NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

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The long-range objective o! this research is to understand how neuronal nicotinic acetylcholine receptors (AChRs) are regulated. Our approach has been to study AChRs in a very simple and tractable system, the frog cardiac ganglion. The parasympathetic neurons in this ganglion are located in the thin interatrial septum, where they can be viewed with exceptional clarity using a variety of microscopic techniques. Ganglionic AChRs can be studied both with ACh iontophoresis and with two kinds of AChR-specific ligands: 1) cross-reacting monoclonal antibodies made against Torpedo AChRs and 2) neural-bungarotoxin (also known as 3.1 bungarotoxin, toxin F, and k-bungarotoxin), which blocks receptor function in the ganglion. The binding of these AChR-specific ligands can be visualized with fluorescence microscopy, autoradiography, and both immunoperoxidase and immunogold electron microscopy. The major questions to be addressed are: 1. What precisely is the distribution of AChRs on the surface of the ganglion cell? Are AChRs as enriched in the postsynaptic membrane of neurons as they are in skeletal muscle? 2. How does the distribution of AChRs change when neurons are denervated? Is the original distribution restored upon reinnervation? Does innervation influence AChR distribution on ganglion cells in a manner similar to that found in skeletal muscle? 3. How do results obtained with AChR-specific ligands compare with those obtained by assaying AChRs functionally via iontophoresis? The functional assay for AChR may be influenced by the presence of acetylcholinesterase on the cell surface, and this enzyme may itself be regulated by innervation. These questions represent a first step toward understanding the mechanism(s) by which AChR distribution is regulated. We also plan an initial foray into more mechanistic studies by searching for other proteins which may be important in AChR clustering on the neuronal surface and by looking for cDNA probes with which receptor expression may be studied. These studies should increase our understanding of how nicotinic AChRs on neurons are regulated. This may, in turn, lead to a better understanding of central disorders in which cholinergic function is disrupted.