The long-term goal of this study is to enhance our understanding of the cause, prevention and cure of neuromuscular junction disorders.
The specific aim of this proposal is to test whether or not the formation of neuromuscular junction during embryonic development depends on the presence of connective tissue cells and their products in the muscle. Connective tissue cells may participate in neuromuscular synapse formation by contributing to the extracellular matrix, including the basal lamina which surrounds each muscle cell. The role of connective tissue in neuromuscular junction formation will be determined by reducing the connective tissue components of the superior oblique muscle and then monitoring the development of pre-and postsynaptic components of the neuromuscular junction. For this, the neural crest cells over the midbrain region which normally provide connective tissue cells to this muscle will be ablated in duck embryos. Ablations will be carried out before neural crest migration and development of neuromuscular junction begin. The reduction of connective tissue cells in the muscle will be verified by electron microscopy. Alterations in the basal lamina of muscle cells resulting from diminished contributions from the neural crest will be ascertained by immunohistochemical localization of its major components. The differentiation of the presynaptic components of the neuromuscular junction will be assessed by monitoring the development of synaptic vesicles and active zones in trochlear nerve terminals with electron microscopy. The differentiation of the postsynaptic components of the neuromuscular junction will be assessed by monitoring the development of acetylcholine receptors, acetylcholinesterase, junctional folds and postsynaptic density with morphological and biochemical techniques. Results of the proposed study should indicate whether or not the development of neuromuscular junction depends on interactions with connective tissue components.
