Sex and β-Endorphin Influence the Effects of Ethanol on Limbic Gabra2 Expression in a Mouse Binge Drinking Model

Abstract

Binge drinking is a widespread problem linked to increased risk for alcohol-related complications, including development of alcohol use disorders. In the last decade, binge drinking has increased significantly, specifically in women. Clinically, sexually dimorphic effects of alcohol are well-characterized, however, the underlying mechanisms for these dimorphisms in the physiological and behavioral effects of alcohol are poorly understood. Among its many effects, alcohol consumption reduces anxiety via the inhibitory neurotransmitter GABA, most likely acting upon receptors containing the α-2 subunit (Gabra2). Previous research from our laboratory indicates that female mice lacking the endogenous opioid peptide β-endorphin (βE) have an overactive stress axis and enhanced anxiety-like phenotype, coupled with increased binge-like alcohol consumption. Because βE works via GABA signaling to reduce anxiety, we sought to determine whether sexually dimorphic binge drinking behavior in βE deficient mice is coupled with differences in CNS Gabra2 expression. To test this hypothesis, we used βE knock-out mice in a "drinking in the dark" model where adult male and female C57BL/6J controls (βE +/+) and βE deficient (βE -/-; B6.129S2-Pomc^tm1Low/J) mice were provided with one bottle of 20% ethanol (EtOH) and one of water (EtOH drinkers) or two bottles of water (water drinkers) 3 h into the dark cycle for four consecutive days. Following a binge test on day 4, limbic tissue was collected and frozen for subsequent qRT-PCR analysis of Gabra2 mRNA expression. Water-drinking βE +/+ females expressed more Gabra2 in central nucleus of the amygdala and the bed nucleus of the stria terminalis than males, but this sex difference was absent in the βE -/- mice. Genotype alone had no effect on alcohol consumption or drug-induced increase in Gabra2 expression. In contrast, βE expression had bi-directional effects in females: in wildtypes, Gabra2 mRNA was reduced by binge EtOH consumption, while EtOH increased expression in βE -/- females to levels commensurate with drug-naïve βE +/+ females. These results support the contention that βE plays a role in sexually dimorphic binge-like EtOH consumption, perhaps through differential expression of GABA_A α2 subunits in limbic structures known to play key roles in the regulation of stress and anxiety.

Keywords: BNST; CeA; GABAA; POMC; alcohol; sex differences; stress.

FIGURE 1

βE masks sex differences in binge-like EtOH consumption. (A) Average 2 h intake across 4 day drinking in the dark (DID). (B) Preference for EtOH solution during the 4 h binge test. (C) Consumption of EtOH during the 4 h binge test on day 4 of the DID procedure. A two-way ANOVA revealed a main effect of sex (female mice > male mice) and a sex by genotype interaction. Post hoc analyses indicated that the βE –/– female mice consumed more EtOH than βE –/– male and βE +/+ female mice. Data are presented as means ± SEM.

FIGURE 2

Effects of ethanol on Gabra2 gene expression in stress- and reward-related brain regions of βE +/+ and βE –/– female mice. Gabra2 mRNA expression following binge-like consumption of either EtOH and water or water only in the DID paradigm. Two-way ANOVAs were used to examine the main and interaction effects of genotype (βE +/+, βE –/–) and treatment (EtOH drinker, water drinkers) in each brain region, results of which are depicted within each graph. (A) In the BNST, post hoc analysis indicated that, under basal conditions (water drinkers), βE –/– females have less Gabra2 expression, relative to βE +/+ females. Further, EtOH consumption reduced Gabra2 expression in βE +/+ females, but increased expression in βE –/– females, such that βE –/– females exhibited higher Gabra2 expression than βE +/+ females who engaged in binge-like EtOH consumption. (B) In the CeA, post hoc analysis indicated that, under basal conditions, βE –/– females have lower Gabra2 expression, relative to βE +/+ females. Similar to the BNST, EtOH also reduced Gabra2 expression in the CeA of βE +/+ females. (C) In the NAc, post hoc analysis indicated that, under basal conditions, βE –/– females have lower Gabra2 expression, relative to βE +/+ females. (D) In the VTA, post hoc analysis indicated that, under basal conditions, βE –/– females have lower Gabra2 expression, relative to βE +/+ females. Following EtOH consumption, βE +/+ females exhibited lower Gabra2 expression than EtOH-drinking βE –/– females due to an EtOH-induced reduction in Gabra2 in βE +/+ females. ^∗p < 0.05 compared with the water drinkers within the same genotype and ^#p < 0.05 compared with the βE +/+ genotype group that received the same treatment. Data are presented as means ± SEM; Bonferroni correction for multiple comparisons.

FIGURE 3

Effects of ethanol on Gabra2 gene expression in stress- and reward-related brains regions of βE +/+ and βE –/– male mice. Gabra2 mRNA expression following binge-like consumption of either EtOH and water or water only in the DID paradigm. Two-way ANOVAs were used to examine the main and interaction effects of genotype (βE +/+, βE –/–) and treatment (EtOH drinker, water drinkers) in each brain region, results of which are depicted within each graph. (A) In the BNST, there was a significant interaction, but post hoc analysis did not reveal any significant differences between groups. (B) In the CeA, post hoc analysis indicated that binge-like EtOH consumption increased Gabra2 expression in βE –/– males. (C) In the NAc, there was a significant interaction, but post hoc analysis revealed no significant differences between groups. (D) Similar to the NAc, in the VTA there was a significant interaction, but post hoc analysis revealed no significant differences between groups. ^∗p < 0.05 compared with the water group within the same genotype. Data are presented as means ± SEM; Bonferroni correction for multiple comparisons.