Chronic Intermittent Ethanol Exposure and Withdrawal Alters (3α,5α)‐3‐Hydroxy‐Pregnan‐20‐One Immunostaining in Cortical and Limbic Brain Regions of C57BL/6J Mice

Abstract

Background The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP, allopregnanolone) has been studied during withdrawal from ethanol in humans, rats and mice. Serum 3α,5α-THP levels decreased and brain levels were not altered following acute ethanol administration (2 g/kg) in male C57BL/6J mice, however the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary ethanol drinking in a time-dependent fashion following repeated cycles of ethanol exposure, we conducted a time-course analysis of CIE effects on 3α,5α-THP levels in specific brain regions known to influence drinking behavior. Methods Adult male C57BL/6J mice were exposed to four cycles of CIE to induce ethanol dependence. All mice were sacrificed and perfused at one of two time points, 8 hr or 72 hr following the final exposure cycle. Free floating brain sections (40 μm; 3-5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α-THP. Results Withdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3α,5α-THP levels across cortical and limbic brain regions. A transient reduction in 3α,5α-THP immunoreactivity was observed in the central nucleus of the amygdala 8 hr after withdrawal from CIE (-31.4 ± 9.3). Decreases in 3α,5α-THP immunoreactivity were observed 72 hr following withdrawal in the medial prefrontal cortex (−25.0 ± 9.3%), nucleus accumbens core (−29.9 ± 6.6%), and dorsolateral striatum (−18.5 ± 6.0%), while an increase was observed in the CA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α-THP immunoreactivity were observed at both time points in the lateral amygdala (8 hr −28.3 ± 12.8%; 72 hr −27.5 ± 12.4%) and in the ventral tegmental area (8 hr −26.5 ± 9.9%; 72 hr −31.6 ± 13.8%). Conclusions These data suggest that specific neuroadaptations in 3α,5α-THP levels may be present in regions of brain that mediate anxiety, stress and reinforcement relevant to ethanol dependence. The changes that occur at different time points likely modulate neurocircuitry involved in ethanol withdrawal as well as the elevated drinking observed after CIE exposure.

Ethanol withdrawal-induced dysregulation of neurosteroid levels in plasma, cortex, and hippocampus in genetic animal models of high and low withdrawal

“…It is interesting that the WSR-1 mice, which were selectively bred for low WD-induced convulsive activity, exhibited the most consistent decrease in cortical neurosteroid levels at 8 h WD; 8 neurosteroids, including ALLO, were significantly decreased. This result is consistent with the decrease in ALLO immunoreactivity in the mPFC of low WD C57 mice at 8 h WD (Maldonado-Devincci et al, 2014). However, the WD-induced decrease in ALLO immunoreactivity in the mPFC persisted at 72 h WD in the Maldonado-Devincci et al (2014) study, whereas cortical ALLO levels were not significantly altered at 48 h WD in WSR-1 mice.…”

“…This result is consistent with the decrease in ALLO immunoreactivity in the mPFC of low WD C57 mice at 8 h WD (Maldonado-Devincci et al, 2014). However, the WD-induced decrease in ALLO immunoreactivity in the mPFC persisted at 72 h WD in the Maldonado-Devincci et al (2014) study, whereas cortical ALLO levels were not significantly altered at 48 h WD in WSR-1 mice. These differences may be due to the differing duration and/or pattern of ethanol vapor exposure between the two studies (72 hrs continuous for WSR-1 mice; 4 bouts of repeated CIE for C57 mice).…”

“…It is likely that the persistent suppression in plasma ALLO and 3α,5β-androstanediol levels occurred at a point downstream of progesterone and testosterone, respectively, given the opposite changes in pregnanolone and 3α,5α-androstanediol that were observed at the 48 h time point. Notably, the finding that different modes of chronic ethanol exposure or consumption and WD produced a consistent reduction in plasma ALLO levels across rodent genotypes, in monkeys, and in humans (Beattie et al, 2017; Cagetti et al, 2004; Hill et al, 2005; Maldonado-Devincci et al, 2014; Romeo et al, 1996; Tanchuck et al, 2009) suggests that adrenal ALLO biosynthesis is suppressed during the acute phase of WD.…”

“…We and others found that WD is associated with a decrease in GABA A R inhibition mediated by a variety of factors that includes functional changes in GABA A R properties, a reduction in the steroidogenic effect of acute ethanol administration, and a decrease in endogenous ALLO levels (see Finn et al, 2004; Kumar et al, 2009). In rodents, monkeys, and humans, WD decreases ALLO levels in plasma and several brain regions (Beattie et al, 2017; Cagetti et al, 2004; Hill et al, 2005; Maldonado-Devincci et al, 2014; Romeo et al, 1996; Snelling et al, 2014; Tanchuck et al, 2009). In small cohorts of male and female alcoholics, the decrease in ALLO and 3α,5α-THDOC levels corresponded to an increase in the subjective ratings of anxiety and depression during days 4 – 5 of WD, versus controls (Hill et al, 2005; Romeo et al, 1996).…”

“…While the pattern of changes in steroid levels imply that ethanol WD is exerting independent effects at several steps of the steroidogenic pathway in plasma (discussed in Snelling et al, 2014), it is not known if similar results would be observed in discrete brain regions. So, cortex and hippocampus were examined in the present studies, based on evidence that basal neurosteroid levels in plasma do not fully reflect levels in cortex and hippocampus (Caruso et al, 2013) and that divergent changes in ALLO immunoreactivity were observed during WD in mPFC and CA3 (Maldonado-Devincci et al, 2014). Cortical and hippocampal regions also exhibit increased spike and sharp wave activity in the electroencephalogram (e.g., Veatch and Becker, 2002; Veatch and Gonzalez, 1996) and increased immediate early gene expression (Vilpoux et al, 2009) that was greater in DBA versus C57 mice (Chen et al, 2009) during WD, highlighting the importance of these brain regions in WD circuitry.…”