THE JOURNAL OF COMl'ARA'IlVE NEUROLOGY 304261-274 (1991)
Representation of the Cecum in the Lateral Dorsal Motor Nucleus of the Vagus Nerve and Commissural Subnucleus of the Nucleus Tractus Solitarii in Rat STEVEN M. ALTSCHULER, DAVID A. FERENCI, RICHARD B. LYNN, AND RICHARD R. MISELIS Division of Gastroenterology and Nutrition, Children's Hospital of Philadelphia (S.M.A., D.A.F.) and Institute of Neurological Sciences and Animal Biology, University of Pennsylvania (R.B.L., R.R.M.), Philadelphia, Pennsylvania 19104
ABSTRACT Motor fibers of the accessory celiac and celiac vagal branches are derived from the lateral columns of the dorsal motor nucleus of the vagus nerve. These branches also contain sensory fibers that terminate within the nucleus of the tractus solitarii. This study traces the innervation of the intestines by using the tracer cholera toxin-horseradish peroxidase. In 53 rats, the tracer was injected into either the stomach, duodenum, jejunum, terminal ileum, cecum, or ascending colon. With all cecal injections, prominent retrograde labeling of cell bodies occurred bilaterally in the lateral columns of the dorsal motor nucleus of the vagus nerve above, at, and below the level of the area postrema. Dendrites of laterally positioned neurons projected medially and rostrocaudally within the dorsal motor nucleus of the vagus nerve and dorsomedially into both the medial subnucleus and parts of the commissural subnucleus of the nucleus of the tractus solitarii. Sensory terminal labeling occurred in the dorsolateral commissural subnucleus a t the level of the rostral area postrema and the medial commissural subnucleus caudal to the area postrema. Additionally, there was sensory terminal labeling within a small confined area of the dorsomedial zone of the nucleus of the tractus solitarii immediately adjacent to the fourth ventricle at a level just anterior to the area postrema. Stomach injections labeled motoneurons of the medial column of the entire rostrocaudal extent of the dorsal motor nucleus of the vagus nerve and a sensory terminal field primarily in the subnucleus gelatinosus, with less intense labeling extending caudally into the medial and ventral commissural subnuclei. Dendrites of gastric motoneurons project rostrocaudally and mediolaterallywithin the dorsal motor nucleus of the vagus nerve and dorsolaterally within the nucleus of the tractus solitarii. They are most pronounced at the level of the rostral area postrema where many dendrites course dorsolaterally terminating primarily within the subnucleus gelatinosus. Injections of the duodenum labeled a small number of the cells within the medial aspects of the dorsal motor nucleus of the vagus nerve. Jejunal, ileal, and ascending colon injections labeled cells sparsely within the lateral aspects of the dorsal motor nucleus of the vagus nerve bilaterally. No afferent terminal labeling was evident after injection of these areas of the bowel. The dorsal vagal complex has a prominent viscerotopography: the dorsal motor nucleus of the vagus nerve has a mediolateral organization corresponding to end-organ innervation; the nucleus of the tractus solitarii has a rostrocaudal axis of visceral representation with some overlap corresponding to rostrocaudal positioning along the alimentary canal and mediolateral separation of terminals within the nucleus.
Accepted October 19,1990, Address reprint requests to Dr. Richard R. Miselis, Dept. of Animal Biology, University of PennsylvaniaVeterinary School, 3800 Spruce Street, Philadelphia,PA 19104-6045.
o 1991 WILEY-LISS, INC.
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262 Key words: intestines, celiac branch of vagus, cholera toxin-horseradishperoxidase, viscerotopy
Progress in topographical studies of visceral innervation has accelerated with the introduction of new tracers and injection strategies to control them. Where there have been ambiguities concerning viscerotopic representation in the vagal complex (Kalia and Mesulam, '80a,b; Hinrichsen and Ryan, '81; Kalia, '81; Yoshida et a]., '81; Rogers and Hermann, '83;Holstege et al., '83;Fryscaket al., '841, there is now more certainty of its existence (Gwyn et al., '79; Katz and Karten, '83;Shapiro and Miselis, '85b; Bieger and Hopkins, '87; Rinaman and Miselis, '87; Pagani et al., '88; Altschuler et al., '89; Okumura and Namiki, '90). With direct intramuscular injection of the tracers (particularly ones with receptor binding properties) or their application to cut nerves, a strong viscerotopic organization for the upper alimentary tract has been established within the NA and NTS of the rat (Bieger and Hopkins, '87; Altschuler et al., '89). In contrast to the viscerotopic organization proposed €or the NA (Lawn, '66a,b; Bieger and Hopkins, '87), the DMN is organized mediolaterallywith respect to specificbranches of the subdiaphragmatic vagus (Fox and Powley, '85; Norgren and Smith, '88). This scheme, which parcels the DMN into a series of longitudinal columns running the entire rostrocaudal extent of the nucleus, has been primarily derived from the application of tracer to the major branches of the subdiaphragmatic vagus. Each DMN is comprised of a large medial column of cells lying immediately adjacent to the midline. These columns of cells provide efferent axons to the two gastric branches. In the lateral aspects of each DMN, a column of cells of much smaller mediolateral extent exists and contributes fibers to the celiac and accessory celiac branches of the subdiaphragmatic vagus. Within the left medial DMN, an additional column exists composed of a few scattered cells providing axons to the hepatic branch. The formation of the rat DMN into longitudinal cell columns raises the issue of how the innervation of specific abdominal viscera is organized within these columns. This question is best answered by direct intramuscular injection of tracer into the viscera under study. To date, the majority of studies utilizing this technique have focused on the stomach and pancreas. Although a number of investigations have suggested that these structures are represented throughout the entire DMN (Weaver, '80; Takayama et al.,
'82; Leslie et al., '82; Sharkey and Williams, '83;Luiten et al., '84), two recent studies employing HRP conjugates, in which adequate measures to prevent spread and leakage of tracer were performed, have demonstrated a far more localized retrograde labeling of cell bodies primarily within the medial columns of the DMN (Shapiro and Miselis, '85b; Rinaman and Miselis, '87). These findings suggest that specific abdominal viscera are localized to either the medial or lateral bilaterally symmetrical columns. We performed a series of intramuscular injections with CT-HRP in different regions of the small and large bowel in order to determine the viscera represented in the lateral columns of the DMN. Preliminary studies revealed that extremely small numbers of cells were retrogradely labeled within the DMN following injection of CT-HRP into most regions of the bowel. The major exception to this observation was the cecum, where injections of CT-HRP resulted in consistent retrograde labeling of cell bodies within the lateral columns of the DMN (Ferenci et al., '89a,b). On the basis of our preliminary results, the majority of injections in this study involved the cecum. Additionally, since CTHRP is very effective in labeling distal dendritic processes, a comparison of the dendritic fields of medial and lateral column motoneurons was undertaken. The distributions of labeled perikarya within the vagal ganglia and afferent terminal fields within the NTS were also investigated. We were most interested in relating their distributions to previously obtained viscerotopic mappings of the vagal ganglia and NTS following injection of tracer into the upper alimentary tract (Altschuler et al., '89). In our experience, CT-HRP is more effective than free HRP and equally effective as the wheat germ agglutinin-HRP conjugate in labeling afferent terminal fields (Shapiro and Miselis, '85b; Altschuler et al., '89).
MATERIALS AND METHODS Fifty-three adult male Sprague-Dawley rats weighing 210-510 g were employed in these experiments. The animals were anesthetized with ketamine (85 mgikg) and xylazine (12 mg/kg) prior to aseptic surgical procedures. CT-HRP, conjugated in our laboratory by a modification of a previously described method (Shapiro and Miselis, '85a), was utilized as the neural tracer in these studies. Briefly,
Abbreviations
AP C cc
CT-HRP d dl DMN lat lat DMN med DMN
HRP MMC NA NTS cen NTS
area postrema caudal central canal cholera toxin-horseradish peroxidase dorsal dorsolateral dorsal motor nucleus of the vagus nerve lateral lateral column of the DMN medial column of the DMN horseradish peroxidase migrating motor complex nucleus ambiguus nucleus of the tractus solitarii subnucleus centralis of NTS
com NTS gel NTS is NTS med med NTS Ng
R
SLN TMB TS 4v
IX X ~~
XI XI1
commissural subnucleus of NTS subnucleus gelatinosus of NTS subnucleus interstitialis of NTS medial medial subnucleus of NTS nodose ganglion rostral superior laryngeal nerve tetramethyl benzidine tractus solitarius fourth ventricle glossopharyngeal nerve vagus nerve accessory nerve hypoglossal nucleus
CENTRAL REPRESENTATION OF CECUM IN RAT CT (supplied by Sigma or List Biological) was covalently attached to HRP (Sigma type VI) to make CT-HRP by using glutaraldehyde as a homobifunctional bridge molecule in a two-step process (Avrameasand Ternynck, '71; Gonatas et al., '79). HRP (153 mg) was incubated with glutaraldehyde [EM grade; 200 pl of a 25% solution in 1,800 p1 of potassium phosphate buffer (pH 6.8)] for 4 hours at 1:lOO molar concentration ratio to activate the molecule (Molin et al., '78). By utilizing gel filtration chromatography (LKB Ultrogel AcA44, 1.6 cm x 90 cm; eluent 0.15 M NACl), monomeric HRP that contains both unreacted and activated HRP was separated from the polymeric byproducts of the activation reaction. Fractions containing 40 kd product were pooled and concentrated to approximately 1 ml (Amicon CF25 concentrating cone) and reacted with CT (4 mg) in 0.1 M carbonate buffer (pH 9.5) for 40 hours at 4°C (molar ratio of HRP to CT was 80:l). Lysine (1 M) was added to the reaction for 6 to 8 hours at 4°C to terminate the reaction and the CT-HRP conjugate was then purified by a second gel filtration. Large molecular weight fractions (84-150 kd) containing the conjugate were pooled and concentrated to a volume of 1.5-2.5 ml using Amicon CF50 concentrating cones. The final protein concentration of the conjugate was adjusted to 0.22-0.44% using phosphate buffered saline as determined by Lowry or Bradford assays (Lowryet al., '51; Bradford, '76). This concentration range is very dilute compared to the concentration of tracer used by others. After abdominal laparotomy and packing the abdomen with dry gauze (precautions to capture leaking tracer), the organ to be injected was exteriorized and placed on a dry gauze and parafilm bed. Most injections involved the cecum (n = 30), as this portion of both the small and large bowel yielded the most consistent retrograde and anterograde labeling in the dorsal vagal complex. Additional injections were made in other regions of the intestine including the duodenum (n = 6),jejunum (n = 31, terminal ileum (n = 4), and ascending colon (n = 4). Six animals received injection of CT-HRP into the stomach and served as controls. The tracer was applied through the serosal surface of the bowel wall in 0.5-3 ~1 aliquots at multiple sites via a glass micropipette affxed with sealing wax to a Hamilton syringe. It should be noted that these injection volumes did not remain within the viscus. They leaked back out the injection tract when the pipette was withdrawn. The great majority of the volume leakage occurred within the first 1 to 2 minutes. In light of this problem, it is difficult to know the amount of tracer that remained and actually contributed to the injection site. Therefore, the volumes we report below (the actual volumes leaving the syringe) are an overestimation of the amount in the injection site. The large volumes of dilute tracer utilized, however, were necessary to inject the appropriate viscus at multiple sites in a reasonable amount of time. It is also obvious that precautions must be taken to collect the leakage (Fox and Powley, '89). Spread was controlled by exteriorizing and isolating the viscus from adjacent structures with gauze and by blotting and mopping up each injection site with cotton swabs. The injected viscus was exteriorized for periods up to 45 minutes postinjection and rinsed frequently in order to dilute any tracer that leaked from the injection site and prevent drying. After replacing the viscus, the peritoneal cavity was flooded with sterile isotonic saline to dilute the tracer concentration further to levels inadequate for binding. The initial low protein concentration of the CT-HRP made this
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possible. The saline was then aspirated and this was repeated two more times. The adequacy of our measures to control spread of the tracer was documented in five animals in which the cecum was injected. Sections and en bloc segments of small intestines (terminal ileum and jejunum) and ascending colon removed from these animals during histological processing did not show evidence of HRP reaction product following their incubation with TMB. Ventral cecal injections were defined as on the surface of the cecum most frequently seen immediately upon opening the abdomen of a rat in dorsal recumbency with the head in a superior position. In general, under these conditions, the ascending colon is to the viewer's left, the ileum is to the viewer's right, and the convex surface of the cecum is toward the viewer. Dorsal cecal injections were made on the opposite surface. Total cecal injections included both ventral and dorsal surfaces with the volumes of tracer from 70 to 105 pl (note qualificationabove). Lateral injections of the cecum were performed over both ventral and dorsal surfaces of the half of the cecum that included the appendix using volumes of tracer up to 80 p1. Medial injections were made over the opposite half of the cecum toward the colon and utilized similar tracer volumes. Proximal duodenal injections were made beginning 0.5 cm distal to the pylorus over a 2 cm segment using volumes up to 45 p1. Distal duodenal injections began between 4 and 7 cm distal to the pylorus. Jejunal injections were made in the area 34-62 cm proximal to the ileocecaljunctions over a segment of 2 cm using volumes up to 80 p1. Terminal ileal injections were just proximal to the ileocecaljunctions with volumes up to 30 pl. Ascending colonic injections were made over a 0.5 cm segment just distal to the cecocolonic junction using volumes up to 40 p1. In the stomach cases, the entire dorsal and ventral surfaces of all regions (forestomach, corpus, and antruml pylorus) were injected with volumes of tracer ranging up to 80 p1. Methods to control spread were similar to those employed with the bowel injections. After survival periods of 3-6 (usually 3) days, the rats were perfused according to the protocol of Mesulam ('82). Frozen serial 40 p,m sections of the brainstem and spinal cord were made in either the transverse, sagittal, or horizontal planes and the tissue was then processed for HRP according to the TMB protocol of Mesulam ('82). All histological material was studied with both bright- and polarized darkfield microscopy and selected sections were counterstained with neutral red. Sections were photographed with an Olympus BH-2 photomicroscope. The analysis of data includes estimates of the number of retrogradely labeled cells in different parts of the DMN made from counts of transverse sections. It should be emphasized that the counts reported represent estimates because of the inherent problems of counting HRP labeled cells in thick sections through the DMN (Norgren and Smith, '88).The intensity of labeling of individual cells was of sufficient magnitude to obscure the presence of the nucleus and nucleolus, thus negating their use in estimating the number of labeled cells (Abercrombie, '46; Gundersen, '86). The most reliable particle (Gundersen, '86) for counting was the discrete profile (Fox and Powley, '85) of a CT-HRP labeled cell apparent under brightfield illumination. Utilizing this technique, we were able to distinguish basal dendrites and fragments of cells, which were excluded from the counts. The size of DMN neuron limited its appearance to at most two serial sections when tissue was
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serially cut into 40 +m sections. Therefore, in order to minimize the double counting of cells (fractionator procedure; Gundersen, '86), every other section was examined and the labeled cells counted. The obex was defined as the opening of the central canal into the fourth ventricle (generally located approximately 250 pm behind the rostral edge of the AP), and this landmark was used to calculate the rostral and caudal extent of neuronal labeling following injection of tracer into the cecum and stomach. Data from a group of animals are always expressed as the mean +- SEM. Statistical analyses were performed by using the independent t test (Croxton and Cowen, '39). In selected animals, en bloc incubations for HRP histochemistry of the sensory ganglia of the IXth and Xth nerves (Altschuler et al., '89) were also performed. Labeled cells were counted from whole mounts of the ganglia and plotted through a camera lucida tube attached to the Olympus BH-2 microscope to aid in the construction of viscerotopic mapping of the sensory ganglia. The distribution of labeled perikarya was evaluated in ten ganglia from six cases in which the entire cecum was injected and four ganglia from two stomach cases.
RESULTS DMN labeling Injections of CT-HRP into different regions of the small and large bowel resulted in central neuronal labeling restricted to the DMN. The labeling of DMN neurons following injections of all regions of the bowel except the cecum was quite sparse with a maximum of 32 labeled neurons seen in any one of these cases. In contrast with all injections of the cecum, a consistent pattern of neuronal labeling of far greater number was observed. Central neuronal labeling was virtually confined to the lateral aspects of the DMN bilaterally following all total cecal injections (Figs. 1-3). Although many labeled dendrites were visible within the medial aspects of the nucleus bilaterally this area conspicuously lacked labeled neurons except in the most anterior aspects of the DMN where there was a sparse distribution of labeled cells (Figs. 2, 3). Labeled neurons were observed extending from approximately 2,000 pm caudal to the obex (defined as opening of the cc) to approximately 900 pm rostral to the obex. However, they were most numerous at the level of the obex (560 pm below to 640 pm above). Medial and lateral injections of the cecum showed less dense neuronal labeling in the same distribution as total cecal injections. Ventral and dorsal surface injections of the cecum showed no difference in the density of neuronal labeling between the right and left sides of the DMN. Following injections of the stomach, the labeled neurons were densely packed medially, occupied a much greater rostrocaudal and mediolateral extent of the DMN bilaterally than observed after cecal injections, and rarely extended into the most lateral portions of the nucleus (Figs. 3, 4, and Table 1).Although the cecum had by far the greatest representation in the DMN of any area of the bowel, it was of much less magnitude in comparison to the stomach (See Table 1for cell counts). Injections of the proximal and distal portions of duodenum labeled a small number of cells within the medial aspects of the DMN. Jejunal, ileal, and ascending colon
injections labeled cells sparsely within the lateral column of the DMN bilaterally.
Dendritic architecture Prominent dendritic labeling within the confines of the DMN was present following total injection of the cecum. A portion of the dendritic arborization projected medially at all rostrocaudal levels into the medial aspects of the DMN where labeled motoneurons were observed following injection of tracer into the stomach and duodenum (Figs. 2-4). A few dendrites appear to cross the midline to the opposite DMN (see Fig. 3 for example). Within the confines of the lateral DMN, there is an extensive arborization of dendritic processes projecting rostrocaudally (Fig. 3A) in a pattern with striking similarities to that seen in the medial aspects of DMN following stomach injections (Fig. 3B). For the lateral column, the rostrocaudal dendrites far outnumber those projecting medially and dorsally. The extranuclear dendritic processes of cecal motoneuions were more limited than those of stomach motoneurons (Figs. 2, 4). They projected dorsomedially into the NTS, terminating within the medial (med NTS) and commissural subnuclei (com NTS). Their orientation was almost perpendicular to the extranuclear dendrites of stomach motoneurons, which exited the DMN in a dorsolateral direction. Rostral to the AP, cecal motoneurons lacked significant extranuclear dendrites. The dendrites of gastric motoneurons terminated within different areas of the NTS, occupying areas where there was a lack of dendrites of cecal motoneurons and cecal afferent terminal labeling (Fig. 4).
NTS terminal labeling Afferent terminal labeling was observed within the NTS in ten animals in which CT-HRP was injected throughout the entire cecum. No afferent terminal labeling was visualized following injection of the tracer into the other areas of the bowel. Although the density of afferent terminal labeling varied between cases, the extent and distribution of labeling was quite consistent (compare Figs. 2 and 4). Anterior to AP, light afferent terminal labeling was bilaterally localized to the periventricular portion of the NTS in an area just medial and dorsal to the gel NTS (Figs. 2A, 4A). Heavier labeling was evident caudally within the com NTS. This labeling extended caudally to a level approximately 500 +m caudal to the obex where it rapidly disappeared.At the level of the anterior AP, afferent labeling was concentrated dorsolaterally within the com NTS occupying an area immediately subjacent to the Ap. (Figs. 2, 4). Very light afferent labeling extended into adjacent areas of the med NTS. In case IN66 where the density of terminal labeling was the heaviest of the cases surveyed, light terminal labeling extended into the marginal aspects of the AP; however, there was no spread to stomach afferent terminal
Fig. 1. A Low power photomicrograph of a dorsal view of the rat brainstem with the cerebellum removed. The white lines that cross it indicate the approximate rostrocaudal level of each transverse section in Fig. 2. B and C: Darkiield photomicrographs of transverse and horizontal sections, respectively, through the DMN of rats injected with CT-HRP throughout the cecum. Labeled cells are present in the lateral column of the DMN. The boxes outlined in B and C are comparable regions appearing in higher magnification in Figs. 2 and 3, respectively. Scale bar in A = 1 mm, in B and C = 0.5 mm.
Figure 1
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Fig. 2. A-F: Darkfield photomicrographs of the DMN at the rostrocaudal levels indicated in Fig. lA, following injection of CT-HRP into the cecum (case IN52, 100 p1). The distances in microns above and below the obex are noted. Heavy neuronal labeling is restricted t o the
lateral DMN and dendritic labeling is present in the medial DMN. The filled arrows indicate dendrites projecting into the medial DMN; unfilled arrows indicate dendrites projecting into the NTS. The thin arrow indicates axon terminal labeling. Scale bar = 0.25 mm.
fields (Fig. 4).Toward the caudal aspects of the AP and extending caudally beyond it, afferent terminals tended to occupy a more localized area within the com NTS close to the midline but dorsal to sites penetrated by gastric motoneuron dendrites (Figs. 2,4). Injection of the stomach with CT-HRP resulted in heavy afferent terminal labeling within the gel NTS and lighter labeling within the medial subnucleus and midline com NTS at the level of the AP (Fig. 4). Gastric afFerents,
however, did not extend as far caudally in the midline com NTS as those from the cecum.
Vagal ganglia labeling After injection of CT-HRP into either the cecum or stomach, perikaryal labeling was limited to nodose ganglia (Fig. 5). Within this inferior portion of the fused ganglionic mass, the location of labeled perikarya was quite similar following injection of the stomach and cecum. However, the
CENTRAL REPRESENTATION OF CECUM IN RAT
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Fig. 3. A: Brightfield photomicrograph of a horizontal section through the DMN following injection of CT-HRP into the cecum (case IN32, 95 pl). B: Brightfield photomicrograph of a horizontal section through the DMN followinginjection of CT-HRP into the stomach (case IN39, 55 pl). Dendrites (thick arrows) project medially and rostrocaudally within the DMN. Axons are marked by thin arrows. The asterisk
marks the level of the obex, which appears more rostral in A due to a small difference in depth of the section. Note that there is a distinct topography of the labeling of motoneurons in the DMN. The stomach is innervated by neurons of the medial column and cecum by neurons of the lateral column. Scale bar = 0.25 mm.
density of labeling was far greater following injections of the stomach (118 2 10 cells vs. 50 2 6 cells, P < 0.05).
strong representation for the stomach within the DMN (Yamamotoet al., '77; Leslie et al., '82; Scharoun et al., '84; Shapiro and Miselis, '85b; Pagani et al., '88; Okumura and Namiki, 'go), this study provides the first evidence for a significant topographically distinct representation for the cecum within the DMN.
DISCUSSION The present results indicate that the rat small and large intestines receive limited parasympathetic innervation from the DMN with the cecum representing a major exception. Together the stomach and the cecum appear to receive the vast majority of efferent projections from the DMN to the alimentary tract. Although previous studies have shown a
DMN-motor neuronal columns Motoneurons projecting to the cecum were found to occupy an area within the lateral aspects of the DMN
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Stomach
Cecum
Figure 4
CENTRAL REPRESENTATION OF CECUM IN RAT
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TABLE 1. Average Cell Counts and Rostrocaudal Extent of Labeled DMN Neurons (Bilateral)'
Injection site Cecum Stomach
No.
Labeled cells'
10 5
108 -t 5 1,144 -t 41*
Rostral extent ( ~ r n ) ~ 892 + 44 2,336 -t 47'
Caudal extent (&ml3 1,876 -t 85 2,720 -t 84'
'Obtained from analysis of transverse sections.Dataare mean 2 SEM 'Actual counts obtaind from every other section (nowrrection). 3Frornobex (openingof central canal). *Significantlydifferent (P < 0.01) when compared tocecum.
bilaterally. Labeled cells were most numerous and densely packed within the caudal DMN forming a coherent column between the level just caudal to the obex and rostral to the AP. These columns were situated laterally, with little overlap, to the more medial column of labeled DMN cells found following injection of the stomach (Fig. 6). The separation of gastric and cecal representation by columns in the DMN is not surprising considering previous observations in the spinal cord, NA, and hypoglossal nucleus that motoneurons innervating a specific muscle tend to group in a single longitudinal column (Sterling and Kuypers, '67; Odutola, '76; Burke et al., '77; Krammer et al., '79; Kuzahara and Chou, '80; Miyazaki et al., '81; Goshgarian and Rafols, '81; Bieger and Hopkins, '87; Furicchia and Goshgarian, '87; Anderson et al., '88; Bao et al., '88). The cecum appears to be specifically innervated by the celiac and accessory celiac branches of the subdiaphragmatic vagus since these branches carry axons from motoneurons of the lateral columns on the DMN (Fox and Powley, '85; Norgren and Smith, '88). The rostrocaudal distribution of retrograde labeling within the DMN found following injection of the cecum in the present study closely matches that obtained previously after incubation of the celiac and accessory celiac branches of the subdiaphragmatic vagus with true blue or free HRP (Fox and Powley, '85; Norgren and Smith, '88). Additionally, cecal afferents terminate within the NTS in a pattern with striking similarities to that found following incubation of the celiac and accessory celiac branches with HRP (Norgren and Smith, '88) (see below for details). The virtual lack of labeled cells within the medial columns of the DMN and terminal labeling within the gel NTS (gastric afferent terminal area) following injection of the cecum indicates that vagal innervation of the cecum is limited to these two branches of the subdiaphragmatic vagus. In contrast, the distribution of labeled DMN cells and pattern of terminal labeling seen following injection of the stomach conforms to the pattern of labeling observed previously with incubation of the two gastric branches of the subdiaphragmatic vagus with tracer (Fox and Powley, '85; Norgren and Smith, '88).
Fig. 5. A and B Photomicrographs of ganglionic labeling following injection of CT-HRP into the cecum (IN67,lOO p1) and stomach (IN68, 75 pl), respectively. Scale bar = 1mm.
Fig. 4. A-D, E-H. Series of darkfield photomicrographs at four rostrocaudal levels through the dorsal vagal complex followinginjection of CT-HRP into the cecum (IN66, 100 p1) and stomach (IN72, 65 pl). The areas of afferent terminal labeling appear topographically distinct for the cecum and stomach. In general, afferent terminal labeling following stomach injections is more lateral to that following injection of the cecum. Note in A a small area of cecal terminal labeling (thin arrow) lying dorsomedial to gel NTS, which is the site of stomach afferent terminal labeling. In E and F, dendrites of stomach projecting motoneurons appear to encircle but not penetrate the area of cecal afferent terminals within the corn NTS (area enclosed by broken line). The arrow in C indicates a dendrite from a cecal motoneuron that projects and terminates within the com NTS (cecal afferent terminal field). Scale bar = 0.25 mm.
The present results support a mediolateral organization within the DMN correspondingto the rostrocaudal positioning along the alimentary canal (Fig. 6). DMN cells projecting to the stomach and duodenum were limited to the medial columns, while those projecting to the jejunum, ileum, and ascending colon were located within the lateral columns. A similar mediolateral distribution of labeled cells within the DMN following injection of free HRP into different regions of the bowel has been previously reported in the cat (Satomi et al., '78). This distribution of DMN labeling suggests the duodenum receives innervation from either the gastric or hepatic branches while the more distal area of the small intestine and proximal large bowel are
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C
A
C
3
R
D corn NTS gel NTS isNTS.
C
D E X
E
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::
Representation of the Cecum in the Lateral Dorsal Motor Nucleus of the Vagus Nerve and Commissural Subnucleus of the Nucleus Tractus Solitarii in Rat STEVEN M. ALTSCHULER, DAVID A. FERENCI, RICHARD B. LYNN, AND RICHARD R. MISELIS Division of Gastroenterology and Nutrition, Children's Hospital of Philadelphia (S.M.A., D.A.F.) and Institute of Neurological Sciences and Animal Biology, University of Pennsylvania (R.B.L., R.R.M.), Philadelphia, Pennsylvania 19104
ABSTRACT Motor fibers of the accessory celiac and celiac vagal branches are derived from the lateral columns of the dorsal motor nucleus of the vagus nerve. These branches also contain sensory fibers that terminate within the nucleus of the tractus solitarii. This study traces the innervation of the intestines by using the tracer cholera toxin-horseradish peroxidase. In 53 rats, the tracer was injected into either the stomach, duodenum, jejunum, terminal ileum, cecum, or ascending colon. With all cecal injections, prominent retrograde labeling of cell bodies occurred bilaterally in the lateral columns of the dorsal motor nucleus of the vagus nerve above, at, and below the level of the area postrema. Dendrites of laterally positioned neurons projected medially and rostrocaudally within the dorsal motor nucleus of the vagus nerve and dorsomedially into both the medial subnucleus and parts of the commissural subnucleus of the nucleus of the tractus solitarii. Sensory terminal labeling occurred in the dorsolateral commissural subnucleus a t the level of the rostral area postrema and the medial commissural subnucleus caudal to the area postrema. Additionally, there was sensory terminal labeling within a small confined area of the dorsomedial zone of the nucleus of the tractus solitarii immediately adjacent to the fourth ventricle at a level just anterior to the area postrema. Stomach injections labeled motoneurons of the medial column of the entire rostrocaudal extent of the dorsal motor nucleus of the vagus nerve and a sensory terminal field primarily in the subnucleus gelatinosus, with less intense labeling extending caudally into the medial and ventral commissural subnuclei. Dendrites of gastric motoneurons project rostrocaudally and mediolaterallywithin the dorsal motor nucleus of the vagus nerve and dorsolaterally within the nucleus of the tractus solitarii. They are most pronounced at the level of the rostral area postrema where many dendrites course dorsolaterally terminating primarily within the subnucleus gelatinosus. Injections of the duodenum labeled a small number of the cells within the medial aspects of the dorsal motor nucleus of the vagus nerve. Jejunal, ileal, and ascending colon injections labeled cells sparsely within the lateral aspects of the dorsal motor nucleus of the vagus nerve bilaterally. No afferent terminal labeling was evident after injection of these areas of the bowel. The dorsal vagal complex has a prominent viscerotopography: the dorsal motor nucleus of the vagus nerve has a mediolateral organization corresponding to end-organ innervation; the nucleus of the tractus solitarii has a rostrocaudal axis of visceral representation with some overlap corresponding to rostrocaudal positioning along the alimentary canal and mediolateral separation of terminals within the nucleus.
Accepted October 19,1990, Address reprint requests to Dr. Richard R. Miselis, Dept. of Animal Biology, University of PennsylvaniaVeterinary School, 3800 Spruce Street, Philadelphia,PA 19104-6045.
o 1991 WILEY-LISS, INC.
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262 Key words: intestines, celiac branch of vagus, cholera toxin-horseradishperoxidase, viscerotopy
Progress in topographical studies of visceral innervation has accelerated with the introduction of new tracers and injection strategies to control them. Where there have been ambiguities concerning viscerotopic representation in the vagal complex (Kalia and Mesulam, '80a,b; Hinrichsen and Ryan, '81; Kalia, '81; Yoshida et a]., '81; Rogers and Hermann, '83;Holstege et al., '83;Fryscaket al., '841, there is now more certainty of its existence (Gwyn et al., '79; Katz and Karten, '83;Shapiro and Miselis, '85b; Bieger and Hopkins, '87; Rinaman and Miselis, '87; Pagani et al., '88; Altschuler et al., '89; Okumura and Namiki, '90). With direct intramuscular injection of the tracers (particularly ones with receptor binding properties) or their application to cut nerves, a strong viscerotopic organization for the upper alimentary tract has been established within the NA and NTS of the rat (Bieger and Hopkins, '87; Altschuler et al., '89). In contrast to the viscerotopic organization proposed €or the NA (Lawn, '66a,b; Bieger and Hopkins, '87), the DMN is organized mediolaterallywith respect to specificbranches of the subdiaphragmatic vagus (Fox and Powley, '85; Norgren and Smith, '88). This scheme, which parcels the DMN into a series of longitudinal columns running the entire rostrocaudal extent of the nucleus, has been primarily derived from the application of tracer to the major branches of the subdiaphragmatic vagus. Each DMN is comprised of a large medial column of cells lying immediately adjacent to the midline. These columns of cells provide efferent axons to the two gastric branches. In the lateral aspects of each DMN, a column of cells of much smaller mediolateral extent exists and contributes fibers to the celiac and accessory celiac branches of the subdiaphragmatic vagus. Within the left medial DMN, an additional column exists composed of a few scattered cells providing axons to the hepatic branch. The formation of the rat DMN into longitudinal cell columns raises the issue of how the innervation of specific abdominal viscera is organized within these columns. This question is best answered by direct intramuscular injection of tracer into the viscera under study. To date, the majority of studies utilizing this technique have focused on the stomach and pancreas. Although a number of investigations have suggested that these structures are represented throughout the entire DMN (Weaver, '80; Takayama et al.,
'82; Leslie et al., '82; Sharkey and Williams, '83;Luiten et al., '84), two recent studies employing HRP conjugates, in which adequate measures to prevent spread and leakage of tracer were performed, have demonstrated a far more localized retrograde labeling of cell bodies primarily within the medial columns of the DMN (Shapiro and Miselis, '85b; Rinaman and Miselis, '87). These findings suggest that specific abdominal viscera are localized to either the medial or lateral bilaterally symmetrical columns. We performed a series of intramuscular injections with CT-HRP in different regions of the small and large bowel in order to determine the viscera represented in the lateral columns of the DMN. Preliminary studies revealed that extremely small numbers of cells were retrogradely labeled within the DMN following injection of CT-HRP into most regions of the bowel. The major exception to this observation was the cecum, where injections of CT-HRP resulted in consistent retrograde labeling of cell bodies within the lateral columns of the DMN (Ferenci et al., '89a,b). On the basis of our preliminary results, the majority of injections in this study involved the cecum. Additionally, since CTHRP is very effective in labeling distal dendritic processes, a comparison of the dendritic fields of medial and lateral column motoneurons was undertaken. The distributions of labeled perikarya within the vagal ganglia and afferent terminal fields within the NTS were also investigated. We were most interested in relating their distributions to previously obtained viscerotopic mappings of the vagal ganglia and NTS following injection of tracer into the upper alimentary tract (Altschuler et al., '89). In our experience, CT-HRP is more effective than free HRP and equally effective as the wheat germ agglutinin-HRP conjugate in labeling afferent terminal fields (Shapiro and Miselis, '85b; Altschuler et al., '89).
MATERIALS AND METHODS Fifty-three adult male Sprague-Dawley rats weighing 210-510 g were employed in these experiments. The animals were anesthetized with ketamine (85 mgikg) and xylazine (12 mg/kg) prior to aseptic surgical procedures. CT-HRP, conjugated in our laboratory by a modification of a previously described method (Shapiro and Miselis, '85a), was utilized as the neural tracer in these studies. Briefly,
Abbreviations
AP C cc
CT-HRP d dl DMN lat lat DMN med DMN
HRP MMC NA NTS cen NTS
area postrema caudal central canal cholera toxin-horseradish peroxidase dorsal dorsolateral dorsal motor nucleus of the vagus nerve lateral lateral column of the DMN medial column of the DMN horseradish peroxidase migrating motor complex nucleus ambiguus nucleus of the tractus solitarii subnucleus centralis of NTS
com NTS gel NTS is NTS med med NTS Ng
R
SLN TMB TS 4v
IX X ~~
XI XI1
commissural subnucleus of NTS subnucleus gelatinosus of NTS subnucleus interstitialis of NTS medial medial subnucleus of NTS nodose ganglion rostral superior laryngeal nerve tetramethyl benzidine tractus solitarius fourth ventricle glossopharyngeal nerve vagus nerve accessory nerve hypoglossal nucleus
CENTRAL REPRESENTATION OF CECUM IN RAT CT (supplied by Sigma or List Biological) was covalently attached to HRP (Sigma type VI) to make CT-HRP by using glutaraldehyde as a homobifunctional bridge molecule in a two-step process (Avrameasand Ternynck, '71; Gonatas et al., '79). HRP (153 mg) was incubated with glutaraldehyde [EM grade; 200 pl of a 25% solution in 1,800 p1 of potassium phosphate buffer (pH 6.8)] for 4 hours at 1:lOO molar concentration ratio to activate the molecule (Molin et al., '78). By utilizing gel filtration chromatography (LKB Ultrogel AcA44, 1.6 cm x 90 cm; eluent 0.15 M NACl), monomeric HRP that contains both unreacted and activated HRP was separated from the polymeric byproducts of the activation reaction. Fractions containing 40 kd product were pooled and concentrated to approximately 1 ml (Amicon CF25 concentrating cone) and reacted with CT (4 mg) in 0.1 M carbonate buffer (pH 9.5) for 40 hours at 4°C (molar ratio of HRP to CT was 80:l). Lysine (1 M) was added to the reaction for 6 to 8 hours at 4°C to terminate the reaction and the CT-HRP conjugate was then purified by a second gel filtration. Large molecular weight fractions (84-150 kd) containing the conjugate were pooled and concentrated to a volume of 1.5-2.5 ml using Amicon CF50 concentrating cones. The final protein concentration of the conjugate was adjusted to 0.22-0.44% using phosphate buffered saline as determined by Lowry or Bradford assays (Lowryet al., '51; Bradford, '76). This concentration range is very dilute compared to the concentration of tracer used by others. After abdominal laparotomy and packing the abdomen with dry gauze (precautions to capture leaking tracer), the organ to be injected was exteriorized and placed on a dry gauze and parafilm bed. Most injections involved the cecum (n = 30), as this portion of both the small and large bowel yielded the most consistent retrograde and anterograde labeling in the dorsal vagal complex. Additional injections were made in other regions of the intestine including the duodenum (n = 6),jejunum (n = 31, terminal ileum (n = 4), and ascending colon (n = 4). Six animals received injection of CT-HRP into the stomach and served as controls. The tracer was applied through the serosal surface of the bowel wall in 0.5-3 ~1 aliquots at multiple sites via a glass micropipette affxed with sealing wax to a Hamilton syringe. It should be noted that these injection volumes did not remain within the viscus. They leaked back out the injection tract when the pipette was withdrawn. The great majority of the volume leakage occurred within the first 1 to 2 minutes. In light of this problem, it is difficult to know the amount of tracer that remained and actually contributed to the injection site. Therefore, the volumes we report below (the actual volumes leaving the syringe) are an overestimation of the amount in the injection site. The large volumes of dilute tracer utilized, however, were necessary to inject the appropriate viscus at multiple sites in a reasonable amount of time. It is also obvious that precautions must be taken to collect the leakage (Fox and Powley, '89). Spread was controlled by exteriorizing and isolating the viscus from adjacent structures with gauze and by blotting and mopping up each injection site with cotton swabs. The injected viscus was exteriorized for periods up to 45 minutes postinjection and rinsed frequently in order to dilute any tracer that leaked from the injection site and prevent drying. After replacing the viscus, the peritoneal cavity was flooded with sterile isotonic saline to dilute the tracer concentration further to levels inadequate for binding. The initial low protein concentration of the CT-HRP made this
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possible. The saline was then aspirated and this was repeated two more times. The adequacy of our measures to control spread of the tracer was documented in five animals in which the cecum was injected. Sections and en bloc segments of small intestines (terminal ileum and jejunum) and ascending colon removed from these animals during histological processing did not show evidence of HRP reaction product following their incubation with TMB. Ventral cecal injections were defined as on the surface of the cecum most frequently seen immediately upon opening the abdomen of a rat in dorsal recumbency with the head in a superior position. In general, under these conditions, the ascending colon is to the viewer's left, the ileum is to the viewer's right, and the convex surface of the cecum is toward the viewer. Dorsal cecal injections were made on the opposite surface. Total cecal injections included both ventral and dorsal surfaces with the volumes of tracer from 70 to 105 pl (note qualificationabove). Lateral injections of the cecum were performed over both ventral and dorsal surfaces of the half of the cecum that included the appendix using volumes of tracer up to 80 p1. Medial injections were made over the opposite half of the cecum toward the colon and utilized similar tracer volumes. Proximal duodenal injections were made beginning 0.5 cm distal to the pylorus over a 2 cm segment using volumes up to 45 p1. Distal duodenal injections began between 4 and 7 cm distal to the pylorus. Jejunal injections were made in the area 34-62 cm proximal to the ileocecaljunctions over a segment of 2 cm using volumes up to 80 p1. Terminal ileal injections were just proximal to the ileocecaljunctions with volumes up to 30 pl. Ascending colonic injections were made over a 0.5 cm segment just distal to the cecocolonic junction using volumes up to 40 p1. In the stomach cases, the entire dorsal and ventral surfaces of all regions (forestomach, corpus, and antruml pylorus) were injected with volumes of tracer ranging up to 80 p1. Methods to control spread were similar to those employed with the bowel injections. After survival periods of 3-6 (usually 3) days, the rats were perfused according to the protocol of Mesulam ('82). Frozen serial 40 p,m sections of the brainstem and spinal cord were made in either the transverse, sagittal, or horizontal planes and the tissue was then processed for HRP according to the TMB protocol of Mesulam ('82). All histological material was studied with both bright- and polarized darkfield microscopy and selected sections were counterstained with neutral red. Sections were photographed with an Olympus BH-2 photomicroscope. The analysis of data includes estimates of the number of retrogradely labeled cells in different parts of the DMN made from counts of transverse sections. It should be emphasized that the counts reported represent estimates because of the inherent problems of counting HRP labeled cells in thick sections through the DMN (Norgren and Smith, '88).The intensity of labeling of individual cells was of sufficient magnitude to obscure the presence of the nucleus and nucleolus, thus negating their use in estimating the number of labeled cells (Abercrombie, '46; Gundersen, '86). The most reliable particle (Gundersen, '86) for counting was the discrete profile (Fox and Powley, '85) of a CT-HRP labeled cell apparent under brightfield illumination. Utilizing this technique, we were able to distinguish basal dendrites and fragments of cells, which were excluded from the counts. The size of DMN neuron limited its appearance to at most two serial sections when tissue was
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serially cut into 40 +m sections. Therefore, in order to minimize the double counting of cells (fractionator procedure; Gundersen, '86), every other section was examined and the labeled cells counted. The obex was defined as the opening of the central canal into the fourth ventricle (generally located approximately 250 pm behind the rostral edge of the AP), and this landmark was used to calculate the rostral and caudal extent of neuronal labeling following injection of tracer into the cecum and stomach. Data from a group of animals are always expressed as the mean +- SEM. Statistical analyses were performed by using the independent t test (Croxton and Cowen, '39). In selected animals, en bloc incubations for HRP histochemistry of the sensory ganglia of the IXth and Xth nerves (Altschuler et al., '89) were also performed. Labeled cells were counted from whole mounts of the ganglia and plotted through a camera lucida tube attached to the Olympus BH-2 microscope to aid in the construction of viscerotopic mapping of the sensory ganglia. The distribution of labeled perikarya was evaluated in ten ganglia from six cases in which the entire cecum was injected and four ganglia from two stomach cases.
RESULTS DMN labeling Injections of CT-HRP into different regions of the small and large bowel resulted in central neuronal labeling restricted to the DMN. The labeling of DMN neurons following injections of all regions of the bowel except the cecum was quite sparse with a maximum of 32 labeled neurons seen in any one of these cases. In contrast with all injections of the cecum, a consistent pattern of neuronal labeling of far greater number was observed. Central neuronal labeling was virtually confined to the lateral aspects of the DMN bilaterally following all total cecal injections (Figs. 1-3). Although many labeled dendrites were visible within the medial aspects of the nucleus bilaterally this area conspicuously lacked labeled neurons except in the most anterior aspects of the DMN where there was a sparse distribution of labeled cells (Figs. 2, 3). Labeled neurons were observed extending from approximately 2,000 pm caudal to the obex (defined as opening of the cc) to approximately 900 pm rostral to the obex. However, they were most numerous at the level of the obex (560 pm below to 640 pm above). Medial and lateral injections of the cecum showed less dense neuronal labeling in the same distribution as total cecal injections. Ventral and dorsal surface injections of the cecum showed no difference in the density of neuronal labeling between the right and left sides of the DMN. Following injections of the stomach, the labeled neurons were densely packed medially, occupied a much greater rostrocaudal and mediolateral extent of the DMN bilaterally than observed after cecal injections, and rarely extended into the most lateral portions of the nucleus (Figs. 3, 4, and Table 1).Although the cecum had by far the greatest representation in the DMN of any area of the bowel, it was of much less magnitude in comparison to the stomach (See Table 1for cell counts). Injections of the proximal and distal portions of duodenum labeled a small number of cells within the medial aspects of the DMN. Jejunal, ileal, and ascending colon
injections labeled cells sparsely within the lateral column of the DMN bilaterally.
Dendritic architecture Prominent dendritic labeling within the confines of the DMN was present following total injection of the cecum. A portion of the dendritic arborization projected medially at all rostrocaudal levels into the medial aspects of the DMN where labeled motoneurons were observed following injection of tracer into the stomach and duodenum (Figs. 2-4). A few dendrites appear to cross the midline to the opposite DMN (see Fig. 3 for example). Within the confines of the lateral DMN, there is an extensive arborization of dendritic processes projecting rostrocaudally (Fig. 3A) in a pattern with striking similarities to that seen in the medial aspects of DMN following stomach injections (Fig. 3B). For the lateral column, the rostrocaudal dendrites far outnumber those projecting medially and dorsally. The extranuclear dendritic processes of cecal motoneuions were more limited than those of stomach motoneurons (Figs. 2, 4). They projected dorsomedially into the NTS, terminating within the medial (med NTS) and commissural subnuclei (com NTS). Their orientation was almost perpendicular to the extranuclear dendrites of stomach motoneurons, which exited the DMN in a dorsolateral direction. Rostral to the AP, cecal motoneurons lacked significant extranuclear dendrites. The dendrites of gastric motoneurons terminated within different areas of the NTS, occupying areas where there was a lack of dendrites of cecal motoneurons and cecal afferent terminal labeling (Fig. 4).
NTS terminal labeling Afferent terminal labeling was observed within the NTS in ten animals in which CT-HRP was injected throughout the entire cecum. No afferent terminal labeling was visualized following injection of the tracer into the other areas of the bowel. Although the density of afferent terminal labeling varied between cases, the extent and distribution of labeling was quite consistent (compare Figs. 2 and 4). Anterior to AP, light afferent terminal labeling was bilaterally localized to the periventricular portion of the NTS in an area just medial and dorsal to the gel NTS (Figs. 2A, 4A). Heavier labeling was evident caudally within the com NTS. This labeling extended caudally to a level approximately 500 +m caudal to the obex where it rapidly disappeared.At the level of the anterior AP, afferent labeling was concentrated dorsolaterally within the com NTS occupying an area immediately subjacent to the Ap. (Figs. 2, 4). Very light afferent labeling extended into adjacent areas of the med NTS. In case IN66 where the density of terminal labeling was the heaviest of the cases surveyed, light terminal labeling extended into the marginal aspects of the AP; however, there was no spread to stomach afferent terminal
Fig. 1. A Low power photomicrograph of a dorsal view of the rat brainstem with the cerebellum removed. The white lines that cross it indicate the approximate rostrocaudal level of each transverse section in Fig. 2. B and C: Darkiield photomicrographs of transverse and horizontal sections, respectively, through the DMN of rats injected with CT-HRP throughout the cecum. Labeled cells are present in the lateral column of the DMN. The boxes outlined in B and C are comparable regions appearing in higher magnification in Figs. 2 and 3, respectively. Scale bar in A = 1 mm, in B and C = 0.5 mm.
Figure 1
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Fig. 2. A-F: Darkfield photomicrographs of the DMN at the rostrocaudal levels indicated in Fig. lA, following injection of CT-HRP into the cecum (case IN52, 100 p1). The distances in microns above and below the obex are noted. Heavy neuronal labeling is restricted t o the
lateral DMN and dendritic labeling is present in the medial DMN. The filled arrows indicate dendrites projecting into the medial DMN; unfilled arrows indicate dendrites projecting into the NTS. The thin arrow indicates axon terminal labeling. Scale bar = 0.25 mm.
fields (Fig. 4).Toward the caudal aspects of the AP and extending caudally beyond it, afferent terminals tended to occupy a more localized area within the com NTS close to the midline but dorsal to sites penetrated by gastric motoneuron dendrites (Figs. 2,4). Injection of the stomach with CT-HRP resulted in heavy afferent terminal labeling within the gel NTS and lighter labeling within the medial subnucleus and midline com NTS at the level of the AP (Fig. 4). Gastric afFerents,
however, did not extend as far caudally in the midline com NTS as those from the cecum.
Vagal ganglia labeling After injection of CT-HRP into either the cecum or stomach, perikaryal labeling was limited to nodose ganglia (Fig. 5). Within this inferior portion of the fused ganglionic mass, the location of labeled perikarya was quite similar following injection of the stomach and cecum. However, the
CENTRAL REPRESENTATION OF CECUM IN RAT
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Fig. 3. A: Brightfield photomicrograph of a horizontal section through the DMN following injection of CT-HRP into the cecum (case IN32, 95 pl). B: Brightfield photomicrograph of a horizontal section through the DMN followinginjection of CT-HRP into the stomach (case IN39, 55 pl). Dendrites (thick arrows) project medially and rostrocaudally within the DMN. Axons are marked by thin arrows. The asterisk
marks the level of the obex, which appears more rostral in A due to a small difference in depth of the section. Note that there is a distinct topography of the labeling of motoneurons in the DMN. The stomach is innervated by neurons of the medial column and cecum by neurons of the lateral column. Scale bar = 0.25 mm.
density of labeling was far greater following injections of the stomach (118 2 10 cells vs. 50 2 6 cells, P < 0.05).
strong representation for the stomach within the DMN (Yamamotoet al., '77; Leslie et al., '82; Scharoun et al., '84; Shapiro and Miselis, '85b; Pagani et al., '88; Okumura and Namiki, 'go), this study provides the first evidence for a significant topographically distinct representation for the cecum within the DMN.
DISCUSSION The present results indicate that the rat small and large intestines receive limited parasympathetic innervation from the DMN with the cecum representing a major exception. Together the stomach and the cecum appear to receive the vast majority of efferent projections from the DMN to the alimentary tract. Although previous studies have shown a
DMN-motor neuronal columns Motoneurons projecting to the cecum were found to occupy an area within the lateral aspects of the DMN
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Stomach
Cecum
Figure 4
CENTRAL REPRESENTATION OF CECUM IN RAT
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TABLE 1. Average Cell Counts and Rostrocaudal Extent of Labeled DMN Neurons (Bilateral)'
Injection site Cecum Stomach
No.
Labeled cells'
10 5
108 -t 5 1,144 -t 41*
Rostral extent ( ~ r n ) ~ 892 + 44 2,336 -t 47'
Caudal extent (&ml3 1,876 -t 85 2,720 -t 84'
'Obtained from analysis of transverse sections.Dataare mean 2 SEM 'Actual counts obtaind from every other section (nowrrection). 3Frornobex (openingof central canal). *Significantlydifferent (P < 0.01) when compared tocecum.
bilaterally. Labeled cells were most numerous and densely packed within the caudal DMN forming a coherent column between the level just caudal to the obex and rostral to the AP. These columns were situated laterally, with little overlap, to the more medial column of labeled DMN cells found following injection of the stomach (Fig. 6). The separation of gastric and cecal representation by columns in the DMN is not surprising considering previous observations in the spinal cord, NA, and hypoglossal nucleus that motoneurons innervating a specific muscle tend to group in a single longitudinal column (Sterling and Kuypers, '67; Odutola, '76; Burke et al., '77; Krammer et al., '79; Kuzahara and Chou, '80; Miyazaki et al., '81; Goshgarian and Rafols, '81; Bieger and Hopkins, '87; Furicchia and Goshgarian, '87; Anderson et al., '88; Bao et al., '88). The cecum appears to be specifically innervated by the celiac and accessory celiac branches of the subdiaphragmatic vagus since these branches carry axons from motoneurons of the lateral columns on the DMN (Fox and Powley, '85; Norgren and Smith, '88). The rostrocaudal distribution of retrograde labeling within the DMN found following injection of the cecum in the present study closely matches that obtained previously after incubation of the celiac and accessory celiac branches of the subdiaphragmatic vagus with true blue or free HRP (Fox and Powley, '85; Norgren and Smith, '88). Additionally, cecal afferents terminate within the NTS in a pattern with striking similarities to that found following incubation of the celiac and accessory celiac branches with HRP (Norgren and Smith, '88) (see below for details). The virtual lack of labeled cells within the medial columns of the DMN and terminal labeling within the gel NTS (gastric afferent terminal area) following injection of the cecum indicates that vagal innervation of the cecum is limited to these two branches of the subdiaphragmatic vagus. In contrast, the distribution of labeled DMN cells and pattern of terminal labeling seen following injection of the stomach conforms to the pattern of labeling observed previously with incubation of the two gastric branches of the subdiaphragmatic vagus with tracer (Fox and Powley, '85; Norgren and Smith, '88).
Fig. 5. A and B Photomicrographs of ganglionic labeling following injection of CT-HRP into the cecum (IN67,lOO p1) and stomach (IN68, 75 pl), respectively. Scale bar = 1mm.
Fig. 4. A-D, E-H. Series of darkfield photomicrographs at four rostrocaudal levels through the dorsal vagal complex followinginjection of CT-HRP into the cecum (IN66, 100 p1) and stomach (IN72, 65 pl). The areas of afferent terminal labeling appear topographically distinct for the cecum and stomach. In general, afferent terminal labeling following stomach injections is more lateral to that following injection of the cecum. Note in A a small area of cecal terminal labeling (thin arrow) lying dorsomedial to gel NTS, which is the site of stomach afferent terminal labeling. In E and F, dendrites of stomach projecting motoneurons appear to encircle but not penetrate the area of cecal afferent terminals within the corn NTS (area enclosed by broken line). The arrow in C indicates a dendrite from a cecal motoneuron that projects and terminates within the com NTS (cecal afferent terminal field). Scale bar = 0.25 mm.
The present results support a mediolateral organization within the DMN correspondingto the rostrocaudal positioning along the alimentary canal (Fig. 6). DMN cells projecting to the stomach and duodenum were limited to the medial columns, while those projecting to the jejunum, ileum, and ascending colon were located within the lateral columns. A similar mediolateral distribution of labeled cells within the DMN following injection of free HRP into different regions of the bowel has been previously reported in the cat (Satomi et al., '78). This distribution of DMN labeling suggests the duodenum receives innervation from either the gastric or hepatic branches while the more distal area of the small intestine and proximal large bowel are
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C
A
C
3
R
D corn NTS gel NTS isNTS.
C
D E X
E
Soft palate r,:.i '-l Pharynx ...... :A: ...... Esophagus Stomach
::
