Sam A. Golden

A standardized protocol for repeated social defeat stress in mice

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.

Individual differences in the peripheral immune system promote resilience versus susceptibility to social stress

Significance Depression and anxiety have been linked to increased inflammation. However, we do not know if inflammatory status predates onset of disease or whether it contributes to depression symptomatology. We report preexisting individual differences in the peripheral immune system that predict and promote stress susceptibility. Replacing a stress-naive animal’s peripheral immune system with that of a stressed animal increases susceptibility to social stress including repeated social defeat stress (RSDS) and witness defeat (a purely emotional form of social stress). Depleting the cytokine IL-6 from the whole body or just from leukocytes promotes resilience, as does sequestering IL-6 outside of the brain. These studies demonstrate that the emotional response to stress can be generated or blocked in the periphery, and offer a potential new form of treatment for stress disorders. Depression and anxiety disorders are associated with increased release of peripheral cytokines; however, their functional relevance remains unknown. Using a social stress model in mice, we find preexisting individual differences in the sensitivity of the peripheral immune system that predict and promote vulnerability to social stress. Cytokine profiles were obtained 20 min after the first social stress exposure. Of the cytokines regulated by stress, IL-6 was most highly up-regulated only in mice that ultimately developed a susceptible behavioral phenotype following a subsequent chronic stress, and levels remained elevated for at least 1 mo. We confirmed a similar elevation of serum IL-6 in two separate cohorts of patients with treatment-resistant major depressive disorder. Before any physical contact in mice, we observed individual differences in IL-6 levels from ex vivo stimulated leukocytes that predict susceptibility versus resilience to a subsequent stressor. To shift the sensitivity of the peripheral immune system to a pro- or antidepressant state, bone marrow (BM) chimeras were generated by transplanting hematopoietic progenitor cells from stress-susceptible mice releasing high IL-6 or from IL-6 knockout (IL-6−/−) mice. Stress-susceptible BM chimeras exhibited increased social avoidance behavior after exposure to either subthreshold repeated social defeat stress (RSDS) or a purely emotional stressor termed witness defeat. IL-6−/− BM chimeric and IL-6−/− mice, as well as those treated with a systemic IL-6 monoclonal antibody, were resilient to social stress. These data establish that preexisting differences in stress-responsive IL-6 release from BM-derived leukocytes functionally contribute to social stress-induced behavioral abnormalities.

HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT2A receptor–dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

IκB Kinase Regulates Social Defeat Stress-Induced Synaptic and Behavioral Plasticity

The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase–related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat–induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.Depression involves plasticity of brain reward neurons, although the mechanisms and behavioral relevance are unknown. Transcriptional profiling of nucleus accumbens (NAc) for RhoGTPase related genes, known regulators of synaptic structure, following chronic social defeat stress, revealed a long-term reduction in Rac1 transcription. This was marked by a repressive chromatin state surrounding its proximal promoter. Inhibition of class 1 HDACs with MS-275 rescued both decreased Rac1 transcription and social avoidance behavior. A similar repressive chromatin state was found surrounding the Rac1 promoter in human postmortem NAc from depressed subjects, which corresponded with reduced Rac1 transcription. We show Rac1 is necessary and sufficient for social avoidance and anhedonia, and the formation of stubby excitatory spines by redistributing synaptic cofilin, an actin severing protein downstream of Rac1. Our data identifies epigenetic regulation of Rac1 in NAc as a bona fide disease mechanism in depression and reveals a functional role in regulating stress-related behaviors.

Structural and synaptic plasticity in stress-related disorders

Abstract Stress can have a lasting impact on the structure and function of brain circuitry that results in long-lasting changes in the behavior of an organism. Synaptic plasticity is the mechanism by which information is stored and maintained within individual synapses, neurons, and neuronal circuits to guide the behavior of an organism. Although these mechanisms allow the organism to adapt to its constantly evolving environment, not all of these adaptations are beneficial. Under prolonged bouts of physical or psychological stress, these mechanisms become dysregulated, and the connectivity between brain regions becomes unbalanced, resulting in pathological behaviors. In this review, we highlight the effects of stress on the structure and function of neurons within the mesocorticolimbic brain systems known to regulate mood and motivation. We then discuss the implications of these spine adaptations on neuronal activity and pathological behaviors implicated in mood disorders. Finally, we end by discussing recent brain imaging studies in human depression within the context of these basic findings to provide insight into the underlying mechanisms leading to neural dysfunction in depression.

Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Stress and CRF gate neural activation of BDNF in the mesolimbic reward pathway

Mechanisms controlling release of brain-derived neurotrophic factor (BDNF) in the mesolimbic dopamine reward pathway remain unknown. We report that phasic optogenetic activation of this pathway increases BDNF amounts in the nucleus accumbens (NAc) of socially stressed mice but not of stress-naive mice. This stress gating of BDNF signaling is mediated by corticotrophin-releasing factor (CRF) acting in the NAc. These results unravel a stress context–detecting function of the brains mesolimbic circuit.

Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress

Depression and anxiety disorders are more prevalent in females, but the majority of research in animal models, the first step in finding new treatments, has focused predominantly on males. Here we report that exposure to subchronic variable stress (SCVS) induces depression-associated behaviors in female mice, whereas males are resilient as they do not develop these behavioral abnormalities. In concert with these different behavioral responses, transcriptional analysis of nucleus accumbens (NAc), a major brain reward region, by use of RNA sequencing (RNA-seq) revealed markedly different patterns of stress regulation of gene expression between the sexes. Among the genes displaying sex differences was DNA methyltransferase 3a (Dnmt3a), which shows a greater induction in females after SCVS. Interestingly, Dnmt3a expression levels were increased in the NAc of depressed humans, an effect seen in both males and females. Local overexpression of Dnmt3a in NAc rendered male mice more susceptible to SCVS, whereas Dnmt3a knock-out in this region rendered females more resilient, directly implicating this gene in stress responses. Associated with this enhanced resilience of female mice upon NAc knock-out of Dnmt3a was a partial shift of the NAc female transcriptome toward the male pattern after SCVS. These data indicate that males and females undergo different patterns of transcriptional regulation in response to stress and that a DNA methyltransferase in NAc contributes to sex differences in stress vulnerability. SIGNIFICANCE STATEMENT Women have a higher incidence of depression than men. However, preclinical models, the first step in developing new diagnostics and therapeutics, have been performed mainly on male subjects. Using a stress-based animal model of depression that causes behavioral effects in females but not males, we demonstrate a sex-specific transcriptional profile in brain reward circuitry. This transcriptional profile can be altered by removal of an epigenetic mechanism, which normally suppresses DNA transcription, creating a hybrid male/female transcriptional pattern. Removal of this epigenetic mechanism also induces behavioral resilience to stress in females. These findings shed new light onto molecular factors controlling sex differences in stress response.

Long-lasting incubation of conditioned fear in rats

BACKGROUND In 1937, Diven reported that human fear responses to cues previously paired with shock progressively increase or incubate over 24 hours. Since then, fear incubation has been demonstrated in both humans and nonhumans. However, the difficulty of demonstrating long-lasting fear incubation in rodents has hampered the study of the underlying mechanisms of this incubation. Here, we describe a rat procedure where fear reliably incubates over time. METHODS We trained food-restricted rats to lever-press for food pellets in daily 90-min sessions. We then gave each rat 100 30-sec tones co-terminating with a .5-sec .5-mA footshock over 10 days (10 pairings/day). Groups of rats (n = 10-15) were then given four presentations of the tone (the fear cue) 2, 15, 31, or 61 days after fear conditioning training and were assessed for conditioned suppression of lever-pressing. RESULTS We found that conditioned fear responses were significantly higher 31 and 61 days after fear training than after 2 or 15 days. In control experiments, we showed that extensive tone-shock pairing is necessary for the emergence of fear incubation and that it is unlikely that non-associative factors contribute to this incubation. CONCLUSIONS We describe a procedure for generating reliable and long-lasting conditioned fear incubation. Our procedure can be used to study mechanisms of fear incubation and might provide a model for studying the mechanisms of delayed-onset posttraumatic stress disorder that occur in a sub-population of people previously exposed to chronic stressors.