The long term objectives of this research plan are to develop immunological probes to specific synaptic glycoproteins in the chicken retina in order to determine the functional significance of these molecules with respect to the formation of and maintenance of synapses. These objectives are of special relevance to studies concerned with the normal development of neural pathways and also studies concerned with neural regeneration following recovery from various neurological disorders. Specifically, the aims of the project are to determine the molecular weights and lectin specificities of individual glycoproteins associated with synaptic plasma membranes and synaptic junctions isolated from the chick retina. Further aspects of this application include the description of cytoskeletal proteins involved in the organization of particular surface glycoproteins in the synapse and the isolation of glycoprotein-binding molecules or endogenous lectins that are associated with specific glycoproteins. Essentially, the methods involve a characterization of lectin-binding affinities of individual glycoproteins within the synaptic array of proteins observed in polyacrylamide gel-nitrocellulose paper blots which we have shown to be a suitable substrate for this purpose. This will provide us with information concerning the presence of and types of sugar residues that are accessible to lectin probes in individual synaptic proteins. Secondly, glycoproteins, endogenous lectins, and glycoprotein associated cytoskeletal proteins will be identified and characterized through use of affinity column chromatography techniques. Affinity isolated glycoproteins and endogenous lectins will be used to produce rabbit antisera against these purified molecules. The antisera will be used first to localize through electron microscope immunocytochemistry these specific molecules in tissue sections of the retina. This localization can be matched to specific molecules identified with lectins and antisera on gel-blots of the isolated synaptic fractions. In the future these immunological probes can be used to study transport and turnover of particular synaptic molecules. They will also be essential for the development of assays of functional relationships between specific molecules and synapse formation during development and regeneration.
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