The main objective of the proposed research is to identify chemically the presumptive primary afferent sensory transmitter(s) of the cochlea of the guinea pig and of the lateral-line organ of Xenopus laevis, the African clawed frog, 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, for determination of ortho-phthaldialdehyde/2-mercaptoethanol adducts of primary amines, 2) mass spectroscopy, for confirmation of chemical composition of hplc fractions, 3) two-dimensional, polyacrylamide-gel electrophoresis and column chromatography, for purification of laterl-line proteins, 4) radioligand-receptor binding assays, for characterization of neurotransmitter receptors of hair-cell fractions, 5) spectrophotometric and fluorometric assays, for determination of transmitter-related enzymes of hair cell fractions, and 6) a bioassay using 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 normal and hair-cell deficient (Waltzing) guinea pigs in the presence and absence of noise, before determining small-molecule content of the fluids, 2) dissection of stimulated or non-stimulated lyophilized Xenopus neuromast and guinea-pig auditory fractions, prior to determination of their small-molecule content, enzyme activity, and receptor content, and 3) the isolation, for chemical analysis, of synaptic vesicles from neuromasts of Xenopus. Using these methods, we plan to establish the identity, and presence in the lateral-line neuromast, of presumptive Xenopus lateral-line transmitter(s), it (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, and possibly efferent-transmitter candidates, that are released 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.
