Journal of Physiology (1989), 411, pp. 195-205 195With 5 text-figuresPrinted in Great Britain

A STUDY OF SYNCHRONIZATION OF QUANTAL TRANSMITTERRELEASE FROM MAMMALIAN MOTOR ENDINGS BY THE USE OF

BOTULINAL TOXINS TYPE A AND D

BY J. MOLGO*, LYNN S. SIEGELt, NACIRA TABTI* AND S. THESLEFFtFrom the *Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS,

91190 Gif sur Yvette, France, tPathology Division, US Army Medical ResearchInstitute of Infectious Diseases, Fort Detrick, Frederick, MD 21701, USAand tDepartment of Pharmacology, University of Lund, S-223 62, Sweden

(Received 20 September 1988)

SUMMARY

1. The effects of botulinum toxin (BoTx) types A and D on spontaneous andevoked phasic transmitter release were studied in the isolated extensor digitorumlongus muscle of the rat or the levator auris longus muscle of mice.

2. The toxins were injected subcutaneously into the hindleg of adult rats or thedorsal aspect of the neck of mice. At various times after the injection the muscleswere removed from the anaesthetized animal and neuromuscular transmissionexamined in vitro by conventional intracellular techniques.

3. Both toxins reduced spontaneous transmitter release recorded as the frequencyof miniature end-plate potentials but BoTx type D was less effective in that respectthan the type A toxin.

4. With both toxins the block of evoked phasic transmitter release, recorded asend-plate potentials, was almost complete. As previously reviewed by Simpson(1986) the block produced by BoTx type A was partially reversed by procedureswhich elevate the intraterminal level of calcium ions. However, in BoTx type D-paralysed muscles such procedures failed to restore phasic transmitter release butcaused a period of high-frequency asynchronous transmitter release following eachnerve impulse.

5. To investigate if the lack of synchronization of evoked transmitter releaseobserved in BoTx type D-paralysed muscles was due to alterations in presynapticcurrents we examined, by perineural recordings, the Na+, fast K+, slow K+, K+-Ca2+-dependent and the Ca2+ currents in BoTx type D-paralysed muscles. These pre-synaptic currents were not altered as compared to unpoisoned controls.

6. We suggest that there exists a presynaptic process, which in addition to Ca2+influx participates in transmitter synchronization and which is a main target forBoTx type D action.

INTRODUCTION

Clostridium botulinum neurotoxins (BoTx) exist in several immunologicallydistinct forms, all of which have been shown to block neuromuscular transmission bypreventing transmitter release (for reviews see Habermann & Dreyer, 1986; Simpson,

7-2

J. MOLGO, L. S. SIEGEL, K. TABTI AND S. THESLEFF

1986; Dolly, Ashton, Evans, Richardson, Black & Melling, 1987; Sellin, 1987). Theseneurotoxins are extremely potent and a study of their mode of action could beexpected to provide valuable information regarding the transmitter release process.

In the present study we compared the effects of BoTx type D (BoTx-D) with BoTxtype A (BoTx-A) on spontaneous and on evoked quantal transmitter release at themammalian neuromuscular junction. Some of the results have been published inabstract form (Molgo, Tabti & Thesleff, 1988).

METHODS

The experiments were performed on either the isolated extensor digitorum longus (EDL) or onthe levator auris longus (LAL) muscle (Angaut-Petit, Molg6, Connold & Faille, 1987) removedunder diethylether anaesthesia from adult male Sprague-Dawley rats (120-180 g) or adult femaleNMRI mice (20-25 g) respectively.Botulinum toxin types A and D were used. Clostridium botulinum type A toxin was the same as

that previously used in several studies (Cull-Candy, Lundh & Thesleff, 1976; Sellin & Thesleff,1980). Clostridium botulinum type D, strain 1873, was grown in the medium described by Miyazaki,Iwasaki & Sakaguchi (1977), which consisted of 2-0% peptone, 05% yeast extract, 05% glucoseand 0-025% sodium thioglycolate, pH 7 0. The culture was incubated at 30 °C for 5 days. Toprecipitate the toxin from the culture fluid, 0-02% (w/v) RNA was added and the pH adjusted to3-5 with 3 N-H2504 (Miyazaki et al. 1977). The precipitate was collected by centrifugation and thetoxin extracted with 0-2 M-phosphate buffer at pH 6-0. The extract was dialysed against 0-2 M-succinate, pH 5-5. After dialysis, the preparation was tested for toxicity in mice and it containedapproximately 103 LD5O/ml.Both toxins were dissolved in phosphate buffer as described by Ambache (1949) and injected

subcutaneously close to the muscle as a single dose of 0-25 ml for rats or 005 ml for mice. The0 05 ml doses used corresponded to about 1 mouse LD50 for BoTx-A and for BoTx-D. The 0-25 mldose was equivalent to about 5 mouse LD50 for BoTx-A and 50 LD50 for BoTx-D. The doses oftoxins used produced a localized muscle paralysis in the area of injection with little or nodisturbance in the general condition of the animals. At various times after injection, usually at 2or 5 days, the muscles and their motor nerves were removed and placed in an organ bath. Thenormal Krebs-Ringer solution had the following composition (in mM): NaCl, 151; KCI, 5; MgCl2,1; CaCl2, 2; glucose, 11; HEPES buffer, 5 (pH = 7 3). The solution was bubbled with pure 02. Insome experiments we used bicarbonate-phosphate buffer bubbled with 95% 02 and 5% CO2 givingit a pH of 7-2. When changes were made in the ionic composition of the bathing solution, osmolaritywas maintained by changing NaCl concentration. Hypertonicity was induced by the addition of150 mM-sucrose to the standard solution. Unless otherwise stated the experiments were performedat 30+0 5 °C.

In a few experiments a freeze-dried crude homogenate of venom glands of Lactrodectus mactanstredecimguttatus was dissolved in physiological saline and added to the bathing solution. Theoptimal amount of the toxin was considered that which in normal muscle, within a few minutes,caused a massive quantal release of acetylcholine recorded as miniature end-plate potentials(MEPPs) at frequencies above 100 Hz.

Resting membrane potentials, MEPPs and end-plate potentials (EPPs) were recorded byintracellular glass capillary microelectrodes, filled with 2 M-potassium citrate, of 4-8 MQ resistance.The nerve was stimulated with supramaximal current pulses of 0 05-0 1 ms duration through asuction electrode at frequencies indicated in the Results.

Synaptic potentials were amplified and stored on videotape with the aid of a modified digitalaudio processor (Sony PCM 701 ES) and a video-recorder. The data were subsequently analysedwith the aid of a microcomputer.

Presynaptic currents were elicited by nerve stimulation and recorded at high-resolution visualcontrol from the perineural space of small superficial nerve bundles supplying several end-plates inthe LAL muscle. A glass microelectrode filled with 2 M-NaCl (resistance 15-20 MO) was used forrecording and an Ag-AgCl electrode as reference. By this method both the sodium component andthe conductance changes at the nerve terminals are detected (Gundersen, Katz & Miledi, 1982;Mallart, 1985). The latter currents appear with reversed polarity when recorded at the perineural

196

BOTl 'Ll\AL TO.XINS AND TRA.VMAIITTER RELEASE

site (outwar(l potassium currents as downward, inward calcium currents as upward deflections).After (onxventional arnplification the signals were displayed on a storage oscilloscope. TheseexpIeriments were ma(le at 20-22 0(1. Because of the neuromuscular block caused by BoTx-D it wastunneessarv to use curare as in normal preparations. Procaine hydrochloride was occasionally usedto p)revent spontaneous repetitive nerve firing in the presence of tetraethylammonium.

In a few instances the LAI, muscle was fixed and stained as a whole-mount preparation using thesilver impregnation method (see Angaut-Petit, Mallart & Faille, 1982).

D)rucs utise( were: tet rodotoxin. 3.4-diaminopyridine (Sigma. USA), procaine hydrochloride(Specia. France). tetraethylammonium bromide (BDH. UK). All salts were of analytical grade.

Statistical analysis of data was performned using Student's t test (two-tailed). Values areexl)resse(l as mean +S. E.M.: data were considered significanit at P < 005.

RESULTS

Effects onl spontaneous quantal transmitter releaseIn norinal (untreated) EDL junctionis spontaneous quantal transmitter release

recorded as MEPP frequency in a normal Krebs-Ringer solution was 29 + 03 s-'(forty-three junctions from seven muscles). In both BoTx-A- and BoTx-D-poisonedjunctions MEPP fre(ueincy was drastically reduced. However, as shown in Fig. IA,the effect of BoTx-D was less marked than that of BoTx-A.

In the early stages of poisoning with both toxins (2-5 days) the majority ofspontaneous MEPPs were of small amplitude; however, at later stages larger thanniormal AIEPPs appeared. Those MEPPs were frequently characterized by a slowtime course as previously described for BoTx-A (Cull-Candy et al. 1976; Kim, Lomo,Lupa & Thesleff, 1984). Since they apparently represent a calcium-insensitive formof quantal ACh release (Thesleff, 1986) we preferred to study transmitter releasebefore the appearance of these MEPPs, i.e. during the period of 2-5 days after toxininjection.As shown in Fig. IA and B, junctions paralysed by BoTx-A or BoTx-D responded

in a qualitatively similar way to procedures that increase MEPP frequency.Elevation of the extracellular calcium concentration from 2-0 to 8-0 mm had littleeffect on MEPP frequencies in both types of paralysed muscles as did nerve terminaldepolarization by high K+ (15-20 mM). Similarly, hypertonicity (150 mM-sucrose)failed to markedly affect MEPP frequencies (Fig. IA). Substitution of extracellularcalcium bv 01-1 mM-lanthanum, however, caused a marked increase in MEPPfrequency in both types of poisoned junctions (Fig. 1B).

Black Widow spider venom, whose major component is a-latrotoxin, inducedwithin minutes of its application to BoTx-A- and to BoTx-D-paralysed muscles amassive release of acetyleholine quanta recorded as MEPP frequencies further above100 Hz. Thus, this toxin enhanced transmitter release in both types of BoTx-paralysed muscles to about the same extent as in normal muscle.

Effects of nerve stimulation on quantal transmitter releaseIn both types of paralysed muscles nerve stimulation at low frequencies (0 1-1 Hz)

failed to induce phasic transmitter release. In BoTx-A-poisoned junctions increasedstimulation frequency (up to 30 Hz), elevated extracellular calcium (up to 8 mM) orblockade of presyna)tic potassium channels by 10-100 /tM- 3,4-diaminopyridine (3,4-DAP) and/or 1-15 mMv-tetraethylammonium (TEA) reduced or abolished failures of

197

198 J. MOLG6, L. S. SIEGEL, N. TABTI AND S. THESLEFF

2-o A 50 8

41-2

401.5

_..ii........i,i,;)- i1 .....,jj30

5T

231.0. 5! ...-._l...iO , . , O5,.

0~~~~~ ~72.L mM- 8 mM- 20 mM- 150 mM 20

340.5

105~~iIj ~5 5

0 2 mm 8mm 20mm 150 mm- 0.l mrvi-lOmm-Ca2+ Ca2+ K+ sucrose La3+ La3+

Fig. 1. The diagram illustrates mean+S.E.M. changes in MEPP frequency in BoTx-A-(open bars) and BoTx-D- (stippled bars) paralysed muscles fibres. The changes are causedby increasing the extracellular calcium concentration from 2 to 8 mm, by 20 mM-K+, byhypertonic solution (A) and by lanthanum 0-1 and 1 mm (B). Note the different scales inA and B. In high K+ and in the hypertonic solution 2 mM-calcium was present while in thepresence of lanthanum no calcium was added. The number above each bar denotes thenumber of junctions investigated.

evoked release, increased the amplitude of EPPs (see Fig. 3) and restoredneuromuscular transmission.

In BoTx-D-poisoned junctions, all the aforementioned procedures failed to affectevoked phasic transmitter release which remained almost completely blocked.However, as shown in Fig. 2, a high frequency of nerve stimulation (10 Hz) andfurther addition of 3,4-DAP (10 ,UM) markedly enhances MEPP frequency.At high external Ca2+ concentration (8 mM), only occasionally did nerve stimuli in

the presence of 3,4-DAP (100 /tM) and/or TEA cause the summation of a few quantaas illustrated in Fig. 3. Increased stimulation frequency under these conditionscaused a period of high-frequency asynchronous quantal transmitter release aftereach nerve impulse. These results are in marked contrast to those obtained withBoTx-A, in which nerve stimulation in the presence of 3,4-DAP mainly caused anincrease in phasic transmitter release, as illustrated by the recordings in Fig. 3.

Cooling the BoTx-D-paralysed preparation from 30 to 20 °C further reduced thespontaneous frequency of MEPPs. However, stimulation of the nerve (1 and 10 Hz),particularly in the presence of 10 ,tM-3,4-DAP, still increased MEPP frequency toabout the same extent as at 30 'C. After a period of nerve stimulation at 10 Hz,

BOTULINAL TOXINS AND TRANSMITTER RELEASE

30

8

20

c_j

10

0)

0 10 10 Hz

Fig. 2. The diagram illustrates changes in mean MEPP frequency in BoTx-D-paralysedjunctions during nerve stimulation at 10 Hz before (open bar) and afterthe addition of 10 4Um-3,4-DAP (stippled bar) to the normal Krebs-Ringer solution. Mean values for MEPPfrequency in unstimulated poisoned junctions are also shown. The number above each barindicates the number of junctions recorded.

MEPP frequency declined towards its low prestimulation value within a few secondsat 30 'C while the elevated frequency lasted for 30-60 s at 20 0C.

Silver impregnation studies on BoTx-D-paralysed muscles revealed that the grossmorphology of the end-plate was normal and no signs of nerve sprouting wereobserved at times when the blockade of phasic transmitter release and theappearance of asynchronous release in response to nerve stimuli were prominent.The marked differences on evoked phasic transmitter release between BoTx-D and

BoTx-A could have presynaptic origins such as: (i) alterations in presynapticcurrents or (ii) changes in the process of synchronization of quantal release.

Presynaptic currents

Presynaptic currents were recorded in the LAL muscle of the mouse after theapplication of 1 mouse LD50 of BoTx-D. This amount of toxin caused within 24 hcomplete paralysis of the muscle and made the use of curare to block neuromusculartransmission unnecessary even in the presence of potassium channel blockers. Thisis in contrast to normal muscle and to BoTx-A-paralysed muscles where under thoseconditions curare (20 /sm) always is required to block transmission.

It is worth noting that curare (20 ,tm) did not induce any noticeable effect on therecorded presynaptic currents if added to BoTx-D-poisoned preparations.

199

J. MOLGO, L. S. SIEGEL, -N. TART! AND S. THE'SLEFF

A1 2

\ A AmV i2 mVt \ i ~~~~10ms 10 Ms

B1 2

1 mV

~~~~ ~~20 ms;

Fig. 3. Intracellular recordings obtained from two differetnt nerve-ED)L, musclepreparations poisoned with BoTx-A (A) or BoTx-1) (B). Tracings in A. I andl 2. showsuperimposed EPPs recorded following nerve stimulation at 1 and( 10 Hz respectively.The Krebs-Ringer solution contained 8 mM-Ca2+ ,3,4-l)AP (10 /IM) was present in .4 2.Notice the synchronized EPPs an(d the absence of failures of release in.4 2 (arrows ini(ficatestimuli artifacts). Tracings in B were obtained durinig 1 (1) and 5 Hz (2) nerve stimulationlin a medium containing 8 rmM-Ca2' and 100 pM-3,4-DJAP. Notice that even in theseconditions phasic EPPs remain of lower amplitudle an(1 are followed by a high rate ofasynchronous M EPPl1s. Calibratiot in B appflies to I atn(d 2.

Figure 4 shows representative perineural recordings effected in a normal ionicmedium in unpoisoned (AI) and in BoTx-D-poisoned (Bi) preparations. The signalsdid not differ from one another and similar results were obtained in six differentBoTx-D-poisoned neuromuscular preparations. The two negative deflections reflectthe Na+ current ('Na) followed by a fast, voltage-dependent outward potassiumcurrent (IK,,) generated at the nerve endings (Mallart, 1985; Dreyer & Penner,1987).

It is possible to further dissect the recorded presynaptic signals into individualcurrents by the use of different potassium channel blockers. The application to thepreparation of 0 1-0 5 mM-3,4-DAP blocked the fast, voltage-dependent outwardpotassium current ('Kr) in the nerve endings revealing a calcium-dependentpotassium current ('K(ca)) (Mallart, 1985). As shown by Fig. 4A2 and B2 IK((.a) coUldbe elicited in poisoned preparations with no difference from normal.As has been reported by Dreyer & Penner (1987), blockade of both IK4r and 'K(ca)

by TEA (10-30 mM) reveals a short-lasting calcium current (Fig. 4A3). The calciumcurrent (ICa) was enhanced and prolonged to a plateau by 3,4-DAP (0A1-0 2 mM)which blocked a slow, voltage-dependent potassium current ('Ks Fig. 4A4). Asillustrated by the recordings in Fig. 4B3 and B4 short- and long-lasting 'Ca could beelicited as in normal unpoisoned preparations. In conclusion, we have not been ableto show that BoTx-D poisoning affects the presynaptic currents: I a IK r, 'K,s IK(C a)and 'ca*

200

BOTULINAL TOXINS AND TRANSMITTER RELEASE

A2

4

40 ms

B

1 2 _

2 mV

3

iIS

.'.i,.

4 ms

4

1

10 ms

Fig. 4. Presynaptic currents recorded from the perineural space in normal (A) and BoTx-D-poisoned nerve-LAL muscle preparation (B). In A, 1 illustrates recordings made innormal Krebs-Ringer solutions showing the sodium- (INa) and the voltage-dependentpotassium (IK..) components; 2 shows the calcium-dependent K+ current IK(ca) revealedafter the addition of 0 5 mM-3,4-DAP (external calcium, 2 mM); 3, the upward deflectionsignals the calcium current (ICa) recorded in the presence of 10 mM-TEA; 4, calciumplateau response induced by combined application of TEA (10 mM) and 3,4-DAP(100,M). In 3 and 4, procaine (100,UM) was added to avoid repetitive firing occurringunder these conditions. In B, tracings 1-4 were obtained under the same conditions as

in A (1-4), except for curare which was not necessary and therefore not used.

In order to check the presynaptic conduction of action poteiti-als, the nerve was

stimulated at three different frequencies (1, 10 and 50 Hz) as shown by perineuralrecordings in Fig. 5. In BoTx-D-poisoned preparations nerve conduction was wellmaintained and the terminals were normally invaded by action potentials both innormal Krebs-Ringer solution as well as in a solution containing 3,4-DAP (0 5 mm)and followed the frequencies of nerve stimulation. A feature to be mentioned was thedecrease of IK(Ca) which occurred when stimulating at a frequency of 50 Hz. The

201

3

1

J. MOLGO, L. S. SIEGEL, N. TABTI AND S. THESLEFF

A -

1 Hz 10 Hz

2

4 ms

50 Hz

40 ms

B C

Fig. 5. Presynaptic currents recorded during repetitive nerve stimulation at differentfrequencies in BoTx-D-poisoned LAL muscles. A, in normal Krebs-Ringer solution; B, inthe presence of 0-5 mM-3,4-DAP and 6 mM-calcium.

decrease presumably resulted from the voltage-dependent action of 3,4-DAP whichreduces the potassium channel block during high rates of stimulation (Kirsch &Narahashi, 1978).

DISCUSSION

The present results show that BoTx-A and BoTx-D block spontaneous quantaltransmitter release recorded as MEPPs. However, the blocking effect of BoTx-Dseems to be less efficient than that of BoTx-A since the frequency of spontaneousMEPPs in paralysed muscles is about 10 times higher after BoTx-D poisoning.Furthermore, procedures which elevated the intracellular Ca2+ concentration orcations which increase transmitter release are less effective in enhancing MEPPfrequency in BoTx-A-poisoned junctions than in BoTx-D-poisoned ones.The opposite is, however, true when considering the effects of the two toxins on

stimulus-evoked phasic transmitter release. In that instance BoTx-D causes acomplete blockade of release which could not be restored by increasing phasic Ca2+entry into the nerve terminal. BoTx-A also blocks phasic release but the block isreadily overcome by enhanced Ca2+ entry. Thus, it seems that BoTx-D selectivelyblocks synchronous phasic transmitter release while both toxins reduce spontaneousquantal transmitter release.A notable feature in BoTx-D-poisoned junctions was the observation that nerve

stimulation (1-30 Hz) evoked a period of high-frequency asynchronous quantaltransmitter release after each stimulus. Similar results have been described in frogneuromuscular junctions by Harris & Miledi (1971). An enhanced probability ofrelease has also been observed at normal unpoisoned junctions (Zengel & Magleby,1981).

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BOTULINAL TOXINS AND TRANSMITTER RELEASE

The possibility that blockade of phasic transmitter release and subsequentappearance of asynchronous quantal release in BoTx-D-poisoned junctions reflectsalterations in presynaptic currents was investigated. No such alterations were,however, observed and it is notable that calcium-activated potassium currents, andcalcium currents, are similar to normal controls and to BoTx-A-paralysed junctions(Gundersen et al. 1982; Dreyer, Mallart & Brigant, 1983; Angaut-Petit, Molgo &Thesleff, 1988). Furthermore, nerve conduction was not affected and spontaneousnerve firing between stimuli not observed in BoTx-D-poisoned preparations.Morphological examination revealed no signs of nerve sprouting nor of other changesthat might affect transmitter release. Consequently, it appears that BoTx-D blocksa specific intraterminal process involved in the synchronization of transmitterquanta without preventing Ca2+ entry into the motor nerve terminals.

Similar effects on evoked phasic transmitter release have previously been reportedfor muscles paralysed by tetanus toxin (Dreyer & Schmitt, 1983), BoTx type B(Sellin, Thesleff &_ DasGupta, 1983; Gansel, Penner & Dreyer, 1987) and for BoTxtype F (Kauffman, Way, Siegel & Sellin, 1985). In those studies, however, except fortetanus toxin (Dreyer et al. 1983) presynaptic currents were not investigated.

Little is known about the mechanisms by which Ca2+ influx following a nerveimpulse triggers synchronous transmitter release. Our results indicate that a specialmechanism exists for transmitter quanta synchronization and that this mechanismis the main target for BoTx-D action.

It is interesting that BoTx-D but not BoTx-A has been shown to ADP-ribosylatea 21-24 kDa protein (Ohashi & Narumiya, 1987) and this effect has been related toits blocking action since it parallels the inhibition of noradrenaline release in adrenalmedulla secretory cells (Adam-Vizi & Knight, 1987). Recent studies by Rdsener,Chhatwal & Aktories (1987) indicate that the ADP-ribosylation observed is not theresult of BoTx-D but caused by the action of a botulinum ADP-ribosyltransferase C3which was present in BoTx-D. We do not know if the BoTx-D preparation used inour study contains this ADP-ribosyltransferase. However, a recent report by Adam-Vizi, Aktories, Knight & Rosener (1988) indicates that inhibition of transmitterrelease and ADP-ribosylation are separate unrelated events.Lack of detectable ADP-ribosylation in brain synaptosomal preparations in which

transmitter release was abolished by BoTx types A and B has been recently reportedby Ashton, Edwards & Dolly (1988). It should be mentioned that tetanus toxin andBoTx types B and F which apparently affect phasic transmitter release similarly toBoTx-D have so far not been shown to have ADP-ribosylating activity.

In conclusion, BoTx-D and BoTx-A both reduce the calcium sensitivity of thetransmitter process. BoTx-D in addition blocks a presynaptic step beyond the influxof calcium that may be a part of the mechanism by which synchronization of phasictransmitter release is achieved.

This study was supported in part by grants from Direction des Recherches Etudes et Techniques(85/017; 88/096) and the Swedish Medical Research Council, Stockholm (14X-3112). N.T. wassupported by Association Francaise contre les Myopathies. We wish to thank Ms B. Hansson andMs L. Faille for excellent technical assistance.

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204 J. MOLGO, L. S. SIEGEL, N. TABTI AND S. THESLEFF

REFERENCES

ADAM-VIZI, V., AKTORIES, K., KNIGHT, D. E. & R6SENER, S. (1988). Botulinum toxin inducedADP-ribosylation and inhibition of catecholamine secretion may be unrelated events. Journal ofPhysiology 403, 107P.

ADAM-VIZI, V. & KNIGHT, D. E. (1987). Does botulinum toxin type D inhibit exocytosis by ADP-ribosylation? Journal of Physiology 394, 96P.

AMBACHE, N. (1949). The peripheral action of Clostridium botulinum toxin. Journal of Physiology108, 127-141.

ANGAUT-PETIT, D., MALLART, A. & FAILLE, L. (1982). Role of denervated sheaths and end-platesin muscle reinnervation by collateral sprouting in the mouse. Biology of the Cell 46, 277-290.

ANGAUT-PETIT, D., MOLGO, J., CONNOLD, A. L. & FAILLE, L. (1987). The levator auris longusmuscle of the mouse: a convenient preparation for studies of short- and long-term presynapticeffects of drugs or toxins. Neuroscience Letters 82, 83-88.

ANGAUT-PETIT, D., MOLG6, J. & THESLEFF, S. (1988). Presynaptic study of frog neuromuscularjunctions in vitro poisoned with botulinum A toxin. Journal of Physiology 406, 59P.

ASHTON, A. C., EDWARDS, K. & DOLLY, J. 0. (1988). Lack of detectable ADP-ribosylation insynaptosomes associated with inhibition of transmitter release by botulinum neurotoxin A andB. Transactions of the Biochemical Society 16, 883-884.

CULL-CANDY, S. G., LUNDH, H. & THESLEFF, S. (1976). Effects of botulinum toxin on neuro-

muscular transmission in the rat. Journal of Physiology 260, 177-203.DOLLY, J. 0., ASHTON, A. C., EVANS, D. M., RICHARDSON, P. J., BLACK, J. D. & MELLING, J.

(1987). Molecular action of botulinum neurotoxins: role of acceptors in targetting to cholinergicnerves and in the inhibition of the release of several transmitters. In Cellular and Molecular Basisof Cholinergic Function, ed. DOWDALL, M. J. & HAWTHORNE, J. N., pp. 517-533. Chichester: EllisHorwood.

DREYER, F., MALLART, A. & BRIGANT, J. L. (1983). Botulinum A toxin and tetanus toxin do notaffect presynaptic membrane currents in mammalian motor nerve endings. Brain Research 270,373-375.

DREYER, F. & PENNER, R. (1987). The action of presynaptic snake toxins on membrane currentsof the mouse motor nerve terminals. Journal of Physiology 386, 455-463.

DREYER, F. & SCHMITT, A. (1983). Transmitter release in tetanus and botulinum A toxin-poisonedmammalian motor endplates and its dependence on nerve stimulation and temperature. PfuiigersArchiv 399, 228-234.

GANSEL, M., PENNER, R. & DREYER, F. (1987). Distinct sites of action of clostridial neurotoxinsrevealed by double poisoning of mouse motor nerve terminals. Pfliigers Archiv 409, 533-539.

GUNDERSEN, C. B., KATZ, B. & MILEDI, R. (1982). The antagonism between botulinum toxin andcalcium in motor nerve terminals. Proceedings of the Royal Society B 216, 369-376.

HABERMANN, E. & DREYER F. (1986). Clostridial neurotoxins: Handling and action at the cellularand molecular level. Current Topics in Microbiology and Immunology 129, 93-179.

HARRIS, A. J. & MILEDI, R. (1971). The effect of type D botulinum toxin on frog neuromuscularjunctions. Journal of Physiology 217, 497-515.

KAUFFMAN, J. A., WAY JR, J. F., SIEGEL, L. S. & SELLIN, L. C. (1985). Comparison of the actionof types A and F botulinum toxin at the rat neuromuscular junction. Toxicology and AppliedPharmacology 79, 211-217.

KIM, Y. L., LOMO, T., LUPA, M. T. & THESLEFF, S. (1984). Miniature end-plate potentials in ratskeletal muscle poisoned with botulinum toxin. Journal of Physiology 356, 587-599.

KIRSCH, G. E. & NARAHASHI, T. (1978). 3,4-Diaminopyridine: a potent new potassium channelblocker. Biophysical Journal 22, 507-511.

MALLART, A. (1985). Electric current flow inside perineurial sheaths of mouse motor nerves.Journal of Physiology 368, 565-575.

MIYAZAKI, S., IWASAKI, M. & SAKAGUCHI, G. (1977). Clostridium botulinum type D toxin:purification, molecular structure, and some immunological properties. Infection and Immunity17, 395-401.

MOLG6, J., TABTI, N. & THESLEFF, S. (1988). Is there a presynaptic process, in addition to Ca2linflux, which allows synchronization of transmitter release at the rodent neuromuscularjunction? Journal of Physiology 406, 62P.

BOTULINAL TOXINS AND TRANSMITTER RELEASE 205

OHASHI. Y. & NARUMIYA, S. (1987). ADP-ribosylation of a Mr 21000 membrane protein by typeD botulinum toxin. Journal of Biological Chemistry 262, 1430-1433.

ROSENER, S.. CHHATWAL, G. S. & AKTORIES, K. (1987). Botulinum neurotoxins Cl and D ADP-ribosylates low molecular mass GTP-binding proteins. Federation of European BiochemicalSocieties Letters 224, 38-42.

SELLIN, L. C. (1987). Botulinum toxin and the blockade of transmitter release. Asia Pacific Journalof Pharmacology 2, 203-222.

SELLIN, L. C. & THESLEFF, S. (1981). Pre- and post-synaptic actions of botulinum toxin at the ratneuromuscular junction. Journal of Physiology 317, 487-495.

SELLIN, L. C., THESLEFF, S. & DASGUPTA, B. R. (1983). Different effects of type A and B botulinumtoxin on transmitter release at the neuromuscular junction. Acta physiologica scandinavica 119,127-133.

SIMPSON, L. L. (1986). Molecular pharmacology of botulinum toxin and tetanus toxin. AnnualReview of Pharmacology and Toxicology 26, 427-453.

THESLEFF, S. (1986). Different kinds of acetylcholine release from the motor nerve. InternationalReview of Neurobiology 28, 59-88.

ZENGEL, .J. E. & MAGLEBY, K. L. (1981). Changes in miniature endplate potential frequency duringrepetitive nerve stimulation in the presence of Ca2", Ba2+, and Sr2" at the frog neuromuscularjunction. Journal of General Physiology 77, 503-529.