PRE & POSTSYNAPTIC NICOTINIC ACHRS--STRUCTURE & FUNCTION

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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.