IDENTIFICATION OF ACOUSTICO-LATERALIS TRANSMITTERS

本提案的主要目标是继续查明 假定的外周神经递质/神经调质和相关的 哺乳动物和鱼类毛细胞器官的生化系统。 方法 包括1）高分辨率、高效液相色谱法， 通过荧光、电流分析法、放射性和 放射免疫测定，2）推定神经递质的体外释放 以及来自毛细胞片层的神经调质，其中毛细胞是 仅完整细胞类型，来自囊状感觉上皮，包括 传出神经和Corti器官，3）化合物的生物测定 利用非洲爪蟾侧线，4）免疫组化， 小分子结合物和酶免疫细胞化学定位 发射机合成和退化，以及5）分析 神经递质受体的放射性配体结合，免疫细胞化学， 和聚合酶链反应（PCR）。 利用这些方法，计划 化学鉴定和确定化合物的生物活性 以钙依赖的方式从毛细胞片层释放， 并确定相关钙通道的亚型， 药理分析 计划鉴定生物化学 神经活性化合物的合成系统（酶）， 谷氨酸和含谷氨酸和谷氨酸的肽，N- 乙酰组氨酸和其他假定的递质，利用放射性 前体和HPLIC分析。 神经递质合成酶 并且降解将在形态上局部化，并且存在或 将证明相关神经递质受体的缺乏。 的 利用微生物生物化学方法的总体方法，应导致 在下文中，详细说明了外周神经递质系统 听力和平衡，指出了治疗方法的发展， 与发射机相关的听力损失、眩晕和耳鸣。

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.