J. Physiol. (1975), 252, pp. 491-508 With 9 text-ftgure8

491

Printed in Great Britain

DISCRETE AND DISCONTINUOUS ACTION OF BROWN WIDOW SPIDER VENOM ON THE PRESYNAPTIC NERVE TERMINALS OF FROG MUSCLE

BY J. DEL CASTILLO AND D. W. PUMPLIN* From the Laboratory of Neurobiology, Medical Sciences Campus, U.P.R., San Juan, Puerto Rico, South America

(Received 10 April 1975) SUMMARY

1. A study was made of the effects of the venom of the brown widow spider (Latrodectus geometrics) on end-plates of the frog sartorius muscle. 2. The increase in the frequency of the minature end-plate potentials (m.e.p.p.s), elicited by the venom in normal-[Ca2+] Ringer solution, occurs in discrete volleys having a sharp onset and end. The frequency of the m.e.p.p.s is high (up to 300 see-') and relatively constant during the volley. 3. The volleys recur at intervals during a period from 5 to 10 min after addition of the venom until the onset of electrical silence, up to 4 hr later. The activity occurs in groups containing volleys of long and short duration. 4. Simultaneous intracellular and extracellular recording from single end-plates indicates that the volleys originate at highly localized areas of the nerve terminals. The high-frequency release of m.e.p.p.s in hypertonic solutions, which was studied for comparison purposes, occurs randomly over the entire end-plate. Volleys originating simultaneously at different sites are often superimposed in the intracellular recordings. 5. In high-[Ca2+] Ringer solution, the initial frequency of the m.e.p.p.s in a volley is comparatively higher. However, the frequency drops to one half its value in a few seconds. The volley then terminates or else the frequency of m.e.p.p.s remains high for some time and the volley has no sharp end. Activity occurs in groups containing both long and short volleys. Many more short (< 5 see) and long (> 30 see) volleys occur in high-[Ca2+] solution than in normal-[Ca2+] solutions. 6. In low-[Ca2+], high-[Mg2+] Ringer solution, the volleys of m.e.p.p.s are fewer in number and much longer in duration. Intra- and extracellular recording of uninterrupted activity during long periods suggests *

To whom correspondence should be addressed.

492 J. DEL CASTILLO AND D. W. PUMPLIN that in this solution the m.e.p.p.s originate diffusely rather than at discrete areas of the nerve terminals. 7. Implications of the above data on possible modes of action of the venom are discussed. INTRODUCTION

Homogenates of the venom glands of the black widow spider (Latrodectus mactans) contain one or more substances, presumably proteins, which, among other effects, cause release of the acetylcholine (ACh) stored in the cytoplasm of the motor nerve terminals of amphibian and mammalian skeletal muscle. In their initial report on the effects of black widow spider venom (BWSV) on the neuromuscular junction of the frog, Longenecker, Hurlbut, Mauro & Clark (1970) described how, from 5 to 10 min after addition of the venom to a Ca-free bathing solution containing ethyleneglycol-bis(,6-aminoethylether) N,N'tetra-acetic acid (EGTA), the frequency of the miniature end-plate potentials (m.e.p.p.s) rose from about 0-5-1 5 sec-1 to a peak value of 300-1000 sec-1. From such a maximum, the frequency of the m.e.p.p.s declined to control levels following a roughly exponential course over a period of 50 min. Observations in the electron microscope of junctions which had been fixed with glutaraldehyde 1-4 hr after addition of BWSV, revealed that the cytoplasm of the motor terminals contained almost no synaptic vesicles (Clark, Mauro, Longenecker & Hurlbut, 1970). On the basis of this evidence, it was suggested that BWSV acts on the presynaptic membrane to cause the release of transmitter. A subsequent study (Clark, Hurlbut & Mauro, 1972) showed that BWSV induced the synaptic vesicles to fuse with the presynaptic membrane, leading to an increase of both the surface area and the volume of the nerve terminals. We have recently performed experiments similar to those of Longenecker et al. (1970) and Clark et al. (1970, 1972), using homogenates of the venom glands of the brown widow spider (Latrodectus geometricus), a locally available species which is closely related to the black widow. In accordance with the literature, these homogenates will be termed brown widow spider venom (BrWSV), although it should be recognized that they contain many more components than the true venom which the spider injects into its prey. The final results of the action of BrWSV on the motor nerve terminals appeared to be the same as those of BrWSV, but in the course of the experiments some interesting information was obtained regarding the mode of action of the venom on the motor nerve terminals. Indeed, our observations have shown that in the presence of normal concentrations of Ca, the depletion of ACh stored in the motor nerve terminals, which is

43 BROWN WIDOW SPIDER VENOM ON MUSCLE 493 elicited by BrWSV, does not take place in a continuous and diffuse manner, but is intermittent and highly localized. Acetylcholine escapes from the presynaptic cytoplasm as well defined bursts or volleys of m.e.p.p.s which may indicate the interaction of molecules of the venom with discrete sites of the surface of the motor nerve endings. Our experiments show also that the effects of BrWSV on the presynaptic membrane, as reflected in the shape and duration of the volleys of m.e.p.p.s, are markedly dependent upon the concentration of the calcium in the surrounding medium. METHODS

All electrophysiological experiments were performed at 20' C on end-plates of surface fibres in the isolated sartorius muscle of the frog (Rana pipien&8). After dissection, the muscles were attached, innermost surface up, with stainless-steel 'insect' pins, to a piece of polyethylene tubing 3-5 cm long and 0-8 cm in diameter which in turn was slipped over a plastic rod in a Plexiglass chamber having a volume of 5 ml. Rotating the rod allowed the end-plates to be easily placed in position for recording. The electrical activity at single end-plates was recorded with conventional glass micro-electrodes. In some experiments the dual channel recording technique described by del Castillo & Katz (1956) was used. One glass micro-electrode, filled with 3 m-KCl and having a resistance of 10-15 fn, was inserted in the end-plate region to record intracellularly the spontaneous end-plate activity. A similar microelectrode, filled with 4 m-NaCl and having a resistance of 2-3 MO2 was used to explore the outer surface of the end-plate to locate 'active spots'; i.e. sites at which the synaptic currents produced by the liberation of single ACh packets could be recorded as the so-called 'external m.e-p-p-s'. Each micro-electrode was connected to an amplifier having a high input impedance. The output of the amplifier was a.c. -coupled to one channel of a Tektronix type 502 cathode ray oscilloscope. Intracellular activity was displayed in the upper beam while the external m.e.p.p.s were shown in the lower beam. To study the action of BrWSV, the glands of one to three spiders (depending on the size of the individuals) were homogenized in 0-2 ml. Ringer solution and the homonogenate was added to the chamber. In the absence of a purified toxin, or of sufficient spiders for tests of toxicity, we used the admittedly crude measure of gland size in an attempt to employ approximately the same amount of toxin in each

experiment. Three types of Ringer solution were used. The normal.[Ca2+] Ringer solution had the following composition (mm): NaCl, 116; KCI, 2; CaCl2, 1-8; Na2HPO4,, 2; NaH2PO4, 1. For some experiments, this Ringer solution was made hypertonic by the addition of 5 % (w/v) sucrose. The high-[0a2+] Ringer solution contained (nmm): NaCl, 96; KCI, 2; CaCl2, 18; Na2HPO4, 0-3. The low-[Ca2+], high-[Mg2+] Ringer solution contained (mM): NaCl, 89; KC1, 2; CaCl2, 0-6; MgCl2, 21P4; Na2HPO4, 0*3.. All solutions were adjusted to pH 7-15-7-20. Neostigmine (10-6 M) was added to the bath at least 30 min before the addition of venom.

494 494

J. DELCGASTILLO AND D. W. PUMPLIN RESULTS

Liberation of m.e.p.p.s induced by Br WS V in normal Ringer solution In marked contrast to the progressive and relatively uniform increase in the frequency of m.e.p.p.s described by Lo.ngenecker et al. (1970), based on experiments performed in Ca-free Ringer solution containing EGTA we observed, working in normal Ringer solution, that BrWSV acts in a discontinuous or intermittent manner. During the first 5-10 min after the addition of venom to the bath, the rate of discharge of the m.e.p.p.s remained unchanged. The first sign of the action of BrWSV was the

Fig. 1. Volleys of miniature end-plate potentials (m.e.p.p.s) recorded 22 and 27 min, respectively, after addition of BrWSV in normal Ringer solution. Glands from one spider were used. Intracellular activity is displayed in the upper trace, while 'focal extracellular' activity recorded at the same end. plate is displayed in the lower trace. The intracellular activity begins and ends abruptly with no corresponding increase in the activity recorded extracellularly. Moving film recording (0.25 em/sec); pH 7-2; 200 C. Calibration marks: 1 mV; 2 sec.

appearance of well defined volleys of m.e.p.p.s. These volleys were characterized by a sharp onset and a sudden end. Within a volley, the frequency of m.e.p.p.s was relatively constant, having a rate of 100-300 sec-. The volleys reappeared at intervals over a period of 1-4 hr, after which the fibre became electrically silent. Three such well defined volleys, all recorded from the same fibre in the same experiment, are shown in Fig. 1. In some instances, the onset of a new volley occurred prior to the end of the previous volley, resulting in a correspondingly increased frequency of m.e.p.p.s. Outside of the volleys, the frequency of m.e.p-p-s increased by

495 BROWN WIDOW SPIDER VENOM ON MUSCLE a factor of 2 or less over the control frequency observed before the addition of the venom. It seems clear from these records, and from similar observations made on the cutaneous pectoris muscle, that the main action of BrWSV on the nerve terminals is to induce discrete discharge or volleys of m.e.p.p.s.

Characteristics of the volleys The number, durations, and temporal occurrences of the volleys were determined from analyses of the film records. The early portion of the m.e.p.p. activity in a single fibre is shown in Fig. 2. Volleys occurred at a mean rate of 098 min' in this experiment. The duration of the volleys is 30 U

.~20 0 C

0

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0

-

10

20

30

Time after addition of venom (min)

Fig. 2. Time course of the volleys of m.e.p.p.s recorded intracellularly from a single muscle fibre during the early portion of exposure to BrWSV in normal Ringer solution. Each bar indicates the duration and the time of onset of a single volley. The fibre was impaled 5 min after the venom was added, as indicated by the arrow. The volleys apparently occur in groups, and there is a latent period between the addition of venom and appearance of the first volley. Two volleys from this experiment are illustrated in Fig. 1.

plotted vs. the time at which they occurred, as measured from the time of addition of venom to the bath. This plot was chosen as a convenient way to illustrate both the relatively short durations of the volleys and the relatively long period over which the venom acts. The activity occurred in groups containing both long and short volleys. The number of volleys originating during each 5 min interval would have a Poisson distribution if the volleys were completely independent of one another. In fact, the distribution differed significantly from the best Poisson fit (X2 = 2O*2; d.f. = 7; 099 < P < 0995), indicating that the volleys were not completely independent. The durations of the volleys, however, were independent of the times at

J. DEL CASTILLO AND D. TV. PUMPLIN 496 which they originated (correlation coefficient r = 0.098). Volleys having short or long durations occurred throughout the action of the venom. In normal-[Ca2+] Ringer solution, the volleys had duration of 3 6-30 sec. A histogram of durations obtained in two experiments in this Ringer solution is shown in Fig. 7.

Spatial origin of the volleys of m.e.p.p.s One of the first problems posed by such a discontinuous mode of action of the venom concerns the spatial origin of the discharges of m.e.p.p.s. Two extreme possibilities can be considered. Each separate volley may involve the entire surface of the nerve terminals, or may represent a localized event occurring at a small site on the membrane of the motor nerve terminal. An answer to this question could be provided by the dual channel (intraand extracellular) recording technique. If the volleys were diffuse events, they should be accompanied by an increase in the frequency of the m.e.p.p.s recorded externally from any active spot within the end-plate. If, on the contrary, they are highly localized discharges, one should expect to observe an increase in the frequency of the external m.e.p.p.s only if the volley happens to originate at, or in the immediate vicinity of, the ' active spot'.

Control experiments in normal Ringer Before attempting to apply this technique to study the m.e.p.p. activity induced by BrWSV, we performed some control experiments in preparations immersed in normal Ringer solution as well as in Ringer solution made hypertonic by the addition of sucrose. The results of our experiments in normal Ringer solution were in agreement with those of del Castillo & Katz (1956). A comparison of the recordings obtained with the intra- and extracellular micro-electrodes suggests that the site of liberation of ACh packets changes continuously within the nerve terminal and only occasionally was one of the internally recorded m.e.p.p.s produced in the vicinity of the extracellular micro-electrode. In some instances, however, brief bursts of m.e.p.p.s were recorded simultaneously with both microelectrodes. These occurred when the tip of the extracellular micro-electrode first 'hit' an 'active spot' while exploring the surface of the muscle fibre. These 'correlated bursts' did not recur if the extracellular tip was left stationary. Therefore, they can be safely attributed to a highly localized mechanical stimulation of the motor terminal.

BROWN WIDOW SPIDER VENOM ON MUSCLE

497

Control experiments in hypertonic solutions These were done in preparations immersed in Ringer solution made hypertonic by the addition of 5 % sucrose (w/v). The frequency of the m.e.p.p.s was greatly increased in this solution (Fatt & Katz, 1952). Our main purpose in these experiments was to obtain information on both the constancy of the frequencies and the degree of correlation between internal and external activity during rapid discharge of m.e.p.p.s. The results obtained were consistent with those of experiments performed in normal Ringer solution. In the experiment illustrated by Fig. 3A, the m.e.p.p.s released from a junction immersed in hypertonic Ringer solution were recorded continuously from an 'active spot' for 260 sec. The film was then divided into intervals of 4 sec and the internal and external m.e.p.p.s occurring in each interval were counted. The average number of internal m.e.p.p.s per interval (4 sec) was 32-78 + 048 (S.E. of mean: n = 65). The average number of external m.e.p.p.s per interval was 5-58 + 0-38 (S.E. of mean). The average ratio of FextlFint for the 65 intervals was equal to 0-17 + 0411 (s.E. of mean). These results suggest that both the external and internal frequencies, as well as the ratio between them, remained reasonably constant during the observation period. This observation was checked further by examining the distribution of the ratios about their mean. This was done by the method of runs (Dixon & Massey, 1957). The number of runs (consecutive groups of ratios lying above or below the mean ratio) was not significantly different from the number expected for a random distribution of the ratios. In other words, it appears that both the internal and external m.e.p.p.s originated at random both in space and time. A histogram of the number of external m.e.p.p.s recorded per interval is given in Fig. 3 B, together with the curve calculated for a Poisson distribution having the same arithmetical mean. An intriguing feature of the above results is the high ratio observed between the external and internal frequencies, since in this experiment the extracellular electrode recorded 17 % of the total number of m.e.p.p.s. Five other experiments in hypertonic solutions yielded similar results. In the six fibres studied the ratio FextlFint had an average value of 030 (range 0.15-0.43). Since it is extremely unlikely that an external microelectrode could record 43 % of the m.e.p.p.s originating at a junction, we feel inclined to believe that such high ratios represent an artifact due perhaps to pressure exerted by the micro-electrode tip on the nerve terminal. Under the influence of a hypertonic solution, this may increase the probability of ACh release at the active spot. A similar mechanical

498 J. DEL CASTILLO AND D. W. PUMPLIN factor was evoked by Katz & Miledi (1973) to explain the peculiar time courses of m.e.p.p.s which originated in the immediate neighbourhood of the tip of the extracellular micro-electrode. At any rate, both the intracellular and extracellular activity recorded from the six fibres studied was consistently uniform and did not show gross changes in frequency. This was confirmed by the absence of a significantly large or small number of 'runs' of consecutive frequencies which were above or below the mean frequency.

A

B 10

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0

0

5

10 M.e. p. p.s/interval

15

Fig. 3. A, m.e.p.p.s in a muscle treated with normal Ringer solution made hypertonic by the addition of 5 % (w/v) sucrose. Intracellular activity is displayed in the upper trace, while 'focal extracellular' activity, recorded from the same end-plate, is displayed in the lower trace. Both intra- and extracellular activities have relatively constant frequencies, and the ratio of m.e.p.p.s recorded extracellularly to those recorded intracellularly was also constant (0-17+0-011) (s.E. of mean). Moving film recording (0-25 cm/sec). pH 7-2; 200 C. Calibration marks: 1 mV; 2 sec. B, histogram of the number of m.e.p.p.s recorded extracellularly during successive 4 sec intervals of a 260 sec record obtained from a single muscle fibre exposed to normal Ringer solution made hypertonic by the addition of 5 % (w/v) sucrose. A portion of the record is shown in A. The curve indicates the Poisson distribution for data having the same arithmetical mean.

BROWN WIDOW SPIDER VENOM ON MUSCLE

49 499

Dual recording in preparations under the influence of BrWS V The results of experiments in which dual recording was performed on a neuromuscular junction under the influence of BrWSV allowed us to give an unambiguous answer to the problem of the origin of the intermittent discharges of transmitter. The volleys of m.e.p.p.s are strictly localized

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Fig. 4. Volleys of m.e.p.p.s obtained during the action of BrWSV in normal Ringer solution. Glands from three spiders were used. Activity recorded extracellularly (lower traces) was uncorrelated (left) or completely correlated (right) with activity recorded intracellularly (upper traces) from the same end-plate. Successive 0-5 sec sweeps occurring at intervals of 1-6 sec were photographed on moving ifilm. Consecutive traces are displayed from bottom to top of each column. Right column begins immediately after the left column. pH 7-2; 200 C. Calibration marks: upper trace, 0-5 mV; lower trace, 2 mV; 0-1I sec.

J. DEL CASTILLO AND D. W. PUMPLIN 500 events. It can be seen, in Fig. 1, that the well developed volleys of m.e.p.p.s recorded intracellularly were not accompanied by a significant increase in the frequency of the external m.e.p.p.s. In only two instances, in experiments on fibres continuously examined during the action of BrWSV, have we been able to record volleys of m.e.p.p.s originating within the recording radius of the tip of the 'extracellular' micro-electrode. One such instance is shown in Fig. 4. The records of the left column show m.e.p.p. activity recorded intracellularly together with a rather low m.e.p.p. activity recorded extracellularly. In the records of the right column, which is a direct continuation of the left column, a volley of m.e.p.p.s originating near the tip of the extracellular microelectrode is shown. During this volley, the ratio between the externally and internally recorded frequencies of m.e.p.p.s became almost equal to 1; in fact, the extracellular recordings became practically the mirror image of the intracellular recordings. Considering (a) that at least two volleys of m.e.p.p.s originating near the tip of the 'extracellular' micro-electrode were recorded, and (b) that the number of extracellular m.e.p.p.s did not increase during most of the volleys which were observed intracellularly, it can be concluded that the volleys of m.e.p.p.s induced by BrWSV are extremely localized or focal events originating at minute areas of the presynaptic membrane.

Influence of the extracellular concentration of calcium on the discharge of

m.e.p.p.s elicited by BrWSV There were several reasons for studying the influence of the extracellular Ca concentration on the effect of BrWSV. In the first place, we wanted to see whether the differences between our results and those of Longenecker et al. (1970) were related to different modes of action of the venoms obtained from the two different species of spiders, or whether they were due to the different ionic environment in which the experiments were carried out. In the second place, the observations of Pumplin & McClure (1972) indicated that the action of BWSV was somewhat related to the extracellular concentration of Ca, since the ability of the venom to release ACh from the superior cervical ganglia of rats was impaired in a solution containing a low [Ca2+] and a high [Mg2+]. Finally, since the liberation of ACh under physiological conditions appears to be determined by an inward Ca2+ current (Katz & Miledi, 1967), it was interesting to see how the liberation of ACh packets by BrWSV was affected by changes in the extracellular [Ca2+].

BROWN WIDOW SPIDER VENOM ON MUSCLE

501

Characteristics of volleys obtained in high-[Ca2+] Ringer solution In the experiment illustrated in Fig. 5 the calcium concentration in the Ringer solution was increased tenfold, to 18 mm. This change had a definite influence on the number, intensity, and time course of the discharges of m.e.p.p.s as can be seen by comparing Fig. 1 with Fig. 5. Volleys occurring in normal-[Ca2+] Ringer solution (Fig. 1) were characterized by both a sharp onset and end, and by a relatively uniform frequency of discharge during the event. The volleys which were recorded in high-[Ca2+] I'

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.i Fig. 5. Volleys of m.e.p.p.s obtained during the action of BrWSV in Ringer solution containing 18 mM-Ca2+. These volleys occurred 31 and 62 min, respectively, after the addition of venom prepared from two spiders. Intracellular activity is displayed in the upper trace while extracellular activity is displayed in the lower trace. Intracellular activity begins abruptly, then decreases. There may be a long 'tail' of increased frequency of m.e.p.p.s (upper record), or the increased frequency may end rapidly (lower record). No increase in extracellular activity was recorded. Moving film recording (0 25 cm/sec). pH 7 2; 200 C. Calibration marks: 2 mV; 2 sec.

Ringer solution (illustrated in Fig. 5) had an equally sharp onset and a relatively higher initial frequency. However, the frequency declined to one half of the initial value in about 5 sec. In some volleys the frequency remained at this level for a considerable period of time, or declined only slowly. Because of this, the end of some of these volleys was blurred and difficult to define. A plot of the duration of volleys vs. the time at which the volleys started after the initial addition of venom to the bath is shown in Fig. 6.

502 J. DEL CASTILLO AND D. W. PUMPLIN This plot represents the complete activity observed in a single fibre between the addition of venom and the onset of electrical silence. The activity again occurred in groups containing both long and short volleys. The distribution of the number of volleys originating during each ten min period was significantly different from the best Poisson fit (X2 = 29-5; d.f. = 9; P > 0 995), indicating again that the volleys were not completely independent of each other. The durations of the volleys were independent of the times at which they originated. The regression line for durations of volleys on different times after the addition of venom has a slope of - 0-084 (the correlation coefficient is not valid in this case because the durations were not normally distributed). Volleys having a very short duration occurred nearly throughout the action of the venom. An example of these is given in the lower record of Fig. 5. 100 U

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4LL40 60 Time after addition of venom (min)

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Fig. 6. Time course of the volleys of m.e.p.p.s recorded intracellularly from a single muscle fibre exposed to BrWSV in Ringer solution containing 18 mM-Ca2+. The fibre was impaled before the addition of venom and activity was recorded until the onset of electrical silence. There is a latent period between the addition of venom and the initial occurrence of a volley, and the volleys appear to be grouped in time. Volleys may have durations which are much longer or shorter than those of volleys seen in normal Ringer solution.

The distribution of the durations of volleys in Ringer solutions containing normal and raised concentrations of Ca2+ are given in Fig. 7. The proportion of volleys having a short duration (< 5 see) is significantly greater in high-[Ca2+] Ringer (52 of III differs from 8 of 41 at 1 % level; x2 test). Volleys having a duration greater than 30 see did not appear in Ringer solution containing a normal concentration of calcium, but constituted 14 % of the volleys observed in Ringer solution containing 18 mm-Ca2+.

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High-Ca Ringer

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Fig. 7. Histograms of durations of volleys of m.e.p.p.s obtained during the action of BrWSV in normal -[Ca2+] (1-8 mM) and high -[Ca2+] (18 mM) Ringer solution. Many more volleys having short ( < 5 sec) or long (> 30 sec) durations occur in the high -[Ca2+] Ringer solution. Each histogram represents the sum of two experiments.

Characteristics of volleys obtained in low-[Ca2+], high-[Mg2+] Ringer solutions An example of the volleys of m.e.p.p.s observed during the action of BrWSV in Ringer solution containing 0-6 mM-Ca2+ and 21-4 mM-Mg2+ is shown in Fig. 8A. These volleys differed both quantitatively and qualitatively from volleys obtained in normal-[Ca2+] Ringer solution. The onset of a volley was gradual, occurring over seconds. At the end of a volley, the frequency of m.e.p.p.s declined to a base line value over an even longer period. No sharp increases or decreases in the frequency of m.e.p.p.s were observed. The time course of volleys in one experiment in low-[Ca2+], high-[Mg2+] Ringer is shown in Fig. 9. These volleys lasted much longer than those seen in Ringer solutions containing normal or raised concentrations of Ca2+ [130 see (range 44-210) vs. 11 see (range 1-6-28-4) and 14-4 see (range 0-4-100), respectively]. Fewer differentiable volleys were seen (0-12 volleys/min. vs. 0-92 + 0-06 (mean + S.D. of three experiments)). The distribution of volleys per 10 min

J. DEL CASTILLO AND D. W. PUMPLIN interval did not differ significantly from a Poisson distribution (X2 = 4.4, 8 d.f.; P > 0410). The durations of the volleys had a Gaussian distribution, rather than the exponential distribution seen in high-[Ca2+] Ringer solutions. Between volleys, the frequency of m.e.p.p.s rose in the first 40 min after the addition of venom from 2%3/sec to 4'3/sec. This frequency was maintained for the next 40 min, after which it slowly declined. In a second experiment conducted in low-[Ca2+], high-[Mg2+] Ringer, an initial 55 see volley of m.e.p.p.s occurred, beginning 16-8 min after the venom was added (upper record of Fig. 8 A). This was followed immediately by a second rise in the frequency of m.e.p.p.s (lower record of Fig. 8A). 504

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Fig. 8. A, m.e.p.p.s recorded intracellularly 17 and 18 min, respectively, after addition of BrWSV in Ringer solution containing 0-6 mM-Ca2+ and 21-4 mMMg2+. Glands from two spiders were used. The frequency rise was gradual (cf. Figs. 1 and 5). After 18 min, the frequency remained high, then slowly diminished until the fibre became electrically silent, 55 min after 'the addition of BrWSV. Moving film recording (0-25 cm/sec); pH 7-2; 200 C. Calibration marks: 1 mV; 2 sec. Fig. 8. B, activity of m.e.p.p.s recorded intracellularly (upper trace) and extracellularly (lower trace) from an end-plate of an isolated sartorius muscle exposed to BrWSV in Ringer solution containing 0*6 mm-Ca2+ and 21.4 mMMg2+. Glands from two spiders were used. Both intra- and extracellular activities have relatively constant frequencies. No defined volleys of m.e.p.p.s were observed in this fibre. Moving film recording (0-25 cm/sec); pH 7-2; 200 C. Calibration marks: upper trace 2 mV; lower trace, 1 mV; 2 sec.

505 BROWN WIDOW SPIDER VENOM ON MUSCLE The frequency of m.e.p.p.s remained elevated over the next 28 min. A slow decline led to electrical silence at approximately 55 min. In a third experiment conducted in this Ringer solution m.e.p.p. activity was recorded both intra- and extracellularly (Fig. 8B). The frequency of external m.e.p.p.s remained nearly constant at 2-5 sec-1 during the period of high activity recorded intracellularly. No period of particularly high or low externally recorded activity could be discerned in the record. Although the majority of externally recorded m.e.p.p.s 200

:-0 100 0

0

80 60 20 40 Time after addition of venom (min)

Fig. 9. Time course of volleys of m.e.p.p.s recorded intracellularly from a single muscle fibre during exposure to BrWSV in Ringer solution containing 0-6 mM-Ca2+ and 21-4 mM-Mg2+. The fibre was impaled before the addition of the venom and activity was recorded until the onset of electrical silence. In this Ringer solution, the volleys are fewer in number, but generally longer in duration. Compare Figs. 2 and 6.

were quite small, a few were large enough to indicate that the electrode was, indeed, very close to the end-plate membrane. The constant frequency of externally recorded m.e.p.p.s (Fig. 8B) resembled that seen in hypertonic solutions (Fig. 3), indicating that in low-[Ca2+], high-[Mg2+] Ringer solution, the release of m.e.p.p.s may be distributed randomly over the entire surface of the end-plate. Although it is clear that BrWSV, like BWSV, is effective in solutions containing little or no Ca2 , this ion is required for the firing of m.e.p.p.s in discrete volleys which are discontinuous in space and time.

506

J. DEL CASTILLO AND D. W. PUMPLIN DISCUSSION

An important objective in the study of presynaptically acting neurotoxins is the finding of compounds which specifically interact with the normal processes of excitation-dependent release, or the uptake and preparation for release, of neurotransmitter molecules. In the absence of further experimental evidence, the mechanism of action of the venom of Latrodectus spiders on the membrane of motor nerve terminals, and the relationship of this mechanism to the normal excitation-dependent release of neurotransmitter, must remain matters of speculation. The ultrastructural evidence (Clark et al. 1972) indicates that, under the action of the venom, synaptic vesicles fuse with the presynaptic membrane, leading to infolding of this membrane due to its increased area. Presumably fusion of a synaptic vesicle leads to release of its contents, resulting in the production of a miniature end-plate potential. We have shown that the production of m.e.p.p.s, under the action of BrWSV, occurs as a series of events which are localized in both time and place. Since an increase and subsequent fall in the frequency of m.e.p.p.s, and a depletion of synaptic vesicles can be the result of such general treatments as depriving the tissue of glucose (Nicolescu, Dolivo, Rouiller & Foroglou-Kerameus, 1966) or using metabolic inhibitors (Atwood, Lang & Morin, 1972), it may be difficult to infer a mechanism of action of BrWSV by analogy with the effects of other agents on nerve endings. Nevertheless, two lines of evidence suggest that the volleys of m.e.p.p.s elicited by BrWSV may be due to the temporary formation of channels in the presynaptic membrane. Firstly, long-lasting bursts of m.e.p.p.s have been observed following the mechanical injury of motor nerve terminals by a recording micro-electrode. Such volleys occur not only in normal preparations in which a focal depolarization of the terminal will cause the m.e.p.p.s to be released, but also in preparations treated with botulinum toxin (Thesleff, 1960; Boroff, del Castillo, Evoy & Steinhardt, 1974), in which strong depolarization of the nerve endings fails to release any m.e.p.p.s. Secondly, bursts of m.e.p.p.s, often followed by long afterdischarges, have been observed during hyperpolarization of the nerve terminals by applied anodic currents (del Castillo & Katz, 1956). These were attributed to a reversible breakdown of the membrane dielectric caused by the increased voltage gradient across the presynaptic membrane. Channels in the presynaptic membrane may be formed by penetration of one or several venom molecules into the membrane lipids. A similar idea was proposed by Davies (1962) as a possible mechanism for olfaction. At least three consequences of opening such channels can be predicted. In the first place, such an event must cause a depolarization of the

507 BROWN WIDOW SPIDER VENOM ON MUSCLE membrane which will spread electrotonically along the terminals. This will lead, in turn, to a release of ACh which would be dependent upon the extracellular concentration of Ca. In the second place, one would expect that the entry of extracellular Ca ions into the presynaptic cytoplasm would induce an additional release of ACh which would be dependent upon the extracellular concentration of this ion. Finally, we know that even in the absence of extracellular Ca and in the presence of a high concentration of Mg a break-down of the membranes induces the release of the ACh, since the bursts of m.e.p.p.s elicited by hyperpolarization of the presynaptic membrane occur in low-[Ca2+], high-[Mg2+] solutions. It is therefore highly probable that the release of m.e.p.p.s which follows the initial membrane event induced by spider venom can be the result of at least three separate factors. The absence of Ca ions does not prevent the occurrence of the initial membrane event induced by the venom. In fact, in low-[Ca2+], high-[Mg2+] solutions, the duration of the membrane breakdown seems to be considerably increased. In the presence of a higherthan-normal concentration of Ca, there is an increase in the amount of ACh released at the beginning of a volley. The concentration of Ca ion also affects the termination of the discharges. An interesting feature of the action of BrWSV is the fact that the binding of venom molecules to the membrane is not followed immediately by the occurrence of volleys. Indeed, volleys of m.e.p.p.s are observed at times up to 4 hr after the addition of venom to the bath, and occur even though the venom solution has been replaced by normal Ringer solution. Since it is unlikely that such a delay could be due to diffusion, it is reasonable to think that the initiation of a volley requires not only the presence of the venom, but also the intervention of some 'triggering event' having a certain activation energy. Another possibility was suggested to us: in order to act, the venom molecules may have to be carried into the presynaptic cytoplasm by coated vesicle formation. The action of.BrWSV is complicated further by the fact that the synaptic vesicles (or at least a large portion of them) are not reformed and refilled following discharge of their ACh. The membrane material of the synaptic vesicles remains incorporated in the ending membrane. Since this incorporation occurs in low-[Ca2+], high-[Mg2+] solutions, it must be the fate of at least those vesicles which are released due to the initial break-down of the nerve terminal membrane. The incorporation may result from a binding of the vesicles during the membrane break-down, or it may have nothing to do with the releasing process, but instead result from the action of a separate toxin contained in the crude venom. Preliminary ultrastructural observations have shown that in terminals which were only partially depleted by BrWSV there is not a uniformly decreased density of vesicles;

J. DEL CASTILLO AND D. W. PUMPLIN instead, adjacent regions contained an apparently normal density of vesicles or no vesicles at all. In agreement with the observations of Clark et al. (1972), the depleted regions were partially surrounded by membranous material, presumably resulting from the fusion of vesicles to the membrane of the terminal. The well defined vesicle-depleted areas may be the result of single volleys of m.e.p.p.s. 508

This research was supported by U.S. Public Health Service Grants nos. NS-07464 and K6N-14938. Contribution no. 36, Laboratory of Neurobiology. We thank F. McKenzie and H. Rodriguez for collecting the spiders, and N. Deliz and G. Garcia for technical assistance.

REFERENCES ATWOOD, H. L., LANG, F. & MORIN, W. S. (1972). Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons. Science, N.Y. 176, 1353-1355. BOROFF, D. A., DEL CASTILLO, J., Evoy, W. H. & STEINHARDT, R. A. (1974). Observations on the action of type A botulinum toxin on frog neuromuscular junction. J. Physiol. 240, 227-253. CLARK, A. W., HURLBUT, W. P. & MAURO, A. (1972). Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom. J. cell Biol. 52, 1-14. CLARK, A. W., MAURO, A., LONGENECKER, H. E., JR. & HURLBUT, W. P. (1970). Effects of black widow spider venom on the frog neuromuscular junction. Effects on the fine structure. Nature, Lond. 225, 703-705. DAVIES, J. T. (1 962). The mechanism of olfaction. In Biological Receptor Mechanisms, Symp. Soc. Exp. Biol. xvi, pp. 170-179. New York: Academic Press. DEL CASTILLO, J. & KATZ, B. (1956). Localization of active spots within the neuromuscular junction of the frog. J. Physiol. 132, 630-649. DIXON, W. J. & MASSEY, F. J. JR. (1957). Introduction to Statistical Analysis, 2nd edn. New York: McGraw-Hill. FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. KATZ, B. & MILEDI, R. (1967). The timing of calcium action during neuromuscular transmission. J. Physiol. 189, 535-544. KATZ, B. & MILEDI, R. (1973). The binding of acetylcholine to receptors and its removal from the synaptic cleft. J. Physiol. 231, 549-574. LONGENECKER, H. E., JR., HURLBUT, W. P., MAURO, A. & CLARK, A. W. (1970). Effects of black widow spider venom on the frog neuromuscular junction. Effects on end-plate potential, miniature end-plate potential and nerve terminal spike. Nature, Lond. 225, 701-703. NicOLESCU, P., DOLIVO, M., ROUILLER, C. & FOROGLOU-KERAAMEUS, C. (1966). The effect of deprivation of glucose on the ultrastructure and function of the superior cervical ganglion of the rat in vitro. J. cell Biol. 29, 267-285. PUMPLIN, D. W. & MCCLURE, W. 0. (1972). Sympathetic ganglion: release of acetylcholine elicited by black widow spider venom. Second Annual Meeting, Society for Neuroscience, abstract 20.5. THESLEFF, S. (1960). Supersensitivity of skeletal muscle produced by botulinum toxin. J. Physiol. 151, 598-607.

Discrete and discontinuous action of brown widow spider venom on the presynaptic nerve terminals of frog muscle.

1. A study was made of the effects of the venom of the brown widow spider (Latrodectus geometricus) on end-plates of the frog sartorius muscle. 2. The...
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