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.
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