Ethanol's effects on receptor- and voltage-gated Ca channels contribute to alcohol intoxication, withdrawal syndromes, and alcohol-related neurologic disorders. The BROAD, LONG-TERM OBJECTIVE of this project is to determine how ethanol affects Ca channel function and Ca-mediated neurotoxicity, and thereby identify pharmacologic approaches for the prevention or treatment of alcohol-related neurologic disease. In previous work, ethanol acutely inhibited N-methyl-D-aspartate receptor- gated Ca channel (NMDA channel) function and NMDA toxicity in cultured cerebral cortical neurons. The proposed research will test the HYPOTHESES that (A) acute inhibition of NMDA-stimulated intracellular Ca (Ca-i) elevation by ethanol correlates with protection from NMDA toxicity; (B) chronic ethanol exposure upregulates NMDA channel function and increases susceptibility to excitotoxic neuronal injury; and (C) upregulation of NMDA channel function by chronic ethanol exposure is associated with increased expression of messenger RNAs coding for one or more channel subunits.
The SPECIFIC AIMS of this project are to: (I) determine the relation between ethanol's acute inhibition of NMDA-stimulated Ca elevations and reduction of NMDA toxicity; (2) determine if chronic exposure to ethanol upregulates NMDA channel binding sites, NMDA-stimulated Ca responses, and NMDA toxicity; and (3) identify the NMDA channel subunits that are upregulated by chronic ethanol exposure. The METHODS to be used are (a) primary culture of embryonic rat cortical neurons; (b) growth of cultures in ethanol-containing medium; (c) spectrophotometric measurement of ethanol concentrations; (d) measurement of Ca by fluo-3 spectrofluorometry, fura-2 photometry, and fura-2 video imaging; (e) assessment of in vitro neurotoxicity by spectrophotometric determination of lactate dehydrogenase release, trypan blue exclusion, and fluorescence microscopy; (f) radioligand binding of 1251-MK-801; (g) isolation of RNA from cortical cultures; (h) preparation of probes for NMDA channel subunit mRNAs from plasmid clones; and (i) Northern analysis. These investigations will yield information that should help to clarify how ethanol alters neuronal function and contributes to neurologic disease.
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