Identification of Acoustico-Lateralis Transmitters

描述(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/调节器，调节其活动的受体，以及在毛细胞突触传递中发生的基本生化事件，这些递质启动八侧肌系统中的传入信号。在这项新的研究中，我们将重点研究5-羟色胺作为Corti听觉器官中的传出递质和前庭毛细胞中的感觉神经递质的作用。我们将研究包含去甲肾上腺素的肾上腺素能传出神经，构成了新发现的在Corti器官内工作的神经系统，并检查新发现的传出神经调节剂PACAP的作用部位和受体。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，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，用Femtomoles灵敏、高分辨率、高效液相色谱。将用分子和免疫组织化学方法分析合成酶和转运蛋白。释放，神经递质识别的标准，将在体内研究对声音刺激的反应。在目标2中，我们将继续通过表面等离子激元共振和下拉结合试验来表征毛细胞电压门控钙通道，这些通道可以引起钙诱导的胞吐信号，并检测蛋白质之间的相互作用。在目标3中，将描述相应的神经递质受体--接收和响应递质的膜结合蛋白--的分子功能域及其神经和毛细胞的免疫定位，定义听觉外周传入/传出信号的机制。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、眩晕和耳鸣的生化和遗传疗法指明方向。在听觉/前庭缺陷如耳聋、耳鸣和眩晕中，对传入信号进行生化/药理学操作的可能性完全依赖于我们对毛细胞感受性神经传递的基本要素的了解，而毛细胞感受性神经传递是由传出神经传递调节的。在毛细胞感觉上皮细胞中识别新的神经递质/神经调节剂方面的最新进展表明，这类研究仍然具有相关性和必要性。目前尚不清楚的外毛细胞递质的鉴定，在建议中被考虑，对外周来源的耳鸣有影响。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在神经肽PACAP的建议中得到了解决，PACAP在兴奋性毒性和噪声性耳聋中很重要。表征电压门控钙通道与突触复合蛋白的相互作用对于提供必要的元件来逆转耳聋具有重要意义，例如DFNB9，由突触复合蛋白Otoferlin的突变引起。

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.