Portal de Periódicos da CAPES

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

1937; The Company of Biologists; Volume: 14; Issue: 2 Linguagem: Inglês

10.1242/jeb.14.2.117

1477-9145

G. P. Wells,

Date Palm Research Studies

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 (3) in high concentrations, the evocation of a regular, continuous rhythm. The first and third effects are the most striking: and in investigating the action of adrenaline on the other preparations employed during this work I have only used those concentrations which evoke continuous rhythm in the isolated extrovert.According to Bacq (1935), cholinesterase is present in polychaete worms. The experiments with acetylcholine were therefore mostly done in the presence of eserine 1 : 500,000. By itself, eserine only slightly affects the activity of the extrovert. It generally calls forth a very slight, gradual rise in tone, and, especially in a weak or irregular preparation, it may increase the vigour of the beats during the active phases ; but the essential activity pattern is unaltered (Fig. 10).The most characteristic effect of acetylcholine is a sharp rise in tone, which falls equally sharply when the drug is withdrawn. Essentially, the behaviour pattern is unaffected. The periods of rhythmic activity and rest succeed each other with the usual frequency (Fig. 10). With high doses of acetylcholine, e.g. 1 : 100,000, a slowing effect is generally perceptible during the rhythmic outbursts, the lever strokes lasting longer and being less numerous than before (Fig. 11).The extrovert is not particularly sensitive to acetylcholine. After exposure to eserine 1 : 500,000 for 2 or 3 hours, a clear tone rise is produced by acetylcholine 1 : 10,000,000 but not 1 : 100,000,000.In a series of experiments, the living extrovert was dissected in various ways in order to localize the site of origin of the excitations. The anatomy of the stomatogastric system has not yet been worked out in Arenicola, so one has no anatomical guide from which to work.As already pointed out, the anatomy of the preparation is complex, and, as dissected out for routine purposes, it includes the following two groups of structures in addition to the gut wall :The following experiments deal respectively with these inclusions :Clearly then, the field of enquiry is restricted to the following parts of the gut wall : the buccal mass, the pharynx, the post-pharyngeal ring, and that part of the oesophagus which lies in front of the first diaphragm.A number of preparations were divided longitudinally into equal halves. Both halves invariably gave the usual activity pattern, whether the extrovert had been divided by a horizontal cut into dorsal and ventral halves, or by a vertical cut into right and left halves. It is even possible to divide a preparation into four longitudinal strips of approximately equal thickness, every one of which shows the characteristic outbursts of rhythmic activity, although in this case, owing to the small size of the strips, the contractions are not as powerful as before (Fig. 12).Evidently we are not dealing with a special ganglion, or other strictly localized centre. The structure from which the excitations emanate is either a ring running round the gut or something diffuse, like a nerve plexus.The experiments on longitudinal localization were done as follows. The extrovert was split longitudinally and pinned out, inner side uppermost, on a disk of cork stuck to the bottom of a finger bowl. If such a preparation is examined under sea water with a low-powered binocular, the different regions of the gut can be readily distinguished (Plate 1b). The extrovert is now divided by a single transverse cut into two halves. The cut edges are pinned to the cork, the rest is freed, and the two ends are connected by threads to fight isotonic levers. The level of the transverse cut was varied in the different experiments, the various levels employed being illustrated in Fig. 13. The effect of cutting at each level was investigated in at least three preparations, and in most cases in six.The specifications of the various cuts are as follows:The results are shown in Figs. 14 and 15, the main points being these:Evidently, the extrovert includes two physiologically distinct regions, the boundary between them being the post-pharyngeal ring. The oesophageal end shows the characteristic intermittent rhythm, and is excited by adrenaline; in both respects it is simply exhibiting properties that are general to the whole of the oesophageal wall and not peculiar to its oral extremity. The proboscis, on the other hand, if isolated from oesophageal influences, shows an entirely different mode of behaviour. Its activity is continuous, not intermittent, and it is inhibited by adrenaline.Finally, if the proboscis is in continuity with oesophageal tissue, the oesophageal rhythm invades the proboscis, and we thus get the behaviour pattern of the entire extrovert.Any remaining doubt that the last statement represents the true course of events is removed by the experiment illustrated in Figs. 16 and 17. An extrovert is split along one side and pinned out on cork. About half of the oesophagus is included in this experiment. Six pins are used, inserted at the points shown in Fig. 16. To the two ends of the preparation, threads are attached so that their movements can be separately recorded, though they are still continuous with each other. The pins immobilize a zone about 6 mm. long, and thus prevent the contraction of either end from affecting the lever attached to the other; While the record is being taken, a sharp knife is run across the junction of the pharynx with the post-pharyngeal ring, so that the two ends are now severed; the four oral pins serve as a guide for this cut. A typical record is reproduced in Fig. 17. Before the knife cut, while the two halves are still continuous, the proboscis shows outbursts of rhythmic activity synchronizing exactly with the oesophageal waves. At the moment of the knife cut the oral end contracts sharply. The oesophageal end contracts little or not at all, the upstroke in its trace on the record being due to fouling by the lower lever. After this, the oesophagus resumes its rhythm, but the associated activity of the proboscis has ceased.This makes it plain that the rhythmic outbursts of the isolated extrovert preparation are due to the waves of the primary rhythm of the oesophagus, flowing forwards and reaching the more vigorous musculature of the proboscis.A word of caution may here be added as to the intimate nature of the oesophageal contraction waves. The term “tone waves” has been used from time to time in describing the primary rhythm of the oesophagus because, in many cases, and especially with the aboral half of the oesophagus, the waves look perfectly smooth on my records. It is, however, not intended to imply that the oesophagus shows smooth, steady waves of excitation which are resolved, on reaching the proboscis, into groups of separate contractions. In many of the records of the oral half of the oesophagus the “tone waves” have numerous minute contractions superposed upon them ; this can be seen for instance in the upper line of Fig. 17, where the appearance strongly suggests that each of the single strokes of the proboscis during the rhythmic outbursts corresponds to a minute contraction of the oesophagus. Unhappily, as already pointed out, the oesophageal contractions are of very small amplitude and my levers were not sufficiently sensitive to enable a definite decision to be reached on this point. That the activity outbursts of the proboscis emanate from the oesophagus is certain; whether the same is true of the single contractions during the outbursts is not yet clear.The preparation of Fig. 16 can be used to analyse further the action of drugs on the extrovert. If such a preparation be adrenalinized (Fig. 18), the oesophageal end only goes into contracture, while the proboscis exhibits a regular rhythm instead of the previous rhythmic outbursts. If now the preparation is severed along the post-pharyngeal ring, the movements of the proboscis cease.In the particular experiment of Fig. 18, the oesophagus shows small tone waves during exposure to adrenaline, and at first glance the proboscis seems to be following the waves as it did before. This is, however, not the case. The undulation of the proboscis trace is now due to variation in amplitude, the frequency being pretty constant throughout; and the relation between the waves is now reciprocal, the amplitude of the contractions of the proboscis diminishing every time the tone of the oesophagus rises. The drug has profoundly altered the behaviour of the system.Clearly, then, the response of the entire extrovert to adrenaline, as seen for instance in Fig. 8, is complex and involves the following factors: (1) contracture of the oesophageal tissue only, and (2) rhythmical contractions of the musculature of the proboscis, which are due to excitations emanating from the oesophagus.Two more points are brought out by Fig. 19. The first is that, before the application of drugs, this particular oesophagus shows secondary in addition to primary rhythm (p. 121). As usual, the proboscis experiences outbursts of rhythmic contraction coinciding with the primary oesophageal waves. The secondary waves have no effect on the proboscis ; while they are in progress, the rhythmic outbursts of the proboscis, and therefore presumably the primary rhythm of the oesophagus, continue with the normal frequency. Evidently, the secondary rhythm differs in nature from the primary rhythm, and is superposed upon it without interrupting it.The second point brought out by Fig. 19 is that the contracture evoked in the isolated extrovert by acetylcholine is limited to its oesophageal end. On adding acetylcholine, the oesophagus goes abruptly into contracture; the proboscis does not, but shows a slow, steady tone rise (compare Fig. 15 C), continuing meanwhile to follow the primary waves which the oesophagus exhibits at the new level.It is interesting to note that the primary waves in the oesophagus resemble the contracture induced in the same organ by high concentrations of adrenaline, in that they are accompanied by rhythmic activity of the proboscis, whereas the secondary waves resemble acetylcholine contracture in having no effect which is transmitted to the proboscis, and in failing to interrupt the primary rhythm.The excitations of oesophageal origin which spread to the proboscis do not end there. By means of the preparation about to be described, their influence can be traced in the movements of the body wall of the worm.A lugworm is pinned out ventral surface upwards, and opened by a longitudinal incision running about 3 mm. to one side of the fine, mid-ventral groove that gives an outwardly visible indication of the position of the nerve cord. 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 freed, and the retractor muscle is divided, as already described on p. 122. During the latter operation care is necessary at the point where the nerve cord perforates the retractor muscle ; the cord must of course not be damaged. By means of two transverse cuts, a strip of body wall from the region of the second chaetigerous annulus is isolated from the rest, retaining, however, its connexion with the nerve cord (Fig. 20). The whole preparation is securely pinned to cork stuck to the bottom of a finger bowl, and threads are tied to the oesophagus, just aboral to the diaphragmatic pouches, and to the tip of the body-wall strip, for connexion to very light isotonic levers.After the dissection, the preparation should be left for hour in sea water, preferably aerated, before the experiment begins. Again, after connecting the threads to the recording levers, it may take another 20 min. or so to “get into its stride”, as it is very sensitive to mechanical disturbance.In a good preparation1, the extrovert shows outbursts of activity as described in the last part. The body-wall strip shows associated outbursts of rhythmic contraction, occurring at the same time as the outbursts of the extrovert (Fig. 21). In between these outbursts, the body wall relapses into a slower rhythm of its own.That the excitations responsible for the outbursts do in fact flow from the extrovert to the body wall is demonstrated by cutting the nerve cord between the two strips. After a sharp contraction of both strips, due to the stimulus of the cut, the activity of the extrovert continues as before, but the outbursts are no longer transmitted to the body wall behind the cut. The latter now steadily maintains its intrinsic rhythm.In a good preparation, the body wall follows the extrovert stroke for stroke during the rhythmic outbursts. The contraction of the two strips is, however, not synchronous, but reciprocal. One lever rises as the other falls. This fact suggests that we have to do with a reciprocal activity of the longitudinal and circular musculature. The upstroke of