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

Effects of EtOH treatment on blood alcohol levels in ovariectomized female rats.

<p>Blood alcohol concentrations (BAC) 1.0 h post-injection in ovariectomized female rats given subcutaneous implants of Ch or E₂ and then treated with saline, acute EtOH or repeated binge-pattern EtOH. Data are expressed as Data are expressed as mean ± SEM of EtOH mg/dl. (_*) indicates a statistically significant difference from saline-treated controls (P<0.05).</p

Effects of EtOH treatment (A) and RU486 pre-treatment (B) on CRH promoter activity in a neuronal cell line.

<p>CRH-luciferase activity was measured in IVB cell line after treatment with 100 mM EtOH for 0.5, 1.0, 2.0 or 4.0 h or media alone (A) and after 16 h pretreatment with 100 nM RU486 and 100 mM ETOH/100 nM RU486 co-treatment for 0.5, 1.0, 2.0 or 4.0 h (B). Data expressed as % change in luciferase activity of vehicle treated control. Dissimilar letters indicate statistically significant difference (P<0.05).</p

Effects of concomitant EtOH/E₂ and EtOH/3βdiol treatment on CRH promoter activity in a neuronal cell line.

<p><i>CRH promoter-l</i>uciferase activity was measured in IVB cells transfected with a CRH promoter-luciferase construct and treated with 100 mM EtOH for 2.0 h in media alone or in the presence of 10 nM E₂ or 100 nM 3βdiol. Data are expressed as % change in luciferase activity of vehicle treated control. (_*) indicates statistically significant difference compared to vehicle treated control (P<0.05).</p

Effects of EtOH treatment on AVP gene expression in the SON of ovariectomized female rats.

<p>AVP mRNA expression in the SON of adolescent Ch- or E₂ - treated female rats given saline, acute, or repeated binge-pattern EtOH. Data are expressed as mean ± SEM of AVP mRNA copies/µg total RNA. (_*) indicates a statistically significant difference from control (p<0.05).</p

Schematic representation of mutant CRH promoter constructs.

<p>Diagrams depicting specific nucleotide sequences deleted within the nGRE site of the CRH promoter located between −249 and −278 bp upstream from the transcription initiation site (arrow).</p

Effects of 100 mM EtOH treatment on cell viability in a neuronal cell line.

<p>Mitochondrial activity was measured in IVB cell line after 0.5, 1.0, 2.0 and 4.0 h of 100 mM EtOH treatment. Data expressed as % change of mitochondrial activity relative to vehicle (10% FBS media) treated controls. Dissimilar letters indicate statistically significant difference (P<0.05).</p