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To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle.