The objective of this proposal is to explore the potential role of endogenous 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha5alpha-THP) in the electrophysiological and behavioral actions of ethanol, including the development of ethanol tolerance. We recently discovered that ethanol administration to rats produces an elevation in plasma and brain levels of the potent GABAA receptor neuroactive steroid, 3alpha5alpha-THP, to pharmacologically relevant concentrations. The effects of ethanol on 3alpha5alpha-THP levels in cerebral cortex are time and dose dependent and sufficient to potentiate GABAA receptor function. Moreover, there is a strong correlation between ethanol sleep time and cerebral cortical levels of 3alpha5alpha-THP. In contrast, brain levels of 3alpha5alpha-THP are not altered by acute ethanol challenge in ethanol dependent rats, therefore, tolerance may develop to the effect of ethanol on the induction of 3alpha5alpha-THP. The loss of ethanol induction of 3alpha5alpha-THP levels may underlie tolerance to the pharmacological effects of ethanol. Therefore, we propose to test the overall hypothesis that 3alpha, 5alpha-THP mediates pharmacological effects of ethanol in vivo. The first goal is to investigate the role of 3alpha5alpha-THP in the behavioral and neurophysiological effects of ethanol. 3alpha5alpha-THP formation will be prevented by pretreatment steroid biosynthesis inhibitors and the effects of ethanol on neuronal firing rates, GABAA receptor-mediated inhibition of spontaneous neuronal activity, intoxication, aerial righting reflex and seizure thresholds will be measured. The role of 3alpha 5alpha-THP will also be investigated in conditional knockout mice that lack 3alpha-hydroxysteroid dehydrogenase-the final step in 3alpha5alpha-THP formation.
The second aim will determine if 3alpha5alpha-THP plays a role in the development of tolerance to ethanol using both knock out mice and steroid biosynthesis inhibitors.
The third aim will focus on the mechanisms of 3alpha5alpha-THP accumulation following ethanol administration. Studies will be conducted to determine if ethanol directly alters the activity of the 3alpha5alpha-THP biosynthetic enzymes. Preliminary results suggest that ethanol may increase 3alpha5alpha-THP biosynthesis, while high dose ethanol may release or uncover a """"""""store"""""""" of 3alpha5alpha-THP. The effect of ethanol on 3alpha5alpha-THP release from cultured astrocytes will be measured. These studies will address the effects of ethanol at rapid time points (seconds to minutes) that may be relevant to the electrophysiological actions of ethanol. These studies may elucidate a new mechanism of ethanol action in the CNS and explain why the effects of ethanol on GABAergic neurotransmission could not be adequately explained by the direct action of ethanol at GABAA receptors. The results of this investigation will extend our knowledge of the potential role of neurosteroids in ethanol action and ethanol tolerance and may identify new factors involved in the etiology of alcoholism.
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