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.
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