Georgia E. Hodes

Paternal Transmission of Stress-Induced Pathologies

BACKGROUND There has been recent interest in the possibility that epigenetic mechanisms might contribute to the transgenerational transmission of stress-induced vulnerability. Here, we focused on possible paternal transmission with the social defeat stress paradigm. METHODS Adult male mice exposed to chronic social defeat stress or control nondefeated mice were bred with normal female mice, and their offspring were assessed behaviorally for depressive- and anxiety-like measures. Plasma levels of corticosterone and vascular endothelial growth factor were also assayed. To directly assess the role of epigenetic mechanisms, we used in vitro fertilization (IVF); behavioral assessments were conducted on offspring of mice from IVF-control and IVF-defeated fathers. RESULTS We show that both male and female offspring from defeated fathers exhibit increased measures of several depression- and anxiety-like behaviors. The male offspring of defeated fathers also display increased baseline plasma levels of corticosterone and decreased levels of vascular endothelial growth factor. However, most of these behavioral changes were not observed when offspring were generated through IVF. CONCLUSIONS These results suggest that, although behavioral adaptations that occur after chronic social defeat stress can be transmitted from the father to his male and female F1 progeny, only very subtle changes might be transmitted epigenetically under the conditions tested.

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.

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.

Brain feminization requires active repression of masculinization via DNA methylation.

The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in female rats. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

Neuroimmune mechanisms of depression

Current diagnosis of depression is based solely on behavioral symptomatology. The available US Food and Drug Administration–approved treatments for depression have come from serendipitous discovery and are ineffective in nearly 30–50% of patients, which is thought to reflect a lack of specificity in targeting underlying pathophysiological mechanisms. Recent evidence has identified depression-related disruptions in a neuroimmune axis that interfaces the immune system and CNS to control behavior. This Review examines the evidence in patients and in animal models of depression that demonstrates how the peripheral immune system acts on the brain to alter an individuals response to stress, ultimately contributing to their vulnerability to mood disorders.

Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons.

Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

Distinctive stress effects on learning during puberty

Puberty is a time of significant change in preparation for adulthood. Here, we examined how stressful experience affects cognitive and related hormonal responses in male and female rats prior to, during and after puberty. Groups were exposed to an acute stressor of brief periodic tailshocks and tested 24 h later in an associative memory task of trace eyeblink conditioning. Exposure to the stressor did not alter conditioning in males or females prior to puberty but enhanced conditioning in both males and females during puberty. The enhancement occurred in pubescent females irrespective of the estrous cycle. In adulthood, sex differences in trace conditioning and the response to stress emerged: females outperformed males under unstressed conditions, but after stressor exposure, trace conditioning in females was impaired whereas that in males was enhanced. These differences were not related to changes in gross motor activity or other nonspecific measures of performance. The effects of acute stress on corticosterone, estradiol, progesterone, and testosterone were also measured. Stressor exposure increased the concentration of corticosterone in all age groups, although sex differences were only evident in adults. All reproductive hormones except estradiol increased with age in a predictable and sex dependent fashion and none were affected by stressor exposure. Estradiol decreased in male rats across age, and remained stable for female rats. Together, these data indicate that males and female respond similarly to learning opportunities and stressful experience before and during puberty; it is in adulthood that sex differences and the opposite responses to stress arise.

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.

Prenatal Stress Induces Schizophrenia-Like Alterations of Serotonin 2A and Metabotropic Glutamate 2 Receptors in the Adult Offspring: Role of Maternal Immune System

It has been suggested that severe adverse life events during pregnancy increase the risk of schizophrenia in the offspring. The serotonin 5-HT2A and the metabotropic glutamate 2 (mGlu2) receptors both have been the target of considerable attention regarding schizophrenia and antipsychotic drug development. We tested the effects of maternal variable stress during pregnancy on expression and behavioral function of these two receptors in mice. Prenatal stress increased 5-HT2A and decreased mGlu2 expression in frontal cortex, a brain region involved in perception, cognition, and mood. This pattern of expression of 5-HT2A and mGlu2 receptors was consistent with behavioral alterations, including increased head-twitch response to the hallucinogenic 5-HT2A agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] and decreased mGlu2-dependent antipsychotic-like effect of the mGlu2/3 agonist LY379268 (1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate) in adult, but not prepubertal, mice born to stressed mothers during pregnancy. Cross-fostering studies determined that these alterations were not attributable to effects of prenatal stress on maternal care. Additionally, a similar pattern of biochemical and behavioral changes were observed in mice born to mothers injected with polyinosinic:polycytidylic acid [poly(I:C)] during pregnancy as a model of prenatal immune activation. These data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for schizophrenia and other psychiatric disorders.

Sex-Specific Effects of Chronic Fluoxetine Treatment on Neuroplasticity and Pharmacokinetics in Mice

Neurogenesis is a mechanism through which antidepressants may produce therapeutic effects. There is a dearth of information regarding the effects of antidepressants on neurogenesis and neurotrophin mobilization in females. This study examined sex differences in the alteration of cell proliferation and survival in multiple regions of the brain. Additional experiments examined brain-derived neurotrophic factor (BDNF) levels and pharmacokinetics of fluoxetine to determine whether they mediate sex differences. MRL/MpJ mice were treated with fluoxetine (5 and 10 mg/kg b.i.d.) for 21 days and received injections of 5-bromo-2′-deoxyuridine (200 mg/kg) to measure DNA synthesis. In the hippocampus, fluoxetine increased cell proliferation at both doses; females treated with 10 mg/kg produced more new cells than males. Fluoxetine did not alter survival in males, but 10 mg/kg reduced survival in females. In the frontal cortex, fluoxetine increased cell proliferation and survival in males treated with 10 mg/kg. In the cerebellum and amygdala, 10 mg/kg fluoxetine increased cell proliferation in both sexes but did not alter the incorporation of the new cells. Fluoxetine increased BDNF levels in the hippocampus of both sexes. BDNF levels correlated with cell proliferation in males but not females. Brain and plasma levels indicated that females metabolized fluoxetine faster than males and produced more of the metabolite norfluoxetine. These data suggest that fluoxetine acts on multiple areas of the brain to increase cell proliferation, and the pattern of activation differs between males and females. Sex-specific effects of fluoxetine on neurotrophin mobilization and pharmacokinetics may contribute to these differences in neural plasticity.