Adolescent intermittent alcohol exposure produces strain-specific cross-sensitization to nicotine and other behavioral adaptations in adulthood in C57BL/6J and DBA/2J mice.

Adolescent alcohol exposure is associated with lasting behavioral changes in humans and in mice. Prior work from our laboratory and others have demonstrated that C57BL/6J and DBA/2J mice differ in sensitivity to some effects of acute alcohol exposure during adolescence and adulthood. However, it is unknown if these strains differ in cognitive, anxiety-related, and addiction-related long-term consequences of adolescent intermittent alcohol exposure. This study examined the impact of a previously validated adolescent alcohol exposure paradigm (2-3 g/kg, i.p., every other day PND 30-44) in C57BL/6J and DBA/2J male and female mice on adult fear conditioning, anxiety-related behavior (elevated plus maze), and addiction-related phenotypes including nicotine sensitivity (hypothermia and locomotor depression) and alcohol sensitivity (loss of righting reflex; LORR). Both shared and strain-specific long-term consequences of adolescent alcohol exposure were found. Most notably, we found a strain-specific alcohol-induced increase in sensitivity to nicotine's hypothermic effects during adulthood in the DBA/2J strain but not in the C57BL/6J strain. Conversely, both strains demonstrated a robust increased latency to LORR during adulthood after adolescent alcohol exposure. Thus, we observed strain-dependent cross-sensitization to nicotine and strain-independent tolerance to alcohol due to adolescent alcohol exposure. Several strain and sex differences independent of adolescent alcohol treatment were also observed. These include increased sensitivity to nicotine-induced hypothermia in the C57BL/6J strain relative to the DBA/2J strain, in addition to DBA/2J mice showing more anxiety-like behaviors in the elevated plus maze relative to the C57BL/6J strain. Overall, these results suggest that adolescent alcohol exposure results in altered adult sensitivity to nicotine and alcohol with some phenotypes mediated by genetic background.

Epigenetic and long-term effects of nicotine on biology, behavior, and health.

Tobacco and nicotine use are associated with disease susceptibility and progression. Health challenges associated with nicotine and smoking include developmental delays, addiction, mental health and behavioral changes, lung disease, cardiovascular disease, endocrine disorders, diabetes, immune system changes, and cancer. Increasing evidence suggests that nicotine-associated epigenetic changes may mediate or moderate the development and progression of a myriad of negative health outcomes. In addition, nicotine exposure may confer increased lifelong susceptibility to disease and mental health challenges through alteration of epigenetic signaling. This review examines the relationship between nicotine exposure (and smoking), epigenetic changes, and maladaptive outcomes that include developmental disorders, addiction, mental health challenges, pulmonary disease, cardiovascular disease, endocrine disorders, diabetes, immune system changes, and cancer. Overall, findings support the contention that nicotine (or smoking) associated altered epigenetic signaling is a contributing factor to disease and health challenges.

Chronic nicotine exposure alters sperm small RNA content in C57BL/6J mouse model.

Multigenerational inheritance is a nongenomic form of heritability characterized by altered phenotypes in the first generation born from the exposed parent. Multigenerational factors may account for inconsistencies and gaps in heritable nicotine addiction vulnerability. Our lab previously found that F1 offspring of male C57BL/6J mice chronically exposed to nicotine exhibited altered hippocampus functioning and related learning, nicotine-seeking, nicotine metabolism, and basal stress hormones. In an effort to identify germline mechanisms underlying these multigenerational phenotypes, the current study sequenced small RNA extracted from sperm of males chronically administered nicotine using our previously established exposure model. We identified 16 miRNAs whose expression in sperm was dysregulated by nicotine exposure. A literature review of previous research on these transcripts suggested an enrichment for regulation of psychological stress and learning. mRNAs predicted to be regulated by differentially expressed sperm small RNAs were further analyzed using exploratory enrichment analysis, which suggested potential modulation of pathways related to learning, estrogen signaling, and hepatic disease, among other findings. Overall, our findings point to links between nicotine-exposed F0 sperm miRNA and altered F1 phenotypes in this multigenerational inheritance model, particularly F1 memory, stress, and nicotine metabolism. These findings provide a valuable foundation for future functional validation of these hypotheses and characterization of mechanisms underlying male-line multigenerational inheritance.

Interstrain differences in adolescent fear conditioning after acute alcohol exposure.

Adolescent sensitivity to alcohol is a predictor of continued alcohol use and misuse later in life. Thus, it is important to understand the many factors that can impact alcohol sensitivity. Data from our laboratory suggested that susceptibility to alcohol-associated contextual fear learning deficits varied among adolescent and adult mice from two mouse strains. To investigate the extent of genetic background's influences on adolescent learning after alcohol exposure, we examined how 9 inbred mouse strains differed in vulnerability to alcohol-induced contextual and cued fear conditioning deficits. We demonstrated significant strain- and sex-dependent effects of acute alcohol exposure on adolescent fear learning, with alcohol having most pronounced effects on contextual fear learning. Female adolescents were more susceptible than males to alcohol-induced impairments in contextual, but not cued, fear learning, independent of genetic background. Heritability for contextual and cued fear learning after alcohol exposure was estimated to be 31 % and 18 %, respectively. Learning data were compared to Blood Ethanol Concentrations (BEC) to assess whether strain differences in alcohol metabolism contributed to strain differences in learning after alcohol exposure. There were no clear relationships between BEC and learning outcomes, suggesting that strains differed in learning outcomes for reasons other than strain differences in alcohol metabolism. Genetic analyses revealed polymorphisms across strains in notable genes, such as Chrna7, a promising genetic candidate for susceptibility to alcohol-induced fear conditioning deficits. These results are the first to demonstrate the impact of genetic background on alcohol-associated fear learning deficits during adolescence and suggest that the mechanisms underlying this sensitivity are distinct from alcohol metabolism.

The effect of acute nicotine administration on human delay cued and context fear conditioning.

Nicotine has been shown to facilitate hippocampal-dependent context fear conditioning (FC), but not hippocampal-independent delay cued fear conditioning. Studies examining the effects of nicotine on learned fear have been exclusively limited to nonhumans. The present study aimed to translate nonhuman findings by investigating the effects of nicotine on cued and context fear in humans using a virtual reality (VR) analog of the fear conditioning task. Sixty-seven nicotine-using undergraduates were randomly assigned to receive either a 2 mg nicotine or placebo lozenge prior to conditioning. During conditioning, participants were confined to a virtual room and were conditioned to green floodlight presentations (conditioned stimulus [CS +]) paired with a wrist shock (unconditioned stimulus [US]). A red floodlight served as the CS- during which no shock occurred. Delay cued and context fear testing immediately followed conditioning. Physiological skin conductance responses (SCRs) were recorded continuously throughout all sessions. Nicotine enhanced context fear conditioning such that SCRs to the shock-paired context were significantly greater for the nicotine group than the placebo group. Nicotine did not enhance delay cued fear. Exploratory analyses examining the relationship between fear conditioning and self-reported anxiety revealed that relative to those with lower levels of trait anxiety, nicotine-treated individuals with higher trait anxiety levels were less likely to demonstrate differential conditioning to the shock-paired cue. These findings support abundant nonhuman literature indicating that nicotine facilitates hippocampus-dependent versions of fear conditioning in humans. Results also suggest a role for dysregulated safety learning in pathological anxiety, which may be exacerbated by nicotine use. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Genetic differences in nicotine sensitivity and metabolism in C57BL/6J and NOD/ShiLtJ mouse strains.

Genetic background impacts sensitivity to nicotine's rewarding and aversive effects and metabolism, which influences susceptibility to nicotine addiction. This is important because sensitivity to nicotine influences susceptibility to nicotine addiction. Thus, understanding genetic contribution to nicotine sensitivity can aid in identifying risk factors for nicotine addiction. Genetic variability in addiction phenotypes can be modeled in rodent systems, and comparisons of nicotine sensitivity in inbred mice can identify contributing genetic substrates. Our laboratory has identified differences in nicotine sensitivity in male mice from two inbred mouse strains, C57BL/6J and NOD/ShiLtJ. We found that the NOD/ShiLtJ strain experienced greater nicotine-induced locomotor depression and hypothermia than the C57BL/6J strain. To investigate possible differences in nicotine metabolism between strains, subjects were treated with acute nicotine and serum and urine samples were analyzed using LC-MS/MS to quantify nicotine and metabolites. This analysis revealed that NOD/ShiLtJ mice had similar serum nicotine but lower cotinine and 3'-hydroxycotinine levels after nicotine treatment when compared to C57BL/6J mice. Possible genetic factors mediating strain differences were identified by surveying nicotine sensitivity- and metabolism-related genes within the Mouse Phenome Database SNP retrieval tool. Polymorphisms were found in 15 of the 26 examined gene sequences. Liver expression levels of nicotine metabolism-related genes (Cyp2a5, Cyp2a4, and Aox1) were measured using qPCR. NOD/ShiLtJ mice showed lower expression of Cyp2a5 and Cyp2a4 and greater expression of Aox1 in liver tissue. These data demonstrate complex differences in nicotine sensitivity and metabolism driven by genetic differences between C57BL/6J and NOD/ShiLtJ inbred mouse strains.

Inbred mouse strain differences in alcohol and nicotine addiction-related phenotypes from adolescence to adulthood.

Understanding the genetic basis of a predisposition for nicotine and alcohol use across the lifespan is important for public health efforts because genetic contributions may change with age. However, parsing apart subtle genetic contributions to complex human behaviors is a challenge. Animal models provide the opportunity to study the effects of genetic background and age on drug-related phenotypes, while controlling important experimental variables such as amount and timing of drug exposure. Addiction research in inbred, or isogenic, mouse lines has demonstrated genetic contributions to nicotine and alcohol abuse- and addiction-related behaviors. This review summarizes inbred mouse strain differences in alcohol and nicotine addiction-related phenotypes including voluntary consumption/self-administration, initial sensitivity to the drug as measured by sedative, hypothermic, and ataxic effects, locomotor effects, conditioned place preference or place aversion, drug metabolism, and severity of withdrawal symptoms. This review also discusses how these alcohol and nicotine addiction-related phenotypes change from adolescence to adulthood.

Genetic influences impacting nicotine use and abuse during adolescence: Insights from human and rodent studies.

Nicotine use continues to be a major public health concern, with an alarming recent rise in electronic cigarette consumption. Heritability estimates of nicotine use and abuse range from 40% to 80%, providing strong evidence that genetic factors impact nicotine addiction-relevant phenotypes. Although nicotine use during adolescence is a key factor in the development of addiction, it remains unclear how genetic factors impact adolescent nicotine use and abuse. This review will discuss studies investigating genetic factors impacting nicotine use during adolescence. Evidence from both rodent and human studies will be summarized and integrated when possible. Human adolescent studies have largely included candidate gene studies for genes identified in adult populations, such as genes involved in nicotine metabolism, nicotinic acetylcholine receptor signaling, dopaminergic signaling, and other neurotransmitter signaling systems. Alternatively, rodent studies have largely taken a discovery-based approach identifying strain differences in adolescent nicotine addiction-relevant behaviors. Here, we aim to answer the following three questions by integrating human and rodent findings: (1) Are there genetic variants that uniquely impact nicotine use during adolescence? (2) Are there genetic variants that impact both adolescent and adult nicotine use? and (3) Do genetic factors in adolescence significantly impact long-term consequences of adolescent nicotine use? Determining answers for these three questions will be critical for the development of preventative measures and treatments for adolescent nicotine use and addiction.

The impact of adolescent stress on nicotine use and affective disorders in rodent models.

Recent findings indicate that stress exposure during adolescence contributes to the development of both nicotine use and affective disorders, suggesting a potential shared biological pathway. One key system that may mediate the association between adolescent stress and nicotine or affective outcomes is the hypothalamic-pituitary-adrenal (HPA) axis. Here we reviewed evidence regarding the effects of adolescent stress on nicotine responses and affective phenotypes and the role of the HPA-axis in these relationships. Literature indicates that stress, possibly via HPA-axis dysfunction, is a risk factor for both nicotine use and affective disorders. In rodent models, adolescent stress modulates behavioural responses to nicotine and increases the likelihood of affective disorders. The exact role that the HPA-axis plays in altering nicotine sensitivity and affective disorder development after adolescent stress remains unclear. However, it appears likely that adolescent stress-induced nicotine use and affective disorders are precipitated by repetitive activation of a hyperactive HPA-axis. Together, these preclinical studies indicate that adolescent stress is a risk factor for nicotine use and anxiety/depression phenotypes. The findings summarized here suggest that the HPA-axis mediates this relationship. Future studies that pharmacologically manipulate the HPA-axis during and after adolescent stress are critical to elucidate the exact role that the HPA-axis plays in the development of nicotine use and affective disorders following adolescent stress.

Genetic Differences in Dorsal Hippocampus Acetylcholinesterase Activity Predict Contextual Fear Learning Across Inbred Mouse Strains.

Learning is a critical behavioral process that is influenced by many neurobiological systems. We and others have reported that acetylcholinergic signaling plays a vital role in learning capabilities, and it is especially important for contextual fear learning. Since cholinergic signaling is affected by genetic background, we examined the genetic relationship between activity levels of acetylcholinesterase (AChE), the primary enzyme involved in the acetylcholine metabolism, and learning using a panel of 20 inbred mouse strains. We measured conditioned fear behavior and AChE activity in the dorsal hippocampus, ventral hippocampus, and cerebellum. Acetylcholinesterase activity varied among inbred mouse strains in all three brain regions, and there were significant inter-strain differences in contextual and cued fear conditioning. There was an inverse correlation between fear conditioning outcomes and AChE levels in the dorsal hippocampus. In contrast, the ventral hippocampus and cerebellum AChE levels were not correlated with fear conditioning outcomes. These findings strengthen the link between acetylcholine activity in the dorsal hippocampus and learning, and they also support the premise that the dorsal hippocampus and ventral hippocampus are functionally discrete.

Terc Gene Cluster Variants Predict Liver Telomere Length in Mice.

Variants in a gene cluster upstream-adjacent to TERC on human chromosome 3, which includes genes APRM, LRRC31, LRRC34 and MYNN, have been associated with telomere length in several human populations. Currently, the mechanism by which variants in the TERC gene cluster influence telomere length in humans is unknown. Given the proximity between the TERC gene cluster and TERC (~0.05 Mb) in humans, it is speculated that cluster variants are in linkage disequilibrium with a TERC causal variant. In mice, the Terc gene/Terc gene cluster are also located on chromosome 3; however, the Terc gene cluster is located distantly downstream of Terc (~60 Mb). Here, we initially aim to investigate the interactions between genotype and nicotine exposure on absolute liver telomere length (aTL) in a panel of eight inbred mouse strains. Although we found no significant impact of nicotine on liver aTL, this first experiment identified candidate single nucleotide polymorphisms (SNPs) in the murine Terc gene cluster (within genes Lrrc31, Lrriq4 and Mynn) co-varying with aTL in our panel. In a second experiment, we tested the association of these Terc gene cluster variants with liver aTL in an independent panel of eight inbred mice selected based on candidate SNP genotype. This supported our initial finding that Terc gene cluster polymorphisms impact aTL in mice, consistent with data in human populations. This provides support for mice as a model for telomere dynamics, especially for studying mechanisms underlying the association between Terc cluster variants and telomere length. Finally, these data suggest that mechanisms independent of linkage disequilibrium between the Terc/TERC gene cluster and the Terc/TERC gene mediate the cluster's regulation of telomere length.

Adolescent Stress Reduces Adult Morphine-Induced Behavioral Sensitization in C57BL/6J Mice.

Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence-a critical period of frontal lobe development-influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex (PFC) miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the PFC compared to control mice. The specificity of the effects for C57BL/6J vs. BALB/cJ mice provides evidence of a gene-environment interaction influencing opioid behaviors. However, this conclusion is dampened by limited locomotor sensitization observed in BALB/cJ mice. It remains possible that results may differ to other doses of morphine or other behavioral responses. Long-term differences in stress reactivity or miRNA expression in C57BL/6J mice suggests two possible biological mechanisms to evaluate in future research.

Therapeutic potential of ketamine for alcohol use disorder.

Excessive alcohol consumption is involved in 1/10 of deaths of U.S. working-age adults and costs the country around $250,000,000 yearly. While Alcohol Use Disorder (AUD) pathology is complex and involves multiple neurotransmitter systems, changes in synaptic plasticity, hippocampal neurogenesis, and neural connectivity have been implicated in the behavioral characteristics of AUD. Depressed mood and stress are major determinants of relapse in AUD, and there is significant comorbidity between AUD, depression, and stress disorders, suggesting potential for overlap in their treatments. Disulfiram, naltrexone, and acamprosate are current pharmacotherapies for AUD, but these treatments have limitations, highlighting the need for novel therapeutics. Ketamine is a N-methyl-D-Aspartate receptor antagonist, historically used in anesthesia, but also affects other neurotransmitters systems, synaptic plasticity, neurogenesis, and neural connectivity. Currently under investigation for treating AUDs and other Substance Use Disorders (SUDs), ketamine has strong support for efficacy in treating clinical depression, recently receiving FDA approval. Ketamine's effect in treating depression and stress disorders, such as PTSD, and preliminary evidence for treating SUDs further suggests a role for treating AUDs. This review explores the behavioral and neural evidence for treating AUDs with ketamine and clinical data on ketamine therapy for AUDs and SUDs.

Systems genetic analysis of nicotine withdrawal deficits in hippocampus-dependent learning.

Cognitive deficits, such as disrupted learning, are a major symptom of nicotine withdrawal. These deficits are heritable, yet their genetic basis is largely unknown. Our lab has developed a mouse model of nicotine withdrawal deficits in learning, using chronic nicotine exposure via osmotic minipumps and fear conditioning. Here, we utilized the BXD genetic reference panel to identify genetic variants underlying nicotine withdrawal deficits in learning. Male and female mice (n = 6-11 per sex per strain, 31 strains) received either chronic saline or nicotine (6.3 mg/kg per day for 12 days), and were then tested for hippocampus-dependent learning deficits using contextual fear conditioning. Quantitative trait locus (QTL) mapping analyses using GeneNetwork identified a significant QTL on Chromosome 4 (82.13 Mb, LRS = 20.03, p < 0.05). Publicly available hippocampal gene expression data were used to identify eight positional candidates (Snacpc3, Mysm1, Rps6, Plaa, Lurap1l, Slc24a2, Hacd4, Ptprd) that overlapped with our behavioral QTL and correlated with our behavioral data. Overall, this study demonstrates that genetic factors impact cognitive deficits during nicotine withdrawal in the BXD recombinant inbred panel and identifies candidate genes for future research.

Multigenerational nicotine exposure affects offspring nicotine metabolism, nicotine-induced hypothermia, and basal corticosterone in a sex-dependent manner.

Parental nicotine exposure can impact phenotypes in unexposed offspring. Our laboratory recently published data showing that nicotine reward and hippocampal gene expression involved in stress pathways were perturbed in F1 offspring of male C57BL/6J mice chronically exposed to nicotine. For the current study, we aimed to further test nicotine and stress-sensitivity phenotypes that may predict vulnerability to nicotine addiction in new cohorts of F1 offspring derived from nicotine-exposed males. We tested locomotor and body temperature sensitivity to acute nicotine administration, serum concentration of nicotine and nicotine metabolites after acute nicotine dosing, and serum corticosterone levels in male and female F1 offspring of nicotine- or saline-exposed males. Paternal nicotine exposure reduced sensitivity to nicotine-induced hypothermia in males, altered nicotine metabolite concentrations in males and females, and reduced serum basal corticosterone levels in females. These findings may point to reduced susceptibility to nicotine addiction-related phenotypes as a result of parental nicotine exposure.

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring.

Nicotine use remains highly prevalent with tobacco and e-cigarette products consumed worldwide. However, increasing evidence of transgenerational epigenetic inheritance suggests that nicotine use may alter behavior and neurobiology in subsequent generations. We tested the effects of chronic paternal nicotine exposure in C57BL6/J mice on fear conditioning in F1 and F2 offspring, as well as conditioned fear extinction and spontaneous recovery, nicotine self-administration, hippocampal cholinergic functioning, RNA expression, and DNA methylation in F1 offspring. Paternal nicotine exposure was associated with enhanced contextual and cued fear conditioning and spontaneous recovery of extinguished fear memories. Further, nicotine reinforcement was reduced in nicotine-sired mice, as assessed in a self-administration paradigm. These behavioral phenotypes were coupled with altered response to nicotine, upregulated hippocampal nicotinic acetylcholine receptor binding, reduced evoked hippocampal cholinergic currents, and altered methylation and expression of hippocampal genes related to neural development and plasticity. Gene expression analysis suggests multigenerational effects on broader gene networks potentially involved in neuroplasticity and mental disorders. The changes in fear conditioning similarly suggest phenotypes analogous to anxiety disorders similar to post-traumatic stress.

The effects of adolescent alcohol exposure on learning and related neurobiology in humans and rodents.

Adolescent alcohol use is a widespread problem in the United States. In both humans and rodents, alcohol can impair learning and memory processes mediated by forebrain areas such as the prefrontal cortex (PFC) and hippocampus (HC). Adolescence is a period in which alcohol use often begins, and it is also a time that can be uniquely sensitive to the detrimental effects of alcohol. Exposure to alcohol during adolescence can cause persisting alterations in PFC and HC neurobiology that are linked to cognitive impairments, including changes in neurogenesis, inflammation, and various neurotransmitter systems in rodent models. Consistent with this, chronic adolescent alcohol exposure can cause PFC-dependent learning impairments that persist into adulthood. Deficits in adult HC-dependent learning after adolescent alcohol exposure have also been reported, but these findings are less consistent. Overall, evidence summarized in this review indicates that adolescent exposure to alcohol can produce long-term detrimental effects on forebrain-dependent cognitive processes.

Impact of nicotine, alcohol, and cocaine exposure on germline integrity and epigenome.

Converging evidence suggests that parental exposure to drugs of abuse can affect offspring phenotypes. The impacts of drug abuse on germ cell quality may mediate multigenerational and transgenerational inheritance, although biological pathways underlying this mode of inheritance are not yet characterized. Germline epigenetic marks are modified by drug exposure and have emerged as promising mechanistic candidates in recent work. Drug exposure also impacts overall germline integrity and reproductive functioning, although the role of these consequences in multi/transgenerational inheritance is unclear. This review synthesizes literature on effects of exposure to alcohol, cocaine, and nicotine on the germline with a focus on epigenetic modifications following drug exposure and broader impacts on germline integrity and reproductive functioning. We discuss potential interactions between reproductive functioning, germline integrity, and germline epigenome/transcriptome in pathways underlying multi/transgenerational inheritance. We find that existing data may support independent or interactive contributions of these germline impacts on offspring phenotypes in a manner that may mediate multi/transgenerational inheritance.

Stress and nicotine during adolescence disrupts adult hippocampal-dependent learning and alters stress reactivity.

Adolescence represents increased susceptibility to stress that increases risk for nicotine dependence. The present study examined the interactive effects of brief exposure to stress (shipping/transportation or experimentally induced) and chronic nicotine during adolescence on cognitive function and stress reactivity in adulthood. Adolescent (P31), but not young adult (P47), C57BL/6J mice had higher levels of corticosterone after shipping vs mice bred onsite. Shipped preadolescent (P23) and adolescent (P38) mice, but not those bred onsite, exposed to nicotine showed deficits in contextual fear learning when tested in adulthood. Adult learning deficits were replicated in adolescent mice bred onsite, exposed to experimentally induced stress, and administered chronic nicotine. Stress and nicotine during adolescence resulted in higher expression of hippocampal glucocorticoid receptors and corticotropin-releasing factor receptors and blunted restraint induced CORT release in adulthood. Importantly, studies examining adolescent behavior in mice should consider stress influences outcomes.