Molecular biology of cochlear efferent receptors

耳蜗传出受体的分子生物学

• 批准号: 6660629
• 负责人: ANNE E LUEBKE
• 金额: $ 3.72万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6660629
• 关键词: Xenopus; acetylcholine; antisense nucleic acid; auditory pathways; auditory reflex; auditory stimulus; 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）：本提案的目的是研究 从橄榄耳传出神经释放乙酰胆碱 (ACh) 影响耳蜗外毛细胞 (OHC) 的分子机制。这 OHC 上的烟碱 ACh 受体 (nAChR) 包含 a9 nAChR 亚基，并且可能 还含有新发现的 al0 nAChR。至少有两个假设 关于耳蜗传出器对听力的贡献的建议：1) 它们保护耳蜗免受声音过度刺激，并且 2）它们增强 存在背景噪音时的声音识别。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，发现反射强度为正 与 a9 nAChR 的表达相关。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 噪音暴露。拟议的实验将扩展这些研究以测试 nAChR 的 a9 和 a10 亚基是否形成异聚复合物以及是否 在成年期调节该复合物的表达可以影响 声音过度刺激后的耳蜗功能。第一个具体目标是 确定 a9 和 a10 nAChR 是否在耳蜗中形成功能性异聚受体 使用免疫共沉淀和免疫组织化学法观察体内毛细胞 技术。第二个具体目标将确定是否上调（或 成年豚鼠中 a9、a10 或 a9 + a10 nAChR 表达的下调） 毛细胞增强（或减弱）传出反射并减少（或 增加）对噪声损坏的敏感性。这些实验将使用 修饰的腺病毒可提供 a9 和 a10 过表达、显性失活、 反义构建的 mRNA 直接作用于成年几内亚的鼓阶 猪。在非洲爪蟾卵母细胞中进行的对照研究将用于验证 受体共免疫沉淀技术并验证显性失活 反义受体结构抑制对 ACh 的反应。 从这些研究中获得的信息将有助于未来的药物治疗 以耳蜗传出系统的胆碱能受体为目标 减少人类噪音引起的听力损失。