CHOLINERGIC MECHANISMS AT EFFERENT SYNAPSES

传出突触的胆碱能机制

基本信息

批准号：
6104305
负责人：
BARBARA J MORLEY
金额：
$ 11.9万
依托单位：
FATHER FLANAGAN'S BOYS' HOME
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-02-01 至 2000-01-31
项目状态：
已结题

项目摘要

There are two targets for efferent fibers in the adult cochlea: The outer hair cells (OHCs) and the dendrites of the type I spiral ganglion cells. The medial olivocochlear axons also send collaterals to the ventral cochlear nucleus (VCN). Both the medial and the lateral systems are largely cholinergic, although the receptors in neither system are well-characterized. In this laboratory, we have studied the mRNA expression of all of the nAChR subunits. We have proposed that the NACHR on OHCs may consist of alpha7 in addition to alpha9 subunit, and that the lateral efferent axodendritic receptor may consist of the alpha6alpha7beta2 subunits. In developing rat, the axodendritic receptor may also possess the alpha3 and alpha4 subunits, suggesting a developmental switch in receptor subunits, and a receptor with unique physiological or pharmacological properties. In this application we will test the hypothesis that the axodendritic NACHR consists of alpha6alpha7beta2 by using immunoprecipitation methods and by characterizing the physiology and pharmacology of the receptor using the Xenopus oocyte expression system. We will also extend our previous observations by performing experiments to quantify, with quantitative RT-PCR, and to localize, with in situ-PCR, the alpha7 subunit in the organ of Corti. In addition, we propose to study, using immunoprecipitation methods and the oocyte expression system, the alpha9 homomer and the putative alpha7alpha9 heteromeric receptor. Lastly, we propose to identify the nAChRs in the VCN, the target of the olivocochlear collaterals. Specifically, we will determine if the alpha4 and beta2 subunits are expressed in the same cells using in situ hybridization on adjacent sections from adult rat brain. Recent data from this laboratory indicates that alpha3 decreases in expression and alpha7 increases in expression in the VCN during the early postnatal period, at about the time of synaptogenesis. We hypothesize that alpha3 subunit is replaced by alpha7 and propose determine this with in situ hybridization on adjacent sections from postnatal VCN at ages PN3-21.

成人耳蜗中的传出纤维有两个目标：外毛细胞 (OHC) 和 I 型螺旋神经节的树突细胞。内侧橄榄耳蜗轴突也向耳蜗腹核（VCN）。内侧和外侧系统大部分是胆碱能的，尽管这两个系统中的受体都不是特点鲜明。在这个实验室，我们研究了 mRNA 所有 nAChR 亚基的表达。我们建议 OHC 上的 NACHR 除了 alpha9 亚基外还可能由 alpha7 组成，并且外侧传出轴突受体可能由 α6α7β2 亚基。在发育中的大鼠中，轴突受体也可能具有 α3 和 α4 亚基，这表明受体亚基的发育开关，以及具有独特的受体生理或药理学特性。在这个应用程序中我们将检验轴突 NACHR 组成的假设 α6α7β2 通过使用免疫沉淀方法并通过使用以下方法表征受体的生理学和药理学非洲爪蟾卵母细胞表达系统。我们还将延续之前的通过进行实验来量化观察结果 RT-PCR，并通过原位 PCR 定位 α7 亚基柯蒂氏器。此外，我们建议研究、使用免疫沉淀方法和卵母细胞表达系统，α9 同聚体和假定的 α7α9 异聚受体。最后，我们建议鉴定 VCN 中的 nAChR，这是橄榄耳蜗络脉。具体来说，我们将确定是否 α4和β2亚基使用原位在相同细胞中表达成年大鼠大脑相邻切片的杂交。近期数据该实验室的结果表明，α3 的表达减少，并且产后早期 VCN 中 alpha7 的表达增加期，大约在突触发生的时间。我们假设 alpha3 亚基被 alpha7 取代，并建议用 in situ 确定这一点对 PN3-21 岁时出生后 VCN 的相邻切片进行杂交。