Following trauma to the peripheral nervous system basal lamina is the only identifiable structure to survive axonal and myofiber degeneration, thus molecules in the extracellular matrix and the anatomically identifiable basal lamina are likely to play key roles in cellular adhesion and development in axonal regeneration and muscle reinnervation. How these molecules are organized within extracellular matrix to allow interaction with pre- and postsynaptic membranes is unclear. This proposal is designed to test the hypothesis that a basal lamina skeleton can serve as a suitable substrate for neurite outgrowth and myofiber regeneration, containing the requisite substances for cellular attachment, guidance and local differentiation. There are three specific aims.
The first aim i s to identify and localize molecules associated with myotube-substrate attachment, and, to learn how these components are organized in the extracellular space to promote adhesion. """"""""Post release fracture labelling"""""""", a recently developed immunocytochemical technique allowing antibody penetration in regions of narrow cell-substrate apposition, will be employed.
The second aim i s to examine the role of basal lamina in axonal regeneration and reinnervation of skeletal muscle. Culture terrain composed of in situ basal lamina skeleton devoid of living cells can be produced by freezing or detergent extracting animal tissues. Experiments will evaluate the role of basal lamina skeleton as a structural scaffold for and myofiber regeneration; adhesive molecules associated with attachment of regenerating myotubes and axons :o the basal lamina skeleton will be identified with immunologic and ultrastructural methods.
The third aim i s to investigate the basal lamina's influence on pre- and postsynaptic specialization in synapse formation. Immunofluorescence and electron microscopy will be used to evaluate basal lamina's influence on the morphology and composition of pre- and postsynaptic structures at the neuromuscular junction; and detect the appearance of antigens unique to the developing nerve terminal and motor end plate.
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