Spider Venom* ('Y-Aminobutyric Acid/Brain Cortex/Neurotoxin) MU-CHIN TZENG, ROCHELLE S

Home , Bungarotoxin, Latrotoxin

Proc. Nati. Acad. Sci. USA Vol. 75, No. 8, pp.. 4016-4020, August 1978 Neurobiology Release of neurotransmitters and depletion of synaptic vesicles in cerebral cortex slices by a-latrotoxin from black widow spider venom* ('y-aminobutyric acid/brain cortex/neurotoxin) MU-CHIN TZENG, ROCHELLE S. COHENt, AND PHILIP SIEKEVITZ The Rockefeller University, New York, New York 10021 Contributed by Philip Siekevitz, May 3, 1978

ABSTRACT The effect of a-latrotoxin on cerebral cortex G-200 chromatography was carried out after the DEAE- slices was studied by both biochemical and morphological Sephadex step to ensure homogeneity of the toxin. methods. This toxin greatly stimulates the release of preloaded For each experiment, one male mouse g) was killed -y-amino[3H butyric acid from cortex slices. The response in- (25-30 creases linearly with dose. The release is not dependent on the by decapitation and its brain quickly removed. One slice was presence of extracellular Ca2+, and therefore it is not mediated taken with a blade and blade guide from the top layer of ce- by the release of other transmitters from other types of neurons. rebral cortex of each of the two hemispheres. The two slices In contrast, no significant increase in the release of a non- (30-40 mg) were first incubated for 30 min at 370 in 1.0 ml of transmitter substance a-amino[14Clisobutyric acid is observed. a modified Krebs-Ringer solution with the following compo- Since previously we have shown that a-latrotoxin stimulated sition: 120 mM NaCl, 4 mM KC1, 1.8 mM CaCl2, 0.8 mM the re ease of acetylcholine and norepinephrine from cortex MgSO4, 4 mM Na2HPO4/HCl buffer (pH 7.4), 15 mM slices, it appears that the toxin probably selectively releases all neurotransmitters. The toxin also profoundly depletes the syn- NaHCO&, and 10 mM glucose. The incubation solution was aptic vesicle population in boutons in the cortex slices. The re- equilibrated with a gas mixture of 95% 02/5% CO2 throughout sults suggest that the release of neurotransmitter and the de- the experiment. In experiments assayingfor GABA release, 0.3 pletion of synaptic vesicle in boutons are manifestations of a MAM [3H]GABA (35 Ci/mmol, New England Nuclear) was single action ofthe toxin. Therefore, a-latrotoxin can be used present in the incubation solution, and 0.5 mM (aminooxy)- as a good tool for the identification of neurotransmitters and acetic acid (Eastman Co.) was used to inhibit the metabolism in studies on the mechanism of neurotransmitter release. of GABA by GABA-glutamate transaminase (3). In the experiments with [14C]AIB, a greater concentration, 35 AM, was used Previous work (1) has described the fractionation of an extract because of the lower specific radioactivity (57 mCi/mmol, of the black widow spider venom gland (BWSV) into several Amersham/Searle). After 30 min incubation, the slices were toxic protein fractions. One of the fractions was purified to the removed and washed once by immersion for 10 min in fresh degree of no detectable contaminating proteins and was re- solution. They were then transferred to a perfusion vessel cently named a-latrotoxin (2). This single toxin factor was containing 1.5 ml of solution. The vessel was immersed in a demonstrated to be responsible for all the effects of BWSV on shaker maintained at 370. Fresh oxygenated modified Krebs- frog and mouse neuromuscular junctions, namely, the increase Ringer solution was perfused through the vessel at a rate of 0.5 in the frequency of miniature end-plate potentials, the complete ml/min by a peristaltic pump. The perfusate of the first 30 min depletion of synaptic vesicles in the nerve terminals (boutons), was discarded, and thereafter 1-ml fractions were collected and the ultimate blockage of neuromuscular transmission (1). every 2 min into vials in a fraction collector. At a suitable time Since BWSV has many actions on both vertebrates and inver- during the perfusion, a-latrotoxin was injected into the perfu- tebrates, particularly their nervous systems (reviewed in ref. sion chamber. Radioactivity discharged into the perfusate was 1), and since several toxic fractions have been found in the determined after the addition of Aquasol, and radioactivity venom, it is of interest to investigate the spectrum of action of remaining in the brain slices was determined after solubilization this highly purified a-latrotoxin in a single species. In a previous of the slices with Protosol. In some experiments, Ca2+ was communication (2) we demonstrated that a-latrotoxin caused omitted, 0.5 mM EGTA was present, and the MgSO4 concen- an increase in the release of both acetylcholine and norepi- tration was increased to 3.0 mM. In order to take into account nephrine (NE) from cerebral cortex slices of mouse. We report the variations among the various experiments in the uptake of here: (i) that a-latrotoxin enhances the release of another pu- the radioactive substances and to correct for the diminution of tative neurotransmitter, -y-aminobutyric acid (GABA), from radioactivity in the tissue during the perfusion, we expressed mouse cortical slices, but not the release of a non-transmitter the radioactivity in the perfusate at any given time as a per- substance, a-aminoisobutyric acid (AIB, an analogue of GABA); centage of the radioactivity still present in the tissue at that time and (ii) that, as in the case of neuromuscular junction, it also (4). causes depletion of synaptic vesicles in cerebral cortex slices. We also assayed, by a procedure modified from that of Nadler and Cooper (5), aliquots from some fractions to deter- MATERIALS AND METHODS of a-latrotoxin were Abbreviations: AIB, a-aminoisobutyric acid; BWSV, black widow The purification and assay of purity per- spider venom gland extract; NE, norepinephrine; GABA, 'y-amino- formed as described (1); occasionally another step of Sephadex butyric acid. * This work was presented in abstract form by M. Tzeng and P. The costs of publication of this article were defrayed in part by the Siekovitz at the Seventh Annual Meeting of the Neuroscience Society, payment of page charges. This article must therefore be hereby marked 1977. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: Department of Anatomy, University of Illinois this fact. Medical School, Chicago, IL. 4016 Downloaded by guest on October 1, 2021 Neurobiology: Tzeng et al. Proc. Natl. Acad. Sci. USA 75 (1978) 4017 mine the degree of metabolism of [3H]GABA. The perfusates GABA were first chromatographed through an AG 50W (BisRad) C-on (Na+ form, pH 10) column. Neutral and acidic com s were eluted with three 1-ml H20 washes, and basic compounds were then washed out with 2 M NaOH. The H20 eluates were combined and acidified to pH <2, and then further fraction- ated on an AG SOW column (NH4+ form, pH 2.5). The column 0 was eluted in sequence by two 1-ml washes of each of the fol- a lowing: H20, 0.3 M NH40H, 0.6 M NH40H, 1.0 M NH4OH, 0a.I 1.5 M NH4OH, and 2.0 M NH40H (all solutions of NH40H :t having pH of 2.5). Al the GABA appeared in the 1.0 M and 1.5 .+-OOi M HN40H fractions. In some experiments the amount of 3H 0o in the tissue that remained as GABA was also determined. To ;6 do this, the brain slices were homogenized in 10% (wt/vol) trichloroacetic acid and then centrifuged in a microcentrifuge AIB for 10 min. The supernatant was treated twice with two vol- 0.41 umes of ethyl ether, the water phase was neutralized with NaOH and then analyzed for GABA as described above. For studies by electron microscopy, brain slices were cut into Time, min small strips and then incubated for 1 hr at 370 in 1.0 ml of the modified Krebs-Ringer solution with 10 gg a-latrotoxin added FIG. 1. Effect of a-latrotoxin on the release of [3H]GABA and at zero time 5 min. [14C]AIB from cortical slices. The amount of radioactivity released and another ,ug added at 30 Control samples in 2 min is expressed as a percentage of the radioactivity still in the were handled in the same manner except that no toxin was tissue at that time. The initial amount of radioactivity in the tissue added. The strips were then fixed with 1% glutaraldehyde/1% at zero time was 2.97 X 106 cpm for [3H]GABA and 1.04 X 10r cpm for paraformaldehyde in 0.12 M phosphate buffer, pH 7.2, and [14C]AIB. The arrow indicates the time at which 10 jug ofa-latrotoxin postfixed with 1% OS04 in 0.03 M barbital buffer, pH 7.4. The was added. (Aminooxy)acetic acid was present in both cases. tissue was then stained en bloc with 0.5% uranyl acetate, de- hydrated by standard procedures, and embedded in Epon, of toxin to the perfused tissue, and the toxin in the solution was which was polymerized at 15.50 for 3 days. Thin sections were washed away quickly; therefore those boutons in the interior cut with a Porter-Blum MT2B of the slices might not have been exposed to the toxin. However, microtome, stained sequentially the acetylcholine experiments were done in an incubation vessel with 8% uranyl acetate and 4% lead citrate, and examined with without perfusion. Also, there are rapid uptake mechanisms for both Hitatchi HU-11B and Siemens-Elmiskop 101 electron both NE and GABA, not only by boutons, but also, in the case microscopes. Only the outermost 20-,um layer of the strips was of GABA, by glial elements (6), and this latter pool presumably used for quantitative analysis, because the inner part of the would not be available for release by a-latrotoxin. tissue slices might not have been exposed to the toxin due to Fig. 2 shows the result of an experiment in which increasing diffusion problems. doses of a-latrotoxin were applied to one brain-slice preparation. The application of 1 ,ug of a-latrotoxin increased the efflux RESULTS rate to a maximum of 2.4 fold, and increasing the dose of a- In the various experiments, 30-40% of the [3H]GABA was taken latrotoxin caused an increasing response in the release of GABA. up by the cortical slices. A typical pattern of efflux of radioac- However, 10 jg of a-latrotoxin applied in this experiment in- tivity from these slices is illustrated in Fig. 1. The basal release creased the release rate to a maximum of only 10 fold, which rate of tritiated compounds in the absence of toxin was very is significantly less than the 17-fold increase that was observed slight, less than 0.1%/min. After a single application of 10 i~g (Fig. 1) when the same amount of toxin was applied to a tissue of a-latrotoxin, the release rate began to increase in less than preparation not previously exposed to the toxin. This phe- 3 min and reached a maximum of about 17 fold (average 16.2 nomenon is most probably a consequence of irreversible vesicle + 1 fold in five experiments) at about 8 min, and then it grad- depletion by earlier stimuli. ually returned to the base line over a 30-min interval. At the end, about 85% of the radioactivity originally taken up was still in the tissue. When the perfusates were assayed by ion-exchange 0.8 chromatography, it was found that about 95% of the radioac- 0.7- tivity released into the medium and about 97% of the radioactivity remaining in the tissue at the end of the experiment was 0.6 in GABA. In those experiments in which a Ca2+-free perfusion >0.5- solution containing EGTA was used, 10 ,g of a-latrotoxin elicited a similar increase in the release of GABA (maximum 0.4 14.8 fold in two experiments). When another protein fraction 0 (E) from BWSV (cf. ref. 1) was tested, no stimulation on the v 0.3- release of GABA was found. This finding is consistent with our o 0.2- proposal (2) that a-latrotoxin is perhaps the only component in BWSV that is active towards vertebrates. 0.1 The 85% of [3H]GABA that was still retained in the tissue at tong t 9g t4Mg tl0 g the end of 1-hr exposure to a-latrotoxin is similar to the case of 0 10 20 30 40 50 60 70 80 90 100 110 120 NE but different from that observed with Time, min (2) acetylcholine (2). FIG. 2. Effect of increasing doses of a-latrotoxin (arrows) on the Possible reasons for this high retention of NE and GABA are: release of [3HJGABA from a brain-slice preparation. Radioactivity these experiments were done by a single short-pulse application released is expressed as given in the legend to Fig. 1. Downloaded by guest on October 1, 2021 4018 Neurobiology: Tzeng et al. Proc. Natl. Acad. Sci. USA 75 (1978) A dose-response relationship of GABA efflux by a-latrotoxin of these three classes are shown in Fig. 4; Table 1 shows the is shown in Fig. 3. The data were all from experiments in which distribution of such boutons among the three categories. Most only one addition of toxin was given to the slices. The threshold boutons (87%) in control tissue were full of vesicles, only 5% dose obtained from the graph was about 0.2 ,g. The response were judged to be completely depleted, and these depletions was fairly linear up to the largest dose (10 Mug) of a-latrotoxin could have been caused by tissue damage. In toxin-treatedslices, used. This linear dose-response relationship is in contrast to the over 70% of the synaptic boutons were completely depleted. nonlinear relationship observed electrophysiologically (1). The Treated tissue appeared to have approximately 4 times less reason perhaps is that the electrophysiological method measures synaptic boutons per area than control tissue. This may be ex- the response at one synapse, whereas the present method plained by the enlargement of the boutons due to possible in- measures the summation of the responses at many synapses. corporation of vesicle membrane into synaptic plasma mem- Also, the variation among the present experiments, generally brane and by the swelling induced by the toxin. Based on this within 20%, is less than that obtained by the electrophysiological observation, a correction factor of four was used in a part of method, which had variations as large as 200-300% (1). Table 1 (see below). A comparison of the release of a known nontransmitter amino In many cases synaptic complexes were not observed in ob- acid, AIB, with that of GABA is also shown in Fig. 1. AIB has vious boutons, which were identified by the presence of syn- been shown to be taken up by both neurons and glia (6). About aptic vesicles. Of all the control boutons observed, -%0% were 20% of the [14C]AIB was taken up by the tissue in our experi- without synaptic complexes. This result may be explained by ments. When 10 Mug of a-latrotoxin was applied, only a slight the orientation of sectioning, but also by the actual absence of increase (average 10% in three experiments) in the efflux of AIB these specializations in certain types of boutons. Recent work occurred. This small increase has doubtful significance, for (7, 8) has shown that 5% of the boutons exhibit GABA efflux was increased 16-fold by the same amount of only aminergic toxin. In the case of AIB efflux, the presence or absence of synaptic complexes. Since these types of boutons cannot be (aminooxy)acetic acid did not make any difference in the re- distinguished from other elements of the neurites when they sults. are depleted of vesicles, a different strategy was used for After a 1-hr continuous exposure to a-latrotoxin, brain slices analysis. The micrographs were screened for obvious vesicle- looked swollen even to the naked eye. Electron micrographs containing boutons that also lacked observable synaptic con- (Fig. 4) showed that most terminals were indeed swollen, with nections, and the data are also given in Table 1. swollen mitochondria. Some showed in the plasmalemma lo- In control experiments, in an area of 3500 ,um2, about 380 calized discontinuities, which may have been caused by the boutons without synaptic complexes were found to contain full swelling induced by the toxin (see Discussion). It should be complements of vesicles, while in toxin-treated tissue, only 16 pointed out that the disrupted appearance of even the control such boutons were found in 6400 Am2. When the number per tissue (Fig. 4a) is probably due to the shaking during the ex- um2 of boutons full of vesicles was compared after a 4-fold periment. Many boutons with synaptic vesicles inside were correction had been made in the toxin-treated sample to take prominent in the control samples, whereas in toxin-treated into account the swelling of the tissue due to the toxin, there was tissue, vesicle-containing elements were almost completely a 1:11 decrease by a-latrotoxin treatment. Therefore, it ap- absent (Fig. 4b). A quantitative analysis of the relative popu- peared that a-latrotoxin caused a complete depletion of syn- lation of synaptic vesicles in boutons of control and toxin-treated aptic vesicles in almost all types of boutons in the cerebral cortex slices was attempted. Boutons with identifiable synaptic com- of the mouse, regardless of the transmitters involved. plexes were counted and were classified as either full, partially depleted, or completely depleted of vesicles. Examples of each DISCUSSION Previously (2) we have shown that a-latrotoxin caused increases in the release of acetylcholine and NE from mouse cerebral cortex slices, thus demonstrating that the effect of a-latrotoxin is not limited to cholinergic neuromuscular junctions. In this 16 - report, a-latrotoxin was shown also to enhance greatly the release of another putative transmitter, GABA, from cerebral cortex, but not of a nontramsmitter, AIB. This effect on GABA was of extracellular similar to the <12- release independent Ca2+, effect on acetylcholine and NE release. The absence of a Ca2+-dependence indicates that the enhancement of GABA release is not mediated trans-synaptically by other transmitters released from other neurons by the toxin. While this paper was in preparation, a communication (9) appeared on the release of GABA from synaptosomes by a less well characterized fraction from BWSV. C/ Our morphological studies (2) extended the observation at the neuromuscular junction that a-latrotoxin depletes synaptic vesicles similarly in the cerebral cortex. Boutons with or without synaptic complexes were affected to a similar extent, apparently 0 2 4 6 8 10 regardless of the transmitters involved. These results ,ug strongly ot-Latrotoxin, suggest that a-latrotoxin affects all types of boutons via a FIG. 3. Dose-response relationship for the increase in [3H]GABA common mode of action, probably by inducing the fusion of efflux by a-latrotoxin. The data are taken from experiments such as vesicles with the presynaptic membrane to release vesicle given in Fig. 1 but with varying doses. Trhe level of GABA release in- Previous creased by va-larotoxinstimidAtionnat.the-peak of the efflux curve was contents and thus lead to eventual vesicle depletion. expressed in terms of multiples of the basal rate. The line was fitted investigators failed to find depletion of vesicles in synaptosomes by the least mean square method. prepared from cerebral cortex (10), thalamus and basal ganglia Downloaded by guest on October 1, 2021 Neurobiology: Tzeng et al. Proc. Natl. Acad. Sc. USA 75(1978) 4019

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FIG. 4. Electron micrographs of brain slices incubated for 1 hr in modified Krebs-Ringer solution with (b, d, e) or without (a, c) a-latrotoxin. (a,b)1 Double arrows point to boutons having distinct synaptic complexes (single arrow). Arrow heads point to boutons lacking recognizable synaptic complexes. (X14,250.) (c) Higher magnification view of some boutons with full complement of synaptic vesicles (v).(X28,500.), (d) A partially depleted synaptic bouton. (x28,500.) (e) A synaptic bouton completely depleted of synaptic vesicles. The synaptic projections seem to be denser than usual. (x28,500.) (11), and Torpedo electric organ (12) treated with BWSV. The is correct, to aid the identification of transmitters. One im- reason for this discrepancy between our work and these reports portant criterion for a substance to qualify as neurotransmitter is not known. is that it should be released by direct nerve stimulation in a If our results are substantiated, a-latrotoxin could be used as Ca2+-dependent manner. But in many systems, it is not possible a tool first to determine whether a substance is stored in vesicles, to isolate the nerve for stimulation, and, as a substitute method, and second, if the hypothesis of transmitter storage in vesicles indiscriminate electrical stimulation or high concentrations of Downloaded by guest on October 1, 2021 4020 Neurobiology: Tzeng et al. Proc. Natl. Acad. Sci. USA 75 (1978)

Table 1. Effect of a-latrotoxin on the morphology of boutons in cortex slices State of synaptic vesicle population Boutons with synaptic complexes Area Partially Completely Boutons without synaptic complexes examined, Full, depleted, depleted, Partially Am2 Total % % % Full depleted Control 3500 265 87 8 5 376 (0.11 ;m-2)* 42 (0.012 Mm-2)* a-Latrotoxin-treated 6400 121 9 17 74 16 (0.010 Mm-2)*t 34 (0.021 Am-2)*t Mousecerebral cortex slices were incubated for 1 hr at 370 in modified Krebs-Ringer buffer, pH 7.4; to the experimental sample were added 10 jtg a-latrotoxin at zero time and 5 ,ug more at 30 min. * Figure in parentheses is the number of boutons per square micrometer. t The value in the toxin-treated case has been corrected to the original area, to take into account the swelling due to the toxin (see text). K+ and other depolarizing agents have been used. However, presence of Ca2+ occur all over the presynaptic face of the ax- nontransmitter substances are also released by high concen- olemma. Our interpretation of these findings is that the toxin trations of K+ from neuronal (13, 14) as well as from non-neu- does act like Ca2+ at the active zone but possibly at a receptor ronal (15) elements. Although well controlled electrical stim- site different from that of Ca2+. Whatever the mechanism of ulation should be more specific, it has been observed that action, the use of a-latrotoxin is one of the better means to electrical stimulation is unable to release GABA from cerebral clarify the relationship between synaptic vesicles and trans- cortex slices at stimulating potentials adequate for the release mitter release. of other putative transmitters. Enhanced release of GABA occurred only with applied potentials that were high enough to We thank Mr. B. Capparella and Dr. N. Frontali for help in ob- taining the spiders, Lois Lynch for aid in the electron microscopy, and also enhance release of nontransmitter substances (16). Drs. W. P. Hurlbut, N. H. Chua, and A. Gorio for their editing of the Some snake toxins such as f3-bungarotoxin have also been manuscript. This research was supported in part by U.S. Public Health found to cause depletion of synaptic vesicles at neuromuscular Service Grant NS 12726 to P.S. junctions (17), but it was found that nontransmitter substances were also released by this toxin (18). Because the nontransmitter, 1. Frontali, N., Ceccarelli, B., Gorio, A., Mauro, A., Siekevitz, P., AIB, was not released by a-latrotoxin, it appears that a-latro- Tzeng, M. & Hurlbut, W. P. (1976) J. Cell Biol. 68,462-479. toxin is unique as a transmitter 2. Tzeng, M. & Siekevitz, P. (1978) Brain Res. 139, 190-196. releasing agent. 3. Gelder, N. M. (1966) Biochem. Pharmacol. 15,533-539. The mechanism of action of a-latrotoxin is still unknown. It 4. Hopkin, J. & Neal, M. J. (1971) Br. J. Pharmacol. 42, 215- has been reported (19) to increase cation premeability in arti- 223. ficial lipid bilayers, which suggests that the toxin molecule can 5. Nadler, J. V. & Cooper, J. R. (1972) J. Neurochem. 19, 2091- insert itself into lipid bilayers. However, this would not explain 2105. the selectivity of the toxin towards its target tissue. Thus, con- 6. Hamberger, A. (1971) Brain Res. 31, 169-178. sistent with the selective action of the toxin to neural tissue, we 7. Descarries, L., Beaudet, A. & Watkins, K. C. (1975) Brain Res. have demonstrated recently a very high-affinity specific 100,563-588. binding of iodinated a-latrotoxin to protein receptor in synaptic 8. Descarries, L., Watkins, K. C. & Lapierre, Y. (1977) Brain Res. membrane fractions from cerebral cortex but not in liver plasma 133, 197-222. membrane preparations (unpublished results). However, the 9. Grasso, A., Rutini, S. & Senni, I. (1978) FEBS Lett. 85, 241- retains its 244. possibility exists that a-latrotoxin still ionophore-like 10. Baba, A., Sen, I. & Cooper, J. R. (1977) Life Sci. 20, 833-842. activity after binding to its receptor. A cation permeability 11. Kornguth, S. E. (1974) Rev. Neurosci. 1, 63-114. increase could be responsible for the depolarization (20) and 12. Granata, F., Traina, M. E., Frontali, N. & Bertolini, B. (1974) swelling of the boutons (1, 21, 22), and for the anomalous effect Comp. Biochem. Physiol. 48A, 1-7. of high concentrations of extracellular Ca2+ (23). Conceivably, 13. Roberts, P. J. (1974) Brain Res. 67, 419-428. the ionophore-like activity may stimulate transmitter release, 14. Vargas, 0. & Orrego, F. (1976) J. Neurochem. 26,31-34. but, since the effect of a-latrotoxin is independent of extra- 15. Sellstrom, A. & Hamberger, A. (1977) Brain Res. 119, 189- cellular Ca2+ (this paper and refs. 1, 20) and of Na+ (24), it 198. seems that this activity is not sufficient to account for all the 16. Orrego, F. & Miranda, R. (1976) J. Neurochem. 26, 1033- effects of the toxin. Another hypothesis based on the interaction 1038. 17. Chen, I. L. & Lee, C. Y. (1970) Virchows Arch. Abt. B. Zellpath. of the toxin or its receptor with presynaptic contractile elements 6, 318-325. has been proposed (25), and it is a candidate for further test- 18. Wernicke, J. F., Vanker, A. D. & Howard, B. D. (1975) J. Neu- ing. rochetn. 25, 483-496. It is not clear at present what is the relevance of transmitter 19. Finkelstein, A., Rubin, L. L. & Tzeng, M. (1976) Science 193, release evoked by a-latrotoxin to that evoked by nerve stimu- 1009-1011. lation. The latter process depends on the presence of extracel- 20. Longenecker, H. E., Jr., Hurlbut, W. P., Mauro, A. & Clark, A. lular Ca2:+, whereas the former does not. This difference would W. (1970) Nature 225,701-703. seem to argue that toxin-induced release occurs via a mecha- 21. Clark, A. W., Mauro, A., Longenecker, H. E., Jr. & Hurlbut, W. nism that is different from normal release. But, because we do P. (1970) Nature 225, 703-705. not know how Ca2+ acts to release transmitters, it is possible that 22. Clark, A. W., Hurlbut, W. P. & Mauro, A. (1972) J. Cell. Biol. and a-latrotoxin both activate the same release mecha- 52, 1-14. Ca2+ 23. Smith, J. E., Clark, A. W. & Kuster, T. A. (1977) J. Neurocytol. nism but by different means. B. Ceccarelli, F. Grohovaz and 6,519-539. W. P. Hurlbut (personal communication) have found that 24. Gorio, A., Rubin, L. L. & Mauro, A. (1978) J. Neurocytol. 7, plasmalemmal deformations, presumably due to vesicle fusions, 193-205. induced by venom in the presence of Ca2+ occur mainly near 25. Tzeng, M. (1978) Dissertation (The Rockefeller University, New York, the active zones, whereas deformations induced by K+ in the N.Y.). 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