EFFECTS OF TETRODOTOXIN ON THE NEUROMUSCULAR JUNCTION

Transcription

1 EFFECTS OF TETRODOTOXIN ON THE NEUROMUSCULAR JUNCTION TARO FURUKAWA, TADAO SASAOKA AND YUJI HOSOYA* Department of Physiology, Osaka City University Medical School, Abeno-ku, Osaka, Japan Tetrodotoxin is a purified toxin extracted from Japanese "Fugu", i.e., swell fish. Because of its severe toxicity, many pharmacological and toxicological studies have been carried out in Japan since old times. Concerning the action of it on nerve and muscle, the toxin was reported in some of these earlier experiments to produce a neuromuscular block in the animal (7, 13, 14). In the later investigations, however, it has been found that the action of the toxin is not necessarily limited to the neuromuscular junction but is more general, i.e., it also lowers the excitability of the muscle and nerve and leads to a complete narcosis (6, 18). Though some interesting reports have since been made (10), studies so far made were mostly based on classical techniques, the only exception being the one reported by Matsumura and Yamamoto (12). So, it seems of some interest to reinvestigate the action of tetrodotoxin on neuromuscular junction with electrophysiological means. In the present study, recording of the end-plate potential (e.p.p.) have been made by means of an intracellular electrode, and the effects of the toxin on the depolarization of the end-plate produced by acetylcholine (ACh) have also been investigated. METHODS The experiments were performed on the sartorius nerve-muscle preparation of frog, but in some experiments, the sartorius muscle of toad was used. The recording of e.p.p. was made through a microelectrode of Ling and Gerard type (11), inserted into the junctional region of the muscle fiber. In cases where an indirect stimulus was followed by a contraction of the muscle, a flexibly mounted microelectrode (17) was used for the recording of e.p.p. Single stimuli were periodically sent through the nerve during the experiment. Tetrodotoxin and other drugs to be tested were mixed into the solution in which the preparation was immersed. Minimal concentration of drugs needed for the production of a conduction block in a motor nerve fiber was determined on the single nerve fibergastrocnemius preparation of frog and toad, twitchs of the muscle in response to single indirect stimuli being used as an indicator. Received for publication September 23,

2 144 T. FURUKAWA ET AL. Depolarization of the end-plate due to ACh externally applied was measured on the eserinized sartorius muscle of frog by means of the method of moving fluid surface electrode (2). Tetrodotoxin used was supplied by Sankyo Company, Tokyo. Most of the present experiments were carried out with a crystalline tetrodotoxin. But, some of the earlier experiments were carried out with a crude preparation, the potency of which was about 1% of the crystalline one. Crystalline tetrodotoxin is a white powder and is soluble in water. In making a stock solution of tetrodotoxin, the crystals are dissolved in a drop of ice acetic acid. The solution is diluted with water, PH of the solution being adjusted to 4.8. This solution maintains its potency for a very long time at room temperature. RESULTS A. Preliminary experiments with external electrodes A preparation was dipped into the solution of tetrodotoxin, and periodically tested with maximal shocks delivered to the nerve. When 5 ~10-9 (weight/ volume) solution of tetrodotoxin was used, muscle contraction in response to an indirect stimulation became weak in about 30min. At this stage, relatively large e.p.p. superimposed by small action potential of the muscle could be recorded from the surface of the muscle with a suitably located recording electrode. Several minutes later, muscle action potential disappeared and only e.p.p. remained as shown in fig. 1. This state was maintained for about 10min. or so. At this stage, a direct stimulation was effective and gave rise to a twitch of the muscle which was preceded by a propagated action potential, though its amplitude was small. Thereafter, e.p.p. became gradually smaller until it disappeared completely in 30min. electrodes. FIG. 1. E.p.p. recorded with external 35min. after the application of 5 ~10-9 tetrodotoxin. Time mark:1 msec. apart. more, when the muscle was found to be totally paralyzed and showed only a weak local contraction in response to a strong direct stimulation. With a little more diluted solution, e.g., 2.5 ~10-9, time course of changes was delayed, and it took about two hours before the muscle showed no contraction in response to an indirect stimulation. But, general feature was very similar to that described above. Similar results were obtained also in the toad's preparation, though it was necessary to use a solution about 10 times more concentrated than for a frog's preparation. When 5 ~10-8 tetrodotoxin was used, a complete neuromuscular

3 TETRODOTOXIN ON NEUROMUSCULAR JUNCTION 145 block was produced after about an hour, retaining a more vigorous direct contractility. Toad's nerve also seems to be more resistive to tetrodotoxin as will be referred to later. It seems certain that a neuromuscular block is produced by tetrodotoxin, because, as has just been stated above, e.p.p. is recorded and the direct excitability of the muscle is preserved in certain stage of the tetrodotoxin action. But, this does not mean that the action of tetrodotoxin may be selectively localized to the neuromuscular junction. On the contrary, the reverse seems to be true, i.e., the selectivity of action to the junction seems to be slight. Sometimes, a weak local contraction to an indirect stimulation is observed until shortly before the total abolishment of e.p.p. This phenomenon, which is frequent in winter preparation, seems to be attributable to the fact that e.p.p. at each junction is so large that it is followed by a local response which in turn causes a local contraction of the muscle fiber. On the contrary, a preparation which has, from the beginning, a low safety factor of transmission, shows a more typical neuromuscular block which is easily produced by a very diluted tetrodotoxin. Changes produced by tetrodotoxin were mostly reversible when the preparation was returned to Ringer, but the recovery was often incomplete and took rather a long time. B. Experiments with microelectrodes 1. Initial changes following the application of tetrodotoxin When a flexibly mounted microelectrode is used, it is easy to follow changes in the action potential and in e.p.p. produced by the toxin. Fig. 2, A shows a typical end-plate spike recorded in a normal muscle. In this and following records, several traces were superimposed, the stimulus being sent through the nerve every 5 sec. When 10-8 was applied, there occurred a characteristic sequence of changes as shown in fig. 2, B-H, and in fig. 3. First sign of the effect is a decrease in the amplitude and the rate of rise of the spike. The peak comes a little later, so that it roughly coincides with the position of the end-plate hump (fig. 2, B, C, D). On the other hand, the rising phase corresponding to the end-plate step does not seem to suffer noticeable change. Thus the diminution of the spike is especially evident in its initial part. This may be explained on the basis of the following two factors:1. A permeability increase to sodium ions, which forms an essential part for the generation of the action potential, becomes weaker. 2. The short circuit action of the transmitter is maintained well. Gradually, the size of the response decreases and the all or none spike merges into a local response and eventually there remains only e.p.p.(fig. 2, G, H). Thereafter, the size of e.p.p. is maintained for a certain period of time, though there occurs a slight but progressive lengthening in the latent time of e.p.p. Then, e.p.p. is abolished abruptly due perhaps to a blockage of conduction in the motor nerve fiber. Curves in fig. 2 are superimposed on each other and shown in fig. 3. It is to be noted that the transition, from a normal end-plate spike to an

4 146 T. FURUKAWA ET AL. A FIG. 2 B C FIG. 3 FIG. 4 FIG. 2. Changes in the end-plate action potential following the application of 10-8 tetrodotoxin. A:before, B-H (D:30 sec., F:1min., G:2min., H:3min.) after the application. Time mark:1 msec. apart. FIG. 3. Same traces as in fig. 2, but they are superimposed. FIG. 4. A large end-plate response which decrements spatially. A:record at the end-plate center, B:0.5mm., C:1.0mm., D:1.5mm. apart from the center. Tetrodotoxin:1.5 ~10-9. Time mark:1 msec. apart. e.p.p. superimposed by a local response of varying grade, is always gradual. So, it is somewhat difficult to say from the potential record the exact time when the e.p.p. failed in evoking a propagated response of the muscle fiber. Not infrequently, very large end-plate response was found to be non-propagating. Fig. 4 shows an example. In this case, the preparation was initially put into a 5 ~10-9 solution of tetrodotoxin for 90min., then it was moved to another containing 1.5 ~10-9 tetrodotoxin, the recording being made in this solution. The response shown in A has a size of about 80mV, and seems to consist of e.p.p. and a local response of the muscle fiber. Though it looks a little unusual, this response was found to be non-propagating, since a spatial decrement along the length of the muscle fiber was observed as shown in fig. 4, B, C and D.

5 TETRODOTOXIN ON NEUROMUSCULAR JUNCTION Experiments on curarized preparations For a study of the effect of tetrodotoxin on e.p.p., curarized preparation was a convenient material, for the toxin has only very slight, if any, depressing effect on ACh depolarization. A preparation was curarized first with 3 to 5 ~10-6 tubocurarine, and e.p.p. was recorded with an intracellular electrode, the nerve being stimulated periodically every 5 to 20sec. After 5 minutes' control recording, the bathing solution was replaced by the one containing tubocurarine and 3 to 10 ~10-9 tetrodotoxin, and changes in the size of e.p.p. were followed. Fig. 5 shows the time course of an experiment, in which stimuli were delivered periodically to the nerve at every 5sec. throughout the whole period. In A of this figure, mean sizes for every one minute's e.p.p. are shown with filled circles, but when the use of the mean was not feasible on account of large individual variations in the size of successive e.p.p.s, representative single values were selected and they are indicated with open circles. There was some reduction in the size of e.p.p. even before the application of tetrodotoxin, for there was not enough pause between each successive stimulus. But, the reduction stopped after 15min. When 9 ~10-9 tetrodotoxin was applied there immediately followed a decrease in the size of e.p.p. The decrease proceeded gradually until there occurred an abrupt abolishment of the e.p.p. But, when the concentration of the toxin was reduced to 2 ~10-9, the e.p.p. soon reappeared, though it was again abolished after another 5min. When the toxin was removed totally, e.p.p. reappeared and after 6 ~10-9 tetrodotoxin was applied it decreased again in size and was abolished. A similar procedure was once more repeated. FIG. 5. Effects of tetrodotoxin on the size of curarine e.p.p. Stimuli were sent once every 5sec. A:full course of an experiment. B:detail of the part marked in A. Filled circle:mean size of 10 successive e.p.p.s. Open circle:individual size of e.p.p.

6 148 T. FURUKAWA ET AL. It was usual that the size of e.p.p. decreased to some extent after the the addition of tetrodotoxin. In the case shown in fig. 5, A, the decrease continued until e.p.p. was, after a certain period, abolished abruptly. But, in others, the decrease ceased soon and the size of e.p.p. was thereafter maintained for rather a long time. Such differences in the time course of the tetrodotoxin action, however, depend chiefly on the concentration of the toxin used and also on the condition of the preparation. Abrupt abolishment of e.p.p. is clearly due to a conduction block in nerve fiber near its terminal portion, and the decrement in the size of e.p.p. too seems to be attributable to a weakened action current in nerve fiber resulting in a decrease in the released ACh, though another possibility of a direct action on ACh release mechanism can not be excluded yet. The size of e.p.p. just before its abolishment was about 70% of the value before the application of tetrodotoxin, though smaller values were often encountered. Another interesting phenomenon which deserves special mention is stepwise changes in the size of e.p.p.s. Mostly such changes were observed just before the abolishment of e.p.p. and also in the course of recovery from it. An example is shown in the latter half of fig. 5, A. Fluctuations in the size of successive e.p.p.s are especially evident in the 5 minutes' period following the application of 2 ~10-9 tetrodotoxin. Details of this part of the figure are shown in fig. 5, B, in which sizes of successive e.p.p.s are shown without omission. Although, as shown in this figure, the size of e.p.p.s in the stable condition seems to be about 7.5mV, 3 responses have a size of 2.5mV, about 6 responses have a size of 5mV, and also there are many which are situated between 5 and 7.5mV. This phenomenon is similar in its appearance to the quantum fluctuations reported by Del Castillo and Katz (3), but seems to be caused by a quite different origin. The most plausible explanation will be that the fluctuations are due to a blockage of conduction in nerve fiber near its terminal arborization. Motor fibers and their connection with muscle fibers are shown in scheme in fig. 6. Now, it may easily be supposed that a conduction block is liable to occur at bifurcating points of the nerve. Then, e.p.p. in the end-plate E, for example, will be abolished in an all or none manner if the blockage occur at A or B, whereas a stepwise change in the e.p.p. size will be seen when the blockage occurs at C or D. A similar phenomenon has been reported by Krnjevie and Miledi (8). FIG. 6. A scheme representing the motor innervation of muscle fibers. C. Effects on conduction in nerve fibers Liminal concentration of tetrodotoxin needed for producing a conduction block in a motor fiber was measured on a single nerve fiber-gastrocnemius preparation, twitches of the muscle in response to single indirect stimuli being taken as an indicator. In such a preparation, effects of a drug appear very soon, and a conduction block occurs immediately (16). Also, the recovery is

7 TETRODOTOXIN ON NEUROMUSCULAR JUNCTION 149 very rapid when the drug is removed. The result was that the liminal concentration was 5 to 10 ~10-9 in frog's motor fiber and 5 to 10 ~10-8 in toad's fiber. Thus, there seems to exist no marked difference in the susceptibility between the motor fibers in the nerve trunk and those near their terminals. In the experiments stated in previous sections, however, it was the terminal portion of the motor fiber that seemed to be blocked earlier. Perhaps, it is because the fibers in the nerve trunk is protected from the drug action by a connective tissue barrier. Another factor is the fact that motor fibers bifurcate freely near their terminal portion, for it is quite possible that these bifurcating points are more susceptible to various narcotics. By the way, it is to be noted that the liminal concentration of tetrodotoxin for a conduction block differed very much between toad and frog, while such a difference was not found with procaine. On the other hand, it has been reported by Matsumura et al.(12) that the rapidly conducting fibers are more susceptible to tetrodotoxin than the slowly conducting ones. This fact was confirmed in the present study, but any detailed study has not been made. D. Effects on the depolarization of end-plates produced by ACh externally applied For this part of the experiment, excised frog's sartorius muscle was used and it was immersed at least for 30min. in 2 ~10-6 eserinized Ringer before the experiment. The depolarization of the end-plate region following the application of 5 to 10 ~10-7 ACh was measured by comparing the distribution of potentials along the length of the muscle before and after its application. Usually, recordings were made before and 30, 60 and 90 sec. after the application of ACh, but there was only a slight difference between the latter two. Fig. 7 shows an example of the record in which 5 ~10-7 and 1 ~10-6 ACh was applied. The ACh solution was removed immediately after taking the last photograph and the preparation was washed three times with eserinized Ringer and the next measurement was made 30 to 60min. later. When the effect of tetrodotoxin was to be tested, the toxin was mixed at a concentration of 5 to 10 ~10-8 to the eserinized Ringer and the muscle was immersed in it for at least 20min. before the test. FIG. 7. An example of the potential record by the moving fluid surface electrode method. Broken line:in eserinized Ringer. Continuous line:after the application of 5 ~10-7 and 1 ~10-6 ACh. Fig. 8 shows the results of 4 successful measurements, in which sizes of depolarization without tetrodotoxin are indicated with filled circles and those with tetrodotoxin are indicated with open circles. There is a tendency within each one series of experiment for the size of depolarization to become a little

8 150 T. FURUKAWA ET AL. smaller in later measurements. But, in no case does the decrease seem to be attributable to the effect of tetrodotoxin. When the rather high concentration of the toxin used is taken into consideration, it may be safely concluded that tetrodotoxin has only very slight, if any, depressing effect on ACh depolarization. A C B D FIG. 8. Sizes of ACh depolarization measured successively on single muscles. Filled circles:without tetrodotoxin. Open circles:with tetrodotoxin. DISCUSSION 1. Kurose (10) studied the effect of tetrodotoxin on the ACh contracture of the perfused gastrocnemius muscle of the toad and reported that tetrodotoxin had no influence on the ACh contracture of the muscle. He concluded from this and other findings that although tetrodotoxin narcotized twitch fibers of the muscle, it did not interfere with the ACh contracture of tonus fibers. His result suggested that tetrodotoxin had no curariform action, because it has become clear from the study of several authors (1, 9) that the ACh receptor of tonus fibers are of a similar property to that of the end-plate of twitch fibers. These inferences based on indirect evidences have been confirmed in the present study with more direct method, namely, tetrodotoxin has no depressive action on the ACh depolarization of the sartorius muscle. This property of tetrodotoxin seems to deserve special mention, because many local anesthetics, like procaine (4), have depressing effects on the ACh depolarization of the end-plate, though of course there may be a large difference in the strength of the effect. On the other hand, tetrodotoxin was proved to be a very potent narcotic, namely, a crystalline tetrodotoxin narcotizes the nerve and the muscle at a concentration of 5 ~19-9 (in frog) to 5 ~10-8 (in toad), i.e., about 104 to 105 times stronger than procaine. One of the authors has made use of this property of tetrodotoxin in his study on the fibrillation of muscle fibers produced by NH4+(5). Namely, the effect of NH4+ is not suppressed by tetrcdo-

9 TETRODOTOXIN ON NEUROMUSCULAR JUNCTION 151 toxin because the fibrillation is produced through the action of ACh released from the nerve ending, whereas K+ contraction (excluding slow contracture) of the muscle is totally suppressed by tetrodotoxin because of a large loss in the excitability. 2. Tetrodotoxin and neuromuscular block Results obtained in the present study have shown that in most cases a neuromuscular block is produced by tetrodotoxin. However, in some instances, a neuromuscular block is hardly produced by the toxin. Namely, in such a preparation, no e.p.p. is recorded with a surface electrode after a weak twitch of the muscle in response to an indirect shock has ceased. It is because a conduction block of the motor fiber occurs before the neuromuscular block becomes complete. It has been known that there is rather a wide range of variation in the safety factor of the neuromuscular transmission among different muscles or even among different end-plates in a same muscle. For instance, the safety factor is higher in m. sartorius than in m. gastrocnemius and m. semitendinosus, and it is considerably lower in toad's muscle than in frog's muscle (15). Now, the easiness with which a neuromuscular block is produced by tetrodotoxin seems to depend largely on this safety factor of transmission, namely, a neuromuscular block is likely to be produced when the safety factor is low. The safety factor of transmission is low when the amount of ACh released from the motor nerve endings is decreased or when the sensitivity of the end-plate receptor to the ACh is lowered. In both of these cases, it will be clear that a neuromuscular block is easily produced by tetrodotoxin, for it elevates the threshold of the muscle fiber. But, what will be the result when the safety factor of the transmission is high enough? As stated in results section, the size of curarine e.p.p. is decreased to about 70% of its original value by the action of tetrodotoxin before the conduction in its motor nerve fiber is blocked. But, since the decrease of 30% is rather slight, and also since the sensitivity of the end-plate to ACh is scarcely impaired, it is clear that a large e.p.p. will be produced, just until a conduction block occurs in the nerve fiber. In such a case, as shown in fig. 4, the original e.p.p. may be reinforced by the local response of the muscle fiber and there may be produced a local contraction even after the conduction in the muscle fiber is abolished. It is well known that a narcotized muscle fiber can set up a local response and a local contraction so long as a fairly strong stimulus is given. Thus, in such a case, an indirect shock does not cease to produce a weak contraction of the muscle until all the motor nerve fibers are blocked. SUMMARY 1. Effects of tetrodotoxin on the neuromuscular junction have been studied on the nerve sartorius preparation of frog. 2. Tetrodotoxin has a potent narcotic action on nerve and muscle, without depolarizing them. But, it does not suppress the sensitivity of the end-plate to ACh even at much higher concentration than needed for the narcosis of nerve and muscle. This point is rather different from many local anesthetics.

10 152 T. FURUKAWA ET AL. 3. When a dilute solution of tetrodotoxin was applied, the size of curarine e.p.p. (recorded by an intracellular electrode) decreased gradually to about 70% of the initial value, then e.p.p. was abolished abruptly due to a conduction block in the nerve fiber. Sometimes a stepwise change in the size of e.p.p. was observed, which may be explained by a blockage of conduction in the nerve fiber near its terminal arborization (fig. 6). 4. In most cases, a selective neuromuscular block was produced by tetrodotoxin, but not in some cases. A neuromuscular block seemed to be more easily produced in a preparation whose safety factor of transmission was low. Authors' thanks are due to Sankyo Company for their generous supply of crystalline tetrodotoxin. This research work was aided by a grant from the Ministry of Education. REFERENCES 1. BURKE, W. AND GINSBORG, B.L.J. Physiol. 132: 599, DEL CASTILLO, J. AND STARK, L. ibid. 116: 507, DEL CASTILLO, J. AND KATZ, B. ibid. 124: 560, FURUKAWA, T. Jap. J. Physiol. 7: 199, FURUKAWA, T., FURUKAWA, A. AND TAKAGI, T. ibid. 7: 252, ISHIWARA, F. Tokyo-Igakkai-Zasshi 31: 717, 1917 (Japanese). 7. IWAKAWA, K. AND KIMURA, S. Arch. Exper. Path. u. Pharmakol. 93: 305, KRNJEVIC, K. AND MILEDI, R.J. Physiol. 140: 440, KUFFLER, S.W. AND WILLIAMS, E.M.V. ibid. 121: 289, KUROSE, T. Folia Pharmacol. Japan. 38: 441, 1943 (Japanese). 11. LING, G. AND GERARD, R.W.J. Cell. and Comp. Physiol. 35: 39, MATSUMURA, M. AND YAMAMOTO, S. Jap. J. Pharmacol. 4: 62, OSAWA, K. Tokyo-Igakkai-Zasshi 3: 533, 1889 (Japanese). 14. TAKAHASHI, D. AND INOKO, Y. Arch. Exper. Path. U. Pharmacol. 26: 400, TAKEUCHI, A. Seitai-No-Kagaku 8: 133, 1957 (Japanese). 16. TASAKI, I. Nervous Transmission. Springfield: Charles C. Thomas, WOODBURY, J. W. Science 123: 100, YANO, I. Fukuoka-Ikadaigaku-Zasshi 30: 1669, 1937 (Japanese).