Unstable Maternal Environment Affects Stress Response in Adult Mice in a Genotype-Dependent Manner.

Early postnatal events exert powerful effects on development, inducing persistent functional alterations in different brain network, such as the catecholamine prefrontal-accumbal system, and increasing the risk of developing psychiatric disorders later in life. However, a vast body of literature shows that the interaction between genetic factors and early environmental conditions is crucial for expression of psychopathologies in adulthood. We evaluated the long-lasting effects of a repeated cross-fostering (RCF) procedure in 2 inbred strains of mice (C57BL/6J, DBA/2), known to show a different susceptibility to the development and expression of stress-induced psychopathologies. Coping behavior (forced swimming test) and preference for a natural reinforcing stimulus (saccharine preference test) were assessed in adult female mice of both genotypes. Moreover, c-Fos stress-induced activity was assessed in different brain regions involved in stress response. In addition, we evaluated the enduring effects of RCF on catecholamine prefrontal-accumbal response to acute stress (restraint) using, for the first time, a new "dual probes" in vivo microdialysis procedure in mouse. RCF experience affects behavioral and neurochemical responses to acute stress in adulthood in opposite direction in the 2 genotypes, leading DBA mice toward an "anhedonic-like" phenotype and C57 mice toward an increased sensitivity for a natural reinforcing stimulus.

Linking drug and food addiction: an overview of the shared neural circuits and behavioral phenotype.

Despite a lack of agreement on its definition and inclusion as a specific diagnosable disturbance, the food addiction construct is supported by several neurobiological and behavioral clinical and preclinical findings. Recognizing food addiction is critical to understanding how and why it manifests. In this overview, we focused on those as follows: 1. the hyperpalatable food effects in food addiction development; 2. specific brain regions involved in both food and drug addiction; and 3. animal models highlighting commonalities between substance use disorders and food addiction. Although results collected through animal studies emerged from protocols differing in several ways, they clearly highlight commonalities in behavioral manifestations and neurobiological alterations between substance use disorders and food addiction characteristics. To develop improved food addiction models, this heterogeneity should be acknowledged and embraced so that research can systematically investigate the role of specific variables in the development of the different behavioral features of addiction-like behavior in preclinical models.

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.

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.

Evidence for a we-representation in monkeys when acting together.

A hallmark of human evolution resides in the ability to adapt our actions to those of others. This aptitude optimizes collective behavior, allowing to achieve goals unattainable by acting alone. We have previously shown that macaque monkeys are able to coordinate their actions when engaged in dyadic contexts, therefore they offer a good model to study the roots of joint action. Here, we analyze the behavior of five macaques required to perform visuomotor isometric tasks, either individually or together with a partner. By pre-cueing or not the future action condition (SOLO or TOGETHER) we investigated the existence of a 'We-representation' in monkeys. We found that pre-instructing the action context improves the dyadic performance, thanks to the emergence of an optimal kinematic setting, that facilitates inter-individual motor coordination. Our results offer empirical evidence of a 'We-representation' in macaques, that when evoked provides an overall beneficial effect on joint performance.

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.

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.

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.

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.

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.

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.

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.

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.

Parieto-frontal gradients and domains underlying eye and hand operations in the action space.

In monkeys, motor intention in its different forms emerges from a parietal-frontal gradient of visual, eye and hand signals, containing discrete dominant domains. These are formed by areas sharing cortical connections and functional properties. Within this gradient, the combination of different inputs determines the tuning properties of neurons, while local and long cortico-cortical connections shape the structure and temporal delays of the network. The pathways linking similar functional domains in parietal and frontal cortex sculpt information processing systems related to different functions, all requiring eye-hand coordination. fMRI experiments show that similar gradients lay at the core of cognitive-motor control in humans as well. This eye-hand matrix provides a framework to address, within a unitary frame, not only basic forms of motor behavior, such as reaching and grasping, but also actions of increasing complexity, such as interception of moving targets, tool use, construction of complex objects, maze analysis and solution, among others. The organization of the cerebral cortex into functional gradients and domains, beyond frontal and parietal cortices, is common to other brain regions, such as prefrontal cortex and hippocampus, and does not support views of the parieto-frontal operations based on specific and strictly segregated eye and hand modules. These can only be found at the eye and hand motor output domains in the frontal cortex, that is in the frontal eye fields and in the primary motor cortex, respectively.