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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS024207-13
Application #
2635692
Study Section
Neurology B Subcommittee 2 (NEUB)
Program Officer
Baughman, Robert W
Project Start
1988-07-01
Project End
2000-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Dentistry
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Rogers, Marc; Sargent, Peter B (2003) Rapid activation of presynaptic nicotinic acetylcholine receptors by nerve-released transmitter. Eur J Neurosci 18:2946-56
Dourado, Michelle; Sargent, Peter B (2002) Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord. J Neurophysiol 87:3117-25
Nguyen, Don; Sargent, Peter B (2002) Synaptic vesicle recycling at two classes of release sites in giant nerve terminals of the embryonic chicken ciliary ganglion. J Comp Neurol 448:128-37
Ullian, E M; McIntosh, J M; Sargent, P B (1997) Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors. J Neurosci 17:7210-9
Horch, H L; Sargent, P B (1996) Effects of denervation on acetylcholine receptor clusters on frog cardiac ganglion neurons as revealed by quantitative laser scanning confocal microscopy. J Neurosci 16:1720-9
Wilson Horch, H L; Sargent, P B (1996) Synaptic and extrasynaptic distribution of two distinct populations of nicotinic acetylcholine receptor clusters in the frog cardiac ganglion. J Neurocytol 25:67-77
Sargent, P B; Garrett, E N (1995) The characterization of alpha-bungarotoxin receptors on the surface of parasympathetic neurons in the frog heart. Brain Res 680:99-107
Ullian, E M; Sargent, P B (1995) Pronounced cellular diversity and extrasynaptic location of nicotinic acetylcholine receptor subunit immunoreactivities in the chicken pretectum. J Neurosci 15:7012-23
Horch, H L; Sargent, P B (1995) Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion. J Neurosci 15:7778-95
Sargent, P B (1994) Double-label immunofluorescence with the laser scanning confocal microscope using cyanine dyes. Neuroimage 1:288-95

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