Chronic variable stress leads to sex specific gut microbiome alterations in mice.

Stress has been implicated in the incidence and severity of psychiatric and gastrointestinal disorders. The immune system is capable of modulating the activity and composition of the gut following stress and vice versa. In this study we sought to examine the sequential relationship between immune signaling and microbiome composition occurring in male and female mice over time using a variable stress paradigm. Tissue was collected prior to, during, and after the stress paradigm from the same mice. Cytokines from plasma and brain were quantified using a multiplexed cytokine assay. Fecal samples were collected at the same timepoints and 16S rRNA amplicon sequencing was performed to determine the relative abundance of microbiota residing in the guts of stressed and control mice. We found sex differences in the response of the gut microbiota to stress following 28 days of chronic variable stress but not 6 days of sub-chronic variable stress. Immune activation was quantified in the nucleus accumbens immediately following Sub-chronic variable when alterations of gut composition had not yet occurred. In both sexes, 28 days of stress induced significant changes in the proportion of Erysipelotrichaceae and Lactobacillaceae, but in opposite directions for male and female mice. Alterations to the gut microbiome in both sexes were associated with changes in cytokines related to eosinophilic immune activity. Our use of an animal stress model reveals the immune mechanisms that may underly changes in gut microbiome composition during and after stress. This study reveals potential drug targets and microbiota of interest for the intervention of stress related conditions.

Premotor projections from the locus coeruleus and periaqueductal grey are altered in two rat models with inborn differences in emotional behavior.

Emotionally motivated behaviors rely on the coordinated activity of descending neural circuits involved in motor and autonomic functions. Using a pseudorabies (PRV) tract-tracing approach in typically behaving rats, our group previously identified descending premotor, presympathetic, and dual-labeled premotor-presympathetic populations throughout the central rostral-caudal axis. The premotor-presympathetic populations are thought to integrate somatomotor and sympathetic activity. To determine whether these circuits are dysregulated in subjects with altered emotional regulation, subsequent neuroanatomical analyses were performed in male subjects of two distinct genetic models relevant to clinical depression and anxiety: the Wistar Kyoto (WKY) rat and selectively bred Low Novelty Responder (bLR) rat. The present study explored alterations in premotor efferents from locus coeruleus (LC) and subdivisions of the periaqueductal grey (PAG), two areas involved in emotionally motivated behaviors. Compared to Sprague Dawley rats, WKY rats had significantly fewer premotor projections to hindlimb skeletal muscle from the LC and from the dorsomedial (DMPAG), lateral (LPAG), and ventrolateral (VLPAG) subdivisions of PAG. Relative to selectively bred High Novelty Responder (bHR) rats, bLR rats had significantly fewer premotor efferents from LC and dorsolateral PAG (DLPAG). Cumulatively, these results demonstrate that somatomotor circuitry in several brain areas involved in responses to stress and emotional stimuli are altered in rat models with depression-relevant phenotypes. These somatomotor circuit differences could be implicated in motor-related impairments in clinically depressed patients.

Antibiotic exposure is associated with decreased risk of psychiatric disorders.

Objective: This study sought to investigate the relationship between antibiotic exposure and subsequent risk of psychiatric disorders. Methods: This retrospective cohort study used a national database of 69 million patients from 54 large healthcare organizations. We identified a cohort of 20,214 (42.5% male; 57.9 ± 15.1 years old [mean ± SD]) adults without prior neuropsychiatric diagnoses who received antibiotics during hospitalization. Matched controls included 41,555 (39.6% male; 57.3 ± 15.5 years old) hospitalized adults without antibiotic exposure. The two cohorts were balanced for potential confounders, including demographics and variables with potential to affect: the microbiome, mental health, medical comorbidity, and overall health status. Data were stratified by age and by sex, and outcome measures were assessed starting 6 months after hospital discharge. Results: Antibiotic exposure was consistently associated with a significant decrease in the risk of novel mood disorders and anxiety and stressor-related disorders in: men (mood (OR 0.84, 95% CI 0.77, 0.91), anxiety (OR 0.88, 95% CI 0.82, 0.95), women (mood (OR 0.94, 95% CI 0.89,1.00), anxiety (OR 0.93, 95% CI 0.88, 0.98), those who are 26-49 years old (mood (OR 0.87, 95% CI 0.80, 0.94), anxiety (OR 0.90, 95% CI 0.84, 0.97)), and in those ≥50 years old (mood (OR 0.91, 95% CI 0.86, 0.97), anxiety (OR 0.92, 95% CI 0.87, 0.97). Risk of intentional harm and suicidality was decreased in men (OR 0.73, 95% CI 0.55, 0.98) and in those ≥50 years old (OR 0.67, 95% CI 0.49, 0.92). Risk of psychotic disorders was also decreased in subjects ≥50 years old (OR 0.83, 95 CI: 0.69, 0.99). Conclusion: Use of antibiotics in the inpatient setting is associated with protective effects against multiple psychiatric outcomes in an age- and sex-dependent manner.

Disrupted serotonin system development via early life antidepressant exposure impairs maternal care and increases serotonin receptor expression in adult female offspring.

Manipulating serotonin (5-HT) levels in the developing brain elicits a range of effects on brain function and behavior. For example, early-life exposure to selective 5-HT reuptake inhibitor (SSRI) antidepressants disrupts dorsal raphe function and triggers aberrant adult behaviors such as increased passive stress coping and anhedonia. However, much less is understood about how alterations in 5-HT signaling in early life impact outcomes in female offspring, including critical social functions such as maternal care. The present study shows that early-life SSRI exposure disrupts adult female offspring's maternal behavior. Pregnant/postpartum female Sprague-Dawley rats were treated with the SSRI citalopram in drinking water or provided regular tap water as control. Female offspring were raised to adulthood and mated with treatment-naïve males. Following parturition, we observed maternal behavior during portions of the light and dark phases of postnatal days (P)1-14. Relative to controls, dams with a history of early-life SSRI exposure exhibited decreased maternal care, including diminished arched-back nursing, reduced licking and grooming of pups, and increased behavioral inconsistency. Brains were collected from dams with and without a history of early-life SSRI exposure to measure relative mRNA expression of select 5-HT receptor transcripts (5HTR1A, -1B, -2A, -2C) in regions involved in maternal care. Early-life SSRI exposure augmented expression of 5-HTR1A in the medial preoptic area and 5-HTR1B, 5-HTR2A, and 5-HTR2C in the prefrontal cortex. These results demonstrate that early alterations to 5-HT signaling through SSRI exposure may disrupt nurturing parental behaviors and 5-HT receptor expression in affected female rat offspring.

Structural and metabolic activity differences in serotonergic cell groups in a rat model of individual differences of emotionality and stress reactivity.

Serotonin regulates a diverse set of functions, including emotional behavior, cognition, sociability, appetite, and sleep. Serotonin is also a key trophic factor that shapes neurodevelopmental processes. Genetic and environmental factors that drive individual differences in the serotonergic system have the capacity to impact brain structure and behavior, and likely contribute to pathophysiological processes involved in neuropsychiatric disorders. Using adult rats selectively bred for low novelty exploration (Low Responders, LR), we previously demonstrated pronounced increases in the levels of their anxiety- and depression- relevant behaviors as compared to the selectively bred High Novelty Responder (HR) rats. These behavioral differences were accompanied by alterations in the expression of genes that regulate serotonin synthesis in the brainstem, and its signaling in the forebrain. The present study extends these observations with a focus on the organization and the metabolism of brainstem serotonin cell groups that provide serotonergic innervation of the hippocampus and other limbic regions of male HR/LR rats. Using design-based stereology, we found the median raphe (MnR) in adult male LR rats contains increased number of serotonergic neurons as compared to the HRs. This is preceded by an increase in the metabolic activity of the caudal dorsal raphe (DRC) and the intrafascicular DR (DRI) during early postnatal development. These findings suggest that structural and functional differences in the raphe-limbic projections shape behavioral inhibition throughout the lifespan.

Resilience to Stress: Lessons from Rodents about Nature versus Nurture.

Individual differences in human temperament influence how we respond to stress and can confer vulnerability (or resilience) to emotional disorders. For example, high levels of behavioral inhibition in children predict increased risk of mood and anxiety disorders in later life. The biological underpinnings of temperament are unknown, although improved understanding can offer insight into the pathogenesis of emotional disorders. Our laboratory has used a rat model of temperamental differences to study neurodevelopmental factors that lead to a highly inhibited, stress vulnerable phenotype. Selective breeding for high versus low behavioral response to novelty created two rat strains that exhibit dramatic behavior differences over multiple domains relevant to emotional disorders. Low novelty responder (bLR) rats exhibit high levels of behavioral inhibition, passive stress coping, anhedonia, decreased sociability and vulnerability to chronic stress compared to high novelty responders (bHRs). On the other hand, bHRs exhibit high levels of behavioral dis-inhibition, active coping, and aggression. This review article summarizes our work with the bHR/bLR model showing the developmental emergence of the bHR/bLR phenotypes, the role the environment plays in shaping it, and the involvement of epigenetic processes such as DNA methylation that mediate differences in emotionality and stress reactivity.

Modeling heritability of temperamental differences, stress reactivity, and risk for anxiety and depression: Relevance to research domain criteria (RDoC).

Animal models provide important tools to study biological and environmental factors that shape brain function and behavior. These models can be effectively leveraged by drawing on concepts from the National Institute of Mental Health Research Domain Criteria (RDoC) Initiative, which aims to delineate molecular pathways and neural circuits that underpin behavioral anomalies that transcend psychiatric conditions. To study factors that contribute to individual differences in emotionality and stress reactivity, our laboratory utilized Sprague-Dawley rats that were selectively bred for differences in novelty exploration. Selective breeding for low versus high locomotor response to novelty produced rat lines that differ in behavioral domains relevant to anxiety and depression, particularly the RDoC Negative Valence domains, including acute threat, potential threat, and loss. Bred Low Novelty Responder (LR) rats, relative to their High Responder (HR) counterparts, display high levels of behavioral inhibition, conditioned and unconditioned fear, avoidance, passive stress coping, anhedonia, and psychomotor retardation. The HR/LR traits are heritable, emerge in the first weeks of life, and appear to be driven by alterations in the developing amygdala and hippocampus. Epigenomic and transcriptomic profiling in the developing and adult HR/LR brain suggest that DNA methylation and microRNAs, as well as differences in monoaminergic transmission (dopamine and serotonin in particular), contribute to their distinct behavioral phenotypes. This work exemplifies ways that animal models such as the HR/LR rats can be effectively used to study neural and molecular factors driving emotional behavior, which may pave the way toward improved understanding the neurobiological mechanisms involved in emotional disorders.

Perinatal SSRI Exposure Disrupts G Protein-coupled Receptor BAI3 in Developing Dentate Gyrus and Adult Emotional Behavior: Relevance to Psychiatric Disorders.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are widely prescribed to pregnant women suffering with depression, although the long-term impact of these medications on exposed offspring are poorly understood. Perinatal SSRI exposure alters human offspring's neurodevelopment and increases risk for psychiatric illness in later life. Rodent studies suggest that perinatal SSRI-induced behavioral abnormalities are driven by changes in the serotonin system as well as epigenetic and transcriptomic changes in the developing hippocampus. A major gene altered by perinatal SSRI exposure is the G-protein coupled receptor Brain Angiogenesis Inhibitor 3 (BAI3). Our present study shows that perinatal exposure to the SSRI citalopram increases mRNA expression of Bai3 and related molecules (including its C1ql ligands) in the early postnatal dentate gyrus of male and female offspring. Transient Bai3 mRNA knockdown in perinatal SSRI-exposed dentate gyrus lessened behavioral consequences of perinatal SSRI exposure, leading to increased active stress coping. To determine translational implications of this work, we examined expression of BAI3 and related molecules in hippocampus and prefrontal cortex from patients that suffered with depression or schizophrenia relative to healthy control subjects. We found sex- and region-specific changes in mRNA expression of BAI3 and its ligands C1QL2 and C1QL3 in men and women with a history of psychiatric disorders compared to healthy controls. Together these results suggest that abnormal BAI3 signaling may contribute to molecular mechanisms that drive adverse effects of perinatal SSRI exposure, and show evidence for alterations of BAI3 signaling in the hippocampus of patients that suffer depression and schizophrenia.

Neonatal resource scarcity alters maternal care and impacts offspring core temperature and growth in rats.

Stressful experiences during childhood, including poverty and inconsistent parental care, can enhance vulnerability for worsened physical and mental health outcomes in adulthood. Using Sprague Dawley rats, the present study explored the impact of limited resource availability on maternal behavior and physiological and emotional behavior outcomes in the offspring. Early life adversity was induced by incorporating aspects of the limited bedding and nesting and scarcity models, wherein limited resource availability has previously been shown to provoke unpredictable or adverse maternal care respectively. In our hands, neonatal limited bedding (NLB) stress during postnatal days (P)2-9 altered maternal care, augmenting pup-directed behaviors and reducing self-directed behaviors, and modestly increased the frequency of transitions between discrete behaviors across consecutive timed observations. NLB-exposed pups had lower core body temperatures immediately following the stressful manipulation and exhibited decreased body weight gain across development. However, NLB exposure did not impact adult offspring's social or emotional behavior outcomes in the three-chamber social interaction, novelty-suppressed feeding, splash, or forced swim tests. These findings add to the literature demonstrating that early life adversity impacts maternal care in rodents and can disrupt certain metabolic and thermoregulatory outcomes in the offspring.

The Property-Based Practical Applications and Solutions of Genetically Encoded Acetylcholine and Monoamine Sensors.

Neuromodulatory communication among various neurons and non-neuronal cells mediates myriad physiological and pathologic processes, yet defining regulatory and functional features of neuromodulatory transmission remains challenging because of limitations of available monitoring tools. Recently developed genetically encoded neuromodulatory transmitter sensors, when combined with superresolution and/or deconvolution microscopy, allow the first visualization of neuromodulatory transmission with nanoscale or microscale spatiotemporal resolution. In vitro and in vivo experiments have validated several high-performing sensors to have the qualities necessary for demarcating fundamental synaptic properties of neuromodulatory transmission, and initial analysis has unveiled unexpected fine control and precision of neuromodulation. These new findings underscore the importance of synaptic dynamics in synapse-, subcellular-, and circuit-specific neuromodulation, as well as the prospect of genetically encoded transmitter sensors in expanding our knowledge of various behaviors and diseases, including Alzheimer's disease, sleeping disorders, tumorigenesis, and many others.

Genetic Liability for Internalizing Versus Externalizing Behavior Manifests in the Developing and Adult Hippocampus: Insight From a Meta-analysis of Transcriptional Profiling Studies in a Selectively Bred Rat Model.

BACKGROUND: For more than 16 years, we have selectively bred rats for either high or low levels of exploratory activity within a novel environment. These bred high-responder (bHR) and bred low-responder (bLR) rats model temperamental extremes, exhibiting large differences in internalizing and externalizing behaviors relevant to mood and substance use disorders. METHODS: We characterized persistent differences in gene expression related to bHR/bLR phenotype across development and adulthood in the hippocampus, a region critical for emotional regulation, by meta-analyzing 8 transcriptional profiling datasets (microarray and RNA sequencing) spanning 43 generations of selective breeding (postnatal day 7: n = 22; postnatal day 14: n = 49; postnatal day 21: n = 21; adult: n = 46; all male). We cross-referenced expression differences with exome sequencing within our colony to pinpoint candidates likely to mediate the effect of selective breeding on behavioral phenotype. The results were compared with hippocampal profiling from other bred rat models. RESULTS: Genetic and transcriptional profiling results converged to implicate multiple candidate genes, including two previously associated with metabolism and mood: Trhr and Ucp2. Results also highlighted bHR/bLR functional differences in the hippocampus, including a network essential for neurodevelopmental programming, proliferation, and differentiation, centering on Bmp4 and Mki67. Finally, we observed differential expression related to microglial activation, which is important for synaptic pruning, including 2 genes within implicated chromosomal regions: C1qa and Mfge8. CONCLUSIONS: These candidate genes and functional pathways may direct bHR/bLR rats along divergent developmental trajectories and promote a widely different reactivity to the environment.

Inborn differences in emotional behavior coincide with alterations in hypothalamic paraventricular motor projections.

Integrated behavioral responses to emotionally salient stimuli require the concomitant activation of descending neural circuits that integrate physiological, affective, and motor responses to stress. Our previous work interrogated descending circuits in the brainstem and spinal cord that project to motor and sympathetic targets. The hypothalamic paraventricular nucleus (PVN), a key node of this circuitry, integrates multiple motor and sympathetic responses activated by stress. The present study sought to determine whether descending projections from the PVN to targets in muscle and adrenal gland are differentially organized in rats with inborn differences in emotionality and stress responsivity. We utilized retrograde transsynaptic tract-tracing with unique pseudorabies virus (PRV) recombinants that were injected into sympathectomized gastrocnemius muscle and adrenal gland in two rat models featuring inborn differences in emotional behavior. Our tract-tracing results revealed a significant decrease in the number of PVN neurons with poly-synaptic projections to the gastrocnemius in male Wistar Kyoto [WKY] rats (versus Sprague Dawley rats) and selectively bred Low Novelty Responder [bLR] rats (versus selectively bred High Novelty Responder [bHR] rats). These neuroanatomical differences mirrored behavioral observations showing that both WKY and bLR rats display marked inhibition of emotional motor responses in a variety of settings relative to their respective controls. Our findings suggest that, in male rodents, PVN poly-synaptic projections to skeletal muscle may regulate emotional motor and coping responses to stress. More broadly, perturbations in PVN motor circuitry may play a role in mediating psychomotor disturbances observed in depression or anxiety-related disorders.

Oxidative metabolism alterations in the emotional brain of anxiety-prone rats.

Mood disorders such as anxiety and depression are heterogeneous disorders with many sufferers unresponsive to current pharmacological treatments. Individual differences in temperament represent one factor that may underlie symptom heterogeneity, so understanding its biological underpinnings can help pave the way to personalized therapies and improved patient outcomes. The present study uses a rodent model of temperamental differences to examine whether individual differences in emotional behavior phenotypes correspond to altered limbic brain cellular metabolism, an indicator of neuronal activity. The model uses two selectively bred rat lines - high novelty responder rats (HRs) that show highly exploratory behavior in a novel environment, active coping style and resilience to chronic mild stress compared to low novelty responder rats (LRs), which are inhibited in novel environments, display passive coping style, and are susceptible to chronic stress. Utilizing transcriptome data from a prior study in adult HR/LR rats, we first show that a preponderance of genes differing in the HR vs. LR hippocampus and amygdala are involved in cellular metabolism. This led us to then ask if oxygen consumption was altered in isolated mitochondria of the hippocampus and amygdala of HR/LR rats; here we found increased oxygen consumption reserve capacity in LR amygdala. Our last experiment examined activity of cytochrome c oxidase (COX), an enzyme responsible for ATP production and correlate of metabolic activity, in several brain regions of HR/LR rats. We found that LRs displayed higher COX activity in the dentate gyrus, prefrontal cortex, and dorsal raphe compared to HRs, with no significant HR/LR difference in nuclei of the amygdala. Correlational analyses of COX activity across brain regions suggested divergent connectivity between the prefrontal cortex, amygdala, hippocampus, and dorsal raphe of HR vs. LR rats. Together these studies point to altered cellular metabolism in the limbic brain of LR/HR animals, which may reflect altered neural circuitry that drives their divergent behavioral profiles.

Rats bred for high anxiety exhibit distinct fear-related coping behavior, hippocampal physiology, and synaptic plasticity-related gene expression.

The hippocampus is essential for learning and memory but also regulates emotional behavior. We previously identified the hippocampus as a major brain region that differs in rats bred for emotionality differences. Rats bred for low novelty response (LRs) exhibit high levels of anxiety- and depression-like behavior compared to high novelty responder (HR) rats. Manipulating the hippocampus of high-anxiety LR rats improves their behavior, although no work to date has examined possible HR/LR differences in hippocampal synaptic physiology. Thus, the current study examined hippocampal slice electrophysiology, dendritic spine density, and transcriptome profiling in HR/LR hippocampus, and compared performance on three hippocampus-dependent tasks: The Morris water maze, contextual fear conditioning, and active avoidance. Our physiology experiments revealed increased long-term potentiation (LTP) at CA3-CA1 synapses in HR versus LR hippocampus, and Golgi analysis found an increased number of dendritic spines in basal layer of CA1 pyramidal cells in HR versus LR rats. Transcriptome data revealed glutamate neurotransmission as the top functional pathway differing in the HR/LR hippocampus. Our behavioral experiments showed that HR/LR rats exhibit similar learning and memory capability in the Morris water maze, although the groups differed in fear-related tasks. LR rats displayed greater freezing behavior in the fear-conditioning task, and HR/LR rats adopted distinct behavioral strategies in the active avoidance task. In the active avoidance task, HRs avoided footshock stress by pressing a lever when presented with a warning cue; LR rats, on the other hand, waited until footshocks began before pressing the lever to stop them. Taken together, these findings concur with prior observations of HR rats generally exhibiting active stress coping behavior while LRs exhibit reactive coping. Overall, our current findings coupled with previous work suggest that HR/LR differences in stress reactivity and stress coping may derive, at least in part, from differences in the developing and adult hippocampus.

Altered DNA Methylation in the Developing Brains of Rats Genetically Prone to High versus Low Anxiety.

There is growing evidence of abnormal epigenetic processes playing a role in the neurobiology of psychiatric disorders, although the precise nature of these anomalies remains largely unknown. To study neurobiological (including epigenetic) factors that influence emotionality, we use rats bred for distinct behavioral responses to novelty. Rats bred for low novelty response (low responder [LR]) exhibit high levels of anxiety- and depressive-like behavior compared with high novelty responder (HR) rats. Prior work revealed distinct limbic brain development in HR versus LR rats, including altered expression of genes involved in DNA methylation. This led us to hypothesize that DNA methylation differences in the developing brain drive the disparate HR/LR neurobehavioral phenotypes. Here we report altered DNA methylation markers (altered DNA methyltransferase protein levels and increased global DNA methylation levels) in the early postnatal amygdala of LR versus HR male rats. Next-generation sequencing methylome profiling identified numerous differentially methylated regions across the genome in the early postnatal HR/LR amygdala. We also contrasted methylation profiles of male HRs and LRs with a control rat strain that displays an intermediate behavioral phenotype relative to the HR/LR extremes; this revealed that the LR amygdalar methylome was abnormal, with the HR profile more closely resembling that of the control group. Finally, through two methylation manipulations in early life, we found that decreasing DNA methylation in the developing male and female amygdala improves adult anxiety- and depression-like behavior. These findings suggest that inborn DNA methylation differences play important roles in shaping brain development and lifelong emotional behavior.SIGNIFICANCE STATEMENT Epigenetic changes are biological mechanisms that regulate the expression and function of genes throughout the brain and body. DNA methylation, one type of epigenetic mechanism, is known to be altered in brains of psychiatric patients, which suggests a role for DNA methylation in the pathogenesis of psychiatric disorders, such as depression and anxiety. The present study examines brains of rats that display high versus low levels of anxiety- and depression-like behavior to investigate how neural DNA methylation levels differ in these animals and how such differences shape their emotional behavioral differences. Studying how epigenetic processes affect emotional behavior may improve our understanding of the neurobiology of psychiatric disorders and lead to improved treatments.

Distinct effects of early-life experience and trait aggression on cardiovascular reactivity and recovery.

We previously demonstrated independent effects of early-life experience (ELE) and trait aggression (TA) on resting heart rate (HR) and mean arterial pressure (MAP) in rats. The present study examined the effects of TA and ELE on stress-evoked cardiovascular reactivity and recovery. Pups born to Wistar-Kyoto dams were exposed to daily 180-min periods of maternal separation (MS) during the first two weeks of life, and aggression was assessed in adult offspring using the resident-intruder test. Radiotelemetry was then used to record stress-evoked HR and MAP responses in response to: strobe light, novel environment, intruder rat, or restraint. Maximal HR and MAP responses were quantified as indices of reactivity, and exponential decay curves were fitted to determine decay constants as a measure of recovery. Strobe light was the weakest stressor, evoking the lowest increases in MAP and HR, which were significantly greater in MS-exposed rats irrespective of TA. In contrast, reactivity to and recovery from exposure to a novel environment or an intruder were significantly influenced by TA, but not ELE. TA animals exhibited greater reactivity in both of these paradigms, with either decreased (novel environment) or increased (intruder) recovery. Restraint stress induced the largest changes in HR and MAP with the slowest recovery, and these responses were shaped by a significant ELE x TA interaction. These data indicate that cardiovascular reactivity and recovery are influenced by ELE, TA, or ELE x TA interaction depending on stressor aversiveness as well as its physical and psychological dimensions.

Perinatal exposure to the SSRI paroxetine alters the methylome landscape of the developing dentate gyrus.

Evidence in humans and rodents suggests that perinatal exposure to selective serotonin reuptake inhibitor (SSRI) antidepressants can have serious long-term consequences in offspring exposed in utero or infancy via breast milk. In spite of this, there is limited knowledge of how perinatal SSRI exposure impacts brain development and adult behaviour. Children exposed to SSRIs in utero exhibit increased internalizing behaviour and abnormal social behaviour between the ages of 3 and 6, and increased risk of depression in adolescence; however, the neurobiological changes underlying this behaviour are poorly understood. In rodents, perinatal SSRI exposure perturbs hippocampal gene expression and alters adult emotional behaviour (including increased depression-like behaviour). The present study demonstrates that perinatal exposure to the SSRI paroxetine leads to DNA hypomethylation and reduces DNA methyltransferase 3a (Dnmt3a) mRNA expression in the hippocampus during the second and third weeks of life. Next-generation sequencing identified numerous differentially methylated genomic regions, including altered methylation and transcription of several dendritogenesis-related genes. We then tested the hypothesis that transiently decreasing Dnmt3a expression in the early postnatal hippocampus would mimic the behavioural effects of perinatal SSRI exposure. We found that siRNA-mediated knockdown of Dnmt3a in the dentate gyrus during the second to third week of life produced greater depression-like behaviour in adult female (but not male) offspring, akin to the behavioural consequences of perinatal SSRI exposure. Overall, these data suggest that perinatal SSRI exposure may increase depression-like behaviours, at least in part, through reduced Dnmt3a expression in the developing hippocampus.

A paternal methyl donor depleted diet leads to increased anxiety- and depression-like behavior in adult rat offspring.

Epigenetic mechanisms such as DNA methylation elicit lasting changes in gene expression and likely mediate gene-environment interactions that shape brain development, behavior, and emotional health. Myriad environmental factors influence DNA methylation, including methyl donor content in the paternal diet, could influence methylation in offspring via changes in the paternal germ line. The present study examines the effects of paternal methyl donor dietary deficiency on offspring's emotional behaviors, including anxiety, social interaction, and depression-like behavior. We previously found that rats bred to display high levels of anxiety- and depression-like behavior exhibit diminished DNA methylation in the amygdala. We also observed that depleting dietary methyl donor content exacerbated the rats' already high levels of anxiety- and depression-like behavior. Here we sought to determine whether paternal dietary methyl donor depletion elicits intergenerational effects on first generation (F1) offspring's behavior (potentially triggering a similar increase in anxiety- and/or depression-like behavior). Thus, adult male rats prone to high anxiety/depression-like behavior, were fed either a methyl donor depleted (DEP) or control (CON) diet for 5 weeks prior to mating. They were paired with females and resultant F1 male offspring were subjected to a behavioral test battery in adulthood. F1-DEP offspring showed a similar behavioral profile to the F0 males, including greater depression-like behavior in the forced swim test (FST) and increased anxiety-like behavior in the open field test (OFT). Future work will interrogate molecular changes in the brains of F1 offspring that mediate these intergenerational effects of paternal methyl donor dietary content on offspring emotional behavior.

Amygdalar expression of the microRNA miR-101a and its target Ezh2 contribute to rodent anxiety-like behaviour.

A greater understanding of neural mechanisms contributing to anxiety is needed in order to develop better therapeutic interventions. This study interrogates a novel molecular mechanism that shapes anxiety-like behaviour, demonstrating that the microRNA miR-101a-3p and its target, enhancer of zeste homolog 2 (Ezh2) in the amygdala, contribute to rodent anxiety-like behaviour. We utilized rats that were selectively bred for differences in emotionality and stress reactivity, showing that high-novelty-responding (HR) rats, which display low trait anxiety, have lower miR-101a-3p levels in the amygdala compared to low-novelty-responding (LR) rats that characteristically display high trait anxiety. To determine whether there is a causal relationship between amygdalar miR-101a-3p and anxiety behaviour, we used a viral approach to overexpress miR-101a-3p in the amygdala of HR rats and test whether it would increase their typically low levels of anxiety-like behaviour. We found that increasing miR-101a-3p in the amygdala increased HRs' anxiety-like behaviour in the open-field test and elevated plus maze. Viral-mediated miR-101a-3p overexpression also reduced expression of the histone methyltransferase Ezh2, which mediates gene silencing via trimethylation of histone 3 at lysine 27 (H3K27me3). Knockdown of Ezh2 with short-interfering RNA (siRNA) also increased HRs' anxiety-like behaviour, but to a lesser degree than miR-101a-3p overexpression. Overall, our data demonstrate that increasing miR-101a-3p expression in the amygdala increases anxiety-like behaviour and that this effect is at least partially mediated via repression of Ezh2. This work adds to the growing body of evidence implicating miRNAs and epigenetic regulation as molecular mediators of anxiety behaviour.