Development of Innervation Topography in Muscle
We have shown that the motoneuron pools of the rat diaphragm and SA (Serratus anterior) muscles project onto the surfaces of these muscles forming an orderly rostrocaudal map. Moreover, we have shown that when these muscles are denervated, regenerating motoneurons reinnervate their targets with topographic selectivity. This is especially evident in the neonatal SA muscle where regrowing LT (long thoracic) motoneurons select their appropriate targets whether the nerve was transected, crushed, or frozen. Further studies reveal that regenerating neonatal motoneurons select their appropriate target within the first week with no subsequent refinement. Taken together these observations provide a sufficiently complete picture of the SA muscle to use it as a model to define mechanisms underlying selective reinnervation of muscles in mammals. We have constructed a series of testable hypotheses organized under three specific aims. They are designed first, to address directly the question of whether endoneurial tubes or original end-plates guide axons in reestablishing the map. Second, what are the initial processes at nerve- muscle contacts in reestablishing the map? Third, after perturbing the system how will regenerating neurites respond? In the first series of experiments the LT nerve will be transected in 1 day old rate pups, and the distal stump removed from the muscle. Intracellular recording from motor end-plates will be used to assess whether a topographic bias can be established in the absence of Schwann cell basal lamina scaffolding. Next we will transect the LT nerve and redirect it to an ectopic, end-plate-free region of the SA muscle. Reinnervation topography will again be assessed to evaluate the role of original end-plates in reforming a rostrocaudal map. Following these studies we will examine the initial contacts between regenerating neurites from ventral roots C6 and C7 on their way to reinnervating the SA muscle. Morphological and physiological studies will assist in distinguishing potential 'silent synapses' from those where end-plate potentials can be recorded. Finally, we will conduct three perturbation experiments. First, we will artificially induce a condition of dual innervation in regenerating synapses to enhance direct synaptic competition between terminals of ventral roots C6 and C7. Next we will assess how competing nerve terminals respond to a reduced target size by deleting caudal sectors of the SA muscle. Finally we will begin a series of collaborative studies in which regenerating nerve terminals are stained with transported lipophilic dyes. By following the elimination of such labeled terminals we will develop better insight into the principles of synaptic competition. These specific aims are designed to explore important fundamental mechanisms of reinnervation. The SA muscle is an ideal model to study processes of neuromuscular regeneration based on topographic simplicity, reinnervation specificity and ease of experimental manipulation.