NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

神经元烟碱乙酰胆碱受体

• 批准号: 6093096
• 负责人: PETER B. SARGENT
• 金额: $ 5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6093096
• 关键词: acetylcholine; autoradiography; cell membrane; chickens; ciliary ganglion; confocal scanning microscopy; developmental neurobiology; fluorescence resonance energy transfer; immunofluorescence technique; nicotinic receptors; receptor binding; receptor expression; receptor sensitivity; synapses

The long term goal of our research is to understand the nature and the consequences of diversity in the expression of neuronal nicotinic acetylcholine receptors (AChRs). Recent molecular biological studies have identified a family of putative AchR genes in the nervous system. However, there is little evidence that AchRs generally serve the same function in the central nervous system that they do in the peripheral nervous system, where they underlie fast, excitatory synaptic transmission. We propose to use both structural and functional approaches to examine AchRs in the chicken ciliary ganglion, where monoclonal antibodies specific for each of the known nicotinic AchRs subunits are available and where AchRs have been found both on the presynaptic terminals and on their target neurons. The target ciliary ganglion neurons make several AchRs classes, some of which may serve novel functions. We will use subunit-specific antibodies to chicken AchRs and laser scanning confocal microscopy to examine the distribution of AchRs subunits on the neuronal surface. At the cellular level, we will examine whether there are differences in AchRs expression between the two neuronal types in the ciliary ganglion (ciliary, choroid). At the subcellular level, we will identify which AchRs subunits are located at synaptic sites, which subunits are located extrasynaptically, and which are located presynaptically. This information will be compared with the results of functional studies that will determine the relative importance of different AchRs classes in underlying rapid excitatory synaptic transmission in the two neuronal populations. At the molecular level, we will use fluorophore-tagged antibodies and fluorescence resonance energy transfer (FRET) to examine the proximity of subunits to each other. This technique should allow us to learn which sudunits are assembled into AchR oligomers. We will pay particular attention to presynaptic nicotinic AchRs, which, although virtually uncharacterized to date, may represent the predominant form of AchR in the central nervous system. We will characterize presynaptic AchRs on the large, calyceal endings in the ciliary ganglion by whole-cell recordings to learn whether these receptors are activated by nerve-released transmitter. In addition, we will characterize presynaptic AchRs by immunofluorescence and confocal microscopy to learn what their subunit composition might be. The studies should enhance our understanding of the structure and function of neuronal nicotinic AchRs.

我们研究的长期目标是了解 神经元烟碱表达多样性的后果 乙酰胆碱受体（AChRs）。 最近的分子生物学研究 已经确定了神经系统中一个假定的AchR基因家族。 然而，几乎没有证据表明AchR通常具有相同的功能。 在中枢神经系统中发挥作用 神经系统，在那里它们是快速，兴奋性突触的基础。 传输 我们建议使用结构和功能 研究鸡睫状神经节中AchR的方法，其中 对每种已知的烟碱AchR特异的单克隆抗体 亚基是可用的，其中AchR已被发现， 突触前末梢及其靶神经元上。 目标纤毛 神经节神经元形成几种AchRs类别，其中一些可能 服务于新的功能。 我们将使用亚基特异性抗体 鸡AchRs和激光扫描共聚焦显微镜检查， AchRs亚单位在神经元表面的分布。 在细胞 水平，我们将研究是否有差异，在AchRs 睫状神经节中两种神经元的表达 （睫状、脉络膜）。 在亚细胞水平上，我们将识别出 AchR亚基位于突触位点，所述亚基位于 位于突触外，位于突触前。 这 信息将与功能研究的结果进行比较， 将决定不同AchRs类别的相对重要性 潜在的快速兴奋性突触传递在两个神经元 人口。 在分子水平上，我们将使用荧光团标记 抗体和荧光共振能量转移（FRET）， 检查子单元彼此之间的接近程度。 这项技术应该 使我们能够了解哪些亚单位组装成AchR寡聚体。 我们将特别关注突触前烟碱乙酰胆碱受体， 尽管迄今为止几乎没有特征，但可能代表了 中枢神经系统中AchR的主要形式。 我们将 表征突触前AchRs的大，萼结束在 睫状神经节的全细胞记录，以了解这些 受体被神经释放的递质激活。 另外我们 将通过免疫荧光表征突触前AchR， 共聚焦显微镜来了解它们的亚基组成。 这些研究应加强我们对结构的了解， 神经元烟碱乙酰胆碱受体的功能。