Identification of Acoustico-Lateralis Transmitters

描述(申请人提供)：本提案的目标是继续确定毛细胞器官的外周神经递质/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、突触复合体的蛋白质以及相应的神经递质受体将在哺乳动物和鱼类模型中进行检测。主要假说将涉及5-羟色胺、去甲肾上腺素和多巴胺作为耳蜗囊递质的存在和功能，L型和非L型电压门控钙通道与突触复合体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)电化学和免疫分析检测的高分辨率、高效液相色谱(Hplc)；2)听觉侧方组织成分的分析；体外去极化诱导从毛细胞为唯一完整细胞类型的囊状毛细胞释放假定的神经递质/神经调节剂，以及声音诱导释放到耳蜗外淋巴液；3)RT-pcr(逆转录聚合酶链式反应)，产生序列，用于电压门控钙通道和与递质释放相关的神经递质受体的分子功能测定；4)酵母双杂交和结合分析通道和突触复合体中的目标蛋白-蛋白质相互作用。5)设计针对相关通道和受体序列的定制抗体；6)通过免疫化学和原位杂交对分子实体进行形态定位。使用这些方法，计划通过低水平的钾去极化，以钙依赖的方式显示声外侧毛细胞的含量和从球囊毛细胞膜释放靶向递质，并通过声音刺激将其释放到外淋巴中，并通过传出输入修正的声音刺激。毛细胞相关的钙通道、它们的亚单位和突触复合体的相互作用蛋白的结合域将被确定。分子功能将从通道和受体的分子结构中预测出来。这些方法，利用微生化方法，将导致持续、详细地阐明听力和平衡的外周神经递质系统的结构和分子功能，为开发与递质相关的听力损失、眩晕和耳鸣的治疗方法指明方向。

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.