We carry on basic research into the cellular and molecular mechanisms involved in the development of the mammalian neuromuscular junction. We utilize a novel mammalian nerve-muscle culture system which is characterized by extensive early development of postsynaptic structure at sites of nerve-muscle contact followed later by maturation of the synaptic cleft and the presynaptic apparatus. We have shown that the capacity to induce postsynaptic acetylcholine receptor aggregation is at least ten-fold greater in developing axons than in dendrites of individual ventral spinal cord neurons. This suggests that one or more of the signals for the induction of postsynaptic differentiation has a polarized distribution in the innervating neurons. We are currently exploring the possibility that adhesive interactions with muscle are specific to axons as opposed to dendrites, and that they may be important for the initiation of postsynaptic differentiation. This is being done by scanning and transmission electron microscopy as well as immunocytochemical localization of specific cell-surface adhesion molecules. Our initial results indicate that the most intimate physical interactions between nerve and muscle cells occur at sites of contact-induced acetylcholine receptor aggregation along axons. We are exploring the role of agrin in the formation of neuromuscular junctions in the mammalian nerve-muscle culture system. Initially, the expression of mRNA for nerve specific isoforms of this acetylcholine receptor aggregating protein is being examined by in situ hybridization while the subcellular distribution of agrin is being studied by immunocytochemistry. The localization of several presynaptic plasma membrane and synaptic vesicle-associated molecules, including synapsin, synaptophysin, syntaxin and SNAP-25 is being examined in developing cocultures. Initial results suggest that these proteins become segregated to axons after the first two days of coculture.
