499

J. Phpiol. (1979), 290, pp. 499-506 With 2 text-flgume Printed in Great Britain

PRESYNAPTIC ACTION OF CURARE BY M. I. GLAVINOVIC* From the Department of Anae8the8ia Re8earch McGill University 3655 Drummond Street Montreal, Quebec H3G 1 Y6, Canada

(Received 15 May 1978) SUMMARY

1. As a result of a conditioning phrenic nerve stimulus, end-plate currents (e.p.c.s) in a voltage clamped uncurarized cut diaphragm show a facilitation which reaches its maximum at 30-40 msec and subsequently decays with a time constant from 150 to 200 msec. In curarized (cut or uncut) diaphragms, however, the conditioning stimulus causes a depression which reaches its maximal value at 10 msec and then decays slowly with a time constant of about 3 sec. This indicates that curare strongly interferes with the process of transmitter release. 2. The presynaptic action of curare is also evident if short tetanic trains are given. In uncurarized preparations e.p.c.s decay in size much more slowly than in curarized preparations, and usually show a transient facilitation. 3. These results can be explained in terms of a model where curare blocks presynaptic depolarizing action of ACh. As a result of this presumed curare action a small increase in Ca permeability and subsequent entry of Ca associated with depolarization are also blocked, and the facilitation resulting from that entry of Ca is abolished. INTRODUCTION

While the post-synaptic action of curare is well documented, there is still a controversy about its possible presynaptic action. It has been observed that curarized muscles in vitro are not as able to sustain contractions at frequencies of stimulation from 5 to 25 Hz as non-curarized muscles in vivo (Adrian & Bronk, 1928, 1929; Emmelin & MacIntosh, 1956), indicating possible presynaptic action of curare. Moreover, electrophysiological studies on curarized preparations in vitro at similar frequencies of stimulation indicate that there is a rapid decline in transmitter release (del Castillo & Katz, 1954; Brooks & Thies, 1962; Elmqvist & Quastel, 1965). However, on the basis of release measurements (Dale, Feldberg & Vogt, 1936; Krnjevi6 & Mitchell, 1961; Straughan, 1960) it has been claimed that curare has no marked presynaptic effect. More recently electrophysiological studies on cut muscle preparations (Hubbard, Wilson & Miyamoto, 1969; Hubbard & Wilson, 1973) have suggested that curare does affect the pattern of transmitter release during tetanic stimulation. This has been contested by Auerbach & Betz (1971) who have claimed that there was no * Present address: Department of Biophysics, University College London, Gower Street, London WC1E 6BT

0022-3751/79/3390-0464 801.50 C 1979 The Physiological Society

M. I. GLA VINO VIO presynaptic action of curare. In addition, Auerbach & Betz (1971) also argued that in cut muscle preparations positioning of the micro-electrodes is intrinsically less accurate, and that conclusions based on results obtained from this preparation could be misleading, particularly if voltage clamping was attempted. It was subsequently shown that these fears are exaggerated and that the cut muscle preparation is probably a good model for studying transmitter release at normal levels (Hubbard & Wilson, 1973; Glavinovic, 1979). The following study was performed in order to clarify the existence and nature of any presynaptic action of curare. 500

METHODS All experiments were done on the isolated, cut left hemidiaphragrms of S-D (240-250 g) male rats, using a voltage clamp technique. For further details concerning the preparations, see the accompanying paper (Glavinovi6, 1979).

RESULTS

The influence of a conditioning impulse on a subsequent test impulse The experiments began on an uncut, curarized rat diaphragm preparation. Then, the preparation was 'cut 'and after about 2 hr the tests were repeated, first in the absence of curare, and then in its presence (the same concentration of curare as used initially). The end-plates were clamped and held at the same potential in all three cases (usually near -60 mV). Pairs of shocks, separated by an interval varying between 7 and 260 msec, were given to the phrenic nerve. The resulting first and second responses were averaged separately. The pairs of stimuli were repeated at intervals of 10 sec or more, which were considered long enough for complete recovery of normal conditions (Takeuchi, 1958). The degree of facilitation or depression of the second response was obtained from the ratio of the end-plate currents e.p.c.2/ e.p.c.1, where e.p.c.1 is the mean amplitude of the first response and e.p.c.2 the mean amplitude of the second response. A graph of the fractional change in amplitude after a single conditioning shock versus time is shown in Fig. 1. The data were obtained from three different preparations; in each case, those from curarized, normal uncut muscle are below, from curarized cut muscle in the middle and from uncurarized cut muscle above. It is evident from Fig. 1 that the application of curare greatly alters the nature and time course of the change in amplitude after a single conditioning shock. As a result of the conditioning stimulus in uncurarized cut muscle there was a facilitation which reached its maximum at 30-40 msec and then decayed with a time constant of 150-200 msec. By contrast, in curarized cut and uncut muscle preparations there was a similar very prolonged depression which was maximal at around 10 msec, and then decayed slowly with a time constant of about 3 sec.

Transient decay of e.p.c.s during high frequency stimulation (100-150 Hz) From the above results one would predict that even during short tetanic trains, particularly at high frequencies of stimulation, curare should have a considerable effect on the rate of a fall-off in e.p.c. amplitude.

PRESYNAPTIC ACTION OF CURARE

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Fig. 1. The influence of a conditioning first shock on subsequent test responses as a fimetion of time. Abscissa: time interval between conditioning and test shocks. Ordinate: ratio of the test and conditioning e.p.c.s. Each curve represents a single experiment and each point is an average of ten measurements. The stimulations were repeated at intervals of 10 see or more. Above: cut muscle in solution without blocking agents. Middle: same cut muscle in 0 7 /SM-D-tubocurarine. Below: same muscle before cutting, in 0-7 /SM-D-tubocurarine. In curarized preparations (middle and below) the experimental points at 7 msec intervals are not shown by circles to avoid crowding of the diagram.

Trains of 11 or 9 pulses (at 150 or 100 Hz) were recorded in cut uncurarized, and cut and uncut curarized preparations. Three inset traces in Fig. 2 illustrate the progressive decrease in amplitude of e.p.c.s during such short trains. In the same Figure, averaged values of ten such trains on a semilogarithmic plot for each kind of experiment are shown. As expected, during stimulation at high frequency, synaptic transmission is not sustained as well in curarized preparations as in non-curarized preparations. In this respect there was no significant difference between cut and uncut preparations. Since curare affects the time course of decay of e.p.c.s during short tetanic trains, the estimates of the probability of release were determined in curarized and uncurarized

M. I. GLA VINO VI6

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Fig. 2. End-plate currents, generated by short tetanic train, plotted against stimulus number. The data represent the average of ten such tetanic trains. The trains were separated by at least 90 sec. The bath was held at room temperature (23 0C). The traces of three such tetanic trains in the different experimental situations are also illustrated. Above: short tetanic stimulation ( 11 pulses at 150 Hz) of a cut muscle in a solution without blocking agents. The line represents the best fit from the 2nd to 11th responses, assuming an exponential decay. The responses are normalized to an extrapolated maximum at zero time. The standard errors ( ± ) of the 3rd and 9th pulse are indicated. The second response is larger than the first, indicating early transient facilitation. Middle and below: short tetanic stimulation (9 pulses at 100 Hz) of a normal and a cut muscle respectively, both in 0-7 SM-D-tubocurarine. Note two phases of decay of e.p.c.s; the first line represents the best fit for the 1st to 5th responses and the second for the 6th to 9th, both assuming exponential decay.

preparation from the tetanic rundown of e.p.c.s (cf. Christensen & Martin, 1970) in order to compare with previously obtained values similarly determined. In both cases it was assumed that during tetanic trains: (a) there is no replenishment of the immediately available store, (b) probability of release remains constant and (c) capacity of the immediately available store to contain transmitter remains constant. For data from non-curarized cut preparations, a straight line was fitted by the least squares method to all the points following the first and extrapolated to the maximum value of 100% at zero time. For data from curarized preparations (cut

503 PRESYNAPTIC ACTION OF CURARE and uncut), two lines were necessary to fit the data: the first, through the initial five points, was normalized to a maximum value of 100% at zero time; and the second was fitted to the later responses (from the sixth to ninth). Rates of exponential decay were calculated from the slopes of these lines (in the case of curarized preparations only the first line was used). Note that in uncurarized preparations, the second response was greater than the first, indicating an early transient facilitation which corresponds to the facilitation seen when two pulses were applied at short intervals (see Fig. 1). Estimates of the probability of release at six different junctions in cut uncurarized, uncut curarized and cut curarized muscles were determined. The values of p in cut uncurarized preparations were all between 0-031 and 0 055. The values of p in uncut curarized and cut curarized preparations were consistently higher and alike, all being between 0*14 and 0*24. This is in good agreement with the values obtained by other investigators either from uncut curarized preparations (Christensen & Martin, 1970) or cut curarized and uncurarized preparations (Hubbard & Wilson, 1973). DISCUSSION

In addition to the well known post-synaptic effect of curare, there seems to be a significant presynaptic action. The present results indicate that curare markedly interferes with the process of transmitter release, especially at high frequencies of stimulation. This was demonstrated by analyzing the responses in short highfrequency trains, as well as by mapping the time course of facilitation or depression after a conditioning stimulus in normal and cut muscle, in the presence or absence of curare. It is interesting to consider how the present results compare with already published data. It has long been known that curare is a more efficient blocker at high frequencies of stimulation (Eccles, Katz & Kuffler, 1941; Feng, 1941). Experiments of Masland & Wigton (1940) and Blackman (1963) indicated that, judging from the force of muscle twitch, the degree of neuromuscular block was greater in the presence of curare the higher the frequency of stimulation. The release studies, however, produced controversial results. The experiments of Dale et al. (1936), Krnjevi6 & Mitchell (1961) and Straughan (1960) did not show any significant effect of curarization on transmitter release. This was always considered a strong argument in favour of purely post-synaptic action of curare. However, in those experiments the transmitter release was studied during low frequency of stimulation. In the more systematic studies of Beani, Bianchi & Ledda (1964) it was observed that the release was lower in curarized than in uncurarized preparation, this decline being greater at higher frequencies of stimulation. Repetition of these experiments by Chang, Cheng & Chen (1967) under the same conditions as Beani et al. (1964) did not confirm their findings. It has been observed recently (Katz & Miledi, 1977) that quantal release is only a small fraction of the total release (perhaps as low as 1 %). Moreover, the results of release studies do not necessarily have to agree with electrophysiological results and indeed it is possible that they have different pharmacology. Auerbach & Betz (1971) have reported that, in the frog, curare decreases the

M. I. GLAVINOVI( size of e.p.c.s (obtained with low frequencies of stimulation) and m.e.p.c.s equally, leaving the quantal content unchanged. In addition they claimed that in cut rat diaphragms, an increase in concentration of curare does not change the time course of synaptic output during short tetanic trains. Similarly, Beranek & Vyskocil (1967) observed that in rat Mg-depressed neuromuscular junction curare did not affect quantal content (obtained with low frequency of stimulation). This is not necessarily in disagreement with the present results. Curare seems more potent at high frequencies of stimulation and it is entirely possible that at frequencies as low as 0 2 to 10 Hz it does not affect the transmitter release. It is worth noting, however, that the time course of depression at curarized rat diaphragm (Lundberg & Quilisch, 1953) after a conditioning pulse (using e.p.p.s as indicators of transmitter release) was in good agreement with the present results. Moreover, Wilson's (1974) observations (see his Table 1) show that in cut muscle rat diaphragm preparations, in normal Ringer solution the second e.p.p. of a short tetanic train (40 pulses at 100 Hz) was larger than the first; but in the presence of curare the second e.p.p. was smaller than the first. This agrees well with the present results. The experiments of Hubbard & Wilson (1973) also indicated that the pattern of e.p.p.s recorded in cut rat diaphragm preparations during repetitive stimulation at 150 Hz is different in the presence and absence of curare. Evidently, the curarized preparations are less able to maintain a high level of transmitter output during tetanic stimulation. Moreover, Galindo (1971) reported that curare (10-7) g/ml.) slows down the recovery of e.p.p. amplitudes following tetanic stimulation and at still lower concentrations (104 g/ml.) it significantly reduces the frequency of m.e.p.p. Therefore one can say that the present results are supported by much of the published evidence, and conclude that curare does affect the ability of the presynaptic terminal to sustain high levels of synaptic output during stimulation at high frequency. The following is further evidence that curare interferes with the activity of the presynaptic terminal: (1) it abolished the ACh-induced decrease in threshold for antidromic action potentials evoked by stimulating current pulses (Hubbard, Schmidt & Yokota, 1965) and in addition (2) Masland & Wigton (1940) and Standaert (1964) reported that is suppresses repetitive antidromic discharges of motor nerves. This, however, was contested by Chang et al. (1967). Although the mechanism of the possible curare action on the presynaptic terminal is not known, various hypotheses have been postulated. It may prevent or reduce the release of transmitter (Riker & Okamoto, 1969) or alternately it may interfere with the metabolic turnover of ACh (Bhatnagar & MacIntosh, 1967). Katz & Miledi (1965) suggested that curare prevents ACh from acting on the same nerve terminal to release more transmitter. It seems probable that curare prevents a presynaptic action of ACh by blocking the depolarizing action of ACh on the presynaptic terminal. The effect of curare on the threshold of ACh-induced antidromic action potentials (Hubbard et al. 1965), on repetitive antidromic discharge of motor nerves (Masland & Wigton, 1940; Standaert, 1964), as well as on the frequency of m.e.p.p.s (Galindo, 1971) can be explained on these grounds. As a result of this presumed action of curare, a small increase in Ca permeability and influx of Ca associated with this and which normally causes facilitation (Katz & Miledi, 1968) are eliminated, making subsequent responses relatively depressed.

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I am indebted to Dr K. Krnjevi6 for many helpful comments on this manuscript. Many ideas given by Dr A. Padjen are greatly appreciated. This paper is based in part on a thesis presented to McGill University in partial fulfilment of requirements for the degree of Doctor of Philosophy. This work was supported by the Medical Research Council of Canada. REFERENCES ADRIAN, E. D. & BRONK, D. W. (1928). The discharge of impulses in motor nerve fibres. Part 1. Impulses in single fibres of the phrenic nerve. J. Physiol. 66, 81-101. ADRIAN, E. D. & BRONK, D. W. (1929). The discharge of impulses in motor nerve fibres. Part II. The frequency of discharge in reflex and voluntary contractions. J. Physiol. 67, 119-151. AUERBACH, A. & BETZ, W. (1971). Does curare affect transmitter release? J. Phy8iol. 213, 691-705. BEANI, L., BiRNcmI, C. & LEDDA, F. (1964). The effect of tubocurarine on acetylcholine release from motor nerve terminals. J. Physiol. 174, 172-183. BERANEK, R. & VYsEOCIL, F. (1967). The action of tubocurarine and atropine on the normal and denervated rat diaphragm. J. Phyaiol. 188, 53-66. BHATNAPAR, S. P. & MAcINTOSH, F. C. (1967). Effects of quaternary bases and inorganic cations on acetylcholine synthesis in nervous tissue. Can. J. Physiol. Pharmac. 45, 249-268. BLACKMAN, J. G. (1963). Stimulus frequency and neuromuscular block. Br. J. Pharmac. 20, 5-16. BROOKS, V. B. & THIEs, R. E. (1962). Reduction of quantum content during neuromuscular transmission. J. Phy"iol. 162, 298-310. CHANG, C. C., CHENG, H. C. & CHEX, T. F. (1967). Does D-tubocurarine inhibit the release of acetylcholine from motor nerve endings? Jap. J. Physiol. 17, 505-515. CHRISTENsEN, B. N. & MARTn, A. R. (1970). Estimates of probability of transmitter release at the mammalian neuromuscular junction. J. Physiol. 210, 933-945. DALE, H. H., FELDBERG, W. & VOGT, M. (1936). Release of acetylcholine at voluntary motor nerve endings. J. Phygiol. 86, 353-380. DEL CASTILLO, J. & KATZ, B. (1954). Statistical factors involved in neuromuscular facilitation and depression. J. Phyiiol. 124, 574-585. ECCLES, J. C., KATZ, B. & KUFFLER, S. W. (1941). Nature of the end-plate potential in curarized muscle. J. Neurophy8iol. 4, 363-387. ELMQVIST, D. & QUASTEL, D. M. J. (1965). A quantitative study of end-plate potentials in isolated human muscle. J. Phy8iol. 178, 505-529. EMMELIV, N. & MACINTOSH, F. C. (1956). The release of acetylcholine from perfused sympathetic ganglia and skeletal muscles. J. Phygiol. 131, 477-496. FENG, T. P. (1941). Studies on the neuromuscular junction XXVI. The changes of the end-plate potential during and after prolonged stimulation. Chin. J. Physiol. Rep. Ser. 16, 341-372. GA~nmo, A. (1971). Prejunctional effect of curare: its relative importance. J. Neurophysiol. 34, 289-301. GLAvINovI6, M. I. (1979). Voltage clamping of unparalysed cut rat diaphragm for study of transmitter release. J. Phyajol. 290, 467-480. HUBBARD, J. I., SCHMIDT, R. F. & YOKOTA, T. (1965). The effect of acetylcholine upon mammalian motor nerve terminals. J. Physiol. 181, 810-829. HUBBARD, J. I., WIrSON, D. F. & MIYAMOTO, M. (1969). Reduction of transmitter release by D-tubocurarine. Nature, Lond. 223, 531-533. HUBBARD, J. I. & WILSON, D. F. (1973). Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of D-tubocurarine. J. Phyaiol. 228, 307-325. KATZ, B. & MILEDI, R. (1965). Propagation of electric activity in motor nerve terminals. Proc. R. Soc. B 161, 453-482. KATZ, B. & MILEDI, R. (1968). The role of calcium in neuromuscular facilitation. J. Physiol. 195, 481-492. KATZ, B. & MILEDI, R. (1977). Transmitter leakage from motor nerve endings. Proc. R. Soc. B

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KRNJEVI6, K. & MrcinLL, J. F. (1961). The release of acetylcholine in the isolated rat diaphragm. J. Phyjiol. 155, 246-262. LUNDBERG, A. & QuLsG, H. (1953). Presynaptic potentiation and depression of neuromuscular transmission in frog and rat. Acta phy8iol. 8cand. 30, supply. 111, 111-120. MASITAND, R. L. & WIGTON, R. 8. (1940). Nerve activity accompanying fasciculation produced by prostigmine. J. Neurophysiol. 3, 269-275. Rnim, W. F., JR. & OKA o, M. (1969). Pharmacology of motor nerve terminals. Ann. Rev. Pharmac. 9, 173-208. STANDAERT, F. G. (1964). The action of D-tubocurarine on the motor nerve terminal. J. Pharmacy. exmp. Thor. 143, 181-186. STRAUGHAN, D. W. (1960). The release of acetylcholine from mammalian motor nerve endings. Br. J. Pharmac. 15, 417-424. TAxEcucH, A. (1958). The long lasting depression in neuromuscular transmission of frog. Jap. J. Phyeiol. 8, 102-113. WILmoN, D. F. (1974). Facilitation of transmitter release at the mammalian neuromuscular junction. Am. J. Phyaiol. 227, 1098-1102.

Presynaptic action of curare.

499 J. Phpiol. (1979), 290, pp. 499-506 With 2 text-flgume Printed in Great Britain PRESYNAPTIC ACTION OF CURARE BY M. I. GLAVINOVIC* From the Depar...
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