ON THE SPONTANEOUS MOVEMENTS OF AMPHIBIAN SKELETAL MUSCLE IN SALINE SOLUTIONS, WITH OBSERVATIONS ON THE INFLUENCE OF POTASSIUM AND CALCIUM CHLORIDES ON MUSCULAR EXCITABILITY. BY GEORGE RALPH MINES, Sidney Sussex College, Allen Scholar in the University of Cambridge. (From the Physiological Laboratory, Cambridge.) I. On the spontaneous movements of amphibian skeletal muscle in saline solutions. HERING (11)1 in 1879 published an account of the behaviour of the frog's sartorius when partly or wholly immersed in a solution of sodium chloride, and apparently he was the first to call attention to the fact that an isolated skeletal muscle in saline solution may exhibit rhythmic contractions. He regarded the solution simply as a liquid conductor, which by closing the circuit caused stimulation of the muscle by its demarcation current. In the following year Biedermann(2) gave an exhaustive description of the movements as studied by direct observa- tion of the muscle placed in a mixture of sodium chloride, sodium phosphate and sodium carbonate. He mentioned that contractions were obtained by placing the sartorius in *6 0/o sodium chloride, but spoke of them as comparatively weak and of short duration. The contractions of frogs' muscles in sodium chloride solution were more adequately described by Ringer(29), who also was the first to note the influence of calcium and potassium salts on the movements. Ringer's work was published in 1886 and since that time the action of salts in producing and stopping contractions has been dealt with by several observers, especially by Loeb (23). Graphic recQrds of the movements have been given by Ringer, Locke (21) and Zenneck k') but till recently little attention has been paid to the form of the curves. The more 1 The numbers refer to the list at the end of the paper.
Transcript
ON THE SPONTANEOUS MOVEMENTS OF AMPHIBIANSKELETAL MUSCLE IN SALINE SOLUTIONS,WITH OBSERVATIONS ON THE INFLUENCE OFPOTASSIUM AND CALCIUM CHLORIDES ONMUSCULAR EXCITABILITY. BY GEORGE RALPHMINES, Sidney Sussex College, Allen Scholar in the Universityof Cambridge.
(From the Physiological Laboratory, Cambridge.)
I. On the spontaneous movements of amphibian skeletal musclein saline solutions.
HERING (11)1 in 1879 published an account of the behaviour of the frog'ssartorius when partly or wholly immersed in a solution of sodiumchloride, and apparently he was the first to call attention to the factthat an isolated skeletal muscle in saline solution may exhibit rhythmiccontractions. He regarded the solution simply as a liquid conductor,which by closing the circuit caused stimulation of the muscle by itsdemarcation current. In the following year Biedermann(2) gave anexhaustive description of the movements as studied by direct observa-tion of the muscle placed in a mixture of sodium chloride, sodiumphosphate and sodium carbonate. He mentioned that contractionswere obtained by placing the sartorius in *6 0/o sodium chloride, butspoke of them as comparatively weak and of short duration. Thecontractions of frogs' muscles in sodium chloride solution were moreadequately described by Ringer(29), who also was the first to note theinfluence of calcium and potassium salts on the movements. Ringer'swork was published in 1886 and since that time the action of salts inproducing and stopping contractions has been dealt with by severalobservers, especially by Loeb(23). Graphic recQrds of the movementshave been given by Ringer, Locke (21) and Zenneck k') but till recentlylittle attention has been paid to the form of the curves. The more
1 The numbers refer to the list at the end of the paper.
CON1RACTION OF SKELETAL MUSCLE.
delicate movements are so feeble that it is difficult to get an accuraterecord of them on a smoked surface. Last year Samoj loff (34)described records of the movemnents in Biedermann's fluid obtained byPh eoph il aktoff with the aid of a photographic registering apparatus.Six months before the ptublication of Samoj loff's paper I had madeuse of the photographic method for the same purpose.
The records which are here discussed were obt-ained with the aid ofthe photographic apparatus described by Keith Lucas (12). The pieceof bone from which the muscle gains its origin was transfixed by a glasshook; a fine silk thread was tied to the tendon at the other end,and this was attached to a short light lever of glass or straw havinga small mirror attached at the axis. The movement of the musclewas magnified about fifteen times. The sensitiveness of the leverwas adjusted by altering the counterpoise so as to obtain a suitableexcursion of the spot of light; usually the muscle was subjected to atension of one or two decigrams. The records were taken on rapidbromide paper.
Hering, Biedermann and Ringer all called attention to thedefinitely rhythmic character often assumed by the movements, and thetwo former observers compared their regularity to that of the heart beat.I take the expression "rhythmic" to imply the recurrence of someevent or group of events at equal intervals of time; the interval betweensimilar points in two successive cycles being the period of the rhythm.Physiological rhytlhtns often show some deviation from absolute time-accuracy, which may be ascribed to the ever varying conditions underwhich living matter, through the very fact of its own mnetabolism, is placed.
Samojloff, in the paper already referred to, states that the rhythmof the sartorius in saline solution is of quite another grade of regularityfrom that of the heart. It is true that the activity of any particulargroup of fibres is seldom maintained for more than a few minutes. ButSamojloff seems to imply that the series of contractions never exhibita true period at all. Measuring the intervals between the members ofhis most regular series he finds that they vary to a considerable extentas in the following instance:
1-04 0 9 0O88 085 0,83 0-88 0 9 099 1-04 1P17.
In this series the variation in the intervals between the contractionsis equal to 640/0 of the shortest interval. It cannot properly be calleda rhythmic series at all, the intervals between the contractions do notapproximate to one constant value, btut they decrease to a minimum
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and then increase again. I have recorded groups of this type, of whichan instance is given in Fig. 1. The appearance of the tracingsuggests a kind of distortion of an essentially rhythmic series, just as
Fig. 1. Frog's sartorius in Biedermann's fluid. Time is marked in seconds.All tracings in this paper are to be read from left to right.
in music there may be gradual variations in time which do not destroythe underlying sense of rhythm. In passing it may be noted thatthese curves do not bear out Samojloff's statement' that the con-tractions of the sartorius, unlike those of the heart, remain unchangedin magnitude when the interval between them alters. In Fig. 1 it isseen that after a shorter interval the contraction is smaller, and viceversa.
I find that the sartorius in saline solutions often exhibits series ofcontractions of a degree of regularity much higher than this. Theappearance of some of the curves given in Figs. 2 and 3 suggests aregularity scarcely inferior to that of the heart beat. Careful measure-ment with the aid of a comparator fully bears out this idea.
AJVAJA&vA,'~fv~VWVI I I I I I I I I I
Fig. 2. Frog's sartorius in Biedermann's fluid. Time marked in seconds.
Distances between apices of curves in original from which Fig. 2 a isreduced (in millimetres):
In this series of 21 contractions there is an extreme variation from6,68 to 7 mm. in the interval between the contractions, i.e. a variation
1 loc. cit.
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of less than 5 0/0. Taking a series of ten contractions from this,beginning at the fourth in the series quoted we have an extremevariation from 6-8 to 6-99, i.e. rather less than 2-8 0/0.
Another series of measurements may be given.
I I I a
Fig. 3. Toad's sartorius in 1 I/o sodium bromide. After six hours' immersion.Time tracing marks intervals of ten seconds.
Toad's sartor'ius in *7 °/o sodium chloride:6 6-1 6-15 5-92 6 6-05 6-1 6-19 6-09 6-2 6-14 6X126-12 6-24 6X17 6-0 6'3 6-24 6-36 6-46 6,24 6-19 6,29.Here in a series of ten contractions (beginning with the fifth quoted)
there is a variation from 6 to 6-24-a variation of 40/o. In these andmany other instances there is no gradual increase or decrease in theinterval between the contractions, but it oscillates within narrow limitsabout a mean value which may reasonably be called the period of therhythm
For the sake of comparison some.measurements were made of theperiod of the frog's heart (after pithing) and of the lymph hearts (afterdestruction of the brain). In both cases, for greater convenience inmeasurement, the movement of the pulsating organ was made to openand close an electric circuit (by moving a wire in and out of a pool ofmercury) in which a signal was included. The electric signal marked adefinite point in each systole on the blackened surface of a Sandstromdrum, moving at the rate of 50 mm. per second. The measurement oftime tracings taken simultaneously showed that the speed of the drumwas constant.
In the case of the heart a series of ten contractions could notinfrequently be found separated by intervals which varied only about11. On the other hand the instances given below show a variation ofperiod of the same order of magnitude as that found for the sartoriuswhen contracting most regularly.
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Distance (in mm. of tracing) between similar points in successivesystoles of frog's heart, circulation intact, in two experiments:
In the first of these instances the extreme variation of period in aseries of ten conitractions is 3'2 0/0; in the second it is 3'1 0/o In thecase of the lymph hearts greater variation was founid.
Intervals between successive contractions of lymph heart, spinalfrog, in different experiments:
(1) 47'5 44'5 42-5 47 44'5 45 47 45 44 46'5.(2) 44 455 46 45s5 47 46'5 44 45 47-5 45a5.(3) 46'5 42'5 43-5 48 45'S 47'5 46'5 45'S 455 44'5.In the first of these instances the variation amounts to 11 0/,,, in the
second it is 7'9 0/0 and in the third 103 0/0 of the shortest interval.It seems then that under certain conditions skeletal nmuscle fibres
can originate, and maintain for a sufficient time to rule out merecoincidence of period, a rhythm at any rate comparable to that of theheart in point of regularity and distinctly superior to that of the lymphhearts.
In other words, skeletal muscle fibres may exhibit a rhythm of thesame order of accuracy as other generally accepted physiologicalrhythms. The fact that the sartorius may also give irregular sequencesof contractions is quite apart from the point that I wish here toestablish.
These rhythmic series are of very diverse frequencies. The slowestthat I have recorded showed one contraction in 6 seconds. Otherseries were recorded with periods of 3, 2, 1, '5, '25, '2, '17 seconds andmany intermiediate values. Sometimes a rhythm of much higherfrequency was noticed, namely one of 13 or 15 contractions per second.Quick rhythmic tremors of this type were seen on numerous occasionsin muscles placed in '7 o/o sodium chloride to which about '05 0/o potassiumchloride was added. They were rarely found-but were recorded onceat least in pure sodium chloride, in sodium bromide and in Biedermann'sfluid.
It has often been stated that the muscle in saline solution does nottwitch with all its fibres simultaneously. The activity of one fibre does
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CONTRACTION OF SKELETAL MUSCLE.
not of necessity excite contraction in the surrounding fibres, for there isnot the same physiological continuity between them that exists betweenthe contractile elements of the heart. Thus it often happens that themovements of the muscle as a whole are irregular. This irregularitydoes not necessarily imply a-rhythmic behaviour on the part of anyindividual contractile element. The mechanical summation or inter-ference of a number of rhythmic units with unlike periods wouldproduce a complex result, the graphic record of which might easily failto reveal any of the component rhythms. It must further be borne inmind that during the inscription of such a curve various fibres may
start or stop their activity and some may give series of contractionswith the distorted rhythm already mentioned. I have obtained manycurves which at first sight appear irregular, but on closer inspection areseen to consist of two or more rhythmic series. Fig. 4 is an instanceof the compounding of two regular rhythmic series. The small twitch
Fig. 4. Toad's sartorius in .70/0 sodium chloride. Temp. 80 C.Time marked in seconds.
recurs once a second-sometimes it appears by itself, at other times itis partly or wholly fused with the larger twitch, which possesses a
slower period. The two movements are quite independent in theirorigin, and their combination is purely mechanical. There is thereforereason to think that the irregular curve which is generally obtainedsoon after the immersion of the muscle (of which an instance is givenin Fig. 5 a), is in reality the result of the interference of regularmovements of different regions of the muscle. The condition whichleads to the appearance of spontaneous movement persists longer in
some fibres than in others, and thus it happens that after a while (as inFig. 5 b) simple rhythmic movements. make their appearance in therecord.
a s
Fig. 5. Toad's sartorius in *7 0/0 sodium chloride. a, after 2j hours' immersion.b, after 5 hours. Time marked in seconds.
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Sometimes a regular series of double peaked contractions is recorded(Fig. 6); this was predicted by Biedermann (2). It is clear that theymight be due to the independent contractions of two different regionsof the muscle which chanced to have the same period. More probably
.s * | . . , . . . I . . . ..~~~~~~~~~aI
Fig. 6. . Double peaked contractions from same muscle as in Fig. 5.Time marked in seconds.
the second contraction is provoked by the first; that is to say, anadjacent fibre in a state of high excitability is stimulated by themovement (or possibly by the electric variation accompanying it) of thefibre which first contracts, for while the double peaked curve is notuncommon, the alternation of two series having the same frequency,but not partly fused in this way, is extremely rare.
With more perfect fusion of the twitches the appearance shown inFig. 7 is produced, resembling a rhythmically repeated tetanus.
* I 1 II I IJ I i l 1
Fig. 7. Toad's sartorius in *7O/0 sodium chloride. Time marked in seconds.
The movements of the sartorius as a rule become most active aboutfive minutes after it is placed in a *7 0/0 sodium chloride. After a fewhours it often shows long periods of rest; in this condition its activitycan readily be started again for a time by the application of a stimulus,which may be mechanical or electrical.
A weak galvanic current makes the movements more vigorous andusually entirely changes their character. An induction shock sometimeshas a similar effect, but as Samojloff found, it may cause an extracontraction without producing any further disturbance in a series ofspontaneous contractions. The significance of this. difference in theaction of the two varieties of electrical stimuli will appear later.
Difference in reaction of diferent muscles in the frog. The move-ments have been studied chiefly in the leg muscles of the frog, thoughGarrey () has found similar twitches in the skeletal muscles ofmammals and many invertebrates. There is a decided difference inthe ease with which the movements are elicited in different muscles ofthe frog and toad. Thus while they constantly appear in the sartorius
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CONTRACTION OF SKELETAL MUSCLE.
within a few minutes of its immersion, the gastrocnemius does nottwitch as a rule till after an hour's immersion. In this case thedifference may perhaps be ascribed to the very different thickness ofthe muscles, but there are other instances in which such an explanationseems insufficient. I never observed twitching in the coraco-radialis orin the flexor carpi radialis when these muscles were simply immersed inthe sodium chloride solution, though the genio-hyo-glossus and thesternohyoid muscles readily gave rhythmic twitches in this way.
By perfusing the blood-vessels of the frog with *7 0/0 sodium chlorideby means of a cannula tied in one of the aortae, it is possible to arousetwitches in apparently the whole skeletal musculature. The movementswere observed under these conditions in the skin muscles, the scapular,pectoral, and other arm muscles, the muscles of the leg and thigh, thesmall muscles of the manus and pes, the muscles of the tongue and thefloor of the mouth. The rectus abdominis gave only slight andoccasional evidence of twitching when immersed in the saline solutionafter perfusion, though it often exhibited slow curling movements.This behaviour of the rectus abdominis recalls its reaction to nicotine.Professor Langley (19) found that this muscle gave twitches with nicotinefar less readily than any other muscle tested.
Influence of temperature. The activity of the muscle in salinesolution is greater as the temperature is higher, within physiologicallimits. The effect of raising the temperature of the solution is as arule to increase both the frequency and the amplitude of the move-ments. It is practically impossible to determine the tetnperaturecoefficient of the rhythm of any particular region. The mere act ofchanging the temperature generally results in a complete alteration inthe character of the movements, which even under the most favourableconditions mianifest a remarkable diversity.
The influence of calcium and pottssium on the contractions. Ringer (29)showed that the addition of a small amnount of a lime salt prevented thetwitching in saline solution. Loeb states that fromn *026 0/0 to -038 0/0of calcium chloride is required to abolish the movements in a solutionof sodium bromide. Using a *7 '/I solution of sodium chloride I find asomewhat wider variation in the amount of calcium needed to stop thetwitches. Thus in October, *01 0/o calcium chloride sufficed to bring themuscles to rest, whereas in May and June, .04 0/o or .05 I/0 was requiredeven if the solutions were cooled to 10°. The effect of calcium even inminimal doses is always to diminish the movements.
With potassium the case is different. Though potassium chloride
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in large amount abolishes the twitches, the first effect of a small doseis to greatly increase them. A muscle giving feeble twitches in sodiumchloride solution becomes far more active on the addition of a smallamount (O505/0 to *1 o/0) of potassium chloride. It has been mentionedthat a very quick rhythm often makes its appearance in muscles placedin such a solution. This is illustrated by Fig. 8.
TV0; 0, _R R PIK-gS
Fig. 8. Biceps cruris of frog in sodium chloride *650/0, potassium chloride *05 /0.A period of one second is marked below.
The effect of curare. The movements of the sartorius in salinesolutions were discovered in a curarised muscle. Ringer found thatthe addition of a small quantity of cuiare (about 003 0/o) to the salinesolution did not prevent them. Zenneck(3) found the contractionsgreater though less frequent in the curarised muscle. Carslaw (6) usinga hypotonic of sodiumn chloride found that curare delayed the appearanceof the twitches, but did not prevent them. I have made some experi-ments with a view to determining whether a stronger solution of curarewill abolish the movements. They generally continue for over anhour in *7 0/0 sodium chloride containing 1 0/o curare. *2 0/0 curareusually stops the twitches in a few minutes. It occurred to me thatpossibly the organic constituents of the curare might be partly respon-sible for this action. I therefore calcined a small quantity of the cuCareand fouind that it yielded 9-31 /0 of a brownish-grey ash. A solution ofthis ash was made up in *7 0/0 sodium chloride of a strength correspond-ing to a -2 0/0 solution of curare. This solution was found to stop themovements of muscles already twitching in *7 0/. sodiunm chloride just in
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CONTRACTION OF SKELETAL MUSCLE.
the same way as the *2 0/, curare solution, though it was entirely withouteffect when injected into a decerebrate frog. It should be mentionedthat the injection of *25 c.c. of a 1 0/o solution of the same specimen ofcurare caused complete paralysis in a spinal frog in about half an hour.Mr J. E. Purvis kindly made a spectroscopic exarnination of the ashand informed me that it consists largely of lime, together with soda anda little potasb.
I conclude that the abolition of the twitches by curare in large dosesis due, not to the alkaloids on which its specific action on the neuro-muscular mechanism depends, but to the presence of inorganic salts,and in particular calcium salts.
II. The cause of the movements.
Hering("1) ascribed the movements of the sartorius immersed insodium chloride solution to electrical stimulation through closure of thedemarcation current by the conducting solution. He stated that themovements did not as a rule occur in uninjured muscles, but that verysimilar movements could be produced in them by the use of a weakconstant current. Ringer(29) mentions that the movements begin laterbut persist longer in the uninjured muscle.
The idea that mechanical injury of the muscle is a necessary con-dition for the appearance of the twitches, is at once set aside bv the factthat on perfusion of the blood-vessels with sodium chloride solutionvigorous twitches soon begin in the muscles and can be seen withouteven removing the skin.
As will be seen shortly, the muscle in sodium chloride solution ispeculiarly susceptible to the influence of weak constant currents, and itis not unlikely that the action of the demarcation current may accountfor the observation of Ringer just referred to.
It is important to determiine what part of the neuro-muscu'larapparatus is responsible for the origination of the movements. Themethod of punctiform application, which Professor Langley(19) has shownto give such definite results in the case of nicotine, is not adapted to thesolution of this problem. Here the factor which leads to the appearance ofthe movements seems to be the reduction in the concentration of calciuim.The application of a small drop of sodium chloride solution is unableto effect this reduction to a sufficient extent. If a larger quantity ofthe solution is applied, or if the nerveless end of the muscle is immersedin the solution, it is impossible to be sure that there is no alteration
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in the concentration of salts by diffusion in the regions which containnerve endings.
Garrey(7) mentions that the twitches can be obtained from musclesafter section and degeneration of their nerves. In the present state ofour knowledge this can only be taken to prove that the nerve fibres arenot concerned in the production of the movements.
It has beeni stated already that the twitches may continue in a solu-tion containing 1 0/0 curare. Now Keith Lucas (4) has- shown that thepresence of 03 0/0 curare in the fluid soaking the muscle is sufficient tomake the whole of it react towards currents of different durations in thesame way as the pelvic region of the normal muscle. Curare in thisamount appears to throw out of action the receptive substance whichis found in the neural region of the muscle, in addition to preventingstimulation of the nerve fibres from affecting the muscle. It may beinferred that the processes which lead to the appearance of muscularmovements in saline solution are seated very near the actual coutractilemechanism; this view receives further support from what follows.
Loeb(25) maintains that the movements are produced by the replace-ment of calcium ions by sodium or potassium ions in the muscle. Heconsiders that a certain balance of electrolytes is a necessary conditionfor the exhibition of rhythm in any contractile tissue, and that in thecase of skeletal muscle this particular equilibrium is arrived at by re-ducing the amount of calcium present.
Now in the case of the heart it is observable that the irritability ofthe muscle towards electric stimulation increases continuously duringthe interval between two contractions. The contractile substance, orthe substance whose break-down discharges the contractile substance,becomes more and more unstable until at last it suddenly gives way.An essential condition for this explosive spontaneous katabolism is theexistence of a very high degree of instability. Indeed, it may be saidthat the stimulus needed to fire off the contraction becomes smaller andsmnaller until a point is reached where no stimulus at all is required.Doubtless the discharge is often effected by minute accidental stimulishortly before this vanishing point is reached, and this is probably oneof the causes of deviation from absolute time accuracy. If the wholelevel of irritability is lowered the contractions cease.
One would expect that the irritability of a skeletal muscle showingspontaneous' movement would be higher than that of one in the ordinary
I It is convenient to speak of the movements as spontaneous. During a period whichmay extend over two days at room temperature, twitches are liable to occur from time to
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condition. Biedermann () indeed classed the sodium salts as chemicalstimuli which acted by causing an increase in the excitability of themuscle, but apparently he demonstrated this for electrical stimuli onlyin the case of sodium carbonate. Loeb (0 on the contrary statesexplicitly that the rhythmic movements in solutions of sodium saltsare not associated with a condition of increased irritability of themuscle. He quotes experiments in which the irritability of a musclein a solution containing calcium in addition to sodium (the muscleshowing no spontaneouts movement) was greater than that of anothercontracting rhythmically in a pure solution of a sodium halide. Loebused break induction shocks as test stimuli. Now Keith Lucasand J(16) have shown that the influence of temperature on the excita-bility of a muscle may be apparently in opposite directions according tothe duration of the current employed to test it. An induction shockis a current of very brief duration, and it seemed to me not unlikelythat some change in the irritability of the muscle towards a galvaniccurrent might be found on treatment with saline solutions, even inthe absence of change towards faradic stimuli.
EXCITABILITY OF THE MUSCLE IN SODIUM CHLORIDE SOLUTION.
Two methods have been employed in investigating changes in theexcitability of muscles. The first is that of finding the current strengthneeded to produce a just perceptible twitch. This method gives an in-dication of the current strength required to excite the fibres which atthe time are most irritable. It has the advantage of disturbing themuscle very little, as the twitch is so small that there is no danger ofshifting the region of the muscle which is stimulated in the successivetests. The second method is that of observing the effect of a changed
time, and the appearance of a twitch or group of twitches is not the immediate result ofany change in the environment of the muscle, but is due to processes going on within it.There is really no justification at present for calling sodium chloride the stimulus for themovements. Its presence is merely one of the conditions requisite for their appearance,just as is the presence of water and the maintenance of the temperature within certainlimits. Moreover the presence of sodium chloride is a normal condition; the abnormalityis the absence of calcium. The whole question turns on the sense in which the term
stimulus is used. If all factors tending to increase the instability of the excitablesubstance are called stimuli, then in the present case sodium chloride is only one of a
number of stimuli. If, however, the term is reserved for a changing factor which evokesa corresponding response, we know nothing of any such stimulus leading to those move-
ments which occur from time to time after the muscle has presumably come intoequilibrium with the surrounding fluid.
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condition on the height of the response to a uniform submaximalstimulus: it will be discussed later.
The essential deviation from the normal surroundings of the musclewhich is followed by the appearance of the spontaneous movements hereconsidered is the substitution of a solution containing sodium salts onlyfor the normal fluid which bathes the muscle and which contains calciumand potassium in addition to sodium. A frog's sartorius placed in Ringer'ssolution will maintain a constant degree of irritability towards electricalstimuli for a time which depends on the temperature to a great extent,but which often lasts more than a day. My experiments were directedin the first instance to ascertaining whether the sartorius in Ringer'ssolution, exhibiting no spontaneous movement, changed its excitabilitytowards electrical stimuli when the solution was changed to one ofsodium chloride in which the muscle from time to time showedspontaneous movements.
The sartorius was dissected with care to avoid injury to its fibres; athin silk thread was tied to the tibial tendon so as to form a loop: thepelvic end remained attached to the Os pubis which was cut out andfastened to a hook at the end of a glass rod. A small glass weight washung on the loop and the muscle was suspended in the fluid electrodesintroduced by Keith Lucas, of the form figured in a previous paper(16)*The electrodes were provided with side tubes to admit of changing thecontained fluid, and they were used in the manner described by Lucasin a more recent paper (15). The muscle was immersed in fluid through-out its length, and stimulation was brought about by the increase incurrent density produced by a constriction of the tube around the re-quired region of the muscle. Lucas (15) has shown that in this way inthe case of nerve the localisation of stimulus is effected with greatprecision. In these experiments the pelvic region of the muscle wasarranged for stimulation.
Further evidence as to the validity of this method of localisa-tion of stimulus will be brought forward in a later section of thispaper.
A Ringer's solution was prepared with distilled water, containing*63 0/0 sodium chloride, *02 S/b potassium chloride, and 05 0/0 calciumchloride, and a solution of pure sodium chloride was prepared of ap-proximately the same electrical conductivity. In the earlier experimentsthe precaution was taken of determining the electrical resistance of theelectrodes with the included muscle, so as to make sure that anyapparent change in the excitability of the muscle was not in reality due
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CONTRACTION OF SKELETAL MUSCLE.
to an alteration in the resistance of the circuit. (See Gotch andMacdonald(8).)
The solutions were allowed to assume the temperature of the roombefore the experiment, and the electrodes were shielded from draughtsby being surrounded with a large glass vessel.
The record of one such experiment is given below.
Sartorius, frog. Stimulation at pelvic end. Closure of constant current. OneDaniell cell.
Solution in PotentiometerTime electrodes min. twitch Temperature Resistance
This experiment shows that a much smaller current was needed toexcite the muscle when in sodiumn chloride solution than when in Ringer,even though in this particular case the actual electrical resistance of thesodium chloride was slightly higher than that of the Ringer and soproportionately a rather smaller current flowed in the former case. Itwas found in subsequent experiments that the result was qualitativelythe same when a solution of higher (-65 5/o) or lower (175 0/0) resistancethan the Ringer was used: the solution generally employed contained71 °/o sodium chloride. Slight alterations of temperature did not affectthe result.
When tested with induction shocks the change from Ringer to sodiumchloride did not as a rule appreciably affect the excitability.
The following table gives a summary of a number of experimentsmade on the lines indicated.
Observations were made alternately in Ringer and in sodium chloride: when two ormore numbers are given in the "Ringer" column, it must be understood that thesereadings alternate with those given in the corresponding "Isodium chloride" column, andvice ver8d.
29-2
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Stimulation with break induction shocks. Stimulation with galvanic current. TheThe numbers represent the distance be- numbers indicate the position oftween the coils in mm. giving minimal the slider of the potentiometertwitch. (maximum 100).
The spontaneous movements invariably occurred when the Ringer'ssolution was replaced by sodium chloride, but they did not constitute aserious difficulty in the way of finding the minimal twitch. Theactivity of the muscle in sodium chloride is intermittent, and in theintervals of rest it is easy to make the tests. The application of aweak constant current is very often followed by a vigorous series oftwitches.
The experiments show that while the excitability of the pelvicend of the sartorius towards break induction shocks is not as a rule atfirst altered by the substitution of sodium chloride for Ringer's solution,the excitability towards the galvanic current is miarkedly increased.
The same fact was demonstrated by the second method alluded to,namely by recording the mechanical response of the muscle to regular,submaximal stimuli under the two different conditions. A word maybe said here as to the validity of this nmethod, which has often beenemployed in experiments on excitability (cf. Gotch and Macdonald(8)).The submaximal response of a muscle has been shown by KeithLuicas (13) to depend largely, and possibly entirely, upon a contraction ofsome of the fibres only. The submaxinial stimulus is above the thresholdof some, and below that of other fibres. Any factor which raises theexcitability, or in other words, which lowers the threshold of stimulation,will bring a larger numiber of the fibres under the influence of the stimnu-lus and thus the magnitude of the contraction will be increased. A con-ceivable objection to the method is that a great rise in excitability of thefibre, towards mechanical stimuli might lead to the discharge of fibres not
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CONTRAQTION O SKELETAL MUSCLE.
directly excited by the electrical stimulus, through the mechanicalagitation of the muscle due to the contraction of fibres electricallyexcited. In the present case it will be seen that such an objectioncannot be maintained. For these experiments I devised the modifica-tion of Lucas' fliid electrodes shown in Fig. 9. The illustrationsufficiently explains the construction of the apparatus. The liquidsurrounding the muscle was changed by admitting the fresh solutionby the tube A and allowing an over-flow at B. Trials with colouredliquids showed that the liquid in the region surrounding the musclecould be quickly and effectually changed in this way. As a rule agentle stream of the solution was kept up during the experimen1l, toavoid admixture with the fluid remaining in the tubes C and,D as 'theresult of diffusion.
Fig. 9. Modification of Lucas' fluid electrodes.
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The silk thread tied to the tendon of the muscle was attached to theshort arm of a lever which exerted a tension of about four grams on themuscle. This was generally found sufficient to prevent the spontaneoustwitches in NaCl from affecting the record. Fig. 10 shows that a changein the fluid from Ringer to NaCl did not affect the response of themuscle to break induction shocks of submaximal intensity, while itgreatly increased the response to the galvanic current.
Fig. 10. Sartorius of frog. Stimulated at pelvic end at intervals of ten seconds alter-nately with single break induction shocks and galvanic currents of i" duration.A, in Ringer. B, after four minutes in NaCi .70/0. C, after four minutes inRinger.
It must be stated that this absence of change in excitability towardsinduction shocks is not an invariable result. After more prolongedimmersion in the sodium chloride, particuLlarly if no stimulus is appliedfor a considerable time, it is often found that the first induction shockapplied evokes a much greater response than when the muscle was inRinger; a second sbock sent in a minute later causes a much smallercontraction. The inqrease in excitability towards the galvanic currentcomes on at once and the condition persists when a number of successivetests are made.
There is no aoubt that the change in excitability brought about byplacing the musole in sodium chloride after Ringer's solution differsconsiderably according to whether the stimulus used for testing it isa current of-long duration and low potential or one of very short dura-tion and relatively high potential (the break induction shock).
How is this difference in reaction to be interpreted? Do bothvarieties of stimulus attack the same "substance" in the muscle?Taking into consideration the view maintained by Griitzner andZenneck(35), that the sartorius contains fibres of two kinds, the thin,
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CONTRACTION OF SKELETA L MUSCLE.
protoplasmic or " slow " fibres and the clear, thick, " quick " fibres, andthat these are possessed of different " excitability-relations " some suchhypothesis as this might be put forward, viz. that the induction shockstimuilates the " quick " fibres, while the galvanic current picks out the" slow " fibres, and that the change of solution affects the " slow " fibresto a much greater extent than the " quick " fibres. Such an hypothesis,though it involves assumptions which are at present unfounded, mightbe held to explain the phenomena observed.
If it were correct, one would expect to find that on testing the musclewith electric currents of diminishing duration the excitability in sodiumchloride remained constantly higher than that in Ringer, until a certaincritical value of current duration was reached at which the quick fibreswere affected, when the excitability in both cases would be the same. Inother words, the curves relating the current strength and current dura-tion for the muscle in Ringer and in sodium chloride would be expectedto run parallel until they abruptly coalesced. Through the kindness ofMr Keith Lucas I was enabled to test this point with the aid of hisnew apparatus' for obtaining currents of different durations. Themuscle was arranged in the usual way for observing the minimal twitch.The testing currents were thrown in at intervals of ten seconds begin-ning with the shortest current used. When a set of observations inRinger's solution had been completed, the fluid was changed to sodiumchloride solution and after about ten or fifteen minutes a fresh set ofobservations was made. Fig. 11 illustrates the results which were ob-tained by this method. Each point in the curves defines the strength andduration ofa current just capable of exciting the muscle. The upper curveexpresses the mean of two sets of observations made respectively beforeand after the set plotted in the lower curve. It is clear that, within therange examined, the muscle was always more excitable in sodium chloridethan in Ringer's solution. But this difference is proportionately greaterfor currents of long duration than for currents of short duration. Thusas the current duration diminishes there is a convergence of the curves,indicating that the difference in excitability brought about by thechange in solution is smaller as the testing current is shorter. It is alsoto be noted that while the curve denoting the excitability of the musclein Ringer's solution becomes parallel to the horizontal axis at about -08",the curve plotted from observations made with the muscle when insodium chloride continues to descend.
1 A description of the apparatus, which was exhibited at the recent InternationalCongress, will be published shortly in this Journal.
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G. R. MINES.
We have, then, no reason to believe that the long and the shortcurrents affect different kinds of fibres. The alternative view is thatboth stimuli excite fibres of the same type, and that the substitution ofsodium chloride for Ringer's solution affects them in such a way thatthey become much more responsive to currents of long duration, butthat their reaction towards very brief currents is not altered in the sameway, or at any rate not to the same extent-in other words that whathas been shown to be true for the muscle taken as a whole is true of itsindividual fibres.
Jo
30
/0 ~A ~ cLi
I I I I 1- I 9 U I I*O,g" .0/" 'CA-"/
Fig. 11. Frog's sartorius. Ordinates represent relative current strengths.Abscissae show current duration.
Certain conclusions as to the causation of the twitches which occurin sodium chloride solution may be drawn from the experimental resultswhich have been described. The occurrence of the movements isassociated with a condition of increased excitability towards currents oflong duration, and presumably this is one of the factors leading to theirappearance. It cannot, however, be the sole factor, since a fall in tempera-ture diminishes the twitches although it is known to cause an increasein excitability towards galvanic currents (8, 16). Presumably then, thereexists another factor which regulates the appearance of the movements,which is reduced in efficiency by a fall of temperature. Possibly thisfactor may be the ionic velocity of the electrolyte or the condition
426
CONTRACTION OF SKELETAL MUSCLE.
of viscosity of the muscle substance or the permeability of the membraneenclosing it.
The fact that the non-neural region of the muscle shows a markedchange in the direction of heightened excitability when the muscle isplaced in sodium chloride lends support to the view that the seat oforigin of the spontaneous twitches is in the actual contractile substanceof the muscle fibres.
A relation between rhythm and "refraetory period " has beensought in most instances of rhythmic discharge. Obviously the periodsof the rhythms exhibited by the sartorius are of quite a different orderof magnitude from the " refractory period " of the muscle as tested byinduction shocks, which, as Bazett (1) has shown, has a value of about*006 of a second.
Carlson (4) has drawn attention to the fact that there may be aperiod of diminished excitability in a rhythmically contracting organ,which he terms the "relative" refractory period-and indeed the"absolute" refractory period mneans no more than an insusceptibilityof the organ to the strongest stimulus tried.
In the case of a structure showing rhythmic contractions it ispresumable that the molecular complex, whose break-down is signalisedby a contraction, must be in a more unstable condition when acontraction is about 'to take place than just after it has occurred.In the light of what has been said as to the differences manifestedin the excitability relations towards different kinds of stimuli, it isclear that such alterations in stability may not affect the reactiontowards the particular electric stimulus used for exploration.
This disparity between rhythm and "refractory period" finds aparallel in the case of the motor cells of the rabbit's spinal cord.Under anesthesia the limbs are often observed to give regularrhythmic movements with a period of a second or more. Thesemovements are of central origin, but their rate is independent ofthe respiratory rhythm, and indeed two limbs may sometimes be seento move at the same time with unlike rhythms. Cells in the spinalcord are here giving rhythmic discharges at intervals very muchgreater than that usually understood as their refractory period.
The rhythms of short period which I have mentioned as occurringmost frequently in solutions containing a little potassium, seem to callfor special consideration. It may be that the maintenance of suchrhythms is to be explained by auto-excitation (cf Gotch and Burch (9)).Kuhne (18) showed that in an injured sartorius a single stimulus might
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G. R. MINES.
be followed by a multiple response, the action current accompanyingeach contraction serving as a stimulus for the next. Possibly in thesamiie way a single spontaneous contraction may be followed by a rapidseries of twitches, each twitch being directly provoked by the electricalvariation due to its predecessor.
III. The influence of calcium and potassium chlorides on the electricalirritability of the non-neural region of the frog's sartorius.
Previous work on the influence of salts on the excitability ofmuscle has been chiefly directed towards ascertaining thie relativelength of time during which excitability is maintained in the varioussolutions rather than to the determination of the more immediatealterations in degree of excitability produced by modification of thesolution bathing the muscle. The object of my experiments has beento determine the influence of the addition or removal of calcium and
Fig. 12. Fluid electrodes, with device for bringing successive regions of the sartoriuswithin the constricted tube. The millimetre scale is set with the pointer at zerowhen the extreme pelvic end of the muscle lies in the constriction.
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CONTRACTION OF SKELETAL MUSCLE.
of potassium chlorides on the electrical irritability of the muscle fibres.The method of localisation of the stimulus must be first considered.The " direct " excitability of muscles has often been tested by applyingmetallic electrodes to the muscle in any region. Our present knowledgeof the physiological complexity of the sartorius suggests that sucha method will give results difficult of interpretation. Another methodof excitation commonly used is that of sending the current throughoutthe length of the muscle, the electrodes taking the form of metallichooks inserted into the bony attachments of the muscle at each end.Since the effective excitation is at the kathode, it might be claimedthat this method ensures stimulation of the non-neural region of themuscle alone. But when the currents used for stimulation are of anyconsiderable strength it is open to question whether regions, other thanthe non-neural ends, may not be directly influenced by the currenit.It is conceivable that some histological discontinuity, such as thatintroduced by the intra-muscular nerves, may lead to tne formationof internal physiological kathodes, after the manner in which tendinousintersections are known to act. This possibility is clearly eliminatedby placing both electrodes in the non-neural region at the same end ofthe muscle. The electrodes which I employed for this purpose havebeen figured on a previous page. In order to test whether, under theconditions of experiment, the use of this form of apparatus wouldindeed secure the localisation of stimulation required, I made somepreliminary experiments in the following way.
The sartoriuis was supported by a glass hook and so arrangedthat successive portions of the muscle could be brought within theconstriction. The device by which this was accomplished will bereadily understood by a glance at Fig. 12. Beginning at the pelvicend the muscle was raised one millimetre at a time, and the positionof the secondary coil of the inductorium needed to evoke a minimaltwitch was determined with the various regions of the muscle lyingin the constricted part of the tube. At the end of each experimentthe muscle was stained with osmic acid and gold chloride by Mays'method (26) to show the distribution of the intra-muscular nerves.The preparation was drawn on a magnified scale with the aid of aprojection apparatus and this drawing was compared with the plottedelectrical observations.
The nature of the results obtained in this way is illustrated byFigs. 13 to 16. These figures are photographs of the drawings placedin position beneath charts indicating the coil distances necessary to
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G. R. MINES.
just produce a twitch with the various regions of the muscle in theconstriction.
Fig. 13. Described in text. Fig. 14. Described in text.
The chart shows the upper extremities of lines which if continueddownwards for about three and a qjuarter centimetres wouild be of lengthsproportional to the distances between the primary and secondary coils
Fig. 15. Described in text. Fig. 16. Described in text.
of the inductorium. I desire in this place merely to point out that inthe region corresponding to the junction of the neural and non-neuralregions there is a definite and often abrupt change in the currentneeded to excite. The sartorii used were taken from rather smallspecimens of Rana temporaria, and were about twenty millimetres inlength. The histological and experimental methods seem to findmutual support. The apparent irregularity, sometimes to be seenat the extreme pelvic end of the muscle, may, I think, be due tothe expansion of the muscle in this region, or perhaps to the partialobtrusion of the bony pelvic attachment into the orifice of the tubecausing some alteration in the distribution of the lines of currentdensity in the fluid, resulting in the passage of an increased fraction
430
CONTRACTION OF SKELETAL MUSCLE.
of the current through the muscle fibres. The distribution of nervefibres within the muscle varies considerably in different cases, but as arule there appears to be a length of muscle not less than four orsix millimetres free from nerves at the pelvic end. The figures givenby Kuh ne (17) and Mays (26), showing the distribution of nerves in thesartorius, have been for the most part from preparations made fromRana esculenta; it may therefore be not superfluous to add here somefurther photographs of drawings showing the nerve distribution inRana temporaria. (See Figs. 17 to 20.)
Fig. 17. Sartorii of Rana temporaria. Fig. 18. Bartorii of Rana temporaria.Treated by Mays' method. Treated by Mays' method.
Fig. 19. Sartorii of Rana temporaria. Fig. 20. Bartorii of Rana temporaria.Treated by Mays' method. Treated by Mays' method.
In the experiments about to be described the sartorius was arrangedso that a region one or two millimetres from the pelvic end lay in theconstricted part of the electrodes, and it may fairly be taken thatstimulation was confined to the non-neural part of the muscle.
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G. R. MINES.
The relative excitability of the muscle in each of various pairs ofsolutions was examined, for the most part by the method of sub-maximal stimulation already discussed. The stimuli used weregalvanic currents of about i second duration, and single breakinduction shocks. Both kinds of stimuli were thrown in automaticallyby means of a Brodie clock, which in the former case closed a simplerelay in the potentiometer circuit, and in the latter case operateda key which I devised for the purpose and which I have describedelsewhere (2n). The arrangement of the electrical circuits is indicatedin Fig. 21.
Fig. 21. Diagram of electrical circuits. Described in text.
A represents the Brodie clock. By means of the switch B thecurrent from the clock could be sent into the key 1P, resulting in thedelivery of a single break induction shock, or into the key C, theclosure of which caused a current to flow through the potentiometerwire E. The Gotch key C allowed either of these currents to be ledto the electrodes H.
To avoid fatigue the stimuli were delivered not more frequentlythan once. in thirty seconds.
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CONTRACTION OF SKELETAL MUSULE.
The solutions whose effects were tested were prepared by adding.1 0/0 solutions of calcium or potassium chloride to a 7 0/ solution ofsodium chloride, all in distilled water. As the amount of calcium orpotassium chloride used never exceeded 10/0, there was little variationin the osmotic pressures of the solutions and they always containedfrom *6 0/o to *7 0/0 of sodium chloride.
That the changes in height of contraction following the substitutionof one solution for another were not due to alterations in the intensityof the stimulating current (such as might be produced by change inthe resistance of the fluid column) could be inferred from the fact thatthese changes were progressive, but as a control in many cases I usedthe fluid electrodes in duplicate, arranging them in series so that thesame current passed through both. The solution surrounding one ofthe muscles was left unchanged, and it was found that when the sub-stitutions of solutions were made in the other electrodes there was nochange in the contractions of the first muscle. The two levers werearranged to write one above the other on the same drum. Beforebeginning an experiment the solutions were always allowed to assumethe temperature of the room ; in the experiments to be described thetemperature lay between 15' and 200 C., the actual variation in tempe-rature during the course of an experiment was negligible.
The influence of potassium chloride.
It has been shown by many workers that potassium salts exert a de-pressantactionon muscular excitability. Thus Ringer and Murrell(32)found that treatment with potassium chloride caused an early disap-pearance of irritability from the musculature of frogs. Locke (20)observed that this salt reduced and finally abolished the response of thesartorius to induction shocks. Biedermann (3) speaks of potassium saltsas muscle poisons exerting a highly depressant and inhibitory action onthe excitability of both skeletal and cardiac muscle. On the other handRinger('*) discovered later that the contractions of a sartorius whichhad been frequently stimulated in sodium chloride were increased bythe additioD of potassium chloride. Carlson(3) states that althoughpotassium chloride depresses the activity of the muscular tissue of theheart of Limulus, it exalts that of the isolated ganglion. It has beenpointed out by Griitzner('0), Zenneck(35), Zoethout(36) and others thatpotassium chloride in isotonic solution causes an immediate and pro-longed contraction when applied to striated inuscle, and this fact in
4%33
G. R. MINES.
itself renders it probable that the salt in smaller doses will have someaction other than depressant'.
I find that the primary effect of potassium chloride, in sufficientlysmall quantities, is to produce a great increase in the excitability of themuscle towards both the galvanic current and induction shocks. This isillustrated by the record shown in Fig. 22. When tested on the same
Fig. 22. Sartorius of frog. Stimulated alternately with galvanic current and inductionshock at 30 second intervals. Each response to an induction shock is marked with across. At the beginning of the tracing the muscle was in NaCl *7 /0/; at the arrowthe fluid was changed to NaCl *65 0/0, KCl 135 0/0.
preparation the increase in excitability towards the galvanic currentappears greater than that towards induction shocks.
The magnitude of the effect produced by a given dose varies in dif-ferent muscles, but a quite appreciable exaltation has been observedwhen the proportion of potassium used amounted to only .01 %.
The secondary effect of potassium chloride is a reduction in excitabilitywhich goes on till there is a complete abolition of the twitch in responseto any kind of stimulus (Fig. 23 b). This stage is quickly reached %A hen
1 In a recent conversation Dr F. S. Locke informed me that in 1894 he observed anincrease in the excitability of the sartorius towards induction shocks as the primaryresult of adding either potassium or calcium chloride to a solution of sodium chloridesurrounding the muscle. The stimulation was not confined to the non-neural region ofthe muscle, These experiments were not published.
434
CONTRACTION OF SKELETAL MUSCLE.
the solution contains *10/0 potassium; it is also arrived at by the moreprolonged action of weaker solutions.
Sodium chloride restores the excitability after its depression orabolition by potassium chlorides (see Fig. 24). If the potassium
Fig. 23. Sartorius of frog. Stimulated with single break induction shocks at intervalsof 30 seconds. a shows the effect of 05 0/0 KCI, b that of 1°0/0 KOC. At thebeginning of a and b and during the five minutes which elapsed between them themuscle was in NaCl 7 0/0.
chloride has not been allowed to act too long, it is fouind that the changeto sodium chloride may be followed by a rise in excitability which reachesa higher value than that shown by the muscle when it-was in sodiumchloride before the treatment with potassium. The probable explanationof this phenomenon is that as the potassium diffuses out of the muscleits concentration becomes reduced to that at which it exerts a beneficialeffect on the excitability.
Fig. 24. Simultaneous records of contractions of right and left sartorii of frog, stimulatedby the same induction shook at intervals of 30 seconds. The irritability of theupper muscle had been abolished by three hours' immersion in KCl -05°/0, NaCl .65o/o.This was replaced by NaCl .70/0 five minutes before the beginning of the tracing.The control muscle was in the latter solution throughout.
The twofold effect of potassium chloride on the excitability of thesartorius was equally evident when sought for by the method referred tobefore as that of the minimal twitch. An instance may be quoted.
PH. XXXVII. .30
435
G. R. MINES.
Frog's sartorius e-xcited at pelvic end by closure of constant current.Position of slider ofpotentiometer forminimal twitch
Muscle in *7 0/a NaCl ... ... ... ... 30After 5 minutes in /0BC/K ... ... 20
20 ,, ,, ,.90
A second dose of potassium chloride, after the effect of the first hasbeen removed by sodium chloride, produces its depressant effect morequickly; the primary exaltation is less pronounced and may even beabsent.
Certain points in connection with the effect of this salt on the formof the contraction curve in response to electrical stimuli may be mentionedhere.
Ringer and Buxton (n) showed long ago that the form of the muscletwitch evoked by an induction shock is greatly modified when the muscleis kept in sodium chloride solution the relaxation being much pro-longed. I find that the addition of a little potassium chloride constantlyhas the effect of converting this contracture into a rapid twitch.Fig. 25 will serve to illustrate this.___Fig. 25. Response of frog's sartorius to single break induction shocks. a in NaCl .7 e/0,
b one minute after the fluid had been changed to KCI .1 0/,, NaCl *6 e/o. Timemarked in seconds.
When the muscle is stimulated with the galvanic current anotherfeature of the action of potassium chloride becomes apparent. As thetwitch which occurs on closure of the circuit becomes first quickenedand then abolished, the kathodic shortening which persists during thewhole time the current is flowing becomes more prominent, and whenthe twitch has completely disappeared the kathodic shortening remainsevideut (cf. Fig. 29).
436
CONTRACTION OF SKELET'AL MUSCLE.
Saito0(") found that the prolonged contraction in response to agalvanic current was reduced in muscles that had been exposed to theaction of moderately small doses of potassium chloride.
His "dauerverkiirzung" is not identical with the kathodic shortening,since in the first place it may occur either at the commencement ortermination of a galvanic current and secondly it may pass off duringthe passage of the current which elicits it. Saito, however, appears notto clearly distinguish the two reactions, which are both to be seen in hisgraphic records. His failure to obtain any evidence of the increase inthe kathodic shortening as the result of the action of potassium must beascribed to the fact that he left the muscles for several hours in thesolution before the tests were made.
The influence of calcium chloride.
It is well known from the work of Ringer A "1) that the addition of acalcium salt to sodium chloride solution enables it to maintain muscu-lar irritability for a much longer time. Lock e (22) showed that calciumchloride would restore the indirect ex-citability of muscles after it had dis-appeared through immersion in sodiumchloride. Carlson(5) mentions that theaction of calcium on the heart muscle ofLimulus is to produce immediate de-pression of excitability.
By the method of submaximal stimu-lation I find that the addition of a smallamount of calcium chloride to the sodiumchloride solution bathing a sartoriuscauses an immediate diminution in theexcitability towards currents of longduration (see Fig. 27).
The addition of *1 0/0 calcium chloridemay cause abolition of the twitch leaving Fig. 26. Sartorii of frog, seriesa somewhat diminished kathodic con- arrangement. Stimulated at
intervals of 30 seconds withtraction. single break induction shocks.
When the stimulus used is an in- Lower muscle in NaCl .70/O,duction shock the effect of calcium changed at arrow to CaCl2chloride on the excitability is variable. 05 /0, NaCl -65°/. Uppermuscle in the latter solutionWhen the muscle has been in sodium throughout.
3Q-2
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G. R. MINES.
chloride for a comparatively short time the addition of a small doseof calcium sometimes produces no apparent change in the excitability:more frequently it causes a slight fall. This effect is shown in Fig. 26.
The different degree of effect on excitability towards galvanic andtowards induction currents is clearly seen in Fig. 27.
Fig. 27. Sartorius of frog. Stimulated at intervals of 30 seconds alternately withgalvanic current and break induction shock. The responses to induction shocks aremarked with crossies.
When the muscle has been in sodium chloride for some considerabletime, and particularly if it has been frequently stimulated, the additionof a little calcium chloride distinctly increases the excitability towardsinduction shocks. Fig. 28 illustrates this action and provides a good
Fig. 28. Sartorius-of frog. Stimulated with break induction shocks at intervals of30 seconds. The muscle had been in NaCi .7 e,0 for 11 hours. Between the ar'rowsa solution was substituted containing CaCl2 -05 e,0, NaCi -65 0/e.
instance of the frequently observed fact thiat calcium makes the responsesmuch more regular. Attention has been called earlier in this paper to theirregularity commonly shown in a series of contractions in response toregular stimuli by the sartorius in sodium chloride. I have not observedany increase in excitability towards galvanic currents on the addition ofcalcium chloride, even after prolonged immersion of the muscle in sodiumchloride.
A few experiments were made to test the excitability towardscurrents of various durations in the manner referred to in the section
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CONTRACTION OF SKELETAL MUSCLE.
dealing with the excitability in Ringer's solution and sodium chloride.The results obtained were very similar. Some data may be quoted.
Frog's sartorius. Stimulation near pelvic end. Temp. 15.3-16o C.
Position of slider of potentiometer for minimal twitcl.(Max. 100.) 1 dry cell
It will be seen that there is a slight progressive fall in excitabilityduring the experiment so that in the third column the values are slightlyhigher than in the first. Comparing the mean of the two sets of obser-vations made in the solution containing calcium with the intermediateset in sodium chloride, it appears in this case that the excitability of themuscle towards currents of very brief duration is the same in bothsolutions, whereas with longer currents the excitability in the solutioncontaining calcium is much less.
While the current strength needed to excite the muscle when insodium chloride continues to fall as the current duration is increasedover a range which extends beyond *12", it is noticeable that when thernuscle is in the solution containing calcium chloride the current strengthneeded to excite it remains roughly constant for current durations above'07".
Comparison of the effects of calcium and potassium chlorides on thespontaneous movements of the sartorius with the action of thesesalts on the irritability of the non-neural region of the muscle.
It is of interest to note that the effect of calcium and potassiumchlorides on the irritability of the non-neural region of the sartoriustowards galvanic currents of " long " duration follows the same courseas the influence of these salts on the activity of a muscle exhibitingspontaneous movements in sodium chloride solution.
439
G. B. MINES.
As I have pointed out in the first section of this paper the additionof a small dose of potassium chloride to a solution of sodium chloride inwhich a sartorius is giving spontaneous movements causes a primaryincrease in the movements, followed by a secondary diminution orabolition: in the same way it brings about a primary exaltation anda secondary depression of the irritability towards galvauic currents.Calcium chloride produces an immediate diminution in the activity ofthe spontaneous movements, and similarly an immediate depression ofthe excitability towards galvanic currents.
It has been seen that while the influence of potassium on the excita-bility towards induction shocks is qualitatively the same as its influenceon the excitability towards galvanic currents, a similar relation doesnot obtain in the case of calcium.
It again appears that the excitability of the muscle towards galvaniccurrents affords some criterion of that condition of the muscle whichleads to spontaneous contraction.
Antagonism between calcium and potassium.
The existence of an antagonistic action between the salts of calciumand potassium was pointed out by Ringer(ao) and has since beendescribed by Loeb(24), Overton(21), and others. Loeb has introduced
~--|Fig. 29. Sartorius of frog. Stimulated with galvanic current (potentiometer 90, two
The middle line marks the time in seconds, the lowest line indicates the duration ofpassage of the current.
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CONTRACTION OF SKELETAL MUSCLE.
the conception of a physiological balance existing between the variousions in a saline mixture such as Ringer's solution.
The mutual checking action exercised by calcium and potassiumchlorides, as regards their influence on muscular excitability, is wellbrought out by the method of experimentation which I have used. Theremoval of calcium from a mixture containing calcium, sodium andpotassium is followed by alterations in the excitability of the immersedmuscle exactly similar to those produced by the addition of potassiumto a solution of sodium chloride. Fig. 29 illustrates the results obtainedwhen the stimulus employed was a galvanic current. The removal ofcalcium from the solution is seen to be followed by an increase both inthe twitch and in the kathodic shortening, the twitch subsequentlydiminishes and soon disappears, though the kathodic shortening remainsdistinctly greater than before the removal of the calcium. The removalof potassium quickly reveals the depressant action of calcium (seeFig. 30).
Fig. 30. Sartorius of frog. Stimulated alternately with galvanic currents and inductionshocks at intervals of 30 seconds. A cross is placed beneath each response to aninduction shook. At the beginning of the tracing the muscle was in NaCl -6 0/0,KCI O5 0/0, CaCl2 *05 0/0. As indicated by the arrows, this solution was replaced firstby CaCl2 -05 b/0, NaCl *65 0/0, and afterwards by KCI .05 0/0, NaCl *6.5 0/0.
Similarly the addition of potassium to a saline solution containingcalcium causes an increase in excitability less in degree than would beproduced in the absence of calcium. Moreover an amount of potassiumwhich in the absence of calcium would cause an abolition of excitabilityin the course of a few minutes, may, when calcium is present, manifestits primary exaltatory effect alone. The antagonism is not complete,
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G. R. MINES.
for as Overton (21) has shown, a moderately small dose of potassium canentirely paralyse the muscle no matter how much calcium is present.
SUMMARY AND CONCLUSIONS.
The study of photographic records of the " spontaneous " movementsof amphibian muscles in saline solutions shows that these movementsoften exhibit a definite rhythm, which while it persists is comparablein regularity to accepted physiological rhythms such as that of thebeating heart. Irregular tracings frequently prove on analysis to resultfrom the mechanical interference of several rhythmic components. Theease with which the twitches can be elicited by the use of 7 I/. sodiumchloride varies somewhat in muscles from different regions of the body,but by perfusion of the blood vessels witlh the solution the movementsmay be produced in the whole skeletal musculature. The periods ofthe rhythms recorded vary in different cases from 6 seconds to *06 of asecond.
The movements will continue in the presence of a concentration ofcurare greatly in excess of that required. to disorganise the myo-neuraljunction. Large doses of curare abolish the movements, but this actionis shown to be sufficiently accounted for by the influence of inorganicsalts present in the curare. This tolerance of curare suggests that thesource of the movements lies in the contractile substance of the musclefibres. More conclusive evidence on this point is derived from experi-ments on the electrical irritability of the sartorius, carried out bymethods which are shown to confine the stimulus to the non-neuralregion of the muscle. The appearance of the movements is associatedwith a greatly increased excitability of this region towards galvaniccurrents of long duration. There exists, moreover, a striking parallelismbetween the influence of calcium and potassium chlorides on thespontaneous movements and the effect of these salts on the excitabilityof the non-neural region towards the variety of stimulus mentioned.Thus the addition of calcium chloride to the sodium chloride solutionsurrounding the muscle leads to an immediate diminution or cessationof the movements and a fall in the excitability of the non-neural regiontowards galvanic currents. Potassium chloride at first increases themovements, often causing a very rapid rhythm, and then stops thementirely; its effect on the excitability towards galvanic currents is aprimary exaltation and a subsequent depression.
The truly rhythmic character of the movements taken in conjunction
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CONTRACTION OF SKELETAL MUSCLE. 443
with the strong probability that they are myogenic, emphasises therunidamental similarity between the skeletal and cardiac muscle fibresof vertebrates.
The apparent changes in excitability produced by various salinesolutions may differ according to the duration of the current by whichthe excitability is tested; there may be a change of excitability towardsgalvanic currents of long duration when none is observed towards shortgalvanic currents or induction shocks. In all probability this is alsotrue of the indivi(lual fibres of the muscle.
The antagonism between calcium and potassium is well shown intheir action on muscular irritability.
In conclusion I sincerely thank Professor Langley for his invaluablecriticism and advice, and Mr Keith Lucas for the loan of severalpieces of apparatus without which much of this work could not havebeen done.
REFERENCES.
(The numbers correspond to those in the text.)
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(10) Grrutzner. Pfiluger's Arch. LII. p. 85. 1893.(11) Hering. Sitz. d. Wien. Akad. LXXXIX. Abt. 3, p. 1. 1879.(12) Keith Lucas. This Journal, xxx. p. 443. 1904.(13) Keith Lucas. Ibid. xxxiii. p. 125. 1905.(14) Keith Lucas. Ibid. xxxvi. p. 113. 1907.(15) Keith Lucas. Ibid. xxxviI. p. 112. 1908.(16) Keith Lucas and Mines. Ibid. xxxvi. p. 334. 1907.(17) Kuhne. Muller's Arch. p. 582. 1859.(18) Kiuhne. Zeitschr. f. Biologie, xxvi. p. 203. 1890.(19) Langley. This Journal, xxxvi. p. 364. 1908.(20) Locke. Pfluger's Arch. LIV. p. 501. 1893.(21) Locke. Journ. of Exp. Med. i. No. iv. 1896.(22) Locke. Centralbl. f. Phys. viii. p. 166. 1904.
444 G. R. MINES.
(23) Loeb. Festschrift f. Fick. 1899. Also, Decennial Publications of Univ. ofCalifornia, iI. p. 518.
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