Neuroendocrine Consequences of Binge Alcohol Exposure During Peri-Puberty on Functioning of the Hypothalamo-Pituitary-Adrenal (HPA) Axis

Binge alcohol (EtOH) exposure in adolescence is a fundamental health concern. In 2005, over 20% of teenagers between ages 15 and 17 reported binge drinking behavior within a one month period preceding the survey (Dept. of Health and Human Services: Substance Abuse and Mental Health Services Administration). Binge drinking is defined as consuming enough alcohol within a 2.0 h period to bring blood alcohol concentration above 0.08%. In the adolescent population, this type of alcohol exposure tends to be repeated. In adults, alcohol abuse has been correlated with increased incidence of mood disorders and these disorders are characterized by dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis, a three tiered biological system that mediates physiological stress response. Corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP) localized in the paraventricular nucleus (PVN) of the hypothalamus are the major neuropeptides involved in modulating HPA axis responses to stress. Corticosterone (CORT) in rodents, and cortisol in humans, is a major glucocorticoid hormone released into the circulation upon activation of the HPA axis. Puberty is a period during which extensive maturation occurs, yet our knowledge of the nurobiological consequences of binge EtOH exposure during this time period is severely limited. I sought to investigate the neuroendocrine consequences of EtOH exposure during pubertal maturation and tested the hypothesis that EtOH exposure during puberty has long lasting detrimental consequences for a proper maturation of the HPA axis. My data revealed that repeated binge-pattern EtOH exposure resulted in a sex specific dysregulation of the HPA axis in pubertal rats. This was marked by an increase in the expression of CRH and AVP mRNA in the male, but not female, PVN. Notably, in both sexes, both single and repeated binge EtOH exposures resulted in increased circulating CORT levels and habituation effects. These results suggested that repeated binge-like EtOH exposure differentially dysregulated the HPA axis in males compared to females (Przybycien-Szymanska et al., AJP Endocrin. and Metabol., 2010). My data further showed that the sex steroid hormone, 17β-estradiol, is required for the maintenance of steady state levels of CRH and AVP mRNA in the PVN of pubertal female rats and for the habituation effects observed in CORT responses after repeated binge-pattern EtOH exposure. Most striking, these results showed that in males, binge-pattern EtOH exposure during puberty resulted in the dysregulation of adult HPA axis. This was evidenced by 1) increased adult basal levels of CRH mRNA in the PVN and lower basal circulating CORT levels, 2) differential patterns of CRH and AVP mRNA expression in the PVN after subsequent EtOH exposures in adulthood, 3) enhanced circulating CORT increase after single or repeated binge-like exposures in adulthood, 4) lack of habituation in CORT response after adult repeated binge pattern EtOH exposure (Przybycien-Szymanska et al., PLoS One, 2011). In addition, I investigated the molecular mechanisms involved in the observed EtOH-induced increase in the CRH mRNA in the PVN. Increased gene expression correlates closely with increased gene promoter activity; therefore I tested whether EtOH affects CRH promoter activity and whether glucocorticoid negative feedback at the promoter was dysregulated by EtOH. My data showed that EtOH exerts a biphasic effects on the activity of the CRH promoter and these effects are blocked by glucocorticoid receptor (GR) antagonist and deletion of glucocorticoid response element site (a binding site for GR) on CRH promoter. These results indicated that EtOH dysregulates functioning of the HPA axis by interfering with normal negative glucocorticoid feedback mechanism exerted on CRH promoter. Moreover, I showed that 17β-estradiol prevented the EtOH-induced increase in CRH promoter activity supporting a role of this hormone in the sexually dimorphic changes of CRH mRNA in the PVN after EtOH exposure. In conclusion, my data showed that binge-pattern EtOH exposure during puberty has long lasting effects on the HPA axis. These effects are manifested by alterations in PVN expression of neuropeptides involved in modulating stress responses and glucocorticoid hormone signaling. These effects are caused by EtOH-induced dysregulation of glucocorticoid negative feedback normally exerted at the level of the gene promoter in the PVN and may lead to increased risk for mood disorders in adulthood

17β-Estradiol Is Required for the Sexually Dimorphic Effects of Repeated Binge-Pattern Alcohol Exposure on the HPA Axis during Adolescence

Alcohol consumption during adolescence has long-term sexually dimorphic effects on anxiety behavior and mood disorders. We have previously shown that repeated binge-pattern alcohol exposure increased the expression of two critical central regulators of stress and anxiety, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), in adolescent male rats. By contrast, there was no effect of alcohol on these same genes in adolescent females. Therefore, we tested the hypothesis that 17β-estradiol (E2), the predominant sex steroid hormone in females, prevents alcohol-induced changes in CRH and AVP gene expression in the paraventricular nucleus (PVN) of the hypothalamus. To test this hypothesis, postnatal day (PND) 26 females were ovariectomized and given E2 replacement or cholesterol as a control. Next, they were given an alcohol exposure paradigm of 1) saline alone, 2) acute (single dose) or 3) a repeated binge-pattern. Our results showed that acute and repeated binge-pattern alcohol treatment increased plasma ACTH and CORT levels in both E2- and Ch-treated groups, however habituation to repeated binge-pattern alcohol exposure was evident only in E2-treated animals. Further, repeated binge-pattern alcohol exposure significantly decreased CRH and AVP mRNA in Ch-, but not E2-treated animals, which was consistent with our previous observations in gonad intact females. We further tested the effects of E2 and alcohol treatment on the activity of the wild type CRH promoter in a PVN-derived neuronal cell line. Alcohol increased CRH promoter activity in these cells and concomitant treatment with E2 completely abolished the effect. Together our data suggest that E2 regulates the reactivity of the HPA axis to a repeated stressor through modulation of the habituation response and further serves to maintain normal steady state mRNA levels of CRH and AVP in the PVN in response to a repeated alcohol stressor

Stimuli associated with the presence or absence of amphetamine regulate cytoskeletal signaling and behavior

Drug-paired stimuli rapidly enlarge dendritic spines in the nucleus accumbens (NAcc). While increases in spine size and shape are supported by rearrangement of the actin cytoskeleton and facilitate the synaptic expression of AMPA-type glutamate receptors, it remains unclear whether drug-related stimuli can influence signaling pathways known to regulate these changes in spine morphology. These pathways were studied in rats trained on a discrimination learning paradigm using subcellular fractionation and protein immunoblotting to isolate proteins within dendritic spine compartments in the NAcc shell. An open field chamber was repeatedly associated with amphetamine in one group (Paired) and explicitly unpaired with amphetamine in another (Unpaired). Rats in a third group were exposed to the open field but never administered amphetamine (Control). When administered saline and returned to the open field one week later, Paired rats as expected displayed a conditioned locomotor response relative to rats in the other two groups. NAcc shell tissues were harvested immediately after this 30-minute test. Re-exposing Paired rats to the drug-paired excitatory context significantly decreased p-GluA2(S880), an effect consistent with reduced internalization of this subunit and increased spine proliferation in these rats. In contrast, re-exposing Unpaired rats to the drug-unpaired context, capable of inhibiting conditioned responding in these animals, significantly decreased levels of both actin binding protein Arp2/3 and p-cofilin, consistent with spine volatility, shrinkage, and inhibition of spine proliferation in these rats. These findings show that contextual stimuli previously associated with either the presence or absence of amphetamine differentially regulate cytoskeletal signaling pathways in the NAcc

Alcohol Dysregulates Corticotropin-Releasing-Hormone (CRH) Promoter Activity by Interfering with the Negative Glucocorticoid Response Element (nGRE)

EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter

Binge-Pattern Alcohol Exposure during Puberty Induces Long-Term Changes in HPA Axis Reactivity

Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis

Sustained Action of Developmental Ethanol Exposure on the Cortisol Response to Stress in Zebrafish Larvae and Adults

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis study was supported by the National Centre for the replacement, refinement and reduction of animals in research

Preclinical evidence implicating corticotropin-releasing factor signaling in ethanol consumption and neuroadaptation

The results of many studies support the influence of the corticotropin-releasing factor (CRF) system on ethanol (EtOH) consumption and EtOH-induced neuroadaptations that are critical in the addiction process. This review summarizes the preclinical data in this area after first providing an overview of the components of the CRF system. This complex system involves hypothalamic and extra-hypothalamic mechanisms that play a role in the central and peripheral consequences of stressors, including EtOH and other drugs of abuse. In addition, several endogenous ligands and targets make up this system and show differences in their involvement in EtOH drinking and in the effects of chronic or repeated EtOH treatment. In general, genetic and pharmacological approaches paint a consistent picture of the importance of CRF signaling via type 1 CRF receptors (CRF1) in EtOH-induced neuroadaptations that result in higher levels of intake, encourage alcohol seeking during abstinence and alter EtOH sensitivity. Furthermore, genetic findings in rodents, non-human primates and humans have provided some evidence of associations of genetic polymorphisms in CRF-related genes with EtOH drinking, although additional data are needed. These results suggest that CRF1 antagonists have potential as pharmacotherapeutics for alcohol use disorders. However, given the broad and important role of these receptors in adaptation to environmental and other challenges, full antagonist effects may be too profound and consideration should be given to treatments with modulatory effects.The authors were supported by the Department of Veterans Affairs; NIH NIAAA grants P60AA010760, R24AA020245 and U01AA013519 and NIH NIDA grant P50DA018165, during the writing of this manuscript. The authors have no financial conflict of interest to disclose

Caveats of chronic exogenous corticosterone treatments in adolescent rats and effects on anxiety-like and depressive behavior and hypothalamic-pituitary-adrenal (HPA) axis function

<p>Abstract</p> <p>Background</p> <p>Administration of exogenous corticosterone is an effective preclinical model of depression, but its use has involved primarily adult rodents. Using two different procedures of administration drawn from the literature, we explored the possibility of exogenous corticosterone models in adolescence, a time of heightened risk for mood disorders in humans.</p> <p>Methods</p> <p>In experiment 1, rats were injected with 40 mg/kg corticosterone or vehicle from postnatal days 30 to 45 and compared with no injection controls on behavior in the elevated plus maze (EPM) and the forced swim test (FST). Experiment 2 consisted of three treatments administered to rats from postnatal days 30 to 45 or as adults (days 70 to 85): either corticosterone (400 μg/ml) administered in the drinking water along with 2.5% ethanol, 2.5% ethanol or water only. In addition to testing on EPM, blood samples after the FST were obtained to measure plasma corticosterone. Analysis of variance (ANOVA) and alpha level of <it>P </it>< 0.05 were used to determine statistical significance.</p> <p>Results</p> <p>In experiment 1, corticosterone treatment of adolescent rats increased anxiety in the EPM and decreased immobility in the FST compared to no injection control rats. However, vehicle injected rats were similar to corticosterone injected rats, suggesting that adolescent rats may be highly vulnerable to stress of injection. In experiment 2, the intake of treated water, and thus doses delivered, differed for adolescents and adults, but there were no effects of treatment on behavior in the EPM or FST. Rats that had ingested corticosterone had reduced corticosterone release after the FST. Ethanol vehicle also affected corticosterone release compared to those ingesting water only, but differently for adolescents than for adults.</p> <p>Conclusions</p> <p>The results indicate that several challenges must be overcome before the exogenous corticosterone model can be used effectively in adolescents.</p

High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model

Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon\u27s (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits