In at least some of the muscles in Coelenterates the size of response is controlled by the neuromuscular junction. The sphincter in Calliactis and the longitudinal mesenteric retractor muscles of Metridium senile are served by part of the nerve network which acts as a single excitable system. The response is not graded through a large number of separate motor units. A single stimulus applied to this nerve net is ineffective, or only causes a trivial response ; but it leaves behind it an effect which facilitates the transmission of a second impulse to the muscle. Each impulse in a series facilitates the transmission of excitation to more and more muscle fibres. After each stimulus the facilitating effect gradually dies away (Pantin, 1935 b). In a short discussion, the relation of this “facilitation” process to other cases of junctional transmission was considered (Pantin, 1937). The process by which excitation is only effective if one stimulus follows its predecessor before the period of facilitation has departed, is analogous to the condition of vertebrate skeletal muscle during partial curarization. But at present the nature of the facilitation process is still unknown. A study of the action of ions and other substances throws considerable light upon its nature, and it is the object of this paper to describe and discuss these effects.
The anemones, Calliactis parasitica and Metridium senile, were exposed to mixtures of sea water with various substances or with isotonic solutions of the chlorides of various cations. The contractions of the muscles were recorded in response to a series of condenser shocks applied to the column of the animal (Pantin, 1935 a). The shocks were sent in through Ag-AgCl electrodes.
Fig. 1 a shows a response of the marginal sphincter when Calliactis is given a series of four shocks at frequencies of 2 per sec., 1 per sec. and 1 every 2 sec. As in all normal contractions, there is no response to the first stimulus and the response increases considerably as the frequency rises. In all the experiments that follow, similar series of stimuli at these frequencies were given as controls and to illustrate the effect of various substances. The experiments were conducted at room temperature, 18-21° C.
Saturated solutions of carbon dioxide in sea water were made. The first effect of immersing the anemone in such a solution is to cause protrusion of the mouth and oral disk. Slow peristaltic contractions of the column may then follow. These effects soon pass off and within 10 min. the characteristic anaesthetic action of CO2 becomes apparent and continues gradually to increase, being fairly complete in 1-2 hr. (Fig. 1). On return to natural sea water recovery is complete in less than 1 hr.
When the sphincter no longer responds to stimulation of the anemone on the column, direct stimulation of the muscle still produces a good response. Since the muscle is not itself incapable of contraction the failure to respond must be due either to failure of the nerve net or of the neuromuscular junction. But failure of response develops as a gradual reduction of the size of the contraction, not as its abrupt abolition. Hence the nervous impulses set up by the stimuli are still reaching the muscle as in the normal animal, though the response to them is less. We may conclude that the primary action of CO2 is on the neuromuscular junction by depression of facilitation. Mayer (1916) showed that in Cassiopeia carbon dioxide can prevent muscular response without affecting nervous conduction.
Though the prime effect of carbon dioxide is upon the junction, it has some direct effect on both the nerve net and the muscle. The threshold of excitation steadily rises, reaching two or three times the normal at the point when indirect response of the muscle fails. The effect on the muscle is a slowing of the response, particularly of the relaxation. This is similar to the effect described by Bozler (1931) on the buccal retractor of Helix. These effects appear to be due specifically to carbon dioxide. Aerated sea water brought to the same pH, 6.5, has no obvious action (Fig. 1 e).
The depression of facilitation by carbon dioxide is greatest at the higher frequencies. The muscular response cannot be made as good as a normal one by raising the frequency of stimulation, whereas the maximal interval between stimuli which will just enable a response to take place is not greatly reduced. The depression is thus not due to an increased rate of decay of facilitation but to a reduced responsiveness of the muscle. The effect is strikingly different from that of a rise in temperature when the animal is in sea water. In this case the responses are quite normal, but facilitation decays at a much faster rate so that the frequency at which all reactions take place is higher (Hall & Pantin, 1937).
Even under profound anaesthesia with carbon dioxide the first contraction to a series of stimuli still takes place on the second stimulus as in the normal animal. But after prolonged exposure several stimuli may be required before the muscle responds. A similar effect occurs under excess magnesium and it will be shown that it is due to breakdown of conducting tracts in the nerve net leading to “intemeural facilitation”, and not to modification of the neuromuscular junction.
Excess magnesium depresses the responses reversibly. Within less than a minute superficial sense organs are anaesthetized, especially in the tentacles. The subsequent effects on the neuromuscular system are both slower to appear and slower to show recovery, than those of carbon dioxide. This is perhaps because of difficulty of diffusion into the thick mesogloea. Magnesium slowly lowers the electric excitability of the nerve net. A mixture of 50 % sea water with 50 % 0-4 M MgCl, (pH 8.0) causes little change in the threshold for about half an hour, but then this rises steadily though it only reaches about twice the normal value when anaes-thetization becomes complete.
The greatest effect of magnesium is on facilitation (Fig. 2). In the mixture just referred to the facilitated response of the sphincter in Calliactis begins to fall off progressively within 5 or 10 min. The individual contractions themselves do not become slower as under the influence of carbon dioxide; indeed they often become sharper with more rapid relaxation. But as under carbon dioxide the size of the responses becomes steadily smaller over a period of an hour or so and finally vanishes. This depression of the response is again greatest at the highest frequencies of excitation so that shortening of the interval between the stimuli does not enable the contractions to attain normal size (Fig. 2d). Although magnesium reduces the size of all contractions, these first become visible at about the same frequency of stimulation as in normal conditions. These facts indicate that magnesium, like carbon dioxide, depresses facilitation but does not increase its rate of decay. Until the response has almost disappeared the rule that the first contraction appears on the second stimulus is strictly adhered to. At this stage the sphincter responds well to direct stimulation, and just as with carbon dioxide, the effect of magnesium must be primarily a depression of facilitation at the neuromuscular junction. This agrees with the experiments of Mayer (1916) on conduction in the subumbrella net of Cassiopeia, and of Parker (1932) bn conduction in the filaments of Physalia. In both these cases magnesium can locally abolish the response of the muscle to indirect stimulation without the abolition of conduction through the nerve net. Katz (1936) has shown in the skeletal muscle of Carcinus maenas that magnesium depresses facilitation at the neuromuscular junction.
Prolonged exposure to excess magnesium leads to breakdown of the rule that the response always begins on the second stimulus of a series. There is evidence that this is not due to modification of neuromuscular conduction but to the action of magnesium in breaking down synaptic junctions in the nerve net itself. The nerve net which serves the sphincter normally conducts without hindrance to all parts of this and of certain other muscles. But when exposure to magnesium has reduced the response to the condition shown in Fig. 2c, it is found that contractions even of the sphincter tend to be localized, particularly to that part of the animal nearest the electrodes. This means that barriers have been developed to free conduction within the nerve net itself. A study of a response such as that recorded to a frequency of one shock per second in Fig. 2 c bears this out. No response took place till the third stimulus. Some minute responses then occurred up to the tenth stimulus. But they only occurred to every alternate stimulus, as though the conduction path to the muscle periodically failed. After the tenth stimulus, however, a path of conduction to the muscle became established and each stimulus produced a response.
Recovery from the breakdown of the through conduction of the nerve net itself is much more rapid than recovery frorri the depression of neuromuscular facilitation. Fig. 2d shows that even 10 min. in sea water after over 1 hr. exposure to excess magnesium suffices to restore normal through-conduction in the nerve net, while neuromuscular facilitation is still greatly depressed. Complete recovery from magnesium depression takes over 12 hr.
It is interesting to note in parenthesis that the “after-disc rges” or adventitious responses which at times occur in normal animals during leries of stimuli, still make their appearance both under carbon dioxide and under excess magnesium. This precludes a possibility that they are due to impulses casually arising from superficial sense organs (Pantin, 1935 b), because in Mg at least these are anaesthetized. It also reduces the likelihood that they are due to enhanced excitability of parts of the nerve net, because both these substances diminish excitability. These facts lend colour to the idea that “after-discharges” may be due to nervous impulses which originate from the same stimulus, reaching the muscle by two independent paths in the nerve net of very different conduction rate.
The effects of excess potassium on the responses of CaUiactis are profound and somewhat unusual. They are completely reversible even after several hours’ exposure of the animal to 50 % o-6 M KC1 with sea water. Immersion in sea water containing 5 % of added isotonic KC1 (0.6 M) immediately causes spasmodic contraction of the tentacles and repeated contractions of the sphincter. These soon become less frequent though for some time there is a tendency for adventitious nervous impulses to appear. But the most striking effect is a huge augmentation of the size of the contractions which rapidly, develops during the first 30 min. of immersion (Fig. 3). Recovery is complete on return to natural sea water.
Despite the large size of the contractions there is little change in the character of the facilitation process. Each stimulus still produces a single contraction and the size of the contractions increases as the interval between stimuli is reduced. The only obvious difference is the greater size of the contractions at all frequencies compared with the normal. Occasionally, however, a distinct though minute contraction develops in response to a single stimulus..
Not only is facilitation still present, but the rate of decay of the facilitating effect of a stimulus is little altered from the normal. Fig. 3 a, b shows that the response will vanish when the interval between a pair of stimuli is increased to about the same value both in a normal animal and when subjected to excess potassium. This is the more remarkable because under potassium even the smallest contractions are enhanced in size and are therefore more readily detected.
The large size of the contractions which take place under the influence of potassium is at least partly due to the prolonged maintenance of tension. Maximum tension is only reached several seconds after the stimulus. Indeed the contractions might be thought to be brief tetani through the stimulus setting up not one but several nervous impulses in the nerve net. This, however, cannot be true because if each stimulus gave rise to several impulses it would completely upset the normal facilitation phenomena ; and there would also be a large response to the first stimulus. Moreover, each of the extra impulses would produce its own response in the muscle, and these are not visible (Fig. 3).
The enhanced contractions may be due partly to increased excitability of the muscle fibres so that more are facilitated at each contraction. But their prolonged character is at least partly due to change of the mechanical response of the muscle.
The prolongation of the state of contraction resembles that of frog’ s muscle under veratrine. There dpes not seem to be an abnormally high intensity of contraction in the muscle fibres, for the maximum tension achieved after a series of stimuli when potassium is present is not far above normal, though fewer stimuli are required to reach this maximum.
This “veratrine-like” effect of excess potassium in actinians appears to be unique. In the muscles of many animals excess potassium gives rise to prolonged contracture, especially in unstriated muscle (Wells, 1928; Gasser, 1930). But in these cases there is a direct contraction in response to the solution, not one which waits upon a stimulus for its appearance. Nevertheless, this may only be a difference of degree for very high concentrations of potassium (10-50 % o-6 M KC1 in sea water) do cause direct contraction of the whole musculature.
POTASSIUM AND MAGNESIUM ANTAGONISM
In the skeletal muscle of CarcinusKatz (1936) showed that excitation by excess potassium is prevented by magnesium. In Calliactis the actions of both these ions are intimately connected. Magnesium-free sea water influences the response of the sphincter in the same way as sea water containing excess potassium (Fig. 3 c, d). Potassium-free sea water resembles in its effects sea water to which magnesium has been added. The effect of any solution depends on the balance between these two cations. Excess magnesium diminishes the enhanced responses due to excess potassium. In high concentrations (50 % 0.4 M MgCl2 with sea water) it will prevent the spontaneous contraction caused by high potassium concentrations (12 % 0.6 M KC1 in sea water, and above).
We have already shown that magnesium acts at the neuromuscular junction. We have also shown that enhanced responses under excess potassium are not due to excitation of the nerve net. Since magnesium can diminish or prevent the action of potassium it appears that this also must exert its effect at neuromuscular junction.
THE HYDROGEN ION
Though a moderate increase in acidity (pH 6.5) produces little effect on the response of Calliactis, a point is reached in the neighbourhood of pH 5 at which the contractions of the sphincter in response to stimulation are greatly enhanced in size and duration. The whole action is parallel to that of excess potassium and the process of facilitation is unaltered except for the size of the contractions (Fig. 3 e,f ).
If a resting Calliactis is caused to respond to several batteries of stimuli in succession the response shows fatigue. The contractions become longer and slower and their size may for a time become considerably increased. Repeated excitation of a muscle must lead to some accumulation of hydrogen ions and of carbon dioxide. Excited tissues also tend to lose potassium to the surrounding tissues (Dulière & Horton, 1929). Such an accumulation of carbon dioxide, hydrogen ions and potassium ions would lead to changes in the response like those actually observed in the early stages of fatigue.
Increase in the calcium content of sea water causes large augmentation of the contractions of the sphincter (Fig. 4). The calcium contractions are quite normal in appearance except for their size. There is no prolonged contraction like that caused by the potassium and hydrogen ions. The effects of even high calcium concentrations are completely reversible in normal sea water.
Fig. 4 shows that the facilitation phenomena are normal. The response increases steadily as the stimulation interval is diminished. With excess calcium there is no evidence of even a slight response to a single stimulus. The interval which just suffices to permit the facilitating effect of a stimulus to die completely away, so that a following stimulus is ineffective, is about the same as in normal animal. Calcium does not influence the rate of decay of facilitation.
Calcium seems to exert its effect at the neuromuscular junction. As in the case of potassium, its augmenting effect is greatly depressed by excess magnesium. Since magnesium exerts its effect on the neuromuscular junction, calcium must therefore act either at this point or at some earlier point in the chain of events between stimulation of the nerve net and the contraction of the muscle. But just as in potassium, the character of the responses, particularly the maintenance of facilitation, makes it clear that augmentation by excess calcium is not caused by extra nervous impulses set up in the nerve net. The only other place at which the calcium can be acting is between the nerve net and the muscle.
We have mentioned that facilitated responses of the muscles of anemones bear an analogy to those of vertebrate skeletal muscle during partial curarization. This analogy is more than superficial. Excess calcium enhances the facilitated response while excess magnesium depresses it; that is, calcium decreases the “curarization”. The curarizing action of magnesium on vertebrate muscle is well known, and Feng (1936a, EXBIO_17_1_61C7b) has shown how calcium antagonizes the action of a very large number of curarizing substances, including magnesium. The parallel with augmentation of facilitation by calcium in Calliactis is clear.
The influences of the substances described in this paper are confined almost entirely to the facilitated response. High concentrations of potassium alone directly excite contraction of the muscles. The calcium and hydrogen ions, and the potassium ion in lower concentrations, only augment the response to stimuli, while carbon dioxide and magnesium only depress it. These substances appear to act at the neuromuscular junction, and their mode of action is curiously limited. The rule that a single stimulus fails to produce a response holds in all cases except for the trivial contractions which sometimes occur under the influence of potassium. And in spite of great modification of the size of the contractions no ion seriously changes the rate at which facilitation decays. This contrasts with the immense effect of temperature on the decay.
These facts throw light on the method by which facilitation takes place in sea anemones. There are at present two general views concerning transmission at neuromuscular and other junctions in different animals. According to one, excitation is directly transmitted by the action current. According to the other, the nervous impulse liberates some transmitter substance, which in turn excites the muscle fibre. Monnier (1936) has suggested that at least in some muscles both these methods of excitation may co-exist. But whichever of these views is taken it would appear that excitation of the muscle is brought about by one factor, whether it is the nervous impulse or a chemical transmitter. It may be, however, that the process of transmission sometimes involves more than one factor. In summarising their conclusion on transmission in vertebrate systems Dale, Feldberg & Vogt (1936) suggested two alternative.hypotheses : (1) that a transmitter substance directly excites the muscle cell, and (2) that the nervous impulse excites the muscle cell but cannot reach it unless the latter is sensitized by a chemical substance produced at the nerve ending. It is not intended to discuss the validity of these hypotheses for vertebrate systems, but they illustrate two possible methods of neuromuscular action. The first hypothesis involves only one factor in excitation, the second involves two; a process of sensitization and a process of excitation which it must precede. Clearly there are many conceivable hypotheses of this class in addition to the particular one mentioned above : either or both processes may be either electrical or chemical. Let us consider to which class the facilitated transmission of actinians belongs.
At first sight the step-like rise in tension caused by a series of shocks strongly suggests a response to successive increments of a transmitter. The effect is in a great measure due to simple mechanical summation. Each stimulus in a series adds to the tension in the viscous muscle while relaxation after each increment is slow. Nevertheless a series of stimuli does progressively augment facilitation. Fig. 5 shows the size of the response of the sphincter of Calliactis to the second stimulus of a pair separated by increasing intervals of time. The facilitating effect of the first stimulus (itself ineffective) and its decay are seen. If, however, a rapid series of stimuli is given instead of the single first stimulus of such a pair, facilitation can be piled up to such an extent that it takes a far longer time to decay and causes a much bigger response, except where the facilitated contractions are maximal. The second curve in Fig. 5 shows the response to a stimulus that has been preceded not by a single stimulus as in the first curve but by two stimuli at 0-5 sec. interval. It shows the larger size of the responses and the prolonged decay. If there are more than two preceding stimuli the effect is still greater. Facilitation is therefore added to by each stimulus of a series and dies away between each. How far is it consistent with the simple transmitter theory?
It is remarkable that a single stimulus applied to an anemone is normally without effect upon the muscles. On the basis of a simple transmitter hypothesis, this failure to respond to a single stimulus must mean that the exciting substance is produced in less than threshold quantity. The response to a second stimulus given soon after the first must then be due to the addition of further exciting substance so that the threshold is exceeded. In such a system factors such as excess calcium which enhance facilitation might enable a clear response to take place at the first stimulus, while factors such as excess magnesium which sufficiently depress facilitation must make it necessary for several stimuli to be given before sufficient transmitter accumulates to cause a response. Yet apart from the occasional trivial responses under potassium, neither ionic changes nor many other changes alter the rule that a single stimulus produces no response, and the response begins on the second stimulus if it takes place at all, so long as the pathways in the nerve net remain intact.
These facts suggest that facilitation in anemones is not brought about simply by a transmitter. This can be tested in the following way. Suppose a single stimulus produces a transmitter in less than threshold amount. Each subsequent stimulus of a series will add to it. But since facilitation decays after each stimulus the rate at which transmitter piles up towards the threshold will depend on the frequency of stimulation. When this is high the threshold will be exceeded at the second stimulus. But if the interval between stimuli is lengthened there will come a point at which the amount of transmitter after the second stimulus just fails to reach the threshold, while a third stimulus at the same interval will enable this to be passed. Consequently the response will only begin at the third stimulus of the series.
Table I gives the size of the contractions of the mesenteric retractor of a Metridium senile in sea water to a series of three stimuli at different frequencies. The size of the contraction is measured in arbitrary units (mm. of the smoked record). It represents the added tension following each shock, not the total tension at the moment. The response was recorded with a weak isometric lever.
It will be seen that as the interval between stimuli is lengthened, the response to the third stimulus disappears at the same time as the response to the second. It is not possible to obtain two apparently ineffective stimuli before the first response as the simple transmitter hypothesis would lead one to suppose. On the other hand, these results can be easily explained if it is supposed, in accordance with the second hypothesis, that the first stimulus is ineffective because it finds the neuromuscular junctions unsensitized.
Fig. 6 illustrates the same question in a different way. It shows the size of response of the sphincter of Calliactis to pairs of stimuli of increasing intervals. The response to the second stimulus disappears in this experiment at a time interval of about 3.0 sec. On a simple transmitter hypothesis there should still be sufficient transmitter left after 3.0 sec. to bring the total amount with that added by a second stimulus almost to the threshold value. If therefore a pair of stimuli is preceded by an extra stimulus 3.0 sec. earlier, the response obtained should be substantially higher than without this preceding stimulus ; for in this case the leading stimulus of the pair brings the amount of transmitter just to threshold value, while without any preceding stimulus it is well below it. To be on the safe side, the preceding stimulus was sent in 2.4 sec. before the pair, so that its facilitating effect had not quite vanished. In spite of this the presence of the preceding stimulus makes no obvious difference to the size of response. This is incompatible with the simple transmitter hypothesis.
A further difficulty for this hypothesis depends on the fact that in anemones the contraction is built up by responses of the muscle directly corresponding to each stimulus received. In considering facilitation in Carcinus muscle, Katz (1936) pointed out that accumulation of a transmitter must soon lead to numerous extra responses of the muscle or indeed to continuous activity. Yet in anemones even maximal responses are built up of contractions corresponding to the arrival of each nervous impulse at the muscle. This remains true even when the facilitation is enhanced by calcium or potassium.
All these facts are easily explained if two distinct processes are involved in neuromuscular transmission. In this case a single stimulus fails not because a transmitter is present below threshold concentration, but because the first stimulus does not find the neuromuscular junction prepared for transmission. The stimulus nevertheless leaves behind it a state of sensitization which enables a subsequent stimulus to be effective. Further, each stimulus of a series can increase the state of sensitization of the junction, thus making larger responses possible. But the muscle will only contract in response to each excitation process.
The nature of the excitation and sensitization processes governing transmission in these actinian muscles remains to be determined. The influence of ions like potassium and magnesium on the response is not inconsistent with excitation being due to the action current itself, for these ions affect the conduction of excitation (Cowan, 1934; Katz, 1936). On the other hand the high temperature coefficient of the decay of the facilitation, the lack of influence upon it of ions which affect the electrical conditions at the cell surface, and the fact that facilitation may endure in some cases for more than 100 times the relative refractory period of the nerve net, suggest that the sensitization process may be due to the accumulation of an actual substance rather than an electrical state of the kind found by Bremer (1931) under certain conditions in vertebrate muscle.
In Carcinus, Katz suggested that potassium might take part in neuromuscular transmission. In anemones potassium is certainly not the facilitating substance itself. For in the presence of an excess of that substance a single stimulus must suffice to cause strong contraction of the muscle. But the possibility that a “facilitator” truly exists, and that it is chemically comparable to the “transmitters” found in the tissues of other animals will be discussed by one of us in a subsequent paper.
The anaesthetic effects of carbon dioxide and magnesium added to sea water on Calliactis parasitica and Metridium senile have been studied. Magnesium first paralyses sense organs, but the main effect of both magnesium and carbon dioxide is a depression of neuromuscular facilitation. This prevents conduction of nervous impulses to the muscles in a way analogous to curarization of vertabrate skeletal muscle.
The chief effects of excess calcium, potassium and the hydrogen ion are increases in the size of the facilitated response to stimulation. The responses under potassium and the hydrogen ion are greatly prolonged and resemble the veratrine contracture of vertebrate skeletal muscle.
All the substances studied exert their chief effects at the neuromuscular junction. Analysis of their mode of action indicates that neuromuscular transmission in anemones involves two distinct processes, a process of excitation and a process of sensitization of the neuromuscular junction without which excitation of the muscle by the nervous impulse cannot be effective. This view is confirmed by examination of the relation of the size of the responses in normal animals to successive stimuli. The nature of the sensitization process and of the excitation process is discussed.