Components of the extracellular matrix are likely to be involved in a variety of developmentally important interactions between neurons and their targets. I propose to study some of these processes at the skeletal neuromuscular junction. Each skeletal muscle fiber is ensheathed by a layer of extracellular matrix, including a basal lamina (BL) that passes through the synaptic cleft. Recent experiments have shown that BL plays important roles in the formation, function, and maintenance of this synapse: BL contains acetylcholinesterase that terminates transmitter action, components that attach nerve to muscle, and molecules that regulate the differentiation of pre- and postsynaptic membranes during neuromuscular regeneration in adults. These results raise questions about the nature, development, metabolic regulation and mechanism of action of its components. Antibodies are now available that bind to several components of muscle fiber extracellular matrix: some define synapse-specific antigens in the BL, other define extrasynaptic region-specific antigens, and members of a third set define antigens shared by synaptic and extrasynaptic regions. I will now use these antibodies as immunocytochemical stains in anatomical experiments and as high-affinity ligands in biochemical studies. (i) The development of synaptic and extrasynaptic BL will be studied by light and electron microscopy in rat embryos. (ii) Factors that regulate the metabolism of BL will be studied with histological and biochemical methods in primary cultures of rat myotubes. (iii) Hypotheses generated from experiments in vitro will be tested in vivo. (iv) Antibodies will be used in blocking experiments, to elucidate the roles of individual components of BL. (v) Synaptic and other components of BL will be isolated and identified. (vi) Monoclonal antibodies will be prepared to some of the more interesting antigens that have been defined and initially studied with antisera. Through this work, I hope to learn how BL regulates and is regulated by synaptic interactions at the neuromuscular junction.
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