The regulatory peptide called calcitonin gene-related peptide (CGRP) was detected by immunofluorescence in frog motor neurons and motor nerve terminals. In motor nerve terminals, CGRP-like immunoreactivity was found to be segregated within large dense-core vesicles. To determine whether exocytosis from acetylcholine-containing small synaptic vesicles and from CGRP-containing large dense-core vesicles can be independently stimulated, nerve-muscle preparations were exposed to alpha-latrotoxin. This toxin induced complete depletion of acetylcholine-containing small synaptic vesicles but did not induce a parallel depletion of CGRP-like immunoreactivity and of large dense-core vesicles. These effects were independent of the presence of extracellular Ca2+ and occurred both at room temperature and at low temperature (1-3 degrees C). These findings suggest that exocytosis from the two vesicle populations is mediated by distinct biochemical mechanisms, which might be differentially regulated by physiological stimuli.

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca 2+, and 10 -4 M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of <1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15°K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5-10/~m, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was ~3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released.Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release. The vesicle hypothesis holds that each quantum is confined within one of the synaptic vesicles that populate the nerve terminal and is released by exocytosis when the membrane of that vesicle fuses with the axolemma at one of the many specialized regions called "active zones" (8,13,51).A number of workers have shown that at frog neuromuscular junctions vesicles fuse with the axolemma of stimulated 1386 terminals and some of the remaining vesicles become labeled with extracellular tracers (9)(10)(11)25). Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent...

Abstract. The distribution of two synaptic vesiclespecific phosphoproteins, synaptophysin and synapsin I, during intense quantal secretion was studied by applying an immunogold labeling technique to ultrathin frozen sections. In nerve-muscle preparations treated for 1 h with a low dose of ot-latrotoxin in the absence of extracellular Ca 2+ (a condition under which nerve terminals are depleted of both quanta of neurotransmitter and synaptic vesicles), the immunolabeling for both proteins was distributed along the axolemma. These findings indicate that, in the presence of a block of endocytosis, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and suggest that, under this condition, at least some of the synapsin I molecules remain associated with the vesicle membrane after fusion. When the same dose of c~-latrotoxin was applied in the presence of extracellular Ca ~+, the immunoreactivity patterns resembled those obtained in resting preparations: immunogold particles were selectively associated with the membrane of synaptic vesicles, whereas the axolemma was virtually unlabeled. Under this condition an active recyling of both quanta of neurotransmitter and vesicles operates. These findings indicate that the retrieval of components of the synaptic vesicle membrane is an efficient process that does not involve extensive intermixing between molecular components of the vesicle and plasma membrane, and show that synaptic vesicles that are rapidly recycling still have the bulk of synapsin I associated with their membrane.

Ouabain (0.1 and 0.05 mM) was applied to frog cutaneous pectoris nerve-muscle preparations bathed in modified Ringer's solution containing either 1.8 mM Ca 2+ (and 4 mM Mg 2+) or no added Ca 2+ (4 mM Mg 2+ and 1 mM EGTA). During the intense quantal release of acetylcholine (ACh) induced by ouabain, the parameters of the miniature endplate potentials (mepps) were deduced from the variance, skew, and power spectra of the endplate recordings by applying a recently described modification of classical fluctuation analysis. Often the high frequency of mepps is not stationary; therefore, the signal was high-pass filtered (time constant of the resistance-capacitance filter of 2 ms) to remove the errors introduced by nonstationarity. When ouabain was applied in the presence of Ca 2÷, mepp frequency started to rise exponentially after a lag of 1.5-2 h, reached an average peak frequency of 1,300/s in ~30 min, and then suddenly subsided to low level (10/s). In Ca2+-free solution, after a shorter lag (1-1.5 h), mepp frequency rose to peak rate of 700/s in ~20 min and then gradually subsided. In spite of the different time course of secretion in the two experimental conditions, the cumulative quantal release was not significantly different (7.4 _+ 1.3 x 105 in Ca2+-containing and 8.8 + 2.7 x 105 in Ca2÷-free solutions). 60 min after the peak secretion, the muscles were fixed for observation in the electron microscope. Morphometric analysis on micrographs of neuromuscular junctions revealed in both cases a profound depletion of synaptic vesicles and deep infoldings of presynaptic membrane. This rapid depletion and the lack of uptake of horseradish peroxidase suggest that ouabain impairs the recycling process that tends to conserve the vesicle population during intense secretion of neurotransmitter. The good correlation observed between the reduction in the store of synaptic vesicles and the total number of quanta of ACh secreted in the absence of a vigorous membrane recycling strongly supports the view that the secretion of a quantum of ACh requires the fusion of a synaptic vesicle with the axolemma.The discovery of synaptic vesicles and the evidence for the quantal nature of transmitter release led to the hypothesis that transmitter is stored in and released from the vesicle by a process of exocytosis (15,16). This hypothesis is accepted by many investigators but is challenged by others who suggest that the membrane of the nerve terminal contains channels that remain open for a relatively fixed period of time, allowing the diffusion of a fixed amount of cytoplasmic acetylcholine

Frog neuromuscular junctions were stimulated by different methods to secrete quanta of ACh, and the attendant changes in the ultrastructure of the nerve terminal were assessed by morphometric analysis of electron micrographs. Secretion was stimulated by electrical stimulation at 2 Hz or by application of the secretagogues, lanthanum, ouabain or black widow spider venom, either in the presence or in the absence of extracellular Ca2+. The numbers of synaptic vesicles, coated vesicles and coated pits, and the length of axolemma and area of axoplasm were measured on the micrographs. There was a significant increase (about threefold) in the total number of coated structures (vesicles plus pits) per micron2 of axoplasm, but the fractional increase in the number of coated pits exceeded the fractional increase in the number of coated vesicles. These increases were positively correlated with the increase in the length of axolemma per unit area and negatively correlated with the changes in concentration of synaptic vesicles, suggesting that they were due to the increases in the surface area of the terminal that accompany a loss of vesicles. However, the increase in the concentration of coated structures was not related to the number of quanta secreted or to the estimated number of vesicles recycled. The lack of correspondence between the fractional increases in the coated pits and coated vesicles and the poor correlation between the numbers of these structures and the overall parameters of the secretory process suggest that, in contrast to the situation in other secretory systems, coated pits and coated vesicles may not play a crucial role in maintaining the functional population of synaptic vesicles at rapidly secreting neuromuscular junctions.