Studies on the Physiology of<i>Arenicola Marina</i>L.: I. The Pace-Maker Role of the Oesophagus, and the Action of Adrenaline and Acetylcholine

Abstract

The following communication contributes to our knowledge of two distinct aspects of annelid physiology.The experiments on the isolated oesophagus were made as follows. The oesophagus was cut through immediately posterior to the pouches of the first diaphragm and again immediately anterior to the oesophageal glands, and removed. In this way almost the whole of the oesophagus was isolated, excepting only one short part between the oesophageal glands and the stomach, and another lying in front of the first diaphragm. In most of the experiments the oesophagus was then divided into two by a transverse cut about half-way along its length, and the oral and aboral halves were separately studied. They gave somewhat different results.The pieces of oesophagus thus dissected were tied at both ends and suspended in sea water. Light isotonic levers were used in all the experiments to be described in this paper.The oesophagus preparations were mounted in finger bowls, to the bottoms of which disks of cork had been glued. A loop of thread tied to one end of each preparation was passed round a bent pin stuck into the cork. The action of adrenaline and acetylcholine was investigated by spraying known volumes of I : 1000 solutions of the drugs (made up as described on p. 127) into the finger bowls with a hypodermic syringe, and subsequently computing the resulting concentrations.The behaviour of the aboral half of the oesophagus is straightforward. It shows, more or less distinctly, smooth contraction waves of low frequency, occurring in most cases about once every 5 or 6 min. (Figs. 1, 2). Occasionally the interval may be as short as 3, or as long as 8 or 9 min., in exceptional preparations. In what follows, this rhythm will be termed the primary rhythm.Both adrenaline and acetylcholine cause contracture. In the case of the latter drug, the primary rhythm can usually be seen to continue at the new level ; but with the former the contracture is generally smooth, the rhythmic waves disappearing.The amplitude of the primary rhythm is exceedingly slight. The levers used magnified the movement about eight times. The actual shortening of the oesophagus during a primary wave under the conditions of these experiments is of the order of one-seventieth of its total length.The oral half of the oesophagus also shows the primary rhythm and it reacts in the same way to adrenaline and acetylcholine. The following complicating phenomena may however appear:These various complications, shown only by the oral end, are of interest as indicating a physiological differentiation between the two halves of the oesophagus. The main result of these experiments is, however, the demonstration of the slow primary rhythm, occurring in both halves of the oesophagus and therefore probably a generally diffused property of the oesophageal wall.The excised proboscis, with a short length of oesophagus attached, yields a vigorous muscle preparation, with a highly characteristic intermittent rhythm. The structure of the preparation is rather complicated, and it is necessary, before considering its physiology, to understand the anatomical relations of its component parts.The anatomy of Arenicola has. been described by Gamble & Ashworth (1898, 1900) and by Ashworth (1904). These accounts differ slightly in terminology; e.g. the term “buccal mass” is used in different senses. To avoid ambiguity, the sense in which the various terms will be used in this paper must therefore be specified. The terminology of Ashworth (1904) is followed, with the introduction of a new name.Moreover, as no clear figure of the relations of the parts when the proboscis is retracted has yet been published, and as the animal is in the retracted condition when operated upon, I have included a photograph of a section of a spirit specimen of a worm which is just beginning to extrude the proboscis (Plate la). In extreme retraction, when the body wall of the front end is maximally contracted, the part of the gut which lies anterior to the first diaphragm is thrown into concertina-like folds, and the insertion of the retractor muscle into the pharynx is brought back to the level of its origin from the body wall. The relevant parts of the gut are clearly shown in Plate I b, which is a photograph of the extrovert split up along one side and pinned out with the lining upwards.The parts of the extrovert are, from before backwards in the retracted condition :In what follows, the term mouth will be used to denote the boundary between the buccal mass and body wall. The buccal mass and pharynx together constitute the proboscis.The relations of the following structures must also be noted: the retractor muscle, which forms a complete sheath round the aboral part of the extrovert, and is inserted into the pharynx and into the body wall at the level of the first chaetigerous annulus ; and the first diaphragm, having roughly the form of a flat cone perforated at its apex by the oesophagus,1 and bearing a pair of backwardly directed diverticula, the diaphragmatic pouches, immediately ventral to the oesophagus.The preparation is made as follows :The front half of an Arenicola is pinned out, ventral surface uppermost. The body wall is divided by a cut along the mid-ventral line. This cut should just divide the first chaetigerous annulus, but go no further forwards. The flaps of body wall are pinned out sideways, that part which lies in front of the first chaetigerous annulus being turned forwards. The oesophagus is now turned forwards and the various membranes which suspend it are divided, up to the first diaphragm. At this stage the retractor muscle lies as a thick red sheath round the front part of the gut, concealing the proboscis ; it must be divided right round, close to its origin from the body wall. When this is done properly, the buccal mass can be extended by gently pulling the oesophagus backwards, and the circum-oral nerve ring and the otocysts can be plainly seen. The gut is now ligatured twice, one thread being tied at about the level of the nerve ring and the other just aboral to the diaphragmatic pouches, and cut free.The resulting preparation should be left for at least half an hour in sea water to recover from the dissection. It may conveniently be suspended in a bath of the pattern shown in Fig. 4, which allows the bathing fluid to be changed while the record is being taken.The anatomy of the preparation is somewhat complex. It includes the buccal mass, the pharynx, the post-pharyngeal ring arid that part of the oesophagus which lies oral to the first diaphragm. It may also include the nerve ring and, owing to the way in which the animal commonly humps its mouth up during the operation, a certain amount of body wall from the region round the mouth. Moreover, as the retractor muscle is most conveniently divided along its origin from the body wall, and is not thereby separated from the first diaphragm, these two structures generally form a more or less complete sheath round the aboral half of the preparation.Suspended in sea water, the extrovert shows a highly characteristic behaviour pattern, periods of vigorous rhythmic activity alternating with periods of comparative rest. The “rhythmic outbursts” generally last for 2 or 3 min. and the intervals between them for somewhat longer; but different preparations vary in the duration of the two phases and also in the amount of activity shown in the relatively quiescent intervals. The four records of Fig. 5 are chosen to illustrate the range of variation as regards the latter factor that may be met with.The individual strokes of the lever, during the rhythmic outbursts, are not simple contractions and relaxations of the longitudinal musculature, but are somewhat more complex acts. If a vigorous preparation is watched, two complicating factors are usually evident: firstly, the preparation does not contract simultaneously along its whole length, the oral end contracting first, and secondly, the relaxation of the longitudinal muscles (downstroke of the lever) is apparently accompanied by contraction of the circular fibres. In the present communication, the nature of the individual strokes will not be analysed in detail. Attention will be directed to the general activity pattern, with its characteristic alternation of active and resting phases.The first point to be established is that this behaviour pattern is normal. It is well known that muscles having a regular and continuous functional activity, e.g. hearts, may show, as an abnormality, grouped beats which greatly resemble the intermittent rhythm of the Arenicola extrovert. The point is illustrated by Fig. 6, which was obtained by the writer during an earlier investigation. It shows the behaviour of an atypical lobster heart, which, unlike most lobster hearts, gave grouped beats whenever it was perfused with a potassium-free solution. Except for the great difference of time scale, the picture is extraordinarily like that given by many of the lugworm preparations. Similar grouped beats have been recorded by Hogben (1925) in crustacean hearts in the presence of excess potassium, by Mines (1912) in the dogfish heart after arrest by lack of urea or by magnesium excess, and also by other authors.The suspicion therefore arises that the intermittent activity of the Arenicola extrovert is an artefact, and that sea water is an unsuitable chemical environment for the tissues of this animal. This point is of great importance, because sea water was used as bathing medium throughout this investigation. I have therefore made experiments in which the preparation was suspended in body fluid from Arenicola, to compare its action with that of sea water.It may first be pointed out that where the blood vessels of Arenicola penetrate among the other tissues, they always run in tubular prolongations of the coelom, so that it is the coelomic fluid, and not the blood, which forms the immediate chemical environment of the cells.The experiments were done in July, when germ cells were thickly suspended in the body fluid. About sixty freshly collected lugworms were “bled” by opening the body cavity, and as each worm was opened the fluid was allowed to flow through muslin on to filter paper in a Buchner funnel. In this way it was freed as rapidly as possible from most of the suspended cells, to minimize the possibility of contamination by products of cellular breakdown. Such contamination might, for instance, raise the potassium concentration significantly. The time which elapsed between the shedding of the fluid and its passage through the funnel was always less than 10 min. The pooled fluid, thus collected, was still slightly cloudy; it was therefore filtered a second time. This was done at about 6 o’clock, the “blood-letting” having begun about midday.The final filtrate was perfectly clear, but light scarlet in colour. Had it been pure coelomic fluid it would of course have been colourless, but owing to the fragility of the blood vessels I found it impossible to avoid slight contamination of the outflowing coelomic fluid with blood. This is probably of little importance, as it is very unlikely that significant differences in electrolyte concentration exist between the two. The fluid was used about 4 hours after the final filtration.Preparations suspended in body fluid showed an activity pattern essentially like that already described (Fig. 7). The following differences were noticed: first, the number of lever strokes in each rhythmic outburst is less in body fluid than in sea water and the duration of each individual contraction is longer, and second, on changing from sea water to body fluid there is a sharp rise of tone, which is reversed when the opposite change is made.This reversible tone rise is probably not due to a difference in electrolyte concentrations between the body fluid and sea water. The effects of varying the concentrations of K, Ca and Mg in the medium have been studied in some detail by the writer, and will be described in a separate paper. From the results of those experiments, it is likely that the composition of the body fluid, as regards inorganic salts, is very similar to that of sea water. Certain changes (e.g. Mg lack, K excess) produce reversible tone rises like that evoked by body fluid, but in these cases the rhythmic pattern is conspicuously modified. The same is true of adrenaline (see next section), which produces a tone rise but profoundly alters the behaviour pattern. Of the various substances studied by the writer, only acetylcholine resembles body fluid in producing a tone rise without disturbance of the characteristic intermittent rhythm. It would be unsafe to conclude on this basis alone that the body fluid contains acetylcholine, for the only chemical agents so far studied are adrenaline, eserine, acetylcholine and salts. It does, however, seem clear that none of these agents, except only acetylcholine, could be responsible for the differences observed between sea water and body fluid.The main result of these experiments is to show that sea water does not alter the fundamental nature of the activity pattern of the isolated extrovert. Its use as a bathing medium for Arenicola tissues is therefore legitimate. Indeed, on comparing Figs. 6 and 7, it is tempting to suppose that the two phenomena have essentially the same physiological mechanism, and that the Arenicola extrovert is normally in a condition which appears as an occasional freak in other rhythmic muscles.A large number of experirtients were done to determine the action of adrenaline and acetylcholine on the isolated extrovert. Because of its vigour and its highly specific behaviour pattern, the preparation is well suited for investigations of this kind. It is best to consider the results before passing to the next section, in which the site of origin of the excitations is localized.The experiments were done in the bath illustrated in Fig. 4. The solutions were made up as follows.A M/5 solution of sodium phosphate was acidified with HC1 until just yellow-green to brom cresol green (pH 4-2). This gave a solution containing M/5 acid sodium phosphate and M/5 NaCl, having a pH at which acetylcholine is very stable. “Roche” acetylcholine was made up 1 : 1000 in this acid phosphate, and used, in most cases, on the same day.This drug was made up 1 : 1000 in the acid phosphate diluted tenfold with distilled water. It was used in most cases within 48 hours of making up, being kept overnight in an ice chest, and showed no traces of discoloration.In most of the experiments on this substance, Parke Davis 1 : 1000 adrenaline solution was used. As this solution contains five times as much chloretone as adrenaline, control experiments were carried out with chloretone in appropriate concentrations, and also with a dry preparation of adrenaline (“Adrenalina B. P.” from British Drug Houses Ltd.), to make sure that the adrenaline was in fact responsible for the effects observed.During an experiment, the 1: 1000 solutions were kept in burettes on the bench, and were diluted in sea water (pH 8 · 2) to the required concentration immediately before application to the tissue. To guard against any possibility that the acetylcholine was being inactivated by hydrolysis in the alkaline sea water, the experiments with that drug were checked over, using sea water acidified with HC1 to pH 6 · 7 as bathing fluid. The results were the same as with pH 8 · 2.The most characteristic adrenaline effect is seen when the drug is applied in concentrations from 1 : 1,000,000 to 1 : 100,000—the latter being the highest concentration employed in this research. There is a sharp rise of tone, and a disappearance of the normal behaviour pattern. In most cases, after a period of somewhat confused excitement which lasts for several minutes, the extrovert settles down to beat with a very steady, continuous rhythm (Fig. 8). The amplitude may fluctuate irregularly from beat to beat, but in many experiments the frequency is now as regular as that of a perfused heart. In some preparations, especially when the drug is applied in the highest concentrations, there is a steady falling off in amplitude that rather recalls the well-known action of magnesium excess on the hearts of various animals.With low concentrations of adrenaline, the picture is not always so clear. A tone rise can be detected in concentrations down to i : 100,000,000. In most preparations a definite inhibitory action on the normal behaviour pattern, appearing as a lengthening of the interval between the rhythmic outbursts, is exerted by adrenaline 1 : 50,000,000 or 1 : 100,000,000 (Fig. 9). With concentrations of 1 :500,000,000 or lower there is usually no effect, but sometimes a very slight and ephemeral tone rise, accompanied by slowing of the rhythmic outbursts, can be made out, even in adrena-line 1 : 2,000,000,000. To summarize, the principal adrenaline actions are: (1) a rise of tone, (2) in low concentrations, a lengthening of the interval between the normal rhythmic outbursts, and in concentrations, the of a continuous rhythm. The first and effects are the most and in the action of adrenaline on the other preparations employed during this I have only used those concentrations which continuous rhythm in the isolated to is present in The experiments with acetylcholine were therefore done in the presence of 1 : only slightly the activity of the extrovert. It generally a very rise in tone, and, especially in a or preparation, it may the vigour of the beats during the active phases ; but the activity pattern is (Fig. most characteristic effect of acetylcholine is a sharp rise in tone, which when the drug is the behaviour pattern is The periods of rhythmic activity and each other with the frequency (Fig. With of acetylcholine, e.g. 1 : a slowing effect is generally during the rhythmic outbursts, the lever strokes and being less than before (Fig. extrovert is not to acetylcholine. to 1 : for 2 or 3 a clear tone rise is by acetylcholine 1 : but not 1 : a of experiments, the extrovert was dissected in various in order to the site of origin of the The anatomy of the has not yet been out in Arenicola, so one has no anatomical from which to already pointed out, the anatomy of the preparation is and, as dissected out for it includes the following two of structures in to the gut wall :The following experiments with these the of is to the following parts of the gut wall : the buccal mass, the pharynx, the post-pharyngeal and that part of the oesophagus which lies in front of the first number of preparations were divided into halves gave the activity pattern, the extrovert had been divided by a cut into and ventral or by a cut into right and left It is even possible to divide a preparation into four longitudinal of every one of which shows the characteristic of rhythmic activity, in this owing to the of the the contractions are not as as before (Fig. are not with a or other The structure from which the excitations is a ring round the gut or like a nerve experiments on longitudinal were done as follows. The extrovert was split and pinned out, side on a of cork stuck to the of a finger If a preparation is under sea water with a the different of the gut can be (Plate The extrovert is now divided by a transverse cut into two The cut are pinned to the the is and the two ends are by to isotonic The level of the transverse cut was in the different experiments, the various employed being illustrated in Fig. The effect of at each level was investigated in at least preparations, and in most cases in of the various are as results are shown in Figs. and the main being the extrovert includes two distinct the boundary between them being the post-pharyngeal The oesophageal end shows the characteristic intermittent rhythm, and is by in both it is that are general to the whole of the oesophageal wall and not to its oral The proboscis, on the other isolated from oesophageal shows an different of Its activity is not and it is by the proboscis is in with oesophageal the oesophageal rhythm the proboscis, and thus the behaviour pattern of the that the last the true course of is by the illustrated in Figs. and extrovert is split along one side and pinned out on cork. About half of the oesophagus is included in this are used, inserted at the shown in Fig. To the two ends of the preparation, are so that can be separately they are still continuous with each The a about 6 and thus the contraction of end from the lever to the the record is being a sharp is run the of the pharynx with the post-pharyngeal so that the two ends are now the four oral as a for this A record is in Fig. the while the two halves are still the proboscis shows of rhythmic activity with the oesophageal At the of the cut the oral end The oesophageal end little or not at the in its on the record being due to by the lower the oesophagus its rhythm, but the activity of the proboscis has it that the rhythmic of the isolated extrovert preparation are due to the waves of the primary rhythm of the forwards and the more vigorous of the of may be as to the nature of the oesophageal contraction The term has been used from time to time in the primary rhythm of the oesophagus in many cases, and especially with the aboral half of the the waves perfectly smooth on It is, however, not to that the oesophagus shows smooth, steady waves of which are on the proboscis, into of separate In many of the records of the oral half of the oesophagus the have contractions them ; this can be seen for in the of Fig. where the that each of the strokes of the proboscis during the rhythmic to a contraction of the oesophagus. as already pointed out, the oesophageal contractions are of very amplitude and levers were not to a definite to be on this the activity of the proboscis from the oesophagus is the same is true of the contractions during the is not yet preparation of Fig. can be used to further the action of drugs on the extrovert. If a preparation be (Fig. the oesophageal end only into while the proboscis a regular rhythm of the rhythmic If now the preparation is along the post-pharyngeal the of the proboscis the of Fig. the oesophagus shows tone waves during to adrenaline, and at first the proboscis to be following the waves as it This is, however, not the The of the proboscis is now due to variation in the frequency being and the between the waves is now the amplitude of the contractions of the proboscis every time the tone of the oesophagus The drug has profoundly the behaviour of the the of the extrovert to adrenaline, as seen for in Fig. is complex and the following (1) contracture of the oesophageal and (2) contractions of the of the proboscis, which are due to excitations from the more are brought out by Fig. The first is before the application of this oesophagus shows in to primary rhythm As the proboscis of rhythmic contraction with the primary oesophageal The waves have no effect on the proboscis ; while they are in the rhythmic of the proboscis, and therefore the primary rhythm of the continue with the normal the rhythm in nature from the primary rhythm, and is it without second point brought out by Fig. is that the contracture evoked in the isolated extrovert by acetylcholine is to its oesophageal acetylcholine, the oesophagus into the proboscis does but shows a steady tone rise Fig. to the primary waves which the oesophagus at the new is to that the primary waves in the oesophagus resemble the contracture in the same by concentrations of adrenaline, in that they are accompanied by rhythmic activity of the proboscis, the waves resemble acetylcholine contracture in having no effect which is to the proboscis, and in to the primary excitations of oesophageal origin which to the proboscis not end of the preparation about to be can be in the of the body wall of the lugworm is pinned out ventral surface and opened by a longitudinal about 3 to one side of the mid-ventral that an of the of the nerve This cut should just divide the first chaetigerous annulus, but go no further forwards. The flaps of body wall are then pinned out, the oesophagus is and the retractor muscle is divided, as already described on p. the latter is at the point where the nerve the retractor muscle ; the must of course not be of two transverse a of body wall from the region of the second chaetigerous annulus is isolated from the however, its with the nerve (Fig. The whole preparation is pinned to cork stuck to the of a finger and are tied to the just aboral to the diaphragmatic pouches, and to the of the for to very light isotonic the the preparation should be left for hour in sea water, before the after the to the it may another min. or so to into its as it is very to a the extrovert shows of activity as described in the last The shows of rhythmic occurring at the same time as the of the extrovert (Fig. In between these outbursts, the body wall into a rhythm of its the excitations responsible for the in fact flow from the extrovert to the body wall is by the nerve between the two a sharp contraction of both due to the of the the activity of the extrovert as but the are no to the body wall the The latter now its a preparation, the body wall follows the extrovert for during the rhythmic The contraction of the two is, however, not but lever rises as the other This fact that have to with a activity of the longitudinal and circular The of