PRE & POSTSYNAPTIC NICOTINIC ACHRS--STRUCTURE & FUNCTION

描述（改编自申请人摘要）：我们的长期目标 研究是为了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 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 然而，几乎没有证据表明nAChRs 在中枢神经系统中的作用与 在周围神经系统中，它们是快速，兴奋 突触传递 我们建议使用结构和功能 在来自鸡胚的两种制备物中检测nAChR的方法， 其中特异于每种烟碱受体基因的单克隆抗体 产品是可用的：外侧海马状核（SpL），位于 中脑和睫状神经节，位于眼眶中。 我们将使用 鸡nAChRs亚单位特异性抗体和免疫细胞化学 技术来检查nAChR亚单位之间和内部的分布 神经元 在细胞水平上，我们将检查是否所有细胞内 神经元群体（SpL，或睫状神经节）表达相同的子集 的subunits。 在亚细胞水平上，我们将研究 受体亚单位在神经元表面，以确定 亚基位于突触位点，亚基位于 突触外，并可运输到轴突终端。 最后， 在分子水平上，我们将使用荧光团标记的抗体， 荧光共振能量转移（FRET）检查的接近 子公司彼此。 这项技术可以让我们了解 亚基组装成受体寡聚体。 我们将特别 注意突触前烟碱受体，虽然实际上 迄今为止尚未表征，可能代表了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 SpL轴突表面是否存在突触前烟碱受体 投射到视顶盖的终端以及它们是否存在于 投射到睫状神经节的节前神经元的末端。 如果在睫状神经节中发现突触前受体， 试图通过膜片钳技术来表征这些受体的功能 录音. 这些研究应该会加深我们对结构的理解 和神经元烟碱受体的功能，通过（1）鉴定 亚基结合形成受体寡聚体， 突触后和突触前的功能和（2）检查结构和 烟碱突触前受体的功能。 很可能是一个更完整的 了解突触后和突触后的结构和功能， 突触前烟碱受体对于理解 尼古丁对神经系统的影响和尼古丁的基础 宽容和依赖。

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.