Molecular biology of cochlear efferent receptors

描述（由申请人提供）：本提案的目的是研究 乙酰胆碱（ACh）释放的分子机制 橄榄耳蜗传出神经影响耳蜗外毛细胞（OHC）。的 OHC上的烟碱乙酰胆碱受体（nAChR）含有α 9 nAChR亚单位， 也含有新发现的al0 nAChR至少有两种假设 关于耳蜗传出神经对听力的贡献提出：1） 它们保护耳蜗免受声音过度刺激，2）它们增强 在存在背景噪声的情况下的声音识别。在上一个项目中， 在此期间，我们通过设计耳蜗测试来研究第一个假设， 传出反射强度，并发现反射强度是积极的 与a9 nAChR表达相关。此外，我们发现， 传出反射较强的动物表现出较少的耳蜗损伤， 噪声暴露拟议的实验将扩展这些研究，以测试 nAChR的α 9和α 10亚基是否形成异聚体复合物， 在成年期调节这种复合物的表达可以影响 听觉过度刺激后的耳蜗功能。第一个具体目标是 确定α 9和α 10 nAChR是否在耳蜗中形成功能性异聚体受体 用免疫共沉淀和免疫组织化学方法在体内检测毛细胞 技术.第二个具体目标将确定是否上调（或 a9、a10或a9 + a10 nAChR在成年豚鼠中的表达 毛细胞加强（或减弱）传出反射，减少（或 增加）对噪音损害的敏感性。这些实验将使用 修饰腺病毒以递送A9和A10过表达，显性阴性， 和反义构建mRNA直接到成年豚鼠的鼓阶 猪在非洲爪蟾卵母细胞中进行的对照研究将用于验证 受体免疫共沉淀技术，并验证显性阴性 和反义受体构建体抑制对ACh的应答。 从这些研究中获得的信息将有助于未来的药物治疗 靶向耳蜗传出系统的胆碱能受体， 减少人类噪音引起的听力损失。

DESCRIPTION (provided by applicant): The objective of this proposal is to study the molecular mechanisms by which acetyicholine (ACh) released from olivocochtear efferents influences cochlear outer hair cells (OHCs). The nicotinic ACh, receptor (nAChR) on OHCs contains the a9 nAChR subunit, and may also contain the newly discovered al0 nAChR. At least two hypotheses have been proposed concerning the contribution cochlear efferents make to hearing: 1) they protect the cochlea from acoustic overstimulation, and 2) they enhance sound recognition in the presence of background noises. In the last project period we investigated the first hypothesis by devising a test of the cochlear efferent reflex strength and found that reflex strength is positively correlated to the expression of a9 nAChR. Furthermore, we discovered that animals with stronger efferent reflexes exhibited less cochlear damage upon noise exposure. The proposed experiments will extend those studies to test whether a9 and al0 subunits of the nAChR form a heteromeric complex and whether modulating expression of this complex in adulthood can influence recovery of cochlear function after acoustic overstimulation. The first specific aim is to determine if a9and alO nAChRs form functional heteromeric receptors in cochlear hair cells in vivo using coimmunoprecipitation and immunohistochemical techniques. The second specific aim will determine if upregulation (or downregulation) of a9, al0, or a9 + alO nAChR expression in adult guinea pig hair cells strengthens (or weakens) the efferent reflex and reduces (or increases) susceptibility to noise damage. These experiments will use a modified adenovirus to deliver a9 and a10 overexpression, dominant negative, and antisense constructs mRNAs directly to the scala tympani of adult guinea pigs. Control studies performed in Xenopus oocytes will be used to validate the receptor coimmunoprecipitation techniques and to verify that dominant-negative and antisense receptor constructs inhibit responses to ACh. Information gained from these studies will contribute to future drug therapies that target cholinergic receptors of the cochlear efferent system with the goal of diminishing noise-induced hearing loss in humans.