Environmental stressors influence limited-access ethanol consumption by C57BL/6J mice in a sex-dependent manner

Abstract

Exposure to stress contributes to ethanol consumption in humans, but it produces inconsistent effects on ethanol drinking in rodent models. Therefore, the present study examined the influence of different stressors (restraint, tail suspension, predator odor, foot shock, and tail pinch) on 2-h access to water and 10% ethanol by male and female C57BL/6J mice and determined whether there were sex-dependent differences in response to stress. Plasma corticosterone (CORT) and allopregnanolone (ALLO) were assessed as indexes of hypothalamic-pituitary-adrenal axis activity and of endogenous neurosteroid levels, respectively, following restraint, tail suspension, and predator odor. These stressors increased plasma CORT and ALLO levels, and produced a greater increase in CORT and ALLO levels in females versus males. 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. Predator odor significantly increased ethanol intake on the following two days in females and on the second day after stress in males. Notably, there was a significant positive correlation between CORT levels immediately after predator odor stress and ethanol intake on the following day. In summary, the type of stressor influenced 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 and ALLO levels as well as to increase ethanol intake following predator odor stress. Because predator odor stress is a model of post-traumatic stress disorder (PTSD), the present sex differences have important implications for preclinical studies modeling the comorbidity of PTSD and alcohol use disorders.

Keywords: Allopregnanolone; Corticosterone; Environmental stress; Foot shock; Predator odor; Tail pinch.

Figure 1

Experimental timeline for (A) cohort 1 and (B) cohort 2. Dark arrows refer to days of limited access 10% ethanol (10E) intake without any experimental manipulation. Days of drinking are numbered consecutively, with weekends excluded (since animals had no ethanol access on the weekends). The order and specific day of drinking that each stressor was administered are shown. In cohort 1 (panel A; n=10 Females and n=9 Males), a tail blood sample was taken immediately after the stress exposure on days 19, 34, and 42 for corticosterone (CORT) and allopregnanolone (ALLO) levels and prior to returning the animal to its home cage. A tail blood sample also was taken on day 16 after the 2 hr 10E intake, to assess a pre-stress “baseline”. In cohort 2 (panel B; n=12 Females and n=12 Males), an orbital blood sample was on day 36 to assess blood ethanol concentration (BEC) on a non-stress day.

Figure 2

Blood ethanol concentrations (BECs) and ethanol intake during the 2-hr 2-bottle choice paradigm. Mean (±SEM) BEC (mg/mL) and ethanol intake (g/kg) in female (A) and male (B) mice following 2-hour access to both water and 10% ethanol (10E). Female mice not only consumed greater amounts of 10E during the access period but also had higher BECs when compared to male mice. ***p<0.001, **p<0.01 vs. male mice. (C-E) Regression analysis of 10E intake and BECs, confirming a significant positive relationship between amount of ethanol consumed and the respective BEC in this paradigm. Male mice (n=11) are presented in gray circles, females (n=12) are presented in black squares.

Figure 3

Impact of environmental stressors on ethanol intake. Mean (±SEM) of 10% ethanol (10E) intake (g/kg/2hr) in female and male mice following (A) 30-min restraint (Cohort 1), (B) 6-min tail suspension (Cohort 1), (C) 30-min predator odor (Cohort 1), (D) 30-min predator odor (Cohort 2), (E) 5-min intermittent foot shock (Cohort 2), and (F) 15-min tail pinch stress (Cohort 2). Baseline is the average of the two drinking days immediately prior to the stress day. ***p<0.001, **p<0.01, *p<0.05 and ⁺p≤0.09 vs. respective baseline intake (post hoc t-test). Cohort 1 group size was 9 (males) and 10 (females); Cohort 2 group size was 12 (males) and 12 (females).

Figure 4

Plasma corticosterone (CORT) levels following select stressors. Mean (±SEM) CORT levels (μg/dL) in female and male mice from Cohort 1 at “baseline” and following exposure to 30-min restraint, 6-min tail suspension, and 30-min predator odor. ***p≤0.001 vs. “baseline” level (ANOVA, main effect of stressor), °°°p≤0.001 vs. females (ANOVA, main effect of sex); Group size for females was 10 except for baseline where one sample was lost; Group size for males was 9.

Figure 5

Regression analysis of g/kg ethanol (10E) intake and μg/dL corticosterone (CORT) levels following select stressors in Cohort 1. There was no relationship between CORT levels in male and female mice following restraint or tail suspension stress and 10E intake on the day of (A and C, respectively) or the day after (B and D, respectively) administration of the stressor. However, in female mice, there was a significant positive linear relationship between CORT levels and ethanol intake on the first day post restraint stress (B; r²=0.42, p<0.05), indicating that 42% of the variance in 10E intake on the day after restraint stress could be explained by CORT levels immediately after restraint. Although levels of CORT were not related to 10E intake on the day of predator odor stress (E), there was a significant positive relationship (p<0.05) between CORT levels and ethanol intake on the day after exposure to the predator odor (F) in male and female mice. The r² value indicates that 26% of the variance in 10E intake on the day after predator odor stress could be explained by CORT levels immediately after predator odor exposure.

Figure 6

Plasma allopregnanolone (ALLO) levels following select stressors. Mean (±SEM) ALLO levels (ng/mL) in female and male mice from Cohort 1 at “baseline” and following exposure to 30-min restraint, 6-min tail suspension, and 30-min predator odor. *p<0.05 vs. “baseline” level (ANOVA, main effect of stressor); Group size for females was 5 (baseline), 9 (restraint), and 10 (tail suspension and predator odor); Group size for males was 9 (baseline, restraint, and tail suspension) and 8 (predator odor). A change in group size is reflective of either loss of plasma or levels of ALLO falling below the sensitivity of the assay.

Figure 7

Regression analysis of g/kg ethanol (10E) intake and ng/mL allopregnanolone (ALLO) levels following select stressors in Cohort 1. There was a trend toward a significant negative relationship between ALLO levels following restraint stress and 10E intake on the day of (A), but not the day after (B) administration of the stressor. The r² value indicates that 20% of the variance in 10E intake on the day of restraint stress could be explained by ALLO levels immediately after 30 min of restraint. There was no relationship between ALLO levels following tail suspension or predator odor stress on the day of (C and E, respectively) or the day after (D and F, respectively) administration of the stressor.