Individual neurons in the leech can regenerate severed axons to reconnect with particular neurons that are their normal synaptic targets. Repair occurs in stages, from initial sprouting of the injured axon and growth that may be along selected pathways to recognition of the target, synapse formation, and cessation of growth. The proposed project addresses the questions: (1) what cellular interactions and capabilities control sprouting, (2) which cells and extracellular elements along the pathway might stimulate or direct growth, (3) are inappropriate as well as appropriate contacts made, (4) how do the new synapses compare with the old in distribution and function, (5) why do axons stop growing, and (6) what features of axon growth and synapse formation during development are revived during regeneration? The methodology will include intracellular and extracellular electrophysiological recording and intracellular injection of markers for subsequent light and electron microscopic examination. Growth of axons, filled with fluorescent markers, will be tracked in living preparations, in some cases maintained in culture medium. Properties of pathways and surfaces of particular cells will be examined, using electron microscopy, with cell-specific ligands including monoclonal antibodies and with freeze-fracture. Single cells can be killed with intracellular injection of proteases or focally lesioned using dyes that produce photodynamic damage. The morphology of certain cells and the complex patterns of normal and regenerated synaptic contacts between them will be reconstructed with a computer. Growth of individual neurons will be studied during development and compared with regeneration. An understanding of the mechanisms for accurate regeneration in the leech central nervous system might suggest ways that nerve regeneration in higher animals including humans could be made more reliable and precise.
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