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.

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.

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.

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.

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.

Adolescent fluoxetine treatment mediates a persistent anxiety-like outcome in female C57BL/6 mice that is ameliorated by fluoxetine re-exposure in adulthood.

The objective of this study was to evaluate whether juvenile fluoxetine (FLX) exposure induces long-term changes in baseline responses to anxiety-inducing environments, and if so, whether its re-exposure in adulthood would ameliorate this anxiety-like phenotype. An additional goal was to assess the impact of adolescent FLX pretreatment, and its re-exposure in adulthood, on serotonin transporters (5-HTT) and brain-derived-neurotrophic-factor (BDNF)-related signaling markers (TrkB-ERK1/2-CREB-proBDNF-mBDNF) within the hippocampus and prefrontal cortex. To do this, female C57BL/6 mice were exposed to FLX in drinking water during postnatal-days (PD) 35-49. After a 21-day washout-period (PD70), mice were either euthanized (tissue collection) or evaluated on anxiety-related tests (open field, light/dark box, elevated plus-maze). Juvenile FLX history resulted in a persistent avoidance-like profile, along with decreases in BDNF-signaling markers, but not 5-HTTs or TrkB receptors, within both brain regions. Interestingly, FLX re-exposure in adulthood reversed the enduring FLX-induced anxiety-related responses across all behavioral tasks, while restoring ERK2-CREB-proBDNF markers to control levels and increasing mBDNF within the prefrontal cortex, but not the hippocampus. Collectively, these results indicate that adolescent FLX history mediates neurobehavioral adaptations that endure into adulthood, which are indicative of a generalized anxiety-like phenotype, and that this persistent effect is ameliorated by later-life FLX re-exposure, in a prefrontal cortex-specific manner.

Autophagy Induction and Accumulation of Phosphorylated Tau in the Hippocampus and Prefrontal Cortex of Adult C57BL/6 Mice Subjected to Adolescent Fluoxetine Treatment.

BACKGROUND: Fluoxetine (FLX) represents the antidepressant of choice for the management of pediatric mood-related illnesses. Accumulating preclinical evidence suggests that ontogenic FLX exposure leads to deregulated affect-related phenotypes in adulthood. Mood-related symptomatology constitutes a risk-factor for various neurological disorders, including Alzheimer's disease (AD), making it possible for juvenile FLX history to exacerbate the development of neurodegenerative diseases. OBJECTIVE: Because AD is characterized by the pathological accumulation of hyperphosphorylated tau, which can result from impaired function of protein degradation pathways, such as autophagy and the ubiquitin-proteasome system (UPS), we evaluated the long-term effects of adolescent FLX exposure on these pathways, using mice as a model system. METHODS: We subjected C57BL/6 adolescent male mice to FLX (20 mg/kg/day) from postnatal day (PD) 35 to PD49. Twenty-one days after the last FLX injection (i.e., adulthood; PD70), mice were euthanized and, using immunoblotting analysis, we evaluated protein markers of autophagy (Beclin-1, LC3-II, p62) and the UPS (K48-pUb), as well as AD-associated forms of phosphorylated tau, within the hippocampus and prefrontal cortex. RESULTS: Juvenile FLX pre-exposure mediated long-term changes in the expression of protein markers (increased LC3-II and decreased p62) that is consistent with autophagy activation, particularly in the prefrontal cortex. Furthermore, FLX history induced persistent accumulation of AD-associated variants of tau in both the hippocampus and prefrontal cortexConclusion: Adolescent FLX treatment may have enduring effects in the neuronal protein degradation machinery, which could adversely influence clearance of abnormal proteins, potentially predisposing individuals to developing AD in later life.

Enduring effects of adolescent ketamine exposure on cocaine- and sucrose-induced reward in male and female C57BL/6 mice.

Ketamine has shown promising antidepressant efficacy for adolescent treatment-resistant depression. However, the potential enduring consequences of ketamine exposure have not been thoroughly evaluated. Thus, we examined if juvenile ketamine treatment results in long-lasting changes for the rewarding properties of sucrose and cocaine in adulthood, across three separate experiments. In Experiment 1, adolescent male and female C57BL/6 mice received ketamine (20 mg/kg) for 15 consecutive days (Postnatal Day [PD] 35-49). Twenty-one days later (PD70; adulthood) we examined their behavioral responsivity to sucrose (1%) on a two-bottle choice design, or cocaine (0, 5, 10 mg/kg) using the conditioned place preference (CPP) test. We found that juvenile ketamine-pretreatment increased preference for sucrose and environments paired with cocaine in male, but not female, adult mice. This long-term outcome was not observed when male and female mice received ketamine as adults (PD70-84) and tested for sucrose and cocaine preference 21-days later (Experiment 2). Similarly, in Experiment 3, no long-lasting differences in these measures were observed when adolescent male mice were exposed to concomitant ketamine and social stressors (PD35-44), namely the social defeat or vicarious defeat stress paradigms-procedures that mediated a depression-related phenotype (along with a ketamine antidepressant-like response). Collectively, we demonstrate that in the absence of physical or psychological stress, adolescent ketamine exposure increases later life preference for the rewarding properties of sucrose and cocaine in a sex- and age-specific manner. As such, this preclinical work provides awareness for the potential long-term behavioral consequences associated with juvenile ketamine exposure.

Fluoxetine exposure in adolescent and adult female mice decreases cocaine and sucrose preference later in life.

BACKGROUND: Preclinical evidence from male subjects indicates that exposure to psychotropic medications, during early development, results in long-lasting altered responses to reward-related stimuli. However, it is not known if exposure to the antidepressant fluoxetine, in female subjects specifically, changes sensitivity to natural and drug rewards, later in life. AIMS: The aim of this work was to investigate if exposure to fluoxetine mediates enduring changes in sensitivity to the rewarding properties of cocaine and sucrose, using female mice as a model system. METHODS: We exposed C57BL/6 female mice to fluoxetine (250 mg/L in their drinking water) for 15 consecutive days, either during adolescence (postnatal day 35-49) or adulthood (postnatal day 70-84). Twenty-one days later, mice were examined on their behavioral reactivity to cocaine (0, 2.5, 5, 7.5 mg/kg) using the conditioned place preference paradigm, or assessed on the two-bottle sucrose (1%) test. RESULTS: We found that regardless of age of antidepressant exposure, female mice pre-exposed to fluoxetine displayed reliable conditioning to the cocaine-paired compartment. However, when compared to respective age-matched controls, antidepressant pre-exposure decreased the magnitude of conditioning at the 5 and 7.5 mg/kg cocaine doses. Furthermore, fluoxetine pre-exposure reduced sucrose preference without altering total liquid intake. CONCLUSIONS: The data suggest that pre-exposure to fluoxetine, during adolescence or adulthood, results in a prolonged decrease in sensitivity to the rewarding properties of both natural and drug rewards in female C57BL/6 mice.