Neurobiology of Stress

All in vivo studies using laboratory animals should be guided by the Three Rs: Replacement, Reduction and Refinement. The concept of Reduction is important in sample size estimation; the sample size used should allow the detection of a biologically meaningful effect size using appropriate statistical tests, but not at the expense of animal suffering. Because studies using chronic variable stress (CVS) procedures deliberately impose suffering, we reasoned that Three Rs principles would be a strong consideration in experimental design. To explore this, we conducted a systematic review of CVS studies to ask whether a biologically meaningful effect size was used to determine the sample size. Only one article in our sample of 385 reported doing this. Accordingly, it is questionable whether most of these studies align strongly with the principle of Reduction. While determining a biologically meaningful effect size is not always straightforward, we believe it is central to making biologically informed decisions about study design and interpretation, and we discuss possible ways forward.

Exposure to stress can cause long-lasting enhancement of fear and other defensive responses that extend beyond the cues or contexts associated with the original traumatic event. These nonassociative consequences of stress, referred to as fear sensitization, are thought to underlie some symptoms of trauma-related disorders. Fear sensitization has been predominately studied using the Stress-Enhanced Fear Learning (SEFL) paradigm, which models the stress-induced amplification of fear learning. Less is known about the mechanisms through which unlearned fear responses are sensitized by stress. Here, we investigated the neural mechanisms for sensitization of unlearned fear responses using a paradigm we termed Stress-Enhanced Fear Responding (SEFR). In this model, mice exposed to a single session of footshock stress exhibit enhanced freezing to a novel tone stimulus. To investigate brain regions that might mediate SEFR, we first used c-Fos mapping to identify neural activity changes associated with stress-induced enhancement of unlearned fear. Our c-Fos screen identified the posterior paraventricular thalamus (pPVT) as a region that was persistently hyperactive after footshock stress and whose activity correlated with behavioral expression of SEFR. Using fiber photometry, we observed that SEFR, but not SEFL, was associated with increased activity in the pPVT. Next, we found that chemogenetic inhibition of the pPVT blocked both the induction of SEFR during stress and its later expression, while artificial stimulation of pPVT in stress-naive mice was sufficient to recapitulate SEFR. Interestingly, pPVT inhibition or stimulation did not affect acquisition or expression of SEFL. In conclusion, our results indicate that sensitization of fear learning (SEFL) and sensitization of unlearned fear (SEFR) have distinct neural mechanisms. Our results identify pPVT hyperactivity as a mechanism for stress-induced sensitization of unlearned fear and highlight pPVT as a potential target for treating arousal and reactivity symptoms of trauma- and stressor-related disorders.

Exposure to early life stress (ELS) can exert long-lasting impacts on emotional regulation. The corticolimbic system including the basolateral amygdala (BLA), ventral hippocampus (vHIP), and the medial prefrontal cortex (mPFC) plays a key role in fear learning. Using the limited bedding paradigm (LB), we examined the functional consequences of ELS on excitatory and inhibitory tone in the prelimbic (PL) mPFC after fear conditioning in rats. In adults, LB exposure enhanced in vivo glutamate release in the PL mPFC during fear conditioning in male, but not female offspring. In contrast, the glutamate response to fear conditioning was diminished in LB-exposed pre-adolescent males, but not females. We investigated whether reduced glutamatergic inputs and/or elevated inhibitory tone might contribute to the diminished glutamate response in the mPFC following LB in pre-adolescent male rats. Indeed, we found that LB exposure specifically increased the activation of PV, but not SST interneurons in layer V, but not layer II/III of the PL mPFC in fear-exposed pre-adolescent males. Presynaptic glutamate release probability was reduced by LB exposure in layer V, but increased in layer II/III of the PL mPFC. These functional changes might be related to the LB-induced alterations in the bilaminar distribution of BLA and vHIP projections to the PL mPFC we observed in pre-adolescent males. Overall, our findings suggest that ELS modifies glutamate release and PL mPFC function during fear conditioning in a sex- and age-dependent fashion, likely through layer-specific shifts in excitation/inhibition balance. Significance StatementEarly life stress (ELS) increases the risk of developing affective disorders and long-term emotional dysregulation might arise from disruptions in the development of the fear circuitry. This study examines how ELS modifies fear-induced activity of long-range excitatory projections and local inhibitory microcircuits in the developing prefrontal cortex. We tested whether ELS-induced alterations in prefrontal cortex function are sex- and age-dependent, leading to the well-documented sex differences in emotional behavioral outcome. Studying how ELS differentially modifies regional excitatory inputs and cell type specific activation in the prefrontal cortex during a critical period of brain development will enhance our understanding of the neurobiological mechanisms underlying the pathogenesis of emotional dysregulation and inspire more targeted intervention after exposure to early adversity.

Early life stress can have significant effects on the developing brain and lead to changes in the structure and function of brain regions involved in stress regulation, emotion and cognitive control. Here, we used the social instability stress (SIS) protocol to understand the impact of social stress during mild (PND30) and late (PND45) adolescence. Our results revealed that SIS can compromise the dominance-subordination coping strategy but does not affect social recognition and motivation in rats. Moreover, SIS can lead to subtle modifications at the molecular level that hamper normal development of the prefrontal cortex in a sex- and age-dependent manner. Understanding the impact of early life stress on brain organization is crucial for developing effective prevention and intervention strategies. By identifying those who are most vulnerable to the effects of stress and providing targeted support and resources, it may be possible to mitigate the negative consequences of early adversity and promote healthy brain development.

Social stress is a risk factor for psychiatric disorders and also influences immune function. While it is known that acute social stress impacts the number of immune cells in circulation, the temporal dynamics of stress induced immune-related transcriptional changes in human blood remain unclear. To investigate changes in gene expression, we exposed 26 adults to the Trier Social Stress Test (TSST), and collected blood at baseline, as well as 5, 30, 60 and 90 min after stress. Whole-blood gene expression was profiled using a 5 targeted RNA-sequencing method (STRT). Differential expression was analyzed using linear and cubic models. We observed a total of 54 differentially expressed genes following stress. Fast responses, with a transient peak immediately following stress, were enriched for cytotoxic T cell, NK cell and dendritic cell functions (e.g., GZMB, GNLY, CCL4 and GZMA) and paralleled lymphocyte count changes. In contrast, gradual, linear responses without any evident peak were enriched for neutrophil related genes (e.g., FPR2, PLAUR, CXCR2, AQP9, and QPCT) and did not mirror neutrophil counts, indicating cell intrinsic transcriptional changes. From pathway and transcription factor enrichment analyses, IL-12 family mediated signaling is inferred as a central mechanism linking stress to immune gene regulation. Our results show that acute psychosocial stress induces both fast and slower changes in gene expression in different immune cell populations. The involvement of the IL-12-STAT4 axis and genes such as PLAUR and FPR2 suggests molecular mechanisms through which stress-related immune activation may contribute to vulnerability for anxiety and depressive disorders.

The lateral habenula (LHb) inhibits midbrain monoaminergic neurons, thereby regulating emotion/cognition. Abnormally high activity in the LHb causes behavioral disorders, but how stressful experiences affect neuronal circuits underlying emotion remains poorly understood. Here, we report the effects of chronic stress on the LHb in postnatal day (P)1-9, P10-20, and P36-45 mice in the pre-, early, and late stages of LHb maturation. At P60, only mice exposed during P10-20 exhibited LHb-specific changes: abnormally high-stress reactivity shown by the expression of the immediate-early gene product (Zif268/Egr1) with insufficient number of parvalbumin (PV) neurons containing GABA. Furthermore, these mice showed anxiety/depression-like behaviors in the light-dark box test/forced swim test. Thus, experiences in early-life are essential for the maturation of neuronal circuits underlying emotion. Early-life stress is thought to have caused anxiety/depression in adulthood by disrupting the maturation of inhibitory PV neurons in the LHb in a period-specific manner.

The amygdala is a key brain region that processes stress-related inputs to reshape future behaviors. The lateral amygdala (LA), basolateral amygdala (BLA), and central amygdala (CeA) are important subregions that mediate different aspects of stress experiences from receiving sensory input to memory formation and behavioral responding. The principal neurons in these regions are glutamatergic pyramidal neurons, which are genetically separable into two subpopulations, protein phosphatase 1 regulatory subunit 1B-positive (Ppp1r1b, also known as DARPP-32) parvocellular neurons and R-spondin2-positive (Rspo2) magnocellular neurons. Recent studies show that these two subpopulations of amygdala neurons differentially regulate appetitive versus aversive behaviors. The research goal of this study is to explore whether amygdala Ppp1r1b and Rspo2 neurons are transcriptionally activated by moderate stress experience, such that persistent cellular changes are made to influence future functional output of these two subtypes of neurons. To test transcriptional activation, we focused on c-Fos, one of the early genes that are transiently expressed in response to cellular stimulations to regulate downstream gene transcription. Moderate stress was introduced through brief footshocks, with mice without footshock as controls. Between shocked and control mice, we observed similar numbers of Ppp1r1b or Rspo2 neurons per unit area that expressed c-Fos, which was consistent across LA-BLA and CeA. Moreover, in LA-BLA, Ppp1r1b/c-Fos cells consistently outnumber Rspo2/c-Fos cells across treatment conditions, and the reverse is true in CeA. These results suggest that moderate stress experience is not sufficient to induce robust transcriptional alterations in the two key subpopulations of amygdala neurons, and Ppp1r1b versus Rspo2 neuron activities, as measured by c-Fos expression levels, show differential dominance in amygdala subregions.

Early life stress (ELS) events during sensitive postnatal time periods can recalibrate future stress responsiveness and precipitate mental disorders. Neurovascular adaptations can influence cognition, mood, and stress responses. Disruption of blood-brain barrier (BBB) integrity, which is formed by endothelial cells, astrocytes, and pericytes, has been implicated in affective disorders such as depression, which often arise from chronic stress experiences. Despite the BBB undergoing critical maturation stages during development, it remains poorly known how ELS influences brain vascular function, as previously shown for adult stress, and whether it augments BBB vulnerability to subsequent challenges. First, we took advantage of a public two-hit stress RNA-sequencing dataset and filtered for vascular enriched genes in the prefrontal cortex and nucleus accumbens, the two brain regions where BBB integrity is frequently compromised. This analysis revealed BBB-related gene ontology categories modulated by either ELS alone or its combination with adult stress. Then, using a mouse model combining ELS with chronic social defeat stress (CSDS) in adulthood, we found that ELS did not exacerbate CSDS susceptibility; instead, it increased social interactions and the likelihood of a resilient profile in both males and females. Transcriptomic profiling in our cohort further identified distinct sex- and region-specific BBB gene expression patterns associated with ELS and its interaction with CSDS. Additionally, we observed a reduction of corticosterone levels, the primary stress hormone, following CSDS. Altogether, these results indicate that ELS modulates stress responses when facing emotional challenges in adulthood, possibly through long-lasting changes of BBB function via the glucocorticoid system. HighlightsO_LIRNA-seq vascular filtering reveals BBB distinct ontology categories for ELS and AS C_LIO_LIELS increases the likelihood of a high social and resilient profile. C_LIO_LIPericytes gene expression associated to resilience is sex- and region-specific. C_LIO_LICORT response desensitizes after adult CSDS in both sexes. C_LI

The ability of the medial prefrontal cortex (mPFC) to exert top-down control of behavior is affected by stress. The molecular response of mPFC to stress is incompletely understood, however, in part because of the regions cellular heterogeneity. Here we used single nucleus RNA sequencing (snRNAseq) to map specific molecular cell types within the mPFC and to detect cell-type specific transcriptional changes to foot-shock stress. We identified Ptgs2, encoding cyclo-oxygenase 2, as an important candidate that is upregulated in layer II/III excitatory neurons after stress. Specifically, Ptgs2 was transiently upregulated with shock-induced fear learning and fear expression, along with Bdnf, Nptx2, and Lingo1, in a layer II/III neuronal population marked by the neuronal excitatory gene Slc17a7 and cell-type specific neuropeptide Penk. These dynamic cell-type specific expression patterns identified with snRNAseq were validated with quantitative fluorescent in situ hybridization. Using a pharmacological approach, we found that systemic lumiracoxib, a selective Ptgs2-inhibitor, led to a significant reduction in fear expression. Furthermore, genetic ablation of Ptgs2 in excitatory Camk2a-expressing neurons led to reduced stress-induced anxiety-like behaviors. Together these findings suggest that Ptgs2 is expressed in a dynamic, cell-type specific way in Layer II/III Penk+ neurons in mPFC, and that its role in prostaglandin and /or endocannabinoid regulation within these neurons may be an important mediator of stress-related behavior.

The impact of stress on gene expression in different cell types of the brain remains poorly characterized. Three pioneering studies have recently used translating ribosome affinity purification followed by RNA sequencing (TRAP-seq) to assess the response to stress in CA3 pyramidal neurons of the hippocampus. The results suggest that acute stress alters the translation of thousands of genes in CA3 pyramidal neurons, and that this response is strongly modulated by factors such as sex, genotype and a history of early life stress. However, our reanalysis of these datasets leads to different conclusions. We confirm that acute stress induces robust translational changes in a small set of genes. However, we found no evidence that either early life stress or sex have an effect on gene translation induced by acute stress. Our findings highlight the need for additional studies with adequate sample sizes and proper methods of analysis to assess the impact of stress across cell types in the brain.

Developmental stress is a well-established risk factor for mental health disorders, yet the neural mechanisms underlying these outcomes remain incompletely understood. Inhibitory brain networks, particularly within the amygdala, are disrupted by stress and implicated in stress-related psychopathologies. Using a rodent model, the current study investigated the isolated and combined effects of prenatal and adolescent stress on adult social interactions and GABAergic neurons surrounded by perineuronal nets (PNNs) in the basolateral amygdala (BLA). Male and female rats were exposed to chronic variable stressors (CVS) prenatally (PS), during adolescence (AS), or during both prenatal and adolescent periods (PS+AS). In adulthood, all animals were tested for social behavior with same-sex weight-matched partners, and brains were collected for identification of BLA inhibitory neurons (GAD67 staining) and PNNs (Wisteria Floribunda Agglutinin staining). For social behavior, AS alone robustly increased social investigation in adulthood relative to non-stressed (NS) controls and animals exposed to combined PS+AS. PS+AS subjects did not significantly differ from NS controls, suggesting that prenatal stress exposure prevented adolescent stress-induced increases in adult social investigation. An analogous data pattern was observed in the BLA. AS alone decreased the number GAD67+ neurons surrounded by PNNs (co-labeled) relative to NS controls and subjects exposed to combined PS+AS. When the percentage of total GAD67+ neurons co-labeled with PNNs was assessed, both PS alone and AS alone reduced the proportion of GAD67+ neurons surrounded by PNNs, whereas combined PS+AS had no effect. Overall, these data suggest that prenatal stress exposure prevents adolescent stress-induced disruptions to perineuronal nets surrounding inhibitory neurons in the BLA, potentially conferring resilience to adolescent stress-induced changes in inhibitory function and social behavior. HighlightsO_LIAdolescent stress exposure increased social investigation in adulthood. C_LIO_LIAdolescent stress decreased the number of BLA cells co-labeled with GAD67 and WFA. C_LIO_LIPrenatal or adolescent stress decreased the proportion of inhibitory neurons with PNNs. C_LIO_LIWhen preceded by prenatal stress, effects of adolescent stress were not observed. C_LI

Early life stress (ELS) adversely affects physiological and behavioral outcomes, increasing the vulnerability to stress-related disorders, such as post-traumatic stress disorder (PTSD). PTSD prevalence is significantly higher in women and is partially mediated by genetic risk variants. Understanding how sex influences the interaction of PTSD risk genes, such as FKBP5, with trauma-related behaviors is crucial for uncovering PTSDs neurobiological pathways. The development of in-depth behavioral analysis tools using unsupervised behavioral classification is thereby a crucial tool to increase the understanding of the behavioral outcomes related to stress-induced fear memory formation. The current study investigates the sex-specific effects of ELS exposure by using the limited bedding and nesting (LBN) paradigm. The LBN exposure disrupted different facets of the hypothalamic-pituitary-adrenal (HPA) axis in a sex-specific manner directly after stress and at adult age. Moreover, freezing was altered by LBN exposure in both the acquisition and the retrieval of fear in a sex-dependent manner. Unsupervised behavioral analysis revealed a higher active fear response after LBN exposure during fear acquisition in females, but not in males. The regulation of the HPA axis is closely intertwined with cellular metabolism and core regulatory cascades. To investigate the impact of LBN exposure on tissue-specific metabolism, a metabolomic pathway analysis in the basolateral amygdala revealed a specific sex- and stress-dependent effect on purine, pyrimidine, and glutamate metabolism. The present study highlights the intricate interplay between metabolic pathways and the neurobiological substrates implicated in fear memory formation and stress regulation. Overall, these findings highlight the importance of considering sex-specific metabolic alterations in understanding the neurobiological mechanisms underlying stress-related disorders and offer potential avenues for targeted interventions.

Post-Traumatic Stress Disorder (PTSD) is a debilitating condition in which a traumatic experience triggers symptoms related to re-experiencing, avoidance, arousal, and mood dysregulation. PTSD negatively impacts 6% of people during their lifetime, with women being disproportionally affected and exhibiting different, more severe symptoms than men. Despite this widespread impact, the molecular mechanisms underlying PTSD and its sex differences remain poorly understood. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a neuropeptide which participates in fine-tuning circuitry throughout the brain and has been associated with PTSD in humans, especially in women. Here, we use Single Prolonged Stress (SPS), an animal model of PTSD, to explore the roles of PACAP and sex in PTSD-like behaviors. Specifically, a PACAP agonist or antagonist was infused into the infralimbic (IL) prefrontal cortex, a region key to regulating fear- and anxiety-related behaviors, prior to SPS in male and female rats. One week later, rats were tested in open field/novel object, elevated plus maze, and social interaction. Utilizing a behavioral indexing method, we were able to uncover SPS effects in PTSD-related behavioral domains that were differentially impacted by PACAP manipulations in males and females. While both sexes exhibited increased threat avoidance and decreased threat assessment following SPS, females increased sociability while males decreased sociability. Males also appeared to be protected by IL PACAP antagonism while female SPS phenotypes were exacerbated by IL PACAP agonism. Furthermore, RNAscope revealed that PACAP in the prefrontal cortex responds differently to SPS in males and females. Together, these findings suggest complex relationships between SPS, sex, and IL PACAP which may have important implications for treating PTSD in men and women. HIGHLIGHTSO_LISPS induces different PTSD-like phenotypes in male and female rats C_LIO_LISPS increases threat avoidance and decreases threat appraisal in both sexes C_LIO_LISociability is decreased in males but increased in females following SPS C_LIO_LIIL PACAP manipulation exerts diverging SPS behavioral effects in males and females C_LIO_LIPrefrontal PACAP signaling plays a sex-specific role in SPS molecular mechanisms C_LI

Fear extinction is an adaptive behavioral process critical for organisms survival, but deficiency in extinction may lead to PTSD. While the amygdala and its neural circuits are critical for fear extinction, the molecular identity and organizational logic of cell types that lie at the core of these circuits remain unclear. Here we report that mice deficient for amygdala-enriched gastrin-releasing peptide gene (Grp-/-) exhibit enhanced neuronal activity in the basolateral amygdala (BLA) and stronger fear conditioning, as well as deficient extinction in stress-enhanced fear learning (SEFL). rAAV2-retro-based tracing combined with visualization of the GFP knocked in the Grp gene showed that BLA receives GRPergic or conditioned stimulus projections from the indirect auditory thalamus-to-auditory cortex pathway, ventral hippocampus and ventral tegmental area. Transcription of dopamine-related genes was decreased in BLA of Grp-/- mice following SEFL extinction recall, suggesting that the GRP may mediate fear extinction regulation by dopamine. Impact statementMice deficient for the amygdala-enriched gastrin-releasing peptide gene are susceptible to stress-enhanced fear, a behavioral protocol with relevance to PTSD, and show a decrease in dopamine-related gene transcription.

A single, severe episode of stress can bring about myriad responses amongst individuals, ranging from cognitive enhancement to debilitating and persistent anxiety; however, the biological mechanisms that contribute to resilience versus susceptibility to stress are poorly understood. The dentate gyrus (DG) of the hippocampus and the basolateral nucleus of the amygdala (BLA) are key limbic regions that are susceptible to the neural and hormonal effects of stress. Previous work has also shown that these regions contribute to individual variability in stress responses; however, the molecular mechanisms underlying the role of these regions in susceptibility and resilience are unknown. In this study, we profiled the transcriptomic signatures of the DG and BLA of rats with divergent behavioral outcomes after a single, severe stressor. We subjected rats to three hours of immobilization with exposure to fox urine and conducted a behavioral battery one week after stress to identify animals that showed persistent, high anxiety-like behavior. We then conducted bulk RNA sequencing of the DG and BLA from susceptible, resilient, and unexposed control rats. Differential gene expression analyses revealed that the molecular signatures separating each of the three groups were distinct and non-overlapping between the DG and BLA. In the amygdala, key genes associated with insulin and hormonal signaling corresponded with vulnerability. Specifically, Inhbb, Rab31, and Ncoa3 were upregulated in the amygdala of stress-susceptible animals compared to resilient animals. In the hippocampus, increased expression of Cartpt - which encodes a key neuropeptide involved in reward, reinforcement, and stress responses - was strongly correlated with vulnerability to anxiety-like behavior. However, few other genes distinguished stress-susceptible animals from control animals, while a larger number of genes separated stress-resilient animals from control and stress-susceptible animals. Of these, Rnf112, Tbx19, and UBALD1 distinguished resilient animals from both control and susceptible animals and were downregulated in resilience, suggesting that an active molecular response in the hippocampus facilitates protection from the long-term consequences of severe stress. These results provide novel insight into the mechanisms that bring about individual variability in the behavioral responses to stress and provide new targets for the advancement of therapies for stress-induced neuropsychiatric disorders.

IntroductionAdolescence is a sensitive developmental period during which chronic stress can induce lasting adaptations in corticolimbic circuits involved in stress regulation, cognition, and emotional behavior. We examined the long-term behavioral, endocrine, and molecular consequences of adolescent chronic variable stress (CVS) in male and female rats, focusing on the infralimbic cortex (IL) and basolateral amygdala (BLA) MethodsSprague Dawley rats of both sexes were exposed to CVS during late adolescence and evaluated in adulthood after an extensive recovery period. Behavioral testing included cued fear conditioning and extinction recall, delayed spatial win-shift, novel object recognition, Morris water maze, three-chamber social behavior, and passive avoidance. HPA-axis reactivity to acute restraint was assessed. Targeted qPCR was used to measure stress-related gene expression in the IL and BLA immediately after stress or after a 5-week recovery period ResultsAdolescent CVS did not cause generalized cognitive impairment, but instead produced selective, sex-specific effects. Females had reduced HPA responses to acute stress and mild deficits in delayed spatial win-shift performance, together with long-term IL changes in genes related to adrenergic signaling, plasticity, and GABA clearance. Males showed enhanced Morris water maze probe retention, weaker novel object discrimination, altered passive avoidance with marked inter-individual variability, and enhanced social preference. At the molecular level, males exhibited long-term upregulation of Fkbp5 in IL and downregulation of PACAP, 1D adrenergic receptor, and proenkephalin in BLA, whereas females showed delayed PACAP upregulation in BLA DiscussionAdolescent CVS induces persistent, sex- and region-specific recalibration of corticolimbic function, supporting distinct patterns of vulnerability and resilience, rather than uniform stress pathology.

Traumatic events increase the risk for anxiety disorders, yet knowledge of how trauma modulates neuronal activity to induce anxiety is incomplete. The amygdala, which processes stressful sensory information, is enriched with interneurons that release the anxiolytic neurotransmitter neuropeptide Y (NPY). Amygdala NPY levels are reduced one week after an aversive event, suggesting chronic alteration of NPY+ interneurons; however, studies of in vivo amygdalar NPY+ cell activity during stressors are lacking. Here, we use a genetically encoded calcium sensor together with fiber photometry to investigate in vivo activation of NPY+ cells in basolateral amygdala (BLA) to aversive stimuli in mice. NPY+ cell activation was evaluated in response to two aversive stimuli, air puffs to the face (mild) and footshocks (strong). Air puffs caused a transient elevation of calcium in BLA NPY+ cells, indicating robust neuronal activation, in both male and female mice with no sex-dependent differences. Interestingly, there was habituation of the calcium signal in NPY+ cells to later air puff iterations. Strong footshocks also caused calcium elevation in both male and female mice with no sex-dependent differences. Excitingly, footshock induces a larger calcium response compared to air-puff. In contrast to air puff, the calcium signal to footshock was prolonged in later iterations. BLA NPY+ cell calcium signals were consistent in response to the same footshock protocol delivered 1 week later, indicating that activation of NPY+ cells by footshock is stable across this timeframe. Taken together, these results reveal a potential role for NPY+ interneurons in basolateral amygdala during aversive events.

The amygdala plays an important role in the regulation of stress and anxiety. However, little is known about the relationship between amygdala connectivity and subsequent stress-induced behavior. The current study investigated whether amygdala connectivity measured before experiencing stress is a predisposing neural feature of subsequent stress-induced behavior while individuals face an emergent and unexpected event like the COVID-19 outbreak. Using an fMRI cohort established before the pandemic in Wuhan, Hubei, we found that resting-state functional connectivity (rsFC) of the right amygdala with the dorsomedial prefrontal cortex (dmPFC) was negatively correlated with the stress-induced behavior of these volunteers during the COVID-2019 outbreak in Hubei. Furthermore, the self-connection of the right amygdala, inferred using dynamic causal modeling, was negatively correlated with stress-induced behavior in this cohort. A significant correlation between the right amygdala-dmPFC rsFC and self-connection of the right amygdala was found. Additionally, after three months of the COVID-19 outbreak in Hubei when the stressor weakened - and in another cohort collected in regions outside Hubei where the individuals experienced a lower level of stress - the relationship between the amygdala-dmPFC rsFC and the stress-induced behavior disappeared. Our findings support that amygdala connectivity is a predisposing neural feature of stress-induced behavior in the COVID-19 outbreak in Hubei, suggesting the amygdala connectivity before stress predicts subsequent behavior while facing an emergent and unexpected event. And thus our findings provide an avenue for identifying individuals vulnerable to stress using intrinsic brain function before stress as an indicator.

Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.

Traumatic stress can cause long-lasting changes in cognition and affect, sometimes leading to diagnoses such as post-traumatic stress disorder (PTSD). The stress-enhanced fear learning (SEFL) model recapitulates understudied components of PTSD, such as stress-induced sensitization of fear learning. The SEFL procedure entails exposing mice to footshock stress followed later by fear conditioning in a different context. When tested later for recall of fear conditioning, previously stressed mice exhibit enhanced freezing compared to non-stressed controls. Studies have shown that dorsal and ventral dentate gyrus (DG) generates neural ensemble representations of contextual fear, such that fear recall involves reactivation of a sparse set of "engram cells" that were active during fear memory acquisition. How stress affects these hippocampal ensemble representations is unknown. We used SEFL and activity-dependent neuronal tagging with FosTRAP2 mice to investigate effects of stress on fear memory ensembles in rostral and caudal hippocampal DG. FosTRAP2/Ai6 mice received footshock stress or equivalent context exposure without shock in Context A on day 1. Five days later, mice received 1-shock conditioning in Context B and immediately received an injection of 4-OHT (55mg/kg) to tag fear acquisition neurons with the zsGreen reporter. One day later, mice were tested for fear recall in Context B and were perfused 90 minutes after testing. Confirming prior studies, prior stress potentiated 1-shock conditioning in Context B, with stressed mice displaying higher freezing in the Context B test session than non-stressed mice. At the level of neural activity, results showed stress had no effect on the number of zsGreen+ fear ensemble cells or the number of cfos+ recall-activated cells in rostral or caudal DG. However, stress increased reactivation (percentage of zsGreen+ cells expressing cfos) in the caudal but not rostral DG. The results suggest stress potentiates later fear learning by enhancing fear representations in caudal hippocampus, a region of the hippocampus specialized for integrating emotional and motivational valence into memory.