Molecular biology of cochlear efferent receptors

耳蜗传出受体的分子生物学

• 批准号: 6383266
• 负责人: ANNE E LUEBKE
• 金额: $ 24.28万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6383266
• 关键词: Xenopus; acetylcholine; antisense nucleic acid; auditory pathways; auditory reflex; auditory stimulus; cholinergic receptors; cochlear nerve; ear hair cell; efferent nerve; guinea pigs; immunocytochemistry; immunoprecipitation; neural information processing; nicotinic receptors; noise biological effect; receptor expression; transfection; western blottings

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.

描述（由申请人提供）：该提案的目的是研究 乙炔（ACH）从橄榄石象传出释放的分子机制会影响人工耳蜗外毛细胞（OHC）。这 OHC上的烟碱ACH，受体（NACHR）包含A9 NACHR亚基，可能 还包含新发现的AL0 NACHR。至少有两个假设 关于人工耳蜗对听力的贡献的建议：1） 它们保护耳蜗免受声学过度刺激的影响，2）它们增强 在背景噪音的情况下，声音识别。在最后一个项目中 时期我们通过设计人工耳蜗的测试来研究了第一个假设 反射强度，发现反射强度是积极的 与A9 NACHR的表达相关。此外，我们发现 发出反射较强的动物在 噪音曝光。提出的实验将把这些研究扩展到测试 NACHR的A9和AL0亚基是否形成异元复合物，以及是否形成 调节这种复合物在成年中的表达会影响 声学过度刺激后的耳蜗功能。第一个具体目的是 确定A9和ALO NACHR是否形成人工耳蜗中的功能异源受体 毛细胞使用共免疫沉淀和免疫组织化学 技术。第二个特定目标将确定上调是否上调（或 成年豚鼠中A9，AL0或A9 + ALO NACHR表达的下调） 毛细胞增强（或削弱）传出反射并减少（或 增加）对噪声损害的敏感性。这些实验将使用 修饰的腺病毒以提供A9和A10过表达，主要负面， 反义将mRNA直接构建到成年几内亚的鳞片 猪。在爪蟾卵母细胞中进行的对照研究将用于验证 受体共免疫沉淀技术，并验证该主要阴性 反义受体构建体抑制对ACH的反应。 从这些研究中获得的信息将有助于未来的药物疗法 该目标的靶向人工耳蜗的胆碱能受体 减少噪声引起的人类听力损失的数量。