Me. Velasco et M. Pecotdechavassine, MEMBRANE EVENTS RELATED TO TRANSMITTER RELEASE IN MOUSE MOTOR-NERVE TERMINALS CAPTURED BY ULTRARAPID CRYOFIXATION, Journal of neurocytology, 22(10), 1993, pp. 913-923
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.