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Am J Physiol Gastrointest Liver Physiol 280: G361–G367, 2001. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus JINFENG J. GUO,1 KIRSTEEN N. BROWNING,1 RICHARD C. ROGERS,2 AND R. ALBERTO TRAVAGLI1 1 Neurogastroenterology Research, Henry Ford Health System, Detroit, Michigan 48202; and 2Department of Neuroscience, Ohio State University, Columbus, Ohio 43210 Received 28 June 2000; accepted in final form 25 September 2000 GASTRIC RELAXATION REFERS to the behavior of the stomach during food ingestion and is characterized by a pronounced capacity to receive large increases in volume with only a slight increase in gastric pressure (1). The vagus nerve provides an essential role in the mediation of this relaxation reflex via an increase in the nonadrenergic noncholinergic (NANC) inhibitory innervation as well as a decrease of cholinergic excitatory innervation to the stomach (1, 20, 29, 34). The dorsal motor nucleus of the vagus (DMV) supplies parasympathetic motor preganglionic fibers to the viscera and is involved in the central nervous system (CNS) control of gastric motility (10). Recently, we (29) have provided physiological evidence that at least two types of vagal motoneurons are involved in the gastric relaxation that follows esophageal distension. Several studies (2, 3, 15, 17–19, 21, 22, 28, 35, 40, 43) have shown that the DMV contains neurons with diverse neurochemical phenotypes. These discrete neurochemical subpopulations could potentially provide differential CNS modulation of specific vago-vagal reflexes involved in, for example, gastric relaxation. It is well established that the DMV in general and its caudal portion in particular are composed of neurons that show tyrosine hydroxylase immunoreactivity (TH-IR) (2, 13, 22, 28, 35, 37). The pattern of distribution of dopamine--hydroxylase (DH) activity overlaps the distribution of TH-IR in the caudal dorsal brain stem (6, 13, 28). Recently, Willing and Berthoud (37) reported that the population of DH and TH-IR neurons in the dorsal vagal complex (i.e., DMV and nucleus of the solitary tract, NTS) were almost identical in number and distribution, arguing that the TH-IR positive neurons in the DMV are capable of synthesizing norepinephrine. These TH-IR positive caudal DMV neurons have been reported (2, 22) to display choline acetyltransferase activity, making them likely candidates for vagal motoneurons that project to peripheral targets. Studies (4, 19) have also focused on the presence and role of nitric oxide (NO) in the brain stem vagal nuclei. Anatomic and physiological evidence points to a possible role of the NO synthase (NOS)-IR positive DMV neurons in vagally mediated gastric relaxation. We (43) have shown recently that a subpopulation of DMV neurons containing NOS-IR projects selectively to the gastric fundus. Immunocytochemical detection of the immediateearly genes encoding for the c-Fos protein allows detection of those neurons activated after a wide variety of stimuli. Increased c-Fos expression in response to administration of the gastric relaxant CCK or after gastric distension has been reported (27, 37) in neurons located in areas of the caudal dorsal brain stem that include the DMV and NTS. The goals of this study were threefold. We aimed to 1) investigate whether the TH-IR positive DMV neu- Address for reprint requests and other correspondence: R. A. Travagli, Univ. of Michigan, Division of Gastroenterology, 3912 Taubman Center, Ann Arbor, MI 48109-0362 (E-mail: travagli7 @hotmail.com). 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. stomach; esophagus; receptive relaxation; gastric motility http://www.ajpgi.org 0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society G361 Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 Guo, Jinfeng J., Kirsteen N. Browning, Richard C. Rogers, and R. Alberto Travagli. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus. Am J Physiol Gastrointest Liver Physiol 280: G361–G367, 2001.—Nitric oxide synthase-immunoreactive (NOS-IR) neurons in the rat caudal dorsal motor nucleus of the vagus (DMV) project selectively to the gastric fundus and may be involved in vagal reflexes controlling gastric distension. This study aimed to identify the gastric projections of tyrosine hydroxylase-immunoreactive (TH-IR) DMV neurons, whether such neurons colocalize NOS-IR, and if they are activated after esophageal distension. Gastric-projecting neurons were identified after injection of retrograde tracers into the muscle wall of the gastric fundus, corpus, or antrum/pylorus before removal and processing of the brain stems for TH- and NOS-IR. A significantly higher proportion of corpus- compared with fundus- and antrum/pylorus-projecting neurons were TH-IR (14% compared with 4% and 2%, respectively, P ⬍ 0.05). Colocalization of NOS- and TH-IR was never observed in gastric-projecting neurons. In rats tested for c-Fos activation after intermittent esophageal balloon distension, no colocalization with TH-IR was observed in DMV neurons. These findings suggest that TH-IR neurons in the caudal DMV project mainly to the gastric corpus, constitute a subpopulation distinct from that of nitrergic vagal neurons, and are not activated on esophageal distension. G362 TH-IR IN RAT DMV rons project selectively to specific gastric areas; 2) explore whether TH- and NOS-IR positive neurons are colocalized within the same DMV neurons; and 3) examine whether TH-IR positive neurons are activated on esophageal distension. EXPERIMENTAL PROCEDURES Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 A total of 47 Sprague-Dawley rats (25–35 days old) of either sex were used. Animals were subdivided into the following six groups. In group 1 (n ⫽ 6) or control, no injections were performed on the gastric walls. In group 2 (n ⫽ 8), tetramethylrhodamine isothiocyanate (TRITC)-filled latex microspheres were injected into the wall of the gastric fundus. In group 3 (n ⫽ 10), TRITC-filled latex microspheres were injected into the wall of the gastric corpus. In group 4 (n ⫽ 8), TRITC-filled latex microspheres were injected into the wall of the antral/pyloric area (see below). In group 5 (n ⫽ 10), double immunocytochemistry (TH and NOS) was carried out, and no injections were performed on the gastric walls. In group 6 (n⫽ 5), esophageal distension followed by double immunocytochemistry (TH and c-Fos) took place, and no injections were performed on the gastric walls. Unless otherwise specified, all rats were injected with fluorogold (Fluorochrome, Englewood, CO; 20 g/1 ml saline/rat, ip) to label vagal preganglionic neurons innervating the subdiaphragmatic viscera allowing delineation of the boundaries of the DMV (24, 43). Using a custom-made anesthetic chamber, rats were anesthetized deeply (abolition of foot pinch withdrawal reflex) with 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane), and the abdominal and thoracic areas were shaved and cleaned with 70% ethanol. After an abdominal laparotomy, the stomach was freed from the liver and reflected gently to one side to facilitate access to the gastric wall. With a 5-l Hamilton syringe, rats in groups 2, 3, and 4 were injected with TRITC-filled latex microspheres (1:4 vol/vol dilution with saline; 0.5–1 l per injection, 5–10 sites per area) in the muscular and mucosal layers of the stomach (fundus, corpus, or antrum/pylorus, respectively). The syringe needle was inserted 2–3 mm into the stomach wall at an angle of 2–5° to facilitate the spread of beads within the stomach wall and to reduce the possibility of perforating the mucosa, perceived as a drop in the resistance opposing the needle penetration into the stomach wall. The laparotomy was then closed with 5-0 silk sutures, and the rats were allowed to recover for 5–15 days, permitting retrograde transport of the fluorescent marker as described previously (43). Rats in group 6 were anesthetized with pentobarbital sodium (Nembutal; 75mg/kg, ip). The thoracic esophagus was then cannulated with a distension balloon inserted to the point where its tip meets resistance at the lower esophageal sphincter. Balloon catheters were constructed as previously described (29). The balloon was distended intermittently (final intramural pressure, 14–18 mmHg; 1 s on and 4 s off) for 60–90 min. At the end of the experiment, rats were perfused transcardially with saline, followed by 4% paraformaldehyde in PBS solution (see below). After extraction, brain stems from all groups were fixed in Zamboni’s fixative overnight, rinsed three to five times with a solution of Triton X-100 in PBS (PBS-TX; for composition, see Composition of solutions), and stored in PBS-TX overnight at 4°C. The brain stem was then immersed in PBS containing 2.5% sucrose before cutting coronal sections of 50 m thickness using a cryostat. The brain stem slices were mounted on gelatin-coated coverslips and preincubated in a humid chamber with PBS-TX-BSA at 37°C for 30 min. The slices from rats in groups 1–4 were rinsed with fresh PBS-TX-BSA solution and incubated at 37°C for 2 h with primary antibody (mouse-␣-TH; 1:500 dilution in PBS-TX containing 0.1% BSA). The slices were rinsed three times with PBS-TX-BSA and then incubated again at 37°C for 30 min with secondary antibody (goat-␣-mouse FITC, Sigma Chemical, St. Louis, MO; 1:100 dilution in PBS-TX containing 0.1% BSA). The specimens were again rinsed three times with PBS-TX-BSA solution and once with PBS-TX. Then the specimens were mounted on glass slides and allowed to air dry at room temperature, and mounted with one drop of Fluoromount-G (Southern Biotechnology Associated, Birmingham, AL). Specimens were examined using a Nikon E400 microscope fitted with epifluorescent filters for TRITC, FITC, and ultraviolet light. For TH and NOS dual immunochemical staining (group 5), coverslips with adherent brain stem sections were preincubated at 37°C for 30 min with PBS-TX-BSA. The specimens were rinsed once with fresh PBS-TX-BSA and incubated at 37°C for 2 h with a mixture of the primary antibodies [mouse␣-TH (1:500 dilution) and rabbit-␣-NOS (1:250 dilution) in PBS-TX containing 0.1% BSA]. The specimens were rinsed three times with PBS-TX-BSA, then incubated at 37°C for 30 min with a mixture of secondary antibodies [goat-␣-mouse FITC (1:100 dilution) and goat-␣-rabbit TRITC (1:50 dilution) in PBS-TX containing 0.1% BSA]. The specimens were rinsed three times with PBS-TX-BSA and once with PBS-TX. The specimens were air dried at room temperature, mounted on glass slides in Fluoromount-G, and examined with a fluorescence microscope as described above. The slices from rats in group 6 were rinsed with fresh PBS-TX-BSA solution and incubated overnight at room temperature with a mixture of the primary antibodies [mouse-␣TH (1:500 dilution) and rabbit-␣-c-Fos (1:5,000 dilution) in PBS-TX containing 0.1% BSA]. The slices were rinsed three times with PBS-TX-BSA, then incubated at 37°C for 30 min with a mixture of secondary antibodies [goat-␣-mouse FITC (1:100 dilution) and goat-␣-rabbit TRITC (1:50 dilution) in PBS-TX containing 0.1% BSA]. The specimens were again rinsed three times with PBS-TX-BSA solution and once with PBS-TX before being mounted on glass slides and air dried at room temperature. The specimens were then mounted with one drop of Fluoromount-G and examined with a fluorescence microscope as described above. Cells were counted on alternate brain stem slices by two independent investigators who were unaware of the treatment. If the cell count differed by ⬎10%, a third investigator then analyzed the brain stem slices. The final cell count was the mathematical average of the independent cell counts. To minimize errors in the counting of DMV somata, we counted only those neurons in which the nucleus was clearly visible. Despite this precaution, we have to consider cell counts as best proportional estimates only, rather than absolute values, when comparing labeled subpopulations. This is of paramount importance because the surface of the stomach wall covered by injection of rhodamine-coated beads was limited to ⬃5 mm2. Therefore, this provided only a small, though representative, fraction of labeled DMV neurons projecting to the gastric areas of interest. The portion of the DMV located caudal to obex was defined as the “caudal DMV” and the portion of DMV located rostral to the anterior tip of the area postrema as the “rostral DMV.” The area composed by the extension of the area postrema was defined as the “intermediate DMV.” Cell count values are given as means ⫾ SE. Control experiments were carried out to ensure that the antibody labeling was selective, namely: 1) incubation of primary or secondary antibodies only and 2) reaction of primary antibody with inappropriate secondary antibody. All TH-IR IN RAT DMV RESULTS Sections of brain stem containing the DMV were analyzed from ⬃2 mm caudal to the posterior tip of the area postrema to ⬃3 mm rostral to the anterior tip of the area postrema. The boundaries of the DMV were delineated clearly by the presence of fluorogold-IR neurons (19, 24, 43). Only those neurons with a distinct Fig. 1. Coronal view of the brain stem showing the distribution within the dorsal motor nucleus of the vagus (DMV) of retrogradely labeled neurons after microinjections of tetramethylrhodamine isothiocyanate (TRITC)-filled latex microspheres in the corpus, intraperitoneal injection of fluorogold to label DMV neurons, and immunocytochemical detection of tyrosine hydroxylase (TH). Computergenerated colors indicate the following: blue, fluorogold; green, TH immunoreactive (TH-IR); red, TRITC microspheres; light blue, colocalization of fluorogold and TH-IR. A: caudal DMV. Note the heavy density of TH-IR neurons within the DMV. Thick arrow, a corpusprojecting TH-positive neuron. B: intermediate DMV. Note the larger number of corpus-projecting (rhodamine bead-filled) neurons, 1 of which contains TH-IR. C: rostral DMV. Note that because TH-IR neurons lie outside the DMV, no corpus-projecting neuron contains TH-IR. Thin arrows, fluorogold and TH-IR neurons. Thick arrows, rhodamine-labeled and TH-IR DMV neurons. Thin convex arrows, TH-IR neurons outside the DMV. Thick convex arrows, rhodaminelabeled DMV neurons (for clarity the rhodamine-labeled neurons in B have not been indicated by arrows). CC, central canal; NTS, nucleus of the solitary tract; XII, hypoglossal nucleus; IV, fourth ventricle. fluorogold-stained profile were counted. There were 998 ⫾ 62, 2,283 ⫾ 87, and 1,080 ⫾ 59 fluorogoldpositive neurons in the caudal, intermediate, and rostral DMV, respectively (n⫽ 6 rats). Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 tests proved negative, indicating that the secondary antibodies were selective for their primary antibodies and that the antibodies themselves exhibited neither nonspecific binding nor excessive autofluorescence. Control experiments were also carried out to ensure that there was no leakage or fading of the fluorescence from the retrograde-labeled neurons due to the immunochemical staining process. This was accomplished by comparison of the number of retrograde-labeled neurons counted immediately after slicing and after the immunochemical staining process. The experiments were carried out in accordance with the Henry Ford Health System guidelines for the care and use of laboratory animals. All efforts were made to minimize pain, reduce the number of animals used, and utilize alternatives to in vivo techniques, if available. Photographs were taken using a SPOT digital camera mounted on a Nikon E400 fluorescent microscope. To reduce the fluorescent light exposure time and the consequent fading of the fluorescence labeling, slices were photographed in black and white using the appropriate filters, merged, and assigned compute-generated colors (SPOT software, Diagnostic Instruments, Sterling Heights, MI). Materials. TRITC latex microspheres (rhodamine beads) were purchased from Lumafluor (Naples, FL). Nembutal was purchased from Abbot Laboratories (North Chicago, IL). All other chemicals were purchased from Sigma Chemical. Composition of solutions. The fixative solution was composed of 32 g paraformaldehyde, 240 ml saturated picric acid, 5.25 g KH2PO4, 35.6 g Na2HPO4 䡠 7H2O, and 1,600 ml H2O. The composition of the PBS solution was as follows: 13.5 g NaCl, 40.2 g Na2HPO4 䡠 H2O, 2.04 g KH2PO4, and 1,500 ml H2O. The PBS-TX solution was composed of 2.25 ml Triton X-100 dissolved in 1,500 ml PBS buffer. The PBS-TX-BSA solution composition was 1 g BSA dissolved in 100 ml PBS-TX buffer. Statistical analysis. Neuronal groups were compared using the 2 test. The level of significance was set at P ⬍ 0.05. G363 G364 TH-IR IN RAT DMV Fig. 2. Coronal view of the caudal brain stem showing the distribution within the DMV (outlined by the fluorogold-positive labeling) of TH and nitric oxide synthase (NOS)-positive neurons. Computer-generated colors indicate the following: blue, fluorogold; green, TH-IR; red, NOS-IR; light blue, colocalization of fluorogold and TH-IR. Note that the TH-positive DMV neurons (thick arrows) do not contain NOS-IR (thin arrows). For clarity, only a few selected DMV neurons have been highlighted. of the DMV colocalized with TH-IR (i.e., 13.7 ⫾ 1.1%; n ⫽ 10 rats; P ⬍ 0.05 vs. fundus or antrum/pylorus). Conversely, after injection of rhodamine beads in the antrum/pylorus, 1 of the 41 rhodamine-labeled neurons in the caudal DMV also contained TH-IR (n ⫽ 8 rats); similarly after injection of rhodamine beads in the fundus, 9 of the 216 rhodamine-labeled neurons colocalized with TH-IR (n ⫽ 8 rats). Figure 1 shows the distribution of dye-labeled neurons after injection of tracer in the gastric corpus, intraperitoneal injection of fluorogold, and immunocytochemical detection of TH. As can be noted in the micrograph (Fig. 1), most of the DMV neurons showing colocalization of dye and TH-IR were located in the caudal pole of the DMV whereas the majority of dyelabeled DMV neurons were in the intermediate portion of the DMV. In Fig. 1, A and B (showing caudal and intermediate brain stem section, respectively), DMV neurons show colocalization of rhodamine beads and TH-IR. In the rostral portion of the DMV (Fig. 1C), TH-IR positive neurons are located outside the fluorogold-defined boundaries of the DMV. Localization of TH- and NOS-positive neurons. In the 10 brain stems analyzed, we never found any DMV neurons that contained both NOS- and TH-IR (Fig. 2), even in the caudal DMV, where both TH- and NOS-IR positive neurons are densely located. In fact, despite the observation that TH-IR and NOS-IR positive DMV Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 Localization and projections of TH-IR neurons. TH-IR neurons were distributed within the boundaries of the DMV (as determined by fluorogold-IR) as well as in the commissuralis, centralis, dorsalis, and medialis subnuclei of the NTS. Focusing on the TH-IR neurons in the DMV, the majority of the TH-IR neurons were located in the caudal DMV where they comprised 10.9 ⫾ 0.6% of the total number of DMV neurons (i.e., 108 ⫾ 12 TH-IR neurons of 998 ⫾ 62 fluorogold-positive neurons; n ⫽ 6 rats). Although there were more fluorogold-labeled neurons in the intermediate portion of the DMV, there were fewer TH-IR positive DMV neurons present (i.e., 6 ⫾ 1 TH-IR neurons of 2,283 ⫾ 86 fluorogold-positive neurons; n ⫽ 6 rats). In the rostral portions of the DMV, the percentage of TH-IR DMV neurons was 4.4 ⫾ 0.9% (i.e., 47 ⫾ 7 TH-IR neurons of 1,080 ⫾ 58 fluorogold-positive neurons; n ⫽ 6 rats). The total number of TH-IR neurons in the DMV would then constitute a value similar to that previously found (22), though somewhat lower than in a more recent study (39). By applying retrograde-tracing techniques and immunochemical staining with specific antibodies against TH, we analyzed the rostro-caudal span of the DMV for colocalization of rhodamine-labeled and TH-IR positive neurons, i.e., TH-IR neurons projecting to the gastric regions of interest. After injection of rhodamine in the corpus, 43 of the 368 rhodamine-labeled neurons in the caudal portion TH-IR IN RAT DMV DISCUSSION The present study provides evidence that 1) TH-IR positive neurons in the caudal DMV project selectively to the gastric corpus; 2) the TH-IR positive DMV neurons comprise a neuronal subpopulation distinct from the NOS-IR positive DMV neurons; and 3) TH-IR pos- itive neurons in the DMV are not activated on esophageal distension. Such evidence leads us to the following two conclusions. 1) The TH-IR positive neurons in caudal DMV constitute a subpopulation of preganglionic neurons distinct from the NOS-IR neurons. These TH-IR neurons do not seem to be implicated in the activation of NANC inhibitory pathways of the gastric receptive relaxation reflex activated by esophageal distension (29, 43). 2) The discrete population of TH-IR positive neurons in caudal DMV projecting to the corpus may then comprise the preganglionic motoneurons involved in gastric relaxation obtained via withdrawal of cholinergic tone (29, 34) or could constitute a subpopulation of dopaminergic neurons involved in the attenuation of stress or chemically induced ulcers (see Ref. 11 for review). The percentage of TH-IR positive neurons in the caudal DMV reported in this study, i.e., 11% of the fluorogold-labeled neurons, is similar to that found previously by Manier et al. (22), though slightly lower than in other reports (35, 39), supporting the validity of our labeling techniques and cell-counting methods. In this study, we report that ⬃14% of vagal neurons projecting to the rat gastric corpus contain TH-IR. The importance of such a relatively low percentage of Fig. 3. Coronal view of the caudal brain stem showing the distribution within the DMV (outlined by the fluorogold positive labeling) of TH- and c-Fos-IR positive neurons. Computer generated colors indicate the following: blue, fluorogold; green, TH-IR; red, cFos-IR; light blue, colocalization of fluorogold and TH-IR. Note that c-Fos positive neurons (thin arrows) are either outside the DMV or inside the DMV but do not contain TH (thin convex arrows). In fact, TH-IR neurons in the DMV do not colocalize with c-Fos (thick arrows). Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 neurons were contained within the same region of the caudal DMV, they were never colocalized. Figure 2 is a micrograph showing the distribution of TH- and NOS-IR positive neurons in the caudal portion of the DMV. As can be noted, within the fluorogold-delimited boundaries of the DMV, the TH-IR positive neurons are intermingled but not colocalized with the NOS-IR positive neurons. The separation of TH- and NOS-IR positive neurons is also present in the NTS area adjacent to the DMV where the NOS-IR positive neurons are located in patches dorsal to the TH-IR positive neurons. Esophageal distension activates c-Fos expression. After esophageal distension for a period between 60 and 90 min (1 s on and 4 s off) we observed a consistent pattern of c-Fos expression in the caudal dorsal brain stem. At the level of the caudal DMV in all five brain stems analyzed, although c-Fos-positive neurons were present, we never found a DMV neuron that contained both c-Fos- and TH-IR (Fig. 3). G365 G366 TH-IR IN RAT DMV frequency coding (33, 42), suggests that the vagus nerve may encode the release of different neurotransmitters according to the firing frequency at which it is driven. Indeed, a frequency-response code seems to exist in both the NTS after esophageal distension (see Fig. 2 in Ref. 29) and the DMV after injection of direct current (5). It is then possible that the frequency code necessary to activate TH-IR positive neurons in DMV is not the same as that activated by our esophageal distension paradigm. Second, though less probable, is that TH-IR neurons in DMV are not involved in gastric distension reflexes but rather in the mucosa protection exerted by dopamine (11). Such a scenario is unlikely since previously discussed evidence (27, 37) provides a rather strong case in favor of involvement of DMV catecholaminergic neurons in gastric relaxation. In conclusion, we have shown that a significant percentage of caudal DMV neurons projecting to the stomach corpus contain TH-IR and are not colocalized with NOS-IR positive neurons. In addition, we suggest that the TH-IR positive neurons may be involved in the withdrawal of cholinergic tone to the gastric corpus. This work was supported by National Science Foundation Grant 9816662 (R. A. Travagli) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-56373 (R. Rogers). REFERENCES 1. Abrahamsson H. Studies on the inhibitory nervous control of gastric motility. Acta Physiol Scand 390 Suppl: 1–38, 1973. 2. Armstrong DM, Manley L, Haycock JW, and Hersh LB. Co-localization of choline acetyltransferase and tyrosine hydroxylase within neurons of the dorsal motor nucleus of the vagus. J Chem Neuroanat 3: 133–140, 1990. 3. Baude A and Shigemoto R. Cellular and subcellular distribution of substance P receptor immunoreactivity in the dorsal vagal complex of the rat and cat: a light and electron microscope study. J Comp Neurol 402: 181–196, 1998. 4. Berthoud HR. Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach. J Comp Neurol 358: 428–439, 1995. 5. Browning KN, Renehan WE, and Travagli RA. Electrophysiological and morphological heterogeneity of rat dorsal vagal neurons which project to specific areas of the gastrointestinal tract. J Physiol (Lond) 517: 521–532, 1999. 6. Chamba G and Renaud B. Distribution of tyrosine hydroxylase, dopamine  hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem. Brain Res 259: 95–102, 1983. 7. De Ponti F, Azpiroz F, and Malagelada JR. Relaxatory responses of canine proximal stomach to esophageal and duodenal distension. Importance of vagal pathways. Dig Dis Sci 34: 873–881, 1989. 8. Fogel R, Zhang X, and Renehan WE. Relationships between the morphology and function of gastric and intestinal distentionsensitive neurons in the dorsal motor nucleus of the vagus. J Comp Neurol 364: 78–91, 1996. 9. Galligan JJ and North RA. Opioid, 5HT1A and alpha 2 receptors localized to subsets of guinea-pig myenteric neurons. J Auton Nerv Syst 32: 1–11, 1991. 10. Gillis RA, Quest JA, Pagani FD, and Norman WP. Control centers in the central nervous system for regulating gastrointestinal motility. In: Handbook of Physiology. The Gastrointestinal System. Motility and Circulation. Bethesda, MD: Am. Physiol. Soc., 1989, sect. 6, vol. I, pt. 1, chapt. 17, p. 621–683. Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 TH-IR neurons in the DMV should not be underestimated, because single vagal motoneurons project extensively to adjacent areas of the gastrointestinal tract (14) and could thus influence selectively similar functions. Our data showing lack of colocalization of TH- and NOS-IR in caudal DMV neurons extend a previous observation by Ohta and colleagues (23). In that study (23), evidence was provided that NOS neurons did not colocalize with catecholaminergic neurons in the adjacent medial NTS subnucleus. Using both in vitro and in vivo techniques, several groups have shown recently that a vast array of electrical (36, 38), physiological (27, 29), or mechanical (8, 25, 37) stimuli can activate DMV motoneurons and induce gastric relaxation (8, 27, 29). Some of these stimuli, i.e., peripheral injection of CCK (27) or gastric (37) or esophageal distension (29), selectively activate the caudal DMV neuronal population. Vagally mediated cholinergic stimuli increase gastric functional parameters, including tone and motility (10). In several studies (9, 30, 31, 41, 42), it has been demonstrated that activation of presynaptic ␣2-adrenoceptors decreases vagally evoked ACh release to the stomach and that exogenous application of norepinephrine decreases ACh release from vagal terminals in both the corpal (30) and antral portions of the stomach (32). In addition, the decrease in gastric motility that follows peripheral CCK administration involves the activation of a vagal-dependent mechanism (12, 26, 27). It is established that both endogenous and exogenous CCK stimulate distension-sensitive vagal afferents, producing distension-like effects on vagal neurons (26) and expression of c-FOS in TH-IR positive DMV neurons (27). Using an esophageal balloon distension protocol proven to induce gastric relaxation (29), we were unable to activate c-Fos expression in TH-IR positive caudal DMV neurons. Our data thus suggest that the origin of the adrenergic modulation of ACh release from vagal terminals must be found in areas other than the caudal DMV. Because our esophageal stimulation paradigm did not activate TH-IR caudal DMV neurons (otherwise we would have observed an increase in c-Fos activity), the following two scenarios may be considered. First, TH-IR positive neurons in the caudal DMV may be implicated in withdrawal of cholinergic tone by an activation of GABA terminals via a mechanism unrelated to esophageal distension. In fact, adrenergic blockade with ␣- or -adrenoceptor antagonists did not seem to affect the relaxatory or contractile responses elicited by either electrical vagal stimulation or by esophageal or duodenal distension (7, 33). At the same time, however, nicotine perfusion, in the presence of the muscarinic receptor antagonist atropine, induced a relaxation of the gastric fundus that was mediated largely by NO release and partially by the release of norepinephrine (16). The observation that vagal inhibitory and excitatory fibers are not simultaneously activated by gastric distension (34), but rather require a TH-IR IN RAT DMV 27. Rinaman L, Verbalis JG, Stricker EM, and Hoffman GE. Distribution and neurochemical phenotypes of caudal medullary neurons activated to express cFos following peripheral administration of cholecystokinin. J Comp Neurol 338: 475–490, 1993. 28. Ritchie TC, Westlund KN, Bowker RM, Coulter JD, and Leonard RB. The relationship of the medullary catecholamine containing neurons to the vagal motor nuclei. Neuroscience 7: 1471–1482, 1982. 29. Rogers RC, Hermann GE, and Travagli RA. Brainstem pathways responsible for oesophageal control of gastric motility and tone in the rat. J Physiol (Lond) 514: 369–383, 1999. 30. Schemann M. Excitatory and inhibitory effects of norepinephrine on myenteric neurons of the guinea-pig gastric corpus. Pflügers Arch 418: 575–580, 1991. 31. Slack BE. Pre- and postsynaptic actions of noradrenaline and clonidine on myenteric neurons. Neuroscience 19: 1303–1309, 1986. 32. Tack J and Wood JD. Actions of noradrenaline on myenteric neurons in the guinea-pig gastric antrum. J Auton Nerv Syst 41: 67–77, 1992. 33. Takahashi T and Owyang C. Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat. J Physiol (Lond) 484: 481–492, 1995. 34. Takahashi T and Owyang C. Characterization of vagal pathways mediating gastric accommodation reflex in rats. J Physiol (Lond) 504: 479–488, 1997. 35. Tayo EK and Williams RG. Catecholaminergic parasympathetic efferents within the dorsal motor nucleus of the vagus in the rat: a quantitative analysis. Neurosci Lett 90: 1–5, 1990. 36. 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. 37. Willing AE and Berthoud HR. Gastric distension-induced c-fos expression in catecholaminergic neurons of rat dorsal vagal complex. Am J Physiol Regulatory Integrative Comp Physiol 272: R59–R67, 1997. 38. Willis A, Mihalevich M, Neff RA, and Mendelowitz D. Three types of postsynaptic glutamatergic receptors are activated in DMNX neurons upon stimulation of NTS. Am J Physiol Regulatory Integrative Comp Physiol 271: R1614–R1619, 1996. 39. Yang M, Zhao X, and Miselis RR. The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of the rat. J Auton Nerv Syst 76: 108–117, 1999. 40. Yang SN, Bunneman B, Cintra A, and Fuxe K. Localization of neuropeptide Y Y1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata. Neuroscience 73: 519–530, 1996. 41. Yokotani K, Muramatsu I, and Fujiwara M. Alpha 1 and alpha 2 type adrenoceptors involved in the inhibitory effect of splanchnic nerves on parasympathetically stimulated gastric acid secretion in rats. J Pharmacol Exp Ther 229: 305–310, 1984. 42. Yokotani K, Okuma Y, Nakamura K, and Osumi Y. Release of endogenous acetylcholine from a vascularly perfused rat stomach in vitro: inhibition by M3 muscarinic autoreceptors and alpha2 adrenoceptors. J Pharmacol Exp Ther 266: 1190–1195, 1993. 43. Zheng ZL, Rogers RC, and Travagli RA. Selective gastric projections of nitric oxide synthase-containing vagal brainstem neurons. Neuroscience 90: 685–694, 1999. Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on May 6, 2017 11. Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, and Hernandez DE. The neurobiology of stress ulcers. Brain Res Rev 16: 301–343, 1991. 12. Grider JR. Role of cholecystokinin in the regulation of gastrointestinal motility. Symposium 1 Suppl: 1134S–1339S, 1994. 13. Gwyn DG, Ritchie TC, and Coulter JD. The central distribution of vagal catecholaminergic neurons which project into the abdomen in the rat. Brain Res 328: 139–144, 1985. 14. Holst MC, Kelly JB, and Powley TL. Vagal preganglionic projections to the enteric nervous system characterized with Phaseolus vulgaris-leucoagglutinin. J Comp Neurol 381: 81– 100, 1997. 15. Kalia M, Fuxe K, and Goldstein M. Rat medulla oblongata. II. Dopaminergic, noradrenergic (A1 and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes. J Comp Neurol 233: 308–332, 1985. 16. Kojima S, Ishizaki R, and Shimo Y. Investigation of nicotineinduced relaxation of circular smooth muscle of the guinea pig gastric fundus. Eur J Pharmacol 241: 171–175, 1993. 17. Krowicki ZK and Hornby PJ. Hindbrain neuroactive substances controlling gastrointestinal function. In: Regulatory Mechanism in Gastrointestinal Function, edited by Gaginella TS. Boca Raton: CRC, p. 277–319, 1995. 18. Krowicki ZK and Hornby PJ. Contribution of acetylcholine, vasoactive intestinal polypeptide and nitric oxide to CNS-evoked vagal gastric relaxation in the rat. Neurogastroenterol Motil 8: 307–317, 1996. 19. 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 NO compounds upon gastric motor function. J Comp Neurol 377: 49–69, 1997. 20. Lefebvre RA, De Vriese A, and Smits GJM. Influence of vasoactive intestinal polypeptide and NG-nito-L-arginine methyl ester on cholinergic neurotransmission in the rat gastric fundus. Eur J Pharmacol 221: 235–242, 1992. 21. Lynn RB, Hyde TM, Cooperman RR, and Miselis RR. Distribution of bombesin-like immunoreactivity in the nucleus of the solitary tract and dorsal motor nucleus of the rat and human: colocalization with tyrosine-hydroxylase. J Comp Neurol 369: 552–570, 1996. 22. Manier M, Mouchet P, and Feuerstein C. Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurons of the vagal motor nucleus in the adult rat. Neurosci Lett 80: 141–146, 1987. 23. Ohta A, Takagi H, Matsui T, Hamai Y, Iida S, and Esumi H. Localization of nitric oxide synthase-immunoreactive neurons in the solitary nucleus and ventrolateral medulla oblongata of the rat: their relation to catecholaminergic neurons. Neurosci Lett 158: 33–35, 1993. 24. Powley TL, Fox EA, and Berthoud HR. Retrograde tracer technique for assessment of selective and total subdiaphragmatic vagotomies. Am J Physiol Regulatory Integrative Comp Physiol 253: R361–R370, 1987. 25. Renehan WE, Zhang X, Beierwaltes WH, and Fogel R. Neurons in the dorsal motor nucleus of the vagus may integrate vagal and spinal information from the GI tract. Am J Physiol Gastrointest Liver Physiol 268: G780–G790, 1995. 26. Rinaman L, Baker EA, Hoffman GE, Stricker EM, and Verbalis JG. Medullary c-Fos activation in rats after ingestion of a satiating meal. Am J Physiol Regulatory Integrative Comp Physiol 275: R262–R268, 1998. G367