PRE & POSTSYNAPTIC NICOTINIC ACHRS--STRUCTURE & FUNCTION

预

• 批准号: 2416111
• 负责人: PETER B. SARGENT
• 金额: $ 13.87万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 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 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2416111
• 关键词: brain mapping; chick embryo; ciliary ganglion; developmental neurobiology; immunocytochemistry; mesencephalon; monoclonal antibody; neural transmission; neuropharmacology; nicotinic receptors; protein structure function; synapses; voltage /patch clamp

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of our research is to understand the nature and the consequences of diversity in the expression of neuronal acetylcholine receptors (nAChRs). Recent molecular biological studies have identified a family of putative nAChR genes in the nervous system. However, there is little evidence that nAChRs 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 nAChRs in two preparations from the embryonic chicken, where monoclonal antibodies specific for each of the nicotinic receptor gene products are available: the lateral spiriform nucleus (SpL), located in the mesencephalon, and the ciliary ganglion, located in the orbit. We will use subunit-specific antibodies to chick nAChRs and immunocytochemical techniques to examine the distribution of nAChR subunits among and within neurons. At the cellular level, we will examine whether all cells within the neuronal population (SpL, or ciliary ganglion) express the same subset of subunits. At the subcellular level, we will examine the distribution of receptor subunits at the neuronal surface in order to determine which subunits are located at synaptic sites, which subunits are located extrasynaptically, and which may be transported to axon terminals. Finally, 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 subunits are assembled into receptor oligomers. We will pay particular attention to presynaptic nicotinic receptors, which, although virtually uncharacterized to date, may represent the predominant form of nAChR in the central nervous system. We will examine by immunocytochemical techniques whether presynaptic nicotinic receptors are found on the surface of SpL axon terminals that project to the optic tectum and whether they are present on the terminals of preganglionic neurons that project to the ciliary ganglion. Should presynaptic receptors be found in the ciliary ganglion, we will attempt to characterize these receptors functionally by patch clamp recordings. The studies should enhance our understanding of the structure and function of neuronal nicotinic receptors by (1) identifying which subunits associate to form receptor oligomers likely to serve both postsynaptic and presynaptic functions and (2) examining the structure and function of nicotinic presynaptic receptors. It is likely that a fuller understanding of the structure and function of both postsynaptic and presynaptic nicotinic receptors will be essential for understanding nicotine's effects on the nervous system and of the basis of nicotine tolerance and dependence.

描述（改编自申请人的摘要）：我们的长期目标 研究是了解多样性的性质和后果 神经元乙酰胆碱受体（NACHRS）的表达。 最近的 分子生物学研究已经确定了一个假定的NACHR家族 神经系统中的基因。 但是，几乎没有证据表明NACHRS 通常在中枢神经系统中发挥相同的功能 在周围神经系统中，它们是快速，兴奋性的基础 突触传输。 我们建议同时使用结构和功能 在胚胎鸡的两次制剂中检查NACHR的方法， 其中每个烟碱受体基因特异的单克隆抗体 产品可用：位于 中脑和睫状神经节位于轨道上。 我们将使用 对小鸡NACHR和免疫细胞化学的亚基特异性抗体 检查NACHR亚基之间和内部NACHR亚基的分布的技术 神经元。 在细胞水平上，我们将检查是否所有细胞 神经元种群（SPL或睫状神经节）表达相同的子集 亚基。 在亚细胞级别，我们将检查 神经元表面的受体亚基，以确定哪个 亚基位于突触部位，亚基位于 外触觉，可以将其运输到轴突末端。 最后， 在分子水平上，我们将使用荧光团标记的抗体和 荧光共振能量转移（FRET）以检查 彼此的亚基。 这项技术应该使我们能够学习哪个 亚基组装成受体低聚物。 我们将付款 注意突触前烟碱受体，虽然实际上是 迄今为止未表征，可以代表NACHR的主要形式 中枢神经系统。 我们将通过免疫细胞化学技术检查 是否在SPL轴突的表面上发现突触前烟碱受体 该项目的终端向视神经底漆以及它们是否存在 向睫状神经节投射的前神经神经元的末端。 如果在睫状神经节中发现突触前受体，我们将 尝试通过斑块夹在功能上表征这些受体 录音。 研究应增强我们对结构的理解 通过（1）识别哪个神经元烟碱受体的功能 亚基辅助形成可能服务的受体低聚物 突触后和突触前功能以及（2）检查结构和 烟碱突触前受体的功能。 很可能会更饱满 理解突触后和功能的结构和功能 突触前烟碱受体对于理解至关重要 尼古丁对神经系统的影响和尼古丁的基础 宽容和依赖性。