The development and maintenance of neuromuscular function depends upon reciprocal interactions between motoneurons and their target muscles. Motoneurons regulate the differentiation of muscle fibers and retrograde signals from the muscle fibers may be essential for the presynaptic specialization of motor terminals. Fundamental information about the mechanisms underlying these cellular interactions will contribute to our understanding of how proper neuromuscular function is maintained, and how it is compromised by congenital defects and disease. The insect nervous system has, for many years, provided a powerful model for investigating these mechanisms. During metamorphosis of the moth manduca sexta, larval leg motoneurons persist to innervate newly formed adult muscles following the degeneration of their larval targets. These motoneurons undergo a pronounced regression of their terminal aborizations and loss of presynaptic specializations, followed by regrowth and re-differentiation to allow innervation of the new adult muscles. The myoblasts that generate the adult muscles accumulate around the re-expanding motor terminals, then proliferate, fuse and differentiate. Accompanying these developmental events are precise changes in the titer of the steroid hormone, 20-hydroxyecdysone. The proposed experiments will examine the relative importance of reciprocal interactions between nerve and muscle, and hormonal cues, in regulating muscle development and presynaptic terminal remodeling. Several techniques, including electrophysiology, confocal and electron microscopy, activity-dependent staining of presynaptic terminals and immunolocalization of synaptic vesicle proteins will be used to compare presynaptic structure and function of the same motoneurons in the larva and adult, and the time course of motor terminal remodeling during metamorphosis. Confocal and electron microscopy will be used to determine the time course of myoblast accumulation and differentiation. in vivo manipulations, including denervation and the introduction of exogenous myoblasts, will be used to examine the role and specificity of motoneuron-derived signals in regulating the accumulation and differentiation of myoblasts. A nerve/muscle co-culture system will be used to further probe the mechanisms underlying these signals and to investigate the role of the ecdysteroid hormones in directing muscle development. Similarly, both in vivo and in vitro manipulations will be performed to examine the role of myoblasts and ecdysteroid hormones in regulating presynaptic differentiation. Basic knowledge derived from this well-defined model system will augment information being generated in other systems to provide a firm understanding of how motor systems develop and sustain normal function.
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