The long term goal of this ongoing study is to relate some of the defects in synaptic transmission associated with certain human presynaptic neuromuscular diseases to precise defects in nerve terminal function. 2,4-Dithiobiuret(DTB) is a thiourea derivative which causes a delayed onset neuromuscular weakness that mimics certain human neuromuscular diseases, thus providing a useful experimental model. Specifically, neuromuscular weakness induced by DTB is associated with altered release of acetylcholine (ACh) from motor nerve terminals, possible alterations of ACh receptor/ion channel complexes, and alterations of nerve terminal ultrastructure in treated animals Proposed studies will focus largely on the presynaptic mechanisms underlying DTB's diminution of release of ACh. They will utilize in vitro neuromuscular preparations from rats or frogs, or cultured neuromuscular junctions made by co-culture of L6 rat myoblasts with mouse hybridoma neuroblastoma/glioma NG108-15 cells, or rat PC12 pheochromocytoma cells. Effects of acute and chronic exposure to DTB will be examined using intracellular recording of postsynaptic potentials, fluorescent indicator dyes of Ca 2+ concentrations, and ultrastructural examination of thin sections, and freeze fracture of active zone membranes. Specific questions include: 1) Does DTB alter the availability of releasable transmitter through mechanisms involving either the packaging of ACh into vesicles, the total number of quanta in the releasable store, the mobilization process, or the relative proportions of quantal and nonquantal release? 2) What is the role of the glial cells and of the altered neuromuscular junction structure in the production of miniature endplate potentials with abnormally large amplitudes and aberrant time courses? 3) Are influxes of Ca 2 + and its subsequent buffering or sequestering perturbed in DTB treated terminals (and can these changes be linked to the increase noted in smooth endoplasmic reticulum?) 4) Is disruption of transmitter release by DTB unique to the cholinergic system or do more generic effects on transmitter release occur? 5) Are chronic effects of DTB on nerve terminal structure and function comparable to those seen in other degenerating junctions? Results of these studies using this novel paralytic agent will lead to further understanding of some of the presynaptic processes of neuromuscular transmission which are perturbed in diseased states or after toxic chemical exposure, as well as increasing our understanding of how these processes interact during normal neuromuscular functioning.
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