different vamp/synaptobrevin complexes for spontaneous …different vamp/synaptobrevin complexes for...

Different VAMP/Synaptobrevin Complexes for Spontaneous andEvoked Transmitter Release at the Crayfish Neuromuscular Junction

SHAO-YING HUA,1 DOROTA A. RACIBORSKA,1 WILLIAM S. TRIMBLE,2 AND MILTON P. CHARLTON1

1Physiology Department and 2Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario,M5S 1A8 Canada

Hua, Shao-Ying, Dorota A. Raciborska, William S. Trimble, ous quantal transmitter release is reduced greatly, unlikeand Milton P. Charlton. Different VAMP/synaptobrevin com- evoked release, it is not eliminated altogether (Capognaplexes for spontaneous and evoked transmitter release at the et al. 1997; Dreyer 1989; Dreyer and Schmitt 1981;crayfish neuromuscular junction. J. Neurophysiol. 80: 3233– Duchen and Tonge 1973; Habermann et al. 1980; Her-3246, 1998. Although vesicle-associated membrane protein reros et al. 1995; Kryzhanovskii et al. 1971; Mellanby(VAMP/synaptobrevin ) is essential for evoked neurotransmitter

and Thompson 1972; Sweeney et al. 1995; Weller et al.release, its role in spontaneous transmitter release remains un-1991) .certain. For instance, many studies show that tetanus toxin

Evoked transmitter release consists of the synchronous(TeNT) , which cleaves VAMP, blocks evoked transmitter re-lease but leaves some spontaneous transmitter release. We used occurrence of the same quantal events as spontaneous releaserecombinant tetanus and botulinum neurotoxin catalytic light (Katz 1969). Because evoked release is simply a gross ac-chains (TeNT-LC, BoNT/B-LC, and BoNT/D-LC) to examine celeration of spontaneous release, it is surprising that boththe role of VAMP in spontaneous transmitter release at neuro- events are not affected in the same way by proteolysis of amuscular junctions (nmj) of crayfish. Injection of TeNT-LC into key protein. Such a discrepancy could arise if the spontane-presynaptic axons removed most of the VAMP immunoreactiv-

ous release process does not require VAMP or uses a VAMPity and blocked evoked transmitter release without affectingisoform that lacks the cleavage site for TeNT or if somenerve action potentials or Ca 2/ influx. The frequency of sponta-VAMP is protected from TeNT by interactions with otherneous transmitter release was little affected by the TeNT-LCproteins. In any case, these observations lead to the generalwhen the evoked transmitter release had been blocked by ú95%.

The spontaneous transmitter release left after TeNT-LC treat- hypothesis that evoked and spontaneous transmitter releasement was insensitive to increases in intracellular Ca 2/ . BoNT/ use somewhat different molecular mechanisms.B-LC, which cleaves VAMP at the same site as TeNT-LC but We investigated this discrepancy using new knowledgeuses a different binding site, also blocked evoked release but about the mechanisms by which various clostridial toxinshad minimal effect on spontaneous release. However, BoNT/D-

attack VAMP; these toxins require a binding site on VAMPLC, which cleaves VAMP at a different site from the other twoin addition to the site where enzymatic cleavage occurs.toxins but binds to the same position on VAMP as TeNT,Although TeNT and botulinum toxin-D (BoNT/D) cleaveblocked both evoked and spontaneous transmitter release at sim-VAMP at different sites, their binding sites are similar. Onilar rates. The data indicate that different VAMP complexes are

employed for evoked and spontaneous transmitter release; the the other hand, TeNT and botulinum toxin-B (BoNT/B)VAMP used in spontaneous release is not readily cleaved by cleave VAMP at the same site but their binding sites areTeNT or BoNT/B. Because the exocytosis that occurs after the different (Pellizzari et al. 1996, 1997) (Fig. 1) .action of TeNT cannot be increased by increased intracellular If spontaneous release does not require VAMP, thenCa 2/ , the final steps in neurotransmitter release are Ca 2/ inde- none of the clostridial toxins known to cleave VAMPpendent.

should block spontaneous release. If the VAMP used forspontaneous release lacks the TeNT cleavage or binding

I N T R O D U C T I O N sites or if these sites are occluded by protein interactions,then other toxins that use different sites may be effective

Proteolysis of the vesicle-associated membrane protein in blocking spontaneous release. Indeed VAMP does form( VAMP ) /synaptobrevin ( Baumert et al. 1989; Trimble complexes with other SNAP receptors (SNAREs) that pro-et al. 1988) by tetanus toxin ( TeNT) blocks evoked neu- tect it from cleavage by TeNT but this protection is lost ifrotransmitter release at synapses ( for review, see Monte- the complex is dissociated by the action of other synapticcucco and Schiavo 1994) , and this is strong evidence of proteins, soluble NSF attachment protein (a-SNAP) anda requirement for VAMP in calcium (Ca 2/ ) -triggered N-ethylmaleimide–sensitive factor (NSF) (Hayashi et al.exocytosis. However, there is a curious contradiction in

1995; Otto et al. 1997; Pellegrini et al. 1994, 1995) . Thusthe effects of TeNT on spontaneous transmitter release;investigation of the role of VAMP in spontaneous andmost studies show that while the frequency of spontane-evoked transmitter release with multiple clostridial toxinsmay reveal functional attributes of these complexes or

The costs of publication of this article were defrayed in part by the their intermediate states.payment of page charges. The article must therefore be hereby markedAfter injection of recombinant catalytic light chain of‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact. TeNT or BoNT/B (TeNT-LC, BoNT/B-LC) into presynap-

32330022-3077/98 $5.00 Copyright q 1998 The American Physiological Society

J235-8/ 9k2f$$de01 12-01-98 10:44:19 neupa LP-Neurophys

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S.-Y. HUA, D. A. RACIBORSKA, W. S. TRIMBLE, AND M. P. CHARLTON3234

Electrophysiology

Experiments were conducted on the opener muscle of the firstwalking leg of small (Ç5 cm) crayfish, Procambarus clarkii, atroom temperature (Wojtowicz and Atwood 1984). This muscle isinnervated by a single excitatory axon and a single inhibitory axon,one of which was separated from the rest of the nerve bundle atthe meropodite segment while all the other nerves were cut toensure that only the selected axon would be stimulated. The proxi-mal end of the axon was stimulated electrically by a platinumelectrode. The crayfish saline contained (in mM) 205 NaCl, 5.4KCl, 13.5 CaCl2 , 2.7 MgCl2 , 10 D-glucose, and 10 HEPES. InCa2/-free saline, equinormal NaCl was substituted for CaCl2 , and 1mM of ethylene glycol-bis-(b-aminoethyl ether)-N,N *-tetraacetic

FIG. 1. Binding and cleavage sites of tetanus toxin (TeNT) and botuli- acid (EGTA, Sigma, St. Louis, MO) was added to chelate freenum neurotoxin catalytic light chains (BoNT/B and BoNT/D) on vesicle- Ca2/ . The pH of each saline used was adjusted to 7.4 with NaOH.associated membrane protein (VAMP) (modified from Pellizzari et al. When Ca2/-free saline was applied, the bathing saline was ex-1996, 1997). j, binding sites V1 and V2 [SNARE (SNAP receptor) motif]

changed several times to eliminate Ca2/ as completely as possible.for each toxin. TeNT and BoNT/D use the same binding site (V1) , whereasIn experiments recording spontaneous miniature excitatory post-BoNT/B uses a different site (V2) . r, TeNT and BoNT/B cleave at thesynaptic potentials (mEPSPs), 10 mM picrotoxin (Sigma) wassame site, but BoNT/D cleaves at a different site. Binding and cleavage sitesadded to the saline to block g-aminobutyric acid (GABA) recep-are found within the domain that participates in protein-protein interaction.

Antibody used for Western blots and immunocytochemistry was made using tors responsible for spontaneous miniature inhibitory postsynaptica fragment of VAMP (aa 33–94) encompassing most of the protein interac- potentials (mIPSPs) (Takeuchi and Takeuchi 1969).tion domain and all of the cleavage sites. Amino acid numbers pertain to The preparation was mounted on an upright epifluorescence mi-rat and human VAMP2. croscope (Nikon Optiphot-2, Nikon, Tokyo, Japan), and either

the excitatory or the inhibitory axon was impaled with a glassmicroelectrode for monitoring presynaptic action potentials andtic axons at crayfish neuromuscular junctions, we observed injecting substances into the axon. For pressure injection of toxins,

that evoked transmitter release was blocked, but, surpris- the presynaptic electrode was filled with one of the following solu-ingly, spontaneous release was relatively little affected and tions: 2.4 mg/ml TeNT-LC in 100 mM KCl and 20 mM HEPES,became insensitive to increases of intracellular Ca2/ concen- pH 7.5; 15 mg/ml BoNT/D-LC in 500 mM KCl and 100 mMtration ([Ca2/]i ) . However, injection of BoNT/D-LC re- HEPES, pH 7.4; or 1–2 mg/ml BoNT/B-LC in a buffer of 200

mM ZnSO4, 80 mM EDTA, 2 mM Tris-Cl, 100 mM KCl, and 20duced both the evoked and spontaneous release with aboutmM HEPES, pH 7.4. Protein concentrations were measured withthe same time course. The crayfish neuromuscular junctionthe dotMETRIC assay kit (Geno Technology, St. Louis, MO).thus provides a simplified synapse for studying molecularThe BoNT/B-LC was a gift of Dr. A. Zdanovsky and originallymechanisms of transmitter release, and application of thecontained 1 mM of EDTA. A centrifuge microfilter (cutoff: 10clostridial toxins demonstrates that spontaneous and evokedkDa, Amicon, Beverly, MA) was used to remove the buffer con-transmitter release must use VAMP molecules that are dif- taining the EDTA while retaining the BoNT/B-LC for later dilution

ferent or that are in different complexes. in injection buffer. Up to 50-ms pressure pulses were applied tothe electrode by a Picospritzer (General Valve, Fairfield, NJ) atan interval of 10 s. The electrode solution also contained a fluores-

M E T H O D S cent dye (FITC-dextran 3 kDa, 225 mM, or Calcium Green-1, 100or 250 mM, both from Molecular Probes, Eugene, OR), which

Expression and purification of His6-tagged recombinant allowed visualization of the injection and rough comparisons oflight chain of tetanus toxin and botulinum toxin type D the amount of substance injected.

Short trains of stimuli (3–6 pulses, 0.3 ms, 100 Hz) were appliedto the proximal end of the axon at 3-s intervals. The evoked trans-Recombinant toxin light chain DNA, containing a carboxyl ter-

minal His6 tag in Qiagen Express plasmid pQE3, was generously mitter release was monitored by recording postsynaptic potentialsfrom the muscle fibers with a glass microelectrode filled with 3 Mprovided by Dr. H. Niemann (Eisel et al. 1993). Bacteria harboring

the DNA were grown to log phase (OD600 Å 0.6–0.7) and induced KCl, 2–5 MV. Because these muscle fibers have a space constantthat exceeds their length, the postsynaptic activities from all thewith 0.5 mM isopropyl-b-D-thiogalactopyranoside (Sigma-Ald-

rich, Oakville, ON, Canada). The culture then was grown for an synapses on a fiber can be recorded by a single electrode (Bittner1968). Stimulation and digital recording were controlled by Toma-additional 2.5 h to allow expression of the recombinant protein.

The harvested cells were frozen overnight at 0207C, disrupted hacq, a program for IBM PC written by Thomas A. Goldthorpe,University of Toronto. The presynaptic action potentials and EPSPsby pulse sonication, and centrifuged (20 min at 30,000 g, 47C),

and the supernatant containing the His6-TeNT-LC was circulated were recorded after low-pass filtering (4 pole Bessel) at 3 and 1kHz, respectively. Pre- and postsynaptic recordings were digitizedthrough nickel-agarose beads for 3 h, allowing the binding of the

histidine tag to nickel on the beads. After removing weakly bound (12 bits) at 100-ms intervals and averaged in groups of 10. Thestimulation artifacts in the original recordings presented in thisproteins by washing the beads with 30 mM imidazole, the beads

then were incubated with 100 mM imidazole added to the buffer paper were removed digitally during analysis. To detect spontane-ous quantal transmitter release, the postsynaptic membrane poten-to elute the His6-TeNT-LC protein off the beads. The buffer con-

taining the recombinant protein was collected and dialyzed against tial was recorded continuously on digital tape (VR 10, Instrutech,Great Neck, NY) for later analysis. To measure the frequency ofreaction buffer [20 mM N-2-hydroxyethylpiperazine-N *-2-ethane-

sulfonic acid (HEPES) and 100 mM KCl, pH 7.5; 6 h at 47C). The spontaneous release, 500 ms of the recording was sampled byTomahacq every 1,500 ms and mEPSPs were counted by handdialyzed protein was immediately made into aliquots and frozen at

0807C under nitrogen gas to prevent degradation. excluding evoked EPSPs; the average frequency of mEPSPs was


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DIFFERENT VAMP COMPLEXES IN SPONTANEOUS AND EVOKED RELEASE 3235

anti-guinea pig IgG (1:100, Jackson ImmunoResearch Labora-tories, West Grove, PA). In some experiments, the preparationwas incubated further with rabbit antisynaptotagmin (1:500) (Lit-tleton et al. 1993) and revealed with Texas red-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR). The preparation thenwas scanned with a confocal laser scanning microscope (Bio-RadMRC-600, Watford, UK) using140 water immersion lens (Nikon,0.55 NA). The FITC was excited at 488 nm, and emission lightlonger than 515 nm was collected. For Texas red, excitation was514 nm and the emission light ú630 nm was collected.

Calcium imaging

To examine Ca2/ influx into the nerve terminals, the excitatoryaxon was first penetrated with an electrode filled with 50 mMKCl and 200 mM Calcium Green-1 hexapotassium salt (MolecularProbes) . The dye was iontophoresed into the axon by applyingnegative current (about 05 nA) continuously to the electrode.After the dye had diffused to nerve terminals, the preparation wasscanned by the Biorad 600 confocal microscope using a120 Nikonobjective (0.35 NA, 20-mm working distance) , which is suitablefor simultaneous electrophysiological recordings. The CalciumGreen-1 was excited at 488 nm, and the emission light ú515 nmwas collected. To study Ca2/ influx, a series of 20 scans of thesynaptic boutons on the same muscle fiber penetrated by a re-cording electrode were taken at a rate of 1 image/2.1 s, startingshortly before a 5- or 10-s train of stimulation (30 Hz). Aftercontrol images of stimulus induced Ca2/ signals were obtained,TeNT-LC was injected into the presynaptic axon. When the toxineffect was developed fully, another series of stimulus-dependentCa2/ signals was obtained. The fluorescence intensity of the bou-tons was averaged for the area of the boutons of interest and Ca2/

entry was expressed in relative change in the intensity asFIG. 2. Recombinant TeNT light chain (TeNT-LC) cleaves crayfish and

rat VAMPs. Homogenized crayfish abdominal ganglia and rat brain (2 mg/ DF /F Å (Fstimulation 0 Frest ) /Frestml) were incubated respectively with or without TeNT-LC (2.5 mM) at377C for 1 h, with 10 mM dithiothreitol added to the reaction buffer 45

R E S U L T Smin before the toxin. Fifteen micrograms of incubated protein sample wasloaded into each lane. Western blot assay was performed using antibody

We wanted to examine the molecular mechanisms of exo-(1:1,000) against a peptide corresponding to the central portion of thehuman VAMP protein (aa 33–94) and a secondary antibody conjugated cytosis at a mature synapse in which we could measurewith HRP (1:10,000). Immunoreactive bands were detected by the en- and modify presynaptic membrane potential and [Ca2/]i , inhanced chemiluminescence assay. which we could inject macromolecules into the presynaptic

terminal, and in which we could clearly distinguish and mea-calculated every 75 s. The amplitude of mEPSPs was measured as sure evoked and spontaneous quantal transmitter releasethe difference between the peak of the mEPSP and the base level, from a single identified presynaptic cell. Neuromuscularusing the digital cursor readout in Tomahacq. In these experiments, junctions of some arthropods fulfill these criteria, and wethe presynaptic axons do not fire spontaneously, and this was con-

used the claw-opener muscle of the crayfish leg, in whichfirmed by continuous recording of the presynaptic membrane po-the presynaptic axons are large enough to permit insertiontential. This allowed the study of spontaneous transmitter release inof microelectrodes for recording (Wojtowicz and Atwoodthe complete absence of spontaneous presynaptic action potentials1984) and injection of exogenous molecules (Dixon andwithout requiring their blockade.Atwood 1989).

ImmunocytochemistryTeNT-LC cleaves crayfish VAMP

For immunocytochemical study, the preparation was fixed with3% paraformaldehyde for 1 h, permeabilized with 0.3% Triton The proteolytic activity of TeNT-LC was assayed on cen-X-100, with 1% bovine serum albumin (BSA) added to block tral nervous tissues of crayfish and rat. Samples of homoge-nonspecific binding, and incubated overnight at 47C with antibody nized crayfish abdominal ganglia and rat brain were incu-(1:50) against human VAMP amino acids 33–94 (Fig. 1) raised bated with or without recombinant TeNT-LC, BoNT/B-LCin guinea pig (Boyd et al. 1995; Li et al. 1994; Shone et al. 1993) or BoNT/D-LC at 377C for 1 h and subjected to sodiumin the presence of 0.3% Triton X-100 and 1% BSA. Because the dodecyl sulfate (SDS)–polyacrylamide gel electrophoresisepitope for this antibody (aa 33–94) spans the protein interaction

(PAGE) and Western blotting. Figure 2 shows that an anti-and toxin cleavage sites on VAMP-1 and VAMP-2, the antibodybody against VAMP that recognizes an epitope spanning thedoes not bind to cleaved VAMPs and therefore can be used totoxin cleavage sites (see Fig. 1) (Boyd et al. 1995; Li et al.detect changes in immunoreactivity caused by cleavage (Figs. 11994; Shone et al. 1993) recognized a protein extracted fromand 2) (Boyd et al. 1995; Li et al. 1994; Raciborska et al. 1998).

The primary antibody then was detected with FITC-conjugated crayfish ganglia with apparent molecular mass similar to that


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detected in the rat brain homogenate. Immunoreactivity of axoplasm as seen with fluorescence microscopy. Presynapticaction potentials and EPSPs evoked by stimulating the excit-this band was reduced greatly in extracts from both species

after incubation with recombinant TeNT-LC, indicating that atory axon, and spontaneous mEPSPs were recorded contin-uously. In all the experiments performed on 14 preparations,proteolytic cleavage had occurred. Similar results were ob-

tained with another antibody against rat brain VAMP-1 and pressure injection of TeNT-LC caused a large decrease inEPSP amplitude and, in most cases, totally blocked EPSPsVAMP-2 (Cain et al. 1992). BoNT/B-LC and BoNT/D-

LC also cleaved crayfish VAMP (see Figs. 7 and 8). evoked by trains of three to six stimuli at 100 Hz. A typicalresult is presented in Fig. 4A. Similar results were obtainedWe next asked whether recombinant TeNT-LC could

cleave VAMP in a live presynaptic cell. Proteolytic activity with natural isolated TeNT-LC (not shown). All data pre-sented in this paper were obtained with recombinant toxinwas verified after electrophysiological experiments in which

TeNT-LC was injected into one of the two main branches catalytic light chains.Although the EPSPs were blocked completely after TeNT-of the excitatory axon in the crayfish claw-opener muscle

(Fig. 3A) . The preparation then was fixed and incubated LC injection, the presynaptic action potentials were affectedvery little (Fig. 4B) , confirming that the toxin does notwith anti-VAMP. In the claw-opener muscle, the excitatory

and inhibitory nerves usually run parallel and close to each block action potential generation or propagation. In threeexperiments, injection of boiled TeNT-LC solution with Cal-other, appearing as two strands of boutons. Figure 3B shows

VAMP immunoreactivity in presynaptic boutons on a mus- cium Green-1 (Fig. 4D) or Calcium Green-1 in electrodesolution with no toxin (n Å 1) did not change EPSP ampli-cle fiber the nerve branch of which was far from the toxin

injection site. The anti-VAMP distribution at this distal site tude, thus indicating that the toxin effect is specific andrequires active enzyme.revealed a double strand of nerve terminal boutons corre-

sponding to the closely parallel excitatory and inhibitory The time required for the EPSP amplitude to decrease by50% varied from 19 to 96 min and averaged 49 min (n Åboutons. There was little if any VAMP immunoreactivity in

axons. Figure 3C shows that, near the injection site, only 9). This latency is due partially to diffusion and transportof the enzyme to the most distal boutons on the impaleda single strand of boutons corresponding to the uninjected

inhibitory boutons contained much VAMP immunoreactiv- muscle fiber and to the rate of VAMP cleavage by the en-zyme. Because when FITC-dextran (3 or 70 kDa) was in-ity. There was a parallel strand of faint staining in the poi-

soned excitatory terminals. These results show that VAMP jected we observed that fluorescence reached the most distalboutons on the impaled muscle fiber in this time, it is likelyexists in both the excitatory and the inhibitory nerve termi-

nals (Fig. 3B) and that the VAMP in the excitatory terminals that all boutons received toxin. The results from immunocy-tochemistry showed that VAMP was cleaved at sites distalnear the injection site had been cleaved. We presume that

at the distal site, insufficient TeNT-LC had arrived to cleave to the injection and recording sites during these experiments(Fig. 3) . Therefore the toxin LC should have reached allVAMP noticeably. All postsynaptic electrophysiological re-

cordings of transmitter release were made at muscle sites the boutons on the penetrated muscle fiber that was near thepresynaptic injection site. Some reduction in toxin activityvery close to the presynaptic toxin injection sites.

In some experiments, nerve terminals also were stained occurred after a sample was thawed, and to minimize vari-ability from this source, the toxin was used at most for 3for synaptotagmin immunoreactivity (Cooper et al. 1995a)

after the muscle had been incubated first with anti-VAMP. days after thawing.We also tested whether TeNT-LC could block inhibitoryFigure 3D shows the double-staining image of a muscle in

which the excitatory nerve had been injected with the transmitter release. Taking advantage of the dual excitatoryand inhibitory innervation of the crayfish claw-opener mus-TeNT-LC (same muscle as Fig. 3, B and C ) . The anti-

synaptotagmin revealed both the excitatory and inhibitory cle, we injected TeNT-LC into the inhibitory axon in anothertwo experiments to see if the toxin could block GABA secre-boutons, whereas the anti-VAMP revealed only the unin-

jected inhibitory boutons. The muscle was scanned twice tion. As illustrated in Fig. 4E, the toxin reduced the ampli-tude of IPSPs produced by stimulation of the inhibitory axon.with the confocal microscope, once for anti-VAMP and

once for anti-synaptotagmin distributions. The merged im- The remainder of this paper addresses excitatory synapsesexclusively.age shows the inhibitory boutons that contained both synap-

totagmin and VAMP in yellow. The results indicate that the The total block of EPSPs could not be ameliorated bystimulation at 30 Hz for 10 s, a procedure that normallyVAMP in the poisoned nerve terminals had been cleaved

by TeNT-LC, but distribution of the synaptotagmin was facilitates release several-fold (Atwood and Wojtowicz1986; Zucker 1989). However, facilitation still could beunaffected. Similar results were obtained with BoNT/B-

LC and BoNT/D-LC (Figs. 7 and 8) . demonstrated as long as EPSPs still were produced. To askwhether TeNT-LC affected facilitation, we compared thetoxin’s effect on the first and later EPSPs caused by a trainTeNT-LC inhibits both glutamate and GABA releaseof three pulses at 100 Hz. After the toxin was injected andpartial blockade obtained, all three EPSPs were inhibited toIf VAMP is required for transmitter release in crayfish

synapses, then destruction of VAMP should block release. a similar extent, indicating that the toxin has little effect onshort-term facilitation. Other types of plasticity, such asThe excitatory axon was penetrated with a microelectrode

containing the TeNT-LC and a fluorescent Ca2/ indicator long-term facilitation, could not be studied owing to therelatively rapid decline of transmitter release after TeNT-dye, Calcium Green-1. The electrode solution then was in-

jected into the axon with brief pulses of pressure, which LC injection.To determine whether a major effect of TeNT-LC is acaused the appearance of tiny puffs of fluorescence in the


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FIG. 3. TeNT-LC reduced VAMP im-munoreactivity in synaptic boutons. A : dia-gram of the muscle and innervation fromwhich pictures B–D were taken. TeNT-LCwas injected into the excitor axon throughelectrode ‘‘pre,’’ and excitatory postsynap-tic potentials (EPSPs) were recorded froma nearby muscle fiber by electrode ‘‘post.’’B : anti-VAMP staining of boutons locateddistal to the injection site. As shown in A,this muscle fiber was innervated by theright branch of the axon. TeNT-LC wouldhave had to move from the injection sitedown one branch and up the other to reachthis site. VAMP immunoreactivity shows adouble string of boutons corresponding toboth excitatory and inhibitory boutons. In-sufficient toxin had reached these excit-atory boutons to cleave much VAMP. C :VAMP immunoreactivity near the injectionsite. Here only the uninjected inhibitoryboutons retained normal VAMP immuno-reactivity and a single string of boutons isvisible. Scale is the same as for B. D : dou-ble staining with anti-VAMP (a) and anti-synaptotagmin (b) . After the pictures inB–Da were made, anti-synaptotagmin wasapplied. Color picture (c) is the mergedimage of a and b . Red, anti-synaptotagmin;green, anti-VAMP; yellow, both antibod-ies. VAMP immunoreactivity is not af-fected in the uninjected inhibitory bou-tons, but synaptotagmin immunoreactivityshows the location of all boutons includingthose depleted of VAMP. Arrow heads inC and Da indicate the faint staining of thepoisoned boutons. The preparation wasfixed after an electrophysiological experi-ment had determined that evoked EPSPshad been blocked near the injection site.Note that VAMP immunoreactivity in theinjected excitatory boutons near the injec-tion site was reduced greatly, but at distalsite B, immunoreactivity was not dimin-ished. Similar results were obtained in an-other 2 experiments. Preparations werescanned at vertical intervals of 2 mm for adepth covering all the boutons in the field(8–62 mm for this figure) , and images ofthe z series were projected to create 1 pic-ture.

reduction of Ca2/ entry, we performed Ca2/ imaging experi- tored during 5- or 10-s stimulus trains at 30 Hz to ensureeffective stimulation. The fluorescence intensity of severalments in injected boutons. Calcium Green-1 was injected

into the excitatory axon to monitor changes in [Ca2/]i , and boutons was averaged for each scan and the relative changein the fluorescence (DF /F) was plotted. After TeNT-LCthe boutons on muscle fibers from which EPSPs were re-

corded were scanned shortly before, during, and soon after was injected and EPSPs blocked, Ca2/ signals were similarto those obtained before TeNT-LC treatment (Fig. 4C) . Sim-the nerve stimulation to create Ca2/ images at different

times. Nerve firing and postsynaptic responses were moni- ilar results were obtained in another three experiments. It is


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FIG. 4. TeNT-LC blocks both EPSPsand inhibitory postsynaptic potentials(IPSPs). A : TeNT-LC completely blockedthe EPSPs. Amplitude of the 6th EPSPshown in B was plotted. B : examples ofaction potentials of the excitatory axon( top) and corresponding EPSPs from amuscle fiber (bottom) , taken at differenttimes as indicated in A. Small changes inaction potentials are due to changes in elec-trode resistance. C : TeNT-LC had minimaleffects on Ca2/ influx. Fluorescence of Cal-cium Green-1 in a single bouton wasscanned before TeNT-LC injection andafter EPSPs had been blocked by theTeNT-LC. During the time indicated, theexcitatory axon was stimulated for 5 s at30 Hz. Similar results were obtained in an-other 3 experiments. D : injection of boiledtoxin solution in another 2 preparations didnot change the amplitude of EPSP. Stimu-lation was stopped for a while for miniaturepotential observation, resulting in the dis-continuity of the recording before the injec-tion. E : IPSP also was inhibited by TeNT-LC injection. r, beginning of injection,which was stopped when small branches ofthe presynaptic neuron and its boutons werefilled. A shorter period of injection doesnot necessarily mean that less toxin wasinjected.

thus clear that TeNT-LC has little effect on Ca2/ influx but TeNT-LC injection has no significant effect on spontaneousuncouples Ca2/ influx from transmitter release (Hunt et al. release frequency even though EPSP amplitude evoked by1994; Llinas et al. 1994). three to six stimuli at 100 Hz had been reduced by ú95%.

Several control experiments were performed to verify thatevents counted were mEPSPs and therefore representedSpontaneous mEPSP frequency is relatively insensitive to spontaneous quantal excitatory transmitter release. To ex-TeNT-LCclude the spontaneous mIPSPs, 10 mM picrotoxin routinelywas included in saline (Takeuchi and Takeuchi 1969), andDuring these electrophysiological experiments, mEPSPsthis was sufficient to block evoked IPSPs. Therefore thewere recorded simultaneously with evoked EPSPs. We mea-synaptic events remaining after TeNT-LC were not due tosured the amplitude and frequency of mEPSPs to determinespontaneous GABA release from uninjected inhibitor bou-if they were affected by TeNT-LC. Figure 5, A and B, showstons. To determine whether all the recorded events are gluta-a typical result from a group of 14 experiments. The mEPSPmatergic, we applied JSTX-3 (20 mM, Natural Product Sci-frequency was not significantly reduced, even long after theences, Salt Lake City, UT) a glutamate receptor blocker thatEPSP was blocked. The mean mEPSP frequency of 14 fibersblocks both EPSPs and mEPSPs in this preparation (Kawaiin 14 different muscles was 0.41 { 0.07 (SE) Hz whenet al. 1982). Both EPSPs and mEPSPs disappeared in themeasured for 10 or 15 min before TeNT-LC injection. Afterpresence of JSTX-3, thus indicating that the miniature eventstoxin injection, mean mEPSP frequency in the same fiberscounted were indeed glutamate evoked. To test whether thewas 0.53 { 0.10 Hz, not significantly different from theremaining mEPSPs after TeNT-LC treatment were causedpretreatment control frequency (Pú 0.05, t-test) . The aver-by transmitter release from some type of cell other than theage change in mEPSP frequency after TeNT-LC injectionexcitor axon (Harrington and Atwood 1995), we injectedwas /0.13 { 0.07 Hz (P ú 0.05, t-test) and the average

percent change was /39 { 22% (P ú 0.05, t-test) . Thus trypsin (Sigma) in three experiments into the excitor axon


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FIG. 5. TeNT-LC blocked EPSPs but hadminimal effect on mEPSPs. A : plots of EPSPamplitude, mEPSP frequency and mEPSP ampli-tude simultaneously recorded in an experiment.Pressure injection (bar, TeNT-LC) of the elec-trode solution sometimes caused a transient in-crease in mEPSP frequency as shown here.Graph of mEPSP amplitude is a continuous re-cord of the measured amplitude of all themEPSPs in a 2-h experiment. B : example ofmEPSP recordings before and after TeNT-LC in-jection from same experiment as in A. Rightsweeps taken 47 min after starting the injectionwhen the evoked EPSPs had been blocked. C :trypsin eliminated spontaneous transmitter re-lease. After evoked transmitter release had beenblocked by TeNT-LC injection, trypsin (2% byweight in 150 mM KCl and 50 mM FITC-dex-tran) was injected into the excitatory axon at thetime indicated (r ) .

to destroy its synapses. Figure 5C shows an example in to Ca2/ . To test this hypothesis, we first examined Ca2/

which evoked transmitter release had been blocked by injec- dependency of miniature potentials before the toxin treat-tion of TeNT-LC, at which time trypsin injection abolished ment and found that in intact synapses, mEPSP frequencythe spontaneous transmitter release and caused loss of FITC- was not sensitive to changes in [Ca2/]o . In five experiments,dextran (3 kDa), a fluorescent dye contained in the injection when normal saline was exchanged for Ca2/-free saline con-buffer. Interestingly, trypsin first caused a gross increase in taining 1 mM EGTA, mEPSP frequencies were not signifi-the frequency of spontaneous mEPSPs. Because all synaptic cantly different (0.85 and 0.80 Hz, respectively, P ú 0.4,activity was eventually eliminated by trypsin injection into n Å 5); the average change in frequency was 00.05 { 0.06the excitor axon, all the mEPSPs must have originated from Hz (P ú 0.2, t-test) and the average percent change wastransmitter released from the excitor boutons. 04 { 6% (P ú 0.2, t-test) (cf Zucker and Lando 1986).

Next we asked whether increases in [Ca2/]i caused by Ca2/

entry though Ca2/ channels could affect mEPSP frequency.Spontaneous release is independent of increasedWhen depolarizing current was applied through an electrodeintracellular Ca2/ a fter TeNT-LCimpaling the axon, there was a large increase in mEPSPfrequency (Wojtowicz and Atwood 1984). The increase inThe preceding experiments enable us to separate sponta-mEPSP frequency caused by depolarization was blocked inneous transmitter release from evoked release and suggestCa2/-free saline with 1 mM EGTA, indicating that the effectthat the spontaneous transmitter release may not use exactlyof the depolarizing current was mediated by Ca2/ influxthe same mechanism as evoked release. Because action po-(Fig. 6A) . However, after EPSPs had been blocked withtentials failed to recruit the vesicles responsible for miniatureTeNT-LC injection, depolarizing current in normal [Ca2/]opotentials after TeNT-LC injection, it is possible that the

remaining spontaneous transmitter release is not sensitive saline failed to induce any change in mEPSP frequency.


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FIG. 6. TeNT-LC blocked mEPSPs induced byCa2/ influx. A : mEPSPs induced by presynaptic de-polarizing currents were blocked in Ca2/-free saline.Left : recordings in normal saline ([Ca2/]o Å 13.5mM). Right : from the same muscle fiber but in Ca2/-free saline with 1 mM EGTA. The 2nd and the 3rdsets of sweeps from the top were recorded when theaxon was depolarized by passing currents throughthe electrode inserted into the excitatory axon. Inten-sity of the currents is indicated for each set of sweeps.B : presynaptic depolarization failed to induce anyincrease in mEPSP frequency after TeNT-LC injec-tion in normal [Ca2/]o saline. Procedure was similarto A, and the currents depolarizing the axon were asindicated. A and B are from different experiments.

Figure 6B is typical of three experiments. It also was ob- but mEPSP frequency was not significantly different; theaverage frequency was 0.48 { 0.12 Hz before injection andserved with Ca2/ imaging that, after TeNT-LC injection,

depolarizing current still caused increases in [Ca2/]i , 0.61 { 0.05 Hz, after injection (P ú 0.05, n Å 5) (Fig. 7,A and B) . The average change in frequency waswhereas mEPSP frequencies were not changed (2 experi-

ments, not shown here) . Therefore, depolarization of the /0.12 { 0.12 Hz (P ú 0.05, t-test) and the average percentchange in frequency was /56 { 33% (P ú 0.05, t-test) .terminal membrane had elevated [Ca2/]i , but after TeNT-

LC treatment this Ca2/ could not increase mEPSP frequency. Injection of boiled toxin solution did not change EPSP am-plitude (n Å 1). BoNT/B-LC cleaved crayfish VAMP invitro (Fig. 7D) , and immunostaining of the injected prepara-Botulinum toxins B and D affect neurotransmitter releasetions showed greatly reduced VAMP immunoreactivity indifferentlythe presynaptic boutons (n Å 5) when evoked release hadbeen blocked, thus confirming the cleavage of VAMP inBecause spontaneous release was little affected by TeNT,cells (Fig. 7C) .it is possible that VAMP is not required by spontaneous

Because the insensitivity of spontaneous release to TeNTtransmitter release or that VAMP used for the spontaneousand BoNT/B contradicts results obtained in several othertransmitter release is for some reason relatively resistant topreparations (see INTRODUCTION and DISCUSSION), weTeNT-LC. As reviewed in Fig. 1, VAMP cleavage by TeNT-wanted to confirm that a reduction in mEPSP frequencyLC requires a specific TeNT-LC binding sequence (activa-could have been detected. To demonstrate this, we measuredtion site) as well as a precise cleavage site (Pellizzari et al.the temperature dependence of spontaneous release fre-1996; Rossetto et al. 1994). If the VAMP used for spontane-quency (Barrett et al. 1978). In three experiments, coldous transmitter release lacks either the binding or cleavagesaline of 8–127C was applied after EPSPs had been blockedsite or if one of the sites is not exposed to the toxin, thenat room temperature by BoNT/B-LC injection. This treat-the toxin would have little effect on mEPSP frequency. Wement caused an average reduction in mEPSP frequency ofnext tested whether the VAMP responsible for spontaneous68% (Fig. 7E) . This showed that large changes in mEPSPrelease could be cleaved if a toxin was used that requires afrequency easily could have been detected in experimentsbinding site on VAMP different from that used by TeNT.with TeNT-LC and BoNT/B-LC.We chose BoNT/B, which shares the VAMP cleavage site

In both vertebrates and invertebrates, more than one typewith TeNT but not the binding site (Pellizzari et al. 1996),of VAMP has been detected (Chin et al. 1993; Elferink etand asked whether this toxin could affect the spontaneousal. 1989), some of which do not have the cleavage or bindingtransmitter release. When we injected into the axon a solu-sites for TeNT and BoNT/B and are resistant to these toxinstion of BoNT/B-LC that contained 1 mM EDTA in the(Pellizzari et al. 1996; Schiavo et al. 1992; Yamasaki et al.buffer, neither EPSP amplitude nor mEPSP frequency was1994a,b) . We therefore examined effects of BoNT/D-LC,changed, probably because the EDTA chelated Zn2/ neces-which cleaves at a site found on most of the known VAMPssary for toxin activity (Schiavo et al. 1992). This showed(Schiavo et al. 1993); this is different from the site cleavedthat the toxin has no nonspecific effects. When the EDTAby TeNT and BoNT/B. BoNT/D-LC injected into the excit-concentration was lowered to 80 and 200 mM of ZnSO4 was

added, injection of BoNT/B-LC blocked the evoked release atory axon gradually decreased the amplitude of evoked


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FIG. 7. BoNT/B-LC affects evoked and spontaneous transmitter release differently. A and B: simultaneous recordings of evokedand spontaneous transmitter release at 1 muscle fiber. Toxin was injected during the time indicated by bars. C: immunoreactivity ofVAMP (top) after injection of BoNT/B-LC had blocked evoked transmitter release is reduced in excitor boutons but normal inuninjected inhibitory boutons. Bottom: both boutons are stained with antibody to synaptotagmin. Scale bar (25 mm) is the same forboth panels. D: Western blot of crayfish CNS extract before (0) and after (/) proteolysis by BoNT/B-LC. Methods were the sameas in Fig. 2. E: in another experiment, cold saline (8–127C) was applied after EPSPs had been blocked by BoNT/B-LC (injectedfrom time 0) at room temperature (Ç217C). Frequency of mEPSPs decreased at the lower temperature.

transmitter release (Fig. 8A) , which is similar to the effect (3–6 stimuli at 100 Hz). The average change in mEPSPfrequency was 00.28 { 0.10 Hz (P õ 0.05, t-test) and theof TeNT-LC. Unlike the effects of TeNT or BoNT/B, the

frequency of spontaneous transmitter release was reduced average percent change in frequency was 081 { 8.9%(Põ 0.05, t-test) . In two of these experiments, spontaneouswith a similar time course (Fig. 8B) . In the five preparations

tested, average mEPSP frequency decreased from a control release was blocked totally. When BoNT/D-LC solution wasinjected with 1 mM EDTA (n Å 2), there was no effectvalue of 0.32 { 0.09 Hz to 0.04 { 0.02 Hz (P õ 0.0001)

when EPSP amplitude had been reduced to 5% of the control on either evoked or spontaneous transmitter release, thus


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FIG. 8. BoNT/D-LC inhibits both evoked and spontaneous transmitter release. A and B : from simultaneous recordingsof evoked (A) and spontaneous (B) EPSPs in 1 muscle fiber. Toxin was injected during the time indicated by bars. C :immunoreactivity of VAMP ( left), after injection of BoNT/D-LC had blocked evoked transmitter release, is reduced inexcitor boutons but normal in uninjected inhibitory boutons. Right : both boutons are stained with antibody to synaptotagmin.Scale bar (50 mm) is the same for both panels. D : Western blot of crayfish CNS extract before (0) and after (/) proteolysisby BoNT/D-LC. Methods were the same as in Fig. 2.

indicating that the effect was specific and required active LC expressed in Drosophila (Sweeney et al. 1995). TeNTalso cleaved VAMP in mouse spinal cord cells (Williamsonenzyme. BoNT/D-LC cleaved crayfish VAMP in vitro (Fig.

8D) and immunostaining of the injected preparations et al. 1996), rat hippocampal slice cultures (Capogna et al.1997), and leech neurons (Bruns et al. 1997). Our datashowed greatly reduced VAMP immunoreactivity in the pre-

synaptic boutons, thus confirming that blockade of transmit- show that in addition to blocking excitatory release, TeNT-LC also blocked inhibitory transmitter release.ter release was accompanied by the cleavage of VAMP in

presynaptic terminals (Fig. 8C) .

Persistence of spontaneous release after TeNT and BoNT/BD I S C U S S I O N

TeNT and BoNT/B caused no net reduction in the fre-quency of spontaneous quantal transmitter release (Figs. 5The results show that VAMP is concentrated in presynap-

tic boutons of both excitatory and inhibitory synapses in and 7) when evoked release was blocked by ú95%. Thiscontrasts with previous reports in other synapses in whichcrayfish and that TeNT-LC cleaves crayfish VAMP in vitro.

When injected into presynaptic axons, TeNT-LC blocked TeNT holotoxin blocked evoked release and also causedlarge decreases in the frequency of miniature potentialsevoked transmitter release without affecting presynaptic ac-

tion potentials or Ca2/ influx, and VAMP immunoreactivity (Kryzhanovskii et al. 1971, 95% reduction of frequency;Mellanby and Thompson 1972, total block; Duchen andwas reduced greatly. Thus the physiological effect of TeNT

is most likely due to cleavage of VAMP. This is consistent Tonge 1973, 064%, 093%; Habermann et al. 1980, 097%;Weller et al. 1991, 092%; Herreros et al. 1995, 072%;with results for isolated natural TeNT-LC injected into the

squid giant synapse (Hunt et al. 1994; Llinas et al. 1994) Capogna et al. 1997, 088%). Drosophila lacking neuralsynaptobrevin also have reduced mEPSP frequencyand Aplysia synapses (Mochida et al. 1989) and for TeNT-


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(Sweeney et al. 1995, 050%; Broadie et al. 1995; Deitcher frequency has been accelerated by high resting [Ca2/]i ,cleavage of VAMP by these toxins will reduce the mEPSPet al. 1998, 075%). An exception was observed by Bevan

and Wendon (1984) who observed that at rat soleus neuro- frequency. We simulated this condition by elevating [Ca2/]i

by depolarization; the acceleration of spontaneous transmit-muscular junctions, mEPSP frequency was not affected byTeNT holotoxin. It is reasonable to assume that both evoked ter release caused by depolarization induced Ca2/ entry was

eliminated after TeNT-LC action (Fig. 6) . This type of resultand spontaneous transmitter release use VAMP similarlybecause both are reduced greatly by TeNT. However, in all is typical of many studies on effects of TeNT (except for

TeNT-expressing Drosophila) (Sweeney et al. 1995). Forstudies except that of Mellanby and Thompson (1972), somespontaneous transmitter release still occurred after TeNT instance, elevation of [Ca2/]i by Ca2/ ionophore could not

rescue transmitter release in TeNT-poisoned cultured hippo-poisoning.To reconcile the resistance of spontaneous release to campal neurons from rat. This contrasts with the observation

that the consequences of SNAP-25 cleavage by BoNT/ATeNT and BoNT/B in the crayfish claw-opener neuromus-cular junction (nmj) with the large reductions in frequency can be reversed partially by high-frequency stimulation or

other manoeuvres that increase [Ca2/]i (Capogna et al. 1997seen in other preparations, it is instructive to consider howthe rate of spontaneous transmitter release is controlled. and references therein) . The implication here is that TeNT

and BoNT/B cleave the VAMP, which is responsible forWe propose that, in all intact synapses, there is a basalrate of spontaneous transmitter release that is Ca 2/ inde- both evoked transmitter release and for any Ca2/-stimulated

spontaneous release. If resting [Ca2/]i is too low to havependent. In addition there is a Ca 2/ -dependent rate ofspontaneous release. Thus the total frequency of spontane- accelerated spontaneous release, then the frequency will not

be affected by cleavage of VAMP by TeNT or BoNT/B. Itous release in the intact synapse depends on the resting[Ca 2/ ]i , changes of which can occur if Ca 2/ influx, efflux, is not clear why the contribution of Ca2/-stimulated sponta-

neous release is extremely small relative to Ca2/-insensitiveor release are altered: i.e., spontaneous release Å basalCa 2/ -independent release / Ca 2/-dependent asynchro- spontaneous release in crayfish claw-opener nmjs, but this

could be due to an unusual relationship between [Ca2/]i andnous release.Evoked transmitter release is simply the synchronization transmitter release or could be due to the relative ease with

which metabolic requirements can be met in physiologicalof Ca2/-dependent asynchronous release due to a rapid in-crease in local [Ca2/]i caused by Ca2/ channel opening experiments. Crustacean synapses can be classified as tonic

or phasic; the former include the crayfish claw-opener syn-during an action potential. Under resting conditions, the Ca2/

permeability of nerve terminals may differ in different synapses and are characterized by low probability of release andgreat facilitation. Phasic synapses have a high probability ofapses; this will depend on the details of Ca2/-channel volt-

age dependence, channel modulators and the presynaptic release and low facilitation (Atwood and Wojtowicz 1986;Msghina et al. 1998). It will be interesting to determineresting potential, a variable that is affected easily by many

experimental conditions. When presynaptic resting Ca2/ whether different types of synapses in Crustacea all reactsimilarly to these toxins. Finally, the data indicate that theconductance is high, there will be a chronic Ca2/ influx

that will accelerate the rate of Ca2/-dependent spontaneous final steps in the exocytosis of synaptic vesicles do not re-quire elevation of [Ca2/]i (Quastel et al. 1971); spontaneousrelease. In these preparations, the frequency of spontaneous

release will be somewhat dependent on extracellular Ca2/ release is independent of [Ca2/]i increases after TeNT orBoNT/B.concentration. If for any reason [Ca2/]i is allowed to in-

crease sufficiently, then Ca2/ stimulation of release occursand the spontaneous release rate will be increased. For in- Different VAMP complexes for spontaneous and evokedstance, at the frog nmj, Erulkar et al. (1978) showed that releasestimulation in 0[Ca2/]o saline caused a reduction in sponta-neous release frequency, thus indicating that under normal One hypothesis for the persistence of spontaneous re-

lease after blockade of evoked release by TeNT and BoNT/conditions, some of the spontaneous release requires intra-cellular Ca2/ and that under a reversed Ca2/ gradient some B is that there was residual VAMP that could be cleaved

by these toxins but that survived intact for the lifetime ofof the Ca2/ left the presynaptic terminal. Furthermore, insome experiments the frequency of spontaneous release is these experiments. While we cannot test this hypothesis

directly, it predicts that, if VAMP is used identically forreduced by presynaptic Ca2/ channel blockers (Grinnell andPawson 1989; Losavio and Muchnik 1997), thus indicating both spontaneous and evoked release, then both types of

release should have declined together under attack bythat chronic Ca2/ influx can keep spontaneous release fre-quency elevated. In other preparations such as the crayfish TeNT or BoNT/B. Instead, the rates at which spontaneous

and evoked release are affected by TeNT and BoNT/B arenmj, resting Ca2/ entry may be negligible, and spontaneousfrequency would have little dependence on [Ca2/]o or very different.

BoNT/D, unlike TeNT and BoNT/B, blocked both[Ca2/]i may be regulated to such a low level that it doesnot normally accelerate the Ca2/ sensitive component and evoked and spontaneous transmitter release with about the

same time course. The most obvious explanation for thistherefore does not contribute to the total spontaneous releasefrequency. observation is that BoNT/D cleaves some VAMP necessary

for spontaneous release that is not easily cleaved by TeNTCleavage of VAMP by TeNT and BoNT/B evidentlyblocks evoked transmitter release by removing Ca2/ sensi- or BoNT/B. BoNT/D holotoxin also blocks both evoked

and spontaneous release at the frog nmj (Raciborska et al.tive release and thus reveals the basal Ca2/-insensitive re-lease rate. In those synapses in which the spontaneous release 1998). There are several ways in which differential VAMP


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cleavage could produce the disparate effects of these toxins. is reasonable to assume that the TeNT binding site is avail-able on the VAMP complex responsible for spontaneousFirst, there could be two vesicle populations, one that is

responsible for evoked release and contains a TeNT/BoNT/ release, and therefore it is the TeNT/BoNT/B cleavagesite that is occluded in that complex. In evoked releaseB-sensitive VAMP-like protein and a second population re-

sponsible for basal spontaneous release that has a TeNT/ under our experimental conditions, the binding and cleav-age sites for TeNT, BoNT/B, and BoNT/D are all exposedBoNT/B-resistant VAMP species. However, it is well estab-

lished that evoked release consists of one or more quantal to the toxins because they all block evoked release. Be-cause VAMP interacts with syntaxin and SNAP-25 in aevents identical to those in spontaneous release (Cooper et

al. 1995b; Dudel and Kuffler 1961; Katz 1969), and this domain that encompasses the binding and recognition sitesfor TeNT and BoNT/B (Fig. 1) (Hao et al. 1997; Hayashiindicates that both types of release use identical vesicles.

Although it is difficult to imagine how some vesicles at the et al. 1994) , such interactions might shield VAMP fromattack by TeNT or BoNT/B.active zone could receive a different protein population than

others, this possibility cannot be excluded. Alternatively Although our data cannot distinguish between two differ-ent VAMPs responsible for spontaneous and evoked releaseeach vesicle could have both types of VAMP. After the

present report was prepared, Deitcher et al. (1998) showed and two different complexes of one VAMP, it is clear thatthe data are inconsistent with one static VAMP complex thatthat in Drosophila (another arthropod) with a null mutation

in neuronal VAMP, spontaneous transmitter release still pro- is responsible for all transmitter release.ceeded at 25% of the normal rate, although, unlike most

We thank Dr. A. Zdanovsky for recombinant light chain of botulinumresults with TeNT (see review in Capogna et al. 1997), thetoxin type B, Dr. E. Habermann for isolated LC of tetanus toxin, and Dr.frequency could be increased with increased Ca2/ entry. TheH. Neimann for the LC constructs. We thank Dr. C. C. Shone for the anti-existence of spontaneous release in the absence of neuronal VAMP/synaptobrevin antibody and Dr. H. Bellen for the anti-synaptotag-

VAMP suggests that another VAMP isoform is responsible min antibody. We thank Dr. H. Atwood, Dr. R. Cooper, Dr. D. Elrick, andJ. Georgiou for suggestions on the manuscript.for remaining spontaneous release or that spontaneous re-

S.-Y. Hua and D. A. Raciborska were supported by a National Scienceslease does not require VAMP at all (Deitcher et al. 1998).and Engineering Research Council Grant and a Medical Research CouncilDrosophila has a neuronal VAMP sensitive to TeNT (DiAn-(MRC) Group Grant (Nerve Cells and Synapses) . A Neuroscience Network

tonio et al. 1993; Sweeney et al. 1995) and a ‘‘nonneuronal’’ Grant was awarded to M. P. Charlton. W. S. Trimble was supported by aform resistant to TeNT (Sudhof et al. 1989; Sweeney et al. MRC grant.

Address for reprint requests: M. P. Charlton,, Physiology Dept., MSB,1995) in which both the TeNT and BoNT/B binding sitesRm 3232, University of Toronto, 8 Taddle Creek Rd., Toronto, ON, M5Sare different from comparable regions of mammalian VAMP1A8 Canada.(Pellizzari et al. 1996, 1997; Sweeney et al. 1995). AlthoughE-mail: [email protected] ‘‘nonneuronal’’ form is not abundantly expressed in the

nervous system of Drosophila (Chin et al. 1993), it could Received 30 March 1998; accepted in final form 17 August 1998.be responsible for the spontaneous release that remains inthe null VAMP mutant and in Drosophila that express TeNT REFERENCES(Sweeney et al. 1995). Although we do not yet have the

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D. Temperature-sensitive aspects of evoked and spontaneous transmitteris resistant to both TeNT and BoNT/B and susceptible torelease at the frog neuromuscular junction. J. Physiol. (Lond.) 279: 253–BoNT/D. If such a molecule exists in crayfish, it must per-273, 1978.form differently from the Drosophila homolog because, un- BAUMERT, M., MAYCOX, P. R., NAVONE, F., DE CAMILLI, P., AND JAHN,

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