Sex differences in binge-like EtOH drinking, corticotropin-releasing hormone and corticosterone: effects of β-endorphin

Abstract

Binge drinking is an increasingly common pattern of risky use associated with numerous health problems, including alcohol use disorders. Because low basal plasma levels of β-endorphin (β-E) and an increased β-E response to alcohol are evident in genetically at-risk human populations, this peptide is thought to contribute to the susceptibility for disordered drinking. Animal models suggest that the effect of β-E on consumption may be sex-dependent. Here, we studied binge-like EtOH consumption in transgenic mice possessing varying levels of β-E: wild-type controls with 100% of the peptide (β-E +/+), heterozygous mice constitutively modified to possess 50% of wild-type levels (β-E +/-) and mice entirely lacking the capacity to synthesize β-E (-/-). These three genotypes and both sexes were evaluated in a 4-day, two-bottle choice, drinking in the dark paradigm with limited access to 20% EtOH. β-E deficiency determined sexually divergent patterns of drinking in that β-E -/- female mice drank more than their wild-type counterparts, an effect not observed in male mice. β-E -/- female mice also displayed elevated basal anxiety, plasma corticosterone and corticotropin-releasing hormone mRNA in the extended amygdala, and all of these were normalized by EtOH self-administration. These data suggest that a heightened risk for excessive EtOH consumption in female mice is related to the drug's ability to ameliorate an overactive anxiety/stress-like state. Taken together, our study highlights a critical impact of sex on neuropeptide regulation of EtOH consumption.

Keywords: BNST; CRF; CeA; HPA axis; POMC; stress.

Figure 1

β‐Endorphin (β‐E) masks sex differences in binge‐like EtOH drinking. (a) Daily consumption of 20% EtOH versus water across the 2‐hour sessions (days 1–3) and 4‐hour binge test (day 4) of the drinking in the dark procedure. A repeated measures ANOVA across days 1–3 revealed a significant sex by genotype interaction, and post hoc analysis indicated that β‐E −/− female mice consumed more EtOH than β‐E −/− male mice. A two‐way ANOVA on the day 4 binge test (BT) revealed a main effect of sex (female mice > male mice) and a sex by genotype interaction. There was a main effect of sex and a sex by genotype interaction for the 4‐hour BT. Post hoc analysis indicated that β‐E −/− female mice consumed more EtOH than β‐E −/− male mice. (b) These data are summarized as average consumption across the 4‐day paradigm. There was a main effect of sex (female mice > male mice), and a sex by genotype interaction with post hoc analysis indicating that β‐E −/− female mice consumed more EtOH overall than β‐E +/− and β‐E +/+ female and β‐E −/− male mice. Data are presented as means ± SEM; *P < 0.05 (Bonferroni corrected); n for each group are displayed within their respective bar

Figure 2

Binge‐like EtOH consumption normalizes corticosterone (CORT) in female, but not male, β‐endorphin (β‐E) −/− mice. Plasma CORT levels obtained immediately following the day 4 binge test from β‐E +/+, +/− and −/− female and male mice consuming either EtOH and water (solid bars) or water controls (hatched bars). A three‐way ANOVA revealed a significant genotype by sex by treatment interaction. Post hoc analysis indicated that β‐E −/− female mice exhibit greater CORT than β‐E +/+ female and β‐E −/− male mice under basal conditions, but voluntary binge‐like EtOH consumption significantly reduces β‐E −/− female CORT levels to near wild‐type levels. Data are presented as means ± SEM; *P < 0.05 (Bonferroni corrected); n for each group are displayed within their respective bar

Figure 3

β‐Endorphin (β‐E) −/− female mice exhibit increased sensitivity to EtOH's anxiolytic effects. (a) Linear regressions depicting the relationship between blood ethanol concentrations (BECs) and preference for the light side of the light–dark box (LDB) in β‐E +/+ and −/− female mice immediately following the binge test. There was a significant positive relationship between degree of intoxication (BECs) and reduced anxiety‐like behavior in β‐E −/−, but not β‐E +/+, female mice, suggesting that β‐E −/− female mice exhibited greater EtOH‐mediated anxiolysis. The dashed line represents the value at which equal time is spent in both compartments of the LDB; the asterisk denotes a significant regression and corresponding goodness of fit value. (b) Because the only significant regressions between BECs and corticosterone (CORT) were observed in β‐E −/− female mice (see Results section), we further explored the effect of EtOH consumption on CORT. All β‐E −/− female mice were split into two groups based on intoxication threshold (BECs < 80 mg/dl and BECs > 80 mg/dl), and CORT levels were compared. β‐E −/− female mice that achieved intoxication tended to exhibit reduced CORT, relative to β‐E −/− female mice that did not, although this did not reach statistical significance (P = 0.060). However, an F‐test comparing variances indicated that β‐E −/− female mice with BECs >80 mg/dl exhibited significantly reduced variability in CORT levels, relative to β‐E −/− female mice with BECs <80 mg/dl (#P = 0.05). (c) Mean summary of data in (a) indicating that, overall, both β‐E +/+ and −/− exhibited anxiety‐like behavior in the LDB; however, there was no significant difference between genotypes. The asterisk denotes mean light side preference significantly lower than the null hypothesis of 50%. (d) There was also no significant genotype difference in locomotor activity, assessed by crossings, during LDB testing. Data are presented as means ± SEM

Figure 4

β‐Endorphin (β‐E) −/− mice have elevated bed nucleus of the stria terminalis (BNST) Crh under basal conditions, but binge‐like ethanol consumption normalizes Crh expression. Crh mRNA expression from mice consuming either EtOH and water (solid bars) or water controls (hatched bars) is represented as mean fold change (±SEM) normalized to β‐E +/+ female water mice as indicated by the dashed line. A three‐way ANOVA revealed a genotype by treatment interaction and a sex by treatment interaction. Post hoc analysis following the genotype by treatment interaction indicated that β‐E −/− water controls exhibit increased Crh expression, relative to β‐E +/+ water controls. However, binge‐like EtOH consumption reduced Crh in β‐E −/−, relative to water controls, such that there was no longer a genotype difference in BNST Crh expression. Note that these effects are primarily driven by differences in female mice; however, the lack of a significant 3‐way interaction precluded our ability to statistically assess such comparisons. Although, post hoc analysis following the sex by treatment interaction indicated that, overall, female water controls have lower BNST Crh mRNA than male water controls (*), which provides novel evidence for basal sex differences in BNST Crh expression. Like symbols indicate significant differences between groups (P < 0.05; Bonferroni corrected); n for each group are displayed within their respective bar

Figure 5

Sex differences in CeA Crh mRNA: β‐Endorphin (β‐E) is inversely associated with CeA Crh in female, but not male, mice. Crh mRNA expression from mice consuming either EtOH and water (solid bars) or water controls (hatched bars) is represented as mean fold change (±SEM) normalized to β‐E +/+ female water mice as indicated by the dashed line. A three‐way ANOVA revealed a genotype by sex interaction and a genotype by treatment interaction. Post hoc analysis following the genotype by sex interaction indicated that β‐E −/− and β‐E +/− female mice exhibit higher CeA Crh than β‐E +/+ female mice; however, these effects were not observed in male mice. Post hoc analysis following the genotype by treatment interaction indicated that binge‐like EtOH consumption reduced Crh only in β‐E −/− mice (*), which was likely driven by the magnitude of reduction in β‐E −/− female mice. Like symbols indicate significant differences between groups (P < 0.05; Bonferroni corrected); n for each group are displayed within their respective bar

Figure 6

β‐Endorphin (β‐E) −/− mice exhibit heightened sensitivity to EtOH‐induced changes in adrenal gland size. Total adrenal gland weight (mean ± SEM, normalized by body weight) from mice consuming either EtOH and water (solid bars) or water controls (hatched bars). A three‐way ANOVA revealed a main effect of sex, indicating that, overall, female mice have larger adrenals than male mice. There was also a genotype by treatment interaction. Post hoc analysis following the genotype by treatment interaction indicated that β‐E −/− water controls have smaller adrenals than β‐E +/+ water controls. However, binge‐like EtOH consumption increased adrenal size only in β‐E −/− mice, relative to β‐E −/− water controls, which resulted in β‐E −/− EtOH mice possessing larger adrenals than β‐E +/+ EtOH mice. Like symbols indicate significant differences between groups (P < 0.05; Bonferroni corrected); n for each group are displayed within their respective bar