ETHANOL INHIBITION OF NMDA RECEPTOR MEDIATED RESPONSES

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The goal of this research project is to further our understandingof the neuronal effects of ethanol (EtOH) that contribute toacute intoxication. Ethanol potently inhibits the function of N- methyl-D-aspartate type glutamate receptors (NMDARs). In most neuronal preparations examined to date, this inhibition is selective with respect to other glutamate receptors. Glutamate is the major excitatory neurotransmitter in the mammalian CNS, and activation of NMDARs by this neurotransmitter has been implicated in a number of CNS functions including motor control and information storage. There is a wealth of evidence indicating that EtOH inhibition of NMDAR function contributes to aspects of acute intoxication, including cognitive impairment and sedation. The mechanism of EtOH action on NMDARs is not fully understood. In addition, there is little definitive information in the literature about the molecular properties of NMDARs that confer EtOH sensitivity. The studies proposed in this application will address these issues by testing two hypotheses. The first aim will test the hypothesis that EtOH inhibition of NMDAR function will be altered in neurons isolated from selected brain regions of mice lacking the epsilon1 or epsilon2 subunits or in which the t-terminal region of these subunits has been deleted. This will be tested by examining EtOH inhibition of receptors in whole-cell recordings from CNS neurons acutely isolated or grown in cell culture. Neurons from the neocortex and cerebellar cortex of wild-type and mutant mice will be examined. It is expected that EtOH will more potently inhibit NMDARs in neocortical neurons from wild-type mice relative to epsilon2 knockout and c-terminal truncated animals. The epsilon1 knockout and c-terminal truncated mice should show a loss of developmental changes in EtOH sensitivity of NMDARs in neocortical neurons. Ethanol sensitivity of NMDA receptors is likely to be enhanced in cerebellar granule cells from epsilon1 mutant mice relative to wild-type mice. NMDAR-mediated synaptic transmission in the CA1 region of hippocampal brain slices will also be examined in the wild-type and epsilon1 mutant mice. It is predicted that EtOH will produce greater inhibition of transmission in wild-type than in epsilon1 knockout and c-terminal truncated mice. The second aim will test the hypothesis that key amino acid residues in the pore-loop and third membrane spanning (TMIII) domains of the NMDAR1 subunit confer EtOH sensitivity on the NMDAR. This will be examined by whole cell electrophysiological experiments in HEK 293 cells expressing recombinant receptors containing mutant or wild-type NMDAR1 subunits. Ethanol sensitivity of wild-type and receptors with single-point mutations in the pore-loop and TMIII regions will be determined. Thorough examination of biophysical and pharmacological properties of mutant receptors will be carried out to determine if mutations specifically affect EtOH sensitivity. The proposed experiments will add to ow knowledge of the molecular basis of EtOH effects on NMDARs. It is hoped that the outcome of these experiments will provide a basis for the development of treatments that can counteract some of the damaging neural effects of EtOH.