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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AA010564-07
Application #
6732028
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Twombly, Dennis
Project Start
1996-04-01
Project End
2005-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
7
Fiscal Year
2004
Total Cost
$252,875
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Psychiatry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Cook, Jason B; Nelli, Stephanie M; Neighbors, Mackenzie R et al. (2014) Ethanol alters local cellular levels of (3?,5?)-3-hydroxypregnan-20-one (3?,5?-THP) independent of the adrenals in subcortical brain regions. Neuropsychopharmacology 39:1978-87
Cook, Jason B; Werner, David F; Maldonado-Devincci, Antoniette M et al. (2014) Overexpression of the steroidogenic enzyme cytochrome P450 side chain cleavage in the ventral tegmental area increases 3?,5?-THP and reduces long-term operant ethanol self-administration. J Neurosci 34:5824-34
Cook, Jason B; Dumitru, Ana Maria G; O'Buckley, Todd K et al. (2014) Ethanol administration produces divergent changes in GABAergic neuroactive steroid immunohistochemistry in the rat brain. Alcohol Clin Exp Res 38:90-9
Girdler, Susan S; Lindgren, Monica; Porcu, Patrizia et al. (2012) A history of depression in women is associated with an altered GABAergic neuroactive steroid profile. Psychoneuroendocrinology 37:543-53
Porcu, Patrizia; O'Buckley, Todd K; Song, Soomin C et al. (2011) Genetic analysis of the neurosteroid deoxycorticosterone and its relation to alcohol phenotypes: identification of QTLs and downstream gene regulation. PLoS One 6:e18405
Besheer, Joyce; Lindsay, Tessa G; O'Buckley, Todd K et al. (2010) Pregnenolone and ganaxolone reduce operant ethanol self-administration in alcohol-preferring p rats. Alcohol Clin Exp Res 34:2044-52
Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E et al. (2010) Differential effects of ethanol on serum GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in mice, rats, cynomolgus monkeys, and humans. Alcohol Clin Exp Res 34:432-42
Boyd, Kevin N; Kumar, Sandeep; O'Buckley, Todd K et al. (2010) Ethanol induction of steroidogenesis in rat adrenal and brain is dependent upon pituitary ACTH release and de novo adrenal StAR synthesis. J Neurochem 112:784-96
Boyd, Kevin N; Kumar, Sandeep; O'Buckley, Todd K et al. (2010) Chronic ethanol exposure produces tolerance to elevations in neuroactive steroids: mechanisms and reversal by exogenous ACTH. J Neurochem 115:142-52
Ray, Lara A; Hutchison, Kent E; Ashenhurst, James R et al. (2010) Naltrexone selectively elevates GABAergic neuroactive steroid levels in heavy drinkers with the Asp40 allele of the OPRM1 gene: a pilot investigation. Alcohol Clin Exp Res 34:1479-87

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