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 musclesforming an orderly rostrocaudal map. Moreover, we have shown that whenthese muscles are denervated, regenerating motoneurons reinnervate theirtargets with topographic selectivity. This is especially evident in theneonatal SA muscle where regrowing LT (long thoracic) motoneurons selecttheir appropriate targets whether the nerve was transected, crushed, orfrozen. Further studies reveal that regenerating neonatal motoneuronsselect their appropriate target within the first week with no subsequentrefinement. Taken together these observations provide a sufficientlycomplete picture of the SA muscle to use it as a model to definemechanisms underlying selective reinnervation of muscles in mammals. Wehave constructed a series of testable hypotheses organized under threespecific aims. They are designed first, to address directly the questionof whether endoneurial tubes or original end-plates guide axons inreestablishing the map. Second, what are the initial processes at nerve-muscle contacts in reestablishing the map? Third, after perturbing thesystem how will regenerating neurites respond?In the first series of experiments the LT nerve will be transected in 1day old rate pups, and the distal stump removed from the muscle.Intracellular recording from motor end-plates will be used to assesswhether a topographic bias can be established in the absence of Schwanncell basal lamina scaffolding. Next we will transect the LT nerve andredirect it to an ectopic, end-plate-free region of the SA muscle.Reinnervation topography will again be assessed to evaluate the role oforiginal end-plates in reforming a rostrocaudal map. Following thesestudies we will examine the initial contacts between regeneratingneurites from ventral roots C6 and C7 on their way to reinnervating theSA muscle. Morphological and physiological studies will assist indistinguishing potential 'silent synapses' from those where end-platepotentials can be recorded. Finally, we will conduct three perturbationexperiments. First, we will artificially induce a condition of dualinnervation in regenerating synapses to enhance direct synapticcompetition between terminals of ventral roots C6 and C7. Next we willassess how competing nerve terminals respond to a reduced target size bydeleting caudal sectors of the SA muscle. Finally we will begin a seriesof collaborative studies in which regenerating nerve terminals arestained with transported lipophilic dyes. By following the eliminationof such labeled terminals we will develop better insight into theprinciples of synaptic competition. These specific aims are designed toexplore important fundamental mechanisms of reinnervation. The SA muscleis an ideal model to study processes of neuromuscular regeneration basedon topographic simplicity, reinnervation specificity and ease ofexperimental manipulation.