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

BackgroundThe GABAergic neuroactive steroid (3,5)-3-hydroxy-pregnan-20-one (3,5-THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3,5-THP levels decreased, and brain levels were not altered following acute EtOH administration (2g/kg) in male C57BL/6J mice; however, the effects of chronic intermittent ethanol (CIE) exposure on 3,5-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary EtOH drinking in a time-dependent fashion following repeated cycles of EtOH exposure, we conducted a time-course analysis of CIE effects on 3,5-THP levels in specific brain regions known to influence drinking behavior. MethodsAdult male C57BL/6J mice were exposed to 4 cycles of CIE to induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72hours following the final exposure cycle. Free-floating brain sections (40m; 3 to 5sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3,5-THP. ResultsWithdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3,5-THP levels across cortical and limbic brain regions. A transient reduction in 3,5-THP immunoreactivity was observed in the central nucleus of the amygdala 8hours after withdrawal from CIE (-31.49.3%). Decreases in 3,5-THP immunoreactivity were observed 72hours 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 3,5-THP immunoreactivity were observed at both time points in the lateral amygdala (8hours -28.3 +/- 12.8%; 72hours -27.5 +/- 12.4%) and in the ventral tegmental area (8hours -26.5 +/- 9.9%; 72hours -31.6 +/- 13.8%). ConclusionsThese data suggest that specific neuroadaptations in 3,5-THP levels may be present in regions of brain that mediate anxiety, stress, and reinforcement relevant to EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed after CIE exposure.