Microglia-mediated calcium-permeable AMPAR accumulation in the nucleus accumbens drives hyperlocomotion during cocaine withdrawal.

During withdrawal from cocaine, calcium permeable-AMPA receptors (CP-AMPAR) progressively accumulate in nucleus accumbens (NAc) synapses, a phenomenon linked to behavioral sensitization and drug-seeking. Recently, it has been suggested that neuroimmune alterations might promote aberrant changes in synaptic plasticity, thus contributing to substance abuse-related behaviors. Here, we investigated the role of microglia in NAc neuroadaptations after withdrawal from cocaine-induced conditioned place preference (CPP). We depleted microglia using PLX5622-supplemented diet during cocaine withdrawal, and after the place preference test, we measured dendritic spine density and the presence of CP-AMPAR in the NAc shell. Microglia depletion prevented cocaine-induced changes in dendritic spines and CP-AMPAR accumulation. Furthermore, microglia depletion prevented conditioned hyperlocomotion without affecting drug-context associative memory. Microglia displayed fewer number of branches, resulting in a reduced arborization area and microglia control domain at late withdrawal. Our results suggest that microglia are necessary for the synaptic adaptations in NAc synapses during cocaine withdrawal and therefore represent a promising therapeutic target for relapse prevention.

A mouse model of the 3-hit effects of stress: Genotype controls the effects of life adversities in females.

Helplessness is a dysfunctional coping response to stressors associated with different psychiatric conditions. The present study tested the hypothesis that early and adult adversities cumulate to produce helplessness depending on the genotype (3-hit hypothesis of psychopathology). To this aim, we evaluated whether Chronic Unpredictable Stress (CUS) differently affected coping and mesoaccumbens dopamine (DA) responses to stress challenge by adult mice of the C57BL/6J (B6) and DBA/2J (D2) inbred strains depending on early life experience (Repeated Cross Fostering, RCF). Three weeks of CUS increased the helplessness expressed in the Forced Swimming Test (FST) and the Tail Suspension Test by RCF-exposed female mice of the D2 strain. Moreover, female D2 mice with both RCF and CUS experiences showed inhibition of the stress-induced extracellular DA outflow in the Nucleus Accumbens, as measured by in vivo microdialysis, during and after FST. RCF-exposed B6 mice, instead, showed reduced helplessness and increased mesoaccumbens DA release. The present results support genotype-dependent additive effects of early experiences and adult adversities on behavioral and neural responses to stress by female mice. To our knowledge, this is the first report of a 3-hit effect in an animal model. Finally, the comparative analyses of behavioral and neural phenotypes expressed by B6 and D2 mice suggest some translationally relevant hypotheses of genetic risk factors for psychiatric disorders.

Repeated witness social stress causes cardiomyocyte contractile impairment and intracellular Ca2+ derangement in female rats.

The impact of psychosocial stressors on cardiovascular health in women is of growing interest in both the popular and scientific literature. Rodent models are useful for providing direct experimental evidence of the adverse cardiovascular consequences of psychosocial stressors, yet studies in females are scarce. Here, we investigated the effects of repeated exposure to witness social defeat stress (WS) on cardiomyocyte contractile function and intracellular Ca2+ homeostasis in young adult wild-type Groningen female rats. Female rats bore witness to an aggressive social defeat episode between two males for nine consecutive days or were exposed to a control procedure. Stress-related behaviors were assessed during the first and last WS/control exposure. Twenty-four hours after the last exposure, plasma corticosterone levels were measured, and cardiomyocytes were isolated for analyses of contractile properties and Ca2+ transients, and expression levels of proteins involved in intracellular Ca2+dynamics. The results show an impairment of the intrinsic cardiac mechanical properties and prolonged intracellular Ca2+decay in WS female rats showing social stress-related behavioral (larger amounts of burying behavior) and neuroendocrine (elevated plasma corticosterone levels) phenotypes. Further, the results implicate alterations in the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex in the contractile defects described in cardiomyocytes of WS female rats. In conclusion, this study highlights the utility of the WS model as an ethologically relevant social stressor for investigating pathophysiological processes that occur in the heart of female subjects and may increase vulnerability to social stress-related cardiovascular risk.

GABAergic miR-34a regulates Dorsal Raphè inhibitory transmission in response to aversive, but not rewarding, stimuli.

The brain employs distinct circuitries to encode positive and negative valence stimuli, and dysfunctions of these neuronal circuits have a key role in the etiopathogenesis of many psychiatric disorders. The Dorsal Raphè Nucleus (DRN) is involved in various behaviors and drives the emotional response to rewarding and aversive experiences. Whether specific subpopulations of neurons within the DRN encode these behaviors with different valence is still unknown. Notably, microRNA expression in the mammalian brain is characterized by tissue and neuronal specificity, suggesting that it might play a role in cell and circuit functionality. However, this specificity has not been fully exploited. Here, we demonstrate that microRNA-34a (miR-34a) is selectively expressed in a subpopulation of GABAergic neurons of the ventrolateral DRN. Moreover, we report that acute exposure to both aversive (restraint stress) and rewarding (chocolate) stimuli reduces GABA release in the DRN, an effect prevented by the inactivation of DRN miR-34a or its genetic deletion in GABAergic neurons in aversive but not rewarding conditions. Finally, miR-34a inhibition selectively reduced passive coping with severe stressors. These data support a role of miR-34a in regulating GABAergic neurotransmitter activity and behavior in a context-dependent manner and suggest that microRNAs could represent a functional signature of specific neuronal subpopulations with valence-specific activity in the brain.

Natural killer cells and innate lymphoid cells 1 tune anxiety-like behavior and memory in mice via interferon-γ and acetylcholine.

The mechanisms of communication between the brain and the immune cells are still largely unclear. Here, we characterize the populations of resident natural killer (NK) cells and innate lymphoid cells (ILC) 1 in the meningeal dura layer of adult mice. We describe that ILC1/NK cell-derived interferon-γ and acetylcholine can contribute to the modulation of brain homeostatic functions, shaping synaptic neuronal transmission and neurotransmitter levels with effects on mice behavior. In detail, the interferon-γ plays a role in the formation of non-spatial memory, tuning the frequency of GABAergic neurotransmission on cortical pyramidal neurons, while the acetylcholine is a mediator involved in the modulation of brain circuitries that regulate anxiety-like behavior. These findings disclose mechanisms of immune-to-brain communication that modulate brain functions under physiological conditions.

The long-lasting effects of early life adversities are sex dependent: The signature of miR-34a.

BACKGROUND: Exposure to early life adversities (ELA) can influence a plethora of biological mechanisms leading to stress-related disorders later in life through epigenetic mechanisms, such as microRNAs (miRs). MiR-34 is a critical modulator of stress response and stress-induced pathologies and a link between ELA and miR-34a has been reported. METHODS: Here using our well-established model of ELA (Repeated Cross Fostering) we investigate the behavioral long-term effects of ELA in male and female mice. We also assess basal and ELA-induced miR-34a expression in adult mice and investigate whether ELA affects the later miR-34a response to adult acute stress exposure across brain areas (medial preFrontal Cortex, Dorsal Raphe Nuclei) and peripheral organs (heart, plasma) in animals from both sexes. Finally, based on our previous data demonstrating the critical role of Dorsal Raphe Nuclei miR-34a expression in serotonin (5-HT) transmission, we also investigated prefrontal-accumbal 5-HT outflow induced by acute stress exposure in ELA and Control females by in vivo intracerebral microdialysis. RESULTS: ELA not just induces a depressive-like state as well as enduring changes in miR-34a expression, but also alters miR-34a expression in response to adult acute stress exclusively in females. Finally, altered DRN miR-34a expression is associated with prefrontal-accumbal 5-HT release under acute stress exposure in females. LIMITATIONS: Translational study on humans is necessary to verify the results obtained in our animal models of ELA-induced depression. CONCLUSIONS: This is the first evidence showing long-lasting sex related effects of ELA on brain and peripheral miR-34a expression levels in an animal model of depression-like phenotype.

Decline of cardiomyocyte contractile performance and bioenergetic function in socially stressed male rats.

Chronic social stress has been epidemiologically linked to increased risk for cardiovascular disease, yet the underlying pathophysiological mechanisms are still largely elusive. Mitochondrial (dys)function represents a potential intersection point between social stress exposure and (mal)adaptive cardiac responses. In this study, we used a rodent model of social stress to study the extent to which alterations in the cellular mechanical properties of the heart were associated with changes in indexes of mitochondrial function. Male adult rats were exposed to repeated episodes of social defeat stress or left undisturbed (controls). ECG signals were recorded during and after social defeat stress. Twenty-four hours after the last social defeat, cardiomyocytes were isolated for analyses of mechanical properties and intracellular Ca2+ dynamics, mitochondrial respiration, and ATP content. Results indicated that social defeat stress induced potent cardiac sympathetic activation that lasted well beyond stress exposure. Moreover, cardiomyocytes of stressed rats showed poor contractile performance (e.g., slower contraction and relaxation rates) and intracellular Ca2+ derangement (e.g., slower Ca2+ clearing), which were associated with indexes of reduced reserve respiratory capacity and decreased ATP production. In conclusion, this study suggests that repeated social stress provokes impaired cardiomyocyte contractile performance and signs of altered mitochondrial bioenergetics in the rat heart. Future studies are needed to clarify the causal link between cardiac and mitochondrial functional remodeling under conditions of chronic social stress.

Early life adversity affecting the attachment bond alters ventral tegmental area transcriptomic patterning and behavior almost exclusively in female mice.

Early life experiences that affect the attachment bond formation can alter developmental trajectories and result in pathological outcomes in a sex-related manner. However, the molecular basis of sex differences is quite unknown. The dopaminergic system originating from the ventral tegmental area has been proposed to be a key mediator of this process. Here we exploited a murine model of early adversity (Repeated Cross Fostering, RCF) to test how interfering with the attachment bond formation affects the VTA-related functions in a sex-specific manner. Through a comprehensive behavioral screening, within the NiH RDoC framework, and by next-generation RNA-Seq experiments, we analyzed the long-lasting effect of RCF on behavioral and transcriptional profiles related to the VTA, across two different inbred strains of mouse in both sexes. We found that RCF impacted to an extremely greater extent VTA-related behaviors in females than in males and this result mirrored the transcriptional alterations in the VTA that were almost exclusively observed in females. The sexual dimorphism was conserved across two different inbred strains in spite of their divergent long lasting consequences of RCF exposure. Our data suggest that to be female primes a sub-set of genes to respond to early environmental perturbations. This is, to the best of our knowledge, the first evidence of an almost exclusive effect of early life experiences on females, thus mirroring the extremely stronger impact of precocious aversive events reported in clinical studies in women.

Mouse model of panic disorder: Vulnerability to early environmental instability is strain-dependent.

Early life experiences and genetic background shape phenotypic variation. Several mouse models based on early treatments have evaluated short- and long-term phenotypic alterations and explored their molecular mechanisms. The instability of maternal cues was used to model human separation anxiety in outbred mice, one of the etiopathogenetic factors that predict panic disorder (PD). Application of the repeated cross-fostering (RCF) protocol to inbred strains (C57 and DBA) allowed us to measure differential responses to the same experimental manipulation. Ultrasounds emitted during isolation indicated that after RCF, pups from both strains lose their ability to be comforted by nest cues, but the frequency modulation of separation calls increased in RCF-C57 and decreased in RCF-DBA mice. No strain-specific difference in olfactory ability explained these responses in RCF-exposed mice. Rather, disruption of the infant-mother bond may differentially affect separation calls in the two strains. Moreover, the RCF-associated increased respiratory response to hypercapnia-an endophenotype of human PD documented among mice outbred strains-was replicated in the C57 strain only. We suggest that RCF-induced instability of the early environment affects emotionality and respiratory physiology differentially, depending on pups' genetic background. These strain-specific responses provide a lead to understand differential vulnerability to emotional disorders.

Elevated miR-34a expression and altered transcriptional profile are associated with adverse electromechanical remodeling in the heart of male rats exposed to social stress.

This study investigated epigenetic risk factors that may contribute to stress-related cardiac disease in a rodent model. Experiment 1 was designed to evaluate the expression of microRNA-34a (miR-34a), a known modulator of both stress responses and cardiac pathophysiology, in the heart of male adult rats exposed to a single or repeated episodes of social defeat stress. Moreover, RNA sequencing was conducted to identify transcriptomic profile changes in the heart of repeatedly stressed rats. Experiment 2 was designed to assess cardiac electromechanical changes induced by repeated social defeat stress that may predispose rats to cardiac dysfunction. Results indicated a larger cardiac miR-34a expression after repeated social defeat stress compared to a control condition. This molecular modification was associated with increased vulnerability to pharmacologically induced arrhythmias and signs of systolic left ventricular dysfunction. Gene expression analysis identified clusters of differentially expressed genes in the heart of repeatedly stressed rats that are mainly associated with morphological and functional properties of the mitochondria and may be directly regulated by miR-34a. These results suggest the presence of an association between miR-34a overexpression and signs of adverse electromechanical remodeling in the heart of rats exposed to repeated social defeat stress, and point to compromised mitochondria efficiency as a potential mediator of this link. This rat model may provide a useful tool for investigating the causal relationship between miR-34a expression, mitochondrial (dys)function, and cardiac alterations under stressful conditions, which could have important implications in the context of stress-related cardiac disease.

Resilience to anhedonia-passive coping induced by early life experience is linked to a long-lasting reduction of Ih current in VTA dopaminergic neurons.

Exposure to aversive events during sensitive developmental periods can affect the preferential coping strategy adopted by individuals later in life, leading to either stress-related psychiatric disorders, including depression, or to well-adaptation to future adversity and sources of stress, a behavior phenotype termed "resilience". We have previously shown that interfering with the development of mother-pups bond with the Repeated Cross Fostering (RCF) stress protocol can induce resilience to depression-like phenotype in adult C57BL/6J female mice. Here, we used patch-clamp recording in midbrain slice combined with both in vivo and ex vivo pharmacology to test our hypothesis of a link between electrophysiological modifications of dopaminergic neurons in the intermediate Ventral Tegmental Area (VTA) of RCF animals and behavioral resilience. We found reduced hyperpolarization-activated (Ih) cation current amplitude and evoked firing in VTA dopaminergic neurons from both young and adult RCF female mice. In vivo, VTA-specific pharmacological manipulation of the Ih current reverted the pro-resilient phenotype in adult early-stressed mice or mimicked behavioral resilience in adult control animals. This is the first evidence showing how pro-resilience behavior induced by early events is linked to a long-lasting reduction of Ih current and excitability in VTA dopaminergic neurons.

MicroRNA-34a regulates 5-HT2C expression in dorsal raphe and contributes to the anti-depressant-like effect of fluoxetine.

Selective serotonin reuptake inhibitors (SSRIs) are designed to improve mood by raising extracellular serotonin levels through the blockade of the serotonin transporter. However, they exhibit a slow onset of action, suggesting the involvement of adaptive regulatory mechanisms. We hypothesized that the microRNA-34 family facilitates the therapeutic activity of SSRIs. We show that genetic deletion of these microRNAs in mice impairs the response to chronic, but not acute, fluoxetine treatment, with a specific effect on behavioral constructs that are related to depression, rather than anxiety. Moreover, using a pharmacological strategy, we found that an increased expression of the serotonin 2C (5-HT2C) receptor in the dorsal raphe region of the brain contributes to this phenotype. The onset of the therapeutic efficacy of SSRIs is paralleled by the desensitization of the 5-HT2C receptor in the dorsal raphe, and 5-HT2C is a putative target of microRNA-34. In this study, acute and chronic fluoxetine treatment differentially alters the expression of 5-HT2C and microRNA-34a in the dorsal raphe. Moreover, by in vitro luciferase assay, we demonstrated the repressive regulatory activity of microRNA-34a against 5-HT2C mRNA. Specific blockade of this interaction through local infusion of a target site blocker was sufficient to prevent the behavioral effects of chronic fluoxetine. Our results demonstrate a new miR-34a-mediated regulatory mechanism of 5-HT2C expression in the dorsal raphe and implicate it in eliciting the behavioral responses to chronic fluoxetine treatment.

Interactions Between Experience, Genotype and Sex in the Development of Individual Coping Strategies.

Coping strategies, the first line of defense against adversities, develop through experience. There is consistent evidence that both genotype and sex contribute to the development of dysfunctional coping, leading to maladaptive outcomes of adverse experiences or to adaptive coping that fosters rapid recovery even from severe stress. However, how these factors interact to influence the development of individual coping strategies is just starting to be investigated. In the following review, we will consider evidence that experience, sex, and genotype influence the brain circuits and neurobiological processes involved in coping with adversities and discuss recent results pointing to the specific effects of the interaction between early experiences, genotype, and stress in the development of functional and dysfunctional coping styles.

Long term effects of early life stress on HPA circuit in rodent models.

Adaptation to environmental challenges represents a critical process for survival, requiring the complex integration of information derived from both external cues and internal signals regarding current conditions and previous experiences. The Hypothalamic-pituitary-adrenal axis plays a central role in this process inducing the activation of a neuroendocrine signaling cascade that affects the delicate balance of activity and cross-talk between areas that are involved in sensorial, emotional, and cognitive processing such as the hippocampus, amygdala, Prefrontal Cortex, Ventral Tegmental Area, and dorsal raphe. Early life stress, especially early critical experiences with caregivers, influences the functional and structural organization of these areas, affects these processes in a long-lasting manner and may result in long-term maladaptive and psychopathological outcomes, depending on the complex interaction between genetic and environmental factors. This review summarizes the results of studies that have modeled this early postnatal stress in rodents during the first 2 postnatal weeks, focusing on the long-term effects on molecular and structural alteration in brain areas involved in Hypothalamic-pituitary-adrenal axis function. Moreover, a brief investigation of epigenetic mechanisms and specific genetic targets mediating the long-term effects of these early environmental manipulations and at the basis of differential neurobiological and behavioral effects during adulthood is provided.

Dissecting major depression: The role of blood biomarkers and adverse childhood experiences in distinguishing clinical subgroups.

BACKGROUND: The syndromic diagnosis of major depressive disorder (MDD) is associated with individual differences in prognosis, course, treatment response, and outcome. There is evidence that patients with a history to adverse childhood experiences (ACEs) may belong to a distinct clinical subgroup. The combination of data on ACEs and blood biomarkers could allow the identification of diagnostic MDD subgroups. METHODS: We selected several blood markers (global DNA methylation, and VEGF-a, TOLLIP, SIRT1, miR-34a genes) among factors that contribute to the pathogenetic mechanisms of MDD. We examined their level in 37 MDD patients and 30 healthy subjects. ACEs were measured by the Parental Bonding Instrument and the Childhood Trauma Questionnaire. RESULTS: We found significant differences between patients and healthy subjects in three biomarkers (TOLLIP, VEGF-a, and global DNA methylation), independently from the confounding effect of parental care received. By contrast, SIRT1 differences were modulated by quality of parental care. The lowest levels of SIRT1 were recorded in patients with active symptoms and low maternal/paternal care. miR-34a and SIRT1 levels were associated with MDD symptoms especially in early-life stressed patients. LIMITATIONS: Small sample size, no information on personality comorbidity and suicidal history, cross-sectional definition of remission, and lack of follow-up. CONCLUSIONS: Our findings suggest that the levels of global DNA methylation, TOLLIP, and VEGF-a reflect pathophysiological changes associated with MDD that are independent from the long-term effects of low parental care. This study also suggests that SIRT1 may be an additional variable distinguishing the ecophenotype that includes MDD patients with exposure to ACEs.

RISC RNA sequencing in the Dorsal Raphè reveals microRNAs regulatory activities associated with behavioral and functional adaptations to chronic stress.

The Dorsal Raphe (DR) is the primary source of serotonergic input in the brain and a center for the homeostatic maintenance of the serotonergic tone. Under repeated stimulation, it can undergo adaptive modifications that alter serotonergic neurotransmission, which can lead to behavioral dysfunction. Post-transcriptional regulation by microRNAs is implicated in these adaptations. However, a global microRNA/target network effect on the DR neuroplasticity has yet to be elucidated. Here we investigate the microRNAs/mRNAs regulatory activity in the mouse DR after a chronic stress experience. First, we assessed the behavioral consequences of repeated restraint stress exposure and the functional adaptations of the DR by measuring the change in acute stress-induced serotonin release. Then, through next generation RNA-Seq of Argonaute2-bound RNA (RISC-Seq) we identified microRNAs and their targets that are associated to the RISC complex of the DR in unstressed and stressed mice. We mapped the potential microRNA/mRNA network within the stress-altered transcripts, uncovering new interactions that contribute to the chronic stress-induced DR modifications.

Xlr4 as a new candidate gene underlying vulnerability to cocaine effects.

Although several studies have been performed in rodents, non-human primates and humans, the biological basis of vulnerability to develop cocaine addiction remains largely unknown. Exposure to critical early events (as Repeated Cross Fostering (RCF)) has been reported to increase sensitivity to cocaine effects in adult C57BL/6J female mice. Using a microarray approach, here we report data showing a strong engagement of X-linked lymphocyte-regulated 4a and 4b (Xlr4) genes in cocaine effects. The expression of Xlr4, a gene involved in chromatin remodeling and dendritic spine morphology, was reduced into the Nucleus Accumbens (NAc) of adult RCF C57BL/6J female. We used virally mediated accumbal Xlr4 down-modulation (AAVXlr4-KD) to investigate the role of this gene in vulnerability to cocaine effects. AAVXlr4-KD animals show a potentiated behavioral and neurochemical response to cocaine, reinstatement following cocaine withdrawal and cocaine-induced spine density alterations in the Medium-Sized Spiny Neurons of NAc. We propose Xlr4 as a new candidate gene mediating the cocaine effects.

MicroRNA-34a Regulates the Depression-like Behavior in Mice by Modulating the Expression of Target Genes in the Dorsal Raphè.

Chronic stress exposure is known to increase vulnerability to the expression of psychiatric disorders, such as depression. Clinical and preclinical evidences support the involvement of the microRNA-34 family in stress-related psychiatric conditions and in the regulation of stress responses. However, the mechanism and the multiple targets by which the microRNA-34 family can affect the stress response and stress-related behavioral alteration are not fully known. Here, with the aid of constitutive and conditional genetic strategy, we examined the role of microRNA-34 family in the expression of depression-like phenotype in mice induced by chronic stress exposure, and we identified their "in vivo" targets during the stressful challenge. We found that microRNA-34a, under chronic stress, is significantly up-regulated in the mouse raphe nuclei, where its recruitment is necessary to induce depression-like behavioral alterations and impact the function of the serotonergic system. Moreover, by next-generation RNA-seq of Ago-2-bound mRNAs, we identified genes that are targeted by microRNA-34a in response to chronic stress and that are likely to mediate its effects.

Correction to: MicroRNA-34 Contributes to the Stress-related Behavior and Affects 5-HT Prefrontal/GABA Amygdalar System through Regulation of Corticotropin-releasing Factor Receptor 1.

The original version of this article unfortunately contained a mistake in Figure 3. The drawing superimposed on photomicrographs to identify the region of Dorsal raphè Nuclei was inappropriately positioned. The corrected figure is given below.

Sex-dependent effects of early unstable post-natal environment on response to positive and negative stimuli in adult mice.

Alterations in early environmental conditions that interfere with the creation of a stable mother-pup bond have been suggested to be a risk factor for the development of stress-related psychopathologies later in life. The long-lasting effects of early experiences are mediated by changes in various cerebral circuits, such as the corticolimbic system, which processes aversive and rewarding stimuli. However, it is evident that the early environment is not sufficient per se to induce psychiatric disorders; interindividual (eg, sex-based) differences in the response to environmental challenges exist. To examine the sex-related effects that are induced by an early experience on later events in adulthood, we determine the enduring effects of repeated cross-fostering (RCF) in female and male C57BL/6J mice. To this end, we assessed the behavioral phenotype of RCF and control (male and female) mice in the saccharine preference test and cocaine-induced conditioned place preference to evaluate the response to natural and pharmacological stimuli and in the elevated plus maze test and forced swimming test to measure their anxiety- and depression-like behavior. We also evaluated FST-induced c-Fos immunoreactivity in various brain regions that are engaged in the response to acute stress exposure (FST). Notably, RCF has opposing effects on the adult response to these tests between sexes, directing male mice toward an "anhedonia-like" phenotype and increasing the sensitivity for rewarding stimuli in female mice.