Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons.

Altered activity of the ventral pallidum (VP) underlies disrupted motivation in stress and drug exposure. The VP is a very heterogeneous structure composed of many neuron types with distinct physiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for motivational behavior. While Npas1+ neurons have been characterized in the globus pallidus external, there is limited information on these neurons in the VP. To address this limitation, we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-sequencing on ribosome-associated mRNA from VP Npas1+ neurons to determine their molecular identity. Finally, we used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We used a similar approach in females using the chronic witness defeat stress (CWDS). We identified VP Npas1+ projections to the nucleus accumbens, ventral tegmental area, medial and lateral habenula, lateral hypothalamus, thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct translatome representing distinct biological processes. Chemogenetic activation of hM3D(Gq) receptors in VP Npas1+ neurons increased susceptibility to a subthreshold SDS and anxiety-like behavior in the elevated plus maze and open field while the activation of hM4D(Gi) receptors in VP Npas1+ neurons enhanced resilience to chronic SDS and CWDS. Thus, the activity of VP Npas1+ neurons modulates susceptibility to social stressors and anxiety-like behavior. Our studies provide new information on VP Npas1+ neuron circuitry, molecular identity, and their role in stress response.SIGNIFICANCE STATEMENT The ventral pallidum (VP) is a structure connected to both reward-related and aversive brain centers. It is a key brain area that signals the hedonic value of natural rewards. Disruption in the VP underlies altered motivation in stress and substance use disorder. However, VP is a very heterogeneous area with multiple neuron subtypes. This study characterized the projection pattern and molecular signatures of VP Neuronal PAS 1-positive (Npas1+) neurons. We further used tools to alter receptor signaling in VP Npas1+ neurons in stress to demonstrate a role for these neurons in stress behavioral outcomes. Our studies have implications for understanding brain cell type identities and their role in brain disorders, such as depression, a serious disorder that is precipitated by stressful events.

Extrahypothalamic oxytocin neurons drive stress-induced social vigilance and avoidance.

Oxytocin increases the salience of both positive and negative social contexts and it is thought that these diverse actions on behavior are mediated in part through circuit-specific action. This hypothesis is based primarily on manipulations of oxytocin receptor function, leaving open the question of whether different populations of oxytocin neurons mediate different effects on behavior. Here we inhibited oxytocin synthesis in a stress-sensitive population of oxytocin neurons specifically within the medioventral bed nucleus of the stria terminalis (BNSTmv). Oxytocin knockdown prevented social stress-induced increases in social vigilance and decreases in social approach. Viral tracing of BNSTmv oxytocin neurons revealed fibers in regions controlling defensive behaviors, including lateral hypothalamus, anterior hypothalamus, and anteromedial BNST (BNSTam). Oxytocin infusion into BNSTam in stress naïve mice increased social vigilance and reduced social approach. These results show that a population of extrahypothalamic oxytocin neurons plays a key role in controlling stress-induced social anxiety behaviors.

Acute and long-lasting effects of adolescent fluoxetine exposure on feeding behavior in Sprague-Dawley rats.

The antidepressant medication fluoxetine (FLX) is frequently prescribed for the management of mood-related illnesses in the adolescent population-yet its long-term neurobehavioral consequences are not understood. To investigate how juvenile FLX exposure influences feeding behavior in adulthood, we conducted two experiments. In Experiment 1, adolescent male and female Sprague-Dawley rats were administered with 20 mg/kg/day FLX (postnatal day [PND] 35-49) and exposed to a binge access paradigm in adulthood (PND72+) to evaluate potential alterations for sweetened-fat preference. No long-term FLX-induced differences in preference for sweetened fat versus chow, nor total caloric intake, were noted; however, females displayed higher preference for sweetened fat compared to males. In Experiment 2, PND35 male rats received FLX (PND35-49) and were exposed to chronic variable stress (CVS) in adulthood (PND74-88). During treatment, FLX decreased body weight and intake (meal size), but not total meal number. Also, no differences in meal pattern parameters were observed after FLX completion. Likewise, no differences in meal pattern parameters to a palatable diet (45% fat, 17% sucrose) presented from PND74 to PND88, even after CVS, were observed. Our findings indicate that juvenile FLX reduces body weight gain acutely via reduced meal size intake; however, no long-term changes in ad libitum feeding behavior or binge access to a palatable stimulus are evident.

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility.

Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility.SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The reduction of Slc6a15 occurs selectively in the NAc D2-neurons. Genetic reduction of Slc6a15 induces susceptibility to a subthreshold stress, while genetic overexpression in D2-neurons prevents social avoidance after chronic social defeat stress.

Fluoxetine exposure during adolescence alters responses to aversive stimuli in adulthood.

The mechanisms underlying the enduring neurobiological consequences of antidepressant exposure during adolescence are poorly understood. Here, we assessed the long-term effects of exposure to fluoxetine (FLX), a selective serotonin reuptake inhibitor, during adolescence on behavioral reactivity to emotion-eliciting stimuli. We administered FLX (10 mg/kg, bi-daily, for 15 d) to male adolescent [postnatal day 35 (P35) to P49] C57BL/6 mice. Three weeks after treatment (P70), reactivity to aversive stimuli (i.e., social defeat stress, forced swimming, and elevated plus maze) was assessed. We also examined the effects of FLX on the expression of extracellular signal-regulated kinase (ERK) 1/2-related signaling within the ventral tegmental area (VTA) of adolescent mice and Sprague Dawley rats. Adolescent FLX exposure suppressed depression-like behavior, as measured by the social interaction and forced swim tests, while enhancing anxiety-like responses in the elevated plus maze in adulthood. This complex behavioral profile was accompanied by decreases in ERK2 mRNA and protein phosphorylation within the VTA, while stress alone resulted in opposite neurobiological effects. Pharmacological (U0126) inhibition, as well as virus-mediated downregulation of ERK within the VTA mimicked the antidepressant-like profile observed after juvenile FLX treatment. Conversely, overexpression of ERK2 induced a depressive-like response, regardless of FLX pre-exposure. These findings demonstrate that exposure to FLX during adolescence modulates responsiveness to emotion-eliciting stimuli in adulthood, at least partially, via long-lasting adaptations in ERK-related signaling within the VTA. Our results further delineate the role ERK plays in regulating mood-related behaviors across the lifespan.

Social defeat stress induces a depression-like phenotype in adolescent male c57BL/6 mice.

Abstract Exposure to stress is highly correlated with the emergence of mood-related illnesses. Because major depressive disorder often emerges in adolescence, we assessed the effects of social defeat stress on responses to depressive-like behaviors in juvenile mice. To do this, postnatal day (PD) 35 male c57BL/6 mice were exposed to 10 days of social defeat stress (PD35-44), while control mice were handled daily. Twenty-four hours after the last episode of defeat (PD45), separate groups of mice were tested in the social interaction, forced swimming, sucrose preference, and elevated plus-maze behavioral assays (n = 7-12 per group). Also, we examined body weight gain across days of social defeat and levels of blood serum corticosterone 40 min after the last episode of defeat stress. Our data indicates that defeated mice exhibited a depressive-like phenotype as inferred from increased social avoidance, increased immobility in the forced swim test, and reduced sucrose preference (a measure of anhedonia), when compared to non-defeated controls. Defeated mice also displayed an anxiogenic-like phenotype when tested on the elevated plus-maze. Lastly, stressed mice displayed lower body weight gain, along with increased blood serum corticosterone levels, when compared to non-stressed controls. Overall, we show that in adolescent male c57BL/6 mice, social defeat stress induces a depression- and anxiety-like phenotype 24 h after the last episode of stress. These data suggest that the social defeat paradigm may be used to examine the etiology of stress-induced mood-related disorders during adolescence.

Chlordiazepoxide reduces anxiety-like behavior in the adolescent mouse elevated plus maze: A pharmacological validation study.

This report evaluates the effects of chlordiazepoxide, a benzodiazepine commonly prescribed to manage anxiety-related disorders in adolescent/pediatric populations, on elevated plus maze (EPM) performance in juvenile mice. This approach was taken because chlordiazepoxide produces anxiolytic-like effects in multiple models in adult rodents, however, less is known about the behavioral effects of this benzodiazepine in juveniles. Thus, we administered a single intraperitoneal injection of chlordiazepoxide (0, 5, or 10 mg/kg) to postnatal day 35 male C57BL/6 mice. Thirty minutes later, mice were allowed to explore the EPM for 5-min. We found that chlordiazepoxide-treated mice (5 and 10 mg/kg) spent more time exploring the open arms of the EPM. No differences in velocity (cm/s) or distance traveled (cm) were observed between the groups. These results indicate that chlordiazepoxide induces anxiolytic-related behavior in adolescent male mice.

Chronic social defeat stress in prairie voles (Microtus ochrogaster): A preclinical model for the study of depression-related phenotypes.

BACKGROUND: Stress-induced illnesses, like major depression, are among the leading causes of disability across the world. Consequently, there is a dire need for the validation of translationally-suited animal models incorporating social stress to uncover the etiology of depression. Prairie voles (Microtus ochrogaster) are more translationally relevant than many other rodent models as they display monogamous social and bi-parental behaviors. Therefore, we evaluated whether a novel social defeat stress (SDS) model in male prairie voles induces depression-relevant behavioral outcomes. METHODS: Adult sexually-naïve male prairie voles experienced SDS bouts from a conspecific pair-bonded male aggressor, 10 min per day for 10 consecutive days. Non-stressed controls (same-sex siblings) were housed in similar conditions but never experienced physical stress. Twenty-four h later, voles were evaluated in social interaction, sucrose preference, and Morris water maze tests - behavioral endpoints validated to assess social withdrawal, anhedonia-related behavior, and spatial memory performance, respectively. RESULTS: SDS-exposed voles displayed lower sociability and body weight, decreased preference for a sucrose solution, and impairment of spatial memory retrieval. Importantly, no differences in general locomotor activity were observed as a function of SDS exposure. LIMITATIONS: This study does not include female voles in the experimental design. CONCLUSIONS: We found that repeated SDS exposure, in male prairie voles, results in a depression-relevant phenotype resembling an anhedonia-like outcome (per reductions in sucrose preference) along with social withdrawal and spatial memory impairment - highlighting that the prairie vole is a valuable model with potential to study the neurobiology of social stress-induced depression-related outcomes.

Sex-Specific Alterations in Spatial Memory and Hippocampal AKT-mTOR Signaling in Adult Mice Pre-exposed to Ketamine and/or Psychological Stress During Adolescence.

Background: Ketamine (KET) is administered to manage major depression in adolescent patients. However, the long-term effects of juvenile KET exposure on memory-related tasks have not been thoroughly assessed. We examined whether exposure to KET, psychological stress, or both results in long-lasting alterations in spatial memory in C57BL/6 mice. Furthermore, we evaluated how KET and/or psychological stress history influenced hippocampal protein kinase B-mechanistic target of rapamycin (AKT-mTOR)-related signaling. Methods: On postnatal day 35, male and female mice underwent vicarious defeat stress (VDS), a form of psychological stress that reduces sociability in both sexes, with or without KET exposure (20 mg/kg/day, postnatal days 35-44). In adulthood (postnatal day 70), mice were assessed for spatial memory performance on a water maze task or euthanized for hippocampal tissue collection. Results: Juvenile pre-exposure to KET or VDS individually increased the latency (seconds) to locate the escape platform in adult male, but not female, mice. However, juvenile history of concomitant KET and VDS prevented memory impairment. Furthermore, individual KET or VDS pre-exposure, unlike their combined history, decreased hippocampal AKT-mTOR signaling in adult male mice. Conversely, KET pre-exposure alone increased AKT-mTOR in the hippocampus of adult female mice. Lastly, rapamycin-induced decreases of mTOR in naïve adult female mice induced spatial memory retrieval deficits, mimicking adult male mice with a history of exposure to VDS or KET. Conclusions: Our preclinical model shows how KET treatment for the management of adolescent psychological stress-induced sequelae does not impair spatial memory later in life. However, juvenile recreational KET misuse, like psychological stress history, results in long-term spatial memory deficits and hippocampal AKT-mTOR signaling changes in a sex-specific manner.

Viral-mediated expression of Erk2 in the nucleus accumbens regulates responses to rewarding and aversive stimuli.

Second-messenger signaling within the mesolimbic reward circuit is involved in both the long-lived effects of stress and in the underlying mechanisms that promote drug abuse liability. To determine the direct role of kinase signaling within the nucleus accumbens, specifically mitogen-activated protein kinase 1 (ERK2), in mood- and drug-related behavior, we used a herpes-simplex virus to up- or down-regulate ERK2 in adult male rats. We then exposed rats to a battery of behavioral tasks including the elevated plus-maze, open field test, forced-swim test, conditioned place preference, and finally cocaine self-administration. Herein, we show that viral overexpression or knockdown of ERK2 in the nucleus accumbens induces distinct behavioral phenotypes. Specifically, over expression of ERK2 facilitated depression- and anxiety-like behavior while also increasing sensitivity to cocaine. Conversely, down-regulation of ERK2 attenuated behavioral deficits, while blunting sensitivity to cocaine. Taken together, these data implicate ERK2 signaling, within the nucleus accumbens, in the regulation of affective behaviors and modulating sensitivity to the rewarding properties of cocaine.

Sex and diet-dependent gene alterations in human and rat brains with a history of nicotine exposure.

Introduction: Chronic nicotine exposure induces changes in the expression of key regulatory genes associated with metabolic function and neuronal alterations in the brain. Many bioregulatory genes have been associated with exposure to nicotine, but the modulating effects of sex and diet on gene expression in nicotine-exposed brains have been largely unexplored. Both humans and rodents display motivation for nicotine use and the emergence of withdrawal symptoms during abstinence. Research comparing pre-clinical models with human subjects provides an important opportunity to understand common biomarkers of the harmful effects of nicotine as well as information that may help guide the development of more effective interventions for nicotine cessation. Methods: Human postmortem dorsolateral prefrontal cortex (dLPFC) tissue BA9 was collected from female and male subjects, smokers and non-smokers (N = 12 per group). Rat frontal lobes were collected from female and male rats that received a regular diet (RD) or a high-fat diet (HFD) (N = 12 per group) for 14 days following implantation of a osmotic mini-pump (Alzet) that delivered nicotine continuously. Controls (control-s) received a sham surgical procedure. RNA was extracted from tissue from human and rat samples and reversed-transcribed to cDNA. Gene expression of CHRNA10 (Cholinergic receptor nicotinic alpha 10), CERKL (Ceramide Kinase-Like), SMYD1 (SET and MYD Domin Containing 1), and FA2H (Fatty Acid 2-Hydrolase) in humans was compared to rats in each subset of groups and quantified by qPCR methods. Additionally, protein expression of FA2H was analyzed by immunohistochemistry (IHC) in human dLPFC. Results: Humans with a history of smoking displayed decreased CHRNA10 (p = 0.0005), CERKL (p ≤ 0.0001), and SMYD1 (p = 0.0005) expression and increased FA2H (p = 0.0097) expression compared to non-smokers (p < 0.05). Similar patterns of results were observed in nicotine exposed vs. control rats. Interestingly, sex-related differences in gene expression for CERKL and FA2H were observed. In addition, ANCOVA analysis showed a significant effect of nicotine in a sex-different manner, including an increase in CERKL in male and female rats with RD or HFD. In rats exposed to an HFD, FA2H gene expression was lower in nicotine-treated rats compared to RD rats treated with nicotine. Protein expression of FA2H (p = 0.001) by IHC was significantly higher in smokers compared to non-smokers. Conclusion: These results suggest that a history of long-term nicotine exposure in humans alters the expression of sphingolipid metabolism-related (CERKL, SMYD1, and FA2H) and neuronal (CHRNA10) marker genes similarly as compared to rats. Sex- and diet-dependent differences appear in nicotine-exposed rats, critical in regulating sphingolipid metabolism and nicotinic acetylcholine receptors. This research enhances the construct validity of rat models of nicotine usage by showing a similar pattern of changes in gene expression in human subjects with a smoking history.

Juvenile administration of concomitant methylphenidate and fluoxetine alters behavioral reactivity to reward- and mood-related stimuli and disrupts ventral tegmental area gene expression in adulthood.

There is a rise in the concurrent use of methylphenidate (MPH) and fluoxetine (FLX) in pediatric populations. However, the long-term neurobiological consequences of combined MPH and FLX treatment (MPH + FLX) during juvenile periods are unknown. We administered saline (VEH), MPH, FLX, or MPH + FLX to juvenile Sprague Dawley male rats from postnatal day 20 to 34, and assessed their reactivity to reward- and mood-related stimuli 24 h or 2 months after drug exposure. We also assessed mRNA and protein levels within the ventral tegmental area (VTA) to determine the effect of MPH, FLX, or MPH + FLX on the extracellular signal-regulated protein kinase-1/2 (ERK) pathway--a signaling cascade implicated in motivation and mood regulation. MPH + FLX enhanced sensitivity to drug (i.e., cocaine) and sucrose rewards, as well as anxiety (i.e., elevated plus maze)- and stress (i.e., forced swimming)-eliciting situations when compared with VEH-treated rats. MPH + FLX exposure also increased mRNA of ERK2 and its downstream targets cAMP response element-binding protein (CREB), BDNF, c-Fos, early growth response protein-1 (Zif268), and mammalian target of rapamycin (mTOR), and also increased protein phosphorylation of ERK2, CREB, and mTOR 2 months after drug exposure when compared with VEH-treated rats. Using herpes simplex virus-mediated gene transfer to block ERK2 activity within the VTA, we rescued the MPH and FLX-induced behavioral deficits seen in the forced-swimming task 2 months after drug treatment. These results indicate that concurrent MPH + FLX exposure during preadolescence increases sensitivity to reward-related stimuli while simultaneously enhancing susceptibility to stressful situations, at least in part, due to long-lasting disruptions in ERK signaling within the VTA.

Post-training cocaine exposure facilitates spatial memory consolidation in C57BL/6 mice.

In this study, we examined the ability of post-training injections of cocaine to facilitate spatial memory performance using the Morris water maze (MWM). We also investigated the role that hippocampal protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK) signaling may play in cocaine-mediated spatial memory consolidation processes. Male and female C57BL/6 mice were first trained in a MWM task (eight consecutive trials) then injected with cocaine (0, 1.25, 2.5, 5, or 20 mg/kg), and memory for the platform location was retested after a 24 h delay. Cocaine had a dose-dependent effect on spatial memory performance because only the mice receiving 2.5 mg/kg cocaine displayed a significant reduction in latency to locate the platform. No sex differences in MWM performance were observed; however, females showed higher hippocampal levels of PKA when compared with males. A second experiment demonstrated that 2.5 mg/kg cocaine enhanced MWM performance only when administered within 2, but not 4 h after spatial training. We also found that cocaine (2.5 mg/kg) increased ERK2 phosphorylation within the hippocampus and one of its downstream targets (ribosomal S6 kinase), a mechanism that may be responsible, at least in part, for the enhanced cocaine-mediated spatial memory performance. Overall, these data demonstrate that a low dose of cocaine (2.5 mg/kg) administered within 2 h after training facilitates MWM spatial memory performance in C57BL/6 mice.

Adolescent fluoxetine exposure increases ERK-related signaling within the prefrontal cortex of adult male Sprague-Dawley rats.

There has been a disproportionate increase in fluoxetine (FLX) prescription rates within the juvenile population. Thus, we evaluated how adolescent FLX exposure alters expression/phosphorylation of proteins from the extracellular signal regulated kinase (ERK)-1/2 cascade within the adult prefrontal cortex (PFC). Male Sprague-Dawley rats were exposed to FLX (20 mg/kg) for 15 consecutive days (postnatal-day [PD] 35-49). At PD70 (adulthood), we examined protein markers for ERK1/2, ribosomal S6 kinase (RSK), and mammalian target of rapamycin (mTOR). FLX-pretreatment decreased body weight, while increasing PFC phosphorylation of ERK1/2 and RSK, as well as total mTOR protein expression in adulthood. We provide first-line evidence that juvenile FLX-pretreatment induces long-term decreases in body weight-gain, along with neurobiological changes in the adult PFC - highlighting that early-life antidepressant exposure increases ERK-related signaling markers in later life.

The BDNF-TrkB Pathway Acts Through Nucleus Accumbens D2 Expressing Neurons to Mediate Stress Susceptible Outcomes.

Brain-derived neurotrophic factor (BDNF) has a critical role in stress response including neuropsychiatric disorders that are precipitated by stress, such as major depressive disorder (MDD). BDNF acts through its full-length BDNF receptor tyrosine kinase B (TrkB) to trigger a pro-plasticity effect. In contrast, the truncated isoform of the BDNF receptor (TrkB.t1) triggers an anti-plasticity effect. In stress outcomes, BDNF acting in the hippocampus has a stress resilience effect, and, inversely, in the nucleus accumbens (NAc), BDNF acts as a stress susceptible molecule. It is unknown if BDNF-TrkB acts on a specific NAc projection neuron, i.e., medium spiny neuron (MSN or spiny projection neuron), a subtype in stress outcomes. To determine this, we performed chronic social or vicarious witness defeat stress (CSDS or CWDS) in mice expressing TrkB.t1 in dopamine receptor 1 or 2 containing MSNs (D1- or D2-MSNs). Our results showed that TrkB.t1 overexpression in NAc D2-MSNs prevented the CSDS-induced social avoidance or other stress susceptible behaviors in male and female mice. We further showed that this overexpression in D2-MSNs blocked stress susceptible behavior induced by intra-NAc BDNF infusion. In contrast, our results demonstrate that overexpression of TrkB.t1 on NAc D1-MSNs facilitates the SDS susceptible behaviors. Our study provides enhanced details into the NAc cell subtype role of BDNF-TrkB signaling in stress outcomes.

Effect of early-life stress or fluoxetine exposure on later-life conditioned taste aversion learning in Sprague-Dawley rats.

In rodents, early-life exposure to environmental stress or antidepressant medication treatment has been shown to induce similar long-term consequences on memory- and depression-related behavior in adulthood. To expand on this line of work, we evaluated how juvenile exposure to chronic variable stress (CVS) or the selective serotonin reuptake inhibitor fluoxetine (FLX) influences conditioned taste aversion (CTA) learning in adulthood. To do this, in Experiment 1, we examined how adolescent CVS alone (postnatal day [PND] 35-48), or with prenatal stress (PNS) history (PNS + CVS), influenced the acquisition and extinction of CTA in adult male Sprague Dawley rats. Specifically, at PND70+ (adulthood), rats were presented with 0.15 % saccharin followed by an intraperitoneal (i.p.) injection of lithium chloride (LiCl) to induce visceral malaise. A total of four saccharin (conditioned stimulus) and LiCl (unconditioned stimulus) pairings occurred across the CTA acquisition phase. Next, saccharin was presented without aversive consequences, and intake was measured across consecutive days of the extinction phase. No differences in body weight gain across the experimental days, rate of CTA acquisition, or extinction of CTA, were observed among the experimental groups (control, n = 7; CVS, n = 12; PNS + CVS, n = 9). In Experiment 2, we evaluated if early-life FLX exposure alters CTA learning in adulthood. Specifically, adolescent stress naïve male and female rats received FLX (0 or 20 mg/kg/i.p) once daily for 15 consecutive days (PND35-49). During antidepressant exposure, FLX decreased body weight gain in both male (n = 7) and female rats (n = 7), when compared to respective controls (male control, n = 8; female control, n = 8). However, juvenile FLX exposure decreased body weight-gain in adult male, but not female, rats. Lastly, adolescent FLX history had no effect on CTA acquisition or extinction in adulthood (PND70), in neither male nor female rats. Together, the data indicate that juvenile FLX exposure results in a long-term decrease of body weight-gain in a male-specific manner. Yet, independent of sex, neither early-life stress nor FLX exposure alters CTA learning in adulthood.

Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors.

Adolescent Fluoxetine Exposure Induces Persistent Gene Expression Changes in the Hippocampus of Adult Male C57BL/6 Mice.

Mood-related disorders have a high prevalence among children and adolescents, posing a public health challenge, given their adverse impact on these young populations. Treatment with the selective serotonin reuptake inhibitor fluoxetine (FLX) is the first line of pharmacological intervention in pediatric patients suffering from affect-related illnesses. Although the use of this antidepressant has been deemed efficacious in the juvenile population, the enduring neurobiological consequences of adolescent FLX exposure are not well understood. Therefore, we explored for persistent molecular adaptations, in the adult hippocampus, as a function of adolescent FLX pretreatment. To do this, we administered FLX (20 mg/kg/day) to male C57BL/6 mice during adolescence (postnatal day [PD] 35-49). After a 21-day washout period (PD70), whole hippocampal tissue was dissected. We then used qPCR analysis to assess changes in the expression of genes associated with major intracellular signal transduction pathways, including the extracellular signal-regulated kinase (ERK), the phosphatidylinositide-3-kinase (PI3K)/AKT pathway, and the wingless (Wnt)-dishevelled-GSK3ß signaling cascade. Our results show that FLX treatment results in long-term dysregulation of mRNA levels across numerous genes from the ERK, PI3K/AKT, and Wnt intracellular signaling pathways, along with increases of the transcription factors CREB, ΔFosB, and Zif268. Lastly, FLX treatment resulted in persistent increases of transcripts associated with cytoskeletal integrity (ß-actin) and caspase activation (DIABLO), while decreasing genes associated with metabolism (fucose kinase) and overall neuronal activation (c-Fos). Collectively, these data indicate that adolescent FLX exposure mediates persistent alterations in hippocampal gene expression in adulthood, thus questioning the safety of early-life exposure to this antidepressant medication.

Food-Seeking Behavior Is Mediated by Fos-Expressing Neuronal Ensembles Formed at First Learning in Rats.

Neuronal ensembles in the infralimbic cortex (IL) develop after prolonged food self-administration training. However, rats demonstrate evidence of learning the food self-administration response as early as day 1, with responding quickly increasing to asymptotic levels. Since the contribution of individual brain regions to task performance shifts over the course of training, it remains unclear whether IL ensembles are gradually formed and refined over the course of extensive operant training, or whether functionally-relevant ensembles might be recruited and formed as early as the initial acquisition of food self-administration behavior. Here, we aimed to determine the role of IL ensembles at the earliest possible point after demonstrable learning of a response-outcome association. We first allowed rats to lever press for palatable food pellets and stopped training rats once their behavior evidenced the response-outcome association (learners). We compared their food-seeking behavior and neuronal activation (Fos protein expression) to similarly trained rats that did not form this association (non-learners). Learners had greater food-seeking behavior and neuronal activation within the medial prefrontal cortex (mPFC), suggesting that mPFC subregions might encode initial food self-administration memories. To test the functional relevance of mPFC Fos-expressing ensembles to subsequent food seeking, we tested region-wide inactivation of the IL using muscimol+baclofen and neuronal ensemble-specific ablation using the Daun02 inactivation procedure. Both region-wide inactivation and ensemble-specific inactivation of the IL significantly decreased food seeking. These data suggest that IL neuronal ensembles form during initial learning of food self-administration behavior, and furthermore, that these ensembles play a functional role in food seeking.

Association of ADHD and Obesity in Hispanic Children on the US-Mexico Border: A Retrospective Analysis.

Pediatric obesity and Attention Deficit Hyperactivity Disorder (ADHD) are rising health concerns in the United States, especially among Hispanic children and adolescents. Research on Hispanic children and adolescents indicates disproportionately higher prevalence rates of obesity in this community but scant data on ADHD prevalence rates. In contrast, a plethora of research studies across the general population examines the relationship between childhood obesity and ADHD. In addition, there is a lack of research that examines the role of ethnicity and sub-ethnic group correlations in ADHD, particularly in the Hispanic population. Existing studies in the general population indicate ADHD may be a risk factor for being overweight compared to normal controls. The objective of the present study is to examine the prevalence of obesity in children with ADHD compared to children in the general population in a predominately Hispanic sample on the US-Mexico border. A total of 7,270 pediatric medical records were evaluated. The retrospective analysis included Body Mass Index (BMI) and related health variables, and ethnicity and showed that children with ADHD are more likely to be underweight. In conclusion, no significant relationship existed between obesity and ADHD among Hispanic children on the US-Mexico Border, and instead we found the opposite correlation.