PRE & POSTSYNAPTIC NICOTINIC ACHRS--STRUCTURE & FUNCTION

描述(改编自申请者摘要)：我们的长期目标 研究是为了了解生物多样性的本质和后果 神经元乙酰胆碱受体(NAChRs)的表达。近期 分子生物学研究已经确定了一个可能的nAChR家族 神经系统中的基因。然而，几乎没有证据表明nAChRs 通常在中枢神经系统中起着与它们相同的功能 在外周神经系统中，它们是快速、兴奋的基础 突触传递。我们建议同时使用结构和功能 从胚胎鸡的两种制剂中检测nAChRs的方法， 其中针对每个尼古丁受体基因的单抗 提供的产品有：外侧螺旋状核(SPL)，位于 中脑和睫状神经节，位于眼眶内。我们将使用 鸡nAChRs亚基特异性抗体与免疫细胞化学 检查nAChR亚基在其间和其内分布的技术 神经元。在细胞水平上，我们将检查是否所有的细胞 神经元群(SPL，或睫状神经节)表达相同的亚群 亚基的数量。在亚细胞水平上，我们将研究 在神经元表面的受体亚基，以确定 亚单位位于突触位置，哪些亚单位位于 非突触的，并且可能被运输到轴突终末。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)检测 亚基相互连接。这项技术应该能让我们了解到 亚基被组装成受体寡聚体。我们会特别付钱的 注意突触前尼古丁受体，尽管实际上 到目前为止还没有特征的，可能代表了nAChR在 中枢神经系统。我们将通过免疫细胞化学技术进行检查 SPL轴突表面是否存在突触前烟碱受体 投射到视顶盖的终末以及它们是否存在于 突起至睫状神经节的节前神经元的终末。 如果在睫状神经节中发现突触前受体，我们将 尝试用膜片钳研究这些受体的功能 录音。这些研究应该增进我们对结构的理解 和神经烟碱受体的功能，通过(1)识别 亚基结合形成可能同时服务于两者的受体寡聚体 突触后和突触前功能以及(2)检查结构和 烟碱能突触前受体的功能。很可能是一个更丰满的 对突触后和突触后的结构和功能的理解 突触前尼古丁受体对于理解 尼古丁对神经系统和尼古丁基础的影响 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.