The aims of this research proposal involve: A) Methodological innovations in the use of native and derivatized lectins, especially wheat germ agglutinin (WG), and the bacterial toxin, cholera toxin (CT), as intraneuronally transported probes for studies of neuronal connectivity, endocytosis and transport in the rat CNS and PNS compared with horseradish peroxidase (HRP); B) The cell biology of the fluid phase endocytosis and intraneuronal transport of HRP compared with the adsorptive endocytosis and intraneuronal transport of native and derivatized WG and CT in neurons of the rat CNS and PNS. The methods wil include light (LM) and electron (EM) microscopy to localize conjugates, produced by a variety of methods, and HRP visualized with HRP neurohistochemistry. LM and EM observations will also be carried out on native WG and CT using immunohistochemistry and anti-ligand antibodies. Both qualitative and quantitative studies will be carried out in well characterized CNS and PNS pathways of the rat. The amounts of internalized ligands and their rates of orthograde and retrograde transport will be characterized using radiolabeled WG and CT and these observations will be contrasted with similar observations on radiolabeled HRP. The role of the neuronal Golgi apparatus and cytoskeleton in the endocytosis and transport of WG and CT will be studied by using reagents which selectively perturb these neuronal organelles. Finally, the possibility that WG and CT are toxic to neurons will be evaluated by LM and EM as well as electrophysiological methods. These studies should provide new information concerning the similarities and differences between adsorptive and fluid phase endocytosis in the mammalian nervous system. The significance of these studies derives from the contribution they will make towards understanding endocytosis and intraneuronal transport; two basic phenomena integral to normal neuronal function and to the pathogenesis of certain nervous system diseases. Finally, they should lead to improved methods for studies of neuronal connectivity and neuronal cell biology.
