The main objective of the present proposal is to continue to identify presumptive peripheral neurotransmitters/neuromodulators and associated biochemical systems of hair-cell organs of mammals and fish. Methods include 1) high-resolution, high-performance liquid chromatography with detection by fluorescence, amperometry, radioactivity, and radioimmunoassays, 2) in vitro release of presumptive neurotransmitters and neuromodulators from a hair cell sheet in which the hair cells is the only intact cell type, from a saccular sensory epithelium including efferents, and from the organ of Corti, 3) biological assay of compounds utilizing Xenopus laevis lateral line, 4) immunohistochemical and immunocytochemical localization of small-molecule conjugates and enzymes of transmitter synthesis and degradation, and 5) analysis of neurotransmitter receptors by radioligand binding, immunocytochemistry, and polymerase chain reaction (PCR). Using these methods, it is planned to chemically identify and determine biological activity of compounds that are released in a calcium-dependent manner from the hair-cell sheet, and determine subtypes of the relevant calcium channels by pharmacological analysis. It is planned to identify biochemical synthesizing systems (enzymes) for neuroactive compounds such as glutamate and glutamate- and aspartate-containing peptides, N- acetylhistidine, and other putative transmitters, utilizing radioactive precursors and HPLIC analysis. Enzymes of neurotransmitters synthesis and degradation will be morphologically localized, and the presence or absence of relevant neurotransmitter receptor will be demonstrated. The overall approach utilizing methods of microbiochemistry, should result in continue, detailed elucidation of peripheral neurotransmitter systems of hearing and balance, pointing the way to development of therapies for transmitter-related hearing loss, vertigo, and tinnitus.
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