Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.

Abstract

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified a-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca'-free solution with 1 mM EGTA and 4 mM Mg2+ , the rate of quantal secretion rose to high levels but quickly subsided ; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up . When BWSV was applied to other muscles bathed in a solution with 1 .8 mM Ca t+ and 4 mM Mg 2+ , the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughout the hour of exposure . Neuromuscular transmission was blocked in fewer than 50% of these fibers . The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles . If HRP had been present, most of the synaptic vesicles were labeled with reaction product.These observations suggest that Ca 2+ plays an important role in endocytosis at the frog neuromuscular junction .The secretory products of many secretory cells are released by exocytosis from intracellular storage organelles after the membrane of the organelle has become continuous with the plasmalemma (1) . This exocytotic event is usually followed by an endocytotic event that recovers an equivalent area of membrane from the plasmalemma (1, 2). At the neuromuscular junction, endocytosis occurs rapidly enough to maintain the population ofsecretory organelles, the synaptic vesicles, at near normal levels during long periods of intense secretion (2-7). The factors that control exocytosis have been well studied, and in many systems this process is believed to be triggered by the influx of extracellular Ca" near the sites of secretion (1,2,(8)(9)(10)(11)(12) . The factors that influence endocytosis, on the other hand, have been difficult to determine, perhaps because they involve components within the cytoplasm that cannot be readily controlled by manipulating the composition ofthe bathing solution (12, 13) .Black widow spider venom (BWSV) contains a toxin that appears to interfere with endocytosis as it causes a relatively rapid depletion of synaptic vesicles (14-16). We have found that the vesicle-depleting power ofsmall doses ofcrude BWSV are highly dependent upon the Ca" concentration of the bathing solution, the depletion being greater in the absence of Ca" . This result suggests that Ca" may be an important factor for endocytosis at the neuromuscular junction.THE JOURNAL OF CELL BIOLOGY " VOLUME 87 OCTOBER 1980 297-303 © The Rockefeller University Press -0021-9525/80/10/0297/07 $1 .00 MATERIALS AND METHODSCutaneous pectoris nerve-muscle preparations were dissected from frogs, Rana pipiens, and mounted in a Lucite chamber at room temperature (17) . The composition of the standard Ringer's solution (in mM) was: Na', 115 ; K',...

α‐Latrotoxin stimulates glutamate release from cortical astrocytes in cell culture

“…3a, these calcium imaging experiments demonstrate that c~-latrotoxin exerts effects on astrocytes that are similar to the toxin's actions on neurons, c~-latrotoxin stimulates an increase in basal release from neurons in the presence and absence of external calcium ions [25,26]. In the absence of external calcium ions transmitter release is stimulated through a direct induction of exocytosis.…”

“…In neurons a similar decline in transmitter release has been shown when c~-latrotoxin is applied in calcium-depleted saline. Parallel electron microscopy has indicated that this is due to a depletion of the vesicles in the nerve terminal [25,26]. Similar effects of ~-latrotoxin in neurons and astrocytes raises the possibility that the transmitter release mechanisms of these two cell types share some fundamental properties.…”

“…3a). Previous studies have shown that ~-latrotoxin has two actions, it forms channels in the plasma membrane that cause a calcium elevation, and it can directly stimulate secretory machinery through interactions with c~-latrotoxin receptors [23][24][25][26]. We asked whether c~-latrotoxin directly stimulates the astrocyte glutamate release mechanism by removing external calcium ions.…”

“…In neurons the requirement for external calcium has been identified. Correlated electron microscopy and electrophysiology has demonstrated that in the absence of external calcium, ~-latrotoxin stimulates transmitter release and a loss of synaptic vesicles [25,26]. ~-Latrotoxin stimulates transmitter release transiently because external calcium is needed for endocytosis and the replenishment of the pool of transmitter filled vesicles.…”

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

“…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.…”