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1 22 6I2.743: ACTION POTENTIALS OF NORMAL MAMMALIAN MUSCLE. EFFECTS OF ACETYLCHOLINE AND ESERINE By G. L. BROWN From the National Institute for Medical Research, Hampstead, London, N.W.3 (Received 9 November 1936) IN a recent paper Brown et al. [1936] have shown that injection directly into the arteries of a normal mammalian muscle of small amounts of acetylcholine causes a quick twitch-like contraction only little different in time relations from the contraction evoked by a single volley in the motor nerve. Theoretical considerations led to the deduction that the acetylcholine contraction was probably a short asynchronous tetanus of the muscle fibres and in a footnote (p. 42) reference was made to the confirmation of this by electrical records. In the present paper an account is given of the action potentials associated with the excitation of muscle by injected acetylcholine. Brown et al. also showed that a small dose of eserine potentiated the response of muscle to single maximal volleys in the motor nerve, changing the muscular contraction from a single twitch into a brief tetanus. This phenomenon has subsequently been shown by Bacq & Brown [1937] to be a property common to other substances antagonizing choline esterase. In this paper the mechanical and electrical response of the muscle under eserine has been examined in detail, in relation especially to the frequency at which the poisoned muscle contracts repetitively in response to a single nerve volley. A number of experiments have been done in which the muscle has been subjected to two volleys in the nerve at varying time intervals. This has been considered in the light of the known failure of the eserine-treated muscle to respond to stimuli delivered above a very low frequency [Briscoe, 1936; Bacq & Brown, 1937] and the depressant effects of acetylcholine injected after eserine. Some

2 ACTION POTENTIALS OF MUSCLE preliminary observations have also been made on the effects on the response of muscle to nerve volleys, of exposure to supraliminal and subliminal concentrations of acetylcholine. METHODS Cats have been used throughout this investigation, either spinal or decerebrate or aniesthetized with chloralose, a substance which Bacq & B r o w n found to be without significant effect on the reactions of muscle ~~~~~~~S 221 A _~~~~~~ Fig. 1. Cat. 2-6 kg. Decerebrated. Record of contractions of soleus. (a) Close arterial injection of 2-5yACh., maximal motor nerve twitch and injection of 5y ACh. respectively. (b) Effect of intravenous administration of -8 mg. eserine on response of muscle to maximal nerve volleys at 1 sec. intervals. to eserine and acetylcholine. Most of the experiments on acetylcholine injection have been made on the gastrocnemius muscle with natural circulation by the method of Brown et al. [1936], injectring through the tibial artery, but recently it has been found possible to use the soleus. This muscle, consisting of "red", slowly contracting fibres, responds just as well to injected acetylcholine as does the gastrocnemius. The difference in time relations between the contraction evoked by acetylcholine and the response to a single motor nerve volley is less pronounced than in gastrocnemius on account of the slowness of the nerve twitch (Fig. 1). This figure also shows the very dramatic potentiating effect of eserine

3 222 G. L. BROWN upon the motor nerve twitch of this muscle. The soleus was prepared as follows. The gastrocnemius tendon was severed near the os calcis, leaving the soleus still attached to the bone, and reflected backwards to expose the tibial artery just after its passage between the heads of the muscle. The branches of the tibial artery to the soleus and to the knee joint were defined and the latter were ligated together with all branches of the artery to the soleus, except those actually supplying the muscle. The tibial artery was exposed down to the interosseous membrane, it was tied and a needle cannula was inserted into the central end. Injections were made through this cannula, the tibial artery central to the origin of the vessel supplying the soleus being clamped immediately before the injection was made. The gastrocnemius, tibialis anticus and other muscles of the foot were immobilized by section of the branches of the popliteal and tibial nerves and the leg was fixed for recording by drills in the condyles of the femur and the lower end of the tibia and fibula. In many of the experiments in which the action of eserine was studied, without arterial injections being made, the records were taken from the tibialis anticus. The appropriate drills in the bone for the muscle under experiment were secured to a heavy iron table which carried, on an adjustable vertical pillar, a stout torsion wire myograph of the "frictionless" pattern described by Eccles & Sherrington [193]. In all experiments the stimuli to the nerve were given by glass-shielded silver wire electrodes, placed on the sciatic nerve a few cm. distal to the origin of the nerves to the hamstrings' The stimuli, timed by a Lucas pendulum, were break induction shocks from coreless coils placed far apart and shielded in metal boxes. Theprimary current (2 V.) was varied by a series resistance. In most experiments stimuli of a strength 5-1 p.c. greater than that required to set up a maximal volley were used. When two stimuli were employed, at very short time intervals, preliminary records were taken to ensure that the second, falling in the relative refractory period of the nerve, was sufficiently strong to evoke a second response from the muscle. It was usually found desirable after completion of the preparation to leave the animal with the stimulating electrodes in place for I to 3 of an hour, since the strong stimuli used sometimes caused, when the nerve was freshly dissected, the setting up of a few repetitive discharges. The arrangements for electrical recording were as follows. The metal plate to which the immobilizing drills were attached was insulated from the iron table by fibre discs and the hook connecting tendon to myograph was interrupted by a section of insulating fibre. The leads from the muscle

4 ACTION POTENTIALS OF MUSCLE 223 for the action potential following nerve stimulation were either two silver pins, one in the tendon connected to the input grid and the other, connected to earth, inserted in the belly of the muscle or concentric needle electrodes [Adrian & Bronk, 1929] consisting of a hypodermic needle with a No. 36 gauge enamelled copper wire just appearing at the tip. The needle body was connected to earth and the copper wire to grid. For the action potentials evoked by acetylcholine injection, either the concentric needle electrodes were used or, to obtain closer localization, electrodes consisting of two enamelled No. 47 gauge wires twisted together and passed down a hypodermic needle or a fine glass capillary [Adrian & Bronk]. With the belly-tendon leads and the concentric needle electrodes no electrical screening of the animal was needed, but with the twin wire electrodes it was found necessary to enclose the animal in a large earthed metal box with a lid covering as much of the front as was compatible with manipulating the syringe for injection. It was found possible to effect the arterial injections without introducing artefacts, if the operator earthed himself by grasping a saline pad on the screening box and took care to touch only the glass piston of the syringe at the moment of injecting. The amplifier consisted of four resistance-capacity coupled stages connected to four pentodes in parallel actuating a Matthews' oscillograph. With belly-tendon leads, the last two stages and large coupling condensers, giving a fall to half value in -52 sec. with a rectangular impact potential, were found satisfactory. With the concentric electrodes, when recording the responses to nerve stimulation, three stages were used with small coupling condensers to minimize baseline drift. When recording action potentials from acetylcholine injection, the four stages were found to be necessary, but only seldom was more than half the maximum amplification (1 tv. = 5 mm. on camera) required. Acetylcholine was injected in acid saline (ph 4) in volumes of 5 c.c. for gastrocnemius and in.25 c.c. for soleus. Eserine when used was administered intravenously, the circulation being protected by the previous injection of 1 mg. atropine. RESULTS Brown et al. [1936] dealt in considerable detail with the mechanical response evoked from normal skeletal muscle by the "'close range" injection of small amounts of acetylcholine. The rapidity and general "twitch-like " character of the response to acetylcholine made it appear at first sight that an action potential not grossly different from that

5 224 G. L. BROWN accompanying a motor nerve twitch might be expected. This, however, was found to be incorrect. Leading from belly to tendon, with a degree of amplification greatly in excess of that required to reveal the motor twitch action potential, either failed entirely to show any response when the muscle was excited by acetylcholine, or showed at the best a small, slow, irregular shift of base-line. 2,Lv[ Fig. 2. Cat. Chloralose. Isometric myogram of gastrocnemius. Action potentials with concentric needle electrodes. Effect of closearterial injection of (a) 5y ACh., (b) -5 c.c. acid saline, (c) 25y ACh. 2 hours later. Higher speed recording surface. In this figure and all subsequent optical records the time record shows intervals of 1 msec. It was obvious, therefore, that the acetylcholine contraction, quick as it was, either had no accompanying electrical activity, which was unlikely, or that the contractions of the individual fibres were so out of phase as not to appear as a recognizable potential. Accordingly the lead from the muscle fibres was restricted by employing the conoentric needle electrode. Under these conditions of recording, injection of acetylcholine produces an irregular outburst of activity shortly preceding and during the mechanical response; a similar injection of saline at the same ph as

6 THE JOURNAL OF PHYSIOLOGY, VOL. 89, No. 2 PLATE I -o 4) ce P*4 o4 & o4c; n I 3 C)~ To face p. 224

7 PLATE II THE JOURNAL OF PHYSIOLOGY, VOL. 89, No. 2 Kg Fig. 11. Cat. Decerebrated. Myogram of gastrocnemius. Action potentials with concentric needle electrode. (a) Response to nerve volley. (b) Response to 2y ACh. by close arterial injection and to superimposed nerve volley. (The action potentials of the responses to the nerve volleys are obscured by the stimulation artefacts.) Kg. 3- r, moo, m, m m M. n 2 I O _ Fig. 12. Cat. Decerebrated. Myogram of tibialis anticus. Action potential, belly-to-tendon lead. 2 hours 2 min. after eserine. Response to single maximal nerve volley, (a) before and (b) 1 min. after intravenous injection of 1 mg. ACh.

8 ACTION POTENTIALS OF MUSCLE used for the acetylcholine has no such effect (Fig. 2 a and b). A similar response with a higher speed of recording surface is shown in Fig. 2c. These records establish beyond question that the contraction of normal mammalian muscle evoked by acetylcholine is a propagated disturbance in the muscle fibres, set up irregularly throughout the body of the muscle as the acetylcholine reaches the sensitive region of each fibre in succession. There can be no question of a contracture analogous to that occurring in denervated mammalian muscles and in normal muscles of birds and amphibia. That the individual muscle fibre responds more than once during the flooding of the muscle with acetylcholine is clear from the fact that tensions very greatly in excess of the maximal synchronous motor twitch can be produced, by injection of higher concentrations of acetylcholine. The increased localization provided by the concentric needle electrodes does not, however, permit of the detection of the frequency of the response of individual fibres, since the asynchronism of the contraction of the muscle is between fibre and fibre, and concentric electrodes of the usual size do not give a localization more precise than to a single motor unit, consisting of some 1 fibres. Accordingly a number of records have been made using the twin wire electrodes, which, under favourable conditions, allow of the picking up of the responses of individual fibres. Records obtained in this way are shown in Fig. 3 (Plate I). With these leads, shielding of the preparation has to be as complete as possible, and this precluded the simultaneous recording of the mechanical response of the muscle. The most satisfactory records have been obtained with relatively small doses of acetylcholine, and usually after a number of injections had been made; the frequent repetition of saline injections produces some degree of cedema among the muscle fibres and this, presumably, allows the action potential of a single fibre more easily to be picked out among the large number of excited fibres. There can be little doubt that the actual potentials recorded are those of single muscle fibres. The individual spikes of any one series remain at exactly the same amplitude and start and stop suddenly, but during their discharge the frequency falls along a smooth curve of characteristic shape; it is very improbable that the random excitation of fibres or groups of fibres by slowly diffusing acetylcholine would produce this constancy in amplitude and frequency. In Fig. 4 is shown graphically the frequency of response of single fibres taken from Fig. 3b. 7 msec. after the first irregular deflexion two discrete spikes appear 5 msec. apart. There is then an irregular discharge followed, 15 msec. from the start of the response to the injection, by regular recurring spikes of constant amplitude. This stops suddenly after PH. LXXXIX

9 226 G. L. BROWN some 9 msec. and is followed by the regular discharge of another, more distant fibre, which continues for 1-2 sec. The discharge of this fibre overlaps the last three discharges of the previous fibre and it may be c) * C).q C) a) a) I= 8r Fig. 4. I I I I I I, I I I I I I -2-4 o6-8 1 Time after first visible response in sec Curve showing the interval between the responses of single muscle fibres during stimulation by ACh. Taken from Fig. 3 (b) and (c). a) 6.., " 4 S B a 2 CaS H. * dhi. VSSOIs.* ::' I I I I I I I I I I I *2.4 * Time after first visible response in sec. Fig. 5. As Fig. 4. Composite curve from three separate injections of ACh. noted that the frequencies coincide. The curve shown in Fig. 4 appears to be characteristic of the response to acetylcholine. In Fig. 5 is shown a composite curve obtained from three separate injection responses taken at different times in the same experiment. The general form is much the same as in Fig. 4. *

10 ACTION POTENTIALS OF MUSCLE 227 EFFECT OF ESERINE ON MUSCULAR RESPONSE TO SINGLE MAXIMAL MOTOR NERVE VOLLEYS After the intravenous injection of doses of eserine of about *3 mg. per kg. there is, as Brown et at. showed, a gradually increasing potentiation of the muscular response to a single maximal motor nerve volley. The effect is maximal in about 5-1 min. Examination of the action potential simultaneously recorded from the muscle by belly-to-tendon leads shows that during this potentiation the main negative spike is followed by a series of small waves which are visible as discrete deflexions for as long as 6 msec. after the spike. The successive deflexions diminish progressively in size, the base broadens and the interval between consecutive discharges gradually lengthens until nothing more than a slight irregularity of the base-line remains [see Brown et al. Figs. 12 and 13] The progressive diminution in amplitude and the broadening of the base obviously indicate that the discharges of individual fibres are falling more and more out of phase. A restriction of the lead to a smaller number of muscle fibres by means of concentric needle electrodes- allows the repetitive discharge to be followed more precisely. A record taken in such a manner is shown in Fig. 8. It is clear that there is a progressive increase in the interval between consecutive responses, an increasing asynchronism among the individual fibres comprising the unit and, apart from the first few deflexions, a steady decrease in the total E.M.F. developed. The apparent larger amplitude of the deflexions immediately succeeding the first spike is due to their summing with the negative artefact produced by the concentric electrodes and the small coupling condensers. A number of measurements have been made of the frequency of the discharge. The shortest interval recorded between the first deflexion and the next was 1-7 msec. This was observed at the maximum of the mechanical potentiation in a cat which had received -3 mg. of eserine per kg. With smaller doses of eserine, or when the effects of a large dose are wearing off, the interval between the first two responses may lengthen to as much as 4 msec. The interval between the succeeding spikes shows a small progressive increase until the response becomes so asynchronous as to prohibit accurate measurement. The interval between two successive responses in relation to the time after the stimulus is shown graphically in Fig. 6. It must be noted that measurement is only possible of the intervals between the crests of the main defilexions and that this can, at the best, give only a value of the mean frequency of a large number of 15-2

11 228 G. L. BROWN discharging fibres, the measurement becoming more inaccurate as the temporal dispersion among the fibres increases. Attempts to confine the leads to single muscle fibres, as was done with the acetylcholine injection, C5 d 5. m X24 a3 o a) b a b H Ca I I a I I I a - a -~~~~ a a s..- I a Time after first response in msec S.5 'M J ) ) a) ~ 2 C "a2 Ca I _ W-- I I I I I 5 1 I 15 2 Time after first response in msec. Fig. 6. Curves showing interval between responses in repetitive response of muscle after eserine: (a) Dots (.) response to single maximal volley. Circles (o) response to two volleys 1-6 msec. apart. 6 min. after eserine. (b) Dots (.) response to single maximal volley. Circles (o) response to two volleys 1-6 msec. apart. 2 min. after eserine. (c) Dots (.) response to single volleys. Circles (o) response to two volleys 1-6 msec. apart. Crosses ( x) response to two volleys 4 msec. apart. Squares (m) response to two volleys 8 msec. apart. 27 min. after eserine. proved abortive, probably on account of the fact that, although the discharge becomes asynchronous, there is always sufficient activity throughout the muscle to confuse the record of a single fibre. In most experiments, the measurable, synchronous, repetitive discharge did not

12 ACTION POTENTIALS OF MUSCLE 229 last much more than 3-4 msec., but in one experiment discrete spikes were visible 1 msec. after the stimulus. This provided a graphic record of the later stages of the response not usually visible (Fig. 7). This curve bears a striking resemblance to the curve of the frequency of the fibres after injection of acetylcholine (Figs. 4 and 5), although there is, of course, a big difference in the values of the coordinates. As the experiment proceeds, and the effects of eserine pass off, the interval between the initial 8 C C 6 a) Dt 5 $4 3 I I I I I I I I I Time after first response in msec. Fig. 7. Curve showing interval between responses in long-lasting repetitive response of muscle to single maximal nerve volley after eserine. responses lengthens and there is some flattening of the curve. This effect does not appear to be due entirely to the diminution in the concentration of eserine circulating, since a second dose does not increase the frequency but serves apparently only to increase the number of the synchronous responses. EFFECT OF TWO MAXIMAL VOLLEYS Af-ter the usual dose of eserine, and when this has attained its maximum effectiveness, a second volley set up in the nerve during the synchronous repetitive discharge is without effect (Fig. 8). The myo-

13 23 G. l. ibrown graphic tension is the same as that produced by a single volley and no significant alteration is to be observed in the frequency, rate of decline or duration of the repetitive response. In Fig. 6 are shown three curves from one experiment. In the first (a) the dots show the frequency curve 4. I 1. 7.j.6/r., Fig. 8. Cat. 2-4 kg. Decerebrated. Myogram of tibialis anticus. Action potential with concentric needle electrodes. 27 min. after 72 mg. eserine intravenous. Responses to maximal nerve volleys. (a) Single volley. (b) Two volleys 1-6 msec. apart. (e) Two volleys at 8 msec. (d) Two volleys at 16 msec. in response to single volleys, and the crosses the response to two volleys set up 1-6 msec. apart. The records from which these points were obtained were taken 6 min. after the administration of eserine. The tensions developed in response to single volleys increased from 2-5 to 2-35 kg., whereas the double volleys produced tensions between 2-4 and 2-58 kg. There is clear evidence from the curve that the interval between the first

14 ACTION POTENTIALS OF MUSCLE 231 and second responses is shortened by the second volley. In conditions such as these the accelerated second response frequently has a narrower base than the second response occurring after a single volley only, indicating the production of a greater synchronization among the fibres. The second curve (b) was taken 2 min. after the administration of eserine and there was only a difference of I kg. between the tensions in response I. Kg. 3 p 2 Fig. 9. Cat. 2*7 kg. Chloralose. Myogram of tibialis anticus. Action potentials led from belly to tendon. One hour after eserine 9 mg. intravenous. Responses to maximal nerve volleys. (a) Single volley. (b) Two volleys 35 msec. apart. (c) Two volleys at 5 msec. (d) Two volleys at 2 msec. to the double and the single volleys. This absence of effect of the second volley is reflected in the frequency curve, there being no significant difference between the two sets of points. In the third curve, single volleys and two volleys at 1-6, 4 and 8 msec. apart were used, the record being taken 27 min. after eserine. The double volleys produced no increase in tension over the single. As the interval between the volleys is increased, usually when it reaches about 3 msec., there appears, in response to the second volley,

15 232 G. L. BROWN a visible action potential which is much reduced in size and is followed by no recrudescence of the repetitive activity. As the volleys are separated further the second action potential increases, a visible prolongation of the myogram is seen and, 2 msec. after the first, the second volley causes definite repetitive discharge after its main action potential (Fig. 9). When the first volley is reduced to such a size that little or no repetitive discharge follows, the depression of a subsequent maximal volley can be followed in greater detail. The depression of the second is apparently maximal when the two are separated only enough to allow Kvg._ 4 _ 3 _ Fig. 1. Cat. 2-6 kg. Decerebrated. Myogram of tibialis anticus. Action potential with concentric needle electrode. 1 hour 4 min. after eserine 8 mg. intravenous. (a) Response to single maximal nerve volley. (b) Response to two volleys 1-2 msec. apart. the second to appear as a discrete deflexion, viz. about 8 msec. Separation of the two volleys leads to a steady increase of the second, until some 2 msec. after the first it has reassumed its original amplitude. It is of interest that the total tension produced by the small volley and the succeeding depressed response is equal to the tension produced by the maximal volley alone. When a submaximal dose of eserine (-5-1 -mg. per kg.) is given, or the effects of an originally maximal dose are passing off, a maximal volley does not occlude a subsequent volley, but the two summate to produce an effect similar to that of one volley during full eserine action. Fig. 1 gives an example of such an effect. One hour and 4 min. after the administration of eserine ( 3 mg. per kg.), a single volley produced a response little different from that before eserine, but two volleys set

16 ACTION POTENTIALS OF MUSCLE up 1-2 msec. apart gave rise to a full repetitive response with a myographic tension greatly out of proportion to the summed effect of two such volleys before eserine. EFFECT OF ACETYLCHOLINE ON RESPONSE TO SINGLE VOLLEY 233 Brown et al. [1936] showed that the injection of a stimulating dose of acetylcholine did not materially affect the response of a muscle to a maximal volley falling 1 sec. after the injection, if the disappearance of acetylcholine from the muscle was not impeded by cedema or the presence of eserine. We have now tested the effect of a stimulant injection of acetylcholine on the response to a volley timed to fall during the acetylcholine contraction. The method employed was to arrange a knock-down key so that, at the moment of depressing the syringe piston, a circuit was opened which released the L u c a s pendulum delivering a break shock to the nerve. It was found that the response to a volley set up so close to the acetylcholine contraction as to fall during the decline of tension was not significantly altered (Fig. 11, Plate II). It must be admitted that the measurement of the tension developed is inaccurate, since the twitch superimposed on the acetylcholine contraction starts at a different initial tension; but, as different initial tensions do not affect the response of gastrocnemius very greatly, and a small diminution in tension is to be expected, on the assumption that some fibres are actually refractory at the moment of nerve stimulation, the conclusion appears to be justified that the response to the volley is, at least, not depressed. In the presence of a large dose of eserine, injection of an excitant dose of acetylcholine into the muscle produces, as noted above, a severe depression of the response to subsequent nerve volleys and a similar effect on subsequent volleys is produced by a nerve volley after eserine. If the actions of eserine are in fact due to its effect in causing persistence of liberated acetylcholine, then it might be expected that a small concentration of acetylcholine, insufficient either to excite or depress, circulating through a muscle might produce an effect analogous to that of eserine. This has proved to be the case. For instance, a cat (2-8 kg.) had received an intravenous injection of 2-2 mg. of eserine in all, in two successive doses. Two hours and 2 min. after the second injection, the effects of the eserine had nearly completely passed off. The tension developed in response to a maximal motor nerve volley had declined until it was only 9 p.c. greater than before eserine, and repetitive discharge was no longer visible. The cat then received 1 mg. of acetylcholine intravenously and stimulation of the motor nerve was continued at

17 234 G. l. BROWN 1 sec. intervals. After 1 min., the tension in response to a single maximal volley had increased to 2-5 kg. as compared with -85 kg. before the acetylcholine. Simultaneously a typical repetitive discharge of the muscle was observed, the action potentials appearing exactly like those after a full dose of eserine (Fig. 12, Plate II). The effect gradually passed off and in some 5 min. the contractions were once more of the simple nonrepetitive character. The effect is observable equally well after removal of the suprarenals. DISCUSSION In a preceding paper [Brown et al. 1936], discussion of the manner in which acetylcholine, given by close arterial injection into a normal muscle, will reach and stimulate the separate muscle fibres led to the view that the quick response of the muscle to such an injection would have the nature of a brief, asynchronous tetanus. Electrical records have now demonstrated the correctness of this deduction. Records from single muscle fibres have shown that the response starts at a frequency of some 2 per sec. and then gradually declines. There is no reason to doubt that these responses are anything but "All-or-Nothing", propagated, disturbances initiated at the motor end plate. Brown et al. [1936] showed that a contraction of this type is not followed by any depression of the response of the muscle to a single volley following some 1 sec. afterwards, if the rapid disappearance of acetylcholine is not prevented by cedema or eserine. This observation has been confirmed and extended to show that the effect of a maximal volley d u r i ng the acetylcholine contraction is likewise unaffected. In the paper mentioned above, and in a subsequent one by Bacq & Brown [1937], evidence was given that when eserine is present, acetylcholine, whether liberated by impulses at the nerve endings or injected into the blood vessels, has a depressant after-effect on the response to further nerve impulses. Brown et al. suggested that this depressant action was exerted, not on the motor end plates, but on the general body of the muscle fibres, attributing it to the lingering of acetylcholine in the interstitial spaces. Evidence which I have here put forward makes it unlikely that such an action, if present at all, will account for all the phenomena of depression. Records such as that of Fig. 3 give clear evidence of a stimulation of the end plate of a muscle fibre by acetylcholine lasting certainly more than 1 sec. This must be pictured as due to acetylcholine persisting in the paramuscular spaces, from which it maintains, by diffusion, a stimulant concentration at the muscle end plate, where it excites and is immediately destroyed by a local concentration of cholinesterase, and where, failing such renewal,

18 ACTIbN POTENTIALS OF MUSCLE 235 it would immediately fall below stimulating strength. Yet under these conditions, in which a persistence of acetylcholine in contact with the general body of the muscle fibre must be postulated, no depression is produced of the response to renewed stimulation by a nerve volley. It is only when eserine is present in sufficient strength to weaken this action of the cholinesterase concentrated at the nerve ending that the after depression comes into the picture. The meaning of this depression, and the nature of the excitable structures which it involves, are at present being further investigated. The hypothesis that the repetitive response of the muscle fibre to a single nerve impulse after eserine is due to the persistence of acetylcholine has received further support from these experiments. The introduction into the circulation of amounts of acetylcholine, in themselves insufficient to excite the muscle directly, has been shown to evoke the typical, repetitive response of the muscle produced by a full dose of eserine. Furthermore, it has been shown that when insufficient eserine is present to evoke a repetitive muscular response, two volleys set up close together in the nerve can produce peripherally a summation, which is evidenced by the appearance of long-lasting repetitive contractions. The fact that this phenomenon can be observed when the volleys are separated by as much as 15 msec. makes unfeasible any explanation of the eserine effect based on an augmented irritability of the muscle fibre. The data obtained of the time relation of the multiple response of the muscle to a single nerve volley after eserine are in complete consonance with the conception of the liberation at the nerve ending of a charge of acetylcholine which, normally destroyed during the refractory period, can, after eserine, persist and re-excite the fibres as they recover from their refractory periods. The shortest time at which the second response in a repetitive series has been found to occur is 1-7 msec.; under the conditions of these experiments, the shortest time for muscular summation, in the absence of eserine, has been found to be 8-1 msec. with the stimulating electrodes some 5 cm. from the muscle. Allowing for the delay of the second impulse, travelling in a partially recovered nerve, the figure of 1x7 msec. must be very close to the absolute refractory period of the neuro-muscular system. The visible synchronous repetitive contractions of the muscle last for some 3 msec. after the first response, and considerable irregular activity can often be seen as long as 5 sec. afterwards. It is reasonable to assume, therefore, that the amount of acetylcholine liberated at the nerve ending is greatly in excess of the minimal amount required to set up an "All-or-Nothing" response in the muscle

19 236 G. L. BROWN fibre and that, in normal conditions, either the end plate receives a greatly supraliminal stimulus or, that much of this acetylcholine is actually inactivated by esterase as it is liberated. That the decline in frequency and ultimate cessation of the discharge is to be attributed entirely to the fall of the liberated acetylcholine below the stimulating concentration is open to question. The whole phenomenon is undoubtedly complicated by the depressant effects of the persisting transmitter. A second volley only produces a visible action potential when separated by some 3 msec. from its predecessor and, as B a cq & Brown have shown, successive stimuli so far apart as 5 sec. may eventually fail to give a full response. The failure of a volley following another at such short time intervals as 1-15 msec. to show any separate action potential or to affect materially the mechanical response is understandable, as such a volley probably falls upon refractory muscle fibres, but the. failure of such a volley to alter, in any way, the duration and frequency of the repetitive discharge cannot at present be explained. The phenomenon is part of the complex picture of the depressant effects of acetylcholine and full discussion must be deferred until more facts are available. SUMMARY 1. Records of action potentials from normal mammalian muscle have shown that the quick contraction evoked by the close arterial injection of acetylcholine is a brief asynchronous tetanus. 2. The components of this contraction are propagated "All-or- Nothing " responses, starting at a frequency of some 2 per sec. and then gradually falling in frequency along a characteristic curve. 3. The effect of eserine in producing a repetitive response of mammalian muscle to a single maximal nerve volley has been investigated in greater detail. The initial frequency of the response has been found to be such that successive contractions fall close to the absolute refractory period of the muscle fibre. 4. A second nerve volley, following closely after one which has produced the full repetitive response of the muscle, is without effect if the interval between the volleys is less than 2 msec. Further separation of the volleys produces a gradual recovery of the effect of the second. 5. In the presence of a dose of eserine, insufficient in itself to cause a repetitive response of the muscle to a single nerve volley, the effects of two suitably timed volleys undergo summation, and evoke a repetitive response.

20 ACTION POTENTIALS OF MUSCLE The persistence in a weakly eserinized muscle of acetylcholine, in a concentration insufficient in itself to cause any response, causes the muscle to give a repetitive response to a single nerve volley. I wish to thank Sir Henry Dale for his stimulating interest in this investigation. REFERENCES Adrian, E. D. & Bronk, D. W. (1929). J. Physiol. 67, 119. Bacq, Z. M. & Brown, G. L. (1937). Ibid. 89, 45. Briscoe, G. (1936). Lancet, 1, 469. Brown, G. L., Dale, H. H. & Feldberg, W. (1936). J. Phy8iol. 87, 394. Eccles, J. C. & Sherrington, C. S. (193). Ibid. 69, 1P.