Molecular biology of cochlear efferent receptors

描述(申请人提供)：本提案的目的是研究 从橄榄囊泪液传出的乙酰胆碱(ACh)影响耳蜗外毛细胞的分子机制。这个 毛细胞上的烟碱型ACh受体(NAChR)含有A9 nAChR亚基，可能 也包含新发现的al0 nAChR。至少有两个假设是 关于耳蜗传出对听力的贡献的建议：1) 它们保护耳蜗免受声学过度刺激，2)它们增强了 在存在背景噪声的情况下进行声音识别。在最后一个项目中 期间，我们通过设计一个测试耳蜗来研究第一个假设 传出反射强度，发现反射强度为正 与A9nAChR的表达相关。此外，我们发现， 传出反射较强的动物对耳蜗肌的损伤较小 噪音暴露。拟议的实验将把这些研究扩展到测试 NAChR的A9和Al0亚基是否形成异构体复合体，以及 成年期调节该复合体的表达可影响神经功能的恢复 声学过度刺激后的耳蜗声功能。第一个具体目标是 A9和Alo-nAChRs在耳蜗区是否形成功能性异构体受体 免疫共沉淀和免疫组织化学技术在活体毛细胞研究中的应用 技巧。第二个具体目标将决定是否上调(或 成年豚鼠A9、A10或A9+nAChR表达下调 毛细胞增强(或减弱)传出反射，减少(或 增加)对噪声损害的敏感性。这些实验将使用 改良腺病毒携带A9和A10过表达，显性阴性， 反义mRNAs直接作用于成年豚鼠鼓阶。 猪。在非洲爪哇卵母细胞中进行的对照研究将被用于验证 受体免疫共沉淀技术并验证显性-阴性 反义受体构建抑制ACh的反应。 从这些研究中获得的信息将有助于未来的药物治疗 以耳蜗神经传出系统的胆碱能受体为靶标 减少人类因噪音造成的听力损失。

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.