The long-term goal of this research is to understand from a cellular and molecular perspective how specialized synaptic sites are generated, maintained, and regulated. The specific focus is to elucidate mechanisms underlying neurotransmitter receptor clustering at postsynaptic sites on CNS neurons. Disruptions in synaptic neurotransmitter receptors are associated with long-term pathological effects of seizure and nerve injury, and are partially responsible for the transitory nature of drug therapies for many neurologic diseases, including Parkinson's disease. An understanding of the mechanisms regulating neurotransmitter receptor synthesis and subcellular targeting will be necessary for the development of better molecular therapies for neurologic diseases. The subcellular targeting of neurotransmitter receptors will be studied in cell cultures of embryonic rat hippocampal neurons. Previous work demonstrated that AMPA-selective glutamate and GABAA receptors selectively cluster at postsynaptic sites opposite terminals releasing the corresponding neurotransmitter. Glutamate receptors cluster predominantly on dendritic spines, whereas GABAA receptors cluster predominantly on dendritic shafts and pyramidal cell somata. In the present work, the subcellular distributions of AMPA, NMDA, and metabotropic glutamate receptors will be compared to determine whether these different classes of glutamate receptor cluster at the same synaptic sites or are differentially targeted within individual neurons. Neurons will be specifically denervated of glutamatergic or GABAergic afferents in culture to define the role of the presynaptic terminal in the maintenance of postsynaptic receptor clusters, and to determine whether individual receptor clusters are independently regulated on a one-to-one basis by signals from the apposing presynaptic terminals. A defective herpes simplex virus vector will be used to introduce wild type and deletion mutant cDNA constructs of the metabotropic glutamate receptor mGluR1alpha into primary neurons to define domains requited for somatodendritic targeting and for postsynaptic clustering. The definition of discrete molecular domains required for targeting of a receptor to postsynaptic sites will open up a new approach to elucidating biochemical mechanisms underlying neurotransmitter receptor clustering.
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