Recent research has shown that some of ethanol's effects may be mediated by neuroactive steroid intermediaries. Neuroactive steroids increase in both brain and plasma in response to a modest dose of ethanol. These neuroactive steroids are able to potently modulate several receptors, most notable the K-aminobutyric acid type A receptors (GABAa). Modulation of GABAa receptors by neuroactive steroids such as allopregnanolone (ALLO, 3ohydroxy-5or-pregnan-20-one) has been shown to alter the antidepressant, anxiolytic, sedative and anti-convulsant properties of ethanol. The goal of this proposal is to elucidate the possible role of neurosteroids such as ALLO on acute ethanol withdrawal behaviors in C57BL/6 and DBA/2 mice, two inbred strains that differ markedly in acute ethanol withdrawal severity. Preliminary data has shown that removal of the peripheral sources of pregnane steroids (such as ALLO), primarily the adrenal glands in male mice and the adrenals and ovaries in females mice, increased acute ethanol withdrawal severity. The overall hypothesis is that adrenalectomy (ADX) and gonadectomy (GDX) significantly increase acute ethanol withdrawal by removing an inhibitory neuroactive steroid, such as ALLO. Studies related to Specific Aim 1 will determine which step along the neurosteroid pathway is necessary and/or sufficient to restore the withdrawal profile through systemic administration of neurosteroid precursors in the presence or absence of enzyme inhibitors after ADX and GDX. Once the specific biosynthetic step has been identified, studies related to Specific Aim 2 will microinject the neurosteroid or precursor into specific brain areas in animals that have the sources of neurosteroids removed through ADX and GDX and determine the effect on acute ethanol withdrawal. Concurrently, an analysis of GABAa receptor subunit expression (through RT-PCR) and measurements of hormone concentrations (through radioimmunoassay) will be undertaken in Specific Aim 3 to attempt to further understand the differences in withdrawal severity seen between two different inbred strains. This proposal will investigate the mechanism by which ethanol withdrawal causes rebound neuronal excitability in the hopes that elucidation of this mechanism will open avenues for treatment of ethanol withdrawal. ? ? ?
