The objective of the research is to identify chemically the presumptive peripheral transmitter(s) of the cochlear nucleus of the guinea pig, the lateral-line organ of Xenopus laevis (the African clawed frog), and of labyrinthine organs of fish, and to characterize the biochemical systems associated with the presumptive transmitter(s). Analytical methods include: 1) high-resolution, high-performance liquid chromatography (HPLC) with fluorescence detection and detection by radioactivity, 2) mass spectrometry, for confirmation of chemical composition of HPLC fractions, 3) radioligand-receptor binding assays and autoradiographic localization, for characterization of neurotransmitter receptors of acoustico-lateralis fractions, 4) fluorometric and radioisotopic assays, for determination of transmitter-related enzymes of hair-cell fractions, and 5) a bioassay using the Xenopus lateral line, for detecting transmitter-like activity. Preparative methods include: 1) a surgical approach to the guinea-pig cochlea and temporal bone, for collection of perilymph and cerebrospinal fluid from guinea pigs in the presence and absence of noise, before determining small-molecule content of the fluids, 2) an in vitro system for release of presumptive neurotransmitters from acoustico-lateralis tissues, and 3) dissection of Xenopus lateral line, fish labyrinthine, and guinea-pig auditory fractions, prior to determination of their small-molecule content, enzyme activity, and receptor content. Using these methods, we plan to establish the identity, and presence in the lateral-line neuromast and fish labyrinthine organs, of presumptive acoustico-lateral-is transmitter(s), its (their) associated receptors and synthesizing and degrading enzymes, and its (their) biological activity. We also plan to establish the identity, stimulated release into perilymph, and biological activity of presumptive guinea-pig cochlear transmitter(s). We further plan to identify chemically non-transmitter materials that are releasted into perilymph during exposure of guinea pigs to noise at high levels. This predominantly biochemical approach should lead to the identification of peripheral neurotransmitter(s) of hearing and balance, and will suggest eventual therapies for transmitter-related hearing loss, tinnitus, and dizziness.
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