An investigation has been made of the action potentials of the muscles of the crayfish, Cambarus clarkii. It was found that the polarity of these potentials was in many instances the reverse of that expected.
It is shown that ‘positive’ potentials cannot be explained by injury to the active tissue, by the complex structure of the muscles, or by the direction of the muscle fibres.
Using muscles in which all extraneous connexions could be rigorously avoided, the muscle action potentials were invariably found to be negative.
Direct evidence is presented for the local non-conducted nature of the crustacean muscle action potentials. An hypothesis is offered to explain the monophasicity and polarity of these action potentials on the basis of multiple unilateral innervation of the muscle fibres.
The muscle-action potentials from decapod crustaceans have been recorded many times by different investigators. These observations have established the fact that these potentials differ considerably from the action potentials of vertebrate striated muscles. Attention has been mainly focused on the phenomena of facilitation (Wiersma, 1933; Katz, 1936; van Harreveld & Wiersma, 1936; Wiersma & van Harreveld, 1938), while much less notice has been taken of the electrical sign of these potentials. Biedermann (1888) observed that in the adductor muscle of the claw of Astacus the electrode placed in the muscle substance became positive relative to one placed distal to the tendon of this muscle. This observation has since received special attention only by Serkoff (1935). Biedermann’s explanation was that the electrode in the muscle functioned as the indifferent one under the circumstances, since the hole made for it would cause so much damage that no activity would take place in its neighbourhood. By cutting the distal part of the muscle and applying an indifferent electrode to this cut, he found that the lead in the body of the muscle then became negative. Serkoff observed that a small injured region near the end of the muscle could become even more electro-negative during excitation than during rest and therefore doubted Biedermann’s explanation. He came to the conclusion that the potentials obtained did not arise under the electrodes but some distance away from them, and that the distal parts became,more negative than the proximal ones owing to the peculiar anatomical structure of the adductor muscle. The cause of this asymmetrical distribution of potential is not discussed. During the course of our investigations it became clear that unexpected signs of the action potentials are the rule for crustacean muscles, and in these cases the explanations provided by the papers cited above were inadequate.
The action potentials from muscles of Cambarus clarkii were used throughout this investigation. They were recorded with the aid of a cathode-ray oscillograph connected to the preparation through a high-gain differential amplifier. In all cases the muscles were stimulated indirectly by square-wave electric pulses which also activated the sweep circuit of the oscillograph. Various types of leading-off electrodes were used; generally platinum wires wrapped in a thin layer of cotton soaked in the physiological solution described by van Harreveld (1936) were employed. The muscles investigated included the closer (adductor) and opener (abductor) of the claw, the extensor of the carpopodite of the cheliped, the first medial segment of the anterior thoracic muscle and the posterior adductor muscle of the mandible.
Closer of the claw
To exclude the possibility that muscle damage would be a factor in the results obtained, a series of experiments was performed in which the electrodes were attached to the outside of the shell of the propodite in various positions. It was found that the potentials obtained in this way were smaller but otherwise similar to those obtained from the same placements when holes were subsequently made. Therefore the slight damage to the muscle caused by insertion of the lead wire is insignificant as far as the electrical sign of the action potentials is concerned, confirming Serkoff’s (1935) finding that a small wound does not completely inactivate the surrounding tissue as would be required for Biedermann’s (1888) explanation of the positive-action potentials.
In further confirmation of Serkoff it was found that an electrical gradient existed along the long axis of the muscle during activity. The distal part of the shell became more negative than the proximal part, and this relationship held true for any given electrode distance. The deflexion obtained was mainly monophasic though often complicated by smaller variations.
The potentials thus far described were obtained from the muscle on stimulation by way of the fast-closer nerve fibre, resulting in twitch contractions. The potentials recorded when the muscle was stimulated by means of the isolated slow-closer nerve fibre followed the same rules of polarity, but were smaller in size. These show, of course, in contrast with the fast-twitch action potential, the facilitation previously described by van Harreveld & Wiersma (1936).
Opener of the claw
The action potentials of the opener muscle are known to resemble those of the slow closer in size and shape (Marmont & Wiersma, 1938). When led off through the intact shell from the region about the muscle, results similar to those from the closer were obtained. Again the muscle potentials were preponderantly monophasic and again the distal regions became more electronegative than the proximal ones. The structure of this muscle is much simpler than that of the closer in that the muscle fibres are more restricted to one plane. It therefore follows that the complex structure of the closer muscle has no significance in the peculiarity of the electrical sign of its action potential.
Anterior thoracic muscle
For further study of the action potentials it seemed desirable to exclude the influence of the feather-like arrangement of the muscle fibres. Therefore the next preparation used was that of the first medial segment of the anterior thoracic muscle. After evisceration there is found on the ventral side of the thoracic cavity a long muscle, divided into parts by tendinous tissue, each segment of the thorax from the second being represented by one such division.
The first of these divisions inserts on a ridge of chitin and is clearly split into two parts, a medial and a lateral, which join in the second segment. The muscle fibres in both these parts run nearly parallel. As shown by Wiersma (1938) the whole thoracic muscle can be brought into practically simultaneous action by stimulation of the giant fibres in the central nervous system. These fibres are in this preparation easily accessible in the oesophageal commissures.
It was always found that an electrode placed anywhere on the dorsal surface of this muscle, which is completely exposed on evisceration, became electro-positive relative to an electrode placed on inactive tissue regardless of the inter-electrode distance. Since other parts of the thoracic muscle as well as muscles in the neigh-bourhood attaching to the legs also contract on stimulation of the giant fibres, further analysis proved difficult (see, however, below). The potentials obtained were large, primarily monophasic spikes, but complicated by numerous small variations attributable to the activity of these surrounding muscles. However, the conclusion is warranted that the feather-like arrangement of fibres in other muscles is not the factor responsible for the positivity of their muscle action potentials.
Posterior adductor of the mandible
We turned our attention to the mandible muscle since it is the only large muscle in the crayfish which can be approached easily from all sides and on which electrodes can be placed on any part of its surface (see Pl. 7). It was found that this muscle responds with a twitch contraction on indirect stimulation with a single shock. The nerve runs along the tendon and follows it in its ventro-dorsal course. Preparation of the nerve is difficult but not necessary, as very good results were obtained by placing the stimulating electrodes on the caudal side of the tendon thereby contacting the nerve. The muscle was isolated by cutting away a curved rectangular piece of carapace, including both the origin on the carapace and the insertion on the mandible.
The muscle may be best described as a half-cone, the flat side being the lateral (carapace) side, and the fibres which are the continuation of the tendon meet the carapace at almost a right angle, whereas the fibres on the medial side form a distinctly sharper angle, which diminishes noticeably during contraction.
When the action potentials of this preparation were recorded by placing one electrode at the middle of the muscle and the other on the tendon, the body of the muscle became electropositive with regard to the tendon lead. It thus seemed inevitable that active crustacean muscle tissue became positive with respect to inactive tissue. Careful probing with the electrode on the muscle disclosed, however, that though most of the muscle surface gave positive potentials, one small area became electro-negative during activity. This area was located very near to the carapace on the lateral flat side of the half-cone and registered a negative spike of about 12 mV. compared to the positive spike of 12 mV. recorded from the opposite curved side of the cone (see Pl. 7). When the electrode approaches this negative area the positive spike first diminishes in amplitude, then becomes negative after passing through a stage in which the amplitude is small and the deflexion diphasic.
When a small cut was made in this negative area its action potential immediately became about as strongly positive as it had been negative. Artificially inactivating other areas on the muscle resulted in a further increase of the original electropositivity. These experiments indicated that the tendon lead became actually more negative during activity than most other parts of the muscle surface. At first glance it was not obvious how this was possible. The only other connexion of the tendon with other parts of the muscle was through the shell, as it curved from the origin of the muscle to the tendon, and it was considered that the electrical resistance of this pathway would be much greater than that of the wet muscle surface itself. If this were the case it ought not to have much influence on the electrical recording. However, further isolation by cutting the shell between the origin and the tendon resulted in a striking effect: all muscle action potentials were from that time on negative, the largest arising from the small area which showed originally the electronegativity. By replacing the connexion between the carapace and tendon by means of a piece of cotton-wool soaked in physiological solution the original positive potentials were restored and the sign of the potentials could in this manner be changed at will. Therefore with the shell intact, or with the cotton connexion, the tendon lead is the more electrically active, but becomes the less active when these connexions are broken.
Not only the whole muscle but also each fibre is conical in shape. This may account for the increase in the size of the action potentials observed when, using the completely isolated preparation, the electrode is placed closer to the carapace, where the thicker ends of the fibres are situated.
Extensor of the carpopodite
The inner surface of the extensor muscle can be rather easily exposed without damage by carefully removing the flexor muscles in the meropodite and the part of the shell to which these are attached. Although this muscle lacks the advantage of being approachable from all sides, as in the case of the mandible muscle, most of its fibres are cylindrically shaped. Furthermore, it has the feature that the ramification of its nerve supply takes place on the exposed side (van Harreveld, 1939). Thus the three nerve fibres supplying this muscle can be lifted on micromanipulated electrodes and their branches cut, if desired. The muscle fibres run in feather-like arrangement from the tendon to the shell, and it is possible to probe the exposed surface of a single muscle fibre with a micromanipulated lead.
It was found that with single shocks these fibres gave large monophasic negative spikes relative to the solution in which the ischiopodite was dipped. These potentials diminished in size when the probing electrode was placed near the tendon or near the shell and reversed to positive in many instances on the inactive insertion of the fibre. The shell was positive with respect to the solution as were potentials obtained from the outer surface of the muscle by leads through holes in the shell.
When a part of the muscle was denervated (see Pl. 8) by cutting a small branch of the ramification of the nerve fibres as indicated in the figure by the arrow, the exposed surface of this part (position 1) then became electro-positive relative to the solution during activity. By moving the electrode to the first contracting fibre (position 2) of the innervated area a reversal was immediately obtained, and there was thus a very sharp electrical boundary between these two parts, active and inactive, of the muscle. These experiments showed that in certain muscles it is indeed possible to lead off negative potentials from the exposed surface of muscle fibres; therefore exposure itself is not the reason why in other cases the surface does not become negative.
The nature of the action potentials
In previous publications (see Wiersma, 1941) there have been presented different arguments for the local nature of the crustacean muscle action potentials. Our present experiments are in complete accord with these views. If an action-potential wave were started at one spot on the muscle fibre and conducted from there over the muscle-fibre surface one would expect that with two leads on the fibre diphasic potentials would result. Furthermore, with the leads placed oppositely across the muscle both electrodes should become equally negative at the same time and no potential difference between them should be recorded. However, monophasic potentials were always recorded when the leads were placed on the same side of a muscle fibre in the extensor muscle and crosssectional potentials were, as stated, easily observed in the mandible muscle by placing one electrode on the lateral, flat side and the other on the curved one. In order to confirm this last point on a muscle with parallel fibres, the thoracic muscle was once more turned to. It was found possible to free completely the muscle from all surrounding tissue except the anterior end attachment, though this separation leads to damage of the muscle parts which attach to the thorax wall posteriorly. A sufficiently long section of the nerve innervating the anterior segments from below can be isolated and makes indirect stimulation of this preparation possible. In this way negative monophasic action potentials were obtained from both the under and the upper sides of the muscle against an indifferent electrode, placed either on the posterior inactive part of the muscle or on the insertion. The largest deflexions resulted with the active electrode on the under side. Because of this difference in potential from the two sides it was clear that cross-sectional leading off from this very thin muscle would give a noticeable potential difference. Indeed, this was found to be the case. Even when the electrodes were brought exactly opposite one another and thus were separated by a muscle fibre layer certainly less than 2 mm. thick, a significant deflexion was obtained. Control experiments, using the frog sartorius, gave under the same condition no such cross-section potentials, illustrating the great difference between these two parallel fibred muscles.
Obviously the action potentials recorded from the crustacean muscles are the resultant of the difference in voltage of two spikes from two places, which are not necessarily the places where the electrodes make contact with the tissue. The nerve conduction time is an important factor in determining the shape of the recorded deflexion, and diphasic action potentials are observed when the muscle areas, whose action is registered, are innervated by nerve branches of sufficient difference in length to cause a noticeable time interval between the start of their activities. This explains, for example, the diphasic potentials led off from the closer muscle.
We have, thus far, not succeeded in mapping out the finer potential distribution of a single muscle fibre during contraction. If the muscle potential is really a series of local potentials as our hypothesis asks for, it would be expected that a fibre would be more negative in the neighbourhood of the nerve endings than in the intervening spaces. Careful probing with platinum micro-electrodes of 0.01 mm. diameter failed to reveal any constant differences.
From the data presented it is clear that, when the active tissue can be sufficiently isolated to insure against any possible extraneous connexions between it and the indifferent lead, the action potentials obtained are negative. This does not explain why monophasic positive potentials consistently appear with many different electrode placements. It is our contention that several factors contribute to this phenomenon, the most important of which is the nature of the action potential. If conduction of the action potential took place, then the summation or subtraction of the voltages observed could not occur, and instead of the monophasic, diphasic action potentials should result under these experimental conditions. In previous publications (see Wiersma, 1941) other arguments have been offered why conduction of excitation over the crustacean muscle fibre is by nerve fibre conduction only. Therefore the following hypothesis in which all muscle potentials are considered to be of local origin is proposed.
Crayfish muscle fibres receive their innervation mainly on one side ; this innervation, in contrast to the end-plate formation in one region as present in vertebrate striated muscle fibres, consists of a profuse branching with many endings contacting the muscle fibre on this side. Each one of these many endings upon reception of a nerve impulse causes the immediate surrounding muscle fibre area to depolarize and therefore this whole side becomes negative more or less all over during activity, though intermediate small areas of relatively less negativity between separate nerve endings should be present. The other half of the muscle-fibre surface is either without or at any rate with considerably fewer innervated points and thus becomes overall much less negative. In adjoining muscle fibres the orientation of the innervated and the non-innervated side is usually the same.
Considering this hypothesis, if the local nature of the action potentials is accepted, it follows that, in order to record any potential whatsoever, not all of the muscle fibre can become equally negative; each fibre must somehow provide both source and sink in order that an electric current and voltage exist. In the case of the local or conducted potentials in nerve or other excitable tissues, the depolarization is definitely confined to one small area which acts as a sink, the surrounding polarized region providing the source of the current.
If the crustacean muscle fibre showed an equal distribution of its local potentials by equal distribution of the nerve endings or by a series of rings of negative potential (like the cross-striations), it would be possible to obtain action potentials from them only by probing the fibre with two micro-electrodes and thus locating areas of slightly different potential. No measurable change would exist at any distance. In order to record potential differences such as do appear there must be electrical asymmetry which is possible if crustacean muscle fibres are unilaterally innervated. In that case the fibre itself acts as a small cylindrical battery with the negative pole the innervated side and the positive pole the non- or only slightly innervated side. According to this hypothesis the crustacean muscle fibre would be comparable to a single electroplax of the electric organ in certain fishes (Cox, 1943).
The evidence for the existence of one-sided innervation is at the present time rather indirect, van Harreveld (1939) found innervation on one side only with silver staining; however, he believed that the other side would have a similar number of nerve endings, which had remained unstained. It was found during the present investigation that with methylene blue staining, which does not generally show the finer endings of the sublemnal branches, but which often demonstrates the many places where the nerve branches become sublemnal, these places always appear only on one side of the muscle fibre. In the thoracic muscle the innervation clearly branches on the underside of the muscle and no large branches are present on the upper side, though in the most anterior part the main nerve branches are no longer on the underside itself, but run in the body of the muscle between the fibre layers. The latter situation has also been found in a number of other muscles, and in these cases it is possible that part of the muscle fibres receive their innervation from one side and the other part from the opposite side.
It seems highly significant that negative action potentials are obtained from the muscle surface on the side from which the muscle is innervated. Thus the extensor muscle receives its innervation from the inside and becomes negative on this side ; the anterior thoracic muscle is, as stated, innervated mainly from the underside from which the largest negative spikes are obtained when this muscle is isolated from the surrounding tissue. In further verification of this rule the lateral side from which the nerve enters the mandible muscle is the side on which the negative area is located.
Since we have shown crayfish muscle fibres to be relatively inactive near the insertion end (see Results—Extensor) and yet find that the carapace to which the mandible muscle is attached is more negative than most of the surface of the muscle itself, it follows that the orientation of the innervated sides of the muscle fibres to the shell is of greater consequence than the inactivity of the muscle endings. This same factor accounts for the potential gradient over the claw where near the carpopodite the shell contacts the inactive ends of the fibres at nearly right angles, whereas towards the tip the shell is exposed to the lateral sides of the fibres only.
It is not clear at present why the carapace acts as a better electrical connexion between the body of the mandible muscle and the tendon than does the muscle substance itself. Further experiments are planned to elucidate this question.
EXPLANATION OF PLATES
A transverse section of the mandible muscle is sketched on the left with the tendon dipping into solution. Part of the carapace has been cut away as illustrated by the dashed line A. B is the optional cotton connexion. Electrode 1, indifferent, is immersed in solution. Placement for negative area is designated by electrode 2. Electrode 3 shows normal placement on body of muscle. The action potentials obtained from the two placements are illustrated on the right. A 1-3 was recorded from muscle body with no connexion from carapace to solution. The deflexion is negative, about 20 mV. B 1—3, the same with cotton wick connecting shell to solution. Deflexion is positive, about 15 mV. A 1-2 was recorded from the negative area with no connexion. The deflexion is negative, about 40 mV. B1-2 the same with cotton connexion from shell to solution. Deflexion is negative, about 15 mV.
The inner surface of the extensor muscle of the meropodite showing the nerve ramifications is illustrated on the left. After a nerve branch was cut, as designated in the figure, and probing electrode placed at 1 the upper spike was recorded. It is mostly positive. If the prober were placed at 2 the lower spike, negative, was obtained.