MEMBRANE EVENTS RELATED TO TRANSMITTER RELEASE IN MOUSE MOTOR-NERVE TERMINALS CAPTURED BY ULTRARAPID CRYOFIXATION

The sequence of structural changes occurring in the presynaptic membra ne during transmitter release was studied at the mouse neuromuscular j unction using the combined quick-freezing and cryosubstitution techniq ues. The mouse levator auris longus (LAL) muscle was stimulated by two means: either, chemically, by soaking 5 min before freezing in a phys iological solution containing 25 mM potassium chloride or, electricall y, by applying, 10 ms before freezing, a single supramaximal stimulus to the nerve-muscle preparation treated with 50 mu M 3,4-diaminopyridi ne (3,4-DAP) and 100 mu M (+)tubocurarine. In both cases, the preparat ions were maintained at approximately 5 degrees C, 5 min prior to free zing, in order to prolong nerve membrane changes. In most experiments, tannic acid (0.1%) was added to the substitution medium for better pr eservation of membranes. The different steps of warming in the substit ution medium were strictly controlled from -90 degrees C to 4 degrees C. When fixed under chemical stimulation, the presynaptic membrane app eared very sinuous and synaptic vesicles were seen apposed to speciali zed sites facing subjunctional folds. When submitted to a single elect rical stimulus, after treatment with 3,4-diaminopyridine, features of synaptic vesicle fusion were observed at these specialized sites which appear similar by their morphology, their macromolecular organization (already described) and their functional changes to active zones of t he frog neuromuscular junction. Other images suggested that with 3,4-d iaminopyridine which causes a pronounced and long-lasting release of t ransmitter, some vesicles collapse after exocytosis instead of being l ocally reformed by endocytosis.