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Projections to the alimentary canal from the dopaminergic neurons in the dorsal motor nucleus of the vagus of the rat
Autonomic Neuroscience: Basic and Clinical 123 (2005) 12 – 18 www.elsevier.com/locate/autneu
Projections to the alimentary canal from the dopaminergic neurons in the dorsal motor nucleus of the vagus of the rat Kiyoshi Tsukamoto a, Tetsu Hayakawa b,*, Seishi Maeda b, Koichi Tanaka b, Makoto Seki b, Takehira Yamamura a b
a Second Department of Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan Department of Anatomy, Hyogo College of Medicine, Mukogawa, Nishinomiya, Hyogo 663-8501, Japan
Received 2 May 2005; received in revised form 29 July 2005; accepted 30 July 2005
Abstract The motility of the alimentary canal is regulated not only by neurons that contain acetylcholine or adrenaline, but also by nonadrenergic noncholinergic neurons. There are many neurons containing dopamine in the dorsal motor nucleus of the vagus (DMV). We examined the projections of these dopaminergic neurons to the alimentary canal with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b following its injection into the subdiaphragmatic esophagus, the cardia, the pylorus, the duodenum, the jejunum, and the ascending colon. Almost all double-labeled neurons were found in the half of the DMV caudal to the area postrema. In the caudal half of the DMV, about 58% of the TH-immunoreactive neurons projected to the cardia, about 36% projected to the pylorus, and about 28% projected to the subdiaphragmatic esophagus. Only a few TH-immunoreactive neurons projected to the duodenum, the jejunum, or the ascending colon. As a whole, less than 10% of the neurons in the DMV that projected to the alimentary canal showed THlike immunoreactivity. These results suggest that some of the dopaminergic neurons in the DMV might regulate the activities of the stomach and the subdiaphragmatic esophagus. D 2005 Elsevier B.V. All rights reserved. Keywords: Retrograde tracing; Tyrosine hydroxylase; Gastrointestinal tract; Vagus nerve; Immunohistochemistry
1. Introduction Immunohistochemical studies have shown that there are many neurons that contain catecholamine in the dorsal motor nucleus of the vagus (DMV) (Armstrong et al., 1982). These neurons are mostly immunoreactive for tyrosine hydroxylase (TH), but not for either dopamine-h-hydroxylase or phenylethanolamine-N-methyltransferase, i.e., have a dopaminergic phenotype (Kalia et al., 1985). Most of the TH-containing neurons in the caudal DMV also contained choline acetyl transferase (ChAT) immunoreactivity (Armstrong et al., 1990). Subsequent studies showed that these
* Corresponding author. Tel.: +81 798 45 6484; fax: +81 798 45 9485. E-mail address: [email protected] (T. Hayakawa). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.07.009
dopaminergic neurons projected to the subdiaphragmatic organs (Yang et al., 1999) including the stomach (Tayo and Williams, 1988) just like the cholinergic neurons. Electrophysiological studies have reported that there were several kinds of neurons in the DMV that gastric and intestinal distention-sensitive neurons respond to stimulate and inhibit motility of the stomach or the duodenum (Fogel et al., 1996). Retrograde tracing studies using two kinds of tracers showed that the cardia and the pylorus were innervated by different neurons in the DMV (Hayakawa et al., 2003). The DMV neurons innervate the subdiaphragmatic esophagus and the stomach by the gastric branch, the duodenum by the hepatic branch, and the intestine and the colon by the celiac branch of the vagus nerve, respectively (Norgren and Smith, 1988; Berthoud et al., 1991; Hayakawa et al., 2002). Thus, it is important to clarify what parts of the
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alimentary canal receive projections from the dopaminergic neurons, and where the dopaminergic neurons projecting to the alimentary canal are located in the DMV. In the present study, we determine the number and the distribution of the dopaminergic neurons that project to the subdiaphragmatic alimentary canal of the rat using doublelabeling immunohistochemistry for TH and the retrograde tracer cholera toxin subunit b (CTb).
2. Materials and methods Twenty male Sprague – Dawley rats weighing 250 – 500 g were used. All surgical procedures were carried out with the animals under sodium pentobarbital anesthesia (40 – 50 mg/ kg, i.p.). The Animal Care and Use Committee of Hyogo College of Medicine approved the procedures. To investigate the distribution of TH-immunoreactive (TH-ir) neurons innervating the alimentary canal, we exposed the subdiaphragmatic alimentary canal by opening the abdominal wall and pushing aside the liver in order to inject CTb. Using a glass micropipette (tip diameter = 80 Am) affixed to a 10-Al Hamilton syringe, we injected with pressure five shots of 2 Al of 1% CTb (List Biological Laboratories, Campbell, CA) in distilled water into the anterior wall of the subdiaphragmatic esophagus (3 cases), the cardia (3 cases), the pylorus (3 cases), the duodenum (3 cases), the jejunum (3 cases), and the ascending colon (3 cases), respectively. Cotton swabs were used during the injections to limit the spread of the tracer to adjacent structures. To control for potential labeling due to the spread of tracer, 10 Al of 1% CTb was poured into the abdominal cavity (2 cases). The muscular layer and the skin were sutured to close the abdominal wound, and applied antibiotic ointment. The rats were recovered in an isolated cage on a heating pad without painkiller. When ambulatory and feeding, they were housed with food and water available ad libitum in a temperaturecontrolled room. Four days after the injection, the animals were anesthetized again with sodium pentobarbital (40 –50 mg/kg, i.p.), and perfused transcardially with 100 ml of physiologic saline and then with 500 ml of 4% paraformaldehyde –15% picric acid in 0.1 M phosphate buffer at pH 7.4 (PB). The brain was immediately removed and placed in the same fixative for 1 h. Serial frontal sections through the medulla oblongata including DMV were cut at a thickness of 70 Am with a Vibratome. After being rinsed with PB, the sections were incubated with 1% bovine serum albumin in PB containing 0.3% Triton X-100 and 0.9% NaCl (PBST) for 1 h. The sections were then incubated with a goat antiCTb serum (List Biological Laboratories; 1 : 20,000 dilution) in PBST for one day. The primary antibodies were localized by incubation with biotinylated donkey anti-goat IgG (Jackson Laboratories, West Grove, PA; 1 : 2000) for 4 h, and then with streptavidin-conjugated horseradish peroxidase (Jackson Laboratories; 1 : 2000) for 1 day. The sections were reacted with the Vector SG substrate kit
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(Vector Laboratories, Burlingame, CA) for 2 min to produce blue reaction products. After being rinsed with PB, the sections were incubated with 1% H2O2 in distilled water for 1 h. The sections were then incubated with a monoclonal mouse anti-TH serum (Chemicon, Temecula, CA; 1:400) in 1% bovine serum albumin and 1% normal rabbit serum in 0.1 M Tris buffer at pH 7.4 containing 0.3% Triton X-100 for 1 day. The primary antibodies were localized by incubation with rabbit anti-mouse IgG (Cappel, Durham, NC; 1 : 100) for 4 h, and then with mouse peroxidase antiperoxidase (Dako, Glostrup, Denmark; 1 : 120) for 1 day. The sections were then reacted with a solution of 0.2% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 0.1 M Tris buffer at pH 7.4 (Tris) for 3 min to produce brown reaction products. After rinsing with Tris, the sections were mounted on gelatin-coated glass slides. Then we counted TH-ir, CTb-labeled and double-labeled neurons in the DMV under an Olympus BX51 light microscope using a X20 objective manually. We recognized circular staining including nucleus as neurons, and another staining as nonspecific. The nomenclature for the nuclei in the medulla oblongata follows the atlas of Paxinos and Watson (1997).
3. Results Immunohistochemistry for TH revealed that many TH-ir neurons were located in the central part of the DMV caudal to the area postrema (Fig. 1A). A few TH-ir neurons were found at the ventromedial edge of the DMV at the level of the rostral end of the DMV (Fig. 1F), and in the medial part of the DMV at the level of the opening of the fourth ventricle. The number of TH-ir neurons decreased at the level of the rostral part of the area postrema, then increased from the level of the caudal part of the area postrema through the caudal end of the DMV (Fig. 2). Therefore, we divided the DMV into the caudal half of the DMV caudal to the area postrema and the rostral half of the DMV rostral to the area postrema to see the distribution pattern of the dopaminergic neurons projecting to the alimentary canal. When CTb was injected into the subdiaphragmatic esophagus, the center of the injection site was located below the diaphragm and about 2 cm rostral to the cardia. The vagus trunk innervates the subdiaphragmatic esophagus. Double-labeling immunohistochemistry for TH and CTb revealed double-labeled neurons with a brown cytoplasm for TH-ir that contained granular blue reaction products for the retrogradely transported CTb (Fig. 1B). Thus, double-labeled neurons were clearly distinguished from single CTb-labeled or single TH-labeled neurons. We counted TH-ir, CTb-labeled, and double-labeled neurons including nucleus on the left side of the DMV from every section of all three cases. Many CTb-labeled neurons were found in the DMV. About 80% of them were located in the
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Fig. 1. Color photomicrographs of TH-ir, CTb-labeled and double-labeled neurons in the dorsal motor nucleus of the vagus (DMV). (A) TH-ir neurons (brown) distributed throughout the DMV as well as the nucleus of the solitary tract (NTS). (B) TH-ir neurons (brown), retrogradely CTb-labeled neurons (granular blue) and double-labeled neurons immunoreactive for both TH and retrogradely transported CTb (a granular blue product against a brown cytoplasm) in the caudal half of the DMV after CTb injection into the subdiaphragmatic esophagus. (C) Many double-labeled neurons in the caudal half of the DMV after CTb injection into the cardia. (D) Many double-labeled neurons in the caudal half of the DMV after CTb injection into the pylorus. (E) One double-labeled neuron in the caudal half of the DMV after CTb injection into the ascending colon. (F) Many CTb-labeled neurons and a few TH-ir neurons in the rostral half of the DMV after CTb injection into the cardia. Note that no double-labeled neurons were present in this section. Arrows indicate double-labeled neurons, arrowheads indicate retrogradely CTb-labeled neurons, and double arrows indicate TH-ir neurons. Scale bars = 100 Am in A, 25 Am in B, and 50 Am in C – F.
caudal half of the DMV (Table 1). The distributions of double-labeled neurons in each injection site were showed as the typical cases in Fig. 2. The TH-CTb double-labeled neurons were found exclusively in the caudal half of the DMV (Fig. 2A). The average percentage of double-labeled neurons per TH-ir neuron was 28.6% in the caudal half of the DMV (Table 1). When CTb was injected into the cardia, the injection site was centered at the anterior wall of cardia. The gastric branch of the vagus trunk innervates the cardia. The halo of CTb extended to the oral part of the fundus. Numerous CTblabeled neurons were found throughout the DMV. The number of CTb-labeled neurons was the largest of the cases. Almost all double-labeled neurons were found in the caudal half of the DMV (Fig. 1C), but few were found in the rostral half of the DMV (Figs. 1F and 2B). The average percentages of double-labeled neurons per TH-ir neuron
were 58.3% in the caudal half of the DMV and 7.4% in the rostral half of the DMV (Table 1). When CTb was injected into the pylorus, the injection site was centered at the anterior wall of the pylorus ring. The hepatic and the gastric branches of the vagus trunk innervate the pylorus. Many CTb-labeled neurons were found throughout the DMV. Many double-labeled neurons were also found in the caudal half of the DMV (Fig. 1D), but only a few were in the rostral half of the DMV. The distribution pattern of the double-labeled neurons in the DMV was similar to those in the cases of CTb injections into the subdiaphragmatic esophagus and the cardia (Fig. 2C). The average percentages of double-labeled neurons per TH-ir neuron were 36.5% in the caudal half and 1.9% in the rostral half of the DMV (Table 1). When CTb was injected into the duodenum, the injection site was at the descending part of the duodenum. The
K. Tsukamoto et al. / Autonomic Neuroscience: Basic and Clinical 123 (2005) 12 – 18
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Fig. 2. Histograms showing the distribution of the TH-ir neurons (TH) and the neurons double-labeled for TH and retrogradely transported CTb (TH-CTb) in the DMV obtained from every second section after injection of CTb into the alimentary canal. The arrow indicates the level of the rostral end of the area postrema. (A) The results of CTb injection into the subdiaphragmatic esophagus in case T19. (B) The results of CTb injection into the cardia in case T1. (C) The results of CTb injection of into the pylorus in case T8. (D) The results of CTb injection into the duodenum in case T14. (E) The results of CTb injection into the jejunum in case T9. (F) The results of CTb injection into the ascending colon in case T10.
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Table 1 Distribution of single- and double-labeled neurons in the DMV of the rat Rostral half of DMV
Subdiaphragmatic Esophagus (n = 3) Cardia (n = 3) Pylorus (n = 3) Duodenum (n = 3) Jejunum (n = 3) Ascending colon (n = 3)
Caudal half of DMV
Whole DMV
Double/TH-ir
Double/CTb
Double/TH-ir
Double/CTb
Double/TH-ir
Double/CTb
(%)
(%)
(%)
(%)
(%)
(%)
0.0/36.0 (0.0)
0.0/191.0 (0.0)
67.0/234.0 (28.6)
67.0/933.7 (7.2)
67.0/270.0 (24.8)
67.0/1124.7 (6.0)
5.0/68.0 (7.4) 1.3/70.0 (1.9) 0.3/50.7 (0.5) 0.3/57.7 (0.5) 0.0/61.3 (0.0)
5.0/875.3 (0.6) 1.3/734.3 (0.2) 0.3/161.7 (0.2) 0.3/22.7 (1.3) 0.0/149.3 (0.0)
109.0/187.0 (58.3) 66.0/181.0 (36.5) 8.0/228.3 (3.5) 1.7/215.3 (0.8) 6.3/188.0 (3.3)
109.0/1264.0 (8.6) 66.0/676.3 (9.8) 8.0/189.3 (4.2) 1.7/61.7 (2.8) 6.3/281.7 (2.2)
114.0/255.0 (44.7) 67.3/251.0 (26.8) 8.3/279.0 (3.0) 2.0/273.0 (0.7) 6.3/249.3 (2.5)
114.0/2139.3 (5.3) 67.3/1410.6 (4.8) 8.3/351.0 (2.4) 2.0/84.4 (2.4) 6.3/431.0 (1.5)
The mean numbers of TH-labeled neurons, retrogradely CTb-labeled neurons, and double-labeled neurons; and the percentages of double-labeled neurons per TH-ir neuron and double-labeled neurons per CTb-labeled neuron in the DMV calculated from the values for every section of all three cases of CTb injections into each part of the alimentary canal.
hepatic branch of the vagus trunk innervates the descending part of the duodenum. The CTb-labeled neurons were scattered throughout the DMV. Only a few double-labeled neurons were found in the caudal half of the DMV (Fig. 2D). The average percentages of double-labeled neurons per TH-ir neuron were very low and were about 3.5% in the caudal half of the DMV (Table 1). When CTb was injected into the jejunum, the injection site was at about 10 cm caudal to the duodenojejunal flexure. The celiac branch of the vagus trunk innervates the jejunum. A few CTb-labeled neurons were found in the DMV. There were only a few double-labeled neurons (Fig. 2E). We counted only 6 double-labeled neurons in the DMV from the three cases. The average percentages of doublelabeled neurons per TH-ir neuron were 0.8% in the caudal half and 0.5% in the rostral half of the DMV (Table 1). When CTb was injected into the ascending colon, the injection site was the anterior wall of the colon between the cecum and the hepatic flexure. The celiac branch of the vagus trunk innervates the ascending colon. A moderate number of CTb-labeled neurons was found in the DMV (Table 1). Only a few double-labeled neurons were found in the caudal half (Fig. 1E) and none in the rostral half of the DMV (Fig. 2F). The average percentage of double-labeled neurons per TH-labeled neuron was 3.3% in the caudal half of the DMV (Table 1). In the control experiments of the CTb pouring over the abdominal cavity, no retrogradely labeled neurons were found in the medulla oblongata including the DMV. As a whole, almost all double-labeled neurons were located in the caudal half of the DMV (Table 1). The percentages of double-labeled neurons per CTb-labeled neuron were small and less than 10% in the caudal half of the DMV in all cases. This is because the numbers of CTblabeled neurons were larger than those of TH-ir neurons in
the cases of injections into the esophagus and the stomach (Table 1).
4. Discussion The present results demonstrated that almost all dopaminergic neurons in the caudal half of the DMV project to the subdiaphragmatic esophagus and the stomach, while only a few dopaminergic neurons projected to the duodenum, the jejunum or the colon. Double-labeling studies combined retrograde fluorescent tracers with TH-immunohistochemistry have shown that there are several TH-ir neurons that project to the stomach in the caudal half of the DMV (Tayo and Williams, 1988; Yang et al., 1999; Guo et al., 2001). Our results confirmed that many TH-ir neurons in the DMV project not only to the stomach, but also to the subdiaphragmatic esophagus. The percentages of the double-labeled neurons per TH-ir neuron were less than 10% in the studies using fluorescent tracers. In the present results, about 60% of the TH-ir neurons in the caudal half of the DMV projected to the cardia, about 40% of them to the pylorus, and about 30% of them to the esophagus. These differences may be due to the fact that immunohistochemistry for the retrogradely transported CTb labeled more neurons in the DMV than the retrogradely transported fluorescent tracers. Hayakawa et al. (2004) reported that about 80% of the TH-ir neurons in the caudal half of the DMV projected to the stomach. It is not appropriate to make a simple comparison of the percentages of the double-labeled neurons in the DMV, because the cardia and the pylorus are innervated by different neurons in the DMV, whereas the cardia and the corpus of the stomach receive collateral projections from the DMV neurons (Hayakawa et al., 2003).
K. Tsukamoto et al. / Autonomic Neuroscience: Basic and Clinical 123 (2005) 12 – 18
The ascending colon is also likely to be innervated by neurons which are different from the neurons that project to the stomach (unpublished observations). Anterograde and retrograde tracing studies have revealed that the DMV neurons innervate the stomach by way of the gastric branch, the duodenum by way of the hepatic branch, and the small intestine and the colon by way of the celiac branch of the vagus nerve (Berthoud et al., 1991). These reports together with our present results indicate that the esophagus and the stomach are innervated by neurons in the DMV that contain dopamine or acetylcholine, but the small intestine and the colon are innervated by the neurons that contain only acetylcholine. There are also several TH-ir neurons present in the rostral half of the DMV that do not project to the alimentary canal. The neurons projecting to the heart, the lung and the trachea are located throughout the DMV (Kalia and Mesulam, 1980). However, no TH-ir neurons were found among the neurons projecting to the heart in the DMV (Takanaga et al., 2003). When retrograde tracers were applied to the glossopharyngeal nerve or the lingual branch of the glossopharyngeal nerve, a few retrogradely labeled neurons were recognized in the rostral part of the DMV, which extended to the inferior salivatory nucleus (Matesz and Szekely, 1983; Hamilton and Norgren, 1984; Kim et al., 2004). Thus, the dopaminergic neurons in the rostral half of the DMV may send fibers to these glossopharyngeal nerves. The nonadrenergic noncholinergic inhibitory neurons in the myenteric ganglia seem to play an important role in the regulation of movements in all parts of the alimentary canal, including the relaxation reflex of the stomach (Abrahamsson, 1986; Costa et al., 1996; Furness, 2000). Electrophysiological studies have shown that the relaxation reflex is elicited by the discharge of nitric oxide, vasoactive intestinal polypeptide (VIP) or cholecystokinin (CCK) in the myenteric ganglia (Takahashi and Owyang, 1997). Because there are only a few neurons that contain nitric oxide synthase in the DMV (Hyland et al., 2001; Takanaga et al., 2003), nitric oxide are released mainly from the neurons of the myenteric ganglion of the stomach but from the DMV neurons. The neurons in the myenteric ganglion of the stomach contain many neurotransmitters or neuromodulators such as the calbindin, calretinin, ChAT, enkephalin, neuropeptide Y, VIP, CCK, serotonin, somatostatin, substance P, and nitric oxide (Reiche et al., 2001; Schemann et al., 2001). There are no neurons containing these chemicals in the DMV. On the other hand, the administration of dopamine elicits the relaxation of the lower esophagus or the stomach (Valenzuela, 1976a; Ulvestad and Gerner, 1985; Lefebvre, 1992). There are dopamine receptors in the smooth muscles of the lower esophagus or the stomach (de Carle and Christensen, 1976; Lefebvre, 1992). Our present results revealed that the neurons containing dopamine in the DMV project to the esophagus and stomach, but not project to the small intestine and the colon. The dopamine containing
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neurons in the DMV may innervate the smooth muscles to inhibit the muscle activity. Thus, they contribute to the relaxation reflex of the esophagus and the stomach, but not to the peristalsis of the intestine (Valenzuela, 1976a,b; Bueno et al., 1983; Sorraing et al., 1984). Though most of the dopaminergic neurons in the caudal half of the DMV also expressed ChAT, it is not clear if the terminals of the vagal motor neurons release dopamine and acetylcholine simultaneously from the same terminal. Further studies are needed to address what kinds of terminals release dopamine or acetylcholine, and what kinds of cells in the alimentary canal receive the projections of the dopaminergic neurons in the DMV.
Acknowledgments The authors thank Ms. M. Hatta and Mr. K. Gion for their technical assistance.
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Projections to the alimentary canal from the dopaminergic neurons in the dorsal motor nucleus of the vagus of the rat Kiyoshi Tsukamoto a, Tetsu Hayakawa b,*, Seishi Maeda b, Koichi Tanaka b, Makoto Seki b, Takehira Yamamura a b
a Second Department of Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan Department of Anatomy, Hyogo College of Medicine, Mukogawa, Nishinomiya, Hyogo 663-8501, Japan
Received 2 May 2005; received in revised form 29 July 2005; accepted 30 July 2005
Abstract The motility of the alimentary canal is regulated not only by neurons that contain acetylcholine or adrenaline, but also by nonadrenergic noncholinergic neurons. There are many neurons containing dopamine in the dorsal motor nucleus of the vagus (DMV). We examined the projections of these dopaminergic neurons to the alimentary canal with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b following its injection into the subdiaphragmatic esophagus, the cardia, the pylorus, the duodenum, the jejunum, and the ascending colon. Almost all double-labeled neurons were found in the half of the DMV caudal to the area postrema. In the caudal half of the DMV, about 58% of the TH-immunoreactive neurons projected to the cardia, about 36% projected to the pylorus, and about 28% projected to the subdiaphragmatic esophagus. Only a few TH-immunoreactive neurons projected to the duodenum, the jejunum, or the ascending colon. As a whole, less than 10% of the neurons in the DMV that projected to the alimentary canal showed THlike immunoreactivity. These results suggest that some of the dopaminergic neurons in the DMV might regulate the activities of the stomach and the subdiaphragmatic esophagus. D 2005 Elsevier B.V. All rights reserved. Keywords: Retrograde tracing; Tyrosine hydroxylase; Gastrointestinal tract; Vagus nerve; Immunohistochemistry
1. Introduction Immunohistochemical studies have shown that there are many neurons that contain catecholamine in the dorsal motor nucleus of the vagus (DMV) (Armstrong et al., 1982). These neurons are mostly immunoreactive for tyrosine hydroxylase (TH), but not for either dopamine-h-hydroxylase or phenylethanolamine-N-methyltransferase, i.e., have a dopaminergic phenotype (Kalia et al., 1985). Most of the TH-containing neurons in the caudal DMV also contained choline acetyl transferase (ChAT) immunoreactivity (Armstrong et al., 1990). Subsequent studies showed that these
* Corresponding author. Tel.: +81 798 45 6484; fax: +81 798 45 9485. E-mail address: [email protected] (T. Hayakawa). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.07.009
dopaminergic neurons projected to the subdiaphragmatic organs (Yang et al., 1999) including the stomach (Tayo and Williams, 1988) just like the cholinergic neurons. Electrophysiological studies have reported that there were several kinds of neurons in the DMV that gastric and intestinal distention-sensitive neurons respond to stimulate and inhibit motility of the stomach or the duodenum (Fogel et al., 1996). Retrograde tracing studies using two kinds of tracers showed that the cardia and the pylorus were innervated by different neurons in the DMV (Hayakawa et al., 2003). The DMV neurons innervate the subdiaphragmatic esophagus and the stomach by the gastric branch, the duodenum by the hepatic branch, and the intestine and the colon by the celiac branch of the vagus nerve, respectively (Norgren and Smith, 1988; Berthoud et al., 1991; Hayakawa et al., 2002). Thus, it is important to clarify what parts of the
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alimentary canal receive projections from the dopaminergic neurons, and where the dopaminergic neurons projecting to the alimentary canal are located in the DMV. In the present study, we determine the number and the distribution of the dopaminergic neurons that project to the subdiaphragmatic alimentary canal of the rat using doublelabeling immunohistochemistry for TH and the retrograde tracer cholera toxin subunit b (CTb).
2. Materials and methods Twenty male Sprague – Dawley rats weighing 250 – 500 g were used. All surgical procedures were carried out with the animals under sodium pentobarbital anesthesia (40 – 50 mg/ kg, i.p.). The Animal Care and Use Committee of Hyogo College of Medicine approved the procedures. To investigate the distribution of TH-immunoreactive (TH-ir) neurons innervating the alimentary canal, we exposed the subdiaphragmatic alimentary canal by opening the abdominal wall and pushing aside the liver in order to inject CTb. Using a glass micropipette (tip diameter = 80 Am) affixed to a 10-Al Hamilton syringe, we injected with pressure five shots of 2 Al of 1% CTb (List Biological Laboratories, Campbell, CA) in distilled water into the anterior wall of the subdiaphragmatic esophagus (3 cases), the cardia (3 cases), the pylorus (3 cases), the duodenum (3 cases), the jejunum (3 cases), and the ascending colon (3 cases), respectively. Cotton swabs were used during the injections to limit the spread of the tracer to adjacent structures. To control for potential labeling due to the spread of tracer, 10 Al of 1% CTb was poured into the abdominal cavity (2 cases). The muscular layer and the skin were sutured to close the abdominal wound, and applied antibiotic ointment. The rats were recovered in an isolated cage on a heating pad without painkiller. When ambulatory and feeding, they were housed with food and water available ad libitum in a temperaturecontrolled room. Four days after the injection, the animals were anesthetized again with sodium pentobarbital (40 –50 mg/kg, i.p.), and perfused transcardially with 100 ml of physiologic saline and then with 500 ml of 4% paraformaldehyde –15% picric acid in 0.1 M phosphate buffer at pH 7.4 (PB). The brain was immediately removed and placed in the same fixative for 1 h. Serial frontal sections through the medulla oblongata including DMV were cut at a thickness of 70 Am with a Vibratome. After being rinsed with PB, the sections were incubated with 1% bovine serum albumin in PB containing 0.3% Triton X-100 and 0.9% NaCl (PBST) for 1 h. The sections were then incubated with a goat antiCTb serum (List Biological Laboratories; 1 : 20,000 dilution) in PBST for one day. The primary antibodies were localized by incubation with biotinylated donkey anti-goat IgG (Jackson Laboratories, West Grove, PA; 1 : 2000) for 4 h, and then with streptavidin-conjugated horseradish peroxidase (Jackson Laboratories; 1 : 2000) for 1 day. The sections were reacted with the Vector SG substrate kit
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(Vector Laboratories, Burlingame, CA) for 2 min to produce blue reaction products. After being rinsed with PB, the sections were incubated with 1% H2O2 in distilled water for 1 h. The sections were then incubated with a monoclonal mouse anti-TH serum (Chemicon, Temecula, CA; 1:400) in 1% bovine serum albumin and 1% normal rabbit serum in 0.1 M Tris buffer at pH 7.4 containing 0.3% Triton X-100 for 1 day. The primary antibodies were localized by incubation with rabbit anti-mouse IgG (Cappel, Durham, NC; 1 : 100) for 4 h, and then with mouse peroxidase antiperoxidase (Dako, Glostrup, Denmark; 1 : 120) for 1 day. The sections were then reacted with a solution of 0.2% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 0.1 M Tris buffer at pH 7.4 (Tris) for 3 min to produce brown reaction products. After rinsing with Tris, the sections were mounted on gelatin-coated glass slides. Then we counted TH-ir, CTb-labeled and double-labeled neurons in the DMV under an Olympus BX51 light microscope using a X20 objective manually. We recognized circular staining including nucleus as neurons, and another staining as nonspecific. The nomenclature for the nuclei in the medulla oblongata follows the atlas of Paxinos and Watson (1997).
3. Results Immunohistochemistry for TH revealed that many TH-ir neurons were located in the central part of the DMV caudal to the area postrema (Fig. 1A). A few TH-ir neurons were found at the ventromedial edge of the DMV at the level of the rostral end of the DMV (Fig. 1F), and in the medial part of the DMV at the level of the opening of the fourth ventricle. The number of TH-ir neurons decreased at the level of the rostral part of the area postrema, then increased from the level of the caudal part of the area postrema through the caudal end of the DMV (Fig. 2). Therefore, we divided the DMV into the caudal half of the DMV caudal to the area postrema and the rostral half of the DMV rostral to the area postrema to see the distribution pattern of the dopaminergic neurons projecting to the alimentary canal. When CTb was injected into the subdiaphragmatic esophagus, the center of the injection site was located below the diaphragm and about 2 cm rostral to the cardia. The vagus trunk innervates the subdiaphragmatic esophagus. Double-labeling immunohistochemistry for TH and CTb revealed double-labeled neurons with a brown cytoplasm for TH-ir that contained granular blue reaction products for the retrogradely transported CTb (Fig. 1B). Thus, double-labeled neurons were clearly distinguished from single CTb-labeled or single TH-labeled neurons. We counted TH-ir, CTb-labeled, and double-labeled neurons including nucleus on the left side of the DMV from every section of all three cases. Many CTb-labeled neurons were found in the DMV. About 80% of them were located in the
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Fig. 1. Color photomicrographs of TH-ir, CTb-labeled and double-labeled neurons in the dorsal motor nucleus of the vagus (DMV). (A) TH-ir neurons (brown) distributed throughout the DMV as well as the nucleus of the solitary tract (NTS). (B) TH-ir neurons (brown), retrogradely CTb-labeled neurons (granular blue) and double-labeled neurons immunoreactive for both TH and retrogradely transported CTb (a granular blue product against a brown cytoplasm) in the caudal half of the DMV after CTb injection into the subdiaphragmatic esophagus. (C) Many double-labeled neurons in the caudal half of the DMV after CTb injection into the cardia. (D) Many double-labeled neurons in the caudal half of the DMV after CTb injection into the pylorus. (E) One double-labeled neuron in the caudal half of the DMV after CTb injection into the ascending colon. (F) Many CTb-labeled neurons and a few TH-ir neurons in the rostral half of the DMV after CTb injection into the cardia. Note that no double-labeled neurons were present in this section. Arrows indicate double-labeled neurons, arrowheads indicate retrogradely CTb-labeled neurons, and double arrows indicate TH-ir neurons. Scale bars = 100 Am in A, 25 Am in B, and 50 Am in C – F.
caudal half of the DMV (Table 1). The distributions of double-labeled neurons in each injection site were showed as the typical cases in Fig. 2. The TH-CTb double-labeled neurons were found exclusively in the caudal half of the DMV (Fig. 2A). The average percentage of double-labeled neurons per TH-ir neuron was 28.6% in the caudal half of the DMV (Table 1). When CTb was injected into the cardia, the injection site was centered at the anterior wall of cardia. The gastric branch of the vagus trunk innervates the cardia. The halo of CTb extended to the oral part of the fundus. Numerous CTblabeled neurons were found throughout the DMV. The number of CTb-labeled neurons was the largest of the cases. Almost all double-labeled neurons were found in the caudal half of the DMV (Fig. 1C), but few were found in the rostral half of the DMV (Figs. 1F and 2B). The average percentages of double-labeled neurons per TH-ir neuron
were 58.3% in the caudal half of the DMV and 7.4% in the rostral half of the DMV (Table 1). When CTb was injected into the pylorus, the injection site was centered at the anterior wall of the pylorus ring. The hepatic and the gastric branches of the vagus trunk innervate the pylorus. Many CTb-labeled neurons were found throughout the DMV. Many double-labeled neurons were also found in the caudal half of the DMV (Fig. 1D), but only a few were in the rostral half of the DMV. The distribution pattern of the double-labeled neurons in the DMV was similar to those in the cases of CTb injections into the subdiaphragmatic esophagus and the cardia (Fig. 2C). The average percentages of double-labeled neurons per TH-ir neuron were 36.5% in the caudal half and 1.9% in the rostral half of the DMV (Table 1). When CTb was injected into the duodenum, the injection site was at the descending part of the duodenum. The
K. Tsukamoto et al. / Autonomic Neuroscience: Basic and Clinical 123 (2005) 12 – 18
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Fig. 2. Histograms showing the distribution of the TH-ir neurons (TH) and the neurons double-labeled for TH and retrogradely transported CTb (TH-CTb) in the DMV obtained from every second section after injection of CTb into the alimentary canal. The arrow indicates the level of the rostral end of the area postrema. (A) The results of CTb injection into the subdiaphragmatic esophagus in case T19. (B) The results of CTb injection into the cardia in case T1. (C) The results of CTb injection of into the pylorus in case T8. (D) The results of CTb injection into the duodenum in case T14. (E) The results of CTb injection into the jejunum in case T9. (F) The results of CTb injection into the ascending colon in case T10.
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Table 1 Distribution of single- and double-labeled neurons in the DMV of the rat Rostral half of DMV
Subdiaphragmatic Esophagus (n = 3) Cardia (n = 3) Pylorus (n = 3) Duodenum (n = 3) Jejunum (n = 3) Ascending colon (n = 3)
Caudal half of DMV
Whole DMV
Double/TH-ir
Double/CTb
Double/TH-ir
Double/CTb
Double/TH-ir
Double/CTb
(%)
(%)
(%)
(%)
(%)
(%)
0.0/36.0 (0.0)
0.0/191.0 (0.0)
67.0/234.0 (28.6)
67.0/933.7 (7.2)
67.0/270.0 (24.8)
67.0/1124.7 (6.0)
5.0/68.0 (7.4) 1.3/70.0 (1.9) 0.3/50.7 (0.5) 0.3/57.7 (0.5) 0.0/61.3 (0.0)
5.0/875.3 (0.6) 1.3/734.3 (0.2) 0.3/161.7 (0.2) 0.3/22.7 (1.3) 0.0/149.3 (0.0)
109.0/187.0 (58.3) 66.0/181.0 (36.5) 8.0/228.3 (3.5) 1.7/215.3 (0.8) 6.3/188.0 (3.3)
109.0/1264.0 (8.6) 66.0/676.3 (9.8) 8.0/189.3 (4.2) 1.7/61.7 (2.8) 6.3/281.7 (2.2)
114.0/255.0 (44.7) 67.3/251.0 (26.8) 8.3/279.0 (3.0) 2.0/273.0 (0.7) 6.3/249.3 (2.5)
114.0/2139.3 (5.3) 67.3/1410.6 (4.8) 8.3/351.0 (2.4) 2.0/84.4 (2.4) 6.3/431.0 (1.5)
The mean numbers of TH-labeled neurons, retrogradely CTb-labeled neurons, and double-labeled neurons; and the percentages of double-labeled neurons per TH-ir neuron and double-labeled neurons per CTb-labeled neuron in the DMV calculated from the values for every section of all three cases of CTb injections into each part of the alimentary canal.
hepatic branch of the vagus trunk innervates the descending part of the duodenum. The CTb-labeled neurons were scattered throughout the DMV. Only a few double-labeled neurons were found in the caudal half of the DMV (Fig. 2D). The average percentages of double-labeled neurons per TH-ir neuron were very low and were about 3.5% in the caudal half of the DMV (Table 1). When CTb was injected into the jejunum, the injection site was at about 10 cm caudal to the duodenojejunal flexure. The celiac branch of the vagus trunk innervates the jejunum. A few CTb-labeled neurons were found in the DMV. There were only a few double-labeled neurons (Fig. 2E). We counted only 6 double-labeled neurons in the DMV from the three cases. The average percentages of doublelabeled neurons per TH-ir neuron were 0.8% in the caudal half and 0.5% in the rostral half of the DMV (Table 1). When CTb was injected into the ascending colon, the injection site was the anterior wall of the colon between the cecum and the hepatic flexure. The celiac branch of the vagus trunk innervates the ascending colon. A moderate number of CTb-labeled neurons was found in the DMV (Table 1). Only a few double-labeled neurons were found in the caudal half (Fig. 1E) and none in the rostral half of the DMV (Fig. 2F). The average percentage of double-labeled neurons per TH-labeled neuron was 3.3% in the caudal half of the DMV (Table 1). In the control experiments of the CTb pouring over the abdominal cavity, no retrogradely labeled neurons were found in the medulla oblongata including the DMV. As a whole, almost all double-labeled neurons were located in the caudal half of the DMV (Table 1). The percentages of double-labeled neurons per CTb-labeled neuron were small and less than 10% in the caudal half of the DMV in all cases. This is because the numbers of CTblabeled neurons were larger than those of TH-ir neurons in
the cases of injections into the esophagus and the stomach (Table 1).
4. Discussion The present results demonstrated that almost all dopaminergic neurons in the caudal half of the DMV project to the subdiaphragmatic esophagus and the stomach, while only a few dopaminergic neurons projected to the duodenum, the jejunum or the colon. Double-labeling studies combined retrograde fluorescent tracers with TH-immunohistochemistry have shown that there are several TH-ir neurons that project to the stomach in the caudal half of the DMV (Tayo and Williams, 1988; Yang et al., 1999; Guo et al., 2001). Our results confirmed that many TH-ir neurons in the DMV project not only to the stomach, but also to the subdiaphragmatic esophagus. The percentages of the double-labeled neurons per TH-ir neuron were less than 10% in the studies using fluorescent tracers. In the present results, about 60% of the TH-ir neurons in the caudal half of the DMV projected to the cardia, about 40% of them to the pylorus, and about 30% of them to the esophagus. These differences may be due to the fact that immunohistochemistry for the retrogradely transported CTb labeled more neurons in the DMV than the retrogradely transported fluorescent tracers. Hayakawa et al. (2004) reported that about 80% of the TH-ir neurons in the caudal half of the DMV projected to the stomach. It is not appropriate to make a simple comparison of the percentages of the double-labeled neurons in the DMV, because the cardia and the pylorus are innervated by different neurons in the DMV, whereas the cardia and the corpus of the stomach receive collateral projections from the DMV neurons (Hayakawa et al., 2003).
K. Tsukamoto et al. / Autonomic Neuroscience: Basic and Clinical 123 (2005) 12 – 18
The ascending colon is also likely to be innervated by neurons which are different from the neurons that project to the stomach (unpublished observations). Anterograde and retrograde tracing studies have revealed that the DMV neurons innervate the stomach by way of the gastric branch, the duodenum by way of the hepatic branch, and the small intestine and the colon by way of the celiac branch of the vagus nerve (Berthoud et al., 1991). These reports together with our present results indicate that the esophagus and the stomach are innervated by neurons in the DMV that contain dopamine or acetylcholine, but the small intestine and the colon are innervated by the neurons that contain only acetylcholine. There are also several TH-ir neurons present in the rostral half of the DMV that do not project to the alimentary canal. The neurons projecting to the heart, the lung and the trachea are located throughout the DMV (Kalia and Mesulam, 1980). However, no TH-ir neurons were found among the neurons projecting to the heart in the DMV (Takanaga et al., 2003). When retrograde tracers were applied to the glossopharyngeal nerve or the lingual branch of the glossopharyngeal nerve, a few retrogradely labeled neurons were recognized in the rostral part of the DMV, which extended to the inferior salivatory nucleus (Matesz and Szekely, 1983; Hamilton and Norgren, 1984; Kim et al., 2004). Thus, the dopaminergic neurons in the rostral half of the DMV may send fibers to these glossopharyngeal nerves. The nonadrenergic noncholinergic inhibitory neurons in the myenteric ganglia seem to play an important role in the regulation of movements in all parts of the alimentary canal, including the relaxation reflex of the stomach (Abrahamsson, 1986; Costa et al., 1996; Furness, 2000). Electrophysiological studies have shown that the relaxation reflex is elicited by the discharge of nitric oxide, vasoactive intestinal polypeptide (VIP) or cholecystokinin (CCK) in the myenteric ganglia (Takahashi and Owyang, 1997). Because there are only a few neurons that contain nitric oxide synthase in the DMV (Hyland et al., 2001; Takanaga et al., 2003), nitric oxide are released mainly from the neurons of the myenteric ganglion of the stomach but from the DMV neurons. The neurons in the myenteric ganglion of the stomach contain many neurotransmitters or neuromodulators such as the calbindin, calretinin, ChAT, enkephalin, neuropeptide Y, VIP, CCK, serotonin, somatostatin, substance P, and nitric oxide (Reiche et al., 2001; Schemann et al., 2001). There are no neurons containing these chemicals in the DMV. On the other hand, the administration of dopamine elicits the relaxation of the lower esophagus or the stomach (Valenzuela, 1976a; Ulvestad and Gerner, 1985; Lefebvre, 1992). There are dopamine receptors in the smooth muscles of the lower esophagus or the stomach (de Carle and Christensen, 1976; Lefebvre, 1992). Our present results revealed that the neurons containing dopamine in the DMV project to the esophagus and stomach, but not project to the small intestine and the colon. The dopamine containing
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neurons in the DMV may innervate the smooth muscles to inhibit the muscle activity. Thus, they contribute to the relaxation reflex of the esophagus and the stomach, but not to the peristalsis of the intestine (Valenzuela, 1976a,b; Bueno et al., 1983; Sorraing et al., 1984). Though most of the dopaminergic neurons in the caudal half of the DMV also expressed ChAT, it is not clear if the terminals of the vagal motor neurons release dopamine and acetylcholine simultaneously from the same terminal. Further studies are needed to address what kinds of terminals release dopamine or acetylcholine, and what kinds of cells in the alimentary canal receive the projections of the dopaminergic neurons in the DMV.
Acknowledgments The authors thank Ms. M. Hatta and Mr. K. Gion for their technical assistance.
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