The long-term objective of this research is to characterize neuronal nicotinic acetylcholine receptors (nAChRs) as regards to their antigenic structure, their distribution on the neuronal surface and the control of their expression during development. Neuronal nAChRs in the optic tectum and in autonomic ganglia of the frog will be studied using cross-reacting monoclonal antibodies (mAbs) made against purified nAChRs from electric organ and skeletal muscle and provided by Dr. Jon Lindstrom. In preliminary results a number of these mAbs have been found to bind to that part of the optic tectum of Rana pipiens which receives a major projection from the retina. These results are interesting in light of previous work indicating that Alpha-bungarotoxin and other nicotinic antagonists appear to block retinotectal transmission in several lower vertebrate species. The first specific aim will be to further characterize the molecule(s) in the tectum that are recognized by anti-receptor mAbs. If these mAbs indeed recognize receptors which subserve retinotectal synaptic transmission, they should bind to the surface of tectal neurons immediately beneath ganglion cell terminals. This prediction will be tested at the fine structural level using immunoperoxidase and immunogold techniques while simultaneously labeling ganglion cell afferents to the tectum. The binding of Alpha-bungarotoxin will also be visualized using HRP-labeled Alpha-bungarotoxin, and the sites to which toxin binds will be compared to mAb binding sites. The second specific aim will be to use cross-reacting polyclonal and monoclonal antibodies to characterize nAChRs on the surface of autonomic ganglion cells. Antibodies which bind to the postsynaptic membrane will be assumed to cross-react with nAChRs on the ganglion cell surface. The distribution of ganglionic nAChRs will be measured using immunogold techniques or electron microscopic autoradiography to measure the extent to which neuronal nAChRs are concentrated in the subsynaptic membrane. The influence of the presynaptic nerve upon nAChR distribution will be determined by studying antibody binding before and after preganglionc denervation. These studies will serve to improve our understanding of the structure and regulation of neuronal acetylcholine receptors.
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