The long-term goal of this project is to understand the function and regulation of synaptic components-on neurons since this is essential for understanding how the nervous system develops and generates specific behaviors. One component of critical importance at the synapse is the neurotransmitter receptor. Families of receptors exist for each major transmitter and a single neuron can express multiple members of the family, but the significance of this for neuronal function is unknown. The central purpose of the current project is to determine the composition, function, and regulation of native acetylcholine receptors (AChRs) on a population of primary neurons with the goal of understanding the contribution each major AChR subtype makes to cell-cell signaling. The model system being used for most of these studies is the chick ciliary ganglion. The neurons contain receptors that are primarily synaptic in location and bind the monoclonal antibody mAb 35 (mAb 35- AChRs). They also have large numbers of receptors primarily synaptic in location that bind alpha- bungarotoxin (alphaBgt-AChRs). The subunit composition of mAb 35-AChRs on the neurons will be examined with subunit- specific mAbs in conjunction with solid phase immunoprecipitations and immunoblots. Individual receptor subtypes in the population will be identified, their contribution to the pool of AChRs on the cell surface determined, and the stoichiometry of subunits in the receptor subtype measured. The physiological properties of alphaBgt-AChRs and mAb 35- AChRs will be compared using whole cell voltage clamp and rapid application of agonist to determine their dose-response curves, desensitization profiles, and ion selectivities. The receptors will be tested for their contributions to ganglionic transmission, and will be compared for their abilities to regulate calcium-dependent events in the cells including calcium-activated currents and release of calcium from internal stores. Immunocytochemistry and immunoblots with subunit- specific mbs will be used to determine which AChR subtypes are destined for unique sites on the neurons such as a presynaptic location on axon terminals or a postsynaptic location on the soma. The source of AChRs containing the neuronal alpha7 subunit in embryonic muscle tissue will be identified and their composition determined. Second messenger regulation of neuronal AChRs will be examined including those of cyclic AMP and arachidonic acid, distinguishing effects on receptor number and function. The role of the postsynaptic target tissue in regulating AChRs on the neurons will be studied, including the contribution of a fibroblast growth factor-like component in the tissue. The results will provide new information about the molecular mechanisms responsible for generating and controlling cell-cell signalling in the nervous system. The health relevance of this research derives from the increase in basic knowledge that it will provide about the nervous system. The findings may hake medical benefits in identifying critical elements of synaptic development and function, and may suggest molecular mechanisms underlying neurodegenerative disorders much as Alzheimer's disease where a neuronal AChR deficit has been reported.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurology C Study Section (NEUC)
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University of California San Diego
Schools of Arts and Sciences
La Jolla
United States
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