NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

我们研究的长期目标是了解自然界和 神经元型烟碱能神经元表达多样性的后果 乙酰胆碱受体(AChRs)。最新的分子生物学研究 已经在神经系统中发现了一个可能的ACHR基因家族。 然而，几乎没有证据表明AchRs通常具有相同的作用。 在中枢神经系统中的功能，它们在外周系统中所做的 神经系统，它们是快速兴奋性突触的基础 变速箱。我们建议同时使用结构和功能 检测鸡睫状神经节AchRs的方法，其中 针对已知的每一种烟碱型AchR的单抗 亚基可用，AchR的位置都在 突触前终末及其靶神经元上。靶纤毛 神经节神经元产生几个AchRs类，其中一些可能 服务新功能。我们将使用亚基特异性抗体来 鸡AchRs和激光扫描共聚焦显微镜检测 AchRs亚单位在神经元表面的分布。在手机上 水平，我们将检查AchRs是否存在差异 两种神经元在睫状神经节中的表达 (纤毛、脉络膜)。在亚细胞水平上，我们将确定哪些 AchRs亚基位于突触部位，哪些亚基位于 突触外，并且位于突触前。这 信息将与功能研究的结果进行比较 将决定不同AchRs类在 两个神经元中潜在的快速兴奋性突触传递 人口。在分子水平上，我们将使用标记的荧光团 抗体和荧光共振能量转移(FRET) 检验亚单位之间的接近程度。这项技术应该 让我们了解哪些单元会组装成ACHR低聚物。 我们将特别关注突触前尼古丁AchRs， 虽然到目前为止几乎没有特征，但可能代表了 中枢神经系统中AchR的主要形式。我们会 突触前AchRs在大的肾盏末梢上的特征 通过全细胞记录睫状神经节来了解这些 受体是由神经释放的递质激活的。此外，我们 将通过免疫荧光和免疫荧光表征突触前AchRs 共聚焦显微镜以了解它们可能的亚基组成。 这些研究应该加深我们对结构和 神经元烟碱型AchRs的功能。

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.