The freeze-fracture technique is useful in the elucidation of membrane structure and events. Freeze-fracture has revealed intramembrane particle specialization in pre- and post-synaptic membranes of mature neuromuscular junctions and clarified events in the release of neurotransmitters. The technique has not yet been used to study the development of neuromuscular junctions, although such development has been followed by electrophysiology and electron microscopy of thin sections. The results of these techniques, however, have not been correlated by electron microscopic observation of the same junction studied electrophysiologically. Correlated freeze-fracture and thin-section studies of electrophysiologically-identified neuromuscular junctions are now possible with a method for freeze-fracturing neurites and myotubes developing in tissue culture. Nearly all the myotubes are fractured, and neurite-myotube contacts identified electrophysiologically can be relocated in replicas. The proposed research exploits these techniques to further clarify the stages in development of neuromuscular junction, to determine the minimum morphology concomitant with junctional transmission, and to enhance understanding of the mechanisms involved in turnover and stabilization of intramembrane particle specializations. Mapping techniques are also used to study the degradation of acetylcholine receptors induced by cross-linkage with myasthenic antibodies.
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