VOL. XIV, No. 2 APRIL, 1937 STUDIES ON THE PHYSIOLOGY OF ARENICOLA MARINA L. I. THE PACE-MAKER ROLE OF THE OESOPHAGUS, AND THE ACTION OF ADRENALINE AND ACETYLCHOLINE BY G. P. WELLS Department of Zoology, University College, London (Received 19 September 1936) (With One Plate and Twenty-six Text-figures) CONTENTS PAGE I. Introduction 117 II. The activity of the isolated oesophagus . .118 III. The invasion of the proboscis by oesophageal rhythms 121 (a) The nomenclature of the parts of the extrovert 121 (b) The isolated extrovert preparation 122 (c) Behaviour of the isolated extrovert in sea water 123 (d) Behaviour of the isolated extrovert in body fluid . '. .124 (e) Adrenaline, acetylcholine and the isolated extrovert ...... 127 (/) Site of origin of the activity of the isolated extrovert ..... 130 (g) Site of action of adrenaline and acetylcholine ....... 136 IV. The invasion of the central nervous system and body wall by oesophageal rhythms . 138 (a) The extrovert : body-wall preparation 138 (6) Behaviour of the preparation in sea water 139 (c) The action of adrenaline and acetylcholine 142 V. Oesophageal rhythms in the intact animal 143 (a) Methods 144 (6) The resting rhythm ' 145 (c) The action of adrenaline ........... 147 VI. Discussion . 150 Summary ............... 154 Acknowledgements ............. 157 References . .............. 157 I. INTRODUCTION THE following communication contributes to our knowledge of two distinct aspects of annelid physiology. (1) Since the work of Quatrefages (1844), it has been known that the polychaetes possess a system of nerves and ganglia, often very elaborate, in the walls of the ]EB-xivii 8 n8 G. P. WELLS eversible proboscis. Although the structure of the stomatogastric nervous system has been described in a number of different species, nothing has yet been published on its physiology. The few investigators who have studied the polychaete nervous system experimentally have confined their attention to the supra- and sub-oeso- phageal ganglia and the ventral nerve cord. The experiments here described were carried out on Arenicola marina, a species which, because of its abundance and large size, is well suited for experimental work, but in which, unhappily, the anatomy of the stomatogastric nervous system has not yet been described. Nevertheless, they throw a certain amount of light on the interrelations between the neuromuscular structures in the front part of the gut and those of the body wall of the anterior end. It is shown below that the oesophageal region of Arenicola exerts a remarkable pace-maker action, discharging impulses forwards, via the proboscis, into the central nervous system, and thus determining certain movements of the whole animal. This point is established by considering one by one a series of preparations of ascending complexity, starting with the isolated oesophagus and finishing with the intact worm. (2) Recently, great advances have been made in our knowledge of the part played by humoral agents in the nervous integration of vertebrates. Comparative data on animals of other phyla are, however, very few. As regards the annelids, Gaskell (1919), reviewing his own work and that of earlier investigators, brought forward evidence to show that adrenaline exists in leeches and probably in certain other annelid worms, and studied the action of the drug on the body wall and blood vessels of the medicinal leech. Since that date, very little has been done on the problem of adrenaline in annelids. More recently, Bacq (1935) showed that choline esters and cholinesterase are present in polychaete worms; but, except for the well- known case of the eserinized dorsum of the leech, which contracts in response to minute amounts of acetylcholine (Fuhner, 1918) or of other choline esters (Chang & Gaddum, 1933), nothing is known about the action of this group of substances on the tissues of annelids, or about their possible physiological significance in that phylum. I therefore thought it worth while to study the action of adrenaline and acetylcholine on the different preparations employed during this research. It was originally intended to publish the experiments with drugs in a separate paper; but the results, especially with adrenaline, throw considerable light on the interrelationships of the various preparations, and it is therefore best to incorporate them with the main portion of the work. II. THE ACTIVITY OF THE ISOLATED OESOPHAGUS The experiments on the isolated oesophagus were made as follows. The oeso- phagus 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 Studies on the Physiology of Arenicola marina L. 119 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. Fig. 1. The two halves of an isolated oesophagus. Oral half below; aboral above. At signal, adrenaline is added (i : 250,000). In all records: Read from left to right; upstroke of lever records contraction of preparation; time signal marks once every minute. Fig. 2. The two halves of an isolated oesophagus. Oral half below; aboral above. At first signal, eserine is added (1 : 500,000); at second, about a minute later, acetylcholine (1 : 500,000). 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. 8-2 ito G. P. WELLS 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 : iooo solutions of the drugs (made up as described on p. 127) into the finger bowls with a hypo- dermic syringe, and subsequently computing the resulting concentrations. Fig. 3. Above: oral half of an isolated oesophagus, showing secondary rhythm superposed on the primary rhythm. Below, oral half of an isolated oesophagus, which gave - an unique behaviour pattern. 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, i, 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 Studies on the Physiology of Arenicola marina L. ill 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 pheno- mena may however appear: (1) The primary waves may not be smooth, but accompanied by a series of relatively rapid contractions of small amplitude. (2) In about one-third of the preparations studied, a secondary rhythm was superposed on the primary rhythm of the oral end. This secondary rhythm consists of slow tonic contractions, of much greater amplitude than the primary rhythm and occurring at much longer intervals; they are accompanied by outbursts of relatively rapid and vigorous contraction (Fig. 3, upper half). (3) In one only, of twenty-seven gullets studied, the oral half gave the very curious record of Fig. 3 (lower half). This is possibly an accelerated secondary rhythm. 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. 111. THE INVASION OF THE PROBOSCIS BY OESOPHAGEAL RHYTHMS 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 con- sidering its physiology, to understand the anatomical relations of its component parts. (a) THE NOMENCLATURE OF THE PARTS OF THE EXTROVERT The anatomy of Arenicola has. been described by Gamble & Ash worth (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.