT ligand -CIT.66 However, correlations between the SERT availability results, cumulative ecstasy consumption, and length of abstinence periods suggested a temporary occupation or downregulation of the binding site rather than structural neurotoxic damage.66 Since then there has been some debate on the validity of SPECT and PET techniques with SERT ligands in measuring MDMA-related neurotoxicity and on additional subject-related methodological problems of these early studies. Nevertheless, all but one more recent studies with refined methods67 and larger samples (up to 61 current and former users68) confirmed reduced SERT availability at least in female current users with a relatively heavy use pattern (>50 pills),58-68-72 and only one small study with 12 former MDMA users was negative.73 All in all, alterations were less pronounced in male users, and were absent in former users following abstinence from MDMA use of at least 12 months. A small longitudinal study with two follow-up (+)McN5652-PET examinations confirmed the reversibility of alterations of SERT availability with a decrease in the intensity of MDMA consumption.74 In summary, these studies indicate that women may be more susceptible to MDMAinduced alterations of the serotonergic system than men, and, in addition, they suggest at least some degree of recovery of the assumed serotonergic lesion following abstinence. Interestingly, another SPECT study with the 5-HT 2A receptor ligand [123I]-R91150 demonstrated reduced cortical binding in current ecstasy users with short-term abstinence and increased binding in former users who had not used ecstasy for an average of 5 months.75 This pattern is in line with animal data showing temporary (up to 1 month) downregulation of postsynaptic 5-HT2 receptors resulting from high synaptic 5-HT concentration after administration of MDMA, and long-lasting upregulation of the same postsynaptic receptors following widespread presynaptic damage of serotonergic neurons leading to 5-HT depletion.76-77 Hence, unlike the SERT data, postsynaptic receptor data suggest that alterations of serotonergic systems may persist over long periods of time in abstinent MDMA users. Such subtle residual changes could be functionally important, and might contribute to clinical or subclinical alterations of psychological well-being and behavior of ecstasy users. Serotonin-related functions The neuromodulator 5-HT is involved in several functional systems of the CNS. Consequently, damage to the central serotonergic system could be theoretically followed by disturbances in different fields such as psychological well-being, neuroendocrine secretion, vegetative functions, processing of sensory stimuli, sleep architecture, and cognition. In the last 10 to 12 years there have been numerous studies demonstrating group differences between ecstasy users and controls in virtually all these fields, and differences favor the control groups in almost every study.44 However, results have been inconsistent and several methodological problems (eg, pre-existing differences, polydrug use, differences in lifestyle) make it difficult and sometimes even impossible to draw firm conclusions from the data. The majority of studies report on psychopathology and cognition. Hence, in the following sections we will focus on these subjects. Psychopathology A low serotonergic tone has been widely associated with psychological disturbances, particularly with depression, suicidality, aggressiveness, and impulsiveness. There are several anecdotal reports of depressive syndromes, anxiety, and psychotic episodes associated with ecstasy use,78 and high psychiatric comorbidity was established in studies with large samples of ecstasy-experienced polydrug users.79-80 A causal link between these disorders and ecstasy may exist at least in a predisposed subgroup of users. However, due to the widespread use of ecstasy and the parallel use of other substances no firm conclusion can be drawn from these reports. Moreover, results from a prospective-longitudinal investigation on a large representative sample of adolescents and young adults (n=2462) over 4 years confirmed a high psychiatric comorbidity in MDMA users, but demonstrated that the use of ecstasy started, in most cases, after the onset of the comorbid disorder.81 Several studies used standardized psychometric instruments and demonstrated higher scores for impulsiveness,

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depressive mood, emotional instability, anxiety, novelty seeking, hostility/ aggression, and an overall heightened level of psychological distress in mostly polydrug ecstasy users compared with control groups.44 However, results have not been entirely consistent; for example, one study reported reduced impulsiveness and aggression compared with the control group.63 Two studies suggested a link between high scores and heavy parallel cannabis use.82-83 Moreover, in a recent study with a longitudinal design and a follow-up period of 18 months increases in self-rated psychopathology were associated with continued cannabis rather than continued ecstasy use. 84 Finally, in recent studies with relatively large samples of 234, 61, and 50 polydrug ecstasy users and controls using other drugs only, elevated psychopathology appeared to be associated with polydrug use in general and not specifically with ecstasy use.68,85-86 All in all, it is still unclear whether the frequently reported emotional instability and impulsive features and/ or the overall high level of psychological distress result from ecstasy use or from the combined use of several substances, or whether alternatively these are factors predisposing to a general affinity to drugs. Interestingly, a recent combined SPECT and psychometric study established decreased SERT availability only in current MDMA users, but elevated depression scores in current and former users.87 In this study, higher depression scores were associated with higher lifetime MDMA dose, but there was no association of psychometric scores with SERT availability.87 Finally, another study suggests an interaction between genetic factors and the effects of MDMA use on mood (high depression scores only in ecstasy users carrying the s allele of the SERT encoding gene but not in users with the ll genotype).88 These findings underline the complexity of the issue and are in line with animal data showing different long-term effects of MDMA on anxiety in rats depending on the level of their baseline anxiety, and only a loose association between the neurotoxic effects of MDMA and its long-term impact on anxiety-related behavior.4,27-28,89 In conclusion, the linkage between ecstasy-induced neurotoxicity and psychological problems does not seem to have been established at this stage. Cognition Although our understanding of the role of serotonin in cognitive processes is incomplete, there are indications

that serotonergic neurotransmission may particularly interfere with an individual's cognitive style (impulsive vs systematic) as well as with memory and learning processes.90-91 Indeed, relative deficits of short-term or working memory, episodic memory and learning, as well as increased cognitive impulsivity and diminished executive control, were frequently reported in ecstasy users.44,92 To date, the most consistent finding is that of subtle deficits in episodic memory and learning abilities. Numerous cross-sectional studies demonstrated relative impairments of learning and memory performance and only a small minority of studies reported no differences between ecstasy users and controls or small and insignificant differences after adjusting for possible confounders.44,92-93 In general, poor memory was associated with a heavier pattern of ecstasy use, although a minority of studies reported an association of memory deficits with the extent of the parallel use of cannabis or the combination of ecstasy and cannabis, rather than the use of ecstasy alone.44 Elevated cognitive impulsivity and diminished executive control were also demonstrated in some studies; however, these results have been less consistent.44,94-96 Although several studies and particularly the earlier studies suffered from significant methodological limitations such as polydrug use, short abstinence periods, poorly matched control groups, and lack of toxicological analyses for verification of the subjects reports, a number of more recent investigations were carefully designed and conducted, and their results have been similar.44-92 The consistency of the data on memory functions and the association of performance with the extent of previous ecstasy use are highly suggestive of a residual neurotoxic effect of MDMA. It is possible that the hippocampus may be particularly vulnerable to the neurotoxic effects of MDMA, and this may explain why residual effects are most consistent in the memory domain.97-98 This interpretation is in line with the animal experimental data, which demonstrated particularly strong and long-lasting neurotoxic effects of MDMA in the hippocampus,11 and a stimulatory role of 5-HT for neurogenesis in the hippocampus.49 Interestingly, three studies in current and former MDMA users with an abstinence period of several months or even years reported similar or even poorer memory performance in the former MDMA users,68,70,99 although SERT availability was only reduced in current

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users.68-70 Two longitudinal studies yielded conflicting results: a small study in 15 ecstasy users reported memory decline after continued use and improvement after abstinence over 36 months,100-101 but a larger study in 38 ecstasy users reported no further deterioration of memory performance after continued use and no improvement after abstinence over 18 months.102 Although these results may be interpreted as evidence against neurotoxicity-related memory decline, it is still possible that memory deficits in ecstasy users persist even after 18 months of abstinence because, as shown in primate studies,11 regeneration of serotonergic axons may take a long time and may remain incomplete. In addition, the functional consequences of neurotoxic lesions observed following a threshold use of ecstasy may manifest themselves in binary (yes/no) manner. Compensatory neural mechanisms that might develop could possibly explain the absence of functional deterioration despite subsequent enlargement of the neurotoxic lesions. This view would be in line both with findings of a dose-dependent memory deficit in cross-sectional studies comparing ecstasy users with control samples, and with the finding of stable performance in the larger within-subject longitudinal study.102 Finally, findings from the only prospective study to date do support this view (part of the Netherlands XTC Toxicity [NeXT] study). A large number of young subjects who socialized in the drug scene, but had not yet used amphetamines or ecstasy, were followed up and reexamined after a mean period of 3 years' follow-up. 103 Although the 58 novice MDMA users reported only very sporadic and low-dose use of MDMA in the followup period (mean 3.2, median 1.5 tablets) they failed to demonstrate retest improvements in verbal memory shown by the persistent MDMA-naive group of 60 subjects.103 This finding suggests that even very low MDMA doses may be associated with persisting alterations in memory and learning functions. Although the clinical relevance of this subtle finding is clearly limited, longterm negative consequences are conceivable. In conclusion, the linkage between ecstasy use and memory decline is considered probable at this stage. the last few years. Initial small studies with PET (regional glucose metabolic rate (rMRGlu), DAT, and D2 receptor availability), SPECT (DAT availability) and MR spectroscopy techniques suggested that heavy use of stimulants may also be neurotoxic in humans and that alterations may persist over prolonged periods of time.104-109 Reduced levels of striatal DAT were found in former METH users even 3 years or more after last use,104 and they were found to be associated with a longer duration of speed use.106 In a preliminary longitudinal study in five former speed users, rMRGlu was assessed after 6 months and again after 12 to 17 months of abstinence. During this follow-up period the initially reduced MRGlu rose in the thalamus, but remained low in the striatum, caudate, and nucleus accumbens.109 Two recent larger MR spectroscopy studies with 24110 and 36111 currently abstinent METH users reported low levels of the neuronal marker NAA (NAA/creatine ratio) in the anterior cingulate even after very long periods of abstinence of several years.110 In contrast, the choline/NAA values were abnormally high in the users with relative short abstinence time, but they normalized after 1 year of abstinence. This finding suggests that following cessation of METH use, adaptive changes occur, which may contribute to some degree of normalization of neuronal structure and function.110 A structural MRI study in 22 METH users and 21 controls revealed smaller hippocampal volumes and significant white-matter hypertrophy in the METH group.112 Finally, the largest cross-sectional study so far demonstrated enlarged putamen and globus pallidus in 50 METH users compared with 50 controls.113 Interestingly, within the METH group larger basal ganglia volumes were associated with better cognitive performance and less cumulative METH usage. Therefore, the authors argued that the enlarged putamen and globus pallidus might represent a compensatory response to maintain function.113 A recent review of the literature reported enlarged striatal volumes, reduced concentrations of the neuronal marker NAA-acetylasparate and total creatine in the basal ganglia, reduced DAT density, and reduced dopamine D2 receptors in the striatum, lower levels of SERT density and vesicular monoamine transporters (VMAT2) across striatal subregions, and altered brain glucose metabolism in the limbic and orbitofrontal regions of METH users.114 Theoretically, neurotoxic dopaminergic lesions could be associated with motor, cognitive, and psychopathologi-

Studies in amphetamine users

Compared with MDMA, the literature on amphetamine related neurotoxicity in humans is limited, but the number of publications has been constantly increasing over

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cal abnormalities. To date, gross motor disturbances have not been demonstrated in METH users.115 However, more subtle motor deficits were reported in two studies.106,109 The literature on long-term psycho(patho)logical sequelae of stimulant use is inconclusive. Similarly, cross-sectional studies in chronic stimulant users demonstrated relatively low performance in short-term and episodic memory, frontal executive control, and planning abilities.111,116-122 However, it is not clear whether these deficits are a consequence of the use of stimulants or whether they reflect pre-existing low cognitive abilities in people who become drug users later in life. Nevertheless, reduced DAT densities and longer duration of METH use were associated with poorer performance in both fine motor and memory tasks in 15 currently abstinent METH users.106 Also, the normalization of rMRGlu in the thalamus was associated with an improvement of motor and memory performance after long-term abstinence of 1 year and more.109 Finally, reduced attentional control (ie, increased Stroop interference) was shown to correlate with levels of NAA-Cr within the anterior cingulate in METH users, but not in controls.111 In conclusion, the limited evidence to date suggests that persisting neurotoxic brain damage is conceivable in METH users, especially in heavy users with binge use patterns. More studies with longitudinal and prospective designs are clearly needed.

Conclusions
Ecstasy (MDMA) and stimulant amphetamines (METH and AMPH) are popular drugs of abuse and they are neurotoxic in animal studies. High and repeated doses of MDMA cause selective and long-lasting degeneration of 5-HT axon terminals in several brain regions, whereas METH and AMPH damage both serotonergic and dopaminergic neurons. Although the doses taken recreationally are considerably lower than the doses typically given in animal studies, some users exhibit compulsive binge use behaviors that may well correspond to the ani-

mal doses. In addition, polydrug use and the typical environment of use (hot, overcrowded, and noisy rooms, extensive physical exercise in the form of dancing) may well potentiate the neurotoxic effects of the drugs. Studies with drug users demonstrated associations of subtle alterations in brain structure and 5-HT brain parameters with MDMA use. Similarly, subtle cognitive dysfunctions, particularly in the memory and learning domain, were also found to be associated with ecstasy use. Although the results are not entirely consistent, these associations were replicated in many welldesigned, controlled studies including longitudinal and one prospective investigation. Moreover, the only prospective study to date demonstrated structural brain alterations and subtle memory dysfunction, even after minimal exposure to MDMA.59,103 Although most ecstasy users do not suffer cognitive impairment of clinically relevant proportions, and even heavy users initially appear mostly unimpaired in their everyday life, several cases with severe deficits have also been reported. 123-124 Moreover, there is concern that the memory deficits of ecstasy usersalthough subtle and mostly subclinical and the possible underlying hippocampal dysfunction might help accelerate the normal brain ageing process and constitute a risk factor for earlier onset and/or more severe age-related memory decline in later years.44 Regarding METH-induced neurotoxicity, evidence from studies with drug users is relatively scarce and still preliminary. However, there are early indications that at least heavy METH use may also be followed by alterations in brain structure, dopaminergic parameters, and cognitive function. In light of the popularity of ecstasy and stimulants among young people, questions around their neurotoxic effects on the brain remain highly topical. To date, the message we have to convey to young people in information campaigns is: MDMA and amphetamine neurotoxicity for humans is not yet proven, but it is highly likely. Further longitudinal and prospective studies are clearly needed.

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Neurotoxicidad de las drogas de abuso: el caso de las metilendioxianfetaminas (MDMA, xtasis) y las anfetaminas estimulantes
El xtasis (MDMA, 3,4 metilendioximetanfetamina), y los estimulantes metanfetamnicos (METH, speed) y anfetamnicos son drogas frecuentes entre los jvenes, especialmente en lugares de baile. Cuando el MDMA los estimulantes anfetamnicos son administradas en altas dosis a animales de laboratorio resultan claramente neurotxicas. El MDMA produce lesiones selectivas y persistentes de los terminales nerviosos serotoninrgicos centrales, mientras que las anfetaminas daan tanto los sistemas serotoninrgicos como dopaminrgicos. En los ltimos aos la pregunta acerca de la neurotoxicidad inducida por el xtasis y las posibles secuelas funcionales ha sido tema de algunos estudios con usuarios de drogas. A pesar de los grandes problemas metodolgicos, la amplia evidencia sugiere que existen alteraciones residuales de la transmisin serotoninrgica en usuarios de MDMA, aunque puede conseguirse cierta recuperacin parcial despus de una abstinencia prolongada. Sin embargo, las secuelas funcionales pueden persistir aun despus de largos perodos de abstinencia. A la fecha, los hallazgos ms consistentes asocian los deterioros cognitivos leves, especialmente las alteraciones de memoria con el uso de xtasis. En contraste, los estudios acerca de los posibles efectos neurotxicos a largo plazo por el uso de estimulantes han sido relativamente escasos. La evidencia preliminar sugiere que las alteraciones del sistema dopaminrgico pueden persistir aun despus de aos de abstinencia de METH y pueden asociarse con dficit en el rendimiento motor y cognitivo. En este artculo se revisar la literatura dedicada a estudios en humanos.
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Neurotoxicit des substances lorigine dabus: cas des mthylnedioxyamphtamines (MDMA, ecstasy) et des amphtamines stimulantes
Lecstasy (MDMA, 3,4-mthylnedioxymthamphtamine) et les stimulants mthamphtaminiques (METH, speed) et amphtaminiques sont des drogues courantes chez les jeunes, surtout dans les milieux festifs. Administres dose leve des animaux de laboratoire, la MDMA et les amphtamines stimulantes sont clairement neurotoxiques. La MDMA provoque des lsions slectives et persistantes des terminaisons nerveuses centrales srotoninergiques et les amphtamines lsent la fois les systmes srotoninergique et dopaminergique. Ces dernires annes, plusieurs tudes ont trait la question de la neurotoxicit de lecstasy et des squelles ventuelles chez les consommateurs de cette drogue. Malgr dimportants problmes mthodologiques, lessentiel des arguments sont en faveur de modifications rsiduelles de la transmission srotoninergique chez les consommateurs de MDMA, rcuprables en partie aprs une longue abstinence. Cependant, mme aprs un arrt prolong, des squelles fonctionnelles peuvent persister. Aujourdhui les rsultats les plus constants font tat de dficits cognitifs subtils avec lecstasy, surtout mnsiques. loppos, les tudes sur les ventuels effets neurotoxiques long terme de la consommation de stimulant sont relativement rares. Des rsultats prliminaires montrent que les modifications du systme dopaminergique persistent, mme aprs des annes dabstinence de METH, persistance qui peut tre associe des dficits des performances motrices et cognitives. Dans cet article, nous passons en revue la littrature centre sur les tudes chez lhomme.
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Ecstasy use and higher-level cognitive functions: weak effects of ecstasy after control for potential confounds. Psychol Med. 2008;38:1319-1330. 94. Gouzoulis-Mayfrank E, Daumann J, Tuchtenhagen F, et al. Impaired cognitive performance in drug free users of recreational ecstasy (MDMA). J Neurol Neurosurg Psychiatry. 2000;68:719-725. 95. Morgan MJ, Impallomeni LC, Pirona A, et al. Elevated impulsivity and impaired decision-making in abstinent ecstasy (MDMA) users compared to polydrug and drug-naive controls. Neuropsychopharmacology. 2006;31:15621573. 96. Quednow BB, Kuhn KU, Hoppe C, et al. Elevated impulsivity and impaired decision-making cognition in heavy users of MDMA ("Ecstasy"). Psychopharmacology. 2007;189:517-530. 97. Fox HC, McLean A, Turner JJ, et al. Neuropsychological evidence of a relatively selective profile of temporal dysfunction in drug-free MDMA ("ecstasy") polydrug users. Psychopharmacology. 2002;162:203-214. 98. Gouzoulis-Mayfrank E, Thimm B, Rezk M, et al. Memory impairment suggests hippocampal dysfunction in abstinent ecstasy users. Prog Neuropsychopharmacol Biol Psychiatry. 2003;27:819-827. 99. Curran HV, Verheyden SL. Altered response to tryptophan supplementation after long-term abstention from MDMA (ecstasy) is highly correlated with human memory function. Psychopharmacology. 2003;169:91-103. 100.Zakzanis KK, Young DA. Memory impairment in abstinent MDMA ("Ecstasy") users: a longitudinal investigation. Neurology. 2001;56:966-969. 101.Zakzanis KK, Campbell Z. Memory impairment in now abstinent MDMA users and continued users: a longitudinal follow-up. Neurology. 2006;66:740741. 102.Gouzoulis-Mayfrank E, Fischermann T, Rezk M, et al. Memory performance in polyvalent MDMA (ecstasy) users who continue or discontinue MDMA use. Drug Alcohol Depend. 2005;78:317-323. 103.Schilt T, de Win MM, Koeter M, et al. Cognition in novice ecstasy users with minimal exposure to other drugs: a prospective cohort study. Arch Gen Psychiatry. 2007;64:728-736. 104.McCann UD, Wong DF, Yokoi F, et al. Reduced striatal dopamine transporter density in abstinent methamphetamine and methcathinone users: evidence from positron emission tomography studies with [11C]WIN-35,428. J Neurosci. 1998;18:8417-8422. 105.Ernst T, Chang L, Leonido-Yee M, et al. Evidence for long-term neurotoxicity associated with methamphetamine abuse: a 1H MRS study. Neurology. 2000;54:1344-1349. 106.Volkow ND, Chang L, Wang GJ, et al. Loss of dopamine transporters in methamphetamine abusers recovers with protracted abstinence. J Neurosci. 2001;21:9414-9418.

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107.Volkow ND, Chang L, Wang GJ, et al. Low level of brain dopamine D2 receptors in methamphetamine abusers: association with metabolism in the orbitofrontal cortex. Am J Psychiatry. 2001;158:2015-2021. 108.Reneman L, Booij J, Lavalaye J, et al. Use of amphetamine by recreational users of ecstasy (MDMA) is associated with reduced striatal dopamine transporter densities: a [123I]beta-CIT SPECT study--preliminary report. Psychopharmacology. 2002;159:335-340. 109.Wang GJ, Volkow ND, Chang L, et al. Partial recovery of brain metabolism in methamphetamine abusers after protracted abstinence. Am J Psychiatry. 2004;161:242-248. 110.Nordahl TE, Salo R, Natsuaki Y, et al. Methamphetamine users in sustained abstinence: a proton magnetic resonance spectroscopy study. Arch Gen Psychiatry. 2005;62:444-452. 111.Salo R, Nordahl TE, Natsuaki Y, et al. Attentional control and brain metabolite levels in methamphetamine abusers. Biol Psychiatry. 2007;61:12721280. 112.Thompson PM, Hayashi KM, Simon SL, et al. Structural abnormalities in the brains of human subjects who use methamphetamine. J Neurosci. 2004;24:6028-6036. 113.Chang L, Cloak C, Patterson K, et al. Enlarged striatum in abstinent methamphetamine abusers: a possible compensatory response. Biol Psychiatry. 2005;57:967-974. 114.Chang L, Alicata D, Ernst T, et al. Structural and metabolic brain changes in the striatum associated with methamphetamine abuse. Addiction. 2007;102(suppl 1):16-32.

115.Caligiuri MP, Buitenhuys C. Do preclinical findings of methamphetamine-induced motor abnormalities translate to an observable clinical phenotype? Neuropsychopharmacology. 2005;30:2125-2134. 116. Ornstein TJ, Iddon JL, Baldacchino AM, et al. Profiles of cognitive dysfunction in chronic amphetamine and heroin abusers. Neuropsychopharmacology. 2000;23:113-126. 117. Simon SL, Domier C, Carnell J, et al. Cognitive impairment in individuals currently using methamphetamine. Am J Addict. 2000;9:222-231. 118. Simon SL, Domier CP, Sim T, et al. Cognitive performance of current methamphetamine and cocaine abusers. J Addict Dis. 2002;21:61-74. 119. Salo R, Nordahl TE, Possin K, et al. Preliminary evidence of reduced cognitive inhibition in methamphetamine-dependent individuals. Psychiatry Res. 2002;111:65-74. 120. Lawton-Craddock A, Nixon SJ, Tivis R. Cognitive efficiency in stimulant abusers with and without alcohol dependence. Alcohol Clin Exp Res. 2003;27:457-464. 121.Woods SP, Rippeth JD, Conover E, et al. Deficient strategic control of verbal encoding and retrieval in individuals with methamphetamine dependence. Neuropsychology. 2005;19:35-43. 122. Scott JC, Woods SP, Matt GE, et al. Neurocognitive effects of methamphetamine: a critical review and meta-analysis. Neuropsychol Rev. 2007;17:275-297. 123.Spatt J, Glawar B, Mamoli B. A pure amnestic syndrome after MDMA ("ecstasy") ingestion. J Neurol Neurosurg Psychiatry. 1997;62:418-419. 124.Soar K, Parrott AC, Fox HC. Persistent neuropsychological problems after 7 years of abstinence from recreational Ecstasy (MDMA): a case study. Psychol Rep. 2004;95:192-196.

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The impact of neuroimmune dysregulation on neuroprotection and neurotoxicity in psychiatric disordersrelation to drug treatment
Norbert Mller, MD, PhD, DipPsych; Aye-Mu Myint, MD, PhD, Markus J. Schwarz, MD, PhD
here is no doubt that dopaminergic, serotonergic, and/or noradrenergic neurotransmission play an important role in the pathophysiology of major depression (MD) and schizophrenia. Although the roles of dopamine in schizophrenia and of serotonin and noradrenaline in depression have been studied intensively, the exact underlying pathological mechanisms of both disorders are still unclear. In MD, glutamatergic hyperfunction seems to be closely related to the lack of serotonergic and noradrenergic neurotransmission. Altered glutamate levels have been observed in the plasma, serum, cerebrospinal fluid (CSF), and in imaging and postmortem studies of depressed An inflammatory pathogenesis has been postulated for schizophrenia and major depression (MD). In schizophrenia and depression, opposing patterns of type-1 vs type-2 immune response seem to be associated with differences in the activation of the enzyme indoleamine 2,3-dioxygenase and in the tryptophan-kynurenine metabolism, resulting in increased production of kynurenic acid in schizophrenia and decreased production of kynurenic acid in depression. These differences are associated with an imbalance in the glutamatergic neurotransmission, which may contribute to an excessive agonist action of N-methyl-D-aspartate (NMDA) in depression and of NMDA antagonism in schizophrenia. Regarding the neuroprotective function of kynurenic acid and the neurotoxic effects of quinolinic acid (QUIN), different patterns of immune activation may also lead to an imbalance between the neuroprotective and the neurotoxic effects of the tryptophan/kynurenine metabolism. The differential activation of microglia cells and astrocytes may be an additional mechanism contributing to this imbalance. The immunological imbalance results in an inflammatory state combined with increased prostaglandin E2 production and increased cyclo-oxygenase-2 (COX-2) expression. The immunological effects of many existing antipsychotics and antidepressants, however, partly correct the immune imbalance and the excess production of the neurotoxic QUIN. COX-2 inhibitors have been tested in animal models of depression and in preliminary clinical trials, pointing to favorable effects in schizophrenia and in MD.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:319-332.

Keywords: schizophrenia; major depression; kynurenine; inflammation; therapy Author affiliations: Department of Psychiatry and Psychotherapy, LudwigMaximilians-Universitt Mnchen, Germany Copyright 2009 LLS SAS. All rights reserved

Address for correspondence: Prof Dr med Dipl-Psych Norbert Mller, Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-Universitt, Nubaumstr. 7, 80336 Mnchen, Germany (e-mail: norbert.mueller@med.uni-muenchen.de) www.dialogues-cns.org

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Selected abbreviations and acronyms
COX IDO IL KYN KYNA MD QUIN TDO TNF cyclo-oxygenase indoleamine 2,3-dioxygenase interleukin kynurenine kynurenic acid major depression quinolinic acid tryptophan 2,3-dioxygenase tumor necrosis factor types and all cellular components of the immune system, including the innate immune system. Helper T-cells are of two types, T-helper-1 (TH-1) and T-helper-2 (TH-2). TH-1 cells produce the characteristic type-1 activating cytokines such as interleukin (IL) -2 and interferon (IFN)-. However, since not only TH-1 cells, but also certain monocytes/macrophages (M1) and other cell types produce these cytokines, the immune response is called the type-1 immune response. The humoral, antibodyproducing arm of the adaptive immune system is mainly activated by the type-2 immune response. TH-2 or certain monocytes/macrophages (M2) produce mainly IL4, IL-10, and IL-13.6 Further terminology separates the cytokines into proinflammatory and anti-inflammatory types. Proinflammatory cytokines, such as tumor necrosis factor (TNF-) and IL-6 are primarily secreted from monocytes and macrophages, activating other cellular components of the inflammatory response. While TNF- is an ubiquitiously expressed cytokine mainly activating the type-1 response, IL-6 activates the type-2 response including the antibody production. Antiinflammatory cytokines such as IL-4 and IL-10 help to downregulate the inflammatory immune response. The type-1 immune system promotes the cell-mediated immune response directed against intracellular pathogens, whereas the type-2 response helps B-cell maturation and promotes the humoral immune response, including the production of antibodies directed against extracellular pathogens. Type-1 and type-2 cytokines antagonize each other in promoting their own type of response, while suppressing the immune response of the other; therefore the term polarized can be used.

patients.1 In schizophrenia, in contrast, dopaminergic hyperfunction in the limbic system and dopaminergic hypofunction in the frontal cortex are thought to be the main neurotransmitter disturbances. Recent research provides further insight that glutamatergic hypofunction might be the cause for this dopaminergic dysfunction in schizophrenia,2 whereas glutamatergic hyperfunction acts through low NMDA antagonism in the kynurenine pathway in MD.3 Glutamatergic dysfunction seems to be a common pathway in the neurobiology of schizophrenia and depression. The glutamatergic system is closely related in function to the immune system and to the tryptophankynurenine metabolism, which both seem to play a key role in the pathophysiology of schizophrenia and MD.4,5

The immune response and type-1 and type-2 polarization

The innate immune system is phylogenetically the oldest part of the immune response, natural killer (NK) cells and monocytes as the first barrier of the immune system being part of this. The adaptive immune response with the antibody-producing B-lymphocytes, the T-lymphocytes and their regulating immunotransmitters, the cytokines, is the specifically acting component of the immune system. (Tables I and II). Cytokines regulate all
Components Cellular Innate Monocytes Makrophages Granulocytes Natural killer cells /-cells Complement, acute-phase protein, mannose-binding lectin Adaptive T- and B-cells

Inflammation in schizophrenia and depression

Infection during pregnancy in mothers of offspring who later develop schizophrenia has been repeatedly described, in particular in the second trimester. 7,8 The
Type-1 IL-2 IL-12 IFN- IL-18 (TNF-) Type-2 IL-4 IL-13 [IL-10]

Cytokines

Humoral

Antibodies

Table I. Components of the unspecific innate and the specific adaptive immune systems in humans.

Table II. Cytokines of the polarized immune response. IL, interleukin; IFN, interferon; TNF, tumor necrosis factor.

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maternal immune response itself, as opposed to any single pathogen, may be related to the increased risk for schizophrenia in the offspring.9 Indeed, increased IL-8 levels of mothers during the second trimester were associated with an increased risk for schizophrenia in the offspring.7 A fivefold increased risk for developing psychoses later on was detected after infection of the central nervous system (CNS) in early childhood. 7,10 These data were confirmed in recent studies.11,12, 13 Signs of inflammation were found in schizophrenic brains,14 and the term mild localized chronic encephalitis to describe a slight but chronic inflammatory process in schizophrenia was proposed.15 An inflammatory model of MD is sickness behavior, the reaction of the organism to infection and inflammation. Sickness behavior is characterized by weakness, malaise, listlessness, inability to concentrate, lethargy, decreased interest in the surroundings, and reduced food intake all of which are depression-like symptoms. The sicknessrelated psychopathological symptoms during infection and inflammation are mediated by proinflammatory cytokines such as IL-1, IL-6, TNF-, and IFN-. The active pathway of these cytokines from the peripheral immune system to the brain is via afferent neurons and through direct targeting of the amygdala and other brain regions after diffusion at the circumventricular organs and choroid plexus. Undoubtedly, there is a strong relationship between the cytokine and the neurotransmitter systems, but the specific mechanisms underlying the heterogeneous disease MD are not yet fully understood. In humans, the involvement of cytokines in the regulation of the behavioral symptoms of sickness behavior has been studied by application of the bacterial endotoxin lipoploysaccharide (LPS) to human volunteers. 16 LPS, a potent activator of proinflammatory cytokines, was found to induce mild fever, anorexia, anxiety, depressed mood, and cognitive impairment. The levels of anxiety, depression, and cognitive impairment were found to be related to the levels of circulating cytokines.17 Mechanisms that may contribute to inflammation and cause depressive states are: A direct influence of proinflammatory cytokines on the serotonin and noradrenaline metabolism An imbalance of the type-1type-2 immune response leading to an increased tryptophan and serotonin metabolism by activation of indoleamine 2,3-dioxygenase (IDO) in the CNS, which is associated with:

-A decreased availability of tryptophan and serotonin -A disturbance of the kynurenine metabolism with an imbalance in favour of the production of the NMDA receptor agonist quinolinic acid (QUIN) -An imbalance in astrocyte and microglial activation associated with increased production of QUIN. Effects of antidepressants on the immune function support this view. The mechanisms and the therapeutic implications will be discussed below. Inflammation, caused by infection or by other mechanisms, seems to play a role in schizophrenia and in MD. Type-1 and type-2 immune responses in schizophrenia A well established finding in schizophrenia is the decreased in vitro production of IL-2 and IFN-,18,19 reflecting a blunted production of type-1 cytokines. Decreased levels of neopterin, a product of activated monocytes/macrophages, also point to a blunted activation of the type-1 response.20 The decreased response of lymphocytes after stimulation with specific antigens reflects a reduced capacity for a type-1 immune response in schizophrenia, as well.21 intracellular adhesion molecule (ICAM)-1 is a type-1 related protein and a celladhesion molecule expressed on macrophages and lymphocytes. Decreased levels of the soluble (s) intercellular adhesion molecule-1 (ICAM-1), as found in schizophrenia, also represent an underactivation of the type-1 immune system.22 Decreased levels of the soluble TNFreceptor p55mostly decreased when TNF- is decreasedwere observed, too.23 A blunted response of the skin to different antigens in schizophrenia was observed before the era of antipsychotics.24 This finding could be replicated in unmedicated schizophrenic patients using a skin test for the cellular immune response.25 However, there are some conflicting results regarding increased levels of Th1 cytokines in schizophrenia.26 The latest meta-analysis showed dominant proinflammatory changes in schizophrenia but not involving Th2 cytokines.27 After including antipsychotic medication effects into the analysis, only increases of IL1 receptor antagonist serum levels and of IL-6 serum levels were found. Type-1 parameters, hypothesized to be downregulated in schizophrenia, were not included in the meta-analysis, because only a few studies have been performed in unmedicated patients. Several reports described increased serum IL-6 levels in schizophrenia.28 IL-6 serum levels might be especially

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high in patients with an unfavorable course of the disease.29 IL-6 is a product of activated monocytes, and some authors refer to it as a marker of the type-2 immune response. Moreover, several other signs of activation of the type-2 immune response are described in schizophrenia, including increased Th2 type of lymphocytes in the blood,30 increased production of immunoglobulinE (IgE), and an increase in IL-10 serum levels.31,32 In the CSF, IL-10 levels were found to be related to the severity of the psychosis.32 The key cytokine of the type-2 immune response is IL4. Increased levels of IL-4 in the CSF of juvenile schizophrenic patients have been reported,33 which indicates that the increased type-2 response in schizophrenia is not only a phenomenon of the peripheral immune response. However, the data show that the immune response in schizophrenia can be confounded partly by factors specific to the disease such as its duration, chronicity, or therapy response, and partly by other factors such as antipsychotic medication, smoking, etc. Increased proinflammatory type-1 cytokines in major depression Characteristics of the immune activation in MD include increased numbers of circulating lymphocytes and phagocytic cells, upregulated serum levels of indicators of activated immune cells (neopterin, soluble IL-2 receptors), and higher serum concentrations of positive acute phase proteins (APPs), coupled with reduced levels of negative APPs, as well as increased release of proinflammatory cytokines, such as IL-1, IL-2, TNF- and IL6 through activated macrophages and IFN- through activated T-cells.32-39 Increased numbers of peripheral mononuclear cells in MD have been described by different groups of researchers.40 Neopterin is a sensitive marker of the cell-mediated type-1 immunity. The main sources of neopterin are monocytes/macrophages. In accordance with the findings of increased monocytes/macrophages, an increased secretion of neopterin has been described by several groups of researchers.41, 42 The increased plasma concentrations of the proinflammatory cytokines IL-1 and IL-6 observed in depressed patients was found to correlate with the severity of depression and with measures of the hypothalamus-pituitary-adrenal (HPA)-axis hyperactivity.43, 44 As genetics plays a role in MD, the genetics of the immune system in relation to MD has also been investigated. Particular cytokine gene polymorphisms, eg, in genes coding for IL1 and TNF- may confer a greater susceptibility to develop MD, although studies are conflicting.45, 46 The production of IL-2 and IFN- is the typical marker of a type-1 immune response. In contrast to schizophrenia, IFN- is produced in greater amounts by lymphocytes of patients with MD than of healthy controls.42, 45 Higher plasma levels of IFN- in depressed patients, accompanied by lower plasma tryptophan availability were described,42 and the IFN/IL-4 ratio, a marker for Th1/Th2 balance is also higher in depressed patients.45 Data on IL-2 in MD are mainly restricted to the estimation of its soluble receptor sIL-2R in the peripheral blood. Increased sIL-2R levels reflect an increased production of IL-2. The blood levels of sIL-2R were repeatedly found to be increased in MD patients.39 Increased expression of ICAM-1 is observed in inflammatory processes, and promotes the influx of peripheral immune cells through the blood-brain barrier.47 By this mechanism, macrophages and costimulatory lymphocytes can invade the central nervous system (CNS), further increasing the proinflammatory immune response. The plasma levels and CNS expression of ICAM-1 are associated with depressive symptoms in patients treated with IFN-. Increased sICAM-1 levels were observed in patients with more depressive symptoms,48 and increased expression of ICAM-1 was found in the prefrontal cortex of elderly depressed patients.49 In late-life depression, however, there are conflicting results.50 Since different pathologies may underlie the syndrome of depression, different immunological states might be involved. Indeed, different types of MD were observed to exhibit different immune profiles: the subgroup of melancholic depressed patients showed a decreased type-1 activationas observed in schizophrenic patients40while the nonmelancholic depressed patients showed signs of inflammation such as increased monocyte count and increased levels of 2-macroglobulin.40 Suicidality, observed in a very high proportion of depressed patients, seems to be an example of the immune activation pattern in depression, since clinical studies have observed higher levels of type-1 cytokines in suicidal patients. In a small study, distinct associations between suicidality and type-1 immune response and a predominance of type-2 immune parameters in nonsuicidal patients were observed.51 An epidemiological study

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hypothesized that high IL-2 levels are associated with suicidality.52 Increased levels of serum sIL-2R have been described in medication-free suicide attempters, irrespective of the psychiatric diagnosis,53 and treatment with high-dose IL-2 has been associated with suicide in a case report.54 These data show that possible different immune states within the category of MD need to be better differentiated. The predominant proinflammatory, type-1 dominated immune state described in MD may be a kind of model state state restricted to a majority of patients suffering from MD. Therefore, these and other methodological concerns have to be considered carefully in future studies.

Therapeutic techniques in depression are associated with downregulation of the proinflammatory immune response Antidepressant pharmacotherapy A modulatory, predominantly inhibitory effect of selective serotonin reuptake inhibitors (SSRIs) on activation of proinflammatory immune parameters was demonstrated in animal experiments.62, 63 Several antidepressants seem to be able to induce a shift from type 1 to type 2, in other words from a proinflammatory to an anti-inflammatory immune response, since the ability of three antidepressants (sertraline, clomipramine, and trazodone) to greatly reduce the IFN/IL-10 ratio was shown in vitro. These drugs reduced the IFN- production significantly, while sertraline and clomipramine additionally raised the IL-10 production.61 Regarding other in-vitro studies, a significantly reduced production of IFN-, IL-2, and sIL-2R was found after antidepressant treatment compared with pretreatment values.63 A downregulation of the IL-6 production was observed during amitriptyline treatment; in treatment responders, the TNF- production decreased to normal.66 There are also studies, however, showing no effect of antidepressants to the in-vitro stimulation of cytokines (overview, ref 67) but methodological issues have to be taken into account. There is significant evidence suggesting that antidepressants of different classes induce downregulation of the type 1 cytokine production in vitro,67 including noradrenaline reuptake inhibitors68 and the dual serotonin and noradrenalin reuptake inhibitors.69 Several researchers have observed a reduction of IL-6 during treatment with the serotonin reuptake inhibitor fluoxetine.70 A decrease of IL-6 serum levels during therapy with different antidepressants has been observed by other researchers.71 The shift of imbalanced IFN/IL-4 towards normal after 6 weeks' antidepressant treatment has also been reported.41 On the other hand, other groups did not find any effect of some antidepressants on serum levels of different cytokines.61, 72 Since IL-6 stimulates PGE2 and antidepressants inhibit IL6 production, an inhibiting action of antidepressants on PGE2 would be expected, too.73 Over 30 years ago it was suggested that antidepressants inhibit PGE2.74 A recent invitro study showed that both tricyclic antidepressants and selective serotonin inhibitors attenuated cytokine-induced PGE2 and nitric oxide production by inflammatory cells.75

Therapeutic mechanisms and the type-1/ type-2 imbalance in schizophrenia and depression
Schizophrenia: antipsychotic drugs correct the type-1/type-2 imbalance In-vitro studies show that the blunted IFN- production becomes normalized after therapy with neuroleptics.18 An increase of memory cells (CD4+CD45RO+) cells one of the main sources of IFN- productionduring antipsychotic therapy with neuroleptics was observed by different groups.55 Additionally, an increase of sIL-2R the increase reflects an increase of activated, IL-2 bearing T-cellsduring antipsychotic treatment was described.56 The reduced sICAM-1 levels show a significant increase during short-term antipsychotic therapy,22 and the ICAM-1 ligand leukocyte function antigen-1 (LFA-1) shows a significantly increased expression during antipsychotic therapy.57 The increase of TNF- and TNF- receptors during therapy with clozapin was observed repeatedly.58 Moreover, the blunted reaction to vaccination with Salmonella typhii was not observed in patients medicated with antipsychotics.59 An elevation of IL-18 serum levels was described in medicated schizophrenics.60 Since IL-18 plays a pivotal role in the type-1 immune response, this finding is consistent with other descriptions of type-1 activation during antipsychotic treatment. Regarding the type-2 response, several studies point out that antipsychotic therapy is accompanied by a functional decrease of the IL-6 system.19,61 These findings provide further evidence that antipsychotics have a balancing effect on cytokines.

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Nonpharmacological therapies: electroconvulsive therapy and sleep deprivation Electroconvulsive therapy (ECT) was found to downregulate increased levels of the proinflammatory cytokine TNF- in patients with MD.76 An immune analysis during sleep showed an increase in the type-1 monocyte derived cytokines TNF- and IL12 and a decrease of the type-2 IL-10 producing monocytes.77 In contrary, continuous wakefulness blocked the increase of type-1 and decrease of type-2 cytokines (T. Lange and S. Dimitrov, personal communication). Thus, sleep deprivation may exert therapeutic effects through a low suppression of type-1 cytokines. Antidepressant pharmacotherapy, but also other antidepressant therapeutic agents or techniques, have a downregulating effect on proinflammatory cytokines. ferent types of CNS cells, TDO was thought for many years to be restricted to liver tissue. It is known today, however, that TDO is also expressed in CNS cells, probably restricted to astrocytes.82 The type-2 or Th-2 shift in schizophrenia may result in a downregulation of IDO through the inhibiting effect of Th2 cytokines. TDO, on the other hand, was shown to be overexpressed in postmortem brains of schizophrenic patients.82 The type-1/type-2 imbalance with type-2 shift is therefore associated with overexpression of TDO. The type 1/type 2 imbalance is associated with the activation of astrocytes and an imbalance in the activation of astrocytes/microglial cells.83 The functional excess of astrocytes may lead to a further accumulation of KYNA. Indeed, a study referring to the expression of IDO and TDO in schizophrenia showed exactly the expected results. An increased expression of TDO compared with IDO was observed in schizophrenic patients and the increased TDO expression was found, as expected, in astrocytes, not in microglial cells.82 However, it is necessary to note that the above proposed
Neuroprotection

Divergent effects of type-1 type-2 immune activation are associated with different effects on the kynurenine metabolism in schizophrenia and depression
Schizophrenia The only known naturally occurring NMDA receptor antagonist in the human CNS is kynurenic acid (KYNA). KYNA is one of the several neuroactive intermediate products of the kynurenine pathway (Figure 1). Kynurenine (KYN) is the primary major degradation product of tryptophan (TRP). While the excitatory KYN metabolites 3-hydroxykynurenine (3HK) and QUIN are synthesized from KYN in the process toward NAD formation, KYNA is formed in a dead-end side arm of the pathway.78 KYNA acts both as a blocker of the glycine coagonistic site of the NMDA receptor and as a noncompetitive inhibitor of the 7 nicotinic acetylcholine receptor.79 The production of KYN metabolites is partly regulated by IDO and tryptophan 2,3-dioxygenase (TDO). Both enzymes catalyze the first step in the pathway, the degradation from tryptophan to kynurenine. Type-1 cytokines, such as IFN- and IL-2, stimulate the activity of IDO.80 There is a mutual inhibitory effect of TDO and IDO: a decrease in TDO activity occurs concomitantly with IDO induction, resulting in a coordinate shift in the site (and cell types) of tryptophan degradation.81 While it has been known for a long time that IDO is expressed in dif-

IFN- IL-4 IDO Kynurenine KMO 3-OH-Kynurenine

Tryptophan

Kynurenine acid
= NMDA-R antagonist

KAT

Serotonin IFN- IL-4 IDO

Neurotoxic radicals

Quinolinic acid
= NMDA-R agonist

Neurodegeneration + apoptosis

Figure 1. Neuroimmune interactions of kynurenine intermediates. Metabolism of tryptophan via the kynurenine pathway leads to several neuroactive intermediates: kynurenic acid (synthesised by kynurenine aminotransferase, KAT) has neuroprotective properties through antagonism at the N-methyl-Daspartate (NMDA) receptor. Quinolinic acid (QUIN), in contrast, is an NMDA receptor agonist. Both 3-hydroxykynurenine (3OH-kynurenine) and QUIN can induce neurodegeneration and apoptosis through induction of excitotoxicity and generation of neurotoxic radicals, respectively. Activity of the key enzyme of the kynurenine pathway, indoleamine 2,3-dioxygenase (IDO), and of the 3-OH-kynurenine forming enzyme kynurenine monoxygenase (KMO) is induced by proinflammatory cytokines like interferon- (IFN-) and inhibited by anti-inflammatory cytokines like interleukin-4 (IL-4). Serotonin is normally degraded to 5-hydroxyindoleacetic acid (5-HIAA), but the indole ring of serotonin can also be cleaved by IDO. (blue arrows = activation; red arrows = inhibition).

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mechanism would fit only for the subpopulation of schizophrenic patients with Th2 dominant immune response. In those schizophrenics with Th1 dominant immune response, the kynurenine pathway changes would be more similar to those changes in MD.84, 85 Major depression Two directing enzymes of the kynurenine metabolism, IDO and kynurenine monoxygenase (KMO), are induced by the type-1 cytokine IFN-. The activity of IDO is an important regulatory component in the control of lymphocyte proliferation, the activation of the type-1 immune response, and the regulation of the tryptophan metabolism.85 It induces a halt in the lymphocyte cell cycle due to the catabolism of tryptophan.87 In contrast to the type-1 cytokines, the type-2 cytokines IL-4 and IL-10 inhibit the IFN--induced IDO-mediated tryptophan catabolism.87 IDO is located in several cell types, including monocytes and microglial cells.88 An IFN-induced, IDO-mediated decrease of CNS tryptophan availability may lead to a serotonergic deficiency in the CNS, since tryptophan availability is the limiting step in serotonin synthesis. Other proinflammatory molecules such as PGE2 or TNF-, however, induce synergistically with IFN- the increase of IDO activity.89 Therefore, not only IFN- and type-1 cytokines, but also other proinflammatory molecules induce IDO activity. Since increased levels of PGE2 and TNF- were described in MD, other proinflammatory molecules also contribute to IDO activation and tryptophan consumption, (eg, ref 39). An imbalance between the NMDA antagonist action by KYNA and the NMDA agonist action by QUIN has been proposed to be involved in the pathophysiology of MD90; a recent study demonstrated this imbalance in patients with MD.3 Accordingly, since the activity of the enzyme kynurenine 3 mono-oxygenase (KMO), directing the production of QUIN, is inhibited by type-2 cytokines but activated by proinflammatory type-1 cytokines,91 an increased production of QUIN in depressive states would be expected. The role of QUIN in depression is discussed in more detail below. One of the more consistent findings is that patients with low 5-hydroxyindoleacetic acid (5-HIAA), the metabolite of serotonin, in CSF are prone to commit suicide.92, 93 This gives further indirect evidence for a possible link between the type-1 cytokine IFN- and the IDO-related reduction of serotonin availability in the CNS of suicidal patients.

A study in patients suffering from hepatitis C showed that immunotherapy with IFN- was followed by an increase of depressive symptoms and serum kynurenine concentrations on the one hand, and a decrease in serum concentrations of tryptophan and serotonin on the other hand.94 The kynurenine/tryptophan ratio, which reflects the activity of IDO, increased. Changes in depressive symptoms were significantly positively correlated with kynurenine and negatively correlated with serotonin concentrations.94 This study and others95 clearly show that the IDO activity is increased by IFN, leading to an increased kynurenine production and a depletion of tryptophan and serotonin. The further metabolism of kynurenine, however, seems to play an additional crucial role for the psychopathological states. In addition to the effects of the proinflammatory immune response on the serotonin metabolism, other neurotransmitter systems, in particular the catecholaminergic system, are involved in depression, too. Although the relationship of immune activation and changes in catecholaminergic neurotransmission has not been well studied, an increase in monoamino-oxidase (MAO) activity, which leads to decreased noradrenergic neurotransmission, might be an indirect effect of the increased production of kynurenine and QUIN.45 The proinflammatory immune state in MD leads on the one hand to a lack of serotonin and on the other hand to an overproduction of the neurotoxic and depressiogenic metabolite QUIN by induction of the directing enzymes of the kynurenine metabolism. Two depressiogenic components result from the IDO activation.

Astrocytes, microglia, and type-1/type-2 response

The cellular sources for the immune response in the CNS are astrocytes and microglia cells. Microglial cells, deriving from peripheral macrophages, secrete preferantially type-1 cytokines such as IL-12, while astrocytes inhibit the production of IL-12 and ICAM-1 and secrete the type-2 cytokine IL-10.96 Therefore, the type-1/type-2 imbalance in the CNS seems to be represented by the imbalance in the activation of microglial cells and astrocytes, although it has to be taken into consideration that the production of cytokines by astrocytes and microglial cells depends on activation conditions. The hypothesis of an overactivation of astrocytes in schizophrenia is supported by the finding of increased CSF levels of S100B

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a marker of astrocyte activationindependent of the medication state of the schizophrenic patients.97 Microglia activation was found in a small percentage of schizophrenics and is speculated to be a medication effect.98 A type-1 immune activation as an effect of antipsychotic treatment has repeatedly been observed. Since the type-1 activation predominates in the response of the peripheral immune system in depression, a dominance of microglial activation compared with astrocyte activation should be observed in depression. Glial reductions were consistently found in brain circuits known to be involved in mood disorders, such as in the limbic and prefrontal cortex.99, 100 Although several authors did not differentiate between microglial and astrocytic loss, this difference is crucial due to the different effects of the type-1/type-2 immune response. Recent studies, however, show that astrocytes are diminished in patients suffering from depression,101 although the data are not entirely consistent.102 A loss of astrocytes was in particular observed in younger depressed patients: the lack of glial fibrillary acid protein (GFAP)-immunoreactive astrocytes reflects a lowered activity of responsiveness in those cells.101 A loss of astrocytes was found in many cortical layers and in different sections of the dorsolateral prefrontal cortex in depression.103 A reduction of astrocytes has also been observed in the dentate gyrus of an animal model of IFN- induced depression (Myint et al, personal communication). Moreover, a loss of astrocytes is associated with an impaired reuptake of glutamate from the extracellular space into astrocytes by high affinity glutamate transporters.104 Impaired glutamate reuptake from the synaptic cleft by astroglia prolongs synaptic activation by glutamate.105 Accordingly, increased glutamatergic activity has been observed in patients with depression.106 Neuroprotective and neurotoxic metabolites of the tryptophan-kynurenine metabolism in psychiatric disorders In contrast to microglial cells which produce QUIN, astrocytes play a key role in the production of KYNA in the CNS. Astrocytes are the main source of KYNA.107 The cellular localization of the kynurenine metabolism is primarily in macrophages and microglial cells, but also in astrocytes.108 KMO, a critical enzyme in the kynurenine metabolism, is absent in human astrocytes, however.109 Accordingly, it has been pointed out that astrocytes cannot produce the product 3-hydroxykynurenine (3-HK), but they are able to produce large amounts of early kynurenine metabolites, such as KYN and KYNA.109 This supports the observation that inhibition of KMO leads to an increase in the KYNA production in the CNS.110 The complete metabolism of kynurenine to QUIN is observed mainly in microglial cells, only a small amount of QUIN is produced in astrocytes via a side-arm of the kynurenine metabolism. Therefore, due to the lack of kynurenine-hydroxylase (KYN-OHse), in case of high tryptophan breakdown to KYN, KYNA may accumulate in astrocytes. A second key player in the metabolization of 3-HK are monocytic cells infiltrating the CNS. They help astrocytes in the further metabolism to QUIN.109 However, the low levels of sICAM-1 (ICAM-1 is the molecule that mainly mediates the penetration of monocytes and lymphocytes into the CNS) in the serum and in the CSF of nonmedicated schizophrenic patients,22 and the increase of adhesion molecules during antipsychotic therapy indicate that the penetration of monocytes may be reduced in nonmedicated schizophrenic patients.57 Quinolinic acid as a depressiogenic and neurotoxic substance Apart from certain liver cells, only macrophage-derived cells are able to convert tryptophan into quinolinic acidolonic acid.111 Interestingly, in a model of infection, the highest concentrations of QUIN are found in the gray and white matter of the cortex, not in subcortical areas. This finding points out that high levels of QUIN therefore may be associated with cortical dysfunction.112 The strong association between cortical QUIN concentrations and local IDO activity supports the view that the induction of IDO is an important event in initiating the increase of QUIN production.113 In the CNS, invaded macrophages and microglial cells are able to produce QUIN.111 During a local inflammatory CNS process, the QUIN production in the CNS might increase without changes of the peripheral blood levels of QUIN. The local QUIN production correlates with the level of 2 microglobulin, an inflammatory marker. Local CNS concentrations of QUIN are able to exceed the blood levels by far.112 Peripheral immune stimulation, however, under certain conditions also leads to increased CNS concentration of QUIN.111 A recent study showed that depressive symptoms are related to an high ratio of KYN/KYNA in depression.114

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The increase of this ratio reflects that in depressed states KYN may be preferentially metabolized to QUIN, while the KYNA pathway is neglected. The increase of QUIN was observed to be associated with several prominent features of depression: decrease in reaction time115 and cognitive deficits, in particular difficulties in learning.112 In an animal model, an increase of QUIN and 3-hydroxykynurenine was associated with anxiety.116 QUIN was shown to cause an over-release of glutamate in the striatum and in the cortex, presumably by presynaptic mechanisms.117 The QUIN pathway of the kynurenine metabolismdirected by proinflammatory cytokinesmight be the key mechanism involved in the increased glutamatergic neurotransmission in MD, 106 while it is unclear whether QUIN itself has depressiogenic properties. Thus, an excess of QUIN might be associated with excess glutamatergic activation. COX-2 inhibition as a therapeutic approach in schizophrenia and depression COX inhibition provokes differential effects on kynurenine metabolism: while COX-1 inhibition increases the levels of KYNA, COX-2 inhibition decreases them.118 Therefore, psychotic symptoms and cognitive dysfunctions, observed during therapy with COX-1 inhibitors, were assigned to the COX-1 mediated increase of KYNA. The reduction of KYNA levels, by a prostaglandin-mediated mechanism, might be an additional mechanism to the above-described immunological mechanism for therapeutic effects of selective COX-2 inhibitors in schizophrenia.118 Indeed, in a prospective, randomized, double-blind study of therapy with the COX-2 inhibitor celecoxib added on to risperidone in acute exacerbation of schizophrenia, a therapeutic effect of celecoxib was observed. 119 Immunologically, an increase of the type-1 immune response was found in the celecoxib treatment group.120 The finding of a clinical advantage of COX-2 inhibition, however, could not be replicated in a second study. Further analysis of the data revealed that the outcome depends on the duration of the disease.121 This observation is in accordance with results from animal studies showing that the effects of COX-2 inhibition on cytokines, hormones, and particularly on behavioral symptoms are dependent on the duration of the preceding changes and the time point of application of the

COX-2 inhibitor.122 In subsequent clinical studies following a similar randomized double-blind placebo-controlled add-on design of 400 mg celecoxib to risperidone (in one study risperidone or olanzapine) in partly different patient populations, similar positive results of cyclo-oxygenase inhibition were able to be obtained: in a Chinese population of first-manifestation schizophrenics,123 and in an Iranian sample of chronic schizophrenics.124 In continuously ill schizophrenics, however, no advantage of celecoxib could be found.125 In schizophrenia, COX-2 inhibition showed beneficial effects preferentially in early stages of the disease, the data regarding chronic schizophrenia are controversial, possibly in part due to methodological concerns. The data are still preliminary and further research has to be performed, eg, with other COX-2 inhibitors. COX-2 inhibition as a possible anti-inflammatory therapeutic approach in depression Due to the increase of proinflammatory cytokines and PGE2 in depressed patients, anti-inflammatory treatment would be expected to show antidepressant effects also in depressed patients. In particular, COX-2 inhibitors seem to show advantageous results: animal studies show that COX-2 inhibition can lower the increase of the proinflammatory cytokines IL-1, TNF-, and of PGE2, but it can also prevent clinical symptoms such as anxiety and cognitive decline, which are associated with this increase of proinflammatory cytokines.122 Moreover, treatment with the COX-2 inhibitor celecoxibbut not with a COX-1 inhibitorprevented the dysregulation of the HPA-axis, in particular the increase of cortisol, one of the biological key features associated with depression.122, 126 This effect can be expected because PGE2 stimulates the HPA axis in the CNS,127 and PGE2 is inhibited by COX2 inhibition. Moreover, the functional effects of IL-1 in the CNSsickness behavior being one of these effects were also shown to be antagonized by treatment with a selective COX-2 inhibitor.128 Additionally, COX-2 inhibitors influence the CNS serotonergic system. In a rat model, treatment with rofecoxib was followed by an increase of serotonin in the frontal and the temporoparietal cortex.129 A possible mechanism of the antidepressant action of COX-2 inhibitors is the inhibition of the release of IL-1 and IL-6. Moreover, COX-2 inhibitors also protect the CNS from effects of QUIN, ie, from neurotoxicity.130 In the depression model

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of the bulbectomized rat, a decrease of cytokine levels in the hypothalamus and a change in behavior have been observed after chronic celecoxib treatment.131 In another animal model of depression, however, the mixed COX1/COX-2 inhibitor acetylsalicylic acid showed an additional antidepressant effect by accelerating the antidepressant effect of fluoxetine.132 Moreover, we were able to demonstrate a significant therapeutic effect of the COX-2 inhibitor on depressive symptoms in a randomized, double-blind pilot add-on study using the selective COX-2 inhibitor celecoxib in MD.133 Also in a clinical study, the mixed COX-1/COX2 inhibitor acetylsalicylic acid accelerated the antidepressant effect of fluoxetine and increased the response rate in depressed nonresponders to monotherapy with fluoxetine in a open-label pilot study.134 Currently, a large study with the COX-2 inhibitor cimicoxib is ongoing. For ethical reasons, clinical trials so far have been performed in an add-on design; no monotherapy with a COX-2 inhibitor was studied. metabolism result in alterations of the serotonergic, glutamatergic, and dopaminergic neurotransmissions; these alterations are typically found in schizophrenia and MD. The tryptophan/kynurenine metabolism, however, generates neurotoxic and neuroprotective metabolites, an imbalance in this metabolism contributes to the production of either the neurotoxic metabolite QUIN or the neuroprotective metabolite KYNA, both exhibiting different effects on the glutamatergic neurotransmission. Additionally, a direct influence of cytokines on neurotransmitters has been noted. Moreover, cytokines can also act in a neurotoxic and neuroprotective manner. Anti-inflammatory drugs, however, are candidates for antidepressants and antipsychotics, which might be more related to the pathophysiology of these disorders compared with the neurotransmitter disturbances. The neurotransmitter disturbances might be a final common pathway of different pathological pathways in schizophrenia and depression, the immunological pathway might be true for a subgroup of patients suffering from these disoders. COX-2 inhibitorsmost studies have been performed with celecoxibhave been shown in invitro experiments, animal studies, and clinical trials by several groups of researchers to exhibit antidepressant and antipsychotic properties. Other anti-inflammatory therapeutic approaches will be of interest in the future, and possibly support the hypothesis that inflammation is an important pathogenetic factor in depression and schizophrenia.
9. Zuckerman L, Weiner I. Maternal immune activation leads to behavioral and pharmacological changes in the adult offspring. J Psychiatr Res. 2005;39:311-323. 10. Gattaz WF, Abrahao AL, Foccacia R. Childhood meningitis, brain maturation and the risk of psychosis. Eur Arch Psychiatry Clin Neurosci. 2004;254:2326. 11. Koponen H, Rantakallio P, Veijola J, Jones P, Jokelainen J, Isohanni M. Childhood central nervous system infections and risk for schizophrenia. Eur Arch Psychiatry Clin Neurosci. 2004;254:9-13. 12. Brown AS. The risk for schizophrenia from childhood and adult infections. Am J Psychiatry. 2008;165:7-10. 13. Dalman C, Allebeck P, Gunnell D, et al. Infections in the CNS during childhood and the risk of subsequent psychotic illness: a cohort study of more than one million Swedish subjects. Am J Psychiatry. 2008;165:59-65. 14. Krschenhausen DA, Hampel HJ, Ackenheil M, Penning R, Mller N. Fibrin degradation products in post mortem brain tissue of schizophrenics: a possible marker for underlying inflammatory processes. Schizophr Res. 1996;19:103-109. 15. Bechter K. Mild encephalitis underlying psychiatric disorders - A reconsideration and hypothesis exemplified on Borna disease. Neurol Psychiatry Brain Res. 2001; 9:55-70. 16. Reichenberg A, Kraus T, Haack M, Schuld A, Pollmacher T, Yirmiya R. Endotoxin-induced changes in food consumption in healthy volunteers are associated with TNF-alpha and IL-6 secretion. Psychoneuroendocrinology. 2002;27:945-956.

Conclusion
A large number of findings point out that inflammation plays a pivotal role in the pathogenesis of major psychiatric disorders, in particular in MD and in schizophrenia. The differential influence of cytokines and proinflammatory mediators, which are altered in schizophrenia and MD, on the enzyme IDO and the tryptophan/kynurenine REFERENCES
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El impacto de la falta de regulacin neuroinmune sobre la neuroproteccin y la neurotoxicidad en los trastornos psiquitricos: su relacin con tratamientos farmacolgicos
Se ha postulado una patognesis inflamatoria para la esquizofrenia y la depresin mayor (DM). En la esquizofrenia y la depresin, patrones opuestos de respuesta inmune tipo 1 versus tipo 2 parecen estar asociados con diferencias en la activacin de la enzima indolamina 2,3 dioxigenasa y en el metabolismo triptfano-kinurenina, lo que lleva a un aumento de la produccin de cido kinurnico en la esquizofrenia y disminucin de la produccin de este cido en la depresin. Estas diferencias estn asociadas con un desequilibrio en la neurotransmisin glutamatrgica, el cual puede contribuir a una excesiva accin agonista del N-metil-D-aspartato (NMDA) en la depresin y otra antagonista del NMDA en la esquizofrenia. Respecto a la funcin neuroprotectora del cido kinurnico y a los efectos neurotxicos del cido quinolnico (QUIN), los diferentes patrones de activacin inmune tambin pueden llevar a un desequilibrio entre los efectos neuroprotectores y neurotxicos del metabolismo triptfano/kinurenina. La activacin diferencial de las clulas de la microgla y los astrocitos puede ser un mecanismo adicional que contribuya a este desequilibrio. El desequilibrio inmunolgico se traduce en un estado inflamatorio combinado con un aumento de la produccin de prostaglandina E2 y aumento de la expresin de ciclo-oxigenasa-2 (COX2). Sin embargo, los efectos inmunolgicos de muchos de los antipsicticos y antidepresivos existentes corrigen parcialmente el desequilibrio inmune y el exceso de produccin del neurotxico QUIN. Los inhibidores de la COX-2 se han evaluado en modelos animales de depresin y en ensayos clnicos preliminares, y orientan a efectos favorables en la esquizofrenia y en la DM.
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Impact dune dysrgulation neuro-immune sur la neuroprotection et la neurotoxicit dans les troubles psychiatriques- relation avec le traitement mdicamenteux
Lhypothse dune pathogense inflammatoire a t avance pour la schizophrnie et la dpression majeure (DM). Dans la schizophrnie et la dpression, lopposition des rponses immunes de type 1 vs type 2 semble tre associe des diffrences dans lactivation de lenzyme indoleamine 2,3dioxygnase et dans le mtabolisme tryptophanekynurnine, la production dacide kynurtique tant augmente dans la schizophrnie et diminue dans la dpression. Ces diffrences sont associes un dsquilibre de la neurotransmission glutamatergique qui peut entraner une action agoniste excessive du NMDA (N-mthyl-Daspartate) dans la dpression et une action antagoniste dans la schizophrnie. En ce qui concerne la fonction neuroprotectrice de lacide kynurtique et les effets neurotoxiques de lacide quinolinique (QUIN), diffrents schmas dactivation immunitaire peuvent aussi conduire un dsquilibre entre les effets neuroprotecteurs et neurotoxiques du mtabolisme tryptophane/kynurnine, auquel peut contribuer lactivation diffrentielle des cellules de la microglie et des astrocytes. Le dsquilibre immunologique provoque un tat inflammatoire associ une production augmente de prostaglandine E2 et une expression augmente de la COX-2 (cyclooxygnase-2). Les effets immunologiques de nombreux antipsychotiques et antidpresseurs existants corrigent cependant en partie ce dsquilibre immunitaire et lexcs de production du neurotoxique QUIN. Les inhibiteurs de la COX-2 ont t tests dans des modles animaux de dpression et dans des tudes cliniques prliminaires, montrant des effets favorables dans la schizophrnie et la dpression.
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An acute phase protein response in the olfactory bulbectomised rat: effect of sertraline treatment. Med Sci Res. 1994;22:313314. 63. Zhu J, Bengtsson BO, Mix E, Thorell LH, Olsson T, Link H. Effect of monoamine reuptake inhibiting antidepressants on major histocompatibility complex expression on macrophages in normal rats and rats with experimental allergic neuritis (EAN). Immunopharmacology. 1994;27:225-244. 64. Maes M, Song C, Lin AH, Bonaccorso S, et al. Negative immunoregulatory effects of antidepressants: inhibition of interferon-gamma and stimulation of interleukin-10 secretion. Neuropsychopharmacology. 1999;20:370379. 65. Seidel A, Arolt V, Hunstiger M, Rink L, Behnisch A, Kirchner H. Cytokine production and serum proteins in depression. Scand J Immunol. 1995;41:534538. 66. Lanquillon S, Krieg JC, Bening-Abu-Shach U, Vedder H. Cytokine production and treatment response in major depressive disorder. Neuropsychopharmacology. 2000;22:370-379.

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67. Kenis G, Maes M. Effects of antidepressants on the production of cytokines. Int J Neuropsychopharmacol. 2002;5:401-412. 68. O'Sullivan JB, Ryan KM, Curtin NM, Harkin A, Connor TJ. Noradrenaline reuptake inhibitors limit neuroinflammation in rat cortex following a systemic inflammatory challenge: implications for depression and neurodegeneration. Int J Neuropsychopharmacol. 2009;12:687-699. 69. Vollmar P, Nessler S, Kalluri SR, Hartung HP, Hemmer B. The antidepressant venlafaxine ameliorates murine experimental autoimmune encephalomyelitis by suppression of pro-inflammatory cytokines. Int J Neuropsychopharmacol. 2009;12:525-536. 70. Sluzewska A, Rybakowski JK, Laciak M, et al. Interleukin-6 serum levels in depressed patients before and after treatment with fluoxetine. Ann N Y Acad Sci. 1995;762:474-6.:474-476. 71. Frommberger UH, Bauer J, Haselbauer P, Fraulin A, Riemann D, Berger M. Interleukin-6-(IL-6) plasma levels in depression and schizophrenia: comparison between the acute state and after remission. Eur Arch Psychiatry Clin Neurosci. 1997;247:228-233. 72. Maes M, Meltzer HY, Bosmans E, Bergmans R, Vandoolaeghe E, Ranjan R, Desnyder R. Increased plasma concentrations of interleukin-6, soluble interleukin-6, soluble interleukin-2 and transferrin receptor in major depression. J Affect Disord. 1995;34:301-309. 73. Pollak Y, Yirmiya R. Cytokine-induced changes in mood and behaviour: implications for 'depression due to a general medical condition', immunotherapy and antidepressive treatment. Int J Neuropsychopharmacol. 2002;5:389-399. 74. Mtabaji JP, Manku MS, Horrobin DF. Actions of the tricyclic antidepressant clomipramine on responses to pressor agents. Interactions with prostaglandin E2. Prostaglandins. 1977;14:125-132. 75. Yaron I, Shirazi I, Judovich R, Levartovsky D, Caspi D, Yaron M. Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synovial cells and synovial tissue cultures. Arthritis Rheum. 1999;42:2561-2568. 76. Hestad KA, Tonseth S, Stoen CD, Ueland T, Aukrust P. Raised plasma levels of tumor necrosis factor alpha in patients with depression: normalization during electroconvulsive therapy. J ECT. 2003;19:183-188. 77. Dimitrov S, Lange T, Tieken S, Fehm HL, Born J. Sleep associated regulation of T helper 1/T helper 2 cytokine balance in humans. Brain Behav Immun. 2004;18:341-348. 78. Schwarcz R, Pellicciari R. Manipulation of brain kynurenines: glial targets, neuronal effects, and clinical opportunities. J Pharmacol Exp Ther. 2002;303:1-10. 79. Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schwarcz R, Albuquerque EX. The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J Neurosci. 2001;21:7463-7473. 80. Grohmann U, Fallarino F, Puccetti P. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 2003;24:242-248. 81. Takikawa O, Yoshida R, Kido R, Hayaishi O. Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase. J Biol Chem. 1986;261:3648-3653. 82. Miller CL, Llenos IC, Dulay JR, Barillo MM, Yolken RH, Weis S. Expression of the kynurenine pathway enzyme tryptophan 2,3-dioxygenase is increased in the frontal cortex of individuals with schizophrenia. Neurobiol Dis. 2004;15:618-629. 83. Aloisi F, Ria F, Adorini L. Regulation of T-cell responses by CNS antigenpresenting cells: different roles for microglia and astrocytes. Immunol Today. 2000;21:141-147. 84. Kim YK, Myint AM, Verkerk R, Scharpe S, Steinbusch H, Leonard B. Cytokine changes and tryptophan metabolites in medication-naive and medication-free schizophrenic patients. Neuropsychobiology. 2009;59:123129. 85. Mellor AL, Munn DH. Tryptophan catabolism and T-cell tolerance: immunosuppression by starvation? Immunol Today. 1999;20:469-473. 86. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med. 1999;189:1363-1372. 87. Weiss G, Murr C, Zoller H, et al. Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells. Clin Exp Immunol. 1999;116:435-440.

88. Alberati GD, Ricciardi CP, Kohler C, Cesura AM. Regulation of the kynurenine metabolic pathway by interferon-gamma in murine cloned macrophages and microglial cells. J Neurochem. 1996;66:996-1004. 89. Braun D, Longman RS, Albert ML. A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation. Blood. 2005;106:2375-2381. 90. Myint AM, Kim YK. Cytokine-serotonin interaction through IDO: a neurodegeneration hypothesis of depression. Med Hypotheses. 2003;61:519-525. 91. Chiarugi A, Calvani M, Meli E, Traggiai E, Moroni F. Synthesis and release of neurotoxic kynurenine metabolites by human monocyte-derived macrophages. J Neuroimmunol. 2001;120:190-198. 92. Lidberg L, Belfrage H, Bertilsson L, Evenden MM, Asberg M. Suicide attempts and impulse control disorder are related to low cerebrospinal fluid 5-HIAA in mentally disordered violent offenders. Acta Psychiatr Scand. 2000;101:395-402. 93. Mann JJ, Malone KM. Cerebrospinal fluid amines and higher-lethality suicide attempts in depressed inpatients. Biol Psychiatry. 1997;41:162171. 94. Bonaccorso S, Marino V, Puzella A, et al. Increased depressive ratings in patients with hepatitis C receiving interferon-alpha-based immunotherapy are related to interferon-alpha-induced changes in the serotonergic system. J Clin Psychopharmacol. 2002;22:86-90. 95. Capuron L, Neurauter G, Musselman DL, et al. Interferon-alphainduced changes in tryptophan metabolism. relationship to depression and paroxetine treatment. Biol Psychiatry. 2003;54:906-914. 96. Aloisi F, Penna G, Cerase J, Menendez IB, Adorini L. IL-12 production by central nervous system microglia is inhibited by astrocytes. J Immunol. 1997;159:1604-1612. 97. Rothermundt M, Falkai P, Ponath G, et al. Glial cell dysfunction in schizophrenia indicated by increased S100B in the CSF. Mol Psychiatry. 2004;9:897899. 98. Bayer TA, Buslei R, Havas L, Falkai P. Evidence for activation of microglia in patients with psychiatric illnesses. Neurosci Lett. 1999;271:126-128. 99. Cotter D, Pariante C, Rajkowska G. Glial pathology in major psychiatric disorders. In Agam G, Belmaker RH, Everall I, eds. The Post-Mortem Brain in Psychiatric Research. Boston, MA: Kluwer Acad Pub; 2002;291-324. 100. Rajkowska G. Depression: what we can learn from postmortem studies. Neuroscientist. 2003;9:273-284. 101. Miguel-Hidalgo JJ, Baucom C, Dilley G, et al. Glial fibrillary acidic protein immunoreactivity in the prefrontal cortex distinguishes younger from older adults in major depressive disorder. Biol Psychiatry. 2000;48:861-873. 102. Davis S, Thomas A, Perry R, Oakley A, Kalaria RN, O'Brien JT. Glial fibrillary acidic protein in late life major depressive disorder: an immunocytochemical study. J Neurol Neurosurg Psychiatry. 2002;73:556-560. 103. Rajkowska G. Astroglia in the cortex of schizophrenics: histopathology finding. World J Biol Psychiatry. 2005;6:74. 104. Choudary PV, Molnar M, Evans SJ, et al. Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression. Proc Natl Acad Sci U S A. 2005;102:15653-15658. 105. Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001;65:1-105. 106. Sanacora G, Gueorguieva R, Epperson CN, et al. Subtype-specific alterations of gamma-aminobutyric acid and glutamate in patients with major depression. Arch Gen Psychiatry. 2004;61:705-713. 107. Heyes MP, Chen CY, Major EO, Saito K. Different kynurenine pathway enzymes limit quinolinic acid formation by various human cell types. Biochem J. 1997;326:351-356. 108. Kiss C, Ceresoli-Borroni G, Guidetti P, Zielke CL, Zielke HR, Schwarcz R. Kynurenate production by cultured human astrocytes. J Neural Transm. 2003;110:1-14. 109. Guillemin GJ, Kerr SJ, Smythe GA, et al. Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection. J Neurochem. 2001;78:842-853. 110. Chiarugi A, Carpenedo R, Moroni F. Kynurenine disposition in blood and brain of mice: effects of selective inhibitors of kynurenine hydroxylase and of kynureninase. J Neurochem. 1996;67:692-698. 111. Saito K, Crowley JS, Markey SP, Heyes MP. A mechanism for increased quinolinic acid formation following acute systemic immune stimulation. J Biol Chem. 1993;268:15496-15503.

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112. Heyes MP, Saito K, Lackner A, Wiley CA, Achim CL, Markey SP. Sources of the neurotoxin quinolinic acid in the brain of HIV-1-infected patients and retrovirus-infected macaques. FASEB J. 1998;12:881-896. 113. Heyes MP, Saito K, Crowley JS. Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease. Brain. 1992;115:1249-1273. 114. Wichers MC, Koek GH, Robaeys G, Verkerk R, Scharpe S, Maes M. IDO and interferon-alpha-induced depressive symptoms: a shift in hypothesis from tryptophan depletion to neurotoxicity. Mol Psychiatry. 2005;10:538-544. 115. Heyes MP, Brew BJ, Martin A, et al. Quinolinic acid in cerebrospinal fluid and serum in HIV-1 infection: relationship to clinical and neurological status. Ann Neurol. 1991;29:202-209. 116. Lapin IP. Neurokynurenines (NEKY) as common neurochemical links of stress and anxiety. Adv Exp Med Biol. 2003;527:121-125. 117. Chen Q, Surmeier DJ, Reiner A. NMDA and non-NMDA receptor-mediated excitotoxicity are potentiated in cultured striatal neurons by prior chronic depolarization. Exp Neurol. 1999;159:283-296. 118. Schwieler L, Erhardt S, Erhardt C, Engberg G. Prostaglandin-mediated control of rat brain kynurenic acid synthesis--opposite actions by COX-1 and COX-2 isoforms. J Neural Transm. 2005;112:863-872. 119. Mller N, Riedel M, Scheppach C, et al. Beneficial antipsychotic effects of celecoxib add-on therapy compared to risperidone alone in schizophrenia. Am J Psychiatry. 2002;159:1029-1034. 120. Mller N, Ulmschneider M, Scheppach C, et al COX-2 inhibition as a treatment approach in schizophrenia: immunological considerations and clinical effects of celecoxib add-on therapy. Eur Arch Psychiatry Clin Neurosci. 2004;254:14-22. 121. Mller N, Riedel M, Dehning S, et al. Is the therapeutic effect of celecoxib in schizophrenia depending from duration of disease? Neuropsychopharmacology. 2004;29:176. 122. Casolini P, Catalani A, Zuena AR, Angelucci L. Inhibition of COX-2 reduces the age-dependent increase of hippocampal inflammatory markers, corticosterone secretion, and behavioral impairments in the rat. J Neurosci Res. 2002;68:337-343. 123. Zhang Y, Chun Chen D, Long Tan Y, Zhou DF. A double-blind, placebocontrolled trial of celecoxib addes to risperidone in first-episode and drugnaive patients with schizophrenia [abstract]. Eur Arch Psychiatry Clin Neurosci. 2006;256(suppl 2):II/50. 124. Akhondzadeh S, Tabatabaee M, Amini H, Ahmadi Abhari SA, Abbasi SH, Behnam B. Celecoxib as adjunctive therapy in schizophrenia: a doubleblind, randomized and placebo-controlled trial. Schizophr Res. 2007;90:179185. 125. Rapaport MH, Delrahim KK, Bresee CJ, Maddux RE, Ahmadpour O, Dolnak D. Celecoxib augmentation of continuously ill patients with schizophrenia. Biol Psychiatry. 2005;57:1594-1596. 126. Hu F, Wang X, Pace TW, Wu H, Miller AH. Inhibition of COX-2 by celecoxib enhances glucocorticoid receptor function. Mol Psychiatry. 2005;10:426428. 127. Song C, Leonard BE. Fundamentals of Psychoneuroimmunology. Chichester, NY: J Wiley and Sons; 2000. 128. Cao C, Matsumura K, Ozaki M, Watanabe Y. Lipopolysaccharide injected into the cerebral ventricle evokes fever through induction of cyclooxygenase-2 in brain endothelial cells. J Neurosci. 1999;19:716-725. 129. Sandrini M, Vitale G, Pini LA. Effect of rofecoxib on nociception and the serotonin system in the rat brain. Inflamm Res. 2002;51:154-159. 130. Salzberg-Brenhouse HC, Chen EY, Emerich DF, et al. Inhibitors of cyclooxygenase-2, but not cyclooxygenase-1 provide structural and functional protection against quinolinic acid-induced neurodegeneration. J Pharmacol Exp Ther. 2003;306:218-228. 131. Myint AM, Steinbusch HW, Goeghegan L, Luchtman D, Kim YK, Leonard BE. Effect of the COX-2 inhibitor celecoxib on behavioural and immune changes in an olfactory bulbectomised rat model of depression. Neuroimmunomodulation. 2007;14:65-71. 132. Brunello N, Alboni S, Capone G, et al. Acetylsalicylic acid accelerates the antidepressant effect of fluoxetine in the chronic escape deficit model of depression. Int Clin Psychopharmacol. 2006;21:219-225. 133. Mller N, Schwarz MJ, Dehning S, et al. The cyclooxygenase-2 inhibitor celecoxib has therapeutic effects in major depression: results of a doubleblind, randomized, placebo controlled, add-on pilot study to reboxetine. Mol Psychiatry. 2006;11:680-684. 134. Mendlewicz J, Kriwin P, Oswald P, Souery D, Alboni S, Brunello N. Shortened onset of action of antidepressants in major depression using acetylsalicylic acid augmentation: a pilot open-label study. Int Clin Psychopharmacol. 2006;21:227-231.

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The neurotrophic and neuroprotective effects of psychotropic agents
Joshua Hunsberger, PhD; Daniel R. Austin, BA; Ioline D. Henter, MA; Guang Chen, MD, PhD

Accumulating evidence suggests that psychotropic agents such as mood stabilizers, antidepressants, and antipsychotics realize their neurotrophic/neuroprotective effects by activating the mitogen activated protein kinase/extracellular signal-related kinase, PI3-kinase, and wingless/glycogen synthase kinase (GSK) 3 signaling pathways. These agents also upregulate the expression of trophic/protective molecules such as brain-derived neurotrophic factor, nerve growth factor, B-cell lymphoma 2, serine-threonine kinase, and Bcl-2 associated athanogene 1, and inactivate proapoptotic molecules such as GSK-3. They also promote neurogenesis and are protective in models of neurodegenerative diseases and ischemia. Most, if not all, of this evidence was collected from animal studies that used clinically relevant treatment regimens. Furthermore, human imaging studies have found that these agents increase the volume and density of brain tissue, as well as levels of N-acetyl aspartate and glutamate in selected brain regions. Taken together, these data suggest that the neurotrophic/neuroprotective effects of these agents have broad therapeutic potential in the treatment, not only of mood disorders and schizophrenia, but also neurodegenerative diseases and ischemia.
2009, LLS SAS

istorically, psychiatric disorders such as mood disorders and schizophrenia have been conceptualized as neurochemical illnesses. However, accumulating data from both postmortem and brain imaging studies reveal morphological changes in the brains of individuals with these illnesses. These changes include ventricle enlargement, volumetric reduction, attenuation of neuronal viability marker N-acetyl aspartate (NAA), and atrophy or loss of neurons and glial cells in selective cortical and limbic brain regions. Several psychotropic agents defined as chemical substances that act primarily on the central nervous system (CNS) to alter brain function are used to treat psychiatric disorders. These psychotropic agents include mood stabilizers, antidepressants, and antipsychotic medications. Many of these drugs exert significant effects on signaling pathways enhancing neurotrophic and neuroprotective cellular mechanisms. Loosely defined, neurotrophic effects can be considered a therapeutic strategy intended to augment proliferation, differentiation, growth, and regeneration, whereas neuroprotective effects slow or halt the progression of neuronal atrophy or cell death following the onset of disease or clinical decline. In this article, we review evidence from animal and human studies reporting that psychotropic agents affect molecular targets and signaling cascades associated with enhanced
Keywords: mood stabilizer; antidepressant; antipsychotic; neurotrophic; neuroprotection; neurogenesis; ERK signaling; PI3-kinase signaling; Wnt/GSK-3 signaling Author affiliations: Laboratory of Molecular Pathophysiology and Experimental Therapeutics, Mood and Anxiety Disorders Program, NIMH, NIH, Bethesda, Maryland, USA Address for correspondence: Guang Chen MD, PhD, Mood and Anxiety Disorders Program, NIMH, Bldg 35, 1C-912, 35 Convent Drive. Bethesda, MD 20892, USA (e-mail: guangchen@mail.nih.gov)

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Copyright 2009 LLS SAS. All rights reserved

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Selected abbreviations and acronyms
BDNF CREB ERK MAPK NAA P13K Wnt/GSK brain-derived neurotrophic factor cAMP response element binding extracellular signal-related kinase mitogen activated protein kinase N-acetyl aspartate PI3-kinase wingless/glycogen synthase kinase (MAPK/ERK) pathway, ii) the phosphatidylinositol 3 kinase (PI3K) pathway, and ii) the wingless/glycogen synthase kinase 3 (Wnt/GSK3) pathway. Mood stabilizers activate neurotrophic signaling pathways Mood stabilizers have been reported to activate the intracellular MAPK/ERK signaling pathway (Figure 1).1-3 This pathway is used by neurotrophins, neurotransmitters, and neuropeptides to exert their neurotrophic and neuroprotective effects by specifically enhancing progenitor cell proliferation and differentiation, neuronal process growth and regeneration, neuronal survival, and long-term synaptic remodeling and plasticity.4-7 The key components of the pathway are three serine/threonine-selective kinases: RAF, MEK, and MAPK/ERK. GTP bond RAS, a small G protein, induces RAF activity. RAF then phosphorylates and activates MEK, which in turn phosphorylates and activates MAPK/ERK. The targets of ERK include protein kinases such as RSK and MNK, ion channel, neurotransmitter receptors, and transcription factors. RSK and MNK are thought to phosphorylate and activate transcription factor cAMP response element binding (CREB). CREB regulates the expression of many different genes, including B-cell lymphoma 2 (Bcl-2)1,8 and brain-derived neurotrophic factor (BDNF) 9 to enhance neuroprotection and neuronal survival mechanisms. In SH-SY5Y human neuroblastoma cells, the mood stabilizers lithium and valproate activated AP-1 transcription factors, and that activation was blocked by a MEK inhibitor.10 That study also demonstrated that valproate increased levels of activated phospho-ERK and reporter gene expression driven by ELK, an ERK-regulated transcription factor; that activation was further blocked by RAS and RAF functional null mutant.10 Valproate also promoted neurite outgrowth and expression of GAP-43 in these cells, which could be blocked by an ERK pathway inhibitor.10 Taken together, these data indicate that valproate activates the ERK pathway and produces neurotrophic-like cellular effects through this activation. Follow-up studies showed that in cultured cerebral cortical cells, valproate induced ERK pathway activation in a manner that was more sustainable than activation by growth factors.3 Valproate-induced activation of the ERK pathway has also been identified in primary cortical neurons,11 cerebral progenitor cells,12 hippocampal progenitor cells,13 and endothe-

neurotrophic and neuroprotective mechanisms, as well as reverse or reduce behavioral deficits associated with preclinical animal models of mania and depression and other psychiatric illnesses. While much of this work has focused on the mood stabilizers lithium and valproate, we will also review the available evidence that antidepressants and antipsychotics exert similar neurotrophic effects.

Mood stabilizers
Mood stabilizers are used to treat bipolar disorder (BPD), which is characterized by mood shifts between mania (characterized by elevated mood, increased energy, impaired judgment, and racing thoughts) and depression (characterized by low mood, anhedonia, etc). These therapeutic agents do not simply target a particular neurotransmitter system or cellular signaling cascade, but diverse targets implicated in many signaling pathways. This may be because mood stabilizers were often designed to treat different disorders, and their use in the treatment of BPD frequently arose through serendipity; for instance, the mood stabilizers carbamazepine and valproateboth used to treat the manic symptoms of BPDhave anticonvulsant properties and were developed for the treatment of epilepsy. In addition, our incomplete understanding of the pathophysiology of BPD, in which both genetic and environmental predispositions may impair cellular resilience and lead to dysfunctional circuits and synapses, further supports the notion that these agents affect diverse targets. Indeed, mood stabilizers may achieve their therapeutic effects by working through these diverse targets to restore cellular resilience; notably, however, chronic treatment is necessary for their neurotrophic and neuroprotective actions to improve functional plasticity in cortical and limbic circuits and synapses. Below we focus on several intracellular signaling pathways targeted by mood stabilizers that may underlie these therapeutic mechanisms: i) the mitogen activated protein kinase/extracellular signal-related kinase

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lial cells.14 Lithium similarly increased activation-phosphorylation of ERK in SY5Y cells,15 cerebellar granular cells,16,17 hippocampal progenitor cells,18,19 and primary cortical neurons.11 Lithium inhibited the ERK pathway in cultures of serum-deprived, quiescent astrocytes.17 Furthermore, lamotrigine, an anticonvulsant prescribed to prevent recurrences of depression or mania in BPD, did not affect the ERK pathway in SH-SY5Y cells15 or primary cortical neurons11; however, lamotrigine still showed neuroprotective effects in models of ischemia and kainate (KA)-induced neurotoxicity, perhaps through glutamate release inhibition.20,21 Taken together, these in vitro data suggest that actiBDNF

vation of the ERK pathway is common to only a subgroup of mood stabilizers and is cell-type specific. In a series of in vivo studies, Chen and colleagues found that chronic treatment with lithium or valproate increased levels of activated phospho-ERK, phosphoRSK1, and activated phospho-CREB in prefrontal cortex and hippocampus.2,3 Lithium-induced increases in activated phospho-ERKs were also observed in the caudate/putamen of infant mouse brains.22 Another study found that valproate increased levels of activated phospho-ERK and activated phospho-CREB in mice with intracerebral hemorrhage.23 Another study found that
Wnt Frizzled

TrkB

TrkB

P13K Ras PDK Dishevelled Secretase Raf AKT GSK3 APP-A MEK
BA G 1

Tau

ERK

Mitochondria

Bc l-2

Nucleus

RSK

CBP

RNA POLII

Figure 1. Intracellular signaling pathways targeted by psychotropic agents. The MAPK/ERK, PI3K, and Wnt/GSK3 signaling cascades. Psychotropic agents such as mood stabilizers, antidepressants, and antipsychotics target these signaling cascades. Targets reported to be regulated by antidepressants, antipsychotics, mood stabilizers (red), or multiple types of treatments (orange) are highlighted. Molecules in blue are critical constituents of the selected pathways that have not been found to be affected by any of the treatments discussed in this review. Arrowheads indicate activation; circles indicate inhibition. Akt, serine/threonine protein kinase AKT; BAG-1, Bcl-2 associated athanogene; Bcl-2, B-cell lymphoma 2; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element binding; ERK, extracellular regulated kinase; GSK-3, glycogen synthase kinase 3; HDAC, histone deacetylase; PI3K, phosphatidylinositol 3 kinase; TrkB, neurotrophic tyrosine kinase receptor, type 2; Ras, resistance to audiogenic seizures; Raf, RAF proto-oncogene serine/threonine-protein kinase; RSK, ribosomal protein S6 kinase, 90kDa; MEK or Map2k1, mitogen-activated protein kinase kinase 1; CBP; CREB binding protein; RNA POLII, RNA polymerase II; HAT, histone acetyltransferase; PDK, pyruvate dehydrogenase kinase; APP-AB, amyloid beta (A4) precursor protein; Wnt, wingless

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A CR EB CR EB

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valproate did not induce changes in phospho-ERK levels in the nucleus accumbens, and reduced phosphoERK levels in the amygdala,24 suggesting that mood stabilizer-induced ERK pathway activation/inactivation may be brain region-specific. The phosphatidylinositol 3 kinase (PI3K) pathwaya regulator of neuronal survival and plasticityis also regulated by growth factors (Figure 1).6,25-27 Upon trophic factor stimulation (Figure 1), the regulatory subunit of PI3K is stimulated by the adapter proteins Grb-2 and Grb-2-associated binding protein 1/2 (Gab1/2), resulting in PI3K activation. The catalytic subunit of PI3K is also stimulated by direct interaction with activated RAS. Activated PI3K converts plasma membrane lipid phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 provides docking sites for phosphoinositide-dependent kinase (PDK) and the serine-threonine kinase Akt (also known as protein kinase B, PKB). Simultaneous binding of PDK and Akt at the PI3K activation site facilitates phosphorylation of Akt by PDK1 and enhances Akt activity. Akt then phosphorylates glycogen synthase kinase-3 (GSK-3), which in contrast to most phosphorylations, leads to the inactivation of this enzyme.28 PI3K, PDK, Akt, and GSK-3 are thought to be the major components of the PI3K pathway. Mood stabilizers target several of these major components of the PI3K pathway. Acute (minutes to hours) or subacute (several days) lithium treatment of cerebellar granule cells, for instance, increased levels of activated phospho-Akt as well as phospho-GSK-3, a product of Akt-catalyzed phosphorylation.29 Interestingly, similar effects were noted in human SH-SY5Y cells treated with lithium and valproate.1,30 The increases were blocked by PI3K inhibitors, indicating that they required PI3K activation.29 Chronic lithium and valproate treatment also increased levels of phospho-GSK-3 in mouse cerebral cortex and hippocampus.30-32 Lithium injections (200 mg/kg of body weight, IP) significantly increased levels of phospho-Akt, phospho-GSK-3 and phospho-GSK-3 in the striatum of dopamine transporter knockout (DAT KO) mice within 30 minutes of administration.33 Valproate increased activated brain phospho-Akt in skeletal muscle in a mouse model of Duchennes muscular dystrophy,34 as well as in a mouse model of intracerebral hemorrhage.23 These data demonstrate that lithium and valproate stimulate the PI3K pathway in vivo and subsequently inactivate GSK-3. Mood stabilizers upregulate levels of neurotrophic and neuroprotective molecules Studies show that lithium and valproate increased mRNA and protein levels of neurotrophins such as BDNF, glial cell-line derived neurotrophic factor (GDNF), neurotrophin 3 (NT-3), and vascular endothelial growth factor (VEGF) in cultured cells and brain regions.2,35-46 Furthermore, lithium increased serum BDNF levels in patients with Alzheimers disease.47 The effects of mood stabilizers on BDNF levels are thought to be mediated via several different mechanisms. These mechanisms may include enhancing BDNF promoter activation40,43,45 by stimulating the ERK and PI3K pathways using lithium or valproate, leading to CREB activation and CRE-mediated gene transcription of BDNF. Valproates inhibition of histone deacetylase (HDAC) via an epigenetic mechanisma molecular process that leads to gene activation and deactivationmay also play a role.40,43,45 In addition to targeting neurotrophic mechanisms, mood stabilizers also target neuroprotective molecules such as Bcl-2. Bcl-2 and its family proteins are the major modulators of apoptosis. Notably, numerous studies have shown that chronic treatment with lithium or valproate upregulates Bcl-2 and Bcl-2 associated athanogene (BAG-1) levels in the brain or nerve tissues.23,32,48-54 This upregulation appears to be partially due to activation of the ERK and PI3K pathways, as well as increased transcriptional activity of CREB.1 Mood stabilizers promote neurogenesis and neuronal process growth The discovery that mood stabilizers can regulate growth factors and produce neurotrophin-like molecular effects led investigators to explore whether these agents could augment hippocampal neurogenesis. Lithium and valproate were indeed found to promote hippocampal neurogenesis in neuronal cell culture and rodent studies.3,18,19,32,55 In vitro evidence showed that lithium induced neuronal differentiation of hippocampal neural progenitor cells via a phospho-ERK and phospho-CREB dependent pathway.19 An in vivo study showed that lithium increased survival of newborn cells in hippocampus, and that an ERK pathway inhibitor blocked lithiums survival effects.56 Valproate activated the ERK pathway and promoted differentiation of hippocampal

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neural progenitor cells in culture; however, valproates differentiation effects were thought to be mediated through HDAC inhibition, not ERK pathway activation.13,57 Whether valproate uses multiple mechanisms to induce hippocampal neurogenesis in intact animals remains to be elucidated. Valproate also promoted neurite growth in cultured cells (for reviews see refs 6,7), which was blocked by ERK pathway inhibition.3,10 Animal studies have found that valproate facilitated axonal regeneration and motor function recovery after sciatic nerve axotomy.51,58 Lithium similarly enhanced survival and axonal regeneration of cultured retinal ganglion cells,4 protected retinal ganglion cells following partial optic nerve crush in rats,59 and promoted axonal regeneration of rubrospinal tract (RST) neurons following injury to the spinal cord.53 These neuroprotective findings are, in part, thought to be mediated by Bcl-2. Another recent study showed that lithium facilitated motor function recovery and axonal regeneration after spinal cord injury, and these effects were associated with increased inactivated-phospho-GSK-3. Further support for GSK-3s role in lithiums neuroprotective effects came from a study where lithiums effects were mimicked by the GSK-3 inhibitor SB415286.60 Another area where lithium exerts neuroprotective effects is in stress-induced morphological alterations. Chronic behavioral stress shortens apical dendrites in the CA3 region of the hippocampus in rodents. Lithium treatment initiated 2 weeks before the stress and continued throughout a 3-week period of stress attenuated these stress-induced reductions in apical dendritic lengths.61 Although the molecular mechanisms of this lithium-induced morphological action are still not fully understood, they are particularly important because social-psychological and behavioral stress cause a variety of brain changes and are key contributing factors to mood disorders.62-65 Evidence from human imaging studies for neurotrophic/neuroprotective actions of mood stabilizers As noted previously, brain imaging studies show brain ventricular enlargements,66,67 cortical regional morphometric reductions,68-72 and cerebral and hippocampal level reductions of NAA72-78 in individuals with mood disorders, especially in unmedicated patients with a family history of mood disorders.

Intrigued by the discovery that Bcl-2 is upregulated by mood stabilizers, investigators used imaging tools to assess the effects of mood stabilizers on brain morphometric and neurochemical measures. In a magnetic resonance imaging (MRI) study, Moore and colleagues found that lithium treatment increased cerebral grey matter volume.79 Similar findings were also obtained in other longitudinal and cross-sectional studies of cerebral grey matter volume,80 left anterior cingulate volume,70 right anterior cingulate volume,81 hippocampal volume,82-86 and amygdala volume.86 A cross-sectional study found that valproate similarly increased left anterior cingulate volume in individuals with BPD.87 One initial longitudinal MRS study found brain regional increases in NAA levels in individuals with BPD and healthy subjects treated for 4 weeks with lithium, 79 a finding replicated by other investigators.88-90 NAA levels were also found to be correlated with brain lithium levels in a study of elderly patients with BPD. 91 Valproate was similarly found to increase hippocampal NAA levels.72 Mood stabilizers produce neuroprotective effects in animal models of disease Mood stabilizers are known to protect cultured cells from a variety of insults (for reviews see refs 6,7,92,93). In this section, we review the neuroprotective effects of lithium and valproate in a series of models of brain ischemia, neurodegeneration, and neuroinflammation (eg, cerebral ischemia, Alzheimers disease (AD), Huntingtons disease, amyotrophic lateral sclerosis (ALS), HIV-associated cognitive impairments, and spinocerebellar ataxia). In a seminal study using an animal model of ischemia, Chuang and colleagues found that ischemic infarct size induced by occlusion of the left middle cerebral artery was markedly reduced by lithium treatment administered before94 or after95 the induction of ischemia; these findings have since been replicated by other investigators.96-104 Follow-up studies showed that valproate had similar protective effects on ischemia-induced brain infarction.105,106 ALS is a progressive, lethal neurodegenerative disease with no known cure. Riluzole, which prolongs the survival of patients by several months, is the only FDAapproved treatment for this disease. Interestingly, riluzole itself has been associated with neuroprotective

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properties.107 SOD1-G93A mice, a model for ALS, carry a high copy number of this transgene with the G93A human SOD1 mutation. Studies show that valproate108 and lithium109,110 both delay disease onset and prolong lifespan in SOD1-G93A mice. Furthermore, lithium and valproate together produce an additive protective effect in SOD1-G93A mice compared with either treatment alone.110 Notably, a clinical trial found that lithium, compared with riluzole, further delays disease progression and death in individuals with ALS.109 With regards to AD, diverse studies have suggested that lithiums neuroprotective effects may have a potential role in the therapeutics of this disease. AD is a leading cause of dementia in the aging population and the most common neurodegenerative disease without an effective treatment. Briefly, the histological hallmarks of AD include amyloid plaques, neurofibrillary tangles, and neuronal loss. The plaques consist of insoluble deposits of amyloid-beta (A) protein and cellular material outside and around neurons. A protein is derived from amyloid precursor protein (APP) through an endoproteolytic cleavage catalyzed by - and -secretase. Mutations in the genes of presenilinsthe core component of -secretase, APP, and tau are associated with AD. One series of experiments in cultured cells found that GSK-3 increased A production,111 and that chronic lithium treatment reduced A produced in a genetic mouse model of AD. These mice expressed APPSwedish (Tg2576) and also carried a knock-in mutation of presenilin-1 (PS1P264L). In a transgenic mouse strain overexpressing mutated (London V717I and Swedish K670M/N671L) human APP (hAPP751), lithium treatment reduced A production, improved performance in the water maze, and preserved dendritic structure in the frontal cortex and hippocampus, all of which are associated with decreased APP phosphorylation and increased levels of phospho-GSK-3.112 In another animal model of AD where APP23 transgenic mice carried human APP751 cDNA with the Swedish double mutation at positions 670/671, Qing and colleagues observed that valproate treatment decreased A production, reduced neuritic plaque formation, and improved memory deficits; these effects were also associated with increased phospho-GSK-3.113,114 Neurofibrillary tangles are formed by hyperphosphorylated tau, a microtubule-associated protein. GSK-3 is a major tau kinase and GSK-3 hyperactivity is known to contribute to tau hyperphosphorylation in cell and animal models. Interestingly, lithium treatment reduced tau phosphorylation in the brains of mice overexpressing mutated (London V717I and Swedish K670M/N671L) human APP (hAPP751).112 In another AD model (3xTG-AD), lithium treatment reduced brain tau phosphorylation and increased brain GSK-3 and phosphorylation at the inhibitory sites; however, it did not improve memory or reduce A protection.115 Given these promising preclinical data, studies began to examine the potential long-term neurotrophic/ neuroprotective effects of lithium and valproate in humans. While some studies suggest that naturalistic lithium treatment may indeed be associated with neuroprotective effects in individuals with AD (see, for instance refs 47,116-118), considerably more data are required. Nevertheless, this remains a promising and exciting area for further investigation. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. In a SCA1 mouse model, chronic administration of lithium initiated before or after the deficit onset had a positive effect on multiple behavioral measures and hippocampal neuropathology.119 Indeed, clinical trials of lithium in patients with SCA1 are currently ongoing (see http://clinicaltrials.gov/ for more information). Finally, the neuroprotective effects of lithium and valproate have also been reported in additional disease and insult models, including animal models of Huntingtons disease,120,121 Parkinson's disease,122 HIV-induced encephalitis and dementia,123,124 and aluminum-induced neurodegeneration.50 At least some of these effects are associated with increased Bcl-2 levels.50,120,121,122

Antidepressants
Chemical antidepressants used to treat depressive disorders, or depressive symptoms in other psychiatric disorders, include monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), or selective norepinephrine reuptake inhibitors (SNRIs). These chemical antidepressants act by increasing monoamines (serotonin and/or norepinephrine) in the synaptic cleft, which occurs immediately; however, for most patients, therapeutic effects are observed only after a few days, and often not until 2 weeks or more. This suggests that adaptive changes in cellular signaling cascades may underlie

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their therapeutic effects.125 Two such pathways that will be considered below include the MAPK/ERK and the Wnt/GSK signaling cascade (Figure 1), which may enhance neurotrophic and neuroprotective mechanisms in addition to neurogenesis. Interestingly, nonchemical antidepressants such as electroconvulsive therapy (ECT) and exercise also target these pathways and may employ similar therapeutic mechanisms. Antidepressants affect prominent signaling cascades involved in neuronal protection and survival As noted above, activation of the MAPK/ERK and Wnt/GSK signaling cascades (Figure 1) ultimately targeted by antidepressants may result in enhanced neuroprotective and survival mechanisms. For instance, both chemical antidepressants and ECT increase BDNF levels. In rats, ECT increased BDNF and its receptor (trkB) in the hippocampus.126 A similar effect was also found following chronic (21 days) but not acute treatment with different classes of antidepressants (the MAOI tranylcypromine, the SSRI sertraline, and the TCA desipramine). Furthermore, chronic antidepressant treatment also increased the expression of CREB mRNA in the rat hippocampus,127 suggesting a potential regulatory mechanism for BDNF through CRE-mediated gene transcription. Exercise has also been reported to upregulate many factors in the MAPK signaling pathway including BDNF, trkB, MEK2, and ERK2.128-133 A recent study found that exercise-induced upregulation of BDNF at the mRNA and protein level and phosphorylation of survival factor Akt both occurred via a CREB-dependent mechanism.134 Interestingly, the SNRI reboxetine also depends on CREB activation (phosphorylation) in order to show similar changes in BDNF and Akt. In humans, serum levels of BDNF levels are decreased in unmedicated depressed patients compared with depressed patients currently taking antidepressants or healthy controls.135 BDNF serum levels were also found to be negatively correlated with depression scores as assessed by the Hamilton Depression Rating Scale (HDRS). Interestingly, BDNF itself also possesses antidepressant-like effects in rodent models used to screen antidepressants following direct infusion into either the midbrain136 or hippocampus.137 This enhancement in BDNF by antidepressants may help promote mechanisms of neuronal protection and survival key to reducing stress-induced damage.

Antidepressants have also been found to have neuroprotective effects. For instance, the SSRI fluoxetine prevented the neurotoxic effects of ecstasy (3,4-methylenedioxymethamphetamine, MDMA).138,139 Mechanistically, fluoxetines neuroprotective effects, in addition to restoring serotonin levels, may result from activation of p38 MAPK, BDNF, and GDNF.140 MAOIs (eg, pargyline, nialamide, tranylcypromine) inhibiting both MAO-A and MAO-B protected against 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)induced dopaminergic neural toxicity.141 Interestingly, Ladostigil, a MAOI used to treat both depression and neurodegeneration that has promising neuroprotective effects, reportedly activated Bcl-2 family members and BDNF142 in addition to ERK1/2 (p44/42 MAPK).143 Notably, exercise also possesses neuroprotective effects. Carro and colleagues showed that rodents subjected to treadmill running were protected against various insults ranging from treatment with the neurotoxin domoic acid to inherited neurodegeneration affecting Purkinje cells of the cerebellum.144 These protective effects depended in part on the neurotrophic factor insulin-like growth factor I (IGF-1); infusing a blocking anti-IGF-1 antibody reduced the protective effects of exercise. Effects of antidepressants on neurogenesis in animals Antidepressants increase hippocampal adult neurogenesis following chronic but not acute treatment. Chronic treatment with the SSRI fluoxetine, the MAOI tranylcypromine, or the SNRI reboxetine produced an approximately 20% to 40% increase in bromodeoxyuridine BrdU-labeled hippocampal cells145; at least 2 weeks of fluoxetine treatment was required to enhance neurogenesis. Furthermore, while stress decreases hippocampal neurogenesis, chronic antidepressant treatment prevented these stress-induced changes.146,147 ECT also increased neurogenesis in rodents,148 as well as hippocampal synapse number.149 ECT was similarly found to increase neurogenesis in nonhuman primates,150 and exercise increased hippocampal neurogenesis151 in addition to enhancing hippocampal-dependent learning and long-term potentiation (LTP).151 The molecular mechanisms underlying these antidepressant-induced enhancements in neurogenesis may involve the MAPK/ERK and/or Wnt/GSK-3 pathways. A very recent study found that suppression of the gene disrupted in schizophrenia 1 (DISC1), which has been

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implicated in BPD, major depressive disorder (MDD), and schizophrenia, decreased neurogenesis by acting through GSK3.152 Antidepressants and the reversal of stress-induced changes in neuronal plasticity In terms of clinical implications, a recent meta-analysis found enhanced antidepressant response in the Met variant of the BDNF 66Val/Met polymorphism in individuals with MDD.153 Curiously, 66Met allele carriers have a lower neuronal distribution of BDNF in addition to decreased activity-dependent BDNF secretion. Given the hypothesis that antidepressant effects are partially mediated through enhanced BDNF secretion, it would seem contradictory that 66Met allele carriers, with their attenuated BDNF secretion, have a more robust response to antidepressants. In addition to enhanced antidepressant treatment response, this BDNF polymorphism was also associated with decreased episodic memory performance, lower hippocampal activation (as measured by fMRI), and lower hippocampal NAA levels in humans.154 In a mouse model of the BDNF-Met variant in which BDNF-Met was expressed at normal levels, but regulated secretion from neurons was reduced, fluoxetine was unable to ameliorate a stressinduced anxiety phenotype.155 Taken together, these data suggest a more complicated picture that requires a better understanding of proper BDNF function (and not just its expression); however, normal BDNF function does appear to be important for proper hippocampal function and mood regulation. Notably, severely depressed patients show elevated levels of the stress hormone cortisol, which is thought to result from a dysfunctional hypothalamic-pituitaryadrenal (HPA) axis negative feedback circuit,156,157 and which may ultimately contribute to the hippocampal damage and volumetric changes reported in the literature. Subjects with MDD were found to have significantly smaller hippocampal volumes, and these reductions correlated with total duration of depression but not with age,158,159 suggesting that the stress associated with depression may have contributed to these volumetric changes. Further support for this notion comes from studies reporting that individuals with post-traumatic stress disorder (PTSD) had impaired hippocampal function (deficits in short term memory, total recall, longterm storage, and retrieval) but no overall IQ differences when compared with controls160; MRI studies found that these PTSD patients had an 8% smaller right hippocampus than controls.161 In addition, the polymorphism in the BDNF gene (val(66)met) has also been associated with reduced hippocampal volume.162 Interestingly, antidepressants can reverse some of these changes. In tree shrews, the selective serotonin reuptake enhancer (SSRE) tianeptine prevented the decreased brain metabolites (NAA, creatine, phosphocreatine), suppressed neurogenesis, and reduced hippocampal volume associated with chronic psychosocial stress. 147 In another study, chronic treatment with antidepressants induced hippocampal neurogenesis, blocked inescapable foot shock stress-induced decreases in hippocampal neurogenesis, and normalized corticosterone levels and behavioral deficits.145,163 Finally, repetitive transcranial magnetic stimulation (rTMS) has also shown putative neurotrophic properties in patients with MDD. In one study, rTMS improved refractory depression by augmenting catecholamines and BDNF,164 while another study found that rTMS augmented BDNF in drug-resistant patients.165

Antipsychotics
Antipsychotic medications are traditionally categorized as typical (also known as traditional, conventional, or classic neuroleptics) or atypical (second generation). Several typical antipsychotics have a higher dopamine D2 receptor affinity than atypical antipsychotics, which bind to a broader group of receptors, including dopamine, serotonin, glutamate, histamine, -adrenergic, and muscarinic receptors.166 While antipsychotics can have an immediate impact on symptoms such as agitation, it often takes weeks before improvement is seen in other symptoms, such as delusions; however, recent findings suggest these improvements may emerge more rapidly than previously believed.167,168 As with mood stabilizers and antidepressants, it is likely that these drugs improve many facets of psychosis through mechanisms beyond their fundamental interaction with dopaminergic, serotonergic, muscarinic, and other receptor families. Chronic treatment with conventional antipsychotics can lead to adverse extrapyramidal side effects (EPS), which mimic the neurodegenerative disorder Parkinsons disease, as well as the potentially irreversible condition known as tardive dyskinesia.169 These effects are less common with atypical antipsychotics, which also have

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improved efficacy in treating the negative symptoms associated with schizophrenia, though their overall benefit is still unclear170; atypical antipsychotics also have their own adverse metabolic side effects like weight gain and diabetes.171 As highlighted below, these two classes of antipsychotics show markedly different profiles for activating neuroplasticity cascades, and for enhancing neuroprotection and neurogenesis in both animal studies and patient-based studies. Antipsychotics alter the expression of prominent intracellular cascades and influence neuroplasticity and neuroprotection in animal models Studies conducted in rodents and cell lines have demonstrated that some antipsychotics can induce significant changes in intracellular cascades that are involved in neuroplasticity and neuroprotection against excitotoxic insults, including ERK/MAPK, Akt, Bcl-2, and BDNF pathways. Acute treatment with the atypical antipsychotic clozapine led to increased levels of active (phosphorylated) MEK1/2 in rat prefrontal cortex, 172 while chronic treatment with the atypical antipsychotic olanzapine increased pERK1/2 levels in rat prefrontal cortex (PFC).173 Interestingly, Browning and colleagues observed decreases in pERK1/2 following either a single injection of olanzapine or haloperidol (a typical antipsychotic), but chronic haloperidol did not alter pERK1/2 levels. Multiple studies in phenochromocytoma (PC12) cells have also noted upregulation of pERK1/2, pAkt, and PI3K following olanzapine treatment.174,175 Antipsychotics have also been shown to influence other prominent cascades discussed above, including Bcl-2,176 GSK-3,177 and CREB.178 Many studies have assessed the effects of antipsychotics on neurotrophic factors such as BDNF and nerve growth factor (NGF), and have noted significant differences between typical and atypical antipsychotics. Typical antipsychotics such as haloperidol tend to reduce BDNF expression in regions of the hippocampus 179-181 and striatum.182 Atypical antipsychotics do not consistently downregulate BDNF, and their more diverse set of responses make critical evaluations more challenging (see ref 183). One recent study noted that, after chronic (90-day) treatment with haloperidol, transitioning to the atypical antipsychotics olanzapine or risperidone appeared to rescue BDNF expression back to near baseline levels.182,184

Studies have demonstrated that chronic or high doses of typical antipsychotics, like haloperidol and reserpine, can be neurotoxic, inducing apoptosis and reducing cell viability. Though the mechanism remains unclear, high doses of haloperidol induced apoptosis in the striatum and substantia nigra of rats treated via acute intraperitoneal injection.185 In vivo investigations have further noted that brain regions like the striatum, hypothalamus, and limbic structures were some of the most drastically altered cytoarchitecturally by conventional antipsychotics.186 Macaque monkeys treated for 17 to 27 months with therapeutic levels of either haloperidol or olanzapine had reduced brain volumes by ~10%, most prominently in the parietal and frontal brain lobes.187 Other studies found the opposite effect, that chronic treatment of rats with haloperidol increased striatal volume.188 In contrast, atypical antipsychotics appear to have some neuroprotective functions. For example, pretreatment with the atypical antipsychotics clozapine, quetiapine, or risperidone prevented PC12 cell death following serum withdrawal,189 while olanzapine reduced cell death in PC12, SH-SY5Y, and 3T3 cells following a number of death-inducing treatments.174 Neuroprotective properties have also been demonstrated for the atypical antipsychotic olanzapine against various insults, such as oxidative stressors190 and ischemia.191 Olanzapine also upregulated the expression of Bcl-2 in rat frontal cortex and the hippocampus, as well as the expression of BDNF in the hippocampus.176,181 Studies have suggested that other atypical antipsychotics, such as risperidone and quetiapine, have neuroprotective properties that might be relevant to their clinical efficacy.192,193 For instance, one study found that the effects of stress-induced decreases of BDNF could be prevented by pretreatment with quetiapine.194 Overall, the findings suggest that antipsychotics can alter prominent intracellular cascades and ultimately induce neurotrophic or neurotoxic responses in vivo and in vitro, depending upon the drug conditions, time course, and brain region under consideration. Effects of antipsychotics on neurogenesis in animals Initial studies detected increased neurogenesis in the gerbil hippocampus following haloperidol treatment,195 but not in the rat hippocampus.145 Two more recent studies found that haloperidol did not affect neurogenesis,196,197 although a study that used osmotic pumps

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(instead of daily intraperitoneal injections or delivery in drinking water) found that haloperidol increased neural stem cell (NSC) proliferation in the adult rat forebrain.198 Furthermore, the researchers demonstrated that this proliferation was mediated through D2 receptor stimulation in vitro, suggesting that under certain conditions, haloperidol could promote neurogenesis through its suppression of D2-mediated pathways that normally prevent NSC proliferation. Atypical antipsychotics have shown a more consistent profile of enhancing neurogenesis, but do not necessarily increase neuronal survival or differentiation into adult neurons. Chronic treatment of rats with clozapine or olanzapine, for example, augmented the number of BrdU-labeled cells in the dentate gyrus196 or prefrontal cortex and dorsal striatum.197 Although both studies detected increased proliferation of precursor cells, neither found a significant difference in the number of BrdU-positive, mature neurons in the weeks following treatment with antipsychotics. Quetiapine has also been shown to reverse the inhibition of hippocampal neurogenesis caused by chronic restraint stress, and significantly increase the number of BrdU-labeled immature neurons detected compared with vehicle-treated, stressed rats.199 Effect of antipsychotics on NAA levels, brain volume, and density in patients Studies conducted with schizophrenic patients have examined NAA measures and volumetric brain changes using 1H-MRS and MRI, respectively, to elucidate the effects of chronic antipsychotic treatment. Patients treated with atypical antipsychotics had higher NAA measures in the frontal lobes200 and anterior cingulate gyrus201 than those treated with typical antipsychotics. Another study measured NAA changes during antipsychotic treatment and after cessation for at least 2 weeks in individual patients using a within-subject design and found significant decreases (~9%) in NAA levels in the dorsolateral prefrontal cortex after ending antipsychotic treatment; no differences were found in other brain regions.202 Schizophrenia, the disorder most often treated with antipsychotics, is well-known to be associated with reduced regional volumes, increased ventricle size,203 and deteriorating course,204 making it difficult to distinguish volumetric changes induced by antipsychotic treatment. Overall, studies suggest that there are differences in the brain volumes of patients treated with antipsychotics compared with controls, or within groups of patients treated chronically with typical versus atypical antipsychotics; for a thorough analysis, see ref 186. One study of patients with first-episode psychosis found that treatment with haloperidol reduced grey matter volume; in contrast, olanzapine-treated patients showed no significant reductions compared with controls.205 Another recent study found that olanzapine increased NAA in the prefrontal cortex of remitted adolescent patients with mania compared with nonremitted patients.206 Although this suggests a possible in vivo neurotrophic effect, this finding needs further replication because the primary aim of the studya NAA increase following olanzapine treatment, independent from clinical changewas negative. In fact, it is possible that the NAA increase seen in responders was more closely related to improved mood than to olanzapines neurotrophic properties.

Closing remarks
The growing data from molecular, cellular, animal, and human studies described in this review support the notion that psychotropic agents used to treat the major psychiatric disordersespecially mood stabilizersare associated with significant neurotrophic/neuroprotective effects. These effects may enhance cellular resilience and plasticity in dysfunctional synapses and neural circuitry implicated in psychiatric disorders. The crux of such research is that, in addition to their proven ability to treat psychiatric disorders, these agents may be useful in the treatment of neurodegenerative illnesses and ischemia. Similarly, psychotropic agents developed for the treatment of neurodegenerative illnesses may be beneficial as therapeutics for major psychiatric illnesses. Currently, several clinical trials are being conducted to evaluate the feasibility of using lithium and valproate to treat a variety of neurodegenerative diseases. Indeed, neuroprotection is the most consistent biological outcome associated with lithium treatment. There is hope that these clinically safe and widely used agents will slow disease progression, and perhaps produce functional improvements. Furthermore, because lithium and valproate stimulate the ERK and PI3K pathways, increase BDNF, Bcl-2, and BAG-1 expression, block HDAC activity (valproate only), and inhibit GSK-3 alpha and

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beta activities, continued study of these agents may elucidate other clinically relevant targets, ultimately leading to improved treatments for these devastating disorders. Additional data are also needed to understand whether the neurotrophic and neuroprotective effects of mood stabilizers, antidepressants, and antipsychotics are cell-type or circuitry specific, and to what extent their neurotrophic/neuroprotective effects contribute to their therapeutic action. Finally, gaining insight into rapid-act-

ing versus long-term compensatory changes facilitated by these psychotropic agents will pave the way for the next generation of therapeutics, whose dual nature will provide both a rapid treatment response to restore function, as well as support long-term changes to maintain successful treatment and prevent relapse.
Acknowledgements: Funding for this work was supported by the Intramural Research Program of the National Institute of Mental Health (NIMH). The authors have no conflicts of interest, financial or otherwise, to disclose.

Los efectos neurotrficos y neuroprotectores de los agentes psicotrpicos

La evidencia acumulada sugiere que los agentes psicotrpicos como los estabilizadores del nimo, los antidepresivos y los antipsicticos producen sus efectos neurotrficos/neuroprotectores mediante la activacin de la quinasa relacionada con la seal de la proteinquinasa/extracelular activada por mitgeno, la quinasa PI3 y las vas de seales de la wingless/glicgeno sintetasa quinasa 3 (GSK). Estos agentes tambin regulan hacia arriba la expresin de molculas trficas/protectoras como el factor neurotrfico cerebral, el factor de crecimiento neural, la protena 2 del linfoma de clulas B, la quinasa serina-treonina y el atanogen 1 asociado a Bcl-2, e inactivan molculas proapoptticas como la GSK-3. Ellos tambin promueven la neurognesis y son protectores en modelos de enfermedades neurodegenerativas e isquemia. La mayor parte, sino toda esta evidencia se recolect a partir de estudios animales que utilizaron esquemas teraputicos clnicamente relevantes. Adems, en humanos los estudios de imgenes han encontrado que estos frmacos aumentan el volumen y la densidad del tejido cerebral, como tambin los niveles de N-acetil aspartato y glutamato en determinadas regiones cerebrales. Tomados en conjunto, estos datos sugieren que los efectos neurotrficos/neuroprotectores de estos frmacos tienen un gran potencial teraputico en el tratamiento no slo de los trastornos del nimo y de la esquizofrenia, sino que tambin en enfermedades neurodegenerativas y en la isquemia.

Effets neuroprotecteurs et neurotrophiques des psychotropes

Daprs un nombre croissant darguments, les psychotropes, comme les rgulateurs de lhumeur, les antidpresseurs et les antipsychotiques, exercent leurs effets neurotrophiques et neuroprotecteurs en activant 3 voies de signalisation : la MAP (mitogen activated protein)/ERK (extracellular signal-related) kinase, la kinase PI3 et la Wnt/GSK (wingless/kinase glycogne synthase). Ces voies rgulent galement positivement lexpression des molcules trophiques/protectrices comme le BDNF (brain-derived neurotrophic factor), le NGF (nerve growth factor), la protine BCL2 (B-cell lymphoma 2), la kinase srine-thronine et le BAG-1 (athanogne 1 associ BCL-2), et des molcules proapoptotiques inactives comme la GSK-3. Elles favorisent aussi la neurogense et exercent un effet protecteur dans les maladies neurodgnratives et lischmie. La plupart de ces preuves, si ce nest toutes, sont issues dtudes animales utilisant des schmas thrapeutiques cliniquement pertinents. De plus, des tudes dimagerie sur lhomme ont montr que ces agents augmentaient le volume et la densit du tissu crbral ainsi que les taux de N-actyl aspartate et de glutamate dans les rgions crbrales slectionnes. Au total, ces donnes suggrent que leurs effets neurotrophiques/neuroprotecteurs ont un potentiel thrapeutique large non seulement dans les troubles de lhumeur et la schizophrnie mais aussi dans les maladies neurodgnratives et lischmie.

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157.Carroll BJ, Curtis GC, Mendels J. Neuroendocrine regulation in depression. II. Discrimination of depressed from nondepressed patients. Arch Gen Psychiatry. 1976;33:1051-1058. 158.Sheline YI, Sanghavi M, Mintun MA, Gado MH. Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J Neurosci. 1999;19:5034-5043. 159. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW. Hippocampal atrophy in recurrent major depression. Proc Natl Acad Sci U S A. 1996;93:3908-3913. 160. Bremner JD, Scott TM, Delaney RC, et al. Deficits in short-term memory in posttraumatic stress disorder. Am J Psychiatry. 1993;150:1015-1019. 161. Bremner JD, Randall P, Scott TM, et al. MRI-based measurement of hippocampal volume in patients with combat-related posttraumatic stress disorder. Am J Psychiatry. 1995;152:973-981. 162. Bueller JA, Aftab M, Sen S, Gomez-Hassan D, Burmeister M, Zubieta JK. BDNF Val66Met allele is associated with reduced hippocampal volume in healthy subjects. Biol Psychiatry. 2006;59:812-815. 163. Malberg JE, Duman RS. Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology. 2003;28:1562-1571. 164. Yukimasa T, Yoshimura R, Tamagawa A, et al. High-frequency repetitive transcranial magnetic stimulation improves refractory depression by influencing catecholamine and brain-derived neurotrophic factors. Pharmacopsychiatry. 2006;39:52-59. 165. Zanardini R, Gazzoli A, Ventriglia M, et al. Effect of repetitive transcranial magnetic stimulation on serum brain derived neurotrophic factor in drug resistant depressed patients. J Affect Disord. 2006;91:83-86. 166. Nasrallah HA. Atypical antipsychotic-induced metabolic side effects: insights from receptor-binding profiles. Mol Psychiatry. 2008;13:27-35. 167. Kapur S, Arenovich T, Agid O, Zipursky R, Lindborg S, Jones B. Evidence for onset of antipsychotic effects within the first 24 hours of treatment. Am J Psychiatry. 2005;162:939-946. 168. Agid O, Kapur S, Arenovich T, Zipursky RB. Delayed-onset hypothesis of antipsychotic action: a hypothesis tested and rejected. Arch Gen Psychiatry. 2003;60:1228-1235. 169. Casey DE. Tardive dyskinesia and atypical antipsychotic drugs. Schizophr Res. 1999;35 (suppl):S61-S66. 170.Jones PB, Barnes TR, Davies L, et al. Randomized controlled trial of the effect on Quality of Life of second- vs first-generation antipsychotic drugs in schizophrenia: Cost Utility of the Latest Antipsychotic Drugs in Schizophrenia Study (CUtLASS 1). Arch Gen Psychiatry. 2006;63:1079-1087. 171. Eder U, Mangweth B, Ebenbichler C, et al. Association of olanzapineinduced weight gain with an increase in body fat. Am J Psychiatry. 2001;158:1719-1722. 172. Fumagalli F, Frasca A, Sparta M, Drago F, Racagni G, Riva MA. Longterm exposure to the atypical antipsychotic olanzapine differently up-regulates extracellular signal-regulated kinases 1 and 2 phosphorylation in subcellular compartments of rat prefrontal cortex. Mol Pharmacol. 2006;69:1366-1372. 173. Browning JL, Patel T, Brandt PC, Young KA, Holcomb LA, Hicks PB. Clozapine and the mitogen-activated protein kinase signal transduction pathway: implications for antipsychotic actions. Biol Psychiatry. 2005;57:617-623. 174.Lu XH, Bradley RJ, Dwyer DS. Olanzapine produces trophic effects in vitro and stimulates phosphorylation of Akt/PKB, ERK1/2, and the mitogenactivated protein kinase p38. Brain Res. 2004;1011:58-68. 175.Lu XH, Dwyer DS. Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via PI3K/AKT, ERK, and pertussis toxin-sensitive pathways. J Mol Neurosci. 2005;27:43-64. 176. Bai O, Zhang H, Li XM. Antipsychotic drugs clozapine and olanzapine upregulate bcl-2 mRNA and protein in rat frontal cortex and hippocampus. Brain Res. 2004;1010:81-86. 177. Engl J, Laimer M, Niederwanger A, et al. Olanzapine impairs glycogen synthesis and insulin signaling in L6 skeletal muscle cells. Mol Psychiatry. 2005;10:1089-1096. 178. Pozzi L, Hakansson K, Usiello A, et al. Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum. J Neurochem. 2003;86:451-459.

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179.Lipska BK, Khaing ZZ, Weickert CS, Weinberger DR. BDNF mRNA expression in rat hippocampus and prefrontal cortex: effects of neonatal ventral hippocampal damage and antipsychotic drugs. Eur J Neurosci. 2001;14:135144. 180.Chlan-Fourney J, Ashe P, Nylen K, Juorio AV, Li XM. Differential regulation of hippocampal BDNF mRNA by typical and atypical antipsychotic administration. Brain Res. 2002;954:11-20. 181.Bai O, Chlan-Fourney J, Bowen R, Keegan D, Li XM. Expression of brainderived neurotrophic factor mRNA in rat hippocampus after treatment with antipsychotic drugs. J Neurosci Res. 2003;71:127-131. 182.Pillai A, Terry AV, Jr, Mahadik SP. Differential effects of long-term treatment with typical and atypical antipsychotics on NGF and BDNF levels in rat striatum and hippocampus. Schizophr Res. 2006;82:95-106. 183.Dean CE. Antipsychotic-associated neuronal changes in the brain: toxic, therapeutic, or irrelevant to the long-term outcome of schizophrenia? Prog Neuropsychopharmacol Biol Psychiatry. 2006;30:174-189. 184.Parikh V, Khan MM, Mahadik SP. Olanzapine counteracts reduction of brain-derived neurotrophic factor and TrkB receptors in rat hippocampus produced by haloperidol. Neurosci Lett. 2004;356:135-139. 185.Mitchell IJ, Cooper AC, Griffiths MR, Cooper AJ. Acute administration of haloperidol induces apoptosis of neurones in the striatum and substantia nigra in the rat. Neuroscience. 2002;109:89-99. 186.Navari S, Dazzan P. Do antipsychotic drugs affect brain structure? A systematic and critical review of MRI findings. Psychol Med. In press. 187.Dorph-Petersen KA, Pierri JN, Perel JM, Sun Z, Sampson AR, Lewis DA. The influence of chronic exposure to antipsychotic medications on brain size before and after tissue fixation: a comparison of haloperidol and olanzapine in macaque monkeys. Neuropsychopharmacology. 2005;30:1649-1661. 188.Chakos MH, Shirakawa O, Lieberman J, Lee H, Bilder R, Tamminga CA. Striatal enlargement in rats chronically treated with neuroleptic. Biol Psychiatry. 1998;44:675-684. 189.Bai O, Wei Z, Lu W, Bowen R, Keegan D, Li XM. Protective effects of atypical antipsychotic drugs on PC12 cells after serum withdrawal. J Neurosci Res. 2002;69:278-83. 190.Qing H, Xu H, Wei Z, Gibson K, Li XM. The ability of atypical antipsychotic drugs vs. haloperidol to protect PC12 cells against MPP+-induced apoptosis. Eur J Neurosci. 2003;17:1563-1570. 191.Yulug B, Yildiz A, Hudaoglu O, Kilic E, Cam E, Schabitz WR. Olanzapine attenuates brain damage after focal cerebral ischemia in vivo. Brain Research Bulletin. 2006;71:296-300. 192.Bian Q, Kato T, Monji A, et al. The effect of atypical antipsychotics, perospirone, ziprasidone and quetiapine on microglial activation induced by interferon-gamma. Prog Neuropsychopharmacol Biol Psychiatry. 2007;32:4248. 193.Yulug B, Yildiz A, Guzel O, Kilic E, Schabitz WR, Kilic E. Risperidone attenuates brain damage after focal cerebral ischemia in vivo. Brain Research Bulletin. 2006;69:656-659. 194.Xu H, Qing H, Lu RB, et al. Quetiapine attenuates the immobilization stress-induced decrease of brain-derived neurotrophic factor expression in rat hippocampus. Neurosci Lett. 2002;321:65-68. 195.Dawirs RR, Hildebrandt K, Teuchert-Noodt G. Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. J Neural Transm. 1998;105:317-327. 196.Halim ND, Weickert CS, McClintock BW, Weinberger DR, Lipska BK. Effects of chronic haloperidol and clozapine treatment on neurogenesis in the adult rat hippocampus. Neuropsychopharmacology. 2004;29:1063-1069. 197.Wang HD, Dunnavant FD, Jarman T, Deutch AY. Effects of antipsychotic drugs on neurogenesis in the forebrain of the adult rat. Neuropsychopharmacology. 2004;29:1230-1238. 198.Kippin TE, Kapur S, van der Kooy D. Dopamine specifically inhibits forebrain neural stem cell proliferation, suggesting a novel effect of antipsychotic drugs. J Neurosci. 2005;25:5815-23. 199.Luo C, Xu H, Li XM. Quetiapine reverses the suppression of hippocampal neurogenesis caused by repeated restraint stress. Brain Res. 2005;1063:3239. 200.Heimberg C, Komoroski RA, Lawson WB, Cardwell D, Karson CN. Regional proton magnetic resonance spectroscopy in schizophrenia and exploration of drug effect. Psychiatry Res. 1998;83:105-115. 201.Braus DF, Ende G, Weber-Fahr W, Demirakca T, Tost H, Henn FA. Functioning and neuronal viability of the anterior cingulate neurons following antipsychotic treatment: MR-spectroscopic imaging in chronic schizophrenia. Eur Neuropsychopharmacol. 2002;12:145-152. 202.Bertolino A, Callicott JH, Mattay VS,, et al. The effect of treatment with antipsychotic drugs on brain N-acetylaspartate measures in patients with schizophrenia. Biol Psychiatry. 2001;49:39-46. 203.Lawrie SM, Abukmeil SS. Brain abnormality in schizophrenia. A systematic and quantitative review of volumetric magnetic resonance imaging studies. Br J Psychiatry. 1998;172:110-120. 204.Lieberman JA. Is schizophrenia a neurodegenerative disorder? A clinical and neurobiological perspective. Biol Psychiatry. 1999;46:729-739. 205.Lieberman JA, Tollefson GD, Charles C, et al. Antipsychotic drug effects on brain morphology in first-episode psychosis. Arch Gen Psychiatry. 2005;62:361-370. 206. DelBello MP, Cecil KM, Adler CM, Daniels JP, Strakowski SM. Neurochemical effects of olanzapine in first-hospitalization manic adolescents: a proton magnetic resonance spectroscopy study. Neuropsychopharmacology. 2006;31:1264-1273.

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Brief report
Neuroplasticity in addictive disorders
Charles P. OBrien, MD, PhD

Compulsive drug-taking behavior develops in vulnerable individuals who ingest substances that activate the reward system. This intense activation produces learned associations to cues that predict drug availability. With repetition the reward system becomes reflexively activated by cues alone, leading to a drive toward drug-taking. The central nervous system changes underlying this conditioned behavior are just beginning to be understood. New treatments aimed at this neuroplasticity are being tested in animal models. The clinical significance of these brain changes is that addiction, once established, becomes a chronic illness with relapses and remissions. It therefore requires chronic treatment with medications and behavioral therapies based on an understanding of the fundamental nature of these changes in the brain.
2009, LLS SAS

Dialogues Clin Neurosci. 2009;11:350-353.

Keywords: addiction; dependence; memory; learning; relapse; tolerance; sensitization; conditioning; reinforcement Author affiliations: Department of Psychiatry, University of Pennsylvania, Treatment Research Center, Philadelphia, Pennsylvania, USA Address for correspondence: Prof Charles P. O'Brien, University of Pennsylvania, Department of Psychiatry, Treatment Research Center, 3900 Chestnut Street, Philadelphia, PA 19104-6178, USA (e-mail: obrien@mail.trc.upenn.edu) Copyright 2009 LLS SAS. All rights reserved

ddiction is a disease of neuroplasticity. In the past, clinicians considered detoxification to be the treatment for addiction. However, detoxification is simply removal of the drug from the body and treatment of withdrawal symptoms. Now we know that the essence of addiction continues long after the last dose of the drug, often lasting for years. This was first demonstrated in animal models, and later shown in human addicts more than 30 years ago.1 Addiction is fundamentally a memory trace that manifests itself by reflex activation of brain circuits, especially the reward system, resulting in motivation to resume drug-taking behavior when drugrelated cues are encountered. Drugs that activate the reward system carry liability for the development of addiction, but vulnerability to this disorder is influenced by complex genetic and environmental variables. A characteristic of all drugs that are abused by humans is that they activate dopamine circuits in brain reward systems by a variety of mechanisms. This has been demonstrated directly in animal models and indirectly in human brain imaging studies. Other neurotransmitters are also involved, but dopamine has received the greatest attention. While a given drug of abuse will tend to have very similar immediate effects in all users, only a minority of users progress to the stage of compulsive use or addiction. Two general forms of neuroplasticity can be demonstrated. The first, and most common, is tolerance accompanied by physical dependence. Tolerance is manifested by reduced effects from a given dose that is given repeatedly, and physical dependence (not addiction) is manifested by withdrawal symptoms when the drug is stopped abruptly. This form of plasticity occurs in all individuals when cer-

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tain drugs are taken repeatedly. Examples include prescribed medications such as -blockers, antidepressants, sedatives, and opioids for pain, as well as commonly abused drugs such as alcohol, cocaine, and nicotine. The second form of neuroplasticity is manifested by compulsive drug-seeking behavior. The reward system, which developed early in evolution, reinforces adaptive behavior such as that leading to the acquisition of water, food, and sex. Drugs that directly activate the reward system may produce learning that diverts the individual to those behaviors that repeat the drug-induced feelings of reward. An important feature of this form of neuroplasticity is that it is stable and perhaps permanent. The dopamine release caused by a drug of abuse tends to be greater than that of natural rewards, and to continue with repeated exposure rather than diminish, as is the case with natural, expected rewards.2 Thus, the drug experience becomes associated with environmental cues and acquires increasing salience. Individuals who develop this neuroplasticity tend to suffer from a chronic illness with potential for relapse, even years after the last dose of the drug. Drug-taking then acquires more salience than natural or adaptive behaviors. Evidence of the plasticity that has occurred with the development of addiction can be demonstrated by brain imaging studies that show rapid activation (increased blood flow to reward pathways) when drug-related cues are shown to addicts who have been free of drugs for at least a month.3 Even cues so brief that they do not reach consciousness (33 msec) can produce rapid activation.4 During brain reward system activation, the addict reports drug craving. The strength of the craving is related directly to the amount of endogenous dopamine released in reward structures, as measured by displacement of labeled raclopride in positron emission tomography (PET) studies.5 More direct studies of the plasticity induced by drugs of addiction can be seen in animal models. Shaham and colleagues have studied the relapse or reinstatement of drug-taking in rats trained to self-administer intravenous cocaine.6 Availability of cocaine is signaled by a light that the animal then associates with cocaine. After the behavior is well trained, the cocaine can be turned off; thus, pushing the lever no longer provides cocaine. After the extinction process is complete, the animal can be tested for reinstatement by returning it to the drug-taking environment and giving the light cue. This is considered to be a model of relapse in human addicts. The intensity

of relapse can be measured by the number of times the light causes the rat to press the bar despite not receiving any cocaine. Eventually, the unrewarded bar pressing stops. It was found that reinstatement occurred when rats were tested 1 week after extinguishing cocaine-seeking, but the reinstatement was significantly greater at 4 weeks, and progressively increased further if the rats were allowed to rest in their cages for up to 6 months before relapse testing. The strengthening of relapse tendency over time has been called incubation and is associated with increases in the levels of the growth factor brain-derived neurotrophic factor (BDNF) in the ventral tegmental area and in the nucleus accumbens. The authors also found that exposure to cocaine cues increased extracellular signal-related kinase (ERK) in central amygdala after 30 days of rest, but not after 1 day. This shows that there is an active neuroplastic process in the brain that increases over time and is manifested by increased cocaine-seeking behavior. Transcription factors have been observed to be changed by addictive drugs. Delta Fos B accumulates in dopamine terminals in the cortex and striatum.7 All drugs of abuse tested produce an increase in delta Fos B, which appears to be involved in the development of motivated behaviors. Disruption of this process blocks the development of drug-associated plasticity such as behavioral sensitization. The latter is the increase in motor behavior in response to repeated, fixed doses of a stimulant.8 Genes directly regulated by delta Fos B appear to have different effects and may limit as well as promote drug reinforcement. The delta Fos B changes are temporary, with return to prior levels when the drug is no longer present. Thus, these transcription factor changes do not seem to underlie long-term neuroplasticity. Changes in neuronal morphology have also been noted in animals exposed to drugs that are abused. In the nucleus accumbens, an increase in dendritic spine density has been reported in medium spiny neurons from rats self-administering cocaine. These changes persisted during abstinence, and may be involved in long-term changes associated with drug-seeking behavior.9 Changes in neuronal morphology have also been found in individuals chronically exposed to opioids. Chronic morphine given to rats, for example, has been found to reduce dendritic spines (whereas stimulants increased spines) on ventral tegmental area neurons. Chronic morphine also reduces neurogenesis in the hippocampus.10 These changes may be the basis for the cognitive losses
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seen in some patients receiving chronic opioids for pain. Since the learned addictive behavior is thought to result from neuroplasticity such as that described above, it seems logical to consider reversal of these changes as a target for the treatment of addictive behaviors. A very interesting animal model of this approach has been illustrated by a series of experiments by Kalivas et al. Using rats trained to selfadminister cocaine, they reported a reduction in glutamate in the brains of animals exposed to long-term cocaine and a disruption of glutamate homeostasis. Following withdrawal from chronic cocaine there is a marked imbalance in glutamate homeostasis, with both cystine-glutamate exchange and glutamate uptake being reduced in the nucleus accumbens.11 The imbalance in glutamate homeostasis is associated with a reduction in basal extracellular glutamate levels and a potentiated release of synaptic glutamate during drug-seeking.12 In addition, there is a basal increase in the -amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) to N-methyl-D-aspartic acid (NMDA) current ratio and a loss of both long-term potentiation (LTP) and long-term depression (LTD).13 There are therapeutic implications in these observations on glutamate homeostasis. Cystine can be administered to animals withdrawn from chronic cocaine using Nacetylcysteine as a carrier, or glutamate uptake can be increased by the antibiotic ceftriaxone. By restoring the glutamate homeostasis in this manner, reinstatement of cocaine seeking is prevented. The treated animals also show a restored ability to induce LTP and LTD, as well as a normalization of the AMPA:NMDA ratio. The treatment also prevents changes in spine head diameter induced during cocaine-seeking.13 Taken together, the data above suggest the possibility that normalization of glutamate homeostasis in addicts might restore the ability to induce synaptic plasticity in the nucleus accumbens, which in turn could facilitate establishing behaviors that might compete with drugseeking. Exogenous N-acetyl cysteine is used for the treatment of hepatic failure in acetaminophen overdose. Thus, it was available to be administered to cocaine addicts presented with cocaine-related cues in an attempt to translate findings in the animal model to human addicts. Those treated with N-acetyl cysteine reported reduced desire for cocaine compared with the control group.14 In another human study, N-acetyl cysteine was found to reduce pathological gambling15 and cigarette smoking.16 Further clinical trials are in progress. Another attempt to reverse the learned behaviors seen in addiction involves a new technology of real-time functional magnetic resonance imaging biofeedback of brain activity.17 Addicts have been shown to have poor ability to inhibit impulses, and this correlates with decreased frontal lobe activity. Normal subjects can activate frontal control mechanisms when attempting to inhibit sexual arousal, but cocaine-dependent patients are unable to inhibit craving when shown drug-related stimuli. By providing feedback of frontal activation, the patients will attempt to learn to activate inhibitory structures and inhibit drug craving. This represents a therapeutic attempt to introduce new learning to control addictive behavior. The continued study of the underlying mechanisms of plasticity will undoubtedly produce other novel pharmacological and behavioral treatments.

Copyright 2009 LLS SAS. All rights reserved

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Neuroplasticidad en los trastornos adictivos

La conducta compulsiva de consumir drogas se desarrolla en individuos vulnerables que ingieren sustancias que activan el sistema de recompensa. Esta intensa activacin produce asociaciones aprendidas frente a seales que predicen la disponibilidad de droga. Con la repeticin, el sistema de recompensa se activa en forma refleja slo por las seales, favoreciendo el camino hacia el consumo de drogas. Recin se estn comenzando a comprender los cambios del sistema nervioso central que subyacen a esta conducta condicionada. En modelos animales se estn evaluando nuevos tratamientos orientados a esta neuroplasticidad. El significado clnico de estos cambios cerebrales es que la adiccin, una vez establecida, llega a ser una enfermedad crnica con recadas y remisiones. Esta patologa requiere por lo tanto de un tratamiento crnico con medicamentos y terapias conductuales basadas en una comprensin de la naturaleza fundamental de estos cambios en el cerebro.

Neuroplasticit et troubles addictifs

Les comportements compulsifs de prise de substance se dveloppent chez des individus vulnrables qui ingrent des substances activant le systme de rcompense. Cette activation intense entrane des associations des signaux qui indiquent la disponibilit de la drogue par un phnomne dapprentissage. Avec les rptitions du processus, le systme de rcompense sactive de faon rflexe par les seuls signaux, provoquant une pulsion de prise de drogue. Les modifications du systme nerveux central qui sous-tendent ce comportement conditionn commencent seulement tre comprises. De nouveaux traitements ciblant cette neuroplasticit sont en train dtre tests dans des modles animaux. Ces modifications crbrales signifient cliniquement que laddiction, une fois installe, se comporte comme une maladie chronique avec rechutes et rmissions. Ceci ncessite donc un traitement chronique avec des mdicaments et des thrapies comportementales bases sur la comprhension de la nature fondamentale de ces modifications crbrales.
10. Eisch AJ, Barrot M, Schad, CA, Self DW, Nestler EJ. Opiates inhibit neurogenesis in the adult rodent dentate gyrus. Proc Natl Acad Sci U S A. 2000;97:7579-7584. 11. Baker DA, McFarland K, Lake RW, et al. Neuroadaptations in cystine glutamate exchange underlie cocaine relapse. Nat Neurosci. 2003;6:743 749. 12. McFarland K, Lapish CC, Kalivas PW. Prefrontal glutamate release into the core of the nucleus accumbens mediates cocaine-induced reinstatement of drug-seeking behavior. J Neurosci. 2003;23:35313537. 13. Shen H-W, Toda S, Moussawi K, Bouknight A, Zahm DS, Kalivas PW. Altered dendritic spine plasticity in cocaine-withdrawn rats. J Neurosci. 2009;29;2876-2884. 14. LaRowe SD, Myrick H, Hedden S, et al. Is cocaine desire reduced by N-acetylcysteine? Am J Psych. 2007;164:1115-1117. 15. Grant JE, Kim SW, Odlaug BL. N-acetyl cysteine, a glutamate-modulating agent, in the treatment of pathological gambling: a pilot study. Biol Psych. 2007;62:652-657. 16. Knackstedt LA, LaRowe S, Mardikian P, et al. The role of cystine-glutamate exchange in nicotine dependence in rats and humans. Biol Psych. 2008;65;841-845. 17. deCharms RC. Applications of real-time fMRI. Nat Rev Neurosci. 2008;9:720-729.

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4. Peripheral and Central Nervous System Advisory Committee. Meeting Documents. Available at: http://www.fda.gov/ohrms/dockets/ac/cder01.htm. Rockville, Md: Food and Drug Administration. Accessed October 21, 2004.

Presentation at a conference:
5. McGlashan TH, Zipursky RB, Perkins DO, et al. Olanzapine versus placebo for the schizophrenic prodrome: 1-year results. Paper presented at: 156th Annual Meeting of the American Psychiatric Association; May 17-22, 2003; San Francisco, Calif.

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Submission: Manuscripts may be submitted by e-mail (mail.dialneuro@fr.netgrs.com) or on a CD, double-spaced, with 1-inch/2.5-cm margins. All pages should be numbered. All corresponding authors should supply a black-and-white portrait photograph for inclusion on the Contributors page at the beginning of the issue. This may be sent by e-mail, provided the resolution of the file is at least 300 dpi. Title page: The title page should include a title, the full names of all the authors, the highest academic degrees of all authors (in country-oforigin language), affiliations (names of department[s] and institution[s] at the time the work was done), a short running title (no more than 50 letters and spaces), 5 to 10 keywords, the corresponding authors complete mailing address, telephone, fax, and e-mail, and acknowledgments. Abstract: A 150-word abstract should be provided for all articles. The editorial department will edit abstracts that are too short or too long. Abstracts will be translated into French and Spanish by the publishers editorial department. Authors who are native French or Spanish speakers may choose to provide an abstract in their own language, as well as an English abstract. Text: All texts should be submitted in English. Abbreviations should be used sparingly and expanded at first mention. A list of selected abbreviations and acronyms should be provided (or will be prepared by the editorial department) where necessary. The style of titles and subtitles should be consistent throughout the text. The editorial department reserves the right to add, modify, or delete headings if necessary. Dialogues in Clinical Neuroscience uses SI units and generic names of drugs.

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Figures should be of good quality or professionally prepared, with the proper orientation indicated when necessary (eg, top or left). As figures and graphs may need to be reduced or enlarged, all absolute values and statistics should be provided. Provide each table and figure on a separate sheet. Legends must be provided with all illustrations, including expansion of all abbreviations used (even if they are already defined in the text). All figures and tables should be numbered and cited in the text.

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Editorial: 400 words. State of the art: 7000 words. Pharmacological aspects; Clinical research; Basic research/Translational research: 3000-5000 words. Free papers: 2500 words. Brief report: 1500 words. May be extensively illustrated, according to the topic and the specifications of the Coordinating Editor. Legends must be provided with all illustrations.

EDITORIAL ASSESSMENT AND PROCESSING

Peer review: All contributions to Dialogues in Clinical Neuroscience will be reviewed by members of the Editorial Board and submitted to expert consultants for peer review. All contributions should be original review articles. Editorial processing: All manuscripts are copyedited according to the guidelines of the latest edition of the American Medical Association Manual of Style (Baltimore, Md: Williams & Wilkins); the spelling used is American (reference dictionaries: latest editions of Merriam-Websters Collegiate Dictionary and Stedmans Medical Dictionary). Proofs: Page proofs will be sent to the corresponding author for approval in PDF format by e-mail. Authors who wish to receive a hard copy of their proofs should contact the editorial offices upon receipt of the proofs by e-mail. Author corrections should be returned within 48 hours by e-mail or fax. If this deadline is not met, the editorial department will assume that the author accepts the proofs as they stand. Authors are responsible for all statements made in their work, including changes made by the editorial department and authorized by the author.

REFERENCES
Citation in text: All references should be cited in the text and numbered consecutively using superscript Arabic numerals. Reference list: Presentation of the references should be based on the Uniform Requirements for Manuscripts Submitted to Biomedical Journals. Ann Intern Med. 1997;126:36-47 (Vancouver style). The authordate system of citation is not acceptable. In press references should be avoided. Titles of journals should be abbreviated according to Index Medicus. All authors should be listed for up to six authors; if there are more, only the first three should be listed, followed by et al. Where necessary, references will be styled by the editorial department to Dialogues in Clinical Neuroscience copyediting requirements. Authors bear total responsibility for the accuracy and completeness of all references and for correct text citation. Examples of style for references: Journal article:
1. Heinssen RK, Cuthbert BN, Breiling J, Colpe LJ, Dolan-Sewell R. Overcoming barriers to research in early serious mental illness: issues for future collaboration. Schizophr Bull. 2003;29:737-745.

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Transfer of copyright: Copyright of articles will be transferred to the publisher of Dialogues in Clinical Neuroscience. The Copyright Transfer Agreement must be signed by all authors and returned to the publisher. Permissions: The author should inform the editorial office if any of the figures or tables are reproduced from elsewhere. For reproduction of copyrighted work, the editorial office will obtain authorization from the publisher concerned. Requests for permission to reproduce material published in Dialogues in Clinical Neuroscience should be sent directly to the editorial office (mail.dialneuro@fr.netgrs.com).

Article in a supplement:
2. Greenamyre JT, Betarbet R, Sherer TB. The rotenone model of Parkinsons disease: genes, environment and mitochondria. Parkinsonism Relat Disord. 2003;9(suppl 2):S59-S64.

Chapter in a book:
3. Carpenter WT Jr, Buchanan RW. Domains of psychopathology relevant to the study of etiology and treatment in schizophrenia. In: Schulz SC, Tamminga CA, eds. Schizophrenia: Scientific Progress. New York, NY: Oxford University Press; 1989:13-22.

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Dialogues in Clinical Neuroscience - Vol 11 . No. 3 . 2009

18. Dubal DB, Zhu H, Yu J, et al. Estrogen receptor alpha, not beta is a critical link in estradiol-mediated protection against brain injury. Proc Natl Acad Sci U S A. 2001;98:1952-1957. 19. Vegeto E, Benedusi V, Maggi A. Estrogen anti-inflammatory activity in brain: A therapeutic opportunity for menopause and neurodegenerative diseases. Front Neuroendocrinol. 2008;29:507-519. 20. Toran-Allerand CD. Minireview: a plethora of estrogen receptors in the brain: where will it end? Endocrinology. 2004;145:1069-1074. 21. Morissette M, Le Saux M, D'Astous M, et al. Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain. J Steroid Biochem Mol Biol. 2008;108:327-338. 22. Dhandapani KM, Brann DW. Role of astrocytes in estrogen-mediated neuroprotection. Exp Gerontol. 2007;42:70-75. 23. Butts CL, Sternberg EM. Neuroendocrine factors alter host defense by modulating immune function. Cell Immunol. 2008;252:7-15.

24. Vegeto E, Belcredito S, Etteri S, et al. Estrogen receptor-a mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci U S A. 2003;100:9614-9619. 25. Vegeto E, Belcredito S, Ghisletti S, Meda C, Etteri S, Maggi A. The endogenous estrogen status regulates microglia reactivity in animal models of neuroinflammation. Endocrinology. 2006;147:2263-2272. 26. Kadhim HJ, Duchateau J, Sebire G. Cytokines and brain injury: invited review. J Intensive Care Med. 2008;23:236-249. 27. Czlonkowska A, Ciesielska A, Gromadzka G, Kurkowska-Jastrzebska I. Estrogen and cytokines production - the possible cause of gender differences in neurological diseases. Curr Pharm Des. 2005;11:1017-1030. 28. Suzuki S, Gerhold LM, Bottner M, et al. Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta. J Comp Neurol. 2007;500:1064-1075. 29. McCullough LD, Hurn PD. Estrogen and ischemic neuroprotection: an integrated view. Trends Endocrinol Metab. 2003;14:228-235.

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