Identification of Acoustico-Lateralis Transmitters

描述（由申请人提供）：本提案的目的是继续鉴定毛细胞器官的周围神经递质/神经调节剂和相关的生化系统。最近描述的神经递质系统、电压门控钙通道、突触复合体蛋白质和相应的神经递质受体将在哺乳动物和鱼类模型中进行检查。主要的假设将涉及血清素、去甲肾上腺素和多巴胺作为耳蜗和囊性递质的存在和功能，l型和非l型电压门控钙通道与突触复合物相互作用模式的作用，以及这些神经活性化合物受体的分子功能分析。方法包括:1)高分辨率，高效液相色谱（HPLC），电化学和免疫检测，2)声侧耳组织含量分析，从囊状毛细胞片（毛细胞是唯一完整的细胞类型）中推测的神经递质/神经调节剂的体外去极化诱导释放和声音诱导释放到耳蜗周围，3)RT-PCR（逆转录聚合酶链反应），获得与递质释放相关的电压门控钙通道和神经递质受体的分子功能测定序列；4)通道和突触复合体中靶向蛋白-蛋白相互作用的酵母双杂交和结合分析；5)针对相关通道和受体序列的定制抗体设计；6)通过免疫化学和原位杂交对分子实体进行形态定位。利用这些方法，计划以钙依赖的方式，通过低水平的钾去极化，证明声学侧边的含量和从球囊毛细胞片释放的目标递质候选物，并通过传出输入修改的声音刺激释放到淋巴周围。结合域将被确定为毛细胞相关钙通道，它们的亚基，和突触复合物的相互作用蛋白。从通道和受体的分子结构来预测分子功能。这些方法，利用微生化方法，将导致对听力和平衡的周围神经递质系统的结构和分子功能的持续、详细的阐明，为递质相关性听力损失、眩晕和耳鸣的治疗方法的发展指明方向。

DESCRIPTION (provided by applicant): The objective of the present proposal is to continue to identify peripheral neurotransmitters/ neuromodulators and associated biochemical systems of hair-cell organs. Recently-described neurotransmitter systems, voltage-gated calcium channels, proteins of the synaptic complex, and corresponding neurotransmitter receptors will be examined in mammalian and fish models. The main hypotheses will address the existence and function of serotonin, norepinephrine, and dopamine as cochlear and saccular transmitters, the role of L-type and non-L-type voltage-gated calcium channels relevant to their mode of interaction with the synaptic complex, and the molecular-functional analysis of receptors for these neuroactive compounds. Methods include: 1) high-resolution, high-performance liquid chromatography (HPLC) with detection by electrochemistry and immunoassay, 2) analysis of acoustico-lateralis tissue content, depolarization-induced release in vitro of the presumptive neurotransmitters/neuromodulators from a saccular hair cell sheet for which the hair cell is the only intact cell type and sound-induced release into cochlear perilymph, 3) RT-PCR (reverse transcription polymerase chain reaction), yielding sequence for molecular functional determination of voltage-gated calcium channels and neurotransmitter receptors associated with transmitter release, 4) yeast two-hybrid and binding analysis of targeted protein-protein interactions in the channel and in the synaptic complex, 5) design of custom antibodies against relevant channel and receptor sequences, and 6) morphological localization of molecular entities by immunochemistry and in situ hybridization. Using these methods, it is planned to demonstrate acousticolateralis content and release from the saccular hair-cell sheet, in a calcium-dependent manner, of the targeted transmitter candidates by low-level potassium depolarization and release into perilymph by sound stimulation as modified by efferent input. Binding domains will be identified for hair-cell-associated calcium channels, their subunits, and interacting proteins of the synaptic complex. Molecular function will be predicted from the molecular structure of channels and receptors. These approaches, utilizing microbiochemical methods, 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.