RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Altered gene expression and spine density in nucleus accumbens of adolescent and adult male mice exposed to emotional and physical stress.

Stressful early life experiences are implicated in lifelong health. However, little is known about the consequences of emotional stress (ES) or physical stress (PS) on neurobiology. Therefore, the following set of experiments was designed to assess changes in transcription and translation of key proteins within the nucleus accumbens (NAc). Male adolescent (postnatal day 35) or adult (8-week-old) mice were exposed to ES or PS using a witness social defeat paradigm. Then, 24 h after the last stress session, we measured levels of specific mRNAs and proteins within the NAc. Spine density was also assessed in separate groups of mice. Exposure to ES or PS disrupted extracellular signal-related kinase 2 (ERK2), reduced transcription of ΔFosB and had no effect on cAMP response element-binding protein (CREB) mRNA. Western blots revealed that exposure to ES or PS decreased ERK2 phosphorylation in adolescents, whereas the same stress regimen increased ERK2 phosphorylation in adults. Exposure to ES or PS had no effect on ΔFosB or CREB phosphorylation. ES and PS increased spine density in the NAc of adolescent exposed mice, but only exposure to PS increased spine density in adults. Together, these findings demonstrate that exposure to ES or PS is a potent stressor in adolescent and adult mice and can disturb the integrity of the NAc by altering transcription and translation of important signaling molecules in an age-dependent manner. Furthermore, exposure to ES and PS induces substantial synaptic plasticity of the NAc.

Sex-Specific Regulation of Stress Susceptibility by the Astrocytic Gene Htra1.

Major depressive disorder (MDD) is linked to impaired structural and synaptic plasticity in limbic brain regions. Astrocytes, which regulate synapses and are influenced by chronic stress, likely contribute to these changes. We analyzed astrocyte gene profiles in the nucleus accumbens (NAc) of humans with MDD and mice exposed to chronic stress. Htra1 , which encodes an astrocyte-secreted protease targeting the extracellular matrix (ECM), was significantly downregulated in the NAc of males but upregulated in females in both species. Manipulating Htra1 in mouse NAc astrocytes bidirectionally controlled stress susceptibility in a sex-specific manner. Such Htra1 manipulations also altered neuronal signaling and ECM structural integrity in NAc. These findings highlight astroglia and the brain's ECM as key mediators of sex-specific stress vulnerability, offering new approaches for MDD therapies.

Alprazolam exposure during adolescence induces long-lasting dysregulation in reward sensitivity to morphine and second messenger signaling in the VTA-NAc pathway.

Increased use of benzodiazepines in adolescents have been reported, with alprazolam (ALP) being the most abused. Drug abuse during adolescence can induce changes with lasting consequences. This study investigated the neurobiological consequences of ALP exposure during adolescence in C57BL/6J male mice. Mice received ALP (0, 0.5, 1.0 mg/kg) once/daily (postnatal day 35-49). Changes in responsiveness to morphine (2.5, 5.0 mg/kg), using the conditioned place preference paradigm, were assessed 24-h and 1-month after ALP exposure. In a separate experiment, mice received ALP (0, 0.5 mg/kg) and then sacrificed 24-h or 1-month after treatment to assess levels of extracellular signal regulated kinase 1/2 (ERK1/2) gene expression, protein phosphorylation, and downstream targets (CREB, AKT) within the ventral tegmental area (VTA) and nucleus accumbens (NAc). ALP-pretreated mice developed a strong preference to the compartment(s) paired with a subthreshold dose (2.5 mg/kg) of MOR short-term, and this effect was also present in the 1-month group. Adolescent ALP exposure resulted in dysregulation of ERK-signaling within the VTA-NAc pathway 24-h and 1-month after ALP exposure. Results indicate ALP exposure during adolescence potentiates the rewarding properties of MOR and induces persistent changes in ERK-signaling within the VTA-NAc pathway, a brain circuit highly implicated in the regulation of both drug reward and mood- related behaviors.

Social Buffering is Dependent on Mutual Experience in Adolescent Male Mice Exposed to Social Defeat Stress.

Background: Individuals who experience emotional, physical, or sexual abuse as children suffer from higher rates of major depressive disorder, drug abuse, and suicide. Early life interventions such as peer support groups can be beneficial to adolescents who experience trauma, suggesting that social support is important in facilitating rehabilitation and promoting resiliency to stress. Although there are some animal paradigms that can model how peer-peer interactions influence stress-reactivity, less is known about how individual stress experiences influence the effectiveness of social buffering. Methods: The vicarious social defeat stress (VSDS) paradigm allows for the assessment of two different stress modalities, physical (PS) and emotional (ES) stress, which confer different levels of stress with similar biological and behavioral outcomes. Using a modified VSDS paradigm in which pairs of mice experience ES and PS together we can begin to evaluate how stress exposure influences the buffering efficacy of social relationships. Adolescent mice (postnatal day 35) were randomly combined into dyads and were allocated into either mutual experience or cohabitation pairs. Within each dyad, one mouse was assigned to the physically stressed (PS) condition and was repeatedly exposed to an aggressive CD1 mouse while the other mouse was designated as the partner. In the mutual experience dyads the partner mice witnessed the defeat bout (ES) while in the cohabitation dyads the partner was separated from the PS mouse and returned after the 10 min defeat bout was terminated (non-stressed). After 10 days of defeat, mice were tested in the social interaction test (SIT), the elevated plus maze (EPM), and the forced swim test (FST). Results: PS-exposed mice in the cohabitation dyads, but not those in the mutual experience dyads, showed significantly more avoidance of a novel CD1 aggressor or c57BL/6 mouse, in the SIT. Surprisingly, both partner conditions showed avoidance to a CD1. Interestingly, non-stressed partner mice spent less time in the open arms of the EPM, suggesting increased anxiety; only PS-exposed mice in cohabitation dyads showed more time spent immobile in the FST, indicative of increased learned helplessness. Conclusions: These data suggest that the efficacy of social buffering can be mediated by individual stress experience.

Ptchd1 mediates opioid tolerance via cholesterol-dependent effects on µ-opioid receptor trafficking.

Repeated exposure to opioids causes tolerance, which limits their analgesic utility and contributes to overdose and abuse liability. However, the molecular mechanisms underpinning tolerance are not well understood. Here, we used a forward genetic screen in Caenorhabditis elegans for unbiased identification of genes regulating opioid tolerance which revealed a role for PTR-25/Ptchd1. We found that PTR-25/Ptchd1 controls µ-opioid receptor trafficking and that these effects were mediated by the ability of PTR-25/Ptchd1 to control membrane cholesterol content. Electrophysiological studies showed that loss of Ptchd1 in mice reduced opioid-induced desensitization of neurons in several brain regions and the peripheral nervous system. Mice and C. elegans lacking Ptchd1/PTR-25 display similarly augmented responses to opioids. Ptchd1 knockout mice fail to develop analgesic tolerance and have greatly diminished somatic withdrawal. Thus, we propose that Ptchd1 plays an evolutionarily conserved role in protecting the µ-opioid receptor against overstimulation.

Exposure to Vicarious Social Defeat Stress and Western-Style Diets During Adolescence Leads to Physiological Dysregulation, Decreases in Reward Sensitivity, and Reduced Antidepressant Efficacy in Adulthood.

A dramatic increase in the prevalence of major depression and diet-related disorders in adolescents has been observed over several decades, yet the mechanisms underlying this comorbidity have only recently begun to be elucidated. Exposure to western-style diet (WSD), high in both fats (45% kcal) and carbohydrates (35% kcal): e.g., high fat diet (HFD), has been linked to the development of metabolic syndrome-like symptoms and behavioral dysregulation in rodents, as similarly observed in the human condition. Because adolescence is a developmental period highlighted by vulnerability to both stress and poor diet, understanding the mechanism(s) underlying the combined negative effects of WSDs and stress on mood and reward regulation is critical. To this end, adolescent male C57 mice were exposed to vicarious social defeat stress (VSDS), a stress paradigm capable of separating physical (PS) versus psychological/emotional (ES) stress, followed by normal chow (NC), HFD, or a separate control diet high in carbohydrates (same sucrose content as HFD) and low in fat (LFD), while measuring body weight and food intake. Non-stressed control mice exposed to 5 weeks of NC or HFD showed no significant differences in body weight or social interaction. Mice exposed to VSDS (both ES and PS) gain weight rapidly 1 week after initiation of HFD, with the ES-exposed mice showing significantly higher weight gain as compared to the HFD-exposed control mice. These mice also exhibited a reduction in saccharin preference, indicative of anhedonic-like behavior. To further delineate whether high fat was the major contributing factor to these deficits, LFD was introduced. The mice in the VSDS + HFD gained weight more rapidly than the VSDS + LFD group, and though the LFD-exposed mice did not gain weight as rapidly as the HFD-exposed mice, both the VSDS + LFD- and VSDS + HFD-exposed mice exhibited attenuated response to the antidepressant fluoxetine. These data show that diets high in both fats and carbohydrates are responsible for rapid weight gain and reduced reward sensitivity; and that while consumption of diet high in carbohydrate and low in fat does not lead to rapid weight gain, both HFD and LFD exposure after stress leads to reduced responsiveness to antidepressant treatment.

The Resilient Phenotype Induced by Prophylactic Ketamine Exposure During Adolescence Is Mediated by the Ventral Tegmental Area-Nucleus Accumbens Pathway.

BACKGROUND: Major depressive disorder is prevalent in children and adolescents and is associated with a high degree of morbidity throughout life, with potentially devastating personal consequences and public health impact. The efficacy of ketamine (KET) as an antidepressant has been demonstrated in adolescent rodents; however, the neurobiological mechanisms underlying these effects are unknown. Recent evidence showed that KET reverses stress-induced (i.e., depressive-like) deficits within major mesocorticolimbic regions, such as the prefrontal cortex, nucleus accumbens (NAc), and hippocampus, in adult rodents. However, little is known about KET's effect in the ventral tegmental area (VTA), which provides the majority of dopaminergic input to these brain regions. METHODS: We characterized behavioral, biochemical, and electrophysiological effects produced by KET treatment in C57BL/6J male mice during adolescence (n = 7-10 per condition) within the VTA and its major projection regions, namely, the NAc and prefrontal cortex. Subsequently, molecular targets within the VTA-NAc projection were identified for viral gene transfer manipulations to recapitulate the effects of stress or KET treatment. RESULTS: Repeated KET treatment produced a robust proresilient response to chronic social defeat stress. This effect was largely driven by Akt signaling activity within the VTA and NAc, and it could be blocked or recapitulated through direct Akt-viral-mediated manipulation. Additionally, we found that the KET-induced resilient phenotype is dependent on VTA-NAc, but not VTA-prefrontal cortex, pathway activity. CONCLUSIONS: These findings indicate that KET exposure during adolescence produces a proresilient phenotype mediated by changes in Akt intracellular signaling and altered neuronal activity within the VTA-NAc pathway.

Ketamine: The final frontier or another depressing end?

Two decades ago, the observation of a rapid and sustained antidepressant response after ketamine administration provided an exciting new avenue in the search for more effective therapeutics for the treatment of clinical depression. Research elucidating the mechanism(s) underlying ketamine's antidepressant properties has led to the development of several hypotheses, including that of disinhibition of excitatory glutamate neurons via blockade of N-methyl-d-aspartate (NMDA) receptors. Although the prominent understanding has been that ketamine's mode of action is mediated solely via the NMDA receptor, this view has been challenged by reports implicating other glutamate receptors such as AMPA, and other neurotransmitter systems such as serotonin and opioids in the antidepressant response. The recent approval of esketamine (Spravato™) for the treatment of depression has sparked a resurgence of interest for a deeper understanding of the mechanism(s) underlying ketamine's actions and safe therapeutic use. This review aims to present our current knowledge on both NMDA and non-NMDA mechanisms implicated in ketamine's response, and addresses the controversy surrounding the antidepressant role and potency of its stereoisomers and metabolites. There is much that remains to be known about our understanding of ketamine's antidepressant properties; and although the arrival of esketamine has been received with great enthusiasm, it is now more important than ever that its mechanisms of action be fully delineated, and both the short- and long-term neurobiological/functional consequences of its treatment be thoroughly characterized.

Nicotine treatment buffers negative behavioral consequences induced by exposure to physical and emotional stress in adolescent male mice.

Early life stress influences adult psychopathology and is associated with an increase in the propensity for drug use/seeking throughout the lifespan. Animal models corroborate that stress exposure exacerbates maladaptive reactivity to stressful stimuli while also shifting the rewarding properties of many drugs of abuse, including nicotine (NIC), a stimulant commonly misused by adolescents. Interestingly, NIC treatment can also normalize some stress-induced behavioral deficits in adult rodents; however, little is known about NIC's therapeutic efficacy following stress experienced during adolescence. The goal of the following experiments was to elucidate NIC's ability to buffer the negative consequences of stress exposure, and to further assess behavioral responsivity while on the drug. Given that stress often occurs in both physical and non-physical forms, we employed the vicarious social defeat stress (VSDS) model which allows for investigation of both physical (PS) and emotional stress (ES) exposure. After 10 days, exposure to PS and ES decreased interaction with a social target in the social interaction test (SIT), confirming social avoidance. Groups were further divided and given NIC (0.0 or 160 mg/L) in their drinking water. After 1 month of NIC consumption, the mice were exposed to the SIT, elevated plus maze (EPM), and the forced swim test (FST), respectively. NIC-treated mice showed a reversal of stress-induced deficits in the EPM and FST. Surprisingly, the mice did not show improvement in the SIT regardless of treatment condition. Together, these data confirm NIC's ability to normalize some stress-induced behavioral deficits; however, NIC's effects on social behavior need further investigation.

Vicarious social defeat stress: Bridging the gap between physical and emotional stress.

BACKGROUND: Animal models capable of differentiating the neurobiological intricacies between physical and emotional stress are scarce. Current models rely primarily on physical stressors (e.g., chronic unpredictable or mild stress, social defeat, learned helplessness), and neglect the impact of psychological stress alone. This is surprising given extensive evidence that a traumatic event needs not be directly experienced to produce enduring perturbations on an individual's health and psychological well-being. Post-traumatic stress disorder (PTSD), a highly debilitating neuropsychiatric disorder characterized by intense fear of trauma-related stimuli, often occurs in individuals that have only witnessed a traumatic event. NEW METHOD: By modifying the chronic social defeat stress (CSDS) paradigm to include a witness component (witnessing the social defeat of another mouse), we demonstrate a novel behavioral paradigm capable of inducing a robust behavioral syndrome reminiscent of PTSD in emotionally stressed adult mice. RESULTS: We describe the vicarious social defeat stress (VSDS) model that is capable of inducing a host of behavioral deficits that include social avoidance and other depressive- and anxiety-like phenotypes in adult male mice. VSDS exposure induces weight loss and spike in serum corticosterone (CORT) levels. A month after stress, these mice retain the social avoidant phenotype and have an increased CORT response when exposed to subsequent stress. COMPARISON WITH EXISTING METHOD(S): The VSDS is a novel paradigm capable of inducing emotional stress by isolating physical stress/confrontation in mice. CONCLUSIONS: The VSDS model can be used to study the short- and long-term neurobiological consequences of exposure to emotional stress in mice.

Repeated ketamine exposure induces an enduring resilient phenotype in adolescent and adult rats.

BACKGROUND: Major depressive disorder afflicts up to 10% of adolescents. However, nearly 50% of those afflicted are considered nonresponsive to available treatments. Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist has shown potential as a rapid-acting and long-lasting treatment for major depressive disorder in adults. Thus, the effectiveness and functional consequences of ketamine exposure during adolescence were explored. METHODS: Adolescent male rats (postnatal day [PD] 35) received two ketamine (0, 5, 10, or 20 mg/kg) injections, 4 hours apart, after exposure to day 1 of the forced swim test (FST). The next day, rats were reexposed to the FST to assess ketamine-induced antidepressant-like responses. Separate groups were exposed to chronic unpredictable stress to confirm findings from the FST. After these initial experiments, adolescent naive rats were exposed to either 1 or 15 consecutive days (PD35-49) of ketamine (20 mg/kg) twice daily. Ketamine's influence on behavioral reactivity to rewarding (i.e., sucrose preference) and aversive (i.e., elevated plus-maze, FST) circumstances was then assessed 2 months after treatment. To control for age-dependent effects, adult rats (PD75-89) were exposed to identical experimental conditions. RESULTS: Ketamine (20 mg/kg) reversed the chronic unpredictable stress-induced depression-like behaviors in the FST. Repeated ketamine exposure resulted in anxiolytic- and antidepressant-like responses 2 months after drug exposure. None of the ketamine doses used were capable of inducing drug-seeking behaviors as measured by place preference conditioning. CONCLUSIONS: Repeated ketamine exposure induces enduring resilient-like responses regardless of age of exposure. These findings point to ketamine, and its repeated exposure, as a potentially useful antidepressant during adolescence.