Identification of Acoustico-Lateralis Transmitters

描述（由申请人提供）：该提案的总体目标是确定外周毛细胞/传出递质/调节剂，介导其作用的受体，以及在毛细胞突触传递中发生的基本生物化学事件，这些事件由在octavolateralis系统中启动传入信号的这些递质调节。在这项新的研究中，我们将集中在5-羟色胺的作用，作为一个传出递质内的听觉器官的Corti和作为一个receptoneural递质在前庭毛细胞。我们将研究含有去甲肾上腺素的肾上腺素能传出神经，构成一个新认识的神经系统内运作的Corti器官，并检查新发现的传出神经调节剂PACAP的作用和受体的网站。毛细胞电压门控钙通道（VGCC）与突触蛋白和支架蛋白的分子相互作用将被研究。在目标1中，我们将继续鉴定毛细胞和内耳感觉上皮细胞中的神经递质/神经调质，采用飞摩尔敏感的高分辨率高效液相色谱法。将用分子和免疫组织化学方案分析递质的合成酶和转运蛋白。释放，神经递质识别的标准，将在响应声音刺激在体内进行研究。在目标2中，我们将继续表征毛细胞电压门控钙通道，通过表面等离子体共振和下拉结合试验，检查蛋白质-蛋白质相互作用，产生钙诱导的胞吐信号。在目标3中，相应的神经递质受体-膜结合蛋白质，接收和响应的递质-将其特征在于其分子功能域和它们的神经和毛细胞免疫定位，定义传入/传出信号在acoustico-lateralis外围的机制。这些方法，利用微生物化学方法，应导致持续的，详细的结构和分子功能的外周神经递质系统的听力和平衡的阐明，指出的方式来发展的生化和遗传疗法的发射机相关的听力损失，眩晕，耳鸣。在听觉/前庭缺陷如耳聋、耳鸣和头晕中，对传入信号进行生物化学/药理学操纵的可能性完全取决于我们对毛细胞受体神经传递基本要素的了解，这些基本要素由传出神经传递调节。最近的进展，新的神经递质/神经调质在毛细胞的感觉上皮细胞的识别点持续的相关性和需要这种调查。鉴定目前未知的外毛细胞递质，在建议中考虑，有影响外周源性耳鸣。5-羟色胺能和肾上腺素能传出神经参与调节耳声发射，并可能参与确定外毛细胞的设定点。I型传入树突的天然神经保护机制是神经肽PACAP的一个问题，在兴奋性毒性和噪声性耳聋中很重要。电压门控钙通道与突触复合体蛋白的相互作用的表征对于提供必要的元件以逆转由突触复合体蛋白otoferlin中的突变引起的耳聋（例如DFNB 9）具有意义。

DESCRIPTION (provided by applicant): The overall objective of the proposal is to identify hair cell/efferent transmitters/modulators at the periphery, the receptors mediating their actions, and the basic biochemical events occurring in hair-cell synaptic transmission as modulated by these transmitters that initiate afferent signaling in octavolateralis systems. In this new investigation, we will focus on the role of serotonin as an efferent transmitter within the auditory organ of Corti and as a receptoneural transmitter in vestibular hair cells. We will investigate norepinephrine-containing adrenergic efferents constituting a newly-recognized neural system operating within the organ of Corti and examine the newly-discovered efferent neuromodulator PACAP as to its sites of action and receptors. Molecular interactions of hair cell voltage-gated calcium channels (VGCC) with synaptic proteins and with scaffolding proteins will be investigated. In Aim 1, we will continue the identification of neurotransmitters/neuromodulators in hair cells and inner-ear sensory epithelia, with femtomole-sensitive, high-resolution, high-performance liquid chromatography. Enzymes of synthesis and transporters for the transmitters will be analyzed with molecular and immunohistochemical protocols. Release, a criterion of neurotransmitter identification, will be investigated in response to sound stimulus in vivo. In Aim 2, we will continue to characterize hair-cell voltage-gated calcium channels that give rise to the calcium-induced exocytotic signal via surface plasmon resonance and pull-down binding assays, examining protein-protein interactions. In Aim 3, the corresponding neurotransmitter receptors - membrane-bound proteins that receive and respond to transmitters - will be characterized as to their molecular functional domains and their neural and hair-cell immunolocalizations, defining mechanisms for afferent/efferent signaling in the acoustico-lateralis periphery. 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 biochemical and genetic therapies for transmitter-related hearing loss, vertigo, and tinnitus. The possibility of biochemical/pharmacologic manipulation of afferent signaling in auditory/vestibular deficits such as deafness, tinnitus, and dizziness is completely dependent upon our knowledge of basic elements of hair-cell receptoneural transmission, as modulated by efferent neurotransmission. Recent advancements in the identification of new neurotransmitters/neuromodulators in hair cell-containing sensory epithelia point to continuing relevance and need for this kind of investigation. Identification of the presently unknown outer hair cell transmitter, considered in the proposal, has implications for peripherally-derived tinnitus. Serotonergic and adrenergic efferents are implicated in modulating otoacoustic emissions and are likely to be involved in determining the set point for the outer hair cells. Native neuroprotective mechanisms for the type I afferent dendrites is an issue addressed in the present proposal for the neuropeptide PACAP, important in excitotoxicity and noise-induced deafness. Characterization of the interactions of voltage-gated calcium channels with synaptic complex proteins has implications for providing the necessary elements to reverse deafness, such as DFNB9, caused by mutations in the synaptic complex protein, otoferlin.