AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 282: R818-R827, 2002; doi:10.1152/ajpregu.00180.2001
0363-6119/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kobashi, M.
Right arrow Articles by Matsuo, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kobashi, M.
Right arrow Articles by Matsuo, R.
Vol. 282, Issue 3, R818-R827, March 2002

Suppression of vagal motor activities evokes laryngeal afferent-mediated inhibition of gastric motility

Motoi Kobashi1, Tomoshige Koga2, Masatoshi Mizutani3, and Ryuji Matsuo1

1 Department of Oral Physiology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700 - 8525; 2 Department of Restorative Science, Kawasaki University of Medical Welfare, Kurashiki 701 - 0193; and 3 Okayama Prefectural University Junior College, Soja 719 - 1197, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that the activation of water-responsive afferents in the superior laryngeal nerve was responsible for the inhibition of gastric motility. The present study was undertaken to clarify the roles of the vagal preganglionic neurons responsible for laryngeal afferent-mediated inhibition of gastric motility. Intravenous injection of atropine abolished the inhibition of motility in both the distal and the proximal stomach induced by water administration into the larynx. The neurons in the dorsal motor nucleus of the vagus (DMV), which project to the abdominal viscera, were exclusively inhibited by water administration. Taken together, inhibition of neurons in the DMV induces inhibition of gastric motility evoked by laryngeal water-responsive afferents via a cholinergic pathway. Because chemical lesions of the intermediate DMV, but not the caudal DMV, abolished the inhibition of the distal stomach motility induced by water administration, the intermediate DMV is responsible for the inhibition shown in the distal stomach.

stomach; relaxation; superior laryngeal nerve; dorsal motor nucleus of the vagus; atropine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE AFFERENT NEURONS in the superior laryngeal nerve (SLN), which bifurcates from the vagal nerve, are responsible for protective reflexes such as apnea (45) and reflex swallowing (44). The SLN, which innervates taste buds distributed on the laryngeal surface of the epiglottis and on the larynx, responds to mechanical and chemical stimulation of the larynx and epiglottis (45). Individual fibers of the SLN are broadly chemosensitive, as are other gustatory nerves (6, 43). The most obvious difference between the sensitivities of chemosensitive fibers of the SLN and those in other gustatory nerves is in their response to water (6, 17, 43). Several studies have suggested that afferent neurons responding to water (water fiber) in the SLN are involved in important functions such as diuresis, prandial drinking, and cardiovascular responses (16, 33, 41). Our previous study revealed that water fiber in the SLN induced inhibition of gastric motility (21). This response may have a role in facilitating the reservoir function of the stomach. The inhibitory responses of the stomach have been abolished by sectioning of the SLN or cervical vagal nerve, suggesting the presence of vagovagal reflexes (21). However, the central processes and vagal motor output to induce this reflex are not yet known. Excitation of vagal motor fibers may cause either a facilitation or an inhibition of motility, suggesting two kinds of motor fibers (28). In fact, inhibition of vagal preganglionic fibers, whose excitation induces facilitation of motility, and excitation of vagal preganglionic fibers, whose excitation induces inhibition of motility, were observed during gastric relaxation caused by swallowing (31).

The consensus is that neuroeffector transmission from the excitatory vagal pathway is cholinergic and that it is consequently blocked by atropine or hyoscine (39). Because much of the tonic input to the stomach is excitatory, the ablation of tonic inputs to the stomach causes the inhibition of gastric motility (29, 32). Indeed, reduction of gastric tone induced by the antrum stretch receptor appears to be caused by the inhibition of vagal preganglionic neurons (29). In addition, nonadrenergic and noncholinergic (NANC) myenteric neurons, activated by excitation of vagal preganglionic neurons, induce the inhibition of gastric motility (1). Thus inhibition of gastric motility can occur because of the inhibition of vagal preganglionic neurons connected to cholinergic myenteric neurons and/or the excitation of vagal preganglionic neurons connected to NANC myenteric neurons. Anatomic studies revealed that the cell bodies of the vagal preganglionic neurons innervating the stomach are predominantly located at the dorsal motor nucleus of the vagus (DMV) (12, 35). Therefore, the DMV neurons have a role in the extrinsic control of gastric functions.

Recently, the heterogeneity of the specific pathway for the inhibition of gastric motility was clarified (38, 49). Neurophysiological recordings identified two subpopulations of DMV neurons (38). The intermediate DMV contains neurons that were excited in response to esophageal distension and also contained neurons that were inhibited. In contrast, the caudal DMV exclusively contained neurons that were excited in response to esophageal distension. An anatomic study showed that fundic projecting neurons, which revealed nitric oxide synthase immunoreactivity (NOS-IR), were exclusively observed in the caudal DMV (49). Thus the intermediate DMV and caudal DMV appear to be different in regulating gastric motor function.

The present study was undertaken to clarify the vagal output responsible for the inhibition in gastric motility induced by water fibers in the SLN found previously (21). The effects of separate lesions of the different portions of the DMV on the inhibitory response of the gastric motility induced by water-responsive afferents in the SLN were investigated. The effect of peripheral muscarinic blockade on the inhibition of gastric motility induced by the administration of water was also investigated. In addition, the response characteristics of the DMV neurons to the administration of water into the posterior oral cavity were investigated.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and common preparation. Animal care was in accordance with the guidelines of the Physiological Society of Japan. Male Sprague-Dawley rats (260-350 g) were used. Each animal was anesthetized with an intraperitoneal injection of urethane-chloralose (urethane, 0.8 g/kg; chloralose, 65 mg/kg body wt). Subsequent anesthesia was administered as required through Silastic tubing (OD 1.0 mm, ID 0.5 mm) inserted into the right jugular vein. Each animal had a tracheal cannula made from polyethylene tubing (OD 2.07 mm). The esophagus was ligated to prevent the test solutions from entering the stomach. In this study, the portion of the DMV located at the point of exit of the obex or located caudal to the obex was defined as the "caudal DMV"; the portion of the DMV located between the obex and anterior tip of the area postrema (AP) was defined as the "intermediate DMV" according to a previous study (49).

Recordings of distal stomach motility and the administration of water. The procedures for recording intragastric pressure and stimulating water fibers in the SLN were reported previously (21). Each animal was fasted for 1 day before starting the experiment to empty the stomach. After anesthesia, an abdominal incision was made. An intragastric balloon, created from thin latex rubber and plastic tubing (OD 1.7 mm), was introduced into the stomach from the greater curvature just proximal to the limiting ridge toward the antrum. The balloon was secured by purse sutures around the gastric wall using 4-0 silk thread. Another tubing (OD 2.0 mm) was also introduced into the fundus to drain the gastric juices and was ligated with the gastric wall around the tubing. The gastric balloon was inflated with warm water at 37°C to a volume of 3.0 ml/kg body wt. The distal end of this tubing was connected with a strain-gauge pressure meter (NEC-Sanei, 6M82) to measure intragastric pressure. To administer the test solutions, an incision was made between the thyroid and circular cartilage after intubation of the intragastric balloon. Polyethylene tubing (PE-50, Intramedic) connected to a syringe was inserted into the larynx. Test solutions (0.1 ml) were administered manually for ~10 s at room temperature. Delivered solutions probably spread into the larynx, onto the epiglottis and also onto the pharynx. Ten minutes after each administration of water, the larynx was rinsed twice with 0.15 M saline. The interval between the stimuli was at least 20 min. The integrity of the success of the stimuli has been reported previously (21).

Chemical lesions of the DMV. To evaluate the participation of the DMV neurons in the inhibition of gastric motility and to clarify the regional heterogeneity of the DMV, chemical lesions of the left DMV were performed. Separate lesions of the intermediate DMV and caudal DMV were made. To avoid difficulties producing symmetrical bilateral lesions, lesions were restricted to the left DMV. Before the lesions, animals underwent sectioning of the right cervical vagus to prevent efferent signals from preganglionic neurons of the right DMV. The right vagus was carefully separated from the right carotid artery, and then sectioning was performed as described previously (21). The vagus was sectioned at the cervical level below where the SLN entered the vagal trunk, allowing afferent signals into the central nervous system. After vagotomy, the fourth ventricle was opened, removing the occipital bone. The inhibitory responses of the gastric motility of the distal stomach were confirmed by the administration of water (0.1 ml) into the larynx in the supine position. After evaluation of the inhibitory response of the stomach, kainic acid (4.7 mM dissolved in 2% Pontamine sky blue and 0.9% saline solution, 30 or 60 nl) was injected using a microinjector (Nihon Kohden, XF-320J) in the prone position. A glass micropipette (5- to 10-µm tip diameter) was inserted into the left DMV through the dorsal surface of the medulla. Kainic acid was injected into the intermediate DMV (11 animals) or the caudal DMV (7 animals). The coordinates used relative to the obex were as follows: intermediate DMV, 0.5 mm rostral, 0.5 mm lateral, 0.5 mm ventral; caudal DMV, 0 mm rostral, 0.3 mm lateral, 0.5 mm ventral. After each injection, the animal was again secured in the supine position. Thirty minutes after the injection of kainic acid, water was again administered to evaluate the inhibitory response of the stomach. At the end of each experiment, the injection site was confirmed histologically by observing the extent of Pontamine sky blue. In six animals, saline was injected into the intermediate DMV as a control experiment (sham lesion). The contractile response was quantified by measuring the area under contractions (kPa × min) using the Flextrace program (Tree Star) on a personal computer. The area was measured at 1-min intervals starting at the beginning of the injection of the test solution. The area before injection was also measured as a control. The mean value of the area 2 min before administration of water and 2 min after was used for comparison between pre- and posttreatment (motility area). To normalize the data, the ratio of the area between 2 min before injection and 2 min after injection was also used for analyses (motility ratio). All numerical values are represented as means ± SE. Significant differences among the mean motility areas were evaluated using the paired t-test (P < 0.05 for significance). Significant differences between each mean motility ratio and 1.0 were also evaluated using the paired t-test (P < 0.05 for significance).

Effect of atropine on the inhibition of gastric motility. To demonstrate the cholinergic neurotransmission to the reflex in the periphery, the effects of atropine on the inhibition of distal stomach motility induced by the administration of water were investigated in eight animals. Atropine methyl nitrate, a peripherally acting muscarinic cholinergic receptor antagonist, was used (37). Atropine methyl nitrate (1.0 mg/kg body wt, Sigma), which was dissolved in 0.15 M saline, was administered intravenously. This dose of atropine completely blocked the increase in intragastric pressure induced by the electrical stimulation of the peripheral cut end of the cervical vagus nerve in the rat (26). Before the administration of atropine, the inhibitory response to the administration of water (0.1 ml) was confirmed. Intravenous injection of atropine reduced gastric tone and inhibited contractile activity. After the appearance of phasic contraction, the response of the stomach to the administration of water was again investigated (10-20 min after the administration of atropine). Significant differences among the mean motility areas were evaluated using the paired t-test (P < 0.05 for significance). Significant differences between each mean motility ratio and 1.0 were also evaluated using the paired t-test (P < 0.05 for significance).

The effects of atropine on the relaxatory response of the proximal stomach were also observed. Nine animals were used for this series of experiments. Each animal received both an administration of water and electrical stimulation of the central cut end of the SLN to elicit the relaxatory responses of the proximal stomach. The left SLNs were isolated from the surrounding tissue and sectioned as described previously (21). The central cut end of the SLN was stimulated using a 20-Hz repeat electric pulse (0.1 ms in duration, 0.1 mA in intensity) for 20 s using platinum bipolar electrodes, because swallowing appears to be optimally evoked at a frequency of 20-30 Hz (11). The gastric balloon was introduced into the proximal part of the stomach from the tip of the fundus. The balloon was inflated with 0.4-0.6 ml of water (37°C). The initial intragastric pressure was 0.5-0.7 kPa. After evaluation of the relaxation induced by the administration of water (0.1 ml) and electrical stimulation, atropine methyl nitrate (1.0 mg/kg body wt) was injected manually. Five minutes after the administration of atropine, the left SLN was electrically stimulated. Twelve minutes after the administration of atropine, water was administered. The degree of the effect of each stimulus was evaluated by the maximal fall in the baseline of the intragastric pressure. For statistical analysis, analysis of variance was used (P < 0.05 for significance). Fisher's post hoc test was used to make comparisons between each value.

Recordings of neural activity in the DMV. Neural activities of the DMV neurons were recorded in 50 animals. After an abdominal incision, a cuff stimulating electrode (Unique Medical, Japan) was attached to the (anterior) ventral subdiaphragmatic vagus without sectioning to identify the DMV neurons that innervate the abdominal viscera by antidromic stimulation. Another catheterization was done to measure arterial blood pressure. In 10 experiments, a cuff stimulating electrode was also attached to the left SLN without sectioning. Electrical stimulations were delivered at a rate of 0.5 per second. Each animal received bilateral sectioning of the chorda tympani and the glossopharyngeal nerve. Both sides of the tympanic membrane were removed, and the chorda tympani nerves were sectioned. Both sides of the glossopharyngeal nerve were also sectioned between the external and internal carotid artery as described previously (21). Polyethylene tubing (OD 2.3 mm, ID 1.5 mm) attached with a 15-gauge injection needle was introduced into the trachea rostrally to inject the test solutions. A suction system was used to remove the test solutions as described previously (6, 34, 43). The animals were neuromuscularly blocked using gallamine triethiodide (10 mg/kg iv, Sigma) and ventilated with O2-enriched room air and positive end-expiratory pressure using a positive pressure ventilator (Narishige, AR-2) to exclude the influence of swallowing movements. Arterial blood pressure was measured through a catheter inserted into the left carotid artery. During neuromuscular blockade, the depth of anesthesia was assessed by monitoring the stability of the arterial blood pressure and the heart rate and the cardiovascular responses to pinching the paws. Neural activity was recorded in the DMV through a glass microelectrode filled with a 2% solution of Pontamine sky blue in 1.0 M sodium acetate (impedance, 10-20 MOmega at 130 Hz). To minimize movements of the medulla caused by respiration and blood pulsation, a small metal ring was pressed onto the recording site. Test solutions were 0.15 M NaCl and water. The test solutions (1.0 ml) were administered manually for ~5 s via a syringe. The oral cavity was rinsed with isotonic saline (2.0 ml) three times 1 min after the administration of the test solutions. After recording of the antidromic response, test solutions were administered through the catheter manually. The penetrations were attempted in an area between 0.4 mm posterior and 1.0 mm anterior to the obex. Antidromic responses to the electrical stimulation of the anterior subdiaphragmatic vagus were recorded in the caudal and the intermediate DMV (see Fig. 6A). At the end of the experiments, recording sites were marked by Pontamine sky blue. Injection sites were confirmed histologically, observing the extent of Pontamine sky blue. The discharges for 5 s immediately before the administration of water were compared with those for 5 s after the administration. When the discharges changed by 50% or more compared with the prestimulus values, the neuron was designated responsive. Significant differences among the discharges for 5 s were evaluated using the paired t-test (P < 0.05 for significance).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of the DMV on the gastric response. The effects of the lesion of the intermediate DMV on the gastric motility of the distal stomach were investigated. Injection of kainic acid into the intermediate DMV were performed in 11 animals. Successful lesions of the intermediate DMV were confirmed histologically in seven animals. In the other four animals, injection sites were identified in the NST just dorsal to the DMV (2 examples) and the reticular formation lateral to the hypoglossal nucleus (2 examples). In these unsuccessful examples, a significant decrease in mean motility area induced by the administration of water was observed before the injection of kainic acid (t = 8.24, P < 0.05, n = 4) and after the injection (t = 5.45, P < 0.05, n = 4). Injection of kainic acid induced a transient increase in intragastric pressure followed by the inhibition of phasic contractions for several minutes in all successful examples. Ten minutes after the injection of kainic acid, phasic contractions were stabilized in frequency and in amplitude. Because the excitatory effect of kainic acid disappeared within 20 min as reported previously (22), the effect of the administration of water on the gastric motility was tested >30 min after the injection of kainic acid. Just before the administration of water, the motility area was larger than that before the lesion in three animals. However, no marked change in gastric motility was observed in the other four animals. Table 1 shows the analysis based on motility area. No significant differences were observed between mean motility area before the lesion and that after the lesion (n = 7). Before the lesion of the intermediate DMV, the mean motility area for 2 min after the administration of water was significantly smaller (t = 9.40, P < 0.05, n = 7) than that before the administration. After the lesion of the intermediate DMV as shown in Fig. 1A, however, no significant difference was observed (n = 7) between the mean motility area before the administration of water and that after the administration. The mean motility ratio observed before the lesion of the intermediate DMV was 0.69 ± 0.03 and that after the lesion was 0.98 ± 0.02 (Fig. 1B). A significant difference in the mean motility ratio compared with 1.0 was observed before the lesion (t = 10.72, P < 0.05, n = 7); however, no significant difference was observed after the lesion (n = 7). An injection of isotonic saline into the intermediate DMV was given to the other animals. Sham lesions of the intermediate DMV did not affect the inhibitory response of the stomach induced by the administration of water (Table 1). A significant difference in the mean motility ratio was observed before the sham lesion (t = 7.10, P < 0.05, n = 6) and after the sham lesion (t = 14.71, P < 0.05, n = 6).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effect of caudal or intermediate DMV lesions on the gastric motility induced by the administration of water



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 1.   Effects of dorsal motor nucleus of the vagus (DMV) lesions on the inhibitory response of motility in the distal stomach. Kainic acid was independently injected into 2 different sites of the left DMV. As controls, 0.15 M saline was also injected into the intermediate DMV (sham lesion). A: response of gastric motility to the administration of water before the injection of kainic acid or saline (Pre) and that after the injection (Post). The lesion of the intermediate DMV abolished the inhibitory response to the administration of water. Dashed lines show the baseline of the intragastric pressure. Filled circles show the administration of water. B: mean motility ratios of the responses are shown. * Significant difference in the value compared with 1.0. NS, not significant. C: locations of injection sites of kainic acid are indicated by filled circles. The areas that were marked by Pontamine sky blue are plotted on drawings of frontal sections from an atlas (36): 12, hypoglossal nucleus; AP, area postrema; DMV; NST, nucleus of the solitary tract; sol, solitary tract.

The effect of the lesion of the caudal DMV on the gastric motility of the distal stomach was investigated. Injection of kainic acid into the intermediate DMV was performed in seven animals. Successful lesions of the caudal DMV were confirmed histologically in six animals. The lesion of the caudal DMV reduced the contractility of the stomach in all animals tested (Fig. 1). Ten minutes after the injection of kainic acid, phasic contraction was stabilized. A significant difference was observed between the mean motility area before the lesion and that after the lesion (t = 7.06, P < 0.05, n = 6) (Table 1). Before the lesion, the mean motility area for 2 min after the administration of water was significantly smaller (t = 6.51, P < 0.05, n = 6) than that before the administration. After the lesion of the caudal DMV, as shown in Fig. 1A and Table 1, the lesion of the caudal DMV did not impair the inhibitory response in motility (t = 13.11, P < 0.05, n = 6). A significant difference was also observed (t = 13.11, P < 0.05, n = 6) between the mean motility area before the administration of water (1.58 ± 0.05 kPa × min, n = 6) and that after the administration (1.21 ± 0.06 kPa × min, n = 6). The mean motility ratio observed before the lesion of the caudal DMV was 0.65 ± 0.04 and that after the lesion was 0.77 ± 0.02 (Fig. 1B). A significant difference in the mean motility ratio compared with 1.0 was observed before the lesion (t = 7.98, P < 0.05, n = 6) and after the lesion (t = 12.58, P < 0.05, n = 6). The injection sites of the kainic acid are presented in Fig. 1C.

Cholinergic contribution to the gastric response. After the identification of the inhibitory response of gastric motility induced by the administration of water, an injection of atropine was given. The injection of atropine abolished the inhibition of contractile activity induced by the administration of water (Fig. 2). Before the injection of atropine, the mean motility area for 2 min after the administration of water (1.00 ± 0.20 kPa × min, n = 8) was significantly smaller (t = 5.490, P < 0.05, n = 8) than that before the administration of water (1.60 ± 0.27 kPa × min, n = 8). After the injection of atropine, however, no significant difference was observed (n = 8) between the mean motility area before the administration of water (0.78 ± 0.074 kPa × min, n = 8) and that after the administration of water (0.77 ± 0.077 kPa × min, n = 8). The mean motility ratio observed before the injection of atropine was 0.61 ± 0.055 and that after the injection of atropine was 0.99 ± 0.020. A significant difference in the mean motility ratio compared with 1.0 was observed before the injection of atropine (t = 6.98, P < 0.05, n = 8); however, no significant difference was observed after the injection of atropine (n = 8).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   Effects of injection of atropine on inhibitory response of motility in the distal stomach. The responses of gastric motility to the administration of water before the injection of atropine (preatropine; top) and after (postatropine; bottom) are shown. Filled circles show the administration of water. Dashed lines show the baseline of the intragastric pressure.

The effects of atropine on the relaxatory response of the proximal stomach induced by the administration of water into the larynx and the electrical stimulation of the central cut end of the left SLN were examined. Intravenous injection of atropine tended to reduce intragastric pressure transiently. However, the mean intragastric pressures immediately before the stimuli were not significantly different [F3,32 = 0.86, not significant (NS)] between the values obtained from four different conditions (the electrical stimulation of the SLN preatropine, 0.55 ± 0.039 kPa; the administration of water preatropine, 0.59 ± 0.040 kPa; the electrical stimulation of the SLN postatropine, 0.51 ± 0.042 kPa; the administration of water postatropine, 0.51 ± 0.044 kPa). Both the administrations of water and electrical stimulation induced relaxation of the proximal stomach. Figure 3 shows a typical example (Fig. 3A) and mean value (Fig. 3B). The administration of water and the electrical stimulation decreased the mean intragastric pressure by 0.12 ± 0.026 kPa (n = 9) and by 0.13 ± 0.020 kPa (n = 9), respectively. After the administration of atropine, the mean reduction in intragastric pressure induced by the administration of water was only 0.01 ± 0.006 kPa, and that induced by the electrical stimulation was 0.06 ± 0.014 kPa. The mean reduction in intragastric pressure was significantly different between the values obtained from four different conditions (F3,32 = 8.77, P < 0.05). The post hoc test showed that the administration of atropine decreased the magnitude of the mean reduction in intragastric pressure induced by the administration of water (P < 0.05) and that induced by the electrical stimulation of the SLN (P < 0.05). After the administration of atropine, the mean reduction in intragastric pressure induced by the electrical stimulation of the SLN was significantly larger than that induced by the administration of water (P < 0.05).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of the injection of atropine on relaxation in the proximal stomach. A: a sample recording of intragastric pressure in the proximal stomach. The magnitude of the reduction in intragastric pressure induced by the electrical stimulation of the superior laryngeal nerve (SLN-ES) decreased, and similar reductions induced by the administration of water (filled circles) were not observed after the injection of atropine. B: mean change in intragastric pressure induced by SLN-ES or the administration of water. * Significant differences compared with the mean decrease in intragastric pressure before the administration of atropine. dagger  Significant difference compared with the mean reduction in intragastric pressure induced by the administration of water after the administration of atropine.

Neural response of the DMV neurons. Antidromic responses to the electrical stimulation of the anterior subdiaphragmatic vagus of 101 neurons were recorded in the DMV. Nineteen and 82 were recorded in the caudal and in the intermediate DMV, respectively. The antidromic responses were ascertained by a cancellation of the elicited response by collision with spontaneous discharges, a constant latency, and the ability to follow double pulse stimulations with intervals <10 ms as described previously (19, 20). The cancellation of the antidromic response was always observed when the stimulus pulse triggered by the spontaneous impulse was delivered (Fig. 4A). Latencies of antidromic responses varied from 90 to 205 ms (140.9 ± 2.7 ms, n = 99). There were no marked differences between the mean latency of neurons that responded to the administration of water into the posterior oral cavity (water-responsive neurons: 137.9 ± 4.4 ms, n = 44) and that of neurons that did not respond to the administration of water (nonresponsive neurons: 143.3 ± 3.3 ms, n = 55).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4.   An example recording of a DMV neuron. Responses shown in A-C were obtained from 1 neuron. A: antidromic responses to electrical stimulation of the anterior subdiaphragmatic vagus (left) are shown. The electrical stimulation triggered by the spontaneous impulse did not elicit the antidromic response by collision (right). Filled circles show the electrical stimulation. B: response to the administration of water or 0.15 M NaCl. Filled circles show the administrations of the test solution. C: response to electrical stimulation of the left SLN. A lack of spontaneous discharges was observed after electrical stimulation. One-hundred sweeps were superimposed. Filled circles show the electrical stimulation.

Of 101 neurons that showed an antidromic response to electrical stimulation of the anterior subdiaphragmatic vagus, a change in the firing rate in response to the administration of water was observed in 10 caudal and 36 intermediate DMV neurons. All water-responsive neurons showed a decrease in firing rate after the administration of water but not 0.15 M NaCl (Fig. 4B). In five neurons (2 in the caudal and 3 in the intermediate DMV), 0.15 M saline could not be administered. No neuron in the DMV showed increases in the discharge rate in response to the administration of water. The other 55 neurons (46 in the caudal and 9 in the intermediate DMV) did not show changes in their firing rates after the administration of water. Figure 5 shows the mean discharges of the DMV neurons after the administration of water and 0.15 M NaCl in water-responsive neurons. In water-responsive neurons, the mean discharge for 5 s immediately after the administration of water (2.09 ± 0.38, n = 46) was significantly smaller (t = 11.42, P < 0.05, n = 46) than that immediately before the administration of water (7.26 ± 0.80, n = 46). No significant difference was observed between the mean discharge for 5 s immediately before the administration of 0.15 M NaCl (6.56 ± 0.783, n = 41) and that immediately after the administration of 0.15 M NaCl (6.37 ± 0.84, n = 41). Nonresponsive neurons did not show a significant difference between the mean discharge for 5 s immediately before the administration of water (11.78 ± 1.23, n = 55) and that immediately after the administration of water (11.20 ± 1.17, n = 55). The effect of electrical stimulation on the left SLN was observed in 10 neurons that showed decreased firing rates in response to the administration of water. All neurons tested showed a lack of a spontaneous discharge, suggesting inhibitory inputs from SLN afferent neurons (Fig. 4C).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5.   Mean discharges of the DMV neurons in response to the administration of water and 0.15 M NaCl. Mean impulse numbers every 5 s were plotted. Arrow indicates the time for the administration of the test solutions.

The recording sites of antidromic response to the electrical stimulation of the subdiaphragmatic vagus were plotted on the horizontal plane of the caudal medulla (Fig. 6A). Example stainings of recording sites of the neurons that responded to a laryngeal administration of water were plotted on drawings of the frontal sections taken from an atlas (36) (Fig. 6B). Eighteen sites in the left DMV were stained. No staining outside of the DMV was observed. Thus the recording positions were restricted to the DMV.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   A: recording sites for antidromic response to the electrical stimulation of the anterior subdiaphragmatic vagus are plotted on the horizontal plane of the caudal medulla. Open circles, recording sites of neurons that did not respond to the administration of water; filled circles, recording sites of neurons that responded to the administration of water. Numerals with each circle show the number of units recorded. B: locations of recording sites of the neurons that responded to the administration of water into the posterior oral cavity. The positions that were marked by Pontamine sky blue are plotted on drawings of frontal sections from an atlas (36). Abbreviations are as in Fig. 1.

In addition, the response to the administration of water into the posterior oral cavity was examined in 17 neurons that did not show an antidromic response to the electrical stimulation of the anterior subdiaphragmatic vagus. Among them, nine neurons increased their firing rate in response to the administration of water, four neurons decreased, and the other four neurons did not respond to the administration of water. Recording sites were confirmed in nine neurons. The recording sites marked by Pontamine sky blue were in the lateral part of the nucleus of the solitary tract (NST) (4 neurons), the hypoglossal nucleus (1 neuron), or the reticular formation lateral to the DMV (4 neurons). No staining was observed in the DMV.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The findings of the present study showed that the inhibition of vagal preganglionic neurons was responsible for the inhibition of gastric motility induced by the administration of water into the larynx. Chemical lesions of the intermediate DMV abolished the inhibition of the phasic contraction of the distal stomach induced by the administration of water. The neurons that showed an antidromic response to the electrical stimulation of the subdiaphragmatic vagus decreased their firing rate in response to the administration of water. The injection of atropine also abolished the inhibition of the phasic contraction of the distal stomach induced by the administration of water. Taken together, the inhibition of preganglionic neurons, whose cell bodies are situated in the intermediate DMV, are responsible for inhibiting contractions in the distal stomach via a cholinergic pathway.

The neuronal diversity of the DMV was demonstrated in previous studies (8, 18). Distension of the esophagus is well known to induce gastric relaxation and the inhibition of contraction (38). The intermediate DMV has at least two types of neurons (38). One is excited and the other is inhibited in response to esophageal distension (38). In contrast, the neurons in the caudal DMV are exclusively excited in response to esophageal distension (38). The caudal and extreme rostral, but not intermediate, DMV neurons revealed NOS-IR (23). Caudal DMV neurons projecting to the gastric fundus revealed NOS-IR, but the intermediate DMV neurons did not (49). Thus the DMV is functionally diverse in the regulation of gastric motility. In the present study, we demonstrated that injections of kainic acid into the intermediate DMV abolished the inhibition of distal stomach contractions induced by the administration of water. In acute experiments, the concentration of kainic acid used in the present research is considered to be adequate to ablate the excitability of neurons (2, 4, 22). In contrast, similar injections into the caudal DMV did not affect the inhibition in motility of the distal stomach. These findings indicate that vagal preganglionic neurons of the intermediate DMV play an essential role at least in the inhibition of distal stomach contractions induced by laryngeal water-responsive afferent neurons. In the present study, the effect of separate lesions of the DMV on the relaxatory response of the proximal stomach was not examined. Therefore, we cannot comment on the regional specificity of the DMV in the regulation of motility in the proximal stomach.

Receptive relaxation (9) is a well-known reflex induced by the extrinsic nervous system to facilitate reservoir function. The inhibitory response of the stomach induced by water fibers in the SLN may have a role in facilitating reservoir function such as receptive relaxation. It is proposed that NANC myenteric neurons are concerned with the gastric inhibitory response induced by the parasympathetic preganglionic neurons (1). The participation of NANC transmitters such as vasointestinal polypeptides and/or nitric oxide in vagi-induced gastric relaxation was demonstrated in rodents (3, 10, 24-27, 47). We did not test the effects of these NANC transmitters on the inhibition of gastric motility caused by the SLN afferents. In the present study, the inhibition of phasic contraction induced by the administration of water was not observed after the injection of atropine. Relaxation in the proximal stomach induced by the administration of water also disappeared after the injection of atropine. Because atropine methyl nitrate, which was used in the present study, does not pass the blood-brain barrier (37), cholinergic myenteric neurons are probable candidates responsible for both the relaxation of the proximal stomach and the inhibition of distal stomach contractions induced by the administration of water. Neural response of the DMV neurons also supports the idea that the ablation of tonic inputs to the stomach is responsible for the inhibitory response of the stomach induced by the administration of water, because DMV neurons were inhibited exclusively in response to the administration of water. However, relaxation of the proximal stomach, induced by electrical stimulation of the SLN, which contains water fibers, was decreased in magnitude, but did not disappear, after the administration of atropine. The afferent neurons in the SLN responded not only to water but also to other stimulants. The SLN afferent fibers also responded to the mechanical stimulation (5) in addition to other taste stimulants (6, 43). Simultaneous activation of these sensations may cause the activation of NANC-myenteric neurons.

Neurons in the DMV that antidromically responded to the electrical stimulation of the subdiaphragmatic vagus exclusively decreased their firing rate in response to the administration of water in animals that underwent denervations of other taste nerves. Therefore, ablation of tonic inputs to the stomach induced relaxation and the inhibition of contraction. Neurons in the NST showed excitation in response to the administration of water and the other taste stimulants (46). Most NST neurons showed an excitatory postsynaptic potential in response to the electrical stimulation of the SLN (30). The DMV neurons might receive superior laryngeal afferent information from the NST neurons via inhibitory synapses. In fact, water-responsive neurons in the DMV were exclusively inhibited in response to electrical stimulation of the SLN in the present study. An inhibitory postsynaptic potential induced by the electrical stimulation of the NST was observed in the DMV (13). Many investigators found that GABAergic inputs play a role in gastric motor functions (7, 42, 48). DMV neurons were almost exclusively inhibited by the distension of the gastric antrum (29). Therefore, GABAergic inputs may have a role in the gastric inhibitory response induced by superior laryngeal afferent inputs.

Perspectives

In the present study, we demonstrated the involvement of peripheral cholinergic neurons in the inhibition of gastric motility induced by SLN afferents. The contribution of NANC myenteric neurons was not verified in the present study. However, a NANC contribution is possible, because gastric relaxation induced by electrical stimulation of the SLN was not completely abolished after the administration of atropine. Although we could not find the excitatory response of DMV neurons induced by laryngeal afferent activation, anatomic studies revealed that the electrical stimulation of the SLN induced c-fos expression in the DMV of cats and mice (14, 40). These anatomic studies did not identify the organ where neurons project. Recently, c-fos expression after esophageal distension was observed in DMV neurons, identifying the projection to the stomach (15). A similar method might be applied to find out whether NANC neurons are involved in laryngeal afferent-mediated gastric inhibition.


    ACKNOWLEDGEMENTS

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 12832065 and 13671937).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Kobashi, Dept. of Oral Physiology, Okayama Univ. Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan (E-mail: mkobashi{at}md.okayama-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpregu.00180.2001

Received 26 March 2001; accepted in final form 15 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abrahamsson, H, and Jansson G. Vago-vagal gastro-gastric relaxation in the cat. Acta Physiol Scand 88: 289-295, 1973[ISI][Medline].

2.   Al-Chaer, ED, Westlund KN, and Willis WD. Nucleus gracilis: an integrator for visceral and somatic information. J Neurophysiol 78: 521-527, 1997[Abstract/Free Full Text].

3.   Boeckxstaens, GE, Pelckmans PA, Bogers JJ, Bult H, De Man JG, Oosterbosch L, Herman AG, and Van Maercke YM. Release of nitric oxide upon stimulation of nonadrenergic noncholinergic nerves in the rat gastric fundus. J Pharmacol Exp Ther 256: 441-447, 1991[Abstract/Free Full Text].

4.   Bonigut, S, Bonham AC, and Stebbins CL. Area postrema-induced inhibition of the exercise pressor reflex. Am J Physiol Heart Circ Physiol 272: H1650-H1655, 1997[Abstract/Free Full Text].

5.   Boushey, HA, Richardson PS, Widdicombe JG, and Wise JC. The response of laryngeal afferent fibres to mechanical and chemical stimuli. J Physiol (Lond) 240: 153-175, 1974[Abstract/Free Full Text].

6.   Bradley, RM, Stedman HM, and Mistretta CM. Superior laryngeal nerve response patterns to chemical stimulation of sheep epiglottis. Brain Res 276: 81-93, 1983[ISI][Medline].

7.   Broussard, DL, Li H, and Altschuler SM. Colocalization of GABA(A) and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat. Brain Res 763: 123-126, 1997[ISI][Medline].

8.   Browning, KN, Renehan WE, and Travagli RA. Electrophysiological and morphological heterogeneity of rat dorsal vagal neurones which project to specific areas of the gastrointestinal tract. J Physiol (Lond) 517: 521-532, 1999[Abstract/Free Full Text].

9.   Cannon, WB, and Lieb CW. The receptive relaxation of the stomach. Am J Physiol 29: 267-273, 1911.

10.   Desai, KM, Sessa WC, and Vane JR. Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature (Lond) 351: 477-479, 1991[Medline].

11.   Dotty, RW. Influence of stimulus pattern on reflex deglutition. Am J Physiol 166: 142-158, 1951.

12.   Ewart, WR, Jones MV, and King BF. Central origin of vagal nerve fibres innervating the fundus and corpus of the stomach in rat. J Auton Nerv Syst 25: 219-231, 1988[ISI][Medline].

13.   Fukuda, A, Minami T, Nabekura J, and Oomura Y. The effects of noradrenaline on neurones in the rat dorsal motor nucleus of the vagus, in vitro. J Physiol (Lond) 393: 213-231, 1987[Abstract/Free Full Text].

14.   Gestreau, C, Bianchi AL, and Grelot L. Differential brainstem Fos-like immunoreactivity after laryngeal-induced coughing and its reduction by codeine. J Neurosci 17: 9340-9352, 1997[Abstract/Free Full Text].

15.   Guo, JJ, Browning KN, Rogers RC, and Travagli RA. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus. Am J Physiol Gastrointest Liver Physiol 280: G361-G367, 2001[Abstract/Free Full Text].

16.   Hanamori, T, and Ishiko N. Cardiovascular responses to gustatory and mechanical stimulation of the nasopharynx in rats. Brain Res 619: 214-222, 1993[ISI][Medline].

17.   Harada, S, and Smith DV. Gustatory sensitivities of the hamster's soft palate. Chem Senses 17: 37-51, 1992[Abstract/Free Full Text].

18.   Jarvinen, MK, and Powley TL. Dorsal motor nucleus of the vagus neurons: a multivariate taxonomy. J Comp Neurol 403: 359-377, 1999[ISI][Medline].

19.   Kobashi, M, and Adachi A. Effect of portal infusion of hypertonic saline on neurons in the dorsal motor nucleus of the vagus in the rat. Brain Res 632: 174-179, 1993[ISI][Medline].

20.   Kobashi, M, and Adachi A. Effects of portal infusion of hypertonic and hypotonic solutions on neuronal activity in the rat dorsal motor nucleus of the vagus. Brain Res 706: 21-29, 1996[ISI][Medline].

21.   Kobashi, M, Mizutani M, and Matsuo R. Water stimulation of the posterior oral cavity induces inhibition of gastric motility. Am J Physiol Regulatory Integrative Comp Physiol 279: R778-R786, 2000[Abstract/Free Full Text].

22.   Koga, T, Qu RY, and Fukuda H. The central pattern generator for vomitting may exist in the reticular area dorsomedial to the retrofacial nucleus in dogs. Exp Brain Res 118: 139-147, 1998[ISI][Medline].

23.   Krowicki, ZK, Sharkey KA, Serron SC, Nathan NA, and Hornby PJ. Distribution of nitric oxide synthase in rat dorsal vagal complex and effects of microinjection of nitric oxide compounds upon gastric motor function. J Comp Neurol 377: 49-69, 1997[ISI][Medline].

24.   Lefebvre, RA, Baert E, and Barbier AJ. Influence of NG-nitro-L-arginine on non-adrenergic non-cholinergic relaxation in the guinea-pig gastric fundus. Br J Pharmacol 106: 173-179, 1992[ISI][Medline].

25.   Lefebvre, RA, De Vriese A, and Smits GJ. Influence of vasoactive intestinal polypeptide and NG-nitro-L-arginine methyl ester on cholinergic neurotransmission in the rat gastric fundus. Eur J Pharmacol 221: 235-242, 1992[ISI][Medline].

26.   Lefebvre, RA, Hasrat J, and Gobert A. Influence of NG-nitro-L-arginine methyl ester on vagally induced gastric relaxation in the anaesthetized rat. Br J Pharmacol 1992: 313-320, 1992.

27.   Li, CG, and Rand MJ. Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur J Pharmacol 191: 303-309, 1990[ISI][Medline].

28.   Martinson, J, and Muren A. Excitatory and inhibitory effects of vagus stimulation on gastric motility in the cat. Acta Physiol Scand 57: 309-316, 1963[ISI].

29.   McCann, MJ, and Rogers RC. Impact of antral mechanoreceptor activation on the vago-vagal reflex in the rat---functional zonation of responses. J Physiol (Lond) 453: 401-411, 1992[Abstract/Free Full Text].

30.   Mifflin, S. Laryngeal afferent inputs to the nucleus of the solitary tract. Am J Physiol Regulatory Integrative Comp Physiol 265: R269-R276, 1993[Abstract/Free Full Text].

31.   Miolan, JP. La motoricite de l'estomac et du sphincter oesophagien: Etude electromyographique: role de l'innervation extrinseque (PhD Thesis). Marseilles, France: Univ. of Aix-Marseille III, 1980.

32.   Miolan, JP, and Roman C. Discharge of efferent vagal fibers supplying gastric antrum: indirect study by nerve suture technique. Am J Physiol Endocrinol Metab Gastrointest Physiol 235: E366-E373, 1978[Free Full Text].

33.   Miyaoka, Y, Sakaguchi T, Yamazaki M, and Shingai T. Changes in water intake following pharyngolaryngeal deafferentation in the rat. Physiol Behav 40: 369-371, 1987[Medline].

34.   Miyaoka, Y, Shingai T, Takahashi Y, Nakamura JI, and Yamada Y. Responses of neurons in the parabrachial region of the rat to electrical stimulation of the superior laryngeal nerve and chemical stimulation of the larynx. Brain Res Bull 45: 95-100, 1998[ISI][Medline].

35.   Okumura, T, and Namiki M. Vagal motor neurons innervating the stomach are site-specifically organized in the dorsal motor nucleus of the vagus nerve in rats. J Auton Nerv Syst 29: 157-162, 1990[ISI][Medline].

36.   Paxinos, G, and Watson C. The Rat Brain in Stereotaxic Coordinates. Sydney, Australia: Academic, 1982.

37.   Rauhofer, EA, Greenberg D, and Smith GP. Peripheral atropinization does not change meal size controlled by prepyloric mechanisms. Physiol Behav 59: 237-240, 1996[Medline].

38.   Rogers, RC, Hermann GE, and Travagli RA. Brainstem pathways responsible for esophageal control of gastric motility and tone in the rat. J Physiol (Lond) 514: 369-383, 1999[Abstract/Free Full Text].

39.   Roman, C, and Gonella J. Extrinsic control of digestive tract motility. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR.. New York: Raven, 1987, p. 507-553.

40.   Sang, Q, and Goyal RK. Swallowing reflex and brain stem neurons activated by superior laryngeal nerve stimulation in the mouse. Am J Physiol Gastrointest Liver Physiol 280: G191-G200, 2001[Abstract/Free Full Text].

41.   Shingai, T, Miyaoka Y, and Shimada K. Diuresis mediated by the superior laryngeal nerve in rats. Physiol Behav 44: 431-433, 1988[Medline].

42.   Sivarao, DV, Krowicki ZK, and Hornby PJ. Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function. Neurogastroent Motil 10: 305-313, 1998[ISI][Medline].

43.   Smith, DV, and Hanamori T. Organization of gustatory sensitivities in hamster superior laryngeal nerve fibers. J Neurophysiol 65: 1098-1114, 1991[Abstract/Free Full Text].

44.   Storey, AT. Laryngeal initiation of swallowing. Exp Neurol 20: 359-365, 1968[ISI][Medline].

45.   Storey, AT, and Johnson P. Laryngeal water receptors initiating apnea in the lamb. Exp Neurol 47: 42-55, 1975[ISI][Medline].

46.   Sweazey, RD, and Bradley RM. Responses of lamb nucleus of the solitary tract neurons to chemical stimulation of the epiglottis. Brain Res 439: 195-210, 1988[ISI][Medline].

47.   Takahashi, T, and Owyang C. Characterization of vagal pathways mediating gastric accommodation reflex in rats. J Physiol (Lond) 504: 479-488, 1997[ISI][Medline].

48.   Travagli, RA, Gillis RA, Rossiter CD, and Vicini S. Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol Gastrointest Liver Physiol 260: G531-G536, 1991[Abstract/Free Full Text].

49.   Zheng, ZL, Rogers RC, and Travagli RA. Selective gastric projections of nitric oxide synthase-containing vagal brainstem neurons. Neuroscience 90: 685-694, 1999[ISI][Medline].

Am J Physiol Regul Integr Comp Physiol 282(3):R818-R827
0363-6119/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Niedringhaus, P. G. Jackson, S. R. T. Evans, J. G. Verbalis, R. A. Gillis, and N. Sahibzada
Dorsal motor nucleus of the vagus: a site for evoking simultaneous changes in crural diaphragm activity, lower esophageal sphincter pressure, and fundus tone
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R121 - R131.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. T. Cruz, E. C. Murphy, N. Sahibzada, J. G. Verbalis, and R. A. Gillis
A reevaluation of the effects of stimulation of the dorsal motor nucleus of the vagus on gastric motility in the rat
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R291 - R307.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Kobashi, Y. Shimatani, K. Shirota, S.-Y. Xuan, Y. Mitoh, and R. Matsuo
Central neuropeptide Y induces proximal stomach relaxation via Y1 receptors in the dorsal vagal complex of the rat
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2006; 290(2): R290 - R297.
[Abstract] [Full Text] [PDF]

Home page
 Chem SensesHome page
M. Kobashi, T. Koga, M. Mizutani, and R. Matsuo
Neural Mechanism and Possible Role of Inhibition of Gastric Motility Induced by Superior Laryngeal Afferents
Chem Senses, January 1, 2005; 30(suppl_1): i72 - i73.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kobashi, M.
Right arrow Articles by Matsuo, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kobashi, M.
Right arrow Articles by Matsuo, R.

HOME HELP FEEDBACK