A major impediment to novel drug development has been the paucity of animal models that accurately reflect symptoms of affective disorders. In animal models, prolonged social stress has proven to be useful in understanding the molecular mechanisms underlying affective-like disorders. When considering experimental approaches for studying depression, social defeat stress, in particular, has been shown to have excellent etiological, predictive, discriminative and face validity. Described here is a protocol whereby C57BL/6J mice that are repeatedly subjected to bouts of social defeat by a larger and aggressive CD-1 mouse results in the development of a clear depressive-like syndrome, characterized by enduring deficits in social interactions. Specifically, the protocol consists of three important stages, beginning with the selection of aggressive CD-1 mice, followed by agonistic social confrontations between the CD-1 and C57BL/6J mice, and concluding with the confirmation of social avoidance in subordinate C57BL/6J mice. The automated detection of social avoidance allows a marked increase in throughput, reproducibility and quantitative analysis. This protocol is highly adaptable, but in its most common form it requires 3–4 weeks for completion.

The nucleus accumbens is a key mediator of cocaine reward, but the distinct roles of the two subpopulations of nucleus accumbens projection neurons, those expressing dopamine D1 vs. D2 receptors, are poorly understood. We show that deletion of TrkB, the brain-derived neurotrophic factor (BDNF) receptor, selectively from D1+ or D2+ neurons oppositely affects cocaine reward. Since loss of TrkB in D2+ neurons increases their neuronal excitability, we next used optogenetic tools to control selectively the firing rate of D1+ and D2+ nucleus accumbens neurons and studied consequent effects on cocaine reward. Activation of D2+ neurons, mimicking the loss of TrkB, suppresses cocaine reward, with opposite effects induced by activation of D1+ neurons. These results provide insight into the molecular control of D1+ and D2+ neuronal activity as well as the circuit level contribution of these cell types to cocaine reward.The nucleus accumbens (NAc) plays a crucial role in mediating the rewarding effects of drugs of abuse (1). However, little is known about the specific function of the two major populations of NAc projection neurons, which together comprise >95% of all NAc neurons, in regulating these behaviors. These neurons, like those in the dorsal striatum, are medium spiny neurons (MSNs) divided into two subtypes based on their distinct projections through cortical-basal ganglia circuits and their differential gene expression, including enrichment of dopamine D1 vs. D2 receptors (2). These two MSN subtypes, in dorsal striatum, exert balanced but antagonistic influences on their downstream outputs and behaviors, most notably motor behaviors (3-5), but their role, in NAc, in regulating reward behaviors still needs to be determined.While activation of both D1 and D2 receptors contributes to the rewarding effects of cocaine (6), current biochemical evidence has focused primarily on cocaine-induced molecular and structural changes in D1+ MSNs (7-11). For example, the extracellular signal-regulated kinase (ERK) pathway is induced in D1+ MSNs after cocaine exposure (8), an effect thought to be mediated directly via activation of D1 receptors (12,13). However, ERK activation by cocaine may occur through other mechanisms, such as brain-derived

Previous work has identified alterations in histone acetylation in animal models of drug addiction and depression. However, the mechanisms which integrate drugs and stress with changes in chromatin structure remain unclear. Here, we identify the activity-dependent class II histone deacetylase, HDAC5, as a central integrator of these stimuli with changes in chromatin structure and gene expression. Chronic, but not acute, exposure to cocaine or stress decreases HDAC5 function in the nucleus accumbens (NAc), a major brain reward region, which allows for increased histone acetylation and transcription of HDAC5 target genes. This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, not acute, cocaine or stress. These findings suggest that proper balance of histone acetylation is a crucial factor in the saliency of a given stimulus and that disruption of this balance is involved in the transition from an acute adaptive response to a chronic psychiatric illness.

Persistent symptoms of depression suggest the involvement of stable molecular adaptations in brain, which may be reflected at the level of chromatin remodeling. We find that chronic social defeat stress in mice causes a transient decrease, followed by a persistent increase, in levels of acetylated histone H3 in the nucleus accumbens, an important limbic brain region. This persistent increase in H3 acetylation is associated with decreased levels of histone deacetylase 2 (HDAC2) in the nucleus accumbens. Similar effects were observed in the nucleus accumbens of depressed humans studied postmortem. These changes in H3 acetylation and HDAC2 expression mediate long-lasting positive neuronal adaptations, since infusion of HDAC inhibitors into the nucleus accumbens, which increases histone acetylation, exerts robust antidepressant-like effects in the social defeat paradigm and other behavioral assays. HDAC inhibitor [N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275)] infusion also reverses the effects of chronic defeat stress on global patterns of gene expression in the nucleusaccumbens, as determined by microarray analysis, with striking similarities to the effects of the standard antidepressant fluoxetine. Stress-regulated genes whose expression is normalized selectively by MS-275 may provide promising targets for the futuredevelopmentofnovelantidepressanttreatments.Together,thesefindingsprovidenewinsightintotheunderlyingmolecularmechanisms of depression and antidepressant action, and support the antidepressant potential of HDAC inhibitors and perhaps other agents that act at the level of chromatin structure.

Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. We identified an essential role for histone 3 lysine 9 (H3K9) dimethylation and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduced global levels of H3K9 dimethylation in the nucleus accumbens. This reduction in histone methylation was mediated through the repression of G9a in this brain region, which was regulated by the cocaine-induced transcription factor ΔFosB. Using conditional mutagenesis and viral-mediated gene transfer, we found that G9a downregulation increased dendritic spine plasticity of nucleus accumbens neurons and enhanced preference for cocaine, thereby establishing a crucial role for histone methylation in the long-term actions of cocaine.Repeated cocaine exposure is characterized by persistent changes in gene expression and altered neuronal morphology within the nucleus accumbens (NAc), a key component of the brain's reward circuitry (1-2). Chromatin remodeling is important in aberrant transcriptional changes in this brain region that may underlie aspects of cocaine addiction (3-9). Cocaine regulation of chromatin structure in the NAc results, in part, from direct cocaine-induced modifications of the chromatin enzymatic machinery, leading to changes in histone acetylation *To whom correspondence should be addressed. eric.nestler@mssm.edu. I certify that none of the materials included within the manuscript entitled Essential Role of the Histone Methyltransferase G9a in Cocaineinduced Plasticity have been previously published or are under consideration elsewhere, including on the Internet.All work involving the use of animals was conducted in accordance with institutional and IACUC guidelines at both The

Despite abundant expression of DNA methyltransferases (Dnmt’s) in brain, the regulation and behavioral role of DNA methylation remain poorly understood. We find that Dnmt3a expression is regulated in mouse nucleus accumbens (NAc) by chronic cocaine and chronic social defeat stress. Moreover, NAc specific manipulations that block DNA methylation potentiate cocaine reward and exert antidepressant-like effects, whereas NAc specific Dnmt3a overexpression attenuates cocaine reward and is pro-depressant. On a cellular level, we show that chronic cocaine selectively increases thin dendritic spines on NAc neurons and that DNA methylation is both necessary and sufficient to mediate these effects. These data establish the importance of Dnmt3a in the NAc in regulating cellular and behavioral plasticity to emotional stimuli.

Brain stimulation and imaging studies in humans have highlighted a key role for the prefrontal cortex in clinical depression; however, it remains unknown whether excitation or inhibition of prefrontal cortical neuronal activity is associated with antidepressant responses. Here, we examined cellular indicators of functional activity, including the immediate early genes (IEGs) zif268 (egr1), c-fos, and arc, in the prefrontal cortex of clinically depressed humans obtained postmortem. We also examined these genes in the ventral portion of the medial prefrontal cortex (mPFC) of mice after chronic social defeat stress, a mouse model of depression. In addition, we used viral vectors to overexpress channel rhodopsin 2 (a light-activated cation channel) in mouse mPFC to optogenetically drive "burst" patterns of cortical firing in vivo and examine the behavioral consequences. Prefrontal cortical tissue derived from clinically depressed humans displayed significant reductions in IEG expression, consistent with a deficit in neuronal activity within this brain region. Mice subjected to chronic social defeat stress exhibited similar reductions in levels of IEG expression in mPFC. Interestingly, some of these changes were not observed in defeated mice that escape the deleterious consequences of the stress, i.e., resilient animals. In those mice that expressed a strong depressive-like phenotype, i.e., susceptible animals, optogenetic stimulation of mPFC exerted potent antidepressant-like effects, without affecting general locomotor activity, anxiety-like behaviors, or social memory. These results indicate that the activity of the mPFC is a key determinant of depression-like behavior, as well as antidepressant responses.

In contrast to the vast literature on stress effects on the brain, relatively little is known about the molecular mechanisms of resilience, the ability of some individuals to escape the deleterious effects of stress. Here we show that the transcription factor, ΔFosB, mediates an essential mechanism of resilience in mice. Induction of ΔFosB in the nucleus accumbens, a key brain reward region, in response to chronic social defeat stress is both necessary and sufficient for resilience. ΔFosB induction also is required for the ability of the standard antidepressant, fluoxetine, to reverse behavioral pathology induced by social defeat. ΔFosB produces these effects through the induction of the GluR2 AMPA glutamate receptor subunit, which decreases the responsiveness of nucleus accumbens neurons to glutamate, and through other synaptic proteins. Together, these findings establish a novel molecular pathway underlying both resilience and antidepressant action.