1. The mechanism of contraction and relaxation in the anterior byssus retractor muscle (A.B.R.M.) of the lamellibranch mollusc Mytilus edulis has been studied with refined stimulating and recording techniques. Two distinct types of response are present: phasic and tonic. The former can be evoked by all kinds of electrical stimulation, but the latter is most readily elicited by continuous direct current.

  2. Serotonin (5-hydroxytryptamine) rapidly abolishes the tonic response, whilst leaving the phasic response practically unaffected for many hours. On electrical stimulation, a serotonin-treated muscle may show spontaneous electrical and mechanical activity, usually of a rhythmical nature. As the muscle is unable to destroy this drug the effects produced by it are likely to be of pharmacological interest only.

  3. Electrical activity in the form of irregular volleys of muscle action potentials can be recorded from the intact A.B.R.M. and even after the nerves to the muscle have been cut. In such (externally) denervated preparations the spontaneous electrical activity is almost unaffected by a.c. stimulation but greatly increased by d.c. stimulation which evokes prolonged tonic contraction.

  4. Stimulation of inhibitory nerves within the muscle quickly terminates a state of tonic contraction and at the same time reduces the electrical activity considerably.

  5. An explanation is offered for the occurrence of electrical activity during relaxation of lamellibranch smooth muscles. The normal functioning of the A.B.R.M.is interpreted, and strong support is given to the tetanus hypothesis of tonic contraction.

The nervous control of muscle has been studied intensively during recent years, particularly in the case of vertebrate striated muscle. Although smooth muscle is much more difficult to investigate, the recording of electrical activity from it (Bozler, 1938, 1946, 1948) offers the prospect of a successful experimental approach here as well, and recently intracellular recording has been carried out from smooth muscle preparations (Bülbring, 1954, 1955). The principal generalization which has emerged is that all muscle fibres, whether striated or smooth, possess a surface boundary which is in a state of electrical polarization, the outside being positive with respect to the inside. When the fibre is inactive the resting membrane potential has a value of 50-100 mV. In all striated muscles, and in those smooth muscles which have been thoroughly investigated, both phasic and tonic activity is associated with some degree of reduction in the resting potential, whether brief, as in the spike potentials which precede twitches and tetanus, or prolonged, as during contracture. This depolarization is effected spontaneously in specialized rhythmically-contracting muscles like heart and gut muscles (Draper & Weidmann, 1951 ; Bozler, 1948), but is otherwise the result of nervous activity.

There remain many unsolved problems in this field, but perhaps none is more paradoxical than that of the tension control in the anterior byssus retractor muscle (A.B.R.M.) of Mytilus edulis. Winton (1937) showed that the muscle relaxes very slowly after it has been induced to contract by continuous direct-current (d.c.) stimulation and quite rapidly after stimulation by alternating current (a.c.). He described the slow relaxation non-committally as being due to an increase in muscle ‘viscosity’ produced by this particular kind of d.c. stimulus. Nevertheless, the implication was that the d.c. stimulus has not only brought about a phasic contraction, but also altered the molecular configuration of the contractile elements in such a way that the resistance to stretch is greatly increased. ‘Relaxation’ is accompanied by a very gradual return to the much lower resting ‘viscosity’. Winton also found that on applying an a.c. stimulus to the muscle during the phase of high ‘viscosity’ the resulting additional contraction was followed by relaxation at the much higher speed normally observed after using a.c. alone. The a.c. had produced a small phasic contraction and abolished the state of high ‘viscosity’ at the same time. These results were confirmed by many subsequent investigations (Fletcher, 1937 a-c ; Nieuwenhoven, 1947; Singh, 1938a, b; Twarog, 1954; Johnson, 1955). Fletcher showed, in addition, that short-duration d.c. pulses act on the muscle in a manner similar to a.c.

All these authors have taken the view that the results of their experiments support the ‘catch mechanism’ hypothesis which maintains that molluscan smooth muscles possess a special tonic contractile mechanism where the molecular elements ‘set’ or ‘catch ‘in the shortened state, thus enabling the muscle to maintain a tonus without doing work (cf. von Uexküll, 1929). Against this view Ritchie (1928), Bozler (1948) and Lowy (1953) have argued that tonic contraction in these muscles is really due to a tetanus. But in the A.B.R.M. no electrical activity has so far been found which would have supported an explanation of its behaviour in terms of the tetanus hypothesis. Fletcher (1937) made an extensive study of this muscle, and reported that although he could detect a large propagated action potential following single shocks to the isolated muscle (each shock resulting in a small twitch) there was no electrical activity at all during prolonged contractions induced by a d.c. stimulus.

On modem views the tetanus hypothesis requires that periodic depolarization of the muscle membranes must occur in order that contraction may be maintained. This should be set up by nervous activity, although myogenic action (i.e. spontaneous depolarization as of heart muscle) would be regarded as equivalent. The ‘catch mechanism’ hypothesis, which reached its most extensive speculations in the thesis by Nieuwenhoven (1947), requires only the set of events occurring at excitation to bring about the state of maintained contraction which is equivalent to Winton’s state of high ‘viscosity’. Special conditions are required to bring such a tonic contraction to an end quickly. In the A.B.R.M. preparation relaxation times have been found to vary from 20 sec. to as long as 4 hr. On the tetanus hypothesis relaxation occurs automatically when the postulated continuous excitation diminishes or disappears—a process in conformity with the evidence about relaxation of striated muscle (Hill, 1950). It is, on the other hand, a necessary corollary of the ‘catch’ mechanism hypothesis that any sort of rapid relaxation such as can be observed after treatment with a.c., various drugs like serotonin, brief pulses and ‘weak’ stimulation of the pedal ganglia (cf. Nieuwenhoven) must be due to an active process, i.e. a process which results in a reversion of the molecular changes or an undoing of the ‘catch’.

One of us has recently used electrical recording technique to demonstrate spontaneous electrical activity in a variety of lameUibranch muscles, particularly in smooth adductors with well-developed tonic properties (Lowy, 1955). These results serve to draw further attention to the Mytilus A.B.R.M. preparation where the isolated muscle, cut and tied at both ends, can be made to show all the phenomena of tonic contraction, rapid and slow relaxation, which are also the properties of this muscle in intact animals. One of us (G.H.) has presented this preparation to honours students in 4 successive years and studied it intensively for several months; J.L. has worked with it intermittently for 5 years. In all, a very large number of preparations have been investigated. In many, including those used by students, results similar to those of Winton were obtained, but at least as many gave anomalous results which are worthy of consideration.

Occasionally a student has reported the exact converse of the Winton results, i.e. rapid relaxation following d.c. and a prolonged contraction following a.c. stimulation. Sometimes a burst of a.c. interposed during a tonic contraction due to d.c. has produced a heightening of the contraction with no subsequent relaxation. On two occasions students obtained relaxation of a tonic contraction during the course of applying a weak a.c. stimulus, with a latent period of about 20 sec., but as soon as the weak a.c. was stopped the muscle contracted again to a greater height than that from which it had relaxed, now to continue to relax at the earlier very slow rate.

These observations appeared to throw doubt on the earlier interpretations and a thorough re-investigation of the preparation was decided upon. This has been carried out independently by the two authors but is here reported jointly.

Animals were kept in fresh aerated sea water before study and opened in the conventional way, i.e. by cutting through the posterior adductor muscle with a knife inserted between the valves. The two halves were gently prised apart, the foot seized with forceps and pulled steadily backwards until it came away completely together with the pedal retractor muscles. This procedure exposes the root of the byssus threads, which are attached to the tendons of the byssus retractor muscles and therefore form a convenient point of attachment for a thread. By cutting out small disks of the shell in the regions to which the anterior ends of the byssus retractor muscles are attached, these muscles can be isolated intact. Following careful ‘cleaning’ to remove the nerves, connective tissue, etc., a very good preparation can be obtained. It was noticed in a large number of animals that the left A.B.R.M. is divided into two almost equal muscles. One of these is attached to the byssus on its own side and the other to the byssus on the opposite side. This arrangement probably helps to strengthen the base of the byssus.

In view of the fact that Winton and most of the previous authors had used muscles which were cut at both ends, many muscles were treated in this way during the present experiments ; some were tested first whilst intact and again after being tied and cut. No serious difference has been observed between the results.

For experiments in which electrical amplification was not required the muscle preparations were mounted in a Palmer muscle-bath and immersed in sea water at about 14° C., contractions of the muscle being recorded isometrically on smoked paper. The muscle was placed so that it ran through a ‘knife-edged’ slot made in a Perspex strip (Fig. 1). Stimulation was accomplished by passing current across the strip using chlorided silver electrodes. Electrical records were obtained after running all the sea water out of the bath. The recording electrodes were cottonwicks, soaked in sea water tied on to chlorided silver wires or platinum electrodes coated with platinum black. Condenser-coupled pre-amplifiers with low noise level were used, and electrical activity displayed after further direct-coupled amplification on an ink-writing oscillograph. No special attempt was made to avoid the stimulus artifact, which was considerable with the high gain employed; a shunt was used to avoid recording it during intensive stimulation. The period of shunting is indicated in the records by a horizontal line under the electrical activity trace. An additional thickening of the line indicates electrical stimulation.

Fig. 1.

Diagram of the preparation to show the method of stimulation in sea water.

Fig. 1.

Diagram of the preparation to show the method of stimulation in sea water.

Direct- and alternating-current stimulation

With the method of stimulation used in the present experiments it is probable that at threshold strength stimulation will occur only at the bridge on the cathodal side. It was found that a direct-current voltage of only 1 or 2 V. applied for 3-4 sec. is usually adequate to produce complete contraction of the muscle. Further stimulation adds nothing to the height, although it frequently increases the subsequent duration of the contraction. When fully contracted the length of the muscle is about a third that of the resting muscle. Winton used more than 10 V. for 10 sec., and even so does not seem to have achieved complete contraction for he obtained a ‘staircase ‘effect with successive stimulations. He was passing current through the whole muscle (immersed in sea water) in an attempt to stimulate it evenly. In the present experiments the character of the response is markedly different from that obtained by Winton. It seemed quite likely that the stimuli were taking effect via the nerve, and this could explain the difference. Such a view is supported by the fact that the low threshold was obtained with the bridge at the byssal end, about 3 mm. from the termination. This is actually the place where the nerves from the pedal ganglia enter the muscle.

If the direction of flow of the stimulating current is now reversed, keeping the bridge close to the byssal end of the muscle, the resulting contraction (even with very large currents) is always minute. This contraction is attributable entirely to the small length of muscle on the cathodal side. When the current ceases there is now often a marked ‘break’ contraction involving the rest of the muscle. The height and duration of this contraction are roughly proportional to the strength and duration of the previous stimulation (Fig. 2). It is interesting to recall that anodal break phenomena contributed greatly to the level of contraction in Winton’s experiments. If the interpretation of the situation in the present experiments is correct, namely, that excitation is effected via the nerve, the impulses set up in the nerve fibres on the cathodal side of the bridge must be blocked on the anodal side, and the anodal break contraction could be due to repetitive firing in these fibres on removal of the block. Unfortunately it does not seem possible to test this possibility satisfactorily. No matter where the bridge is placed on the relaxed muscle, contraction always occurs on the cathodal side—but it is also fairly complete. In Fletcher’s experiments, where cotton wicks carried current to the muscle suspended in air, contraction only occurred in the region of the cathode, and it seemed as though he was stimulating the muscle fibres directly.

Fig. 2.

Anodal break responses obtained with a bridge at the byssal end of the muscle and the major length of the muscle on the anodal side. The drum was stopped between experiments. Time in min.

Fig. 2.

Anodal break responses obtained with a bridge at the byssal end of the muscle and the major length of the muscle on the anodal side. The drum was stopped between experiments. Time in min.

In spite of the localization of his contraction in the cathodal region (1937c, fig. 6), Fletcher claimed that an action potential was propagated along the muscle fibres outwards from the point of stimulation along the whole length of the muscle.

Fig. 6.

Spontaneous tonic contractions. The first six responses were evoked by a.c. In four of them relaxation was interrupted by a spontaneous contraction which was maintained by the tonic system. The last response was evoked by d.c. Time in min.

Fig. 6.

Spontaneous tonic contractions. The first six responses were evoked by a.c. In four of them relaxation was interrupted by a spontaneous contraction which was maintained by the tonic system. The last response was evoked by d.c. Time in min.

The rise-time of the contraction is fairly constant with d.c., 50∼ a.c., and a 20/sec. pulse stimulation, being from 15 to 20 sec. Both the shape and the duration of the relaxation curve are, however, extremely variable following direct-current stimulation. At its fastest, relaxation may occur as quickly as the phasic contraction, so that the whole curve of contraction and relaxation occupies less than a minute. At the other extreme very gradual relaxation may take 3 or 4 hr. and complete tonic contractions may last even longer than this. Sometimes there is an initial rapid relaxation, followed by a prolonged period of maintained tonus, followed finally by slow relaxation. All these various results can often be obtained from the same preparation under identical conditions with similar stimulations. It thus appears that in some experiments a separate tonic response is obtained from the preparation, and in others it is not. On the ‘catch mechanism’ hypothesis it would be argued that on some occasions the ‘catch’ is set by the stimulation (or after about 30 sec. latency), whilst in others it is not. Even if this explanation is accepted it does not satisfactorily account for the great variety of the relaxation curves.

Alternating currents of threshold strength applied across the knife-edge bridge produce similar phasic contractions. The voltages required are much less than those used by Winton, but the results are on the whole rather similar. Relaxation usually occurs immediately, and the whole course of contraction and relaxation seldom takes more than 120 sec. Sometimes, however, prolonged tonic contractions are set up by a.c. (Fig. 3). This has happened even in preparations which did not go into a state of tonic contraction after d.c. Such occasional anomalies are most significant, for they rule out the argument which has been developed following Winton’s work, namely, that the tonic phenomena, Winton’s high ‘viscosities’, are specially related to the continuous d.c. kind of stimulation, and that the a.c. kind of stimulation leads to a state of low ‘viscosity’. The present results of experiments with d.c. and a.c. stimulation show quite clearly that the tonic phenomena must be regarded as special responses of the preparation. They are more readily produced by d.c. stimulation, but there is probably no particular connexion between molecular events in the muscle and the kind of current passing through it. It is possible to divide the responses of the muscle into two separate categories, phasic and tonic. The former is fairly regular in action, while the latter is extremely irregular and requires elucidation.

Fig. 3.

Anomalous responses to d.c. and a.c. stimulation. Time in min.

Fig. 3.

Anomalous responses to d.c. and a.c. stimulation. Time in min.

Inhibition

The presence of inhibitory nerve fibres capable of relaxing tonically-contracted mollusc muscles was first demonstrated by Pavlov in 1885. Nieuwenhoven (1947) showed that the tonically-contracted A.B.R.M. could be made to relax by weak faradic stimulation of the pedal ganglia, thus indicating the possibility of the presence of inhibitory nerves supplying this muscle. Fletcher had shown that under certain conditions electric pulses applied to a tonically-contracted muscle produced relaxation without evoking any additional contraction first. The use of the term ‘inhibition’ for these relaxation phenomena is not strictly justified in the absence of a complete understanding of the processes involved in contraction. ‘Inhibition ‘actually implies an acceptance of the tetanus theory of tonic contraction. Nieuwenhoven correlated his own and Fletcher’s observations, and constructed a hypothesis of an active relaxation process effected by nervous elements. Thus he supposed that tonic contraction involves the operation of a ‘catch’ and that this state is unlocked by the action of ‘inhibitory’ nerve fibres running from the pedal ganglia. Subsequently, this view has received support from the work of Twarog (1954) and Barnes (1955). Twarog extracted from Mytilus the substance 5-hydroxytryptamine (5-H.T.), a pharmacological agent known as serotonin which raises tonus in vertebrate smooth muscle. She showed that concentrations of this drug as low as 10−8 exert a powerful relaxing effect on the tonically-contracted muscle.

With the kind of stimulation used in the present experiments, where it seemed possible that stimulation of the muscle was occurring via the motor nerves, it was reasonable to hope that it would also be feasible to stimulate the postulated ‘inhibitory ‘nerve fibres.

It was found in fact that with the bridge on the point of entry of the nerve from the pedal ganglia weak rectangular pulses, condenser discharges or alternating current can all be used to promote the rapid relaxation of the tonically-contracted muscle. The threshold voltage is approximately half that required to produce contraction. The rate and final extent of relaxation are functions of the duration of the ‘inhibitory’ stimulation. There is a long latent period of 15-20 sec. before relaxation commences, and it usually continues for a similar period after the stimulation. Hence a burst of ‘inhibitory ‘stimulation may produce an effect only after it ceases.

From this evidence it seems probable that nerves from the pedal ganglia contain fibres which liberate a substance, destroyed or lost by diffusion only slowly, which effects relaxation of the tonically-contracted muscle. The relaxing effect can be obtained at any level of tonic contraction (Fig. 4) and almost any degree of relaxation can be achieved depending on the frequency and duration of stimulation and also to a lesser extent on the strength of stimulation. All these effects are, however, so variable that a quantitative appraisal of the phenomena is not possible. Relaxation can also be produced by certain drugs, e.g. L.S.D.* and 5-H.T., but the rate of relaxation which they bring about (even in high concentrations) is always much slower than that produced by stimulation of the ‘inhibitory ‘nerves. In fact stimulation of the ‘inhibitory’ nerve accelerates relaxation started by 5-H.T.

Fig. 4.

Records of relaxation (inhibition) of tonic contractions. The contractions were produced by continuous direct current and the relaxations by rectangular pulses, marked by the first and second stimulus marks respectively in each record. Relaxation without preceding contraction could be effected at any stage during the gradual decline of the tonic response. The last two traces of records b and c show the effects of increased frequency and intensity of inhibitory stimulation. Time in min.

Fig. 4.

Records of relaxation (inhibition) of tonic contractions. The contractions were produced by continuous direct current and the relaxations by rectangular pulses, marked by the first and second stimulus marks respectively in each record. Relaxation without preceding contraction could be effected at any stage during the gradual decline of the tonic response. The last two traces of records b and c show the effects of increased frequency and intensity of inhibitory stimulation. Time in min.

5 -Hydroxytryptamine

Although Twarog (1954) demonstrated the relaxing action of this drug she did not study the effect of stimulating a treated muscle. This has been done in the present experiments and produced most interesting results. After treatment with 10−8 5-H.T. the muscle, although completely relaxed, nevertheless responds to all kinds of electrical stimulation with a powerful phasic contraction. This is always followed by rapid relaxation (Figs. 5, 7). 5-H.T. therefore abolishes the tonic response without diminishing the phasic one. These findings strongly support the contention made above that phasic and tonic contractions are due to two independent systems.

Fig. 5.

Record showing: typical tonic response following d.c. ; stimulation of inhibitory nerve (at I) ; phasic response to a.c.; relaxation of a second d.c. tonic response with 10−5 5-H.T. and final phasic response only of the treated muscle to similar d.c. Time in min.

Fig. 5.

Record showing: typical tonic response following d.c. ; stimulation of inhibitory nerve (at I) ; phasic response to a.c.; relaxation of a second d.c. tonic response with 10−5 5-H.T. and final phasic response only of the treated muscle to similar d.c. Time in min.

Fig. 7.

Spontaneous rhythmic activity. The record show’s first : testing inhibitory stimulation ; then d.c. stimulation followed by a tonic response which was inhibited at I; a second tonic response which was relaxed with 10−6 5-H.T.; phasic response only of the treated muscle but followed by rhythmic phasic contractions. Time in min.

Fig. 7.

Spontaneous rhythmic activity. The record show’s first : testing inhibitory stimulation ; then d.c. stimulation followed by a tonic response which was inhibited at I; a second tonic response which was relaxed with 10−6 5-H.T.; phasic response only of the treated muscle but followed by rhythmic phasic contractions. Time in min.

Following treatment with 5-H.T., muscles were washed at intervals in fresh sea water and subjected to d.c. stimulation. It was found that tonic responses could not be obtained for several hours and in some instances only after 40 hr. Evidently the muscle is unable to destroy the drug, so it seems highly unlikely that it could be identified as the substance naturally responsible for relaxation as Twarog suggested. Following relaxation produced by stimulating the ‘inhibitory’ nerve, powerful tonic contractions can be produced again immediately.

Spontaneous activity

Singh (1943) noted that spontaneous contractions sometimes occur in the isolated A.B.R.M. and they have occasionally been noted by us. Following the rapid relaxation usually obtained after a.c. the muscles often contract again partially, and maintain the new level of contraction by the tonic system (Fig. 6).

After the tonic response has been abolished by 5-H.T. a direct current stimulus may set up the usual contraction and relaxation, but this is often followed by rhythmic contractions (Fig. 7) which occur at the rate of about 1/min. and can persist for 20-30 min. after which they gradually subside. Further d.c. stimulation is now followed by a resumption of the rhythmical activity (Fig. 7).

Electrical activity

If electrical activity were produced by the Mytilus A.B.R.M. as by vertebrate smooth muscles, it might be possible to clarify some of the problems outlined above. There seemed at first little hope of this. Fletcher (1937) had reported electrical activity only in the form of single action potentials following stimulation by a large condenser discharge, with no activity at all during a tonic contraction produced by d.c. But recently one of us (Lowy, 1953) demonstrated that tonic contractions in intact Mytilus adductors is accompanied by continuous electrical activity, and it therefore seemed worth while searching for muscle potentials of small magnitude in the isolated A.B.R.M. This effort was amply rewarded.

It was decided to look first for electrical activity in the intact A.B.R.M. AS described earlier (p. 297), the muscle was exposed in situ and recording electrodes were placed on it. No action potentials resembling the very large ones described by Fletcher have ever been recorded, but many much smaller potentials were always found to be present (Fig. 8). These potentials appear spontaneously in irregular bursts, each composed of spikes of various amplitudes and of low frequency. The more vigorous bursts are associated with small contractions (Fig. 8). At this stage the dissection was carried a step further and the nerves leading to the muscle from the pedal ganglia were cut. Electrical activity in the muscle did not cease, although after a while it subsided considerably. The final stages of the dissection were now carried out and the muscle treated as an isolated preparation. After soaking in oxygenated sea water for about 1 hr. an externally denervated muscle prepared in this way shows plenty of spontaneous electrical activity. Other lamellibranch muscles have recently been found to show spontaneous electrical activity following external denervation (Lowy, 1955).

Fig. 8.

Simultaneous recording of tension (upper trace) and electrical activity (lower trace) occurring spontaneously in the nearly intact preparation.

Fig. 8.

Simultaneous recording of tension (upper trace) and electrical activity (lower trace) occurring spontaneously in the nearly intact preparation.

Electrical stimulation

Application of a.c. or of brief rectangular pulses produces a phasic contraction associated with a short outburst of electrical activity (Fig. 9 a, b). In contrast, a d.c. stimulus gives rise to a longer lasting contraction and the electrical activity does not cease when the current is stopped (Fig. 9 c). Instead, electrical activity continues at a high level for some time (often up to 10 min., depending on the initial duration of the d.c. stimulus), whilst the muscle relaxes slowly (Figs. 10, 11).

Fig. 9.

The mechanical (upper trace) and electrical (lower trace) responses of the A-B.R.M. to: a, a.c. ; b, rectangular pulses; c, continuous d.c. The duration of shunting of the amplifier leads is indicated by a horizontal line under the electrical response trace. The duration of stimulation is indicated by a thickening of this line.

Fig. 9.

The mechanical (upper trace) and electrical (lower trace) responses of the A-B.R.M. to: a, a.c. ; b, rectangular pulses; c, continuous d.c. The duration of shunting of the amplifier leads is indicated by a horizontal line under the electrical response trace. The duration of stimulation is indicated by a thickening of this line.

Fig. 10.

The mechanical (upper trace) and electrical (lower trace) responses of the A.B.R.M. to increasing duration of continuous direct current. Stimulation for approximately: a, 2 sec. ; b, 4 sec.; c, 6 sec. ; d, 10 sec.

Fig. 10.

The mechanical (upper trace) and electrical (lower trace) responses of the A.B.R.M. to increasing duration of continuous direct current. Stimulation for approximately: a, 2 sec. ; b, 4 sec.; c, 6 sec. ; d, 10 sec.

Fig. 11.

Prolonged tonic contractions associated with continued electrical activity following d.c. stimulation. Upper trace tension, lower trace electrical activity. Note the variety of electrical activity recorded.

Fig. 11.

Prolonged tonic contractions associated with continued electrical activity following d.c. stimulation. Upper trace tension, lower trace electrical activity. Note the variety of electrical activity recorded.

In some preparations electrical activity during tonic contraction is very irregular (Fig. 11 b), and it was discovered at this stage that even a small shift of the recording electrodes could produce a marked change in the pattern of the response recorded : there are sites at which almost no electrical activity of any kind can be detected, although a small shift of the electrodes reveals plenty. In other sites there is complete silence after the initial burst associated with contraction, and instances were observed in which activity occurs only some time after the cessation of the electrical stimulus. Furthermore, electrical activity is often present during relaxation; this will be discussed later. Recordings made using two pairs of similar recording electrodes placed on different regions of the muscle and simultaneously recording reveal that during tonic contraction there is almost no simultaneous or co-ordinated activity in the different regions (Fig. 12).

Fig. 12.

Spontaneous electrical activity in the isolated A.B.R.M. recorded simultaneously during tonic contraction from regions near the byssus end (B)and the shell end (S). Note the localized nature of the activity.

Fig. 12.

Spontaneous electrical activity in the isolated A.B.R.M. recorded simultaneously during tonic contraction from regions near the byssus end (B)and the shell end (S). Note the localized nature of the activity.

Drug action

Drops of 10−7 acetylcholine applied to the isolated A.B.R.M. give small contractions accompanied by short bursts of potentials. Higher concentrations evoke more powerful contractions accompanied by vigorous electrical activity. There is a prolonged tonic contraction as Twarog (1954) demonstrated, and the responses to acetylcholine greatly resemble those produced by direct current.

Treatment with 5-hydroxytryptamine effects a marked reduction in electrical activity which normally follows d.c. stimulation (Fig. 13).

Fig. 13.

The action of 5-H.T. (10−8) on the mechanical (upper trace) and electrical (lower trace) responses of the A.B.R.M. to similar d.c. stimulations, a, before treatment; b, c and d, a few minutes after treatment.

Fig. 13.

The action of 5-H.T. (10−8) on the mechanical (upper trace) and electrical (lower trace) responses of the A.B.R.M. to similar d.c. stimulations, a, before treatment; b, c and d, a few minutes after treatment.

Inhibition

When ‘inhibitory’ stimulation is applied, as described earlier, the spontaneous discharge associated with prolonged tonic contraction disappears.

Before discussing the significance of the present results it is necessary to consider observations made by Fletcher (1937) because on these are based most of the recent speculations which attempt to account for the behaviour of smooth lamellibranch muscles in terms of the ‘catch’ mechanism hypothesis. The potentials recorded by Fletcher had a duration up to fifty times longer than those observed in the present work. More important, however, only a single such potential could be detected by Fletcher in response to a direct current applied to the isolated muscle, whereas our experiments show that prolonged electrical activity invariably follows such a stimulus. These contradictions can be resolved by comparing Fletcher’s figures for the maximum gain and fastest response time of his recording devices with the corresponding figures for our own apparatus : it would appear that Fletcher’s results were due to the relative insensitivity of his amplifier and recording apparatus.

With this difficulty out of the way, a reasonably clear account can now be given of the functioning of the Mytilus A.B.R.M. It is evident from the irregularity of much of the electrical activity and the variable magnitude of the spikes that the innervation of the muscle must be complex. Bowden & Lowy (1955) have recently demonstrated the presence of ganglion cells in the muscle. Furthermore, the different responses obtained from different regions show that there is no uniformity in the innervation of the different parts of the muscle.

As the electromyogram shows, the muscle works by continuous excitation of small groups of fibres, the number of such units in action at any time determining the level of tonus. Changes in the tonus level may be brought about by activation of motor or inhibitory axons via nerve cells located in the pedal ganglia. Phasic contraction could be due to transient stimulation of a large number of muscle units. The observation that the capacity of the muscle for tonus can be abolished, while it continues to give phasic responses, suggests the existence of two independent excitatory systems. It has not yet been possible to show whether the spontaneous activity of the system responsible for tonic contraction is neurogenic or myogenic. At all events, nervous pathways almost certainly serve for co-ordination of the numerous small muscle units during phasic contraction.

On the basis of the above considerations the effects produced by a.c. and d.c. stimulation can be interpreted as follows. Pulses and a.c. would usually bring about only a transitory increase in the rate of firing of the spontaneously active nerve (or muscle) elements. But a d.c. stimulus very often appears to lead to their persistent increased activity, thus giving rise to a prolonged tonic contraction. The d.c. could bring about ionic shifts which might influence spontaneous activity owing to a change in membrane potentials of the automatically-discharging elements.

Activation of ‘inhibitory’ axons reduces electrical activity in the isolated muscle, even when the level of activity is very high, as for instance following application of acetylcholine or d.c. stimulation. Similar results were obtained by stimulating the inhibitory nerves leading to the posterior adductor of Mytilus (Lowy, 1953). It may well be that activation of the inhibitory axons releases a substance which reduces the rate of firing of the spontaneously occurring activity.

There is one puzzling phenomenon which still has to be accounted for, namely, the observation of electrical activity during relaxation of the denervated A.B.R.M. (cf. Fig. 10b) and of the intact Mytilus adductor (Lowy, 1953). One of us (J. L.) has previously suggested that a process analogous to the β-inhibition, described by Marmont & Wiersma (1938) for crustacean muscle, could be responsible for producing this effect. However, in view of the present findings, an alternative explanation is possible. It is obvious that, as the potential recorded is a measure of the electrical unbalance between the recording points, a small single muscle unit firing in a suitable position could produce a greater potential difference than a very large number firing synchronously—if the electrodes chanced to record from regions where similar events were taking place. Now because electrical activity is so widely scattered the observation of large muscle potentials during relaxation (even when few can be observed during contraction) is not really surprising : these spikes probably represent activity in only a few small units, which serves to slow down the rate of relaxation. The almost complete absence of electrical activity during really fast relaxation of intact muscles and on stimulation of inhibitory nerves (see also Lowy, 1953) constitutes good evidence in support of this explanation. The suggestion that electrical activity during relaxation indicates the operation of an active process which switches off the ‘catch mechanism’ (Barnes, 1955) is not likely to be correct.

The picture of the Mytilus A.B.R.M. which now emerges is that of a muscle controlled in many respects like vertebrate visceral smooth muscles: there is a built-in tonic system capable of automatic firing whose activity is normally regulated by motor and inhibitory axons from the central nervous system. The present experiments have shown how these two types of nerve can be brought into action by suitable electrical stimulation to produce muscular responses similar in every way to those observed in intact animals.

Both authors are independently indebted to the Government Grants Committee of the Royal Society for grants towards the purchase of electrical apparatus.

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*

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