The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter receptor in brain, is critical for neuronal development and synaptic plasticity. Recent studies have suggested that the NMDA receptor is an important site of action of ethanol, and has been implicated in the development of alcohol dependence and withdrawal syndrome. Acute exposure to ethanol results in inhibition of NMDA receptor function whereas chronic ethanol treatment upregulates NMDA receptor function and binding. Results from the PI's laboratory demonstrated that exposure to chronic ethanol treatment augments the synthesis of NMDA Rl and R2B subunit polypeptide synthesis with a concomitant increase in only NMDA R2B subunit MRNA levels. Cytoplasmic stability and gene transcription assay results point to a regulation of NMDA RI subunit at the translation level and NMDA R2B subunit at the gene transcription level. To further elucidate the potential molecular mechanisms that underlie this differential regulation, it is necessary to study regulation of the NMDA receptor gene expression at the transcriptional level and at the translation level. In order to undertake such investigations, it is essential to have a neuronal cell line that naturally expresses functional NMDA receptors. In this proposal, the PI's objective is to develop such a cell line by immortalizing murine fetal cortical cells with Simian virus 40 large tumor antigen, an immortalizing oncogene. The PI will generate clonal cell lines which will be extensively characterized 1) by immunocytochemistry to examine the cellular origin of the clonal cell lines; only those that are positive for neuron specific enolase will be further examined for 2) the presence of NMDA receptor subunit polypeptides and mRNAs, 3)the presence of NMDA receptors and 4) the presence of functional NMDA receptors. Cell lines expressing functional NMDA receptors will then be exposed to ethanol to determine if NMDA receptors are regulated in a manner comparable to in vivo. Cell lines expressing functional NMDA receptors will then be used in the future to define the precise mechanism(s) as to how ethanol influences NMDA receptor subunit gene expression.
