Evidence from our laboratory indicates that ethanol has a regionally specific action in brain to antagonize NMDA-induced excitation of neural activity. The NMDA receptor is composed of multiple protein subunits which form a functional ion channel. These NMDA receptor subunits have differing neuroanatomical distributions, thereby providing evidence for heterogeneity of NMDA receptors in mammalian brain. The purpose of this grant is to test the hypothesis that the regionally specific effect of ethanol on responses to NMDA in brain is linked to the expression of different combination of NMDA receptor subunits. Based upon our present observation of ethanol- induced regional inhibition of NMDA (i.e., ethanol-sensitive and ethanol- insensitive brain regions), the first Specific Aim of this proposal will determine the regional localization of the mRNA for the subunits cloned for the NMDA receptor using PCR to identify NMDAR-1 splice variants and the NMDAR-2a,b,c,&d subunits found in micropunches from brain regions shown electrophysiologically to be either insensitive or sensitive to the action of ethanol to antagonize NMDA-induced excitation.
In Specific Aim II, we will determine the sensitivity of dissociated neurons to ethanol's antagonism of NMDA using fluorescent measures of increased intracellular calcium flux induced by NMDA. Subsequently, the subunit mRNA composition of the NMDA receptors in dissociated cells with proven sensitivity to ethanol will be determined with PCR amplification.
In Specific Aim III, patch-clamp electrophysiology as well as changes in fluorescence will be used to compare the structural components of NMDA isoreceptors within individual neurons with their differing sensitivities to ethanol. Additionally, receptor components will be related to phosphorylation drug actions and to ethanol's inhibition of NMDA. Once the subunit composition for an NMDA receptor in individual neurons sensitive to ethanol has been documented in Specific Aim III, Specific Aim IV will map the NMDA subunits important for ethanol's action throughout brain using in situ hybridization. Following this, extracellular electrophysiological studies will be performed to test additional brain sites predicted from the mapping study to be sensitive to ethanol. Thus, with this multidisciplinary effort, it will be determined whether specific structural components making up NMDA isoreceptors predict ethanol's action on NMDA responses and will delineate 'new"""""""" sites where ethanol does or does not affect responses to NMDA. Such data will define the neurobiological and molecular basis of ethanol's regional action on this ligand-gated ion channel.
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