Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

BACKGROUND: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. OBJECTIVE: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. MATERIALS AND METHODS: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. RESULTS: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. CONCLUSIONS: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

Mice with an autism-associated R451C mutation in neuroligin-3 show intact attention orienting but atypical responses to methylphenidate and atomoxetine in the mouse-Posner task.

RATIONALE: Atypical attention orienting has been associated with some autistic symptoms, but the neural mechanisms remain unclear. The human Posner task, a classic attention orienting paradigm, was recently adapted for use with mice, supporting the investigation of the neurobiological underpinnings of atypical attention orienting in preclinical mouse models. OBJECTIVE: The current study tested mice expressing the autism-associated R451C gene mutation in neuroligin-3 (NL3) on the mouse-Posner (mPosner) task. METHODS: NL3R451C and wild-type (WT) mice were trained to respond to a validly or invalidly cued target on a touchscreen. The cue was a peripheral non-predictive flash in the exogenous task and a central spatially predictive image in the endogenous task. The effects of dopaminergic- and noradrenergic-modulating drugs, methylphenidate and atomoxetine, on task performance were assessed. RESULTS: In both tasks, mice were quicker and more accurate in the validly versus invalidly cued trials, consistent with results in the human Posner task. NL3R451C and WT mice showed similar response times and accuracy but responded differently when treated with methylphenidate and atomoxetine. Methylphenidate impaired exogenous attention disengagement in NL3R451C mice but did not significantly affect WT mice. Atomoxetine impaired endogenous orienting in WT mice but did not significantly affect NL3R451C mice. CONCLUSIONS: NL3R451C mice demonstrated intact attention orienting but altered responses to the pharmacological manipulation of the dopaminergic and noradrenergic networks. These findings expand our understanding of the NL3R451C mutation by suggesting that this mutation may lead to selective alterations in attentional processes.

Increased paternal corticosterone exposure influences offspring behaviour and expression of urinary pheromones.

BACKGROUND: Studies have shown that paternal stress prior to conception can influence the innate behaviours of their offspring. The evolutionary impacts of such intergenerational effects are therefore of considerable interest. Our group previously showed in a model of daily stress that glucocorticoid treatment of adult male mouse breeders prior to conception leads to increased anxiety-related behaviours in male offspring. Here, we aimed to understand the transgenerational effects of paternal stress exposure on the social behaviour of progeny and its potential influence on reproductive success. RESULTS: We assessed social parameters including social reward, male attractiveness and social dominance, in the offspring (F1) and grand-offspring (F2). We report that paternal corticosterone treatment was associated with increased display of subordination towards other male mice. Those mice were unexpectedly more attractive to female mice while expressing reduced levels of the key rodent pheromone Darcin, contrary to its conventional role in driving female attraction. We investigated the epigenetic regulation of major urinary protein (Mup) expression by performing the first Oxford Nanopore direct methylation of sperm DNA in a mouse model of stress, but found no differences in Mup genes that could be attributed to corticosterone-treatment. Furthermore, no overt differences of the prefrontal cortex transcriptome were found in F1 offspring, implying that peripheral mechanisms are likely contributing to the phenotypic differences. Interestingly, no phenotypic differences were observed in the F2 grand-offspring. CONCLUSIONS: Overall, our findings highlight the potential of moderate paternal stress to affect intergenerational (mal)adaptive responses, informing future studies of adaptiveness in rodents, humans and other species.

Paternal early life stress exerts intergenerational effects on male C57Bl/6J offspring risk-taking behaviors and predator scent-induced c-Fos expression.

Paternal preconceptional health factors, such as exposures to stress, diet and exercise, have been found to significantly influence offspring phenotypes in a range of animal models. Preclinical studies have provided evidence that paternal stress is associated with increased stress responsivity and anxiety-related traits, particularly in male offspring. It was previously reported that a paternal history of maternal separation (MS) led to male offspring (PatMS) displaying reduced cautious behavior during exploration of a novel environment. The neural basis for that absence of behavioral moderation is unclear. Here, we investigated the adaptive behavioral responses of control and PatMS male offspring in the predator odor risk-assessment task (PORT). PatMS mice failed to moderate their behaviors in the presence of a predator odor 2,4,5-trimethylthiazoline (TMT). c-Fos mapping revealed reduced cellular activation in fear-regulating brain regions of PatMS mice, such as in the cingulate cortex, dentate gyrus of the hippocampus and the basolateral amygdala. Expression of the paternally imprinted gene Grb10 (previously identified as a key molecular regulator of risk-taking behavior) was unaltered in PatMS mice. However, other paternal imprinted genes such as Igf2 and PEG3 were differentially expressed in PatMS mice. Overall, our study provides the first evidence of an intergenerational influence of preconceptional paternal stress exposure on offspring brain zunction relevant to risk-taking behavior, which is also independent of Grb10 gene expression.

Loss of Tyro3 causes anxiety-relevant behavioural changes in female mice.

White-matter brain abnormalities have been found across a variety of psychiatric disorders. The extent of white matter pathology is proposed to be predictive of the severity of anxiety disorders. However, it is still unknown whether disruptions of white matter integrity precede, and are sufficient to give rise to, the behavioural symptoms. Interestingly, mood disturbances feature prominently in central demyelinating diseases such as multiple sclerosis. It is unclear whether the greater frequency of neuropsychiatric symptoms is linked to underlying neuropathology. In this study, we characterised male and female Tyro3 knockout (KO) mice using a variety of behavioural paradigms. Anxiety-related behaviours were assessed with the elevated-plus maze and light-dark box. Fear memory processing was assessed using fear conditioning and extinction paradigms. Finally, we assessed immobility time in the Porsolt swim test as a measure of depression-related behavioural despair. Surprisingly, loss of Tyro3 did not lead to manifestation of major shifts in baseline behaviour. We noted significant differences in habituation to novel environments and post-conditioning freezing levels of female Tyro3 KO mice, which are consistent with the female bias in anxiety disorders and could be indicative of maladaptive stress-responses. This study has demonstrated that white matter pathology related to a loss of Tyro3 is associated with pro-anxiety behavioural responses of female mice. Future studies could probe their contribution to increased risk for neuropsychiatric disorders when combined with stressful triggering events.

Use of a gene expression signature to identify trimetazidine for repurposing to treat bipolar depression.

OBJECTIVES: The aim of this study was to repurpose a drug for the treatment of bipolar depression. METHODS: A gene expression signature representing the overall transcriptomic effects of a cocktail of drugs widely prescribed to treat bipolar disorder was generated using human neuronal-like (NT2-N) cells. A compound library of 960 approved, off-patent drugs were then screened to identify those drugs that affect transcription most similar to the effects of the bipolar depression drug cocktail. For mechanistic studies, peripheral blood mononuclear cells were obtained from a healthy subject and reprogrammed into induced pluripotent stem cells, which were then differentiated into co-cultured neurons and astrocytes. Efficacy studies were conducted in two animal models of depressive-like behaviours (Flinders Sensitive Line rats and social isolation with chronic restraint stress rats). RESULTS: The screen identified trimetazidine as a potential drug for repurposing. Trimetazidine alters metabolic processes to increase ATP production, which is thought to be deficient in bipolar depression. We showed that trimetazidine increased mitochondrial respiration in cultured human neuronal-like cells. Transcriptomic analysis in induced pluripotent stem cell-derived neuron/astrocyte co-cultures suggested additional mechanisms of action via the focal adhesion and MAPK signalling pathways. In two different rodent models of depressive-like behaviours, trimetazidine exhibited antidepressant-like activity with reduced anhedonia and reduced immobility in the forced swim test. CONCLUSION: Collectively our data support the repurposing of trimetazidine for the treatment of bipolar depression.

Effects of wheel-running on anxiety and depression-relevant behaviours in the MCAO mouse model of stroke: moderation of brain-derived neurotrophic factor and serotonin receptor gene expression.

Stroke continues to be a major cause of mortality globally. Post-stroke treatment is complicated by the heterogenous nature of pathology and the emergence of secondary psychological symptoms are an additional challenge to the recovery process. Poststroke depression (PSD) is a common co-morbidity and is a major impediment to recovery. While selective serotonin reuptake inhibitors (SSRIs) have proven to be clinically efficacious in treating PSD, the pathogenic processes that underlie the manifestation of depressive mood post-stroke remains unclear. Furthermore, the use of SSRIs is associated with risks of intracerebral haemorrhage, so alternative treatment options need to be continuously explored. Exercise has been demonstrated to be beneficial for improving mood in humans and preclinical models of neurological conditions. Little is known of the mood-related benefits of physical exercise post-stroke. Using the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischaemia, we investigated whether behavioural deficits emerge post-MCAO and could be rescued by voluntary wheel-running. We report that MCAO induced hypo-locomotion and anhedonia-related behaviours, with some improvements conferred by wheel-running. Serotonin transporter gene expression was increased in the MCAO hippocampus and frontal cortex, but this increase remained despite wheel-running. Wheel-running associated up-regulation of BDNF gene expression was unaffected in MCAO mice, reflecting conservation of key neuroplasticity molecular pathways. Taken together, our results highlight the need for further research into serotonergic modulation of the affective symptoms of stroke.

Sex-dependent effects of chronic exercise on cognitive flexibility but not hippocampal Bdnf in aging mice.

Cognitive impairments associated with advanced age involve alterations in the hippocampus that changes with experience throughout life. The hippocampus is critical for cognitive flexibility involved with extinction and reinstatement of conditioned fear. It is widely accepted that regular exercise can be beneficial for hippocampal function. Therefore, we asked whether chronic voluntary exercise in middle-aged mice can improve extinction and/or reinstatement of conditioned fear compared with standard-housing. Eight-month-old male and female C57Bl/6J mice had access to a running wheel or remained in standard-housing until 11 months of age. Alongside control standard-housed young adult (3-month-old) mice, they received tone-footshock pairings, which were subsequently extinguished with tone-alone presentations the next day. Half of the mice then received a reminder in the form of a single footshock. Male and female 11-month-old mice housed in standard conditions exhibited impaired reinstatement compared with young adult mice. However, for males that had access to a running wheel from 8 months of age, the reminder treatment rescued reinstatement ability. This was not observed in females. Additionally, exercise during middle age in both sexes increased expression of brain-derived neurotrophic factor (Bdnf) mRNA in the hippocampus, specifically exon 4 mRNA. These results show that, at least for males, physical exercise is beneficial for reducing age-related decline in cognitive abilities. Despite not affecting reinstatement, exercise also increased Bdnf gene expression in the female hippocampus, which could potentially benefit other forms of hippocampus-dependent cognition.

Intergenerational effects of a paternal Western diet during adolescence on offspring gut microbiota, stress reactivity, and social behavior.

The global consumption of highly processed, calorie-dense foods has contributed to an epidemic of overweight and obesity, along with negative consequences for metabolic dysfunction and disease susceptibility. As it becomes apparent that overweight and obesity have ripple effects through generations, understanding of the processes involved is required, in both maternal and paternal epigenetic inheritance. We focused on the patrilineal effects of a Western-style high-fat (21%) and high-sugar (34%) diet (WD) compared to control diet (CD) during adolescence and investigated F0 and F1 mice for physiological and behavioral changes. F0 males (fathers) showed increased body weight, impaired glycemic control, and decreased attractiveness to females. Paternal WD caused significant phenotypic changes in F1 offspring, including higher body weights of pups, increased Actinobacteria abundance in the gut microbiota (ascertained using 16S microbiome profiling), a food preference for WD pellets, increased male dominance and attractiveness to females, as well as decreased behavioral despair. These results collectively demonstrate the long-term intergenerational effects of a Western-style diet during paternal adolescence. The behavioral and physiological alterations in F1 offspring provide evidence of adaptive paternal programming via epigenetic inheritance. These findings have important implications for understanding paternally mediated intergenerational inheritance, and its relevance to offspring health and disease susceptibility.

Alternative models for transgenerational epigenetic inheritance: Molecular psychiatry beyond mice and man.

Mental illness remains the greatest chronic health burden globally with few in-roads having been made despite significant advances in genomic knowledge in recent decades. The field of psychiatry is constantly challenged to bring new approaches and tools to address and treat the needs of vulnerable individuals and subpopulations, and that has to be supported by a continuous growth in knowledge. The majority of neuropsychiatric symptoms reflect complex gene-environment interactions, with epigenetics bridging the gap between genetic susceptibility and environmental stressors that trigger disease onset and drive the advancement of symptoms. It has more recently been demonstrated in preclinical models that epigenetics underpins the transgenerational inheritance of stress-related behavioural phenotypes in both paternal and maternal lineages, providing further supporting evidence for heritability in humans. However, unbiased prospective studies of this nature are practically impossible to conduct in humans so preclinical models remain our best option for researching the molecular pathophysiologies underlying many neuropsychiatric conditions. While rodents will remain the dominant model system for preclinical studies (especially for addressing complex behavioural phenotypes), there is scope to expand current research of the molecular and epigenetic pathologies by using invertebrate models. Here, we will discuss the utility and advantages of two alternative model organisms-Caenorhabditis elegans and Drosophila melanogaster-and summarise the compelling insights of the epigenetic regulation of transgenerational inheritance that are potentially relevant to human psychiatry.

Small Non-coding RNAs Are Dysregulated in Huntington's Disease Transgenic Mice Independently of the Therapeutic Effects of an Environmental Intervention.

Huntington's disease (HD) is a neurodegenerative disorder caused by a trinucleotide repeat expansion in the huntingtin gene. Transcriptomic dysregulations are well-documented in HD and alterations in small non-coding RNAs (sncRNAs), particularly microRNAs (miRNAs), could underpin that phenomenon. Additionally, environmental enrichment (EE), which is used to model a stimulating lifestyle in pre-clinical research, has been shown to ameliorate HD-related symptoms. However, the mechanisms mediating the therapeutic effects of EE remain largely unknown. This study assessed the effect of EE on sncRNA expression in the striatum of female R6/1 transgenic HD mice at 12 weeks (prior to over motor deficits) and 20 weeks (fully symptomatic) of age. When comparing wild-type and R6/1 mice in the standard housing condition, we found 6 and 64 miRNAs that were differentially expressed at 12 and 20 weeks of age, respectively. The 6 miRNAs (miR-132, miR-212, miR-222, miR-1a, miR-467a, and miR-669c) were commonly dysregulated at both time points. Additionally, genotype had minor effects on the levels of other sncRNAs, in particular, 1 piRNA was dysregulated at 12 weeks of age, and at 20 weeks of age 11 piRNAs, 1 tRNA- and 2 snoRNA-derived fragments were altered in HD mice. No difference in the abundance of other sncRNA subtypes, including rRNA- and snRNA- derived fragments, were observed. While EE improved locomotor symptoms in HD, we found no effect of the housing condition on any of the sncRNA populations examined. Our findings show that HD mainly affects miRNAs and has a minor effect on other sncRNA populations. Furthermore, the therapeutic effects of EE are not associated with the rescue of these dysregulated sncRNAs and may therefore exert these experience-dependent effects via other molecular mechanisms.

Cardinal role of the environment in stress induced changes across life stages and generations.

The stress response in rodents and humans is exquisitely dependent on the environmental context. The interactive element of the environment is typically studied by creating laboratory models of stress-induced plasticity manifested in behavior or the underlying neuroendocrine mediators of the behavior. Here, we discuss three representative sets of studies where the role of the environment in mediating stress sensitivity or stress resilience is considered across varying windows of time. Collectively, these studies testify that environmental variation at an earlier time point modifies the relationship between stressor and stress response at a later stage. The metaplastic effects of the environment on the stress response remain possible across various endpoints, including behavior, neuroendocrine regulation, region-specific neural plasticity, and regulation of receptors. The timescale of such variation spans adulthood, across stages of life history and generational boundaries. Thus, environmental variables are powerful determinants of the observed diversity in stress response. The predominant role of the environment suggests that it is possible to promote stress resilience through purposeful modification of the environment.

Orexin-1 receptor signaling within the lateral hypothalamus, but not bed nucleus of the stria terminalis, mediates context-induced relapse to alcohol seeking.

BACKGROUND: The lateral hypothalamic orexin (hypocretin) system has a well-established role in the motivation for reward. This has particular relevance to substance use disorders since orexin-1 receptors play a critical role in alcohol-seeking behavior, acting at multiple nodes in relapse-associated networks. AIMS: This study aimed to further our understanding of the role of orexin-1 receptor signaling within the lateral hypothalamus and bed nucleus of the stria terminalis, specifically in context-induced relapse to alcohol-seeking following punishment-imposed abstinence. METHODS: We trained inbred male alcohol-preferring rats to self-administer alcohol in one environment or context (Context A) and subsequently punished their alcohol-reinforced lever presses in a different environment (Context B) using contingent foot shock punishment. Finally, we tested rats for relapse-like behavior in either context following systemic, intra-lateral hypothalamus or intra-bed nucleus of the stria terminalis orexin-1 receptor antagonism with SB-334867. RESULTS/OUTCOMES: We found that systemic orexin-1 receptor antagonism significantly reduced alcohol-seeking in both contexts. Intra-lateral hypothalamus orexin-1 receptor antagonism significantly reduced alcohol-seeking in Context A whereas intra-bed nucleus of the stria terminalis orexin-1 receptor antagonism had no effect on alcohol-seeking behavior. CONCLUSIONS/INTERPRETATION: Our results suggest a role for the orexin-1 receptor system in context-induced relapse to alcohol-seeking. Specifically, intra-lateral hypothalamus orexin microcircuits contribute to alcohol-seeking.

Preconceptual paternal environmental stimulation alters behavioural phenotypes and adaptive responses intergenerationally in Swiss mice.

Experimental research has recently revealed that paternal environmental conditions can influence the offspring phenotype through epigenetic mechanisms. However, it is unclear whether these effects impact adaptive responses in the offspring. Environmental enrichment (EE) is a well-established paradigm that promotes neural plasticity. We investigated whether EE in male mice could modify behaviours that are highly relevant for determining adaptive fitness, i.e. spatial memory, attractiveness and social dominance, in the offspring of outbred mice. Male Swiss mice were housed in either EE or standard housing from post-weaning to adulthood before breeding for offspring. Their offspring were raised in standard housing until adulthood then assessed for behavioural, physiological and molecular parameters. F0 male mice exposed to EE had lower body weight, higher adrenal, spleen and hippocampal weights, better novelty processing and spatial learning, greater hippocampal BDNF levels, and higher social dominance. Unexpectedly, their male offspring (F1) showed spatial memory impairment, lowered social dominance and were less attractive to receptive females, compared to controls. These ethologically relevant measures suggest a maladaptive response in the male F1 offspring. Interestingly, when separate cohorts of male F1 offspring of standard housing or EE fathers were exposed to 8-day EE protocol during adulthood, differences in spatial memory and attractiveness to receptive females were no longer observed between them. These results provide evidence that the paternal environment can influence the offspring's adaptiveness.

Assessment of conditioned fear extinction in male and female adolescent rats.

Pavlovian fear conditioning and extinction have been widely studied across many species to understand emotional learning and memory. Importantly, it is becoming clear that these processes are affected by sex and age. In adult rodents and humans, sex differences are evident in extinction, with estradiol playing a significant role. In adolescence, an extinction deficit has been reported in rodents and humans. However, the influence of sex on extinction during adolescence is unknown. This is surprising, since adolescence coincides with the onset of hormone cycling, and therefore it might be expected that hormones fluctuations exert a more profound effect at this time. Therefore, we examined Pavlovian fear conditioning and extinction in adolescent male and female rats. In experiment 1, 35-day-old male and female rats were exposed to 6 pairings of a conditioned stimulus (CS, a tone) with an aversive unconditioned stimulus (US, a footshock). The next day they were extinguished in a contextually distinct chamber, via 60 presentations of the CS without the US. Extinction recall was tested 24 hours later in the extinction context. Estrous phase was monitored by cytology on vaginal smears taken 1 hour after each behavioral session. In experiment 2, male and female rats were given sham surgery or gonadectomy at 21 days of age. They were then trained and tested as for experiment 1. We observed that females in proestrus or met/diestrus during extinction showed delayed extinction and impaired extinction recall the next day compared to males. Ovariectomy enhanced extinction for female rats, but orchidectomy delayed extinction for males. Plasma analyses showed that met/di/proestrus phases were associated with high estradiol levels. These findings suggest that high plasma estradiol levels impair extinction for adolescent females. These results contradict what is reported in adult animals, suggesting that hormonal influences on extinction are dependent on age. Given that impaired extinction is widely used as a model to understand resistance to exposure-based therapies, our findings have important implications for understanding mental health treatments in adolescents.

HPA axis regulation and stress response is subject to intergenerational modification by paternal trauma and stress.

There is increasing evidence that one's risk for psychiatric disturbances and metabolic syndromes is influenced by their parents' own health history, lifestyle and living environment. For example, paternal high fat diet is strongly linked to neuroendocrine dysregulation in offspring and increased risk for diabetes. The potential intergenerational impact of paternal stress has only just begun to emerge, with the initial evidence suggestive of greater risk for anxiety-related disorders. The hypothalamic-pituitary-adrenal (HPA)-axis is a key neuroendocrine signalling system involved in physiological homeostasis and stress response. In individuals, dysregulation of this system is closely associated with behavioral deficits and mood disorders. Various preclinical models of paternal stress have demonstrated robust behavioral shifts but little is known about the intergenerational modification of HPA axis function. This review will present evidence drawn from a range of laboratory mouse and rat models that the intergenerational influence of paternal stress on offspring behavioral phenotypes involve some level of HPA axis dysregulation. It makes the case that further investigations to comprehensively profile HPA axis function in offspring generations is warranted.

Novel approaches to alcohol rehabilitation: Modification of stress-responsive brain regions through environmental enrichment.

Relapse remains the most prominent hurdle to successful rehabilitation from alcoholism. The neural mechanisms underlying relapse are complex, but our understanding of the brain regions involved, the anatomical circuitry and the modulation of specific nuclei in the context of stress and cue-induced relapse have improved significantly in recent years. In particular, stress is now recognised as a significant trigger for relapse, adding to the well-established impact of chronic stress to escalate alcohol consumption. It is therefore unsurprising that the stress-responsive regions of the brain have also been implicated in alcohol relapse, such as the nucleus accumbens, amygdala and the hypothalamus. Environmental enrichment is a robust experimental paradigm which provides a non-pharmacological tool to alter stress response and, separately, alcohol-seeking behaviour and symptoms of withdrawal. In this review, we examine and consolidate the preclinical evidence that alcohol seeking behaviour and stress-induced relapse are modulated by environmental enrichment, and these are primarily mediated by modification of neural activity within the key nodes of the addiction circuitry. Finally, we discuss the limited clinical evidence that stress-reducing approaches such as mindfulness could potentially serve as adjunctive therapy in the treatment of alcoholism. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment".

Adolescent Inhalant Abuse Results in Adrenal Dysfunction and a Hypermetabolic Phenotype with Persistent Growth Impairments.

BACKGROUND/AIMS: Abuse of toluene products (e.g., glue-sniffing) primarily occurs during adolescence and has been associated with appetite suppression and weight impairments. However, the metabolic phenotype arising from adolescent inhalant abuse has never been fully characterised, and its persistence during abstinence and underlying mechanisms remain unknown. METHODS: Adolescent male Wistar rats (post-natal day 27) were exposed to inhaled toluene (10,000 ppm) (n = 32) or air (n = 48) for 1 h/day, 3 days/week for 4 weeks, followed by 4 weeks of abstinence. Twenty air rats were pair-fed to the toluene group, to differentiate the direct effects of toluene from under-nutrition. Food intake, weight, and growth were monitored. Metabolic hormones were measured after exposure and abstinence periods. Energy expenditure was measured using indirect calorimetry. Adrenal function was assessed using adrenal histology and hormone testing. RESULTS: Inhalant abuse suppressed appetite and increased energy expenditure. Reduced weight gain and growth were observed in both the toluene and pair-fed groups. Compared to the pair-fed group, and despite normalisation of food intake, the suppression of weight and growth for toluene-exposed rats persisted during abstinence. After exposure, toluene-exposed rats had low fasting blood glucose and insulin compared to the air and pair-fed groups. Consistent with adrenal insufficiency, adrenal hypertrophy and increased basal adrenocorticotropic hormone were observed in the toluene-exposed rats, despite normal basal corticosterone levels. CONCLUSIONS: Inhalant abuse results in negative energy balance, persistent growth impairment, and endocrine changes suggestive of adrenal insufficiency. We conclude that adrenal insufficiency contributes to the negative energy balance phenotype, potentially presenting a significant additional health risk for inhalant users.