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Afferent impulses in the gastric and oesophageal branch of the vagal nerve of toad
Phrsioh~.ey am/B~hm'ior. Vol. 2, pp. I-4. Pergamon Press Ltd., 1967 Printed in Great Britain
Afferent Impulses in the Gastric and Oesophageal Branch. of the Vagal Nerve of Toad AKIRA
NIIJIMA
Department of Physiology, Niigata University School o f Medicine, Niigata, Japan (Recgived 20 M a y 1966) NnzIMA, A. Aff'erent hnptdses ht the gastric and oesophageal branch of the vagal nerve of the toad. PHYSIOL.BEHAV. 2 (1) I 4, 1967.--The function of vagal gastric and oesophageal mechano-receptors was studied by means of recording the :tffcrenl discharges from a single or from several afferent nerve fibers dissected from a gastric and oesophageal branch of Ihe wtgal nerve. Three different types of mechano-receptors were found in the gastric wall. Type A: Rapidly-adapting type. situated in the mucous membrane or in the submueosa of the oesophageal border of the cardia, and which do not respond to peristaltic contraction of the stomach wall. Type B: Slowly-adapting type, situated in the muscular layer and responsive to distension and contraction of the stomach. Type C: Slowly-adaptingtype, connected to the non-myelinated liber, situated in the muscular layer, and which respond to distension and contraction of the gastric wall. In the oesophagus the same three types were found. However, the number of rapidly-adapting type receptors is far larger than the other type of receptors. It has been concluded that the oesophageal receptors mainly send information about the passing of food and the vagal gastric receptors send information concerning the degree of fullness or emptiness and motility of the stomach. Vagal afferents
Mechano-receptors
Gastric and oesophageal meehano-receptors
IT ;~AS~EEN SAIDthat the afferent information from peripheral receptors, particularly in the gastro-intestinal organs, plays an important role in the control of food and water intake. From this point of view many workers [6, 7, 10, 11, 13,] studied gastro-intestinai receptors. The authors o f these papers have pointed out that distension of the gastrointestinal canal produces satiation. This indicates that there is a relationship between the satiation mechanism in the central nervous system and the function of the mechanoreceptors in the wall of the gastro-intestinal canal. Afferent impulses from vagal fibers of the stomach have been studied accurately by Paintal [9], Iggo [4] and Niijima [8]. They have reported that there are slowly-adapting and rapidly-adapting types of mechano-receptors in the stomach wall. On the other hand, Andrew [2, 3] recorded the afferent impulse discharges from the superior laryngeal nerve of rats. He reported that the most easily recognized type of afferent was connected to slowly-adapting tension receptors in the wall of the oesophagus. In these studies, however, there was little information about the relationship between characteristics of oesophageal or gastric mechano-receptors and their role in the regulation o f food intake. This report will deal mainly with the afferent impulse discharges from the oesophageal and gastric vagal nerve which connect to the mechano-receptors in the wall of the oesophagus or stomach, differences between them, and their possible role in the regulation of food intake.
Toad
branches from the vagal nerve trunk. They are called R. oesophagus and R. ga#trices N. vagi. The toads were pithed. The stomach and oesophagus were excised from the body together with the above mentioned oesophageal and gastric branches of the vagal nerve. Afferent impulse discharges, usually unitary, were recorded from the fine nerve filament or single nerve fiber dissected from the nerve branch with the aid of a stereo-microscope. The process o f dissection of a single nerve fiber was as follows: First, the nerve trunk was • mounted on a glass plate, then the sheath was extracted and severed by a pair of needles. Next, the nerve fibers were separated from one another. One intact fiber o f the desired size was selected for use, and all remaining ones were cut away. The dissected single nerve fiber was confirmed by a high power microscope, and its diameter was estimated. Some photomicrographs were taken. F o r mechanical stimulation, touch by a nylon hair and pressure by a glass rod were used. Electrical stimulation, rectangular pulses, was produced by means of an electronic stimulator and isolator. Afferent impulses were monitored and recorded by a doublebeam oscilloscope with a long recording camera. RESULTS
1. Afferent Impulses from the Vagal Gastric Fibers Generally, the mechano-receptors belonging to the vagal gastric fiber tended to adapt rapidly to pressure stimulation of the stomach. Irrespective o f the stimulus strength, afferent impulses were observed instantaneously at the beginning and sometimes at the end of the stimulation. This type o f receptor did not respond to the peristaltic contraction of the stomach
METHOD
All the experiments were conducted on toad's viscera. In loads the oesophagus and stomach receive several nerve 1 A
Food intake
2 wall caused by frequent electrical stimulation. After taking off the mucous membrane, no response was observed when mechanical stimulation was applied on the receptive field. This type of receptor is located in the mucous membrane and is capable of responding to phasic mechanical stimuli. This receptor will hereafter be referred to as " A type receptor" (Fig. 1). In addition to A type receptors, a slow-adapting vagal mechano-receptor was found. The number of this kind of receptor, hereafter referred to as "B type", was small. Without mechanical stimulation, spontaneous regular background discharges were observed. When pressure by a nylon hair or a stretch by a pair of forceps was applied to the receptive field, frequent afferent discharges were observed. This observation was also made after cutting off the mucous membrane. The receptive field of this type is usually a little larger than that of A type (Fig. 2). Simultaneously with the peristaltic contraction of the stomach wall which was induced by several seconds of repetitive electrical stimulation, the frequency of the spontaneous afferent impulses was increased. These results show that the B type receptors were situated in the muscular layer of the stomach wall and may respond to passive distension and active contraction of the muscular layer in the stomach wall (Fig. 3). The diameter range of the vagal afferent fiber from the mechano-mceptor in the stomach wall including the myelinsheath, was about 4-13 microns. Usually a nerve fiber from A type receptor was around 10 microns, and from a B type receptor it was about 5 microns in dia. To estimate the conduction velocity of the vagal afferent fiber, the following experiment was carried out: After recording the afferent impulses from a single vagal nerve fiber which had been induced by the application of mechanical stimulation to the meehano-reccptor in the stomach wall, this single nerve fiber was cut off with the nerve trunk. Next, a photomicrograph was taken to estimate the diameter o f the fiber. Then the action potential induced by means of the application of an electrical pulse o f a duration of 0.1 msec to the central end of the nerve trunk was recorded from a single dissected part. As the distance between stimulating and recording electrode was 13.5 mm and conduction time (which was measured from the oscillogram, see Fig. 4, as the time between shock artefact and initiation of the action potential) was 0.75 m s ~ , the calculated conduction velocity was 18 msec at room temperature. The dianmter of this fiber estimated from the photomicrograph was about 8 microns. Conduction velocity could also be calculated from the following formula, V = K × D and K is 2.05 for frog by Tasaki [12]. Therefore, in this case, V = 2.05 × 8 -----16.4 m/see. This was approximately the same value which was calculated from the previous mentioned p r o ~ d u r e (Fig. 4). F r o m this formula, it can he said that the conduction velocity o f the afferent nerve fiber to a B type receptor which is 5 microns in dia. will be 10.25 m/see. The afferent impulses from the mechano-rec~ptors in the stomach were recorded not only from the myolinated fibers but also from non-myelinated fibers as shown in Fig. 5. The fine filament of non-myelinated fibers, which were conth-med by photomicrograph, were dissected from the v a ~ l nerve trunk to the stomach. The afferent i m p u l ~ discharges were recorded from the filament when the ~ e a l stimuli were applied to the stomach. After that the nerve filament was cut off with the nerve trunk. Then the action potential which had been induced by an electrical pulse applied to the central end
NIIJIMA of the nerve trunk for a duration of I msec was recorded from the filament. The distance between stimulating and recording electrodes was 5 ram, and conduction time which was estimated from the lowest record in Fig. 5 was 11 msec. Therefore, the conduction velocity of this filament should be 0.45 m/see. This value is reasonable for the conduction velocity of non-myelinated nerve fibers. The record at the top of Fig. 6 shows the spontaneous background discharge from non-myelinated fibers to the mechano-receptors in the stomach. The second and third records show that the discharge frequency increases with the peristaltic contraction of the stomach wall when it is induced by frequent electrical stimulation to the stomach. The fourth record indicates that these receptors may also respond to pressure stimulation applied to the stomach wall. These will be referred to as "C type" mechano-receptors. Thus it can be said that there are three types of mechanoreceptors in the stomach wall. They are A, B, and C types. Table 1 shows the varying characteristics of these three different types of receptors. TABLE 1 GASTRIC MECHANO-RECEPTORS
Type A
Diameter of nerve fiber (~) Response for pressure stimulus Response for stretch Response for contraction Adaptation Receptive field area Situation of receptor Localization of receptor in the wall
Type B
Type C
10
5
non-myelinated
(+)
(+)
( ~)
(+)
(+)
(4)
(-) (+) (+) rapidly slowly slowly adapting type adapting type adapting type 2 × 2ram ~ cardia mucosa
5 x 5ram 2 corpus muscular layer
3 × 3ram ~ corpus cardia submucosa muscular layer
2. Afferent Impulses from the Vagal Oesophageal Fibers As mentioned previously, the oesophagus in toads receives the nerve supply from the vaBal nerves. The characteristics o f the m e c h a n o - r ~ p t o r s in the o e s o p ~ wall were studied by recording the afferent impulses from the vagal nerve fibers. Figure g shows one example o f the mew.bane-receptors in the wall of the oesophagus. The fiber diartmter was 10 microns, and tim receptive field area was about 5 × 5 m m a. Adaptation for mechanical stimulation was rapid, as shown in the top and middle records in Fig. 7. After removing the mucous nmmbrahe of the receptive field area, no response could be obm~,ed with m ~ h a n i c a l stimulation. This fact indicated that the receptor is situated in tim mucous meambrane of the oesophageal walt. F r o m the above nmntioned results, it can be said that this type of recaptor helongs to the same group as the A type in the stomach wall. Figure 8 shows another example of the mechano-reccptor in the oesophagus. In this case the diameter of the nerve fiber was 5 microns. The receptive field area was about 2 × 2 mmt
. . . . . . . .
~
t
FIG. !. Afferent impulse discharges in a single vagal gastric.,fibe r connected to A type mechano-receptor in the gastric wall. From top to bottom, touch with a nylon hair to the wall Of the stomach, repetitive electrical stimulation to the stomach (I mse¢, 301 C/s), touch to the mucous membrane and touch to the muscular layer after removing the mucous membrane, Horizontal bar shows the signal for mechanical stimulation. Time mark shows 1 sec in all succeeding figures.
FIG. 2. Afferent impulse discharges in a single vaga] gastric fiber connected to B type mechano-receptor in the gastric wall. From top to bottom, touch with a nylon hair to the wall of the stomach, stretch of the stomach wall by a pair of forceps, touch with a hair to the mucous membrane, touch to the muscular layer after removing of the mucous membrane and stretch of the muscular layer by a pair of forceps. Shaded area shows the receptive field area. Horizontal bar shows the signal for mechanical stimulation.
FIG. 3. Afferent impulses from the B type mechano-rcceptor in the gastric wall. From top to bottom, spontaneous afferent discharge, before, during and after the repetitive electrical stimulation (1 msec, 30 c/s) to the wall of the stomach. All the graphs are continued. Decreasing of the impulse frequency at the beginning of the stimulation shows the relaxation of the receptive field area in the muscular layer preceding the arrivai of the peristaltic wave.
(facing page 2)
on
0~!
FIG. 4. Conduction velocity and afferent impulse discharge of a single myelinated afferent ~ber connected to the A type receptor. From top to bottom, afferent discharge from A type receptor according to the mechanical stimulation of the stomach, photomicrograph of the single afferent fiber and action potential originated by an electrical pulse applied to the vagal nerve trunk. In the lowest graph, the arrow shows the shock artefact. ~----t1.1t m
°
FIG. 5. Conduction velocity and afferent impulse discharge of non-myelinatcd fibers connected to the mechano-receptors in the gastric wall. Top: Photomicrograph of a sma|t group of non-myelinated fibers stained by I"~, osmic acid. Middle: Afferent discharge from non-myelinated fibers by mechanical stimulation of the stomach wall. Horizontal line shows signal for stimulation. Bottom: Action potential Originated by an electrical pulse applied to the vagal nerve trunk.
FIG. 6. Afferent impulse discharge from C type mechano-receptors in the gastric wall, Top: Photomicrograph of the nerve fiber bundle from which afferent impulse discharges were recorded, M indicates myelinated nerve fiber and N indicates non-myeiinated fi~r. First record: Afferent impulse discharge before electrical stimulation. Second and third records: Afferent impulse discharge after repetitive electrical stimulation of the gastric wall. Fourth record~ Afferent di~harge caused by mechanical stimulation of the ga.~tric wall. Horizont;d line shows the signal for stimulation.
FIG. 7. Afferent impulse discharge from rapidly adapting oesophageal mechano-receptor. From top to bottom, pressure stimulation to the mucous membrane by a glass rod, squeezing by a nylon hair and pressure stimulation to the same place after removing the mucous membrane'. Shaded area shows the receptive field area. Horizontal line indicates signal for stimulation.
FIG. 8. Afferent impulse discharge from slowly adapting oesophagcal mechano-reecptor. Above: Pressure stimulation to the mucous membrane by a glass rod. Below: Touch to the mucous membrane by a nylon hair. Horizontal line shows signal for stimulation and shaded area shows receptive field area.
FIG. 10. Responseof a rapidly adapting mechanoreceptor in the ccsophagcal wall. Above: Mechanical stimulation by a nylon hair. HorizQnt~al line shows signal for stimulation. Below: Atter the repetitive electrical stimulation. P indicates the peristaltic wave originated by electrical stimulation.
FIG. II. Response of a slowly adapting mechano-receptor in the oesophageal wall. Top: Mechanical stimulation by a nylon hair. Horizontal line shows signal for stimulation. Middle: Before the repetitive electrical stimulation. Bottom: After the electrical stimulation. P indicates the peristaltic wave after the electrical stimulation.
.... . J
FIG. 12. Relationship between insertion of the wax ball into the stomach through the oesophagus and afferent discharge from the oesophageal and gastric mechano-receptors. Horizontal bar shows the signal for passing the wax ball through the oesophagus, P indicates the peristaltic movement of the gastric wall caused by insertion of the ball into the stomach.
FIG. 13. Simultaneous recording of the afferent discharges from the vagal rootlet and sympathetic branch. Arrows show the beginning and end of the mechanical stimulation.
GASTRIC AND OESOPHAGEAL MECHANO-RECEPTORS and was situated in the cardiac site of the oesophagus. Adaptation for mechanical stimulation was slow and it responded only to strong pressure applied by a glass rod and not to the weak touch of a fine nylon hair. This fact tells us that the receptor is probably located in the submucosa or in the muscular layer. The number of this type o f receptor was generally small.
/
; 1
/
3 With the peristaltic contraction of the oesophageal wall caused by several seconds o f repetitive electrical stimulation, no spontaneous afferent discharges were observed from the wagal fiber connected to the rapidly adapting type mechanoreceptors (Fig. 10). On rare occasions, as shown in Fig. 11, extremely slowly-adapting type mechano-receptors were found. F r o m these receptors spontaneous background discharges were usually observed. After several seconds of repetitive stimulation had induced peristaltic contraction of the oesophageal wall, the frequency o f the afferent impulse discharges was increased. This fact reveals that this kind of receptor is situated in the muscular layer in the same manner as the B type receptors are situated in the stomach wail. In addition, mechano-receptors supplied with nonmyelinated nerve fibers were found. The receptive field area was less than 3 x 3 mm ~, and adaptation was generally slow (Table 3).
/ TABLE 3
FIG. 9. Schematic representation of the receptive field area of vagal mechano-receptors on |he ocsophageal wall.
AFFERENT NONMYELINATED FIBERS
Figure 9 is a schematic illustration of the distribution of the mechano-receptors in the wall of the oesophagus which were observed in the above mentioned experiment. Table 2 indicates the characteristics of the mechano-receptors in the wall of the oesophagus and the afferent nerve fibers connecting them. As is shown in the table, it may be conclusively stated that the oesophageal mechano-receptors are mainly situated at the oral site of the oesophagus and localized in the mucous membrane. Further, it can be said that the adaptation of these receptors to mechanical stimulation is generally rapid, therefore a phasic stimulus such as the passing o f food through the oesophagus would be one of the most effective for them.
TABLE 2 OESOPHAGEAL MECHANO-RECEPTOR
DiaAdapSituation Location Fiber meter tation of Receptive of of receptor Adeq. No. (~) receptor field area receptor in the wall stimulus I 2
7 5
rapidly rapidly
3
5
slowly
4 5 6
I0 I0 7
rapidly rapidly rapidly
7 8 9 10 I!
10 8 10 10 8
rapidly rapidly rapidly rapidly rapidly
3 × 3 mm ~oral site 3 × 3 mms middle portion 2x 2 cardiac site 2×2 oral site 3× 3 oral site 2×3 middle portion 2x 2 oral site 3x 3 oral site 5×5 oral site 5× 5 oral site 2x 2 oral site
mucosa submucosa ? submucosa? mucosa mucosa submucosa? rnucosa mucosa mucosa mucosa ? submucosa ?
touch pressure pressure touch touch touch touch touch touch touch pressure
Fiber no.
Adaptation of receptor
i 2 3 4 5 ,6
slow slow slow slow slow slow
Receptive field area 3 3 2 3 3 3
x × × × × x
3 mms 3 mm s 2 mm ~ 3 mm 9 3 mm ~ 3 mm 2
Situation of receptor oesophagus oesophagus oesophagus oesophagus Stomach (cardia) Stomach (cardia)
3. The Role Played by Oesoplu~geal and Gastric Mechanoreceptors in Food Intake To determine the physiological role played by oesophageai and gastric mechano-receptors for food intake, the next experiment was conducted. When a wax ball 10 mm in dia. was inserted into the stomach from the mouth through the oesophagus, the burst discharges were recorded as it passed through the oesophagus, as is indicated by the horizontal line in the top and second records in Fig. 12. After the insertion of the wax ball into the stomach, a number of afferent discharges were observed corresponding to the distension of the stomach wall. The third record shows that the train of the afferent discharges was observed with the peristaltic contraction which had been induced by the distension of the wall after the insertion of the wax ball. The fourth record shows that the push and pull of the wax ball through the oesophagus causes vigorous afferent discharges, but if the ball is held in one position, as is shown in the middle part of the graph, there are no impulse discharges. On the other hand, when the ball was held in the stomach, as is shown in the lowest graph, a train of afferent discharges corresponding to the distension of the wall was observed.
4. Pathway of the Vagal Afferents from the Oesophageal and Gastric Mechano-receptors It is known that the vagal nerve trunk in toads consists of vagal and sympathetic fibers. This is called the vagosympathetic nerve. Vagal rootlets originate from the medulla
4
NIIJtMA
oblongata, and a sympathetic branch comes from the sympathetic chain. These join at the site o f the. jugular ganglion, and from there run together to the oesophagus, stomach and other visceral organs. To find the pathway of the vagal afferents from the oesophageal and gastric mechanoreceptors, one pair of recording electrodes was,place,d o n the vagal rootlets and another pair was placed on the sympathetic branch. Afferent impulse discharges were recorded simultaneously from the vagal rootlets and from the sympathetic branch. When mechanical stimuli were applied to the oesophagus or to the stomach, afferent burst discharges were recorded only from the vagal rootlets. This fact indicates that the great majority of all afferent fibers from the oesophageal and gastric mechano-receptors are contained in the vagal rootlets (Fig. 13). DISCUSSION
From the above mentioned results, it may be concluded that the oesophageal receptors mainly send information about the passing of food, and the vagal gastric receptors send information concerning the degree of fullness or emptiness of the stomach. The burst discharge accompanying the peristalsis of the stomach was observed after food intake, which indicates that the vagal receptors are also able to send information on the motility of the stomach. In a previous paper, the author [8] maintained that the mucosal and submucosal gastric mechano-receptors are sensitive to the passive distension and active contraction of the stomach wall. However results obtained from the present experiments show that this conclusion should be corrected. This is proved by the fact that A type receptors, situated in the mucous membrane, do not respond to the perostaltic contraction, and B type receptors, localized in the muscular layer, do respond to the passive distension and active contraction of the stomach wall. The latter must be situated "in series" in the muscular layer, as Iggo [4] reported in his experiments on the cat and on the goat. Andrew [2, 3] recorded the afferent discharges f r o m the superior laryngeal nerve fibers connected to the tension receptors in the wall of the cervical oesophagus in the rat. He reported that the most easily recognized type of afferent
was connected t o slowly-adapting tension receptors in the wall of the :oesophagus. He, obse~cA that the inflation of the oesophagus with air or the distension of an ending zone with a bolus increased the discharge. Further, he said that contraction of the oesophageal muscle during a propulsive wave was also a potent stimulus. On the contrary, the results obtained here show that in toads most of the vagal oesophageal receptors adapt rapidly to mechanical stimulation. He reports that the number of slowly-adapting oesophageal receptors which are responsive to passive distension and active contraction of the oesophageal wall is generally small. This difference is probably due to the difference of species of animals. Paintal [9] reported that the mean conduction velocity of gastric stretch fibers would be about 9 m/see in the cat. Iggo [4] said that the velocity of four single fibers from gastric tension receptors was 2-12 m/see in the goat. Further, Iggo [5] estimated the conduction velocities of the vagal afferent fibers by means of the collision method and reported that in the cat, with two exceptions, both mucosal units, the conduction receptors were below 2.5 m/see and the majority of the fibers have a conduction velocity less than 1.5 m/see. His conclusion that the slowest vagal fibers are non-myelinated is based on the conduction velocity measurements. It is said that there are species differences in the number of myelinated fibers in the abdominal vagi; they are abundant in the sheep and goat, but are less than 2 per Cent in the cat [1]. From the above mentioned results, it can be said that in the toad there are myelinated and non-myelinated afferent fibers from the oesophageal and gastric mechano-receptors; this was indicated on ttae photomicrographs and was confirmed by the direct estimations of the conduction velocities. It was found that the myelinated afferent fibers usually are of medium or larger size in diameter, and conduction velocities are relatively high. The difference from the results obtained in cats might also be due to the difference of the species. The differentiation between myelinated and non-myelinated fibers has been made by light microscopic examination. However, to reach a definite conclusion an electronmicroscopic examination should be conducted.
REFERENCES
1. Agostoni, E., J. E, Chinnock, M. DeBurgh Daly and J . G . Murray. Functional and histologicad mJdies on the vagus and its branches to the heart, lungs and abdominal viscera in the cat. Y. Physiol. 135: 182-205, 1957. 2. Andrew, B. L. The nervous control of the cervical oesophagus of the rat during swallowin~ J. Physiol. 134: 729--740, 1956b. 3. Andrew, B. L. Activity in afferent nerve fibers from the cervical oetophagus. J. Physiol. 135: 54-55, 1957. 4. Iggo, A. Tension receptors in the stomach and the urinary bladder. J. Physiol. 1 ~ : 593--607, 1955. 5. Iggo, A. The ~ o p h y s i o l o g i ~ ~ o n of sinsle nerve fibers, with ~ r e f e m ~ to the slowest-condu~ang v a ~ afferent fibers in the cat, J. Physiol. i.42:1 |0-126, 1958, 6. Jacobs, H. L. The interaction of hunger and thirst: Experimental separation of osmotic and oral-gastric favors in the regulation of caloric intake. In: Thirst in the Regulation of Body Water, edited by M. J. Wayner. Oxford ~. Press, 1964. 7. Janowitz, H. D. and M. I. G ~ . Some fact0~ ~t~eb~ing the food intake of normal doss a n d : ~ w i t h ~ e ~ p ~ m y and gastric fistula. Am. J. Physiol. 159: 143-148, I940. - ~
8. N~ima, A. Afferent impulses in the vagal and splanckmic nerves of the toad's stomach, and their role in sensory mechanism. Jap. y. Physiol. 12: 25-44, 1962. 9. Paintal, A. S. A study of ~ t r i c stretch receptors, Their role in the peripheral mechanism of satiation of hunger and thirst. J. PhysioL 17,6: 255-270, 1954b. 10. Share, I., K. Martyniuk and M. I. Grossman. Effect of prolonsed intra 8a~ric feeding on oral food intake in dogs. Am. dr. PhyMol. 169: 229--235, 1952. 11. Sharma, K. N., B. K. Anand, S. Sua and B. Singh. Role of stomach in regulation of activities of hypothalamic feeding centers. Am. J, Physiol, 201: 593-598, 1961, 12. Tasaid, I. Nertous transmission. Springfield, Illinois: Thomas, 1953. 13. Towbin, E. J. Gastric distension as a factor in the satiation of thirst in esophagostomized dogs. Am. J. Physiol. 159: 533-~M1, 1949.
Afferent Impulses in the Gastric and Oesophageal Branch. of the Vagal Nerve of Toad AKIRA
NIIJIMA
Department of Physiology, Niigata University School o f Medicine, Niigata, Japan (Recgived 20 M a y 1966) NnzIMA, A. Aff'erent hnptdses ht the gastric and oesophageal branch of the vagal nerve of the toad. PHYSIOL.BEHAV. 2 (1) I 4, 1967.--The function of vagal gastric and oesophageal mechano-receptors was studied by means of recording the :tffcrenl discharges from a single or from several afferent nerve fibers dissected from a gastric and oesophageal branch of Ihe wtgal nerve. Three different types of mechano-receptors were found in the gastric wall. Type A: Rapidly-adapting type. situated in the mucous membrane or in the submueosa of the oesophageal border of the cardia, and which do not respond to peristaltic contraction of the stomach wall. Type B: Slowly-adapting type, situated in the muscular layer and responsive to distension and contraction of the stomach. Type C: Slowly-adaptingtype, connected to the non-myelinated liber, situated in the muscular layer, and which respond to distension and contraction of the gastric wall. In the oesophagus the same three types were found. However, the number of rapidly-adapting type receptors is far larger than the other type of receptors. It has been concluded that the oesophageal receptors mainly send information about the passing of food and the vagal gastric receptors send information concerning the degree of fullness or emptiness and motility of the stomach. Vagal afferents
Mechano-receptors
Gastric and oesophageal meehano-receptors
IT ;~AS~EEN SAIDthat the afferent information from peripheral receptors, particularly in the gastro-intestinal organs, plays an important role in the control of food and water intake. From this point of view many workers [6, 7, 10, 11, 13,] studied gastro-intestinai receptors. The authors o f these papers have pointed out that distension of the gastrointestinal canal produces satiation. This indicates that there is a relationship between the satiation mechanism in the central nervous system and the function of the mechanoreceptors in the wall of the gastro-intestinal canal. Afferent impulses from vagal fibers of the stomach have been studied accurately by Paintal [9], Iggo [4] and Niijima [8]. They have reported that there are slowly-adapting and rapidly-adapting types of mechano-receptors in the stomach wall. On the other hand, Andrew [2, 3] recorded the afferent impulse discharges from the superior laryngeal nerve of rats. He reported that the most easily recognized type of afferent was connected to slowly-adapting tension receptors in the wall of the oesophagus. In these studies, however, there was little information about the relationship between characteristics of oesophageal or gastric mechano-receptors and their role in the regulation o f food intake. This report will deal mainly with the afferent impulse discharges from the oesophageal and gastric vagal nerve which connect to the mechano-receptors in the wall of the oesophagus or stomach, differences between them, and their possible role in the regulation of food intake.
Toad
branches from the vagal nerve trunk. They are called R. oesophagus and R. ga#trices N. vagi. The toads were pithed. The stomach and oesophagus were excised from the body together with the above mentioned oesophageal and gastric branches of the vagal nerve. Afferent impulse discharges, usually unitary, were recorded from the fine nerve filament or single nerve fiber dissected from the nerve branch with the aid of a stereo-microscope. The process o f dissection of a single nerve fiber was as follows: First, the nerve trunk was • mounted on a glass plate, then the sheath was extracted and severed by a pair of needles. Next, the nerve fibers were separated from one another. One intact fiber o f the desired size was selected for use, and all remaining ones were cut away. The dissected single nerve fiber was confirmed by a high power microscope, and its diameter was estimated. Some photomicrographs were taken. F o r mechanical stimulation, touch by a nylon hair and pressure by a glass rod were used. Electrical stimulation, rectangular pulses, was produced by means of an electronic stimulator and isolator. Afferent impulses were monitored and recorded by a doublebeam oscilloscope with a long recording camera. RESULTS
1. Afferent Impulses from the Vagal Gastric Fibers Generally, the mechano-receptors belonging to the vagal gastric fiber tended to adapt rapidly to pressure stimulation of the stomach. Irrespective o f the stimulus strength, afferent impulses were observed instantaneously at the beginning and sometimes at the end of the stimulation. This type o f receptor did not respond to the peristaltic contraction of the stomach
METHOD
All the experiments were conducted on toad's viscera. In loads the oesophagus and stomach receive several nerve 1 A
Food intake
2 wall caused by frequent electrical stimulation. After taking off the mucous membrane, no response was observed when mechanical stimulation was applied on the receptive field. This type of receptor is located in the mucous membrane and is capable of responding to phasic mechanical stimuli. This receptor will hereafter be referred to as " A type receptor" (Fig. 1). In addition to A type receptors, a slow-adapting vagal mechano-receptor was found. The number of this kind of receptor, hereafter referred to as "B type", was small. Without mechanical stimulation, spontaneous regular background discharges were observed. When pressure by a nylon hair or a stretch by a pair of forceps was applied to the receptive field, frequent afferent discharges were observed. This observation was also made after cutting off the mucous membrane. The receptive field of this type is usually a little larger than that of A type (Fig. 2). Simultaneously with the peristaltic contraction of the stomach wall which was induced by several seconds of repetitive electrical stimulation, the frequency of the spontaneous afferent impulses was increased. These results show that the B type receptors were situated in the muscular layer of the stomach wall and may respond to passive distension and active contraction of the muscular layer in the stomach wall (Fig. 3). The diameter range of the vagal afferent fiber from the mechano-mceptor in the stomach wall including the myelinsheath, was about 4-13 microns. Usually a nerve fiber from A type receptor was around 10 microns, and from a B type receptor it was about 5 microns in dia. To estimate the conduction velocity of the vagal afferent fiber, the following experiment was carried out: After recording the afferent impulses from a single vagal nerve fiber which had been induced by the application of mechanical stimulation to the meehano-reccptor in the stomach wall, this single nerve fiber was cut off with the nerve trunk. Next, a photomicrograph was taken to estimate the diameter o f the fiber. Then the action potential induced by means of the application of an electrical pulse o f a duration of 0.1 msec to the central end of the nerve trunk was recorded from a single dissected part. As the distance between stimulating and recording electrode was 13.5 mm and conduction time (which was measured from the oscillogram, see Fig. 4, as the time between shock artefact and initiation of the action potential) was 0.75 m s ~ , the calculated conduction velocity was 18 msec at room temperature. The dianmter of this fiber estimated from the photomicrograph was about 8 microns. Conduction velocity could also be calculated from the following formula, V = K × D and K is 2.05 for frog by Tasaki [12]. Therefore, in this case, V = 2.05 × 8 -----16.4 m/see. This was approximately the same value which was calculated from the previous mentioned p r o ~ d u r e (Fig. 4). F r o m this formula, it can he said that the conduction velocity o f the afferent nerve fiber to a B type receptor which is 5 microns in dia. will be 10.25 m/see. The afferent impulses from the mechano-rec~ptors in the stomach were recorded not only from the myolinated fibers but also from non-myelinated fibers as shown in Fig. 5. The fine filament of non-myelinated fibers, which were conth-med by photomicrograph, were dissected from the v a ~ l nerve trunk to the stomach. The afferent i m p u l ~ discharges were recorded from the filament when the ~ e a l stimuli were applied to the stomach. After that the nerve filament was cut off with the nerve trunk. Then the action potential which had been induced by an electrical pulse applied to the central end
NIIJIMA of the nerve trunk for a duration of I msec was recorded from the filament. The distance between stimulating and recording electrodes was 5 ram, and conduction time which was estimated from the lowest record in Fig. 5 was 11 msec. Therefore, the conduction velocity of this filament should be 0.45 m/see. This value is reasonable for the conduction velocity of non-myelinated nerve fibers. The record at the top of Fig. 6 shows the spontaneous background discharge from non-myelinated fibers to the mechano-receptors in the stomach. The second and third records show that the discharge frequency increases with the peristaltic contraction of the stomach wall when it is induced by frequent electrical stimulation to the stomach. The fourth record indicates that these receptors may also respond to pressure stimulation applied to the stomach wall. These will be referred to as "C type" mechano-receptors. Thus it can be said that there are three types of mechanoreceptors in the stomach wall. They are A, B, and C types. Table 1 shows the varying characteristics of these three different types of receptors. TABLE 1 GASTRIC MECHANO-RECEPTORS
Type A
Diameter of nerve fiber (~) Response for pressure stimulus Response for stretch Response for contraction Adaptation Receptive field area Situation of receptor Localization of receptor in the wall
Type B
Type C
10
5
non-myelinated
(+)
(+)
( ~)
(+)
(+)
(4)
(-) (+) (+) rapidly slowly slowly adapting type adapting type adapting type 2 × 2ram ~ cardia mucosa
5 x 5ram 2 corpus muscular layer
3 × 3ram ~ corpus cardia submucosa muscular layer
2. Afferent Impulses from the Vagal Oesophageal Fibers As mentioned previously, the oesophagus in toads receives the nerve supply from the vaBal nerves. The characteristics o f the m e c h a n o - r ~ p t o r s in the o e s o p ~ wall were studied by recording the afferent impulses from the vagal nerve fibers. Figure g shows one example o f the mew.bane-receptors in the wall of the oesophagus. The fiber diartmter was 10 microns, and tim receptive field area was about 5 × 5 m m a. Adaptation for mechanical stimulation was rapid, as shown in the top and middle records in Fig. 7. After removing the mucous nmmbrahe of the receptive field area, no response could be obm~,ed with m ~ h a n i c a l stimulation. This fact indicated that the receptor is situated in tim mucous meambrane of the oesophageal walt. F r o m the above nmntioned results, it can be said that this type of recaptor helongs to the same group as the A type in the stomach wall. Figure 8 shows another example of the mechano-reccptor in the oesophagus. In this case the diameter of the nerve fiber was 5 microns. The receptive field area was about 2 × 2 mmt
. . . . . . . .
~
t
FIG. !. Afferent impulse discharges in a single vagal gastric.,fibe r connected to A type mechano-receptor in the gastric wall. From top to bottom, touch with a nylon hair to the wall Of the stomach, repetitive electrical stimulation to the stomach (I mse¢, 301 C/s), touch to the mucous membrane and touch to the muscular layer after removing the mucous membrane, Horizontal bar shows the signal for mechanical stimulation. Time mark shows 1 sec in all succeeding figures.
FIG. 2. Afferent impulse discharges in a single vaga] gastric fiber connected to B type mechano-receptor in the gastric wall. From top to bottom, touch with a nylon hair to the wall of the stomach, stretch of the stomach wall by a pair of forceps, touch with a hair to the mucous membrane, touch to the muscular layer after removing of the mucous membrane and stretch of the muscular layer by a pair of forceps. Shaded area shows the receptive field area. Horizontal bar shows the signal for mechanical stimulation.
FIG. 3. Afferent impulses from the B type mechano-rcceptor in the gastric wall. From top to bottom, spontaneous afferent discharge, before, during and after the repetitive electrical stimulation (1 msec, 30 c/s) to the wall of the stomach. All the graphs are continued. Decreasing of the impulse frequency at the beginning of the stimulation shows the relaxation of the receptive field area in the muscular layer preceding the arrivai of the peristaltic wave.
(facing page 2)
on
0~!
FIG. 4. Conduction velocity and afferent impulse discharge of a single myelinated afferent ~ber connected to the A type receptor. From top to bottom, afferent discharge from A type receptor according to the mechanical stimulation of the stomach, photomicrograph of the single afferent fiber and action potential originated by an electrical pulse applied to the vagal nerve trunk. In the lowest graph, the arrow shows the shock artefact. ~----t1.1t m
°
FIG. 5. Conduction velocity and afferent impulse discharge of non-myelinatcd fibers connected to the mechano-receptors in the gastric wall. Top: Photomicrograph of a sma|t group of non-myelinated fibers stained by I"~, osmic acid. Middle: Afferent discharge from non-myelinated fibers by mechanical stimulation of the stomach wall. Horizontal line shows signal for stimulation. Bottom: Action potential Originated by an electrical pulse applied to the vagal nerve trunk.
FIG. 6. Afferent impulse discharge from C type mechano-receptors in the gastric wall, Top: Photomicrograph of the nerve fiber bundle from which afferent impulse discharges were recorded, M indicates myelinated nerve fiber and N indicates non-myeiinated fi~r. First record: Afferent impulse discharge before electrical stimulation. Second and third records: Afferent impulse discharge after repetitive electrical stimulation of the gastric wall. Fourth record~ Afferent di~harge caused by mechanical stimulation of the ga.~tric wall. Horizont;d line shows the signal for stimulation.
FIG. 7. Afferent impulse discharge from rapidly adapting oesophageal mechano-receptor. From top to bottom, pressure stimulation to the mucous membrane by a glass rod, squeezing by a nylon hair and pressure stimulation to the same place after removing the mucous membrane'. Shaded area shows the receptive field area. Horizontal line indicates signal for stimulation.
FIG. 8. Afferent impulse discharge from slowly adapting oesophagcal mechano-reecptor. Above: Pressure stimulation to the mucous membrane by a glass rod. Below: Touch to the mucous membrane by a nylon hair. Horizontal line shows signal for stimulation and shaded area shows receptive field area.
FIG. 10. Responseof a rapidly adapting mechanoreceptor in the ccsophagcal wall. Above: Mechanical stimulation by a nylon hair. HorizQnt~al line shows signal for stimulation. Below: Atter the repetitive electrical stimulation. P indicates the peristaltic wave originated by electrical stimulation.
FIG. II. Response of a slowly adapting mechano-receptor in the oesophageal wall. Top: Mechanical stimulation by a nylon hair. Horizontal line shows signal for stimulation. Middle: Before the repetitive electrical stimulation. Bottom: After the electrical stimulation. P indicates the peristaltic wave after the electrical stimulation.
.... . J
FIG. 12. Relationship between insertion of the wax ball into the stomach through the oesophagus and afferent discharge from the oesophageal and gastric mechano-receptors. Horizontal bar shows the signal for passing the wax ball through the oesophagus, P indicates the peristaltic movement of the gastric wall caused by insertion of the ball into the stomach.
FIG. 13. Simultaneous recording of the afferent discharges from the vagal rootlet and sympathetic branch. Arrows show the beginning and end of the mechanical stimulation.
GASTRIC AND OESOPHAGEAL MECHANO-RECEPTORS and was situated in the cardiac site of the oesophagus. Adaptation for mechanical stimulation was slow and it responded only to strong pressure applied by a glass rod and not to the weak touch of a fine nylon hair. This fact tells us that the receptor is probably located in the submucosa or in the muscular layer. The number of this type o f receptor was generally small.
/
; 1
/
3 With the peristaltic contraction of the oesophageal wall caused by several seconds o f repetitive electrical stimulation, no spontaneous afferent discharges were observed from the wagal fiber connected to the rapidly adapting type mechanoreceptors (Fig. 10). On rare occasions, as shown in Fig. 11, extremely slowly-adapting type mechano-receptors were found. F r o m these receptors spontaneous background discharges were usually observed. After several seconds of repetitive stimulation had induced peristaltic contraction of the oesophageal wall, the frequency o f the afferent impulse discharges was increased. This fact reveals that this kind of receptor is situated in the muscular layer in the same manner as the B type receptors are situated in the stomach wail. In addition, mechano-receptors supplied with nonmyelinated nerve fibers were found. The receptive field area was less than 3 x 3 mm ~, and adaptation was generally slow (Table 3).
/ TABLE 3
FIG. 9. Schematic representation of the receptive field area of vagal mechano-receptors on |he ocsophageal wall.
AFFERENT NONMYELINATED FIBERS
Figure 9 is a schematic illustration of the distribution of the mechano-receptors in the wall of the oesophagus which were observed in the above mentioned experiment. Table 2 indicates the characteristics of the mechano-receptors in the wall of the oesophagus and the afferent nerve fibers connecting them. As is shown in the table, it may be conclusively stated that the oesophageal mechano-receptors are mainly situated at the oral site of the oesophagus and localized in the mucous membrane. Further, it can be said that the adaptation of these receptors to mechanical stimulation is generally rapid, therefore a phasic stimulus such as the passing o f food through the oesophagus would be one of the most effective for them.
TABLE 2 OESOPHAGEAL MECHANO-RECEPTOR
DiaAdapSituation Location Fiber meter tation of Receptive of of receptor Adeq. No. (~) receptor field area receptor in the wall stimulus I 2
7 5
rapidly rapidly
3
5
slowly
4 5 6
I0 I0 7
rapidly rapidly rapidly
7 8 9 10 I!
10 8 10 10 8
rapidly rapidly rapidly rapidly rapidly
3 × 3 mm ~oral site 3 × 3 mms middle portion 2x 2 cardiac site 2×2 oral site 3× 3 oral site 2×3 middle portion 2x 2 oral site 3x 3 oral site 5×5 oral site 5× 5 oral site 2x 2 oral site
mucosa submucosa ? submucosa? mucosa mucosa submucosa? rnucosa mucosa mucosa mucosa ? submucosa ?
touch pressure pressure touch touch touch touch touch touch touch pressure
Fiber no.
Adaptation of receptor
i 2 3 4 5 ,6
slow slow slow slow slow slow
Receptive field area 3 3 2 3 3 3
x × × × × x
3 mms 3 mm s 2 mm ~ 3 mm 9 3 mm ~ 3 mm 2
Situation of receptor oesophagus oesophagus oesophagus oesophagus Stomach (cardia) Stomach (cardia)
3. The Role Played by Oesoplu~geal and Gastric Mechanoreceptors in Food Intake To determine the physiological role played by oesophageai and gastric mechano-receptors for food intake, the next experiment was conducted. When a wax ball 10 mm in dia. was inserted into the stomach from the mouth through the oesophagus, the burst discharges were recorded as it passed through the oesophagus, as is indicated by the horizontal line in the top and second records in Fig. 12. After the insertion of the wax ball into the stomach, a number of afferent discharges were observed corresponding to the distension of the stomach wall. The third record shows that the train of the afferent discharges was observed with the peristaltic contraction which had been induced by the distension of the wall after the insertion of the wax ball. The fourth record shows that the push and pull of the wax ball through the oesophagus causes vigorous afferent discharges, but if the ball is held in one position, as is shown in the middle part of the graph, there are no impulse discharges. On the other hand, when the ball was held in the stomach, as is shown in the lowest graph, a train of afferent discharges corresponding to the distension of the wall was observed.
4. Pathway of the Vagal Afferents from the Oesophageal and Gastric Mechano-receptors It is known that the vagal nerve trunk in toads consists of vagal and sympathetic fibers. This is called the vagosympathetic nerve. Vagal rootlets originate from the medulla
4
NIIJtMA
oblongata, and a sympathetic branch comes from the sympathetic chain. These join at the site o f the. jugular ganglion, and from there run together to the oesophagus, stomach and other visceral organs. To find the pathway of the vagal afferents from the oesophageal and gastric mechanoreceptors, one pair of recording electrodes was,place,d o n the vagal rootlets and another pair was placed on the sympathetic branch. Afferent impulse discharges were recorded simultaneously from the vagal rootlets and from the sympathetic branch. When mechanical stimuli were applied to the oesophagus or to the stomach, afferent burst discharges were recorded only from the vagal rootlets. This fact indicates that the great majority of all afferent fibers from the oesophageal and gastric mechano-receptors are contained in the vagal rootlets (Fig. 13). DISCUSSION
From the above mentioned results, it may be concluded that the oesophageal receptors mainly send information about the passing of food, and the vagal gastric receptors send information concerning the degree of fullness or emptiness of the stomach. The burst discharge accompanying the peristalsis of the stomach was observed after food intake, which indicates that the vagal receptors are also able to send information on the motility of the stomach. In a previous paper, the author [8] maintained that the mucosal and submucosal gastric mechano-receptors are sensitive to the passive distension and active contraction of the stomach wall. However results obtained from the present experiments show that this conclusion should be corrected. This is proved by the fact that A type receptors, situated in the mucous membrane, do not respond to the perostaltic contraction, and B type receptors, localized in the muscular layer, do respond to the passive distension and active contraction of the stomach wall. The latter must be situated "in series" in the muscular layer, as Iggo [4] reported in his experiments on the cat and on the goat. Andrew [2, 3] recorded the afferent discharges f r o m the superior laryngeal nerve fibers connected to the tension receptors in the wall of the cervical oesophagus in the rat. He reported that the most easily recognized type of afferent
was connected t o slowly-adapting tension receptors in the wall of the :oesophagus. He, obse~cA that the inflation of the oesophagus with air or the distension of an ending zone with a bolus increased the discharge. Further, he said that contraction of the oesophageal muscle during a propulsive wave was also a potent stimulus. On the contrary, the results obtained here show that in toads most of the vagal oesophageal receptors adapt rapidly to mechanical stimulation. He reports that the number of slowly-adapting oesophageal receptors which are responsive to passive distension and active contraction of the oesophageal wall is generally small. This difference is probably due to the difference of species of animals. Paintal [9] reported that the mean conduction velocity of gastric stretch fibers would be about 9 m/see in the cat. Iggo [4] said that the velocity of four single fibers from gastric tension receptors was 2-12 m/see in the goat. Further, Iggo [5] estimated the conduction velocities of the vagal afferent fibers by means of the collision method and reported that in the cat, with two exceptions, both mucosal units, the conduction receptors were below 2.5 m/see and the majority of the fibers have a conduction velocity less than 1.5 m/see. His conclusion that the slowest vagal fibers are non-myelinated is based on the conduction velocity measurements. It is said that there are species differences in the number of myelinated fibers in the abdominal vagi; they are abundant in the sheep and goat, but are less than 2 per Cent in the cat [1]. From the above mentioned results, it can be said that in the toad there are myelinated and non-myelinated afferent fibers from the oesophageal and gastric mechano-receptors; this was indicated on ttae photomicrographs and was confirmed by the direct estimations of the conduction velocities. It was found that the myelinated afferent fibers usually are of medium or larger size in diameter, and conduction velocities are relatively high. The difference from the results obtained in cats might also be due to the difference of the species. The differentiation between myelinated and non-myelinated fibers has been made by light microscopic examination. However, to reach a definite conclusion an electronmicroscopic examination should be conducted.
REFERENCES
1. Agostoni, E., J. E, Chinnock, M. DeBurgh Daly and J . G . Murray. Functional and histologicad mJdies on the vagus and its branches to the heart, lungs and abdominal viscera in the cat. Y. Physiol. 135: 182-205, 1957. 2. Andrew, B. L. The nervous control of the cervical oesophagus of the rat during swallowin~ J. Physiol. 134: 729--740, 1956b. 3. Andrew, B. L. Activity in afferent nerve fibers from the cervical oetophagus. J. Physiol. 135: 54-55, 1957. 4. Iggo, A. Tension receptors in the stomach and the urinary bladder. J. Physiol. 1 ~ : 593--607, 1955. 5. Iggo, A. The ~ o p h y s i o l o g i ~ ~ o n of sinsle nerve fibers, with ~ r e f e m ~ to the slowest-condu~ang v a ~ afferent fibers in the cat, J. Physiol. i.42:1 |0-126, 1958, 6. Jacobs, H. L. The interaction of hunger and thirst: Experimental separation of osmotic and oral-gastric favors in the regulation of caloric intake. In: Thirst in the Regulation of Body Water, edited by M. J. Wayner. Oxford ~. Press, 1964. 7. Janowitz, H. D. and M. I. G ~ . Some fact0~ ~t~eb~ing the food intake of normal doss a n d : ~ w i t h ~ e ~ p ~ m y and gastric fistula. Am. J. Physiol. 159: 143-148, I940. - ~
8. N~ima, A. Afferent impulses in the vagal and splanckmic nerves of the toad's stomach, and their role in sensory mechanism. Jap. y. Physiol. 12: 25-44, 1962. 9. Paintal, A. S. A study of ~ t r i c stretch receptors, Their role in the peripheral mechanism of satiation of hunger and thirst. J. PhysioL 17,6: 255-270, 1954b. 10. Share, I., K. Martyniuk and M. I. Grossman. Effect of prolonsed intra 8a~ric feeding on oral food intake in dogs. Am. dr. PhyMol. 169: 229--235, 1952. 11. Sharma, K. N., B. K. Anand, S. Sua and B. Singh. Role of stomach in regulation of activities of hypothalamic feeding centers. Am. J, Physiol, 201: 593-598, 1961, 12. Tasaid, I. Nertous transmission. Springfield, Illinois: Thomas, 1953. 13. Towbin, E. J. Gastric distension as a factor in the satiation of thirst in esophagostomized dogs. Am. J. Physiol. 159: 533-~M1, 1949.