IDENTIFICATION OF ACOUSTICO-LATERALIS TRANSMITTERS

描述（来自申请人的摘要）。的主要目标 目前的建议是确定外围 神经递质/神经调质和相关的生化系统 毛细胞器官外周神经递质电压门控钙 通道和神经递质受体将检查哺乳动物 鱼模型系统。主要的假设涉及存在和 非谷氨酸毛细胞递质的功能，非L型电压- 门控钙通道和最近描述的传出神经递质 乙酰胆碱和多巴胺的受体。方法包括：1）高- 分离度，高效液相色谱（HPLC）检测 通过电化学、荧光、放射性和放射免疫测定，2） 组织含量分析和体外去极化诱导释放 来自囊状毛发的假定神经递质和神经调质 毛细胞是唯一完整细胞类型的细胞片层，以及 声诱导耳蜗外淋巴液释放; 3）生物学测定 化合物利用非洲爪蟾侧线，4）形态 通过免疫化学和原位定位分子实体 杂交方法，5）序列分析后的分子功能 RT-PC（反转录聚合酶链反应）电压门控 与毛细胞的递质释放相关的钙通道，以及6） 传出神经递质的RT-PCR后功能序列分析 乙酰胆碱和多巴胺的受体。使用这些方法， 计划从化学上鉴定并确定 化合物从囊状感觉细胞中以钙依赖性释放， 低水平钾去极化并释放到外淋巴液中 通过声音刺激。神经活性单胺的生物合成及相关 分子将利用放射性前体和HPLC进行研究。我们 将展示毛发的分子特征和定位 细胞相关、非L型电压门控钙通道和传出神经 相关的;烟碱受体和多巴胺D2（ing）和D1受体。这 方法，利用微生物生物化学的方法，应导致 继续，详细阐明的结构和分子功能的 听觉和平衡的外周神经递质系统， 开发与发射机相关的听力损失治疗方法的途径， 眩晕耳鸣

DESCRIPTION (from the Applicant's abstract). The major objective of the present proposal is to identify peripheral neurotransmitters/neuromodulators and associated biochemical systems of hair-cell organs. Peripheral neurotransmitters voltage-gated calcium channels, and neurotransmitter receptors will be examined for mammalian and fish model systems. The main hypotheses address the existence and function of non-glutamate hair cell transmitter(s), non-L-type voltage- gated calcium channel(s), and recently-described efferent neurotransmitter receptors for acetylcholine and dopamine. Methods include: 1) high- resolution, high-performance liquid chromatography (HPLC) with detection by electrochemistry, fluorescence, radioactivity, and radioimmunoassay, 2) analysis of tissue content, and depolarization-induced release in vitro of presumptive neurotransmitters and neuromodulators from a saccular hair cell sheet for which the hair cell is the only intact cell type, and sound-induced release in vivo into cochlear perilymph, 3) biological assay of compounds utilizing Xenopus laevis lateral line, 4) morphological localization of molecular entities by immunochemical and in situ hybridization methods, 5) sequence analysis as to molecular function after RT-PC (reverse transcription polymerase chain reaction) of voltage-gated calcium channels associated with transmitter release of haircells, and 6) functional sequence analysis after RT-PCR of efferent neurotransmitter receptors for acetylcholine and dopamine. Using these methods, it is planned to identify chemically and determine the biological activity of compounds release from saccular sensory cells in a calcium-dependent manner by low level potassium depolarization and released into perilymph by sound stimulation. Biosynthesis of neuroactive monoamines and related molecules will be studied utilizing radioactive precursors and HPLC. We will demonstrate molecular characteristics and localization of a hair cell-associated, non-L-type voltage-gated calcium channel and efferent- related a. nicotinic receptor and dopamine D2(ing) and D, receptors. This approach, utilizing methods of micro-biochemistry, 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.