INDENTIFICATION OF ACOUSTICO-LATERALIS TRANSMITTERS

拟议研究的主要目标是从化学上鉴定 耳蜗的假定初级传入感觉递质 豚鼠和非洲爪蟾的侧线器官 爪蛙，并描述与之相关的生化系统 假定的发射机。 分析方法包括：1） 高分辨率高效液相色谱法（HPLC）， 荧光检测，用于测定 伯胺的邻苯二甲醛/2-巯基乙醇加合物，2）质量 光谱学，用于确认HPLC级分的化学组成， 3)双向聚丙烯酰胺凝胶电泳和柱 层析，用于纯化后系蛋白，4） 放射性配体-受体结合测定，用于表征 毛细胞组分的神经递质受体，5）分光光度法 和荧光测定法，用于测定传递相关酶 的毛细胞部分，和6）使用非洲爪蟾侧线， 检测类似发射器的活动 具体方法包括：1）a 豚鼠耳蜗和颞骨的手术入路， 从正常和毛细胞中收集外淋巴液和脑脊液 缺陷（华尔兹）豚鼠在存在和不存在噪音， 在确定流体的小分子含量之前，2）解剖 刺激或非刺激冻干爪蟾神经肥大和豚鼠 听觉部分，在测定其小分子含量之前， 酶活性和受体含量，以及3）化学分离， 分析，突触囊泡从神经乳头的爪蟾。 使用这些 方法，我们计划建立身份，并在 推测爪蟾侧线递质侧线神经肥大， 它（它们）相关的受体和合成和降解酶， 其生物活性。 我们还计划确定身份， 刺激释放到外淋巴中，以及推定的 豚鼠耳蜗发射器。 我们还计划用化学方法 非发射器材料，以及可能的传出发射器候选物， 在豚鼠暴露于噪声时释放到外淋巴中， 高水平。 这种主要的生物化学方法应该导致 识别听觉和平衡的外周神经递质， 并将为与发射机相关的听力损失提出最终的治疗方法， 耳鸣头晕

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.