Environmental factors, including exposure to stress, are known to contribute to the propensity to consume ethanol. However, stress produces inconsistent effects on ethanol drinking in rodent models. Therefore, the present study examined the impact of different stressors on limited access ethanol consumption and determined whether there were sex-dependent differences in response to stress. To this end, male and female C57BL/6J mice had 2-hr access to a water and 10% ethanol solution, beginning 30 minutes before onset of the dark cycle. Once ethanol intake was stable, the effect of restraint, tail suspension, predator odor, foot shock, and tail pinch on subsequent intake was explored. Both plasma corticosterone (CORT) and allopregnanolone (ALLO) were assessed as indices of hypothalamic-pituitary-adrenal (HPA) axis activity and of endogenous neurosteroid levels respectively, following restraint, tail suspension and predator odor. Ethanol intake was decreased following restraint, tail suspension, foot shock, and tail pinch in both sexes, with stressor-related differences in the duration of the suppression. The effect of predator odor on ethanol intake was biphasic in females; ethanol consumption was significantly reduced on the day of stress but significantly increased on the following two days. In males, predator odor produced a delayed significant increase in ethanol intake on the second day after stress. All three stressors increased plasma CORT, with higher CORT levels in females when compared with males. Notably, there was a significant positive correlation between CORT levels immediately after predator odor stress and ethanol intake on the following day as well as a significant positive linear relationship between CORT levels immediately after restraint stress and ethanol intake on the following day in females. Furthermore, the three stressors produced a greater increase in ALLO levels in female versus male mice, but ALLO levels following predator odor were not correlated with subsequent ethanol intake. In summary, the type of stressor administered had a profound impact on subsequent ethanol consumption, with subtle sex differences in the magnitude and persistence of the effect. These findings are the first to demonstrate that a single, acute exposure to restraint, tail suspension, and predator odor stress increased plasma CORT and ALLO levels in animals with a history of ethanol consumption and that female mice were more responsive than males to the ability of stress to increase CORT levels as well as to the ability of predator odor stress to produce a delayed increase in ethanol intake. Because predator odor stress is a model of posttraumatic stress disorder, the present sex differences have important implications for future preclinical studies modeling the comorbidity of posttraumatic stress disorder and alcohol use disorders.

Rationale The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. Objectives Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABAAR sensitivity to ALLO in Withdrawal Seizure–Prone (WSP) but not in Withdrawal Seizure–Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABAAR-active steroids is not known. Methods After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of 10 neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. Results We quantified levels of 9 neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of 5 neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed 5 neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. Conclusions Due to lower basal levels of some GABAAR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.

Manipulation of endogenous levels of the GABAergic neurosteroid allopregnanolone alters sensitivity to some effects of ethanol. Chronic ethanol withdrawal decreases activity and expression of 5α-reductase-1, an important enzyme in allopregnanolone biosynthesis encoded by the 5α-reductase-1 gene (Srd5a1). The present studies examined the impact of Srd5a1 deletion in male and female mice on several acute effects of ethanol and on chronic ethanol withdrawal severity. Genotype and sex did not differentially alter ethanol-induced hypothermia, ataxia, hypnosis, or metabolism, but ethanol withdrawal was significantly lower in female versus male mice. On the elevated plus maze, deletion of the Srd5a1 gene significantly decreased ethanol’s effect on total entries versus wildtype (WT) mice and significantly decreased ethanol’s anxiolytic effect in female knockout (KO) versus WT mice. The limited sex differences in the ability of Srd5a1 genotype to modulate select ethanol effects may reflect an interaction between developmental compensations to deletion of the Srd5a1 gene with sex hormones and levels of endogenous neurosteroids.