Ethanol Administration Produces Divergent Changes in GABAergic Neuroactive Steroid Immunohistochemistry in the Rat Brain


Jason B. Cook, Ana Maria G. Dumitru, Todd K. O'Buckley, A. Leslie Morrow


Background: The 5α-reduced pregnane neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a potent positive modulator of GABAA receptors capable of modulating neuronal activity. In rats, systemic ethanol (EtOH) administration increases cerebral cortical and hippocampal levels of 3α,5α-THP, but the effects of EtOH on 3α,5α-THP levels in other brain regions are unknown. There is a large body of evidence suggesting that 3α,5α-THP enhances EtOH sensitivity, contributes to some behavioral effects of EtOH, and modulates EtOH reinforcement and motivation to drink. In this study, we used immunohistochemistry (IHC) to determine EtOH-induced changes in cellular 3α,5α-THP expression in brain regions associated with EtOH actions and responses. Methods: Male Wistar rats were administered EtOH (2 g/kg) or saline intraperitoneally and after 60 minutes transcardially perfused. IHC was performed on free-floating sections (3 to 4 sections/animal/brain region) using an affinity purified anti-3α,5α-THP primary antibody, and immunoreactivity was visualized with 3,3′-diaminobenzidine. Results: EtOH significantly increased 3α,5α-THP immunoreactivity by 24 ± 6% in the medial prefrontal cortex, 32 ± 12% in the hippocampal Cornu Ammonis area 1 (CA1) pyramidal cell layer, 52 ± 5% in the polymorph cell layer of the dentate gyrus (DG), 44 ± 15% in the bed nucleus of the stria terminalis, and 36 ± 6% in the paraventricular nucleus of the hypothalamus. In contrast, EtOH administration significantly reduced 3α,5α-THP immunoreactivity by 25 ± 5% in the nucleus accumbens “shore” and 21 ± 3% in the central nucleus of the amygdala. No changes were observed in the ventral tegmental area, dorsomedial striatum, granule cell layer of the DG, or the lateral and basolateral amygdala. Conclusions: The results suggest acute EtOH (2 g/kg) produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. Regional differences in the effects of EtOH suggest there may be regional brain synthesis of 3α,5α-THP independent of the adrenal glands and novel mechanisms that reduce cellular 3α,5α-THP. Regional differences in EtOH-induced changes in 3α,5α-THP levels likely contribute to EtOH effects on neuronal function in brain.