. The major objective of the present proposal is to identify peripheral neurotransmitters/neuromodulators and associated biochemical systems of hair-cell organs. Peripheral neurotransmitters voltage-gated calcium channels, and neurotransmitter receptors will be examined for mammalian and fish model systems. The main hypotheses address the existence and function of non-glutamate hair cell transmitter(s), non-L-type voltage- gated calcium channel(s), and recently-described efferent neurotransmitter receptors for acetylcholine and dopamine. Methods include: 1) high- resolution, high-performance liquid chromatography (HPLC) with detection by electrochemistry, fluorescence, radioactivity, and radioimmunoassay, 2) analysis of tissue content, and depolarization-induced release in vitro of presumptive neurotransmitters and neuromodulators from a saccular hair cell sheet for which the hair cell is the only intact cell type, and sound-induced release in vivo into cochlear perilymph, 3) biological assay of compounds utilizing Xenopus laevis lateral line, 4) morphological localization of molecular entities by immunochemical and in situ hybridization methods, 5) sequence analysis as to molecular function after RT-PC (reverse transcription polymerase chain reaction) of voltage-gated calcium channels associated with transmitter release of haircells, and 6) functional sequence analysis after RT-PCR of efferent neurotransmitter receptors for acetylcholine and dopamine. Using these methods, it is planned to identify chemically and determine the biological activity of compounds release from saccular sensory cells in a calcium-dependent manner by low level potassium depolarization and released into perilymph by sound stimulation. Biosynthesis of neuroactive monoamines and related molecules will be studied utilizing radioactive precursors and HPLC. We will demonstrate molecular characteristics and localization of a hair cell-associated, non-L-type voltage-gated calcium channel and efferent- related a. nicotinic receptor and dopamine D2(ing) and D, receptors. This approach, utilizing methods of micro-biochemistry, should result in continued, detailed elucidation of structure and molecular function 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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