Abstinence from prolonged ethanol exposure affects plasma corticosterone, glucocorticoid receptor signaling and stress-related behaviors

Abstract

Alcohol dependence is linked to dysregulation of the hypothalamic-pituitary-adrenal axis. Here, we investigated effects of repeated ethanol intoxication-withdrawal cycles (using chronic intermittent ethanol vapor inhalation; CIE) and abstinence from CIE on peak and nadir plasma corticosterone (CORT) levels. Irritability- and anxiety-like behaviors as well as glucocorticoid receptors (GR) in the medial prefrontal cortex (mPFC) were assessed at various intervals (2h-28d) after cessation of CIE. Results show that peak CORT increased during CIE, transiently decreased during early abstinence (1-11d), and returned to pre-abstinence levels during protracted abstinence (17-27d). Acute withdrawal from CIE enhanced aggression- and anxiety-like behaviors. Early abstinence from CIE reduced anxiety-like behavior. mPFC-GR signaling (indexed by relative phosphorylation of GR at Ser211) was transiently decreased when measured at time points during early and protracted abstinence. Further, voluntary ethanol drinking in CIE (CIE-ED) and CIE-naïve (ED) rats, and effects of CIE-ED and ED on peak CORT levels and mPFC-GR were investigated during acute withdrawal (8h) and protracted abstinence (28d). CIE-ED and ED increased peak CORT during drinking. CIE-ED and ED decreased expression and signaling of mPFC-GR during acute withdrawal, an effect that was reversed by systemic mifepristone treatment. CIE-ED and ED demonstrate robust reinstatement of ethanol seeking during protracted abstinence and show increases in mPFC-GR expression. Collectively, the data demonstrate that acute withdrawal from CIE produces robust alterations in GR signaling, CORT and negative affect symptoms which could facilitate excessive drinking. The findings also show that CIE-ED and ED demonstrate enhanced relapse vulnerability triggered by ethanol cues and these changes are partially mediated by altered GR expression in the mPFC. Taken together, transition to alcohol dependence could be accompanied by alterations in mPFC stress-related pathways that may increase negative emotional symptoms and increase vulnerability to relapse.

Keywords: Abstinence; Aggression; Anxiety; Corticosterone; Glucocorticoid receptor; Medial prefrontal cortex.

Figure 1. Plasma corticosterone (CORT) concentration and glucocorticoid receptors (GR) expression in the medial prefrontal cortex are modulated by chronic intermittent ethanol experience (CIE) and abstinence from CIE

Rats were housed in reverse light-cycle 12h light/12h dark rooms and were exposed to 7 weeks of CIE followed by 4 weeks of abstinence. (A) For Experiment 1, plasma CORT was measured one hour before the end of the light cycle (peak CORT) and again two hours before the end of the dark cycle (nadir CORT). Peak CORT increased at week 5 and 6 of CIE compared to pre-CIE levels (week 0). Peak CORT levels decreased transiently during early-intermediate abstinence (day 1-11) compared to pre-abstinence (week 5-6) and then increased back to levels seen during CIE. No differences were revealed in nadir CORT between weeks. Values are mean ± S.E.M of plasma CORT (in ng/mL plasma). n=8 during CIE and 4 during abstinence. * p<0.05 compared to CORT before the onset of CIE (Week 0); # p<0.05 compared to peak CORT before the cessation of CIE (Week 5-6). (B-E). For Experiment 2, protein expression of total GR (tGR) and GR phosphorylated at Ser211 (pGR(^Ser211)) was evaluated during various intervals into ethanol abstinence. Representative immunoblots of pGR(^Ser211) (B), tGR (D), with corresponding blots for beta-tubulin (β-Tub) from one control and one CIE rat from 7 d and 21 d time points. Densitometric analysis revealed that GR function (pGR^(Ser211)/tGR) transiently decreased during intermediate abstinence (7-21 d) from CIE (C). In contrast, tGR transiently increased during early-intermediate abstinence (7 d). Values are mean ± S.E.M. for pGR(ser211)/tGR and tGR in the prefrontal cortex of rats withdrawn from CIE presented as a percent of ethanol naïve controls (100 ± 11.8 and 114 ± 13.0, respectively, represented by the dotted line). n=4-12/group, * p<0.05 compared to the 100% expression in the ethanol naïve controls. For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.

Figure 2. Aggression-like behavior and anxiety-like behavior were altered during abstinence from chronic intermittent ethanol exposure (CIE)

(A, B) Agonistic behavior in rats withdrawn from ethanol after 7 weeks of chronic intermittent ethanol exposure (CIE) and in age-matched ethanol naïve controls was measured using bottlebrush irritability test (BBIT). Values are mean ± S.E.M of aggressive score and defensive score at various time points during ethanol abstinence; n=5-24/group. (A) CIE withdrawn rats show greater aggression-like behavior compared to control, $p<0.05 indicates main effect of CIE; *p<0.05 main effect of time, with score at 14 d being greater than 8 h -3 d time-points. (B) Defensive-like behavior was increased over repeated BBIT across treatment groups, but no effect of ethanol abstinence was revealed. *p<0.05 main effect of time such that 7 d and 14 d was greater than 8 h and 1 d. (C, D) Anxiety-like behavior, measured using elevated plus maze (EPM), was increased at 8 h of abstinence and was decreased at 8 d of abstinence. (C) Time spent in the open arm (% open arm time/(open+closed) arm time) was decreased after 8 h, and increased after 8 d of forced abstinence from ethanol. (C) Number of closed arm entries was decreased at only at 8 h of forced abstinence from ethanol. Values are mean ± S.E.M; n=7-12/group, * p<0.05 compared to corresponding value in ethanol naïve control rats, # p<0.05 compared to corresponding value after 8 h of forced abstinence. For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.

Figure 3. Mifepristone (Mif) inhibited escalated ethanol self-administration and reversed ethanol exposure mediated decrease in glucocorticoid receptor (GR) signaling in the medial prefrontal cortex (mPFC)

Schematic representation of the experimental design is presented. Rats were trained to self-administer ethanol (10%w/v; ethanol drinking, ED) and then divided into four groups. Two groups received chronic intermittent ethanol vapor (CIE) treatment (CIE-ED rats), the other group continued to be maintained under standard housing conditions (ED rats). One CIE-ED and one ED group was implanted with mifepristone sustained release pellet (CIE-ED-Mif and ED-Mif) and the others were implanted with placebo pellets (CIE-ED and ED). All groups were tested for seven sessions of ethanol self-administration across the 24 days of the mifepristone release. (A) Mif suppressed escalation of ethanol self-administration in CIE-ED rats such that CIE-ED-Mif were not different from ED and ED-Mif rats. Also, mifepristone did not alter ethanol self-administration in ED rats. Values are mean ± S.E.M. for ethanol intake in g/kg/30-min ethanol self-administration session. n=8-10/group, * p<0.05 increase in CIE-ED compared to ED rats at an equivalent time; ˆ p<0.05 increase in CIE-ED compared to intake during session 1; # p<0.05 decrease in CIE-ED-Mif compared to CIE-ED at an equivalent time. (B) Representative immunoblots of pGR(ser211), tGR, with corresponding blots for b-tubulin (β-Tub) from ethanol naïve control, ED, CIE-ED, ED-Mif and CIE-ED-Mif at 8 h withdrawal from CIE treatment. Densitometric analysis of (C) GR function (pGR(ser211)/tGR) and (D) total GR expression (tGR) showed that both decreased during acute withdrawal, an effect that was reversed by chronic Mif treatment. Values are mean ± S.E.M. for expression of pGR(ser211)/tGR and GR in the medial prefrontal cortex presented as a percent of age-matched, ethanol and behaviorally naïve controls. n=8-10/group, * p<0.05 decreased compared to age-matched ethanol and behaviorally naïve controls; ^# p<0.05 main effect of Mif, i.e., increased by Mif compared to placebo, ^$ p<0.05 main effect of CIE, i.e. decreased in CIE treated rats compared to ED. For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.

Figure 4. Abstinence in rats with ethanol vapor history and those without it demonstrate differential effects on plasma CORT levels but not in glucocorticoid receptor (GR) signaling in the medial prefrontal cortex

Schematic representation of the experimental design is presented. Rats were trained to self-administer ethanol (10%w/v; ethanol drinking, ED) and then divided into two groups. Plasma CORT was evaluated during alcohol experience and again during experimenter imposed abstinence. In the saline self-administering group, plasma CORT levels were determined only once during abstinence. GR expression and function in the mPFC were evaluated in postmortem tissue obtained after four weeks of abstinence. (A) Peak plasma CORT increased in rats self-administering ethanol (ED) and those exposed to chronic intermittent ethanol exposure (CIE) with ethanol self-administration (CIE-ED) at week 1 of CIE compared to ethanol naïve controls that experienced saline self-administration. In CIE-ED rats, peak CORT increased further during weeks 5 and 7 of vapor experience compared to week 1. Values are mean ± S.E.M of peak CORT levels. n=8-10/group; * p<0.05 compared to ethanol naïve saline self-administering rats; # p<0.05 compared to CIE-ED rats at Week 1. (B) Ethanol intake increases over weeks of CIE in CIE-ED compared to ED. Values are mean ± S.E.M. for ethanol intake in g/kg/30-min ethanol self-administration session. n=8-10/group, * p<0.05 compared to ED rats at an equivalent time; ˆ p<0.05 compared to intake before onset of CIE (Wk 0). (C) Ethanol paired cued-context induced reinstatement (Rst) of ethanol seeking in rats self-administering ethanol with and without CIE (CIE-ED and ED, respectively). Values are mean ± S.E.M. for lever presses. ** p<0.001, increased compared to responding during extinction (Ext); ^## p<0.001, increased compared to responding on the inactive lever. (D) Peak plasma CORT was greater in ED compared to CIE-ED after 9 days of forced abstinence from ethanol, although peak CORT was increased in both CIE-ED and ED compared to ethanol naïve saline self-administering rats. Values are mean ± S.E.M of peak CORT levels. n=8-10/group; * p<0.05 compared to ethanol naïve saline self-administering rats; # p<0.05 compared to CIE-ED rats at 9 days of forced abstinence. (E) Representative immunoblots of pGR, tGR, with corresponding blots for b-tubulin (β-Tub) from control, ED and CIE-ED rats 2h after cue-induced reinstatement of ethanol seeking. Densitometric analysis revealed that GR function (pGR(ser211)/tGR, F) was not different between groups, and GR expression (tGR, G) was increased in both CIE-ED and ED compared to ethanol and behaviorally naïve controls. Values are mean ± S.E.M. for expression of GR and pGR(ser211)/tGR in the medial prefrontal cortex of CIE-ED and ED rats presented as a percent of age-matched, ethanol and behaviorally naïve controls. n=8-10/group, * p<0.05 compared to age-matched ethanol and behaviorally naïve controls. For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.