The objective of the present proposal is to continue to identify peripheral neurotransmitters/ neuromodulators and associated biochemical systems of hair-cell organs. Recently-described neurotransmitter systems, voltage-gated calcium channels, proteins of the synaptic complex, and corresponding neurotransmitter receptors will be examined in mammalian and fish models. The main hypotheses will address the existence and function of serotonin, norepinephrine, and dopamine as cochlear and saccular transmitters, the role of L-type and non-L-type voltage-gated calcium channels relevant to their mode of interaction with the synaptic complex, and the molecular-functional analysis of receptors for these neuroactive compounds. Methods include: 1) high-resolution, high-performance liquid chromatography (HPLC) with detection by electrochemistry and immunoassay, 2) analysis of acoustico-lateralis tissue content, depolarization-induced release in vitro of the presumptive neurotransmitters/neuromodulators from a saccular hair cell sheet for which the hair cell is the only intact cell type and sound-induced release into cochlear perilymph, 3) RT-PCR (reverse transcription polymerase chain reaction), yielding sequence for molecular functional determination of voltage-gated calcium channels and neurotransmitter receptors associated with transmitter release, 4) yeast two-hybrid and binding analysis of targeted protein-protein interactions in the channel and in the synaptic complex, 5) design of custom antibodies against relevant channel and receptor sequences, and 6) morphological localization of molecular entities by immunochemistry and in situ hybridization. Using these methods, it is planned to demonstrate acousticolateralis content and release from the saccular hair-cell sheet, in a calcium-dependent manner, of the targeted transmitter candidates by low-level potassium depolarization and release into perilymph by sound stimulation as modified by efferent input. Binding domains will be identified for hair-cell-associated calcium channels, their subunits, and interacting proteins of the synaptic complex. Molecular function will be predicted from the molecular structure of channels and receptors. These approaches, utilizing microbiochemical methods, 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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