The goal of this proposal is to investigate the mechanisms guiding synapse formation in the embryonic nervous system. In addition to its implications for neuronal development, this endeavor is likely to yield insight into many different facets of the adult CNS, such as short and long term synaptic modulation, with their implications for memory and learning respectively. One aspect of synapse formation amenable to analysis is neurotransmitter receptor accumulation. The recent cloning of multiple subunits for each of the two subtypes of excitatory neurotransmitter receptors, NMDA and non-NMDA receptors, has made it possible to study the regulation of these receptor subunits during synaptogenesis. A model system will be developed in which purified cultures of Purkinje cells, taken from the rat, can be grown in the presence and absence of their normal source of excitatory input, granule cells, taken from a different species. Using highly specific molecular techniques such as nuclease protection and Western blotting, combined with electrophysiology, changes in the expression of excitatory transmitter receptors by the post synaptic Purkinje cell during innervation will be quantified. Particular attention will be focused on changes in the number, distribution and phenotype of these receptors. An additional goal will be to correlate the expression of excitatory neurotransmitter receptor subunit mRNA and protein, with the electrophysiological properties of receptors recorded from Purkinje cells. Furthermore, the heterologous expression of subunit cDNA's in transfected cells will allow an estimation of the degree to which receptor phenotype is controlled by transcriptional regulation, and the role, if any, of post-translational modification. The mechanism by which excitatory transmitter receptors are regulated, such as phosphorylation, subunit synthesis, or desensitization, will be investigated. Additionally, the agents mediating these changes, and any correlative changes in cell morphology will be sought. The modulation of excitatory neurotransmitter receptors during synapse formation has important implications for many neurodegenerative processes. Excitatory transmission has been implicated as an etiologic agent of both acute and chronic neurological diseases. The alteration of post-synaptic receptor properties such as conductance and desensitization may increase the susceptibility of CNS neurons to the toxicity of excitatory agonists. The multidisciplinary training emphasized in this proposal will allow great flexibility in approaching this issue, and offer excellent preparation for a career integrating clinical investigation and molecular neuroscience.
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