IDENTIFICATION OF ACOUSTICO-LATERALIS TRANSMITTERS

本提案的主要目标是继续确定 推测的外周神经递质/神经调节剂和相关的 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、高性能高效液相色谱 通过荧光、安培、放射性和 放射免疫测定法，2)推定神经递质的体外释放 和来自毛细胞片的神经调节剂，其中毛细胞是 只有完整的细胞类型，来自球囊感觉上皮，包括 传出和来自Corti器官的，3)化合物的生物测定 利用非洲爪哇侧线，4)免疫组织化学和 小分子偶联物和酶的免疫细胞化学定位 发射机的合成和降解，以及5)分析 神经递质受体的放射配基结合，免疫细胞化学， 聚合酶链式反应(PCR)。使用这些方法，它是有计划的 化合物的化学鉴定和生物活性测定 以依赖钙的方式从毛细胞膜中释放出来， 并通过以下方式确定相关钙通道的亚型 药理分析。计划将生物化学 神经活性化合物的合成系统(酶)，如 谷氨酸、谷氨酸和天冬氨酸多肽，N- 乙酰组氨酸和其他假定的传递体，利用放射性 前兆和HPLIC分析。神经递质合成酶 和退化将在形态上局部化，并且存在或 相关神经递质受体的缺失将被证明。这个 利用微生物化学方法的总体方法应取得成果 继续，详细阐述外周神经递质系统 听力和平衡，为治疗的发展指明了方向 与递质相关的听力损失、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.