A central problem in neurobiology is the elucidation of the mechanisms whereby neurons seek our and make stable connections with their appropriate targets. The objective of this proposal is to identify the specific glycosphingolipids and glycoproteins that are necessary for synapse formation. The strategy to accomplish this long-range goal includes three parts: the choice of an appropriate model system, the identification of changes in glycoconjugates that correlate with synapse formation, and the generation of monoclonal antibodies against the most prominently changing glycolipid or glycoprotein. The clonal cell line, NG108-15, will be used because it is capable of making synapses when cocultured with muscle myotubes, and is part of a panel of well characterized clones that display a variety of neuronal properties and specific synaptic defects. The identification of the appropriate glycoconjugate changes will be undertaken with (a) available antibody probes to specific glycoconjugates, (b) one- and two-dimensional, high performance thin-layer chromatography of isolated, radiolabeled neutral glycolipids and gangliosides and (c) two-dimensional SDS- polyacrylamide gel electrophoresis of radioactive glycoproteins. The glycoconjugate differences or changes that will most strongly correlate with synapse formation will be those which are found in the NG108-15 muscle cocultures but do not appear in any of the four control conditions. The control conditions are: a) NG108-15 cells cultured alone, b) muscle cells cultured alone, c) NG108-15 cells cocultured with fibroblasts derived from the muscle cultures, and d) N18TG-2 cells, the NG108-15 parent, non- synapsing, neuroblastoma clone, cocultured with muscle. Monoclonal antibodies will be generated against the most prominently changing glycoconjugates. These studies will provide the foundation for the long-range goal of establishing the glycoconjugate function in synapse formation. These studies should have important implications for understanding neuronal regeneration and reconnection with their targets after injury or transplantation.
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