The experimental work proposed is a continuation of our research efforts to characterize the effects o chronic alcohol intake on nerve cell function and, especially, on the adaptation made by neurons in the brain to prolonged exposure to ethanol. This proposal has two major themes, a characterization of the molecular events involved in alcohol-induced changes in the expression and function of brain neuronal glutamate/N- methyl-D-aspartate (NMDA) receptors and in the changes in the regulation of intracellular calcium (Ca2+) through altered expression and function of the Na+-Ca2+ exchange proteins (NCX). Ethanol interferes with the normal function of glutamate as an excitatory transmitter in brain and as a result of such acute interference with glutamate neurotransmission, an adaptation occurs in neurons that brings about the appearance of enhanced glutamate receptor activity. This adaptive response is of importance in the manifestation of post-ethanol withdrawal reactions, especially the appearance of seizures. Part of the adaptive response that brain neurons make to the chronic presence of ethanol is to increase the NCX activity by altering the expression of NCX proteins. This altered activity has a protective effect when the cells are confronted with disruption of Ca2+ homeostatic mechanisms. We have provided experimental evidence that NMDA receptors are not a single family of proteins and therefore the complex responses of neurons to ethanol are the result of a great variation of potential macromolecular targets with which ethanol interacts to produce its effects. In addition, we have shown that there are two forms of NCX proteins in brain neurons, therefore the neuronal adaptations may include changes in both types of NCX transporters. The proposed studies are designed to probe the specific changes that occur during chronic ethanol treatment in the expression of the two major forms of NMDA receptor and NCX transport proteins. These studies include the following: an analysis of the expression of the various subunits of the NMDA receptors and of the NCX proteins in brain following chronic ethanol treatment; an analysis of the roles that the NMDAR1 and GBP subunits of the two forms of the receptors play in the development of enhanced sensitivity of neurons chronically treated with ethanol to NMDA; a characterization of the role of two signal transduction pathways in the function of the NMDA receptors in neurons chronically exposed to ethanol; an analysis of the regulation of expression of NMDA receptors and of NCX proteins in cells exposed to ethanol in vivo and in brain in situ; an examination of the effects of ethanol on resting levels and the decay of stimulated elevations in intracellular [Ca2+] in control neurons and neurons exposed to ethanol for several days; and a comparison of the regulatory elements in the promoter regions of the genes for the NMDAR1 and GBP subunits of the two forms of NMDA receptors.
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