(Received 8 July 1957) according to the conditions at the time of observation. They are known to be

Transcription

1 1 J. Physiol. (I958) I4I, I-2I REFLEX STIMULATION AND INHIBITION OF RETICULUM CONTRACTIONS IN THE RUMINANT STOMACH BY D. A. TITCHEN From the Physiological Laboratory, University of Cambridge (Received 8 July 1957) Contractions of the reticulum, one of the anterior divisions of the ruminant stomach, generally occur in conscious animals at regular intervals of sec, according to the conditions at the time of observation. They are known to be abolished by bilateral cervical vagotomy (Mangold & Klein, 1927; Duncan, 1953) and after the administration of atropine. Within the past few years there have been a number of reports that contractions of the reticulum occur in decerebrated or anaesthetized animals and that they may be evoked in these preparations by stimulation, in an afferent sense, of vagal fibres in the abdomen or neck (Iggo, 1951, 1956; Titchen, 1953; Dussardier & Albe-Fessard, 1954). Alterations in the conditions within the different divisions of the stomach have been shown to excite or inhibit such contractions in decerebrate or anaesthetized preparations (Titchen, 1953, 1954; Dussardier, 1955). In this paper an account is given of experiments in which a variety of stimuli applied to different parts of the ruminant stomach resulted in the reflex excitation or inhibition of contractions of the reticulum. Preliminary accounts of some of these experiments have been presented previously (Titchen, 1953, 1954). METHODS The animals used were young and adult goats, sheep and calves 2-10 weeks old. The younger animals were fed on milk and hay, the older animals on grass or hay supplemented with a protein-rich pellet diet. Food but not water was withheld in the hr before an experiment. Anaesthesia was induced with ethyl chloride and maintained with ether. Decerebrate preparations were made as previously described (Comline & Titchen, 1951 a). Contractions of the reticulum were recorded from a water manometer connected by an airfilled tube to a balloon containing water. This balloon was inserted into the reticulum through an incision about 1*5 cm long in the region of its free pole. It was kept in position with a pursestring suture tied around the polythene tube to which the balloon was attached. The capacity of the balloons used varied from 100 to 700 ml. at cm water pressure. In some experiments, and particularly those in which the responses of the reticulum to stretch of itself were studied, 1 PHYSIO. CXLI

2 2 D. A. TITCHEN care had to be taken to avoid the overflow of fluid from the manometer during contractions of the reticulum. For this reason the simple closed system of recording from a balloon containing water andconnected byan air-filled tube to a water manometer was modified to include an open reservoir. This modification is shown in Fig. 1, and has been referred to as the open system of recording. The presence of this reservoir in the recording system protected the water manometer during contractions of the reticulum when large volumes of water were displaced from the balloon. Records obtained with this open system gave a less accurate representation of the form and force of the contractions of the reticulum than those obtained with the simpler closed system. With both systems the records obtained did not measure exactly the actual form and force of the contractions of the reticulum but were probably more accurate than those which would have been obtained from balloons inflated with air. The records were obtained with pressures of 5-15 cm water in the balloons; at these pressures slight relaxations of the reticulum, which could be observed, were sometimes recorded. Writing point - 7 mm bore Glass reservoir glass tubing 250 ml. capacity Air- Wire - cementedtpic into ebonite float--- Meniscu Polythne.nS.. Glass I To balloon (water-filled) in reticulum Fig. 1. The reservoir-manometer system sometimes used to record the reflex contractions of the reticulum, and referred to, in the text, as the open system of recording. Nerve stimulation was carried out in the manner described previously (Comline & Titchen, 1951 a). L-Adrenaline base (British Drug Houses) and the sympatholytic substance phentolamine (Rogitine, Ciba) were dissolved with HCI in 0 9 % NaCl solution and diluted to an appropriate concentration with saline before injection through a cannula into a femoral vein. Atropine sulphate (British Drug Houses) was dissolved in saline. Blood pressure was recorded with a mercury manometer connected to a cannula inserted into a femoral or carotid artery. When required the abomasum was isolated by ligatures tied about the pylorus and at the omaso-abomasal junction. Care was taken to avoid the inclusion of major vessels and nerves in these ligatures. Samples of the contents of the abomasum were obtained through a cannula tied into a relatively avascular part of its ventral surface. The abomasum was washed out at the beginning of an experiment with successive volumes of ml. of 0.9% NaCl at 380 C ml. of saline at 38 C was introduced into the abomasum and withdrawn after mm in experiments in which the secretory activity of the abomasum was followed. These samples were titrated against 0 05N-NaOH using the Topfer-phenolphthalein indicator as described by King (1951) to titrate free and total acidity of the washings. The progressive diminution in the strength of acid introduced into the abomasum was determined by titration against N-NaOH. ph was determined electrometrically.

3 REFLEX RETICULUM CONTRACTIONS 3 RESULTS Contractions of the reticulum which involved the whole of the organ, similar to those recorded in conscious animals, occurred in decerebrate preparations of goats and sheep, and also of calves over 2 weeks of age. The contractions usually began within min of the laparotomy to expose the reticulunm and occurred at regular intervals of sec. When first recorded they were usually diphasic contractions of the musculature of the entire organ. Such contractions, observed in conscious animals, have been referred to in the literature as total contractions (Mangold & Klein, 1927; Phillipson, 1939). These contractions were present in many, but not all, experiments for min after decerebration under ether anaesthesia. After this time they ceased. The contractions diminished in force or frequency, or both, and often changed from their original diphasic to a monophasic form before they ceased. Such a series of contractions was observed in four preparations in which respiration ceased after decerebration and did not return until after a period of min of artificial ventilation. In two decerebrate preparations, one of a calf and one of a goat, the reticulum contractions continued largely undiminished in force and frequency for 300 min after decerebration. The activity of the reticulum was not recorded and the operative procedures undertaken were only those necessary to carry out the decerebration and to observe, but not to record, from the reticulum in these two experiments. In calves the two phases of the diphasic contractions were separated by a complete relaxation, but in sheep and goats there was only a partial relaxation between them. Monophasic contractions were usually complete within 3-5 sec of the start of the contraction; relaxation and contraction occupied about equal times. Diphasic contractions occurred over a period of 7-10 sec. The first and second phases each occupied about 3-5 sec. Direct observation of the reticulum revealed that there were increasingly intense contractions of localized regions of the reticulum before the onset of a total contraction. Localized activity of the reticulum was sometimes recorded as a slight increase in the base line of the manometer tracing for a period of 5-25 sec before the occurrence of the total contraction. The localized contractions of the reticulum were markedly reduced after each total contraction- The effect of afferent stimulation from the gut was first studied on the series of contractions which occurred after decerebration. The contractions were inhibited by manipulation of the abdominal viscera, by laparotomy incisions and by distension of the abomasum. They were increased in force and frequency after the addition of fluid to the balloon in the reticulum, after moderate stretch of the omasum, the introduction of a tube into the oesophagus or the introduction of acid into the abomasum. When the contractions had ceased they reappeared with the delivery of one of these stimuli. 1-2

4 4 D. A. TITCHEN The decline in the force and frequency of the contractions of the reticulum observed in a number of experiments in the period immediately after decerebration was accompanied by a decline in the amount of acid detected in successive samples from the abomasum. The progressive decline in the force and frequency of reticulum contractions after decerebration and the diminution in the amounts of acid in successive samples from the abomasum in one experiment is shown in Fig. 2. Both the reticulum contractions and acid secretion from the abomasum waned over a period of min after decerebration. An examination of the free and total acid content of successive samples obtained from the abomasum revealed that the total amount and duration of acid secretion by the abomasum was smaller in sheep than it was Amounts of Free acid Total acid (ml. 0-1 N acid) Fig. 2. Decerebrate goat (28 kg). Progressive decline in contractions of the reticulum observed after decerebration in the periods 0-10, and min after recording commenced. Closed system recording of reticulum contractions. The amounts of the free and total acid detected in the abomasum in these periods are shown to the right of the tracings.

5 REFLEX RETICULUM CONTRACTIONS 5 in goats after decerebration; the force and frequency of the reticulum contractions declined more rapidly in sheep after decerebration than it did in goats. These observations led to an examination of the effects of the introduction of acid into the abomasum. The conditions under which this proved a stimulus to reticulum contractions are described below. Reflex stimulation of reticulum contractions Vagal stimulation. Contractions of the reticulum could be evoked, or when already present, increased in force or frequency, by stimulation of the central end of an abomasal or a reticulo-omasal branch of the ventral abdominal vagus nerve, or by stimulation of the central end of the ventral abdominal vagus nerve itself (Fig. 3). The reaction to stimulation of these nerve trunks varied. Frequently a short period of stimulation produced a number of contractions of the reticulum, although sometimes only one contraction followed. With either prolonged or repeated short periods of stimulation a series of contractions could be evoked. These contractions rarely occurred at intervals of less than 30 sec and sometimes continued after the stimulation ceased. 'l.' I I- Fig. 3 Decerebrate goat. Effect of stimulation of central end of the main abomasal branch of the ventral abdominal vagus nerve (signal), neon tube stimulator 40/sec. Records from above down: closed system record of reticulum contractions, signal, time marker 30 sec. The first contraction which occurred in response to stimulation of one of these afferent nerves was often monophasic; later contractions were diphasic. There was a variable delay between the application of the stimulus and the response. It was usually greatest when nerve stimulation was first undertaken in an experiment and least after a series of contractions had been evoked or when the effects of afferent nerve stimulation were studied during a preexisting, or previously stimulated, series of contractions. The latent period thus varied from 2 to 20 sec. The response to afferent nerve stimulation also

6 6 D. A. TITCHEN varied according to the frequency of the stimulation. Excitatory effects were obtained with frequencies of stimulation of 20-60/sec, but with higher frequencies and particularly those above 120/sec there was either an inhibition or no response to stimulation of, for example, the abomasal nerve. The responses of the reticulum to stimulation of these nerves were abolished by the administration of atropine ( mg/kg). Reflex contractions of the reticulum were not observed in preparations in which the dorsal abdominal vagus nerve had been cut and only the ventral abdominal vagus nerve left intact. Stimulation of the peripheral end of the ventral vagus nerve elicited slight contractions of the reticulum. Both the response to stimulation of the peripheral end of the ventral abdominal vagus and the greater contraction of the reticulum evoked by stimulation of the peripheral end of the dorsal abdominal vagus nerve were abolished by the administration of atropine ( mg/kg). Acid in the abomasum. The introduction of HCI or H2SO4 into the abomasum elicited contractions of the reticulum when the volume and strength of acid introduced were sufficient to lower the ph of the contents of the abomasum to or less. Thus the acids were not effective in the presence of large volumes of abomasal contents, nor when introduced when phosphate buffer was already present in the abomasum. Their effect was terminated by the introduction of saline or of a buffer solution into the abomasum. The responses of the reticulum to acid introduced into the abomasum usually occurred within sec, rarely up to 180 sec, when the first of a series of contractions of the reticulum was observed. This contraction was usually smaller than those which followed in the next 3-4 min, after which time the contractions declined in force or frequency or both, as the strength of the acid in the abomasum decreased. They usually ceased when the ph of the abomasal contents rose to about 1-1l2 (Fig. 4). Reticulum contractions were not stimulated by the introduction of acid into the abomasum after the abomasal branches of the ventral abdominal vagus nerve had been cut. In these experiments the abomasum had been isolated from the rest of the stomach and from the duodenum by ligatures tied around the pylorus and at the omaso-abomasal junction. Contractions of the reticulum sometimes occurred after the abomasal mucosa had been touched with the end of a glass rod or a polythene tube. The nature of the stimulus was not defined but the response was associated with the movement of the rod or tube rather than with pressure. In a number of experiments movement of the abomasal cannula evoked contractions of the reticulum. Stretching the reticulum. The first contraction of the reticulum observed in an experiment frequently occurred when the organ was stretched as it was lifted in order to introduce a balloon into it. The possibility that stretching the reticulum

7 REFLEX RETICULUM CONTRACTIONS 7 would stimulate its reflex contractions was therefore examined in further detail. A series of contractions of the reticulum followed an increase in the volume of fluid in the reticulum balloon. If contractions of the reticulum had already ceased they were increased in frequency when this procedure was adopted. The effect was usually apparent within sec of the addition of the fluid to the balloon. The contractions usually reached a maximum force and frequency within 5-6 min of the application of the stimulus and continued for min, Fig. 4. Decerebrate sheep. Reflex contractions of the reticulum stimulated by the introduction of HCI into the abomasum. Open system recording of reticulum contractions, time marker 30 sec. At the point marked a, 50 ml. 0-2N-HCI was introduced into the abomasum; contraction of the reticulum occurred 60 sec later. The ph of the samples of the contents of the abomasum withdrawn at the points marked were b, 0-87; c, 1-1; d, 1-4. and in some experiments in which the balloon was distended to a greater degree for up to 150 min, in both cases gradually waning in frequency and sometimes also in force over this period (Fig. 5). In a number of preparations there was an increase in the force of the contractions as they decreased in frequency. The stimulus appeared to be associated with the change in the volume in the balloon. Thus if the balloon were returned to its original volume during a series of contractions, these ceased (Fig. 6). Again, if the fluid was withdrawn from the balloon after the reflex response had ceased, the inflation min later with the same volume would again evoke a series of reflex contractions, if the balloon had in the meantime remained empty or had contained a considerably reduced volume of fluid.

8 8 D. A. TITCHEN Responses of the reticulum to stretch were obtained in preparations in which the ventral abdominal vagus nerves had been cut. The contractions of the reticulum observed were less forceful and sometimes also less frequent than those stimulated by the same degree of stretch before this nerve was cut. After the ventral abdominal vagus had been cut a greater degree of stretch was required to evoke reticulum contractions. They were not observed at all in those preparations in which the dorsal abdominal vagus nerve had been divided. The administration of atropine ( mg/kg) resulted in the cessation of the reticulum contractions stimulated by stretch. *~ ~~~~~~~~~~~~~~~~~~ I -~~~~~~~~~~~ _ ll _ Fig. 5. Decerebrate sheep (35 kg). Records as in Fig. 3. The reflex re-sponse of the reticulum to stretching. At the point marked by the arrow the volume of water in the reticulum balloon was increased from 200 to 400 ml. The first of a series of contractions of the reticulum occurred 35 sec later. The last contraction shown in this record occurred 39 min after the volume of the reticulum balloon was increased. Stimulation of the omasum. In three experiments the inflation of a balloon introduced into the omasal canal was followed by an increase in the force and frequency of reticulum contractions. In decerebrate preparations squeezing the omasum frequently stimulated swallowing movements which were sometimes accompanied by a mono- or di-phasic contraction of the reticulum. The stimulus was difficult to repeat many times in one experiment, as the omasum tended to retain its deformed shape and then further pressure applied to it was ineffective. The responses to this form of stimulation of the omasum were not obtained after section of the ventral abdominal vagus nerve. Stimulation of the oesophagus. When a tube was introduced through the mouth into the termin'al part of the thoracic oesophagus, up to five or six contractions of the reticulum occurredl. The response was associated with the movement of the tube in this terminal part of the oesophagus. Contractions

9 REFLEX RETICULUM CONTRACTIONS 9 were not elicited if the tube remained stationary or was moved in another region of the oesophagus; moderate distension of a balloon introduced into the oesophagus was ineffective. The contractions which occurred in response to this stimulus frequently showed three or more phases and were also usually more forceful than the diphasic contractions elicited with the other stimuli described. Fig. 6. Decerebrate goat. Open system record of reticulum contractions. Effect of a short period of distension of the reticulum balloon. The volume of water in the balloon was increased from 50 to 100 ml. at the first arrow and returned to 50 ml. 8 min later at the second arrow. Time marker 30 sec. Inhibition of reticulum contractions The insertion of cannulae into the abomasum, and other procedures which involved manipulation of the abdominal viscera, particularly of the abomasum or the pylorus, produced a profound inhibition of reticulum contractions which frequently persisted for 20 min or more. It was largely avoided by carrying out such procedures either before or immediately after decerebration whilst the ether anaesthesia persisted. Inhibition due to distension of the abomasum could also be shown by inflation of a balloon in it or by the introduction of ml. 0 9 % NaCl at 38 C, according to the size of the animal. The effect was apparent within sec and persisted throughout periods of distension of up to 20 min. In

10 10 D. A. TITCHEN some experiments the reticulum contractions were completely abolished by the distension. This was the case with greater degrees of distension; a lesser volume in the abomasum caused a reduction of the force or frequency orboth of reticulum contractions (Fig. 7). In two experiments in which the abomasum was divided across its longitudinal axis into two approximately equal parts, with as little interruption of its blood and nerve supply as possible, the introduction of acid into either half of the abomasum elicited contractions of the reticulum, which could be inhibited by distension of the other part. Fig. 7. Decerebrate sheep (17 kgl. Reticulum contractions stimulated by stretch (500 ml. water introduced into balloon at R), inhibited completely by distension of abomasum (signal) with 500 ml. saline at 380 C (A and B), and partially inhibited by distension of the abomasum with 250 ml. saline (C). Closed system of recording of reticulum contractions, time marker 60 sec. Section of the splanchnic nerves affected the responses to distension of the abomasum. In three goats and two sheep, in which the splanchnic nerves had been cut at operations 2 weeks to 3 months previously, distension of the abomasum produced a predominantly excitatory response and no longer caused a complete inhibition of the contractions of the reticulum. An increase in the frequency of the contractions of the reticulum was observed in all five preparations on distension of the abomasum; in two they were slightly reduced in force and in the rest increased in force (Fig. 8). Similar resullts were obtained in three preparations in which the splanchnic nerves were cut during the course of the experiment. The effect of stimulation of the central end of a splanchnic nerve was examined in three experiments. The start of a period of stimulation was marked by a pronounced contraction of the abdominal muscles and succeeded by a period of inhibition of the contractions of the reticulum (Fig. 9). This result

11 REFLEX RETICULUM CONTRACTIONS 11 of stimulation of the central end of a splanchnic nerve was obtained in two preparations in which the other splanchnic nerve had also been cut. In four decerebrate preparations, two of goats, two of sheep, the effect of distension of the abomasum was altered by section of the major abomasal branch of the ventral abdominal vagus nerve. After this nerve had been cut the inhibitory effects of abomasal distension were less marked, and the complete inhibition was replaced by a slight reduction in the force with sometimes a slight increase in the frequency of the reticulum contractions. Fig. 8. Decerebrate goat (21 kg); both splanchnic nerves had been cut at an operation two weeks before the experiment. Effect of distension of abomasum with 300 ml. saline at 380 C (signal). The stimulus to reticulum contractions before distension of the aboma8um was stretch (200 ml.). Closed system recording of reticulum contractions, time marker 30 see. The increase in frequency of reticulum contractions with abomasal distension was not observed in preparations in which the whole ventral abdominal vagus -nerve trunk had been cut in the first part of its course in the abdomen immediately beyond the oesophageal hiatus of the diaphragm. In experiments in which the effects of abomasal distension were examined the abomasum had been isolated between ligatures tied at the pylorus and at the omaso-abomasal junction. It proved important to ensure that these ligatures had been effective. In two cases the ligatures failed to prevent the escape of fluid from the abomasum. In these experiments, after the abomasum was emptied there was a period during which the reticulum contractions

12 12 D. A. TITCHEN occurred at a markedly increased frequency. All the fluid introduced to distend the abomasum was not recovered from it; in both experiments some escaped into the omasum and in one into the duodenum also. The ligature tied at the omaso-abomasal junction was found at the end of one of these experiments to have displaced the fold of abomasum which appears to serve usually as a flap valve to prevent the regurgitation of abomasal contents into the omasum. Fig. 9. Decerebrate sheep (17 kg). Response of reticulum to stretch (400 ml. into balloon at A) showing effect of stimulation of central end of left splanchnic nerve for 5 min, signal (induction coil, 11 cm). Elevation in reticulum record marked by arrow was due to a contraction of abdominal muscles. Records from above down: closed system record of reticulum contractions, carotid arterial blood pressure, signal, time marker 60 sec. Only slight changes in the blood pressure followed distension of the abomasum. These were rarely as great as 10 mm Hg and usually consisted of an initial slight fall, succeeded either by a continued slight depression, or by a return to the previous or a slightly increased level. Stimulation of the central end of a splanchnic nerve was followed by a sustained slight fall in the blood pressure with, initially, a slowing of the heart (Fig. 9). In addition to the immediate effects of a period of distension of the abomasum, changes in the reticulum contractions after its relief were observed. These after-effects were apparent for 3-5 min or more after the withdrawal of fluid from the abomasum. After one or two less forceful contractions of the reticulum, there was an increase in the force with a decrease in the frequency

13 REFLEX RETICULUM CONTRACTIONS 13 of the reticulum contractions (Fig. 7 B), an increase in both force and frequency of reticulum contractions (Fig. 7A), or in their frequency (Fig. 1OA) or in their force. The excitatory effects of abomasal distension in preparations in which the splanchnic nerves had been cut also continued after the abomasum was emptied (Fig. 8). In contrast there was a persistent reduction in both the force and frequency of reticulum contractions after their complete inhibition during stimulation of a central end of a splanchnic nerve (Fig. 9). The persistent after-effects of these stimuli were not accompanied by any similarly prolonged changes in blood pressure. Fig. 10. Decerebrate sheep (15 kg). Response of reticulum to stretch (250 ml.) interrupted by two periods of abomasal distension, each with 250 ml. saline at 380 C (signal) before (A) and after (B) the major abomasal branch of the ventral abdominal vagus nerve was cut at a. Closed system recording of reticulum contractions, time marker 30 sec. Pyloric inhibition. The most profound and persistent form of inhibition -encountered was that which arose from manipulation, or dissection about the region, of the pylorus. In a number of those experiments in which a ligature had been tied about the pylorus there was either no response to stimuli, such as acid introduced into the abomasum or stretching the reticulum, or the -number and force of the reflex contractions was very much smaller than was usually the case. The magnitude and duration of the responses were markedly increased after all the visible nerves to the region of the pylorus had been cut and the pylorus had been separated from its mesenteric attachments, or after the infiltration of 1% procaine beneath the serous surface of the pylorus. The

14 14 D. A. TITCHEN pyloric inhibition was reduced by section of both splanchnic nerves or of the ventral abdominal vagus nerve. It was encountered in two preparations in which both splanchnic nerves and the ventral abdominal vagus nerve had been cut. The differences in the responses of the reticulum to the introduction of acid into the abomasum before and after denervation of the pylorus are shown in Fig Fig. 11. Decerebrate sheep. Pyloric inhibition. A ligature had been tied about the pylorus. The two parts of the record show the response to the introduction of 50 ml. of 0*2 i-hcl into the abomasum before (A) and after (B) denervation of the pylorus. The ph of the first sample of the abomasal contents withdrawn after the introduction of acid into the abomasum (indicated by a on the record) was in the first instance 0-83 and in the second After denervation of the pylorus the duration and magnitude of the responses of the reticulum were increased. Open system of recording from the reticulum, time marker 30 sec. The effect of stimulation of the peripheral end of the splanchnic nerves or of the injection of adrenaline on the contractions of the reticulum. Contractions of the reticulum were completely or partially inhibited by either the intravenous injection of adrenaline or stimulation of the peripheral end of the splanchnio nerves. After the injection of 2-10,tg/kg of adrenaline the inhibition persisted for from 5 to 10 min. That produced by stimulation of the splanchnic nerve was reduced and shortened by removal of the adrenal gland of that side.

15 REFLEX RETICULUM CONTRACTIONS 15 The inhibition from both sources was usually preceded by a slow contraction of the reticulum which resembled those produced by similar stimulation in spinal preparations (Comline & Titchen, 1951b) and can be seen in Fig. 12. Such a slow contraction was also observed in some decerebrate preparations in which contractions of the reticulum had not occurred for some time. It occurred at about the same time as the development of the pressor response (Fig. 13). Fig. 12. Decerebrate goat (12 kg). Effect of adrenaline (2 pg/kg) injected intravenously (signal, Adr) on a series of reticulum contractions stimulated by stretch (250 ml.). Records from above downwards: closed system record of reticulum, femoral arterial blood pressure, time marker 10 sec. In addition to these inhibitory effects, the intravenous injection of adrenaline was, in some preparations, followed by a series of contractions of the reticulum. This was most frequently observed in preparations in which the reticulum had failed to respond to stimuli which were normally effective in the production of reflex contractions. For example in one such experiment there was no response of the reticulum to stretch of itself within 3 min and an intravenous injection of 2 pzg/kg of adrenaline was then made. During the pressor response a contraction of the reticulum occurred which was succeeded by a series of contractions. These re-sponses were accompanied by a slight secretion of acid by the abomasum in two experiments. In one of these before the injection of adrenaline no acid had been detected in saline introduced into, and later removedl from, the abomasum. In the saline left in the abomasum for 17 min after the adrenaline was injected the titration figure for total acidl was 1-5 ml. O*1 N acid, in the next 29 min 2'6 ml., andl in the next 30 min 1*4 ml. 0-1N acid. These figures correspond to the periods between the signal marks Adlr andl A, A andl B, B and C in Fig. 14. Such an excitatory

16 16 D. A. TITCHEN 16D.ATI HE effect of adrenaline was usually observed in preparations in which a profound inhibition was apparently present. It was never seen in preparations in which the vagus nerves had been cut or atropine administered. Fig. 13. Decerebrate goat (18 kg). Adrenaline contraction of the reticulum. Signal marks the injection of 5 jig/kg of adrenaline intravenously. Open system recording, femoral arterial blood pressure, time marker 10 sec. Fig. 14. Decerebrate goat (14 kg). The reticulum stretched (250 ml.) at arrow. Adrenaline (2 ug/kg) injected intravenously (signal, Adr) The points A, B, C (signals) mark the withdrawal of fluid from the abomasum for acid estimations (see text). Records from above downwards: closed system record of reticulum contractions, femoral arterial blood pressure, signal, time marker 30 sec.

17 REFLEX RETICULUM CONTRACTIONS 17 DISCUSSION The evidence obtained in the present experiments indicates that the characteristic diphasic contractions of the reticulum observed in decerebrate preparations are reflex contractions and that they result from the summed effects of afferent stimulation, both excitatory and inhibitory, from different parts of the stomach. This conclusion is based, in part, on the results of stimulation and section of branches of the vagus nerves. Stimulation of the ventral vagus nerve in an afferent sense elicited reflex reticulum contractions mediated by efferent cholinergic parasympathetic fibres distributed to the reticulum in the dorsal vagus nerve. Such experiments demonstrate the reflex stimulation of reticulum contractions, but do not necessarily establish the course of all the afferent and efferent nerve fibres which may be involved in the reflex responses. Afferent and efferent vagal fibres of the reticulum are distributed in both the dorsal and ventral abdominal vagus nerves. The efferent fibres appear to be confined to the vagus nerves. Reflex contractions could not be elicited after both the dorsal and ventral abdominal vagus nerves had been cut. No evidence was obtained that such contractions depended on the integrity of the adrenergic sympathetic innervation of the reticulum. These results are in agreement with those obtained in unanaesthetized animals in which reticulum contractions were absent after the vagus nerves had been cut (Mangold & Klein, 1927; Duncan, 1953), but still present after the splanchnic nerves had been cut (Duncan, 1953). They also indicate that the contractions stimulated were reflex responses and not local responses of the organ, of either a myogenic or neurogenic origin. The reflex centre or centres are probably situated in the medulla oblongata; they are less likely to be in the pons which suffers a variable degree of damage during decerebration by mechanical transection. The contractions described were absent in the spinal preparations used in a study of the efferent innervation of the reticulum (Comline & Titchen, 1951b). They were present in decerebrate preparations from which the cerebellum has been removed (personal observations) and in decerebrate preparations in which the cervical spinal cord has been transected (Iggo, 1951; personal observations). This conclusion on the site of the reflex centre is supported by the evidence obtained by Bell & Lawn (1955) who recorded contractions of the oesophagus, reticulum and rumen following stimulation through electrodes inserted into the medulla oblongata. The stimuli which excite reflex reticulum contractions via vagal afferent fibres include stretching the reticulum, touch applied to the abomasum or to the oesophagus apparently close to the site of the sphincter described by Dougherty & Meredith (1955) and a reduction in the ph of the abomasum to between 09 and 1P0. The ph which is effective in the stimulation of reticulum 2 PHYSIO. CXLI

18 18 D. A. TITCHEN contractions is lower than that of the abomasal contents of the conscious, fed animal; Masson & Phillipson (1952) found this to be 2f It is, however, close to that of 09 advanced by Hollander (1949) and Conway (1953) for the ph of the secretion of the oxyntic cells in a number of species and also to the figure of reported by Masson & Phillipson (1952) for the ph of the secretion obtained from a Hollander pouch prepared in the abomasum of a sheep. The discrepancy between the ph of the abomasal contents and that effective as a stimulus in these experiments might be explained if the receptors sensitive to this acid stimulation were close to the site of its secretion by the oxyntic cells. If this were the case then the acid secretion and not the ph of the abomasal contents would appear likely to be the effective stimulus in the conscious animal. Direct and unequivocal evidence for the existence of acid or other receptors in the stomach has not been provided. The present experiments and those in which the activity in vagal afferent fibres was recorded during changes within the stomach (Paintal, 1953, 1954; Iggo, 1954, 1956) provide indirect but strong evidence for their existence. Striking features of the reflex responses of the reticulum are their enduring nature with certain stimuli, their long latencies and the prolonged course of an inhibition once established. No satisfactory explanation can be offered for these temporal sequences, but the prolonged effects of stretching the reticulum, for example, may be due partly to the properties of the receptors involved. Paintal (1953) showed that the stretch receptors of the cat's stomach adapt slowly with constant maintained stimulation. In contrast the responses of the reticulum to apparently tactile stimulation were difficult to maintain or to repeat with continued or interrupted stimulation. These observations may be an indication that visceral tactile and stretch receptors resemble in their reactions the rapidly adapting tactile receptors of the skin (Adrian, 1928) and the more slowly adapting stretch receptors in skeletal muscle (Matthews, 1931) respectively. The inhibition of reticulum contractions by distension of the abomasum resembles that reported by Phillipson (1939) in the conscious sheep. The present experiments show that the majority of the afferent nerve fibres concerned in this inhibition are contained in the splanchnic nerves, and that in some experiments their removal did not simply lessen or remove the inhibition but revealed an excitation on distension of the abomasum. Thus there appear to be two effects of abomasal distension, an inhibition mediated principally by splanchnic and also by some vagal afferent fibres and an excitation mediated by vagal afferents. Further analysis of this effect is complicated by the difficulty of separating the effects of afferent and efferent fibres in the splanchnic nerves. The possibility cannot be dismissed that abomasal distension inhibited reticulum contractions by a reflex excitation of the sympathetic innervation of the reticulum brought about by splanchnic afferents. The available evidence

19 REFLEX RETICULUM CONTRACTIONS 19 does not suggest that this is the most probable explanation. Alternatively, the inhibition might be ascribed to effects on the reflex centre also mediated by afferent fibres in the splanchnic nerves. Some evidence in favour of this might have been obtained from the experiments in which stimulation of the central end of one splanchnic nerve inhibited reticulum contractions in preparations in which both had been cut. However, afferent fibres other than those from the abomasum would be stimulated under these circumstances; these observations did not provide a precise definition of the nature of the inhibition. The profound inhibition which follows manipulation of the pylorus resembles in many respects that arising from distension of the abomasum and is probably similar in its nature. Section of the splanchnic nerves did not completely remove the inhibition, and the course of all the afferent nerve fibres responsible was not determined. The pyloric inhibition may effectively limit the demonstration of reflex contractions of the reticulum in a decerebrate preparation. In the experiments reported here both excitatory and inhibitory effects of adrenaline on the reticulum were found. In many experiments its administration reduced or abolished reflex contractions; this was to be expected in view of previous reports of its action in the conscious animal (Magee, 1932). Two forms of excitatory response to adrenaline were also observed. One was the comparatively slow single contraction which was described in spinal preparations (Comline & Titchen, 1951b) and appears to be a direct response to adrenaline. The other effect was that of a single intravenous injection of adrenaline being followed by a series of diphasic contractions, which were dependent on the vagal innervation. This last effect might be attributed to circulatory changes either in the central nervous system or in the reticulum. Facilitation by adrenaline of the receptors which must be presumed to be present in the stomach must also be considered as a probable explanation of the effect. It is supported by the observations made by Paintal (1954) that adrenaline may facilitate the stretch receptors of the cat's stomach and by those of Kay (1955) on the parotid secretion of sheep in which the anatomical separation of the afferent and efferent nerves has allowed a more precise definition of such a facilitation of visceral receptors. The reflex effects on the reticulum were obtained in preparations in which previous to the delivery of the stimulus the reticulum had been inactive. The contractions of the reticulum which were observed immediately after decerebration without the application of any definite stimulus resembled the reflex contractions in all respects, and it is possible that their decline in force and frequency and their cessation may be attributed simply to a simultaneous decline in acid secretion by the abomasum which occurred after decerebration. An extension of this argument to ascribe reticulum contractions in the conscious animal purely to reflex excitation requires, however, further experimental evidence. 2-2

20 20 D. A. TITCHEN SUMMARY 1. Reflex reticulum contractions have been evoked in decerebrate preparations of sheep, goats and calves by electrical stimulation of vagal afferent nerve fibres and by alterations in conditions in the stomach. 2. The contractions ceased after the administration of atropine and were absent in preparations in which the dorsal abdominal vagus nerve had been cut. 3. The contractions were stimulated by: Stretching the reticulum, or distension of a balloon in the omasal canal; A reduction in the ph in the abomasum to 0 9-1'0; Touching the lower part of the thoracic oesophagus or the abomasal mucosal surface; or Stretching the abomasum after section of the splanchnic nerves. 4. The reflex contractions were inhibited by distension of the abomasum when the splanchnic nerves remained intact, by manipulation of the pylorus and by stimulation of the central end of a splanchnic nerve. 5. Both excitatory and inhibitory effects of adrenaline on the reticulum contractions were observed. It is my pleasure to acknowledge the assistance, advice and encouragement given by Dr R. S. Comline through whom financial assistance from the Agricultural Research Council was received, and also the technical assistance given by Messrs G. E. Baker, H. V. Sandfield and W. Whybrow, and the advice on the preparation of the manuscript by Drs R. S. Comline and C. L. G. Pratt. REFERENCES ADRIAN, E. D. (1928). The Basis of Sensation, p. 77. London: Christophers. BELL, F. R. & LAWN, A. M. (1955). Localization of regions in the medulla oblongata of sheep associated with rumination. J. Physiol. 128, COMLINE, R. S. & TITCHEN, D. A. (1951 a). Reflex contraction of the oesophageal groove in young ruminants. J. Physiol. 115, COMLINE, R. S. & TITCHEN, D. A. (1951 b). Contractions of the reticulum of the young goat. J. Physiol. 115, 24P. CONWAY, E. J. (1953). The Biochemistry of Gastric Acid Secretion, p. 42. Oxford: Blackwell. DOUGHERTY, R. W. & MEREDITH, C. D. (1955). Cinefluorographic studies of the ruminant stomach and of eructation. Amer. J. vet. Res. 16, DUNCAN, D. L. (1953). The effects of vagotomy and splanchnotomy on gastric motility in the sheep. J. Physiol. 119, DUSSARDIER, M. (1955). Controle nerveux du rythme gastrique des ruminants. J. Physiol. Path. gen. 47, DUSSARDIER, M. & ALBE-FESSARD, D. (1954). Quelques proprietes du centre vagal controlant l'activite reflexe de l'estomac des ruminants. J. Physiol. Path. gqn. 46, HOLLANDER, F. (1949). The composition and mechanism of formation of gastric acid secretion. Science, 110, IGGO, A. (1951). Spontaneous and reflexly elicited contractions of reticulum and rumen in decerebrate sheep. J. Physiol. 115, 74P. IGGO, A. (1954). Receptors in the stomach and the bladder. J. Physiol. 126, 29P. IGGo, A. (1956). Central nervous control of gastric movements in sheep and goats. J. Physiol. 131,

21 REFLEX RETICULUM CONTRACTIONS 21 KAY, R. N. B. (1955). The action of adrenaline on the flow of parotid saliva in sheep. J.Physiol. 130, 15P. KIMG, E. J. (1951). Micro-analysis in Medical Biochemistry, 2nd ed. p London: Churchill. MAGEE, H. E. (1932). Observations on digestion in the ruminant. J. exp. Biol. 9, MANGOLD, E. & KrEIN, W. (1927). Bewegungen und Innervation des Wiederkauermagens. Leipzig: Georg Thieme. MASSON, M. J. & PYmITIPSON, A. T. (1952). The composition of the digesta leaving the abomasum of sheep. J. Physiol. 128, MATTHEWS, B. H. C. (1931). The response of a single end organ. J. Physiol. 71, PAITAL, A. S. (1953). Impulses in vagal afferent fibres from stretch receptors in the stomach and their role in the peripheral mechanism of hunger. Nature, Lond., 172, PAITAL, A. S. (1954). The response of gastric stretch receptors and certain other abdominal and thoracic vagal receptors to some drugs. J. Physiol. 126, PIMLLIPSON, A. T. (1939). The movements of the pouches of the stomach of the sheep. Quart. J. exp. Physiol. 29, TITCHEN, D. A. (1953). Reflex contractions of the reticulum. J. Physiol. 122, 32P. TrrCHEN, D. A. (1954). Inhibition of reflex contractions of the reticulum. J. Physiol. 125, 25P