J. Physiol. (1987), 386, pp. 475-484 475With 6 text-ftguresPrinted in Great Britain

THE EFFECTS OF IN VITRO APPLICATION OF PURIFIED BOTULINUMNEUROTOXIN AT MOUSE MOTOR NERVE TERMINALS

BY J. 0. DOLLY*, S. LANDE AND D. W.-WRAYFrom the Pharmacology Department, Royal Free Hospital Medical School, RowlandHill Street, London NW3 2PF and *Biochemistry Department, Imperial College of

Science and Technology, South Kensington, London SW7 2AZ

(Received 23 July 1986)

SUMMARY

1. Purified botulinum neurotoxin type A (10 nM) was applied in vitro to mousediaphragm muscles. Intracellular micro-electrode recordings were made continuouslyin single fibres.

2. This treatment reduced end-plate potential (e.p.p.) amplitudes with a time tohalf-maximal effect of about 75 min at 22-25 'C. E.p.p. rise-times remained fast andunaffected by the toxin.

3. Miniature end-plate potential (m.e.p.p.) frequency was reduced by the toxin toless than 5% of control frequency, and followed a similar time course to the blockof e.p.p. amplitudes. The m.e.p.p. rise-time and coefficient of variation (c.v.) ofm.e.p.p. amplitude distributions both increased, but the time course ofthese increaseslagged significantly behind the change in frequency.

4. A population of slow rise-time m.e.p.p.s was present in controls at lowfrequency. This population was found to be unaffected by the toxin.

5. The above-detailed in vitro changes could be explained by the toxin acting bya single common mechanism to inhibit the release process underlying both fastrise-time m.e.p.p.s and e.p.p.s. A distinct release process, which leads to slowrise-time m.e.p.p.s, was unaffected by the toxin.

INTRODUCTION

Botulinum neurotoxin (BoNT) acts specifically and irreversibly at the skeletalneuromuscular junction to inhibit spontaneous and evoked acetylcholine (ACh)release (Boroff, del Castillo, Evoy & Steinhardt, 1974; Cull-Candy, Lundh & Thesleff,1976; Simpson, 1981; Tse, Dolly, Hambleton, Wray & Melling, 1982). The toxinappears to achieve this effect by initially binding to specific acceptor sites on themotor nerve terminal membrane followed by internalization (Dolly, Black, Williams& Melling, 1984). Inside the terminal, the toxin, or a part of it, acts on some target(as yet undefined) to cause blockade of ACh release (Simpson, 1980). BoNT does notaffect either propagation of action potentials along nerve terminals (Harris & Miledi,1971) or presynaptic voltage-dependent Ca2+ currents (Gundersen, Katz & Miledi,1982; Dreyer, Mallart & Brigant, 1983; Molgo & Thesleff, 1984).

J. 0. DOLL Y, S. LANDE AND D. W.- WRA Y

In the initial stages of BoNT poisoning, miniature end-plate potentials (m.e.p.p.s)greatly decrease in frequency. The rise-times of the remaining botulinized m.e.p.p.sare slower than in unpoisoned muscles and their amplitude distribution is skewedrather than the normal bell-shape (Harris & Miledi, 1971; Cull-Candy et al. 1976;Kriebel, Llados & Matteson, 1976; Tse et al. 1982). The frequency of these slowrise-time m.e.p.p.s in botulinized muscle is relatively unaffected by most procedureswhich are known to enhance ACh release (Thesleff & Molgo, 1983). Furthermore, apopulation of slow rise-time m.e.p.p.s is also present in unpoisoned muscles at verylow frequency (Kim, Lomo, Lupa & Thesleff, 1984). The quantal content of thenerve-evoked end-plate potential (e.p.p.) is also greatly reduced by the toxin(Cull-Candy et al. 1976). However, e.p.p.s have normal rise-times in both control andbotulinized muscles.The majority of the above-mentioned studies have examined the chronic effects

of the botulinum neurotoxin-haemagluttinin complex on muscles of animals whichhave been injected locally with a non-lethal dose and then examined some days later.Here we have used in vitro application of purified neurotoxin on the mousediaphragm, so avoiding the complication of denervation-like effects appearing (Sellin& Thesleff, 1981). Furthermore, detailed studies of the time course of the effects ofthe toxin (continuously followed in single end-plates) provide information on themechanism of action of the toxin.

Preliminary results have been communicated to the Physiological Society (Dolly,Lande & Wray, 1985).

METHODS

BKTO mice (20-25 g) were used in all experiments. After cervical dislocation, the hemi-diaphragm with the phrenic nerve was removed and transferred to oxygenated (95% 02+ 5% C02)Krebs solution of the following composition (mM): Na+, 143-0; K+, 5.9; Mg2+, 1-2; Ca2+, 2-5; Cl-,127*7; HC03-, 25*0; H2P04-, 1.2; S042-, 1-2; glucose, 11-1; pH, 7-1-7-4; temperature, 22-25 'C.BoNT type A (10 nM), purified to homogeneity as previously described (Tse et al. 1982), wasapplied in the bathing solution for 10 min, followed by perfusion with toxin-free Krebs solution.Continuous intracellular voltage recordings of m.e.p.p.s and e.p.p.s were made using glass

micro-electrodes filled with 3 M-KCl (resistance 10-20 MQ) as previously described (Wray, 1981).E.p.p.s and m.e.p.p.s were recorded on magnetic tape and analysed by computer after digitizingat 10 kHz. End-plates were localized by finding m.e.p.p.s or e.p.p.s with fast rise-times (< 1 ms).Only fibres with resting potentials at all times more negative than -60 mV were used for recordings.If the membrane potential changed by more than 5 mV during the recording, the fibre was rejected.When recording e.p.p.s, tubocurarine was added in the perfusing solution at a concentration(4-3 gM) sufficient to inhibit contraction. The nerve was stimulated at very low frequency(1 pulse/min), so as not to influence significantly the time course of the toxin block (Hughes &Whaler, 1962). When stimulation failed to elicit an e.p.p., the tubocurarine was washed out andrecordings of e.p.p.s were made at 0 5 Hz. Quantal content was then calculated by the failuresmethod (del Castillo & Katz, 1954).

Rise-times calculated by computer were taken as the time taken for the m.e.p.p. or e.p.p. to risefrom 20 to 80% of its full amplitude. M.e.p.p. amplitude distributions were obtained for each fibre,and the coefficient of variation (c.v.) calculated from c.v. = (standard deviation)/mean.

Initial values, before the toxin had begun to act, were used as controls. Test and control meanvalues were compared using two-tailed Student's t test. Paired t tests were used wherever possible.The Kolmogorov-Smirnov and Mann-Whitney tests were used to compare amplitude and rise-timedistributions. Mean +S.E. of the mean are quoted.

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IN VITRO ACTION OF BOTULINUM NEUROTOXIN 477

RESULTS

Examples of e.p.p.s are shown in Fig. 1 while Fig. 2 illustrates the time course ofthe typical effect of BoNT on nerve-evoked release for a single end-plate. Duringcontinuous recordings of e.p.p.s the mean resting potential before and after the actionof the toxin was 77 + 2 and 75 + 2 mV respectively (n = 5 end-plates). After an initial

A B

1 mvl Ii1mH10 ms

Fig. 1. Examples of e.p.p.s recorded in a control diaphragm muscle (A, 4-3 gM-tubocurarine) and after BoNT (B, 10 nM), applied as described in Methods (no tubo-curarine present). Note the failures to nerve stimulation after BoNT, reflectinga decreased quantal content. The mean quantal content in poisoned muscles was0 032 + 0 007 (n = 6 end-plates).

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Fig. 2. Continuous recording of e.p.p.s in a single fibre following application of BoNT(10 nM). The nerve was stimulated once per minute. The Figure shows amplitude (A) andrise-time (B).

-

J. 0. DOLL Y, S. LANDE AND D. W.- WRA Y

A B

2 ms 2 ms

Fig. 3. Examples of m.e.p.p.s recorded in a control muscle (A) and after BoNT (B, 10 nM)in the same fibre.

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Fig. 4. Time course of action of BoNT (10 nM) during continuous recording of m.e.p.p.sin a single fibre. Frequency (A), coefficient of variation (c.v., B) and rise-time (C). Meanand standard errors are shown for each point (vertical bars) as well as the analysis period(horizontal bars). The continuous curve for frequency shown in A was drawn by eye andthis curve was used to obtain the predicted curves (see Results) shown in B and C.

478

IN VITRO ACTION OF BOTULINUM NEUROTOXIN

delay following BoNT application (mean delay 610 + 29 min, n = 5) the e.p.p.amplitude decreased, and at 89-8 + 3-8 min no further e.p.p.s could be detected. Themean time taken for the e.p.p. amplitude to fall by half (ti) was 75-3 + 3-7 min (n = 5).E.p.p. rise-times were unaffected by the toxin (Figs. 1 and 2); the mean rise-timebefore the toxin had begun to act was 0-44 + 001 ms (n = 5) while the rise-time was0-42 + 0 03 ms when the e.p.p. amplitude had been reduced to 20% relative to controlvalues. Furthermore, after washing out tubocurarine for 1 h (see Methods) meane.p.p. rise-time was 0 39+ 0-02 ms (n = 4), which was not significantly different fromthe control values.

Fig. 3 shows examples of m.e.p.p.s while Fig. 4A shows the typical time courseof the effect ofBoNT on spontaneous release recorded at a single end-plate. The meanresting potential before and after the action of the toxin was 69+ 2 and 66+1 mVrespectively (n = 8). After an initial delay (mean 552 + 2-9 min) m.e.p.p. frequencystarted to decrease. Final values of m.e.p.p. frequency (< 5% of controls, Table 1)were attained at 96-6 + 6-9 min with a ti of 723+ 4-6 min (n = 8). These times werenot significantly different from the corresponding values for the fall in e.p.p.amplitude, indicating a similar time course.

TABLE 1. All m.e.p.p.s

Frequency Rise-time Amplitude c.v. of amplitude(S-l) (Ms) (mV) distribution

Control 079+0A13 030+0-02 1-21 +0-13 0-22+O003BoNT 0035 + 0009**** 0-67 + 0-08*** 095 + 0.11 * 0-52 + 0-06**

Values shown are mean + S.E. of mean averaged over eight end-plates.Significant differences compared with controls: *P < 0-02; **P < 0005; ***P < 0002;

****p < 0-001.

M.e.p.p. amplitude distributions were constructed at selected stages of the toxin'saction during continuous recording from single end-plates and pooled data for allexperiments are shown in Fig. 5. The toxin caused a change from the predominantlybell-shaped distribution (Fig. 5A) to a broader distribution (Fig. 5D). This appearedto result from the toxin acting mainly on the normal bell-shaped population ofm.e.p.p.s which was progressively reduced (Fig. 5B and C) leaving the broaderpopulation. As the action of BoNT progressed, the increasingly broad amplitudedistribution is reflected in a significant increase in the coefficient of variation of thesedistributions by a factor of 2-4 (Fig. 4B, Table 1). The ti for the time course of thischange was 871 + 5-4 min (n = 8). Furthermore, BoNT also caused a significantincrease in m.e.p.p. rise-times by a factor of 2-2 (Figs. 3 and 4C, Table 1). The timecourse for this effect had a ti of 89-7 + 50 min (n = 8). Mean m.e.p.p. amplitude wasdecreased by only 20% of initial values (Table 1) and therefore detailed analysis ofthe time course of this small change was not studied further.

It is noteworthy that the ti values for the increase in m.e.p.p. rise-time andcoefficient of variation were each significantly greater (P < 0 05) than the ti valuesfor the decrease in m.e.p.p. frequency and in e.p.p. amplitude. We have investigatedthis further using the hypothesis that there are two independent populations ofm.e.p.p.s in controls (fast rise-time and slow rise-time m.e.p.p.s) and that the soleeffect of the toxin is to markedly reduce the frequency of one of these populations(fast rise-time m.e.p.p.s).

479

480 J. 0. DOLLY, S. LANDE AND D. W.-WRAY

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Fig. 5. Distribution of m.e.p.p. amplitudes during the action of BoNT (10 nM). Control(A), at 44-56% reduction in m.e.p.p. frequency (B), at 71-82% reduction in m.e.p.p.frequency (C) and at end-point (D). The vertical axis shows the frequency of occurrenceof m.e.p.p.s in each amplitude bin (horizontal axis) obtained by dividing the number ofm.e.p.p.s by the total analysis time and the bin width. Data were pooled from eightend-plates as follows. For each individual end-plate m.e.p.p. amplitudes were multipliedby: (mean amplitude averaged over eight end-plates)/(mean amplitude for individualend-plate). This factor attempts to take into account variations in mean amplitudebetween different end-plates.

More specifically, under the assumption of two independent populations of m.e.p.p.s (i.e. a slowrise-time and a fast rise-time population) it can be shown that for the total population, the rise-time,T, mean m.e.p.p. amplitude, x, and the coefficient of variation of the m.e.p.p. amplitudedistribution, c.v., are given by

T {Tf} + {(TS-Tr)fs}/f, (1)

X = {Xf} + {(XS-Xf)fM}/f, (2)

X2(C.V.2 + 1) = {X2(C.V.2 + 1)} + {(X2(C.V.2 + 1) -X2(C.V.2f + '))fs}/f (3)

where we have used f = ff + f, Here f, ff and fr are the total, fast rise-time and slow rise-timem.e.p.p. frequencies respectively, Tf and T. are the rise-times of the fast and slow populationsrespectively, x, and x. are m.e.p.p. amplitudes of fast and slow populations respectively and c.v.fand c.v., are the coefficients of variation of the m.e.p.p. amplitude distributions for the fast andslow rise-time populations respectively.On the assumption that the only effect of the toxin is a reduction in the frequency (f) of the

fast population, this implies that Tf, TS, xf, xs, c.v.f, c.v.s and fs are all constant. The expressionsin curly brackets {} in eqns. (1), (2) and (3) above are then all constants and their values weredetermined for each fibre from the initial and final experimental values of T, x, x2(c.v.2 + 1) andf. Eqns. (1), (2) and (3) then relate T, x and x2(c.v.2 + 1) tof. The time course offwas taken directly

IN VITRO ACTION OF BOTULINUM NEUROTOXIN

from smooth curves through the experimental data for each fibre (e.g. Fig. 4A). Hence, knowingfas a function of time, predicted curves for T, x and x2(c.v.2 + 1) as a function of time were obtainedfor each fibre using eqns. (1)-(3). Fig. 4C shows an example for the predicted time course ofT (fromeqn. (1)) while Fig. 4B shows the predicted time course of c.v. for the same fibre (from eqns. (2)and (3)). The time course for x was not plotted because m.e.p.p. amplitudes were not greatly affectedby the toxin (see above), i.e. xf and x. were not very different (eqn. (2)).

The predicted curves for T and c.v. as a function of time in this model fit the datawell, as shown for the single end-plate illustrated in Fig. 4B and C. Similar good fitswere found for six of the seven other end-plates studied. The mean ti predicted fromthe theoretical curves for all eight end-plates was 88-9+ 4-6 min for rise-times and86-8 + 4-9 min for coefficients of variation of amplitude distributions; both valueswere not significantly different from the corresponding experimental ti values (see

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0 0'0 1.0 2.0 3-0 0 1.0 2.0 3.0

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Fig. 6. Distribution of slow rise-time m.e.p.p. amplitudes: control (A) and at end pointafter application of BoNT (B, 10 nM). The histograms were obtained as in Fig. 5 usingonly the slow rise-time m.e.p.p.s. Statistical comparisons between the two distributionswere made using raw m.e.p.p. amplitude data not corrected for variation in meanamplitude between different end-plates.

above) for these parameters (paired t test). Using as input to the model the m.e.p.p.frequency time course, the model therefore predicts that the ti values for rise-timeand for coefficient of variation are both greater than that for frequency, as observed.The nature of the two populations was further investigated by selecting slow

rise-time m.e.p.p.s, defined for each fibre as those m.e.p.p.s having a rise-time greaterthan the mean control value for that fibre plus three standard deviations of therise-time distribution. This cut-off was chosen because, at three standard deviations,a negligible number of the fast m.e.p.p. population would be found outside this limit(0-1 % assuming a normal distribution). Such slow rise-time m.e.p.p.s were found incontrols as well as in poisoned muscles and the pooled amplitude distributions ofthesem.e.p.p.s are shown in Fig. 6. In the controls, the slow rise-time m.e.p.p.s comprisedonly 1-4% of all the m.e.p.p.s (Tables 1 and 2). However, in botulinized muscles, slow

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J. 0. DOLLY, S. LANDE AND D. W.- WRA Y

rise-time m.e.p.p.s made up 37 % of the total m.e.p.p. population (Tables 1 and 2).For this slow rise-time population, the frequency, rise-time and amplitude were notsignificantly different before and after BoNT (paired t test, eight end-plates,Table 2). Furthermore, the toxin did not change the pooled amplitude distributions(Fig. 6) or rise-time distributions of these slow rise-time m.e.p.p.s (Kolmogorov-Smirnov, Mann-Whitney tests). Thus the slow rise-time m.e.p.p. population was notaffected by the toxin.

TABLE 2. Slow rise-time m.e.p.p.sFrequency Rise-time Amplitude

(8-') (Ms) (mV)Control 0 011 +O0002 086+006 082+ 0-14BoNT 0-013+0004 097+009 086+014

Values shown are mean + S.E. of mean, averaged over eight end-plates. Because of the very lowfrequency of occurrence of slow rise-time m.e.p.p.s, values for c.v. for each end-plate could not bereliably calculated. No significant differences from control were obtained.

DISCUSSION

In this paper we have studied the time course of the in vitro action of BoNT. Ourdata show that there are two populations of m.e.p.p.s present in controls. Onepopulation is unaffected by BoNT (Fig. 6, Table 2) and has slow rise-times, a broadamplitude distribution and comprises only 1-2% of the total m.e.p.p.s in controls.The other population consists of fast rise-time m.e.p.p.s with a bell-shaped amplitudedistribution and is markedly reduced in frequency by the toxin (Fig. 5, Table 1).Furthermore, the detailed time course of the in vitro changes produced by the toxin(Fig. 4) could be fitted quantitatively by this two-component model in which thetoxin acts only on the fast rise-time population. More specifically, the model predicteda lag in changes in rise-time and coefficient of variation as compared with changesin frequency (Fig. 4). This result is to be expected in this model since thecharacteristics of the slow rise-time population are swamped by the fast rise-timepopulation until the latter population has been considerably reduced in frequencyby the toxin.For e.p.p.s, however, in vitro application of BoNT showed that there was only a

single population, with rise-times remaining fast throughout the action of the toxin.The reduction in e.p.p. amplitude followed a similar time course (Fig. 2) to thedecrease in fast rise-time m.e.p.p. frequency, indicating a common mechanism (seealso Dreyer & Schmitt, 1983). E.p.p.s are produced by the near-simultaneous releaseof ACh packets identical with those producing m.e.p.p.s (del Castillo & Katz, 1954),and both e.p.p.s and m.e.p.p.s appear to be produced by release from active zones(e.g. Wray, 1987). However, slow rise-time m.e.p.p.s do not make up the quantalcomponents of e.p.p.s (Cull-Candy et al. 1976). Thus, more precisely, only fastrise-time m.e.p.p.s appear to form the quantal components of e.p.p.s. The similaraction of the toxin on fast rise-time m.e.p.p.s and e.p.p.s suggests that release fromactive-zone sites is blocked by the toxin. On the other hand, slow rise-time m.e.p.p.sare not affected by the toxin and such m.e.p.p.s may originate by release from sitesaway from the active zones (Thesleff & Molgo, 1983). However, these other release

482

IN VITRO ACTION OF BOTULINUM NEUROTOXIN

sites do not appear to be very distant from the normal active zones (Tse, Wray,Melling & Dolly, 1986) and the slow rise-times of these m.e.p.p.s may be due to aprocess of protracted release of the individual ACh packets.A similar population of slow rise-time m.e.p.p.s has been described in the rat by

Thesleff (1982) after chronic treatment by BoNT. It was found that these m.e.p.p.sresponded differently from normal m.e.p.p.s to procedures which increased internalCa2+ (e.g. high K+, ouabain) and to certain drugs (e.g. 4-aminoquinoline; Thesleff &Molgo, 1983). This indicates a different release process for the slow rise-time m.e.p.p.sas compared to the fast rise-time population.Kim et al. (1984) further found that, after chronic BoNT treatment, the frequency

of slow rise-time m.e.p.p.s in rat muscle increased over periods of several days toweeks. On the other hand we found that the frequency of the slow rise-timepopulation found in controls was not changed by in vitro treatment with BoNT. Thechanges seen in chronic BoNT treatment are presumably adaptive, and apparentlydo not have time to occur in vitro.

It is known that after denervation, ACh release can occur spontaneously fromSchwann cells (Bevan, Grampp & Miledi, 1976). There are many similarities betweensuch Schwann cell m.e.p.p.s and the slow rise-time population described here.However, it is unlikely that the slow rise-time m.e.p.p.s originate from Schwann cellssince in mice deficient in Schwann cells, slow rise-time m.e.p.p.s can still be detected(Thesleff & Molgo, 1983).By using mice only 4-28 days old, Kriebel et al. (1976) found that botulinum toxin

reduced a major component of the m.e.p.p. population leaving behind a 'subminia-ture' small amplitude, fast rise-time population which was more resistant to thetoxin. They suggested that simultaneous release of these subminiatures made up thenormal m.e.p.p. However, in our study, the population remaining after the toxin hadslow rise-times and a wide range of amplitudes (mean amplitude similar to controls)and thus cannot simply be considered as a subminiature population.

In summary, BoNT acts by a common mechanism to greatly reduce the populationof fast rise-time m.e.p.p.s and e.p.p.s (which arise by release from active zones). Thepopulation of slow rise-time m.e.p.p.s found in controls is not affected by the toxinand could result from release from sites near to, but not at, the active zones.

We thank the Wellcome Trust for support.

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CULL-CANDY, S. G., LUNDH, H. & THESLEFF, S. (1976). Effects ofbotulinum toxin on neuromusculartransmission in the rat. Journal of Physiology 260, 177-203.

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