The function of the N-methyl-D-asparate (NMDA) subtype of glutamate receptor in the brain is very sensitive to in vitro inhibition by ethanol and initial results show that after animals have ingested ethanol chronically, and are tolerant to and physically dependent on ethanol, the number of NMDA receptors, as measured by binding of the uncompetitive antagonist, MK-801, is increased in several brain areas. This increase is expected to result in increased sensitivity to glutamate and could contribute to CNS hyperexcitability during ethanol withdrawal. In the present proposal, we will investigate in detail the changes in NMDA receptors after chronic ethanol ingestion, and begin to analyze the mechanism of the changes. Quantitative autoradiography and membrane binding studies will be used to analyze the characteristics of the NMDA receptor-gated channel complexes in brain regions of ethanol-fed mice. Agonist and antagonist-preferring forms of the NMDA receptor will be assessed, and binding of ligands to the NMDA receptor site, the ion channel, and the glycine site on the receptor will be assayed. The glycine-NMDA interaction is a focus of the investigations, because of evidence for the importance of the glycine site in the acute effects of ethanol on the NMDA receptor. The functional consequences of alterations in NMDA receptors will be examined by measuring NMDA-stimulated neurotransmitter release in slice preparations from various brain areas of ethanol-fed mice, and by assessing NMDA-stimulated dephosphorylation of the striatal protein, DARPP-32, which can have long-term consequences for CNS function. To determine if receptor 'up-regulation' represents a specific adaptive response to the chronic presence of ethanol, the effects on NMDA receptors of chronic treatment with specific NMDA antagonists will be determined. NMDA receptors will also be characterized in primary cultures of neuronal cells exposed chronically to ethanol, to evaluate the use of these cells as model systems to explore the molecular mechanism of changes in the receptor. These studies will define the changes in NMDA receptor function that may be involved in ethanol withdrawal seizures and ethanol-induced organic brain damage, and will therefore permit a more focused approach to the pharmacotherapy of alcohol withdrawal.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
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Biochemistry, Physiology and Medicine Subcommittee (ALCB)
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University of Colorado Denver
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Bhave, Sanjiv V; Hoffman, Paula L (2004) Phosphatidylinositol 3'-OH kinase and protein kinase A pathways mediate the anti-apoptotic effect of pituitary adenylyl cyclase-activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol. J Neurochem 88:359-69
Wu, P H; Tabakoff, B; Szabo, G et al. (2001) Chronic ethanol exposure results in increased acute functional tolerance in selected lines of HAFT and LAFT mice. Psychopharmacology (Berl) 155:405-12
Snell, L D; Bhave, S V; Tabakoff, B et al. (2001) Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons. J Neurochem 78:396-405
Tabakoff, B; Nelson, E; Yoshimura, M et al. (2001) Phosphorylation cascades control the actions of ethanol on cell cAMP signalling. J Biomed Sci 8:44-51
Bhave, S V; Snell, L D; Tabakoff, B et al. (2000) Chronic ethanol exposure attenuates the anti-apoptotic effect of NMDA in cerebellar granule neurons. J Neurochem 75:1035-44
Bhave, S V; Snell, L D; Tabakoff, B et al. (1999) Ethanol sensitivity of NMDA receptor function in developing cerebellar granule neurons. Eur J Pharmacol 369:247-59
Bhave, S V; Ghoda, L; Hoffman, P L (1999) Brain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action. J Neurosci 19:3277-86
Valenzuela, C F; Bhave, S; Hoffman, P et al. (1998) Acute effects of ethanol on pharmacologically isolated kainate receptors in cerebellar granule neurons: comparison with NMDA and AMPA receptors. J Neurochem 71:1777-80
Bhave, S V; Hoffman, P L (1997) Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA. J Neurochem 68:578-86
Bhave, S V; Snell, L D; Tabakoff, B et al. (1996) Mechanism of ethanol inhibition of NMDA receptor function in primary cultures of cerebral cortical cells. Alcohol Clin Exp Res 20:934-41

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