We have observed that two neuronal plasma membrane activities, the glutamate binding sites/glutamate receptors and the Na+- Ca2+ exchange carriers are quite sensitive to the effects of ethanol in vitro and in vivo. Long-term administration of ethanol to experimental animals also led to an increase in the maximal glutamate binding and Na+ - Ca2+ exchange capacity of synaptic membranes. We believe that these changes represent important biochemical and physiological neuronal adaptations to the long- term effects of ethanol on brain neurons. We propose to examine the following issues: a) the cellular mechanisms that produce these neuronal changes, and b) the biochemical/physiological consequences of the effects of ethanol exposure on these two membrane-related activities. The specific studies that we plan to conduct are: a) To develop internally-labeled monoclonal antibodies and to use these monoclonal antibodies to quantify the GBP units in membrane fractions from several brain regions of controls, chronically ethanol-treated animals and ethanol-withdrawn animals; b) To determine the effect of ethanol on L-glutamate binding and glutamate-activated ion channels in hippocampal primary cell cultures, measure changes in the number of GBP units in cultured neurons, and measure the effects of ethanol treatment on the rate of synthesis and membrane transfer of GBP; c) To measure free (Ca2+) in synaptosomes and hippocampal neurons with Fura-2 and determine to what extent ethanol's effects are due to inhibition of the antiporter; d) To purify the Na+ - Ca2+ exchange carrier protein and raise polyclonal and monoclonal antibodies against the protein which will be used to determine whether chronic ethanol treatment produces quantitative changes in the carrier protein units detected by immunochemical procedures.
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