GASTRIC MUCOSAL CHEMORECEPTORS WITH VAGAL AFFERENT FIBRES IN THE CAT. By A. IGGO. From the Department of Physiology, University of Edinburgh.

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1 GASTRIC MUCOSAL CHEMORECEPTORS WITH VAGAL AFFERENT FIBRES IN THE CAT. By A. IGGO. From the Department of Physiology, University of Edinburgh. (Received for publication 14th May 1957) Electrical activity was recorded in 19 single fibres, with very slow conduction velocities, dissected from the cervical vagi of 10 cats. The nerve endings of these fibres were in the gastric mucosa and were destroyed when the mucosa was scraped away. A brief burst of impulses was recorded when the mucosa was stroked firmly with a smooth rod but there was no response when the stomach was made to contract, when it was compressed firmly between finger and thumb, or when it was lightly distended. Over-distension excited 3 of the mucosal nerve endings. The mucosal nerve endings were specifically sensitive to either acid or alkaline solutions on the mucosa. The discharge of impulses in the afferent fibre persisted for as long as the stronger solutions used (0-1 N-NaOH or 0-1 N-HCR) remained on the mucosa. The threshold of the acid receptors was ph 3 or lower and of the alkali receptors was ph 8 or above. The endings were not affected by hypotonic or hypertonic solutions or by any other chemicals that were tried. A clear distinction is made between the gastric mucosal nerve endings described in the paper and the tension and "stretch" receptors which are found in the deeper layers of the gastric wall. It is concluded that the mucosal nerve endings probably function as slowly-adapting chemoreceptors. A low-threshold gastric "stretch" receptor is described. Both mechanical and chemical stimulation of the gastric mucosa elicit a variety of sensations and reflex responses. Carlson and Braafladt [1915] and Wolf and Wolff [1947] found that there was a sensation of pressure or an undifferentiated awareness when the normal gastric mucosa of their subjects was rubbed or squeezed; touch, however, was not perceived. Gastric movements in sheep and goats may be enhanced by acidification of the gastric contents [Titchen, 1953] or by mechanical stimulation of the gastric mucosa [see Habel, 1956, for review]. There does not seem to have been any previous electrophysiological account of afferent fibres with endings in the mucosa. The gastric afferent fibres already described came from tension and "stretch" receptors [Paintal, 1954 a; Iggo, 1955, 1957 a]. The endings of these fibres were in the outer layers of the gastric wall. The present study is part of a wider investigation of small afferent fibres. It was begun when a gastric afferent fibre was found which did not fit into the category described previously. The ending of the fibre was destroyed when the gastric mucosa was removed. This paper describes the response of similar fibres to mechanical and chemical stimulation. A preliminary account of this work has already appeared [Iggo, 1957 b]. 398

2 Gastric Mucosal Chemoreceptors 399 METHODS The manner in which the cats were prepared for the dissection of single fibres and the electrical recording methods have already been described [Iggo, 1955, 1957 a]. Endings in the mucosa were found by rubbing a smooth probe, introduced into the stomach through a small hole, over the surface of the mucosa and listening to the amplified action potentials picked up from the strand of the cervical vagus. Once a mucosal unit was found, the stomach was cut open so as to expose the appropriate part of the mucosa. The mucosa exposed in this way tended to become colder than the rest of the cat. To prevent this the fluids used to irrigate it were kept at 39-40' C. and warm pads of cotton wool were put on to the mucosa whenever convenient. Histological Examination of the Mucosa.-This was done in three units to see how much of the mucosa had been removed by scraping. The appropriate part of the gastric wall was cut out and fixed in formalin (10 per cent v/v). Sections were cut and stained with heemotoxylin-eosin. Chemical.-The following solutions were used to test the ph sensitivity of the mucosal nerve-endings: 0-1 N-NaOH, diluted as required; 005 M-borax, ph 9-3; 0-2 N-HCI diluted as required; mixtures of 0-2 M-Na2HPO2 and 0-1 M-citric acid to give solutions of different ph, according to Mcllvaine [1921]. The ph of the solutions used was measured with a glass electrode. The ph of the solutions in actual contact with the mucosa was not measured. RESULTS The 19 vagal fibres described in this paper were isolated as single units from the right cervical vagi of 10 cats. The conduction velocities of the fibres ranged from 1 to 5 m./sec. [Iggo, 1957 c]. Location of the Receptors.-The receptors were found in all parts of the stomach. Because of its accessibility the pyloric antral mucosa was explored most thoroughly and the majority of the receptors described were in that region. There were no systematic differences in the properties of receptors in different parts of the stomach. If the afferent unit was still alive after the tests, to be described, were made and the conduction velocity of the centripetal fibre had been measured, an attempt was then made to find the layer of the gastric wall in which the fibre ended. This was done by gradually scraping away the mucosa with a scalpel blade. When the most sensitive spot of the gastric mucosa was scraped there was a brief burst of impulses in the single fibre (fig. la) Vigorous scraping gradually removed the mucosa and at some stage in this denudation the characteristic response to scraping became shorter until only a single impulse was set-up in the fibre (fig. lb). Further scraping abolished even this response but electrical stimulation still excited the fibre. There was no response to a previously effective chemical stimulus when a mechanical stimulus had become ineffective so that it is clear that the chemicals were acting on the nerve ending and not on the axon. Removal of one-third or less of the mucosa was sufficient to destroy the endings of 4 fibres; with 3 units it was necessary to expose the muscularis mucosa before the response

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4 Gastric Mucosal Chemoreceptors 401 was lost. With 1 unit the response to mechanical stimulation was absent after the mucosa had been removed by splitting the gastric wall through the submucosa. It was noticed several times that removal of the mucosa at the centre of the most sensitive area did not prevent a response to mechanical stimulation of the adjacent intact mucosa. Histological examination revealed that the muscularis mucosa was exposed at the centre, the spot which no longer responded to stimulation, and that the surrounding mucosa was undamaged. These results suggest that a single axon innervates an area perhaps as large as 5 cm.2 and does this by sending collateral fibres into the superficial mucosa from a main trunk which ramifies in the deeper layers of the mucosa. TABLE I.-THE RESPONSE OF GASTRIC MucosAL NERVE ENDINGS TO MECHANICAL AND CHEMICAL STIMULATION Fibre No. 15 m 27 m m m 40 m m 53 m 55 m 56 m 58 m 59 m 60 m 61 m 62 Stroke Stretch + +E + _ + _ + _ + _ + + +_ +_ E + + * +* * * t * t * t * t. * * - Contraction Compression *. * - Acid Alkali Ringer. Locke _ *- _ * +_ +_ +. +_ +_ + - _ + = response; - = no response; E = over-distension or firm st: m =mucosal origin confirmed by scraping off the mucosa;.. Threshold 0.01 N-NaOH ph 8-0 ph 8-0 ph 2-2 ph 9-0 ph 9*3 ph N.NaOH ph 3-0 ph 1F5 retch of the mucosa; = not tested. The Response to Mechanical Stimulation.-Light touch or digital compression failed to excite any of the mucosal nerve endings. A burst of impulses was recorded in each of the fibres when a small area of the mucosa was stroked with a smooth probe or cotton wool (fig. la), but not when more distant parts of the mucosa were stroked. A sustained irregular stream of impulses was recorded in 3 of the units when the mucosa was very firmly stretched and when the stomach was over-distended with saline or with an air-filled balloon. The peak frequency of discharge was low; 10/sec. in Fibre 52, fig. ld. Gastric contractions caused by faradic stimulation of the serosa or the mucosa did not excite the mucosal nerve endings (Table I).

5 402 Iggo Gastric tension receptors, which adapt slowly to light gastric distension, gastric contractions and digital compression of the gastric wall [Iggo, 1955, 1957 a], were also excited when the mucosa was stroked with a smooth rod (fig. lc). The response was not so localized as with the mucosal nerve endings and the discharge was easily prolonged by steady, light pressure on the mucosa. These differences in the response to mechanical stimulation made it possible to distinguish the centripetal fibres of the mucosal endings from those of tension receptors. The Action of Chemicals.-When it became clear that there were vagal afferent units with endings in the gastric mucosa an attempt was made to see if they were sensitive to chemicals in solution. The following fluids, when tried, all failed to excite the afferent units; distilled water, Ringer-Locke solution, 0*15 M-NaCl solution, 0*6M-NaCl solution, peptone solution, oleic acid, ethanol (30-70 per cent), glucose or sucrose solution, bile from the gall bladder, culinary mustard suspension (30 per cent w/v). The 4 units tested were insensitive to phenyl diguanide (40-80 pg/kg. intravenously). The mucosal endings were, however, excited by solutions of either high or low H-ion concentration and could be divided into 2 classes on this basis. Acid Sensitive Receptors. (a) Exposed mucosa. Five units out of 14 units tested were excited by acid solutions. The threshold was about ph 3 for the most excitable units (Table I), but at this ph the discharge was erratic and lasted only a short time. The discharge was persistent and the peak frequency was higher when more acidic test solutions were used (fig. 2). In 2 instances, repeated application of strong solutions (0.1 N or 0-2 N-HCR) damaged the mucosa after several hours and destroyed the receptors. No precise measurement of the frequency of discharge at different acidities was made. (b) Intact stomach. The receptors described in (a) were found by mechanical stimulation of the mucosa. This method of detection may have altered the reaction of the endings to chemical stimuli and so 4 experiments were done to see if acid-sensitive units could be found by perfusing the intact stomach with acidic solutions. Only 1 unit (Fibre 62) was isolated inthis way. This low yield of only 1 unit in 4 experiments is attributed to technical difficulties rather than to the sparsity of receptors. The unit was not excited bv Ringer-Locke solution or by 0.03 N-RCl(pH 1-5). It was excited by 0-1 N-HC1 and the discharge was more prolonged, but the peak frequency was only a little higher with 0-2 N-HCl (fig. 3). This unit, therefore, had a higher threshold than the units described in (a), but whether this difference was due to the isolation procedure is not known. Alkali Sensitive Receptors.-Exposed mucosa. Nine of the 13 units tested with both acid and alkaline solutions gave a sustained discharge of impulses when 0-1 N-NaOH was put on the exposed mucosa (fig. 4b and Table I). The peak frequency of the discharge was impulses/sec. When a few drops of solution were used the discharge was brief, but when several ml. were used it persisted for as long as the solution remained on the mucosa. Repeated applications of 0*1 N-NaOH were made, on occasion

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7 404 Iggo for 3 hr., and the receptors continued to respond in a consistent way although the peak frequency of the discharge varied. This treatment did not impair the response of the receptors to less alkaline solutions or to mechanical stimulation. Attempts to establish the threshold of the receptors to alkaline solutions were unsatisfactory because of random fluctuations in sensitivity. The B C D ili IL~JLLJiUI11I1IAUAAliiA Aih ILIi FIas. 3a, b, c, d and e.-the response of an acid-sensitive receptor in the intact stomach to solutions put into the stomach through the cardiac sphincter, Fibre 62. (a) 0 03 N-HCl, ph 1.5; (b) 041 N-HCl; (c) the same unit 30 sec. later with the acid still in the stomach; (d) 0-2 N-HCI; and (e) 30 sec. later as in (c). Time marks, 1 sec. results are given in Table I and, as is shown, the most excitable units were excited at ph 8. At this level the discharge was unpredictable but, when present, was of low frequency and short duration. The frequencv and persistence of the discharge was greater with more alkaline solutions. A layer of mucous which prevented alkalis from exciting 1 receptor did not block mechanical stimulation. Specificity of Reaction.-The 2 classes of receptor described were quite distinct and, as is illustrated in fig. 4c, the discharge in an alkali-sensitive

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9 406 Iggo unit could be abolished pronmptly when acid was put on the mucosa; acid alone did not excite the receptor. The converse held for the acid-sensitive receptors. These results emphasize the need to isolate single units in an analysis of this kind. Gastric "Stretch" Receptor.-The existence of volume receptors in the gastric wall, described by Paintal [1954 a], has been questioned [Iggo, 1955, 1957 a]. Some gastric mucosal endings gave a slowly adapting discharge of impulses when the stomach was over-distended (Table I). Apart from these endings which could be mistaken for "stretch" receptors, 1 solitary example of a low-threshold "stretch receptor was found. The unit was excited by distension, stretch and digital compression of the stomach but was not excited by gastric contractions nor by acid or alkaline solutions. It was, therefore, quite distinct from the gastric tension receptors which were all excited by gastric contractions. Careful dissection showed that the receptor was on the greater curvature of the stomach, probably in the serosa or omentum. The unit was not excited by massive intravenous doses of phenyl diguanide (150 mg./kg.). DISCUSSION The present work shows that vagal afferent fibres with endings in the gastric mucosa can be very clearly distinguished fromi similar afferent fibres with endings in the outer layers of the gastric wall. The distinction is based on the response of the receptors to both mechanical and chemical stimuli. The mucosal receptors were not excited by moderate gastric distension (< 20 mm. Hg), gastric contractions or digital compression of the gastric wall, all of which evoked a persistent slowly adapting discharge of impulses from tension receptors [Iggo, 1955, 1957 a]. Some -of the mucosal receptors yielded a slowly adapting train of impulses to excessive gastric distension or to a powerful stretch of the mucosa. In the absence of further information about the location of the endings or their reaction to chemicals these results could lead to the mistaken conclusion that the gastric wall contains large numbers of " stretch " receptors, i.e., receptors which could function unambiguously as volume-signalling devices. Low-threshold gastric stretch receptors do exist but they are much less numerous than the tension receptors and mucosal receptors; the solitary example found in the current series of 51 single units was in the serosa or omuentum on the greater curvature of the stomach. Receptors of each of the above ciasses were excited when the gastric mucosa was stroked firmly with a smooth glass rod, but whereas the mucosal units responded with a brief burst of 1-10 impulses the tension and stretch receptors responded with a burst of impulses which could easily be prolonged by keeping up a light pressure on the mucosa. This reaction of receptors in the deeper layers of the gastric wall to stimulation of the mucosa shows that caution should be exercised in attributing reflex responses elicited bymucosal

10 Gastric Mucosal Chemoreceptors 407 stimulation to the exclusive excitation of mucosal nerve endings. Receptors in the deeper layers of the stomach appear to be at least as sensitive as mucosal endings to certain forms of mechanical stimulation. Another striking difference between the mucosal receptors and those in the deeper layers of the gastric wall was in the response of receptors to chemicals on the mucosa. The mucosal receptors displayed a characteristic sensitivity to the ph of solutions in contact with the mucosa whereas the tension receptors were unaffected unless gastric movements were aroused. This sensitivity of the mucosal receptors to the ph of fluids on the mucosa is, perhaps, the most interesting finding in the present investigation. The response is not an injury discharge. Repeated stimulation for several hours with 041 N-NaOH or 0-1 N-HC1 gave consistent results and did not interfere with the response of the endings to weaker solutions. The reaction was a property of the nerve endings; when the mucosal surface was scraped off, a previously effective stimulus no longer caused a discharge of impulses even though electrical stimulation was effective. It is clear that the solutions were not exciting the axons directly. The response to acids or alkalis was specific. This is well shown in fig. 4 where the discharge in an alkali-sensitive unit evoked by 0 1 N-NaOH disappeared when 0-1 N-HCl was added. Activity in an acid-sensitive unit could be abolished by alkali. If the discharge was caused by injury then both the acid and the alkali might be expected to arouse a similar response from all the vagal endings in the mucosa. Acid receptors are well known in the sense of taste but there is no agreement on the specificity of response. The single unit studies of Pfaffmcn [1941, 1955] show that some acid receptors can be excited by non-acidic solutions whereas Andersson and Zotterman [1950] found fibres in the frog which were excited by acid but not by water and hypertonic salines on the tongue. The gastric acid receptors isolated as single unit preparations in the present work were specifically sensitive to acid; they were not excited by hypertonic or hypotonic solutions or by Ringer-Locke solution. This specificity suggests that they are concerned with signalling the ph of the gastric contents. Gastric motility is enhanced by low gastric acidity in sheep [Titchen, 1953]. The gastric acid receptors described in this paper may initiate these reflex responses. There is convincing evidence that acid in the duodenum reflexly depresses gastric motility and gastric acid secretion via vagal paths [Thomas, Crider and Mogan, 1934; Pincus, Friedman, Thomas and Rehfuss, 1944; Code and Watkinson, 1955; Sircus, personal communication]. The threshold for all these reflex responses was ph 2-5 or less; this is very similar to the threshold for the gastric mucosal acid receptors. If there are similar acid receptors in the duodenal mucosa they may be the afferent limb of the reflexes from the duodenuin. It is concluded that the acid-sensitive mucosal endings are slowly-adapting chemoreceptors and, as suggested in the preliminary report [Iggo, 1957 b], that they function as ph receptors. The function of the alkali-sensitive mucosal receptors is unknown and,

11 408 Iggo indeed, they are unlikely ever to be excited by alkali in the stomach since the gastric ph rarely rises above 7 [James, 1957]. The histological evidence for afferent nerve endings in the mucosa of the glandular stomach has recently been strengthened by Schofield [personal communication] who found vagal fibres which passed through the enteric plexuses and ramified in the mucosa to send branches to end close to the gastric mucosal surface in the rat. The present results on the location of the mucosal receptors are complementary for they show that the receptors can be destroyed by scraping off the outer layers of the mucosa. Moreover, with some individual afferent units, if the centre of the sensitive area was removed down to the muscularis mucosa, the sensitivity of the adjacent mucosa did not change. Through the kindness of Dr. A. S. Paintal, I have read the manuscript of a paper in which he describes intestinal receptors which he believes to lie in the mucosa. Paintal was unable to alter the activity of one of these endings when he put "dilute acid or a strong solution of soda-lime" on the mucosa. This fact, together with other differences, makes it unlikely that these intestinal receptors are the same as the mucosal chemoreceptors described in this paper. Several types of receptor in the gastric and intestinal wall have now been identified by recording from single afferent fibres dissected from the cervical vagus. One significant feature of the various investigations is that each class of receptor was found by the use of a particular stimulus so that there was an arbitrary, though sometimes intentional, selection of the units examined. The stimuli used to make the initial isolation of the afferent units were: (a) phenyl diguanide and distension [Paintal, 1954 a, b]; (b) mild distension [Iggo, 1955, 1957 a]; (c) rubbing the mucosa combined with insensitivity to distension [this paper]; (d) irrigation of the stomach with solutions of different ph [this paper]; and (e) phenyl diguanide coupled with insensitivity to distension [Paintal, 1957]. The receptors isolated by these means have had some properties in common but there have also been clear-cut differences so that 4 types of behaviour can be recognized, although definite histological structures cannot be assigned to them. They are: (1) tension receptors in series with the contractile elements in the outer muscular layers of the stomach and intestines [Iggo, 1955, 1957 a]; (2) distension-sensitive receptors in the outer layers of the gastric wall which are not excited by gastric contractions or by intravenous phenyl diguanide [this paper]; (3) chemoreceptors in the gastric mucosa [this paper]; and (4) distension-insensitive intestinal receptors [Paintal, 1957] believed to be in the mucosa.

12 Gastric Mucosal Chemoreceptors 409 REFERENCES ANDERSSON, B. and ZOTTERMAN, Y. (1950). "The water taste in the frog", Acta physiol. scand. 20, CARLSON, A. J. and BRAAFLADT, L. H. (1915). "Contributions to the physiology of the stomach. XVIII. On the sensibility of the gastric mucosa", Amer. J. Physiol. 36, CODE, C. F. and WATKINSON, G. (1955). "Importance of vagal innervation in the regulatory effect of acid in the duodenum on gastric acid secretion", J. Physiol. 130, HABEL, R. E. (1956). "A study of the innervation of the ruminant stomach", Cornell Vet. 46, IGGO, A. (1955). "Tension receptors in the stomach and the urinary bladder", J. Physiol. 128, IGGO, A. (1957 a). "Gastro-intestinal tension receptors with unmyelinated afferent fibres in the vagus of the cat", Quart. J. exp. Physiol. 42, IGGo, A. (1957 b). "Gastric ph receptors with slowly conducting afferent fibres", J. Physiol. 137, P. IGo. A, (1957 c). "Conduction velocity in vagal afferent fibres", J. Physiol. [In the press.] JAMES, A. H. (1957). The Physiology of Gastric Digestion. London: Edward Arnold. McILvAINE, T. C. (1921). "A buffer solution for colorimetric comparison", J. biol. Chem. 49, PAINTAL, A. S. (1954 a). "A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst", J. Physiol. 126, PAINTAL, A. S. (1954 b). "The response of gastric stretch receptors and certain other abdominal and thoracic receptors to some drugs", J. Physiol. 126, PAINTAL, A. S. (1957). "Responses from mucosal mechanoreceptors in the small intestine of the cat", J. Physiol. [In the press.] PFAFFMAN, C. (1941). "Gustatory afferent impulses", J. cell. comp. Physiol. 17, PFAFFMAN, C. (1955). "Gustatory nerve impulses in rat, cat and rabbit", J. Neurophysiol. 28, PINcus, I. J., FRIEDMAN, N. H. F., THoOMAS, J. E. and REHFUSS, M. E. (1944). "A quantitative study of the inhibitory effect of acid in the intestines on gastric secretion", Amer. J. dig. Dis. 11, THOMAS, J. E., CRIDER, J. 0. and MOGAN, C. J. (1934). "A study of reflexes involving the pyloric sphincter and antrum and their role in gastric evacuation", Amer. J. Physiol. 108, TITCHEN, D. A. (1953). "Reflex contractions of the reticulum", J. Physiol. 122, 32 P. WOLF, S. and WOLFF, H. G. (1947). Human Gastric Function. London: Oxford University Press. VOL. XLII, NO