MUSCLE. BY C. F. WATTS (Research Student of Gonville

Transcription

1 THE EFFECT OF CURARI AND DENERVATION UPON THE ELECTRICAL EXCITABILITY OF STRIATED MUSCLE. BY C. F. WATTS (Research Student of Gonville and Caius College, Cambridge). (From the Physiological Laboratory, Cambridge.) THE relation between the strength and duration of the current needed to excite a tissue has been studied by Keith Lucas(l), Lapicque(2), and others. The time constants of the strength-duration curve and in particular the "chronaxie" (i.e. the current duration at which the threshold strength of current is doubled) are generally considered as having fixed values for any one tissue. With nerve and heart muscle the results of various observers are in agreement, but in the case of skeletal muscle this is not so. For the non-neural portion of a frog's sartorius Keith Lucas obtained a "chronaxie" of about 003 sec., while Lapicque obtains a value of about sec. as the " chronaxie " of various skeletal muscles. The discrepancy between these results has been explained by the work of Jinnaka and Azuma(3) and H. Davis(4), who showed that the area of the stimulating electrode plays an important part in determining the "chronaxie." With a fluid electrode, they obtain a long value appro.ximating to that found by Lucas with this type of electrode, but when an electrode of small stimulating area, such as the capillary pore electrode devised by Pratt, or the small silver chloride electrode used by Lapicque, is employed, they find a short value of the same order as that obtained from nerve. This result makes it difficult, as Davis pointed out, to assign any definite "chronaxie" to the muscle, and must also be taken into account in any attempt at a physical explanation of the strength-duration curve. The differing results of Lucas and Lap i cq u e are, however, of great interest in connection with the supposed change in the "chronaxie" of the muscle fibres observed under the action of curari, and after section and degeneration of the nerve fibres supplying them. In both cases the muscle becomes inexcitable through its nerve, and in both cases the "chronaxie" of the muscle, taken as a whole, is observed to lengthen.

2 144 C. F. WATTS. How far is this change due merely to the fact that the stimuli formerly took effect on the intra-muscular nerve fibres, and now excite the muscle fibres themselves, and how far does it imply an actual change in the time constants of these muscle fibres? The nature of the change in the case of denervation is important in connection with the electrical testing of muscles, and the action of curari is a point of some theoretical interest. Keith Lucas, using his fluid electrodes (5), found that the non-neural region of a frog's sartorius gave a simple curve with a "chronaxie" of *003 sec. He called this the a curve and regarded it as characteristic of the muscle fibre. In the neural region of the muscle he obtained complex curves, double or even triple in form. One of these complex curves had the same form as the a curve and of the other two, one, y, had the same time constants as the curve obtained from the nerve trunk (" chronaxie " about 0005 sec.), and the other, /, had the shortest "chronaxie" of the three. Doses of curari just strong enough to abolish conduction from nerve to muscle caused the y curve to disappear and slowed the time constants of the /3 curve. Slightly stronger doses abolished the /B curve altogether, leaving the a curve unchanged. L u c as concluded from these results that the very rapid,b curve represented the excitation of an intermediate substance between muscle and nerve-a conception agreeing in general terms with that which had been formed from the action of drugs. According to this view curari does not affect the muscle fibre itself, but breaks the connection between nerve and muscle by its specific action on an intermediate substance localised in the region of the nerve endings and having a very short "chronaxie." Lapicque, using his small silver chloride electrodes, finds the "chronaxie" of normal nerve and muscle to have approximately the same value and he regards this "isochronism" of the two tissues as essential to conduction between them. The effect of curari is, from his point of view, to lengthen the "chronaxie" of the muscle fibre itself, and when this becomes about twice its normal value conduction fails owing to the "heterochronism" of nerve and muscle so produced. Failure of conduction is hence not due to any specific effect upon an intermediate substance but to an effect on the muscle fibre itself causing a slowing of its time constants. The nature of the effect of small doses of curari on the strength-duration curve of a muscle fibre is hence an important point in connection with the supposed mode of action of the drug and I have therefore investigated the strength-duration curves of normal, denervated, and curarised

3 NERVE-MUSCLE CHRONAXIE. 145 muscles with special reference to the type of stimulating electrode employed. Methods. (1) Fluid electrodes of the slot type were employed. Two slots of differing sizes were used, the area of one being 5 x 1 mms. and the other 3-5 times as great-7-5 x 2-5 mms. The smaller slot just accomm,odates a normal sartorius. Both gave curves extending over a similar range for normal muscles but the threshold in the case of the larger slot was materially higher and more variable as the form of the current led in probably varied more than in the case of the smaller slot. (2) For stimulating individual muscle fibres two Pratt electrodes were employed, one having a diameter of 3 5,.u and the other being an ellipse 73 x 44,u. (These pores were made by Dr H. Davis to whom I am indebted for their use.) The smaller pore was capable of stimulating one fibre only, but with the larger one this was not always the case as its dimensions are greater than those of an average muscle fibre ( ,u). Hence the range of values obtained from it is relatively larger but in all cases the curves from muscles under similar conditions are of a similar type. Ag-AgCl electrodes were used for connection with the pore, and for the indifferent electrode which dipped in the Ringer in which the whole frog was immersed during this stage of the experiment. The pores were used on the muscle (sartorius throughout) in situ 1 hour after skin reflection and immersion of the frog in the bathing fluid. The sartorii were then dissected out and after leaving for 1-31 hours in the requisite bathing fluid the strength-duration curves were redetermined with the fluid slot electrode. The spring contact breaker and shortrange pendulum designed by Lucas(1) were both used for obtaining currents of shoit duration. Currents ranging from * sec. could thus be obtained. Normal muscles. To describe a strength-duration curve (e.g. of the complex form shown in Fig. 1 D) it is not sufficient to note only the "chronaxie," and I have taken four points on each curve in comparing those obtained under different conditions. These are: (a) Threshold (0), i.e. the least value of current of infinite duration which will excite; (b) the time of first rise of threshold, i.e. time at which the current required to excite is first greater than (0); (c) the "chronaxie"; (d) the minimum time which a current of infinite strength must last in order to excite. This is called the Minimal Excitation Time. The form of the curves will not be affected by variation in (a), but will be altered by changes in (b), (c) and, to a less extent, (d). The following table gives the values obtained from normal muscles by the different techniques:

4 146 C. F. WATTS. TABLE I. Normal muscles. Time of 1st Min. excitation Threshold (0) rise from (0) Chronaxie time Volts Secs. Secs. Secs. ElEctrode (a) (b) (c) (d) Small pore (1) * * about , dia. (2) *69 * *00004 Large pore (1) * * r x 44 g (2) * *00055 *00005 Slot (1) 064-' * x 1 mms (2) *10 *02 *0027 * In all curves, Case (1) gives the general range of values obtained; (2) gives a representative experimental value commonly obtained. All figures refer to the pelvic end of the muscle. With the fluid slot electrodes a curves are obtained from the pelvic end of the muscle, and fi and y curves appear when the middle region is placed in the slot. The curves obtained with the two pores show an increase in the values obtained with the increased size of the pore, and this corroborates the evidence of Davis that it is the size of the pore which determines the absolute values of the curve. The effect of denervation. The frogs operated upon were anaesthetised and the main sciatic trunk or its deep posterior branch cut high up the thighl. The experiments were performed from days after the operation, but only two of 12 experiments were performed less than 30 days after denervation. That denervation had been effected was proved by observing whether the muscle twitched on severance of its nerve during dissection and further by histological examination after staining with methylene blue at the conclusion of the experiment. In general the curves show a decreased excitability to long currents but the "chronaxie" is not appreciably altered. What alteration there is, is observed mainly with the large pore, and, although in some cases the "chronaxdie" may be as much as doubled, the general range of values obtained with this electrode is not greatly altered. This slight genera decrease of excitability may be attributed to the loss of trophic nerve influences. When fluid electrodes are used, the fi and y curves disappear while the a curve remains practically the same throughout the muscle during the degeneration of the nerve, but may alter when the muscle has been denervated for some time. The values obtained from denervated muscles are given in Table II and a comparison of the curves of normal and denervated muscles may be seen in Fig I am indebted to Dr Adrian for operating on all the frogs used in these experiments and my best thanks are due to him for much helpful criticism and advice throughout this work.

5 NERVE-MUSCLE CHRONAXIE. 147 TABLE II. Denervated muscles. Time of 1st Min. excitation Threshold (6) rise from (6) Chronaxie time Volts Secs. Secs. Secs. Electrode (a) (b) (c) (d) Small pore (1) "575-1'15 *002-'003 *00015-'0003 '00002-' ,u dia. (2) *9 *0025 *00018 *00004 Normal muscle * *00015 *00004 Large pore (1) *487-'75 '01-'025 '00048-'0013 *00008-' x 44, (2) *56 *012 *001 *0001 Normal muscle *45 *005 *00055 *00005 Slot (1) '08-'12 ' '0021-' ' x I mms (2) *10 *02 '003 *0002 Normal muscle '10 *02 ' The very small (or absent) change in the "chronaxie" of a muscle after denervation is, at first sight, difficult to reconcile with the fact that, in the body, a denervated muscle needs a "galvanic" current to excite it, instead of a " faradic " (reaction of degeneration), and with the measurements of Bourguignon(6) and Adrian(7), which show a very great increase in the "chronaxie" of human muscle after nerve injury. But the electrodes used for human muscle testing are of large area, and the denervated muscle will therefore give long values comparable to those we have obtained on excised muscles with the slot electrode. When the nerve supply is intact, however, the current presumably excites the nerve fibres in preference to the muscle, and hence the short " chronaxie " of nerve is obtained. Thus, the great increase in the "chronaxie" observed in human subjects after denervation is due, not to any alteration in the time constants of the muscle fibres, but merely to the point of incidence of the stimulus shifting from nerve to muscle, and we should probably be unable to find such a great change if we were able to use an electrode of sufficiently small area on the human subject. It does not, of course, follow that the "chronaxie" of the muscle remains absolutely unchanged after the degeneration of its nerve. Any change occurring after the nerve has had time to become inexcitable must clearly be due to a change in the time constants of the muscle itself, and such alterations have been reported by Bourguignon(6) and Adrian(7). But these changes are not of the same order of magnitude as the sudden lengthening which occurs in the first fortnight after denervation. This latter change can only be due to the causes mentioned above, i.e. to the fact that we are dealing with two distinct excitable tissues which have different time relations when large electrodes are PH. LIX. 10

6 148 C. F. WATTS. employed, and not with one tissue whose time relations have been modified by nerve injury. 0l Fig9C. c. Fi9.1.D. NORMAL -sx NORMAL &-e CDENERVNATED OO2Bsec. 0 sec. CHROrMIE00283m. DENERVATED x---xc CHRONAXIE 0048sec. ~~~~~~CH.RONAXIE3A000.,c CHRONAXIE DENERVATED X---X OO32sec &cawd01 *015 *001 *005 *01 *015 Fig. 1. Normal and Denervated muscles. A. Small pore electrode. Pelvic end of muscle. Temp. Normal 110 Denervated C. Muscle denervated 32 days. B. Large Pore electrode. Pelvic end of muscle. Temp. Normal 9.80 Denervated C. Muscle denervated 22 days. C. Slot electrode. Pelvic end of muscle. Simple a curves. Temp. Normal Denervated C. Muscle denervated 37 days. D. Slot electrode. Middle region of muscle. Temp. Normal 150 Denervated 110 C. Muscle denervated 32 days. The arrow thus on the normal curve indicates the point at which a change in the nature of the contraction was observed. The curve is not obtained after denervation. The effect of curari. Doses varying from -5-1O mgms were injected under the skin of the back of the frog, and after 3 hour the frog

7 NERVE-MUSCLE CHRONAXIE. was placed in curari-ringer containing from p.c. curari. The smallest dose used was sufficient to paralyse the muscle, as was shown by failure of stimuli on the nerve, and the absence of any twitch in the muscle when its nerve was severed. Small doses of curari do not alter the excitability appreciably, any effect being in a contrary direction to that of denervation. The fact that the values given are below those of normal muscles may in part be due to the fact that the temperature of these experiments was higher, and also they were performed at a different period of the year to those on normal muscles. However, Table III and Fig. 2 show that there is a definite increase of excitability to currents of long duration and the curve is hence more rectangular than it is normally. With the larger doses, all the values begin to increase, and in *2 p.c. curari-ringer the "chronaxie" may be doubled, but yet not outside the normal range. In three experiments the effect of curari on a denervated muscle was studied, and it was observed that the action was similar to that noted above on normal muscles. This indicates that the degeneration of the nerve fibres does not alter the susceptibility of the actual muscle fibre to curari, and confirms the evidence already obtained that the excitability of the muscle is only affected by relatively strong doses of the drug. TABLE III. The effect of curari. Time of 1st Min. excitation Threshold (0) rise from (0) Chronaxie time Volts Secs. Secs. Secs. Electrode (a) (b) (c) (d) Smal Weak curari (1) * About (00004 pore (2) * *00008 J ,u Stronger,, (1) * * About dia. (2) *8 *002 * Large Weak curari (1) * ' * pore (2) *27 *012 *00045 * x Stronger,, (1) * *000O y (2) *637 * *0001 Slot Weak curari (1) * * * x1 (2) * * mms Stronger,, (1) * *042 *001-O0058 * (2) * *0024 *0002 Weak Curari = *55 mgms injected under the skin of the back..01 % curari-ringer as bathing fluid. Stronger,, = 10 mgms injected under the skin of the back. 02 %-3 % curari-ringer as bathing fluid. It seems evident from these results that the time relations of the excitability of the muscle fibres are not radically changed as a result of denervation or mild curarisation. On the other hand, under these conditions, conduction from nerve to muscle is suspended. This seems

8 150 C. F. WATTS. definite evidence against the theory that heterochronism is the main factor which prevents the muscle responding when the nerve is stimulated. 3-0 Fig. 2.A. NORMAL CHRONAXIE 0002seC. Fig.2.B. NOP.MAL &--e CHRONAXIE (00054 sec. 2R0CHER..XICsOOOOSseC CHPR.ONAXIE O0O3s5ec. 25 CURARI 01/O0--X CURARI 01 /O*--X 1-5 L X\ 1-0 s---- -y Seco,rhO0005 * *001 Second 005 *01 F;9 2.C. -Fig 2.D. I NORMAL -t--@3-0 NoRMAL CHRONXIE OO008sec. k UAICHRONAXIE O000545ec. * \ \ CURARI 01, --x CHRONAXIE 001 X CHRONAXIE 0028Osec. k 'S.~~~~~~~~~~~. 01l 05 J I g iis I, *001 Second *001 Second *005 *01 Fig. 2. Normal and Curarised muscle. A. Weak curari. Small pore electrode. Pelvic end of muscle. Temp. Normal 11 Curarised C. Dose. 2 mgms injected. Frog bathed in *01 % curari-ringer. B. Weak curari. Large pore electrode. Pelvic end of muscle. Temp. Normal 9.80 Curarised C. Dose. 2 mgms injected. Frog bathed in *01 % curari-ringer. C. Weak curari. Slot electrode. Middle region of muscle. Temp. Normal 150 Curarised Dose. *5 mgm injected. Muscle bathed in *01 % curari.ringer. Curari abolishes the 8 curve. D. Strong curari. Large pore electrode. Pelvic end of muscle. Temp. Normal 9.80 Curarised C. Dose. 10 mgms injected. Frog bathed in *3 % curari-ringer. Great increase of threshold and chronaxie with this strengti of the drug. For, although the time relations of the muscle may be doubled by curari or denervation, this is by no means a sine qua non for failure of conduction

9 NERVE-MUSCLE CHRONAXIE. 151 from nerve to muscle. The normal isochronism of nerve and muscle seems to depend upon the technique employed in measuring the excitability of these tissues, and may be present after small doses of curari have abolished the possibility of conduction between them. Thus, with small doses we obtain the effects described by Lucas on the strength-duration curves, and by Langley(12) on the action of nicotine, i.e. effects which are only obtained in the neural region of the muscle. With larger doses, however, the muscle itself is affected, as previous observers have noted, but it is not definitely known whether this action is due to the drug itself or to impurities, e.g. salts. This action is, however, subsidiary, and not the main factor involved in the failure of conduction between nerve and muscle. It follows that if curari does not act on the muscle substance to produce paralysis it must act upon something which is distinct from both these substances, and this agrees with the classical theory as to the mode of action of drugs on striated muscle. The intermediate substance may be comparable to the nerve endings which Claude Bernard supposed were the seat of action of curari, or to the neuro-muscular junction postulated as the result of the experiments of Langley(s), Anderson(9), Brodie and Dixon(lo), and Elliot(11). However, Langley, from later observations (12) upon the action of nicotine on striated muscle, developed the theory of a receptive substance situated on the muscle side of the nerve endings and it is generally supposed that the, curve of Lucas represents the time relations of this substance. But whereas Langley found that denervation did not abolish the action of nicotine, as regards tonic contraction, the,b curve of Lucas has not been found in a muscle after denervation. There is little evidence, however, as to the nature of this substance, but the work of Davis suggests that it may have an extremely small structure. Davis used capillary pore electrodes of different sizes and found that the smaller the pore used, if less than about 75,u in diameter, the smaller is the "chronaxie" obtained. Hence when the electrode is of small area relative to the tissue element stimulated, it is the size of the electrode and not the tissue that determines the absolute values obtained. Jinnaka and Azuma(5) using a lo,u pore obtained from the nonneural region of a sartorius muscle a curve similar to the j9 curve of Lucas with a "chronaxie" of about 0003 sec. From what has been said above, however, this does not imply that a substance having these time relations lay under their electrode but rather that the area of stimulation

10 152 C. F. WATTS. employed was such as to give these values when applied to the individual muscle fibre. On the other hand, my experiments with the two fluid slot electrodes of different sizes show that when a relatively large electrode is used any alteration in its size (provided it is large enough to stimulate the whole muscle) does not affect the values of the time constants obtained. When, therefore, a curve is obtained with this type of electrode, such as Lucas' fi curve which differs radically from the normal a curve, it may be accepted as definite evidence of a distinct excitable substance in the region of the muscle which is being stimulated, i.e. the neural region. This conclusion as to the definite existence of an intermediate substance which is the seat of action of drugs, etc., harmonises with the phenomena of decremental conduction, Wedensky inhibition, fatigue, etc., already observed by various authors and explained by them as due to the existence of such a substance between nerve and muscle. SUMMARY. The work of H. Davis has shown that the size of the stimulating electrode relative to that of the tissue elements plays a great part in determining the excitability constants, i.e. " chronaxie " and the strengthduration curve, of that tissue. The changes in "chronaxie" following denervation and curarisation have been investigated in the light of Davis' work. When a muscle is denervated it is found that the "chronaxie" of the muscle fibres themselves does not alter to any great extent, and the lengthening of the "chronaxie" observed is due to the point of effect of the stimulus passing from nerve to muscle. In the case of curari, it is concluded that the effect of the drug in paralysing the muscle is not due to any action on the muscle fibres, though such an action may be observed when larger doses are employed. The bearing of these results on the mechanism of conduction of the impulse from nerve to muscle is briefly discussed. REFERENCES. (1) Keith Lucas. This Journ. 35. p ; and 36. p (2) Lapicque. C. R. Soc. de Biol. p ; and p. 733, etc (3) Jinnaka and Azuma. Proc. Roy. Soc. B. 94. p (4) Davis, H. This Journ. 57; Proc. Physiol. Soc. p. lxxxi (5) Keith Lucas. Ibid. 34. p (6) Bourguignon. Le Chronaxie chez L'Homme. (7) Adrian. Brain 39 p ; Arch. of Radiol. 21. p (8) Langley. This Journ. 27. p (9) Anderson. Ibid. 30. p (10) Brodie and Dixon. Ibid. 30. p (11) Elliot. Ibid. 32. p (12) Langley. Ibid. 33. p ; Proc. Roy. Soc. B. 78. p