Curarized cutaneous pectoris nerve-muscle preparations from frogs were stimulated at 10/s or at 2/s for periods ranging from 20 min to 4 h . End plate potential were recorded intracellularly and used to estimate the quantity of transmitter secreted during the period of stimulation . At the ends of the periods of stimulation the preparations were either fixed for electron microscopy or treated with black widow spider venom to determine the quantities of transmitter remainind in the terminal . Horseradish peroxidase or dextran was added to the bathing solution and used as a tracer to detect the formation of vesicles from the axolemma . During 4 h of stimulation at 2/s many new vesicles were formed from the axolemma and the quantity of transmitter secreted was several times greater than the quantity in the initial store . After this period of stimulation, the terminals were severely depleted of transmitter, but not of vesicles, and their general morphological organization was normal . During 20 min of stimulation at 10/s the nerve terminals swelled and were severely depleted both of vesicles and of transmitter . During a subsequent hour of rest the changes in morphology were largely reversed, many new vesicles were formed from the axolemma and the stores of transmitter were partially replenished, These results suggest (a) that synaptic vesicles fuse with, and re-form from, the membrane of the nerve terminal during and after stimulation and (b), that the re-formed vesicles can store and release transmitter .

A B S T R A C TCurarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/see while recording" from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6--8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of tbeir transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control p;eparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma. I . N T R O D U C T I O NSeveral workers have tried to deplete neuromuscular junctions of their stores of transmitter and of their synaptic vesicles by tetanic stimulation of the nerve (1, 2, 3, 4). Depiction of transmitter has been obtained only when synthesis was inhibited by hemicholinium (2, 3) and, under this condition, a reduction in the number of vesicles occurred only in the regions of the axoplasm immediately adjacent to the axolemma (4). In these previous works the preparations were stimulated for from several minutes to a few hours at frequencies of 10/see or more. We have stimulated a neuromuscular preparation from the frog for 6-9 hr at a rate of 2/see in the absence of hemicholinium and have successfully depleted the terminals of their store of transmitter and of their population of vesicles. M A T E R I A L S A N D M E T H O D SThe cutaneous pectorls muscle of the frog, Rana p¢zens, was used. The muscles were mounted in the chamber described previously (5) and maintained at about 22°C in a Ringer's soIution that contained 116 rn~i NaC1, 2.0 mu KC1, 1.8 m:~ CaC12, 1 rn~ NaH2PO4, and 2 m~ Na2HPO4 (pH 7.0). End plate regions were impaled with mieropipettes filled with 3 • KC1. Conventional recording equipment was used and photographic records of the end plate potentials @.p.p.s) and miniature end plate potenrials (m.e.p.p.s) were obtained. The nerve was stimulated with square pulses 0.1 msec in duration and amplitude three to four times threshold. The muscle twitch was blocked by adding curare to the bath at a concentration of 3 X 10 -6 g/ml, an 30THE JOV~,tL O~" CELL BIOLOGr -VoLtnuE 54~ 197g • pages 30-38 on

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',...

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The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50, pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of at least four protein components that are similar in their molecular weights (about 130,000) and isoclectric points (ranging from pH 5.2 to 5.5) and are immunologicaUy indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.The physiological effects of the extract of the venom glands of the black widow spider, Latrodectus mactans, particularly the variety tredecimguttatus, have been studied extensively and the primary effects seem to be exerted on the nervous system (2-5, 7, 8, 12, 13, 15-18, 20-23, 30, 31, 33, 36-40, 43). Some of the active agents are proteins (18), and they seem to act in at least two basic ways: (a) to depolarize the cell bodies of some neurons and (b) to cause the release of neurotransmitters from a variety of nerve endings. For example, the extract depolarizes the cell body of the crayfish stretch receptor (S. Obara and A. Mauro, unpublished data) and induces a discharge of impulses in the axon (26). This depolarizing action of the extract may be responsible for its ability to induce a discharge of action potentials in the abdominal ganglion of the cockroach (5, 17).In addition to depolarizing excitable cells, the extract causes a release of transmitter from cholinergic nerve endings in brain (21, 24), sympathetic ganglia (38, 39), and Torpedo electric tissue (25), and from adrenergic nerve endings in the iris and other tissues (20,22,24). At neuromuscular junctions, the extract increases the frequency of occurrence of miniature end plate potentials, and blocks neuromuscular transmission. The neuromuscular effects have been observed at the cholinergic junctions of frogs and mammals (31, 37), at adrenergic nerve endings (vas deferens) in mammals (27), at both the inhibitory (3,-aminobutyrate) 462

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.

A method based upon an extension of Campbell's theorem is used to measure the amplitude, waveform, and frequency of occurrence of miniature endplate potentials (mepps) at rapidly secreting neuromuscular junctions of frog cutaneous pectoris muscles. Measurements of the variance, skew, and power spectrum of the fluctuations in membrane potential are used to deduce the mepp parameters. These estimates of mepp amplitude and frequency are insensitive to slow drifts in membrane potential that preclude the conventional application of Campbell's theorem, which uses the mean and variance. The new method becomes unreliable at high mepp frequencies because the distribution of the values of membrane potential approaches a Gaussian thereby reducing the accuracy of skew measurements. Frequencies approaching 10(4) s-1 can be measured, however, if the data are high-pass filtered. The method has been tested with computer simulated data and applied to junctions exposed to La3+; the effects of Ca2+ on the La3+-induced secretion have been explored. Some muscles were fixed after treatment with La3+, and changes in nerve terminal ultrastructure were assessed by morphometric analysis of electron micrographs. Horseradish peroxidase was used to obtain information about vesicle recycling.

Abstract. Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of ct-latrotoxin in Ca2÷-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, ~t-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of a-latrotoxin in the presence of 1.8 mM extracellular Ca 2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.