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The vagal innervation of the different layers of the rat gastrointestinal wall was identified with the fluorescent carbocyanine dye Dil, injected into the dorsal motor nucleus of the vagus (dmnX). Multiple, bilateral injections were used to label all dmnX preganglionic motoneurons, and as a consequence, most of the vagal primary afferents that terminate in the adjacent nucleus of the solitary tract (nts) were also retrogradely and transganglionically labeled. With Fluorogold used to label the enteric nervous system completely and specifically, the Dil-labeled vagal profiles could be visualized and quantified in their anatomical relation to the neurons of the myenteric and submucous ganglia. In the myenteric plexus, vagal fibers and terminals were found throughout the gastrointestinal tract as far caudal as the descending colon, but there was a general decreasing proximodistal gradient in the density of vagal innervation. All parts of the gastric myenteric plexus (fundus, corpus, antrum), as well as the proximal duodenum, were extremely densely innervated, with vagal fibers and terminals in virtually every ganglion and connective. Further caudally, both the percentage of innervated myenteric ganglia and the average density of label within the ganglia rapidly decreased, with the exception of the cecum and proximal colon, where up to 65% of the ganglia were innervated. In the gastric and duodenal submucosa very few and in the mucosa no vagal fibers and terminals were found. With both normal epifluorescence and laser scanning confocal microscopy, highly varicose or beaded terminal structures of various size and geometry could be identified. The Dil injections, which impregnated the dmnX as well as the adjacent nts, resulted in retrograde and anterograde labeling of all the previously reported forebrain connections with the dorsal vagal complex. We conclude that the myenteric plexus is the primary target of vagal innervation throughout the gastrointestinal tract, and that its innervation is more complete than previously assumed. In contrast, vagal afferent (and efferent) innervation of mucosa and submucosa seems conspicuously sparse or absent. Furthermore, the use of more focal injections of Dil offers the prospect to simultaneously identify specific subsets of vagal preganglionics and their central nervous inputs.

The present study provides a LM and EM inventory of the fibers of the rat abdominal vagus, including dorsal and ventral trunks and the five primary branches. Whole mounts (n = 15) were prepared to characterize the branching patterns. A set of EM samples consisting of both trunks and all branches (i.e. dorsal and ventral gastric, dorsal and accessory celiac, and hepatic) were then obtained from each of six additional animals. A complete cross-sectional montage (x 10000) was prepared from each sample. All axons were counted, and greater than 10% of them were evaluated morphometrically. The means of unmyelinated axon diameters for each of the five branches were similar (0.75-0.83 microns). However, the shapes of the fiber size distributions, as summarized by their skew coefficients, revealed that the two gastric branches differed significantly from the two celiac branches; furthermore, the hepatic size distribution differed from all others. Most of the myelinated fibers (85%) in all branches were less than 2.6 microns in diameter and had sheath widths between 0.1 and 0.5 micron. The gastric branches, however, also contained a few larger myelinated fibers with sheath widths as great as 0.85 micron. Whole mounts revealed fibers which were not of supradiaphragmatic origin within all five vagal branches; these adventitial bundles were traced along the perineurium between adjacent branches. The sum of the fibers in the five branches (26930) was 21% more than the number counted in the parent trunks (22272); this excess probably reflects the adventitial fiber content. The whole mounts also showed that a large and regularly positioned paraganglion was associated with the dorsal branches. The structural profiles observed (i.e. unmyelinated and myelinated fibers size distributions, presence of extrinsic fascicles, glomus tissue content, etc.) differentiate the vagal branches into three morphologically distinct sets: a gastric pair, a celiac pair, and a hepatic branch. The fiber counts, when considered with observations of the numbers of efferents and adventitial fibers in the nerve, suggest that the percentage of efferent fibers is much higher than in all the widely accepted estimates found in the literature: efferent fibers may represent over a quarter of the total number of fibers.

In an experiment to investigate the role of the vagus in the lateral hypothalamic (LH) syndrome of body weight loss, male albino rats were divided into four groups: (a) animals with bilateral LH lesions that were subsequently given a bilateral subdiaphragmatic vagotomy, (b) LH animals that received a control vagotomy operation, (c) nonlesion animals that were given a subdiaphragmatic vagotomy operation, (c) nonlesion animals that were given a subdiaphragmatic vagotomy, and (d) nonlesion animals that received a control vagotomy operation. Both LH lesions and vagotomy reduced body weight levels, though the effects differed in terms of the length of time required to reach initial maximal loss, the time required to reach chronic levels of maintenance, and the severity of body weight reduction. Fasting gastric acid secretion was lowered by LH lesions, and the extent of this reduction was positively correlated with the reduction in body weight. Finally, gastric contents after a 24-hr fast were greater in vagotomized rats than in nonvagotomized animals. These data are discussed in relation to the changes in gastric functioning after LH lesions and vagotomy.

A chronically indwelling gastric fistula was implanted into rats. To collect gastric acid, a collection system was coupled to the fistula. This system operated by infusing physiological saline directly into the stomach of unanesthetized rats; the acid content of the perfusate was immediately determined by automatic titration machinery. Using this technique, a value of 3.25 uEq H+/2 minutes was obtained as the basal gastric secretion. For a sham-fed test meal of 2 ml of 36% sucrose, the stimulated phase of gastric acid secretion consisted of a dynamic response (peak 200% of basal) with a brief latency (< 2 minutes) and short duration (< 24 minutes). Results indicated that this response to sham feeding was a cephalic response, since direct infusion of a similar test meal into the stomach did not alter basal gastric acid output. Experience with the perfusion-collection technique indicated that the procedure was a sensitive nondisruptive method for chronic gastric acid collection from unanesthetized animals.

Small injections of True Blue (TB) into 4 different segments of the pancreas of the rat resulted in characteristic and different numbers and distributions of labeled cells within the dorsal motor nucleus of the vagus (DMN). Composites of these patterns of labeled cells in the DMN closely matched the distributions previously reported for more extensive injections of retrograde tracers into the pancreas. However, the application of a diffusion barrier (formed with a plastic wound spray) on the outer surface of the stomach and intestines adjacent to the pancreas segment which contained the TB injection depot prevented virtually all of the labeling of DMN cells. Similarly, applying a diffusion barrier directly to the injected pancreas segment itself prevented all or most (mean greater than 99%) of the DMN labeling. In contrast to this effect on DMN label, the barrier reduced more modestly the labeling of celiac ganglion somata after pancreas injections. Several additional control experiments also suggest that the absence of DMN label after the barrier application resulted from interference with tracer diffusion from the injected organ and not from neurotoxic effects. These include the following demonstrations in the presence of the barrier: (1) observation of unimpaired vagally stimulated insulin secretion, (2) uncompromised cell labeling of DMN from other organs treated with TB plus the barrier, and (3) normal hematoxylin and eosin stained pancreas tissue which had received TB injections and barrier application. It was concluded that both the number of parasympathetic preganglionic neurons that project monosynaptically to the pancreas and their distribution in the medulla may have been very significantly overestimated in previous tracer studies.

In order to examine the role of the pituitary in the obese-hyperglycemic syndrome, obese (ob/ob) and nonobese sibling (OB/?) mice were sham-operated or hypophysectomized by a parapharyngeal method at 60 to 90 days of age and subsequently weighed for nine weeks. Completeness of hypophysectomy was assessed by microscopic examination of stained serial sections of the brain and attached hypophyseal capsule. Complete hypophysectomy resulted in a moderate weight loss in the obese mice and attenuated further increases in adiposity, but it failed to eliminate the adiposity already established prior to surgery as determined from Lee index values. We tentatively conclude that neither the pituitary nor the target organs it controls represent the site of the primary genetic abnormality that maintains the obesity of the ob/ob.

A single injection of N,N'-bis(beta-chloroethyl)-4,4'-bipiperidine (BPM) into 30-day old rats produced brain lesions in the ventromedial hypothalamus, the dorsal vagal complex (dorsal motor nucleus of the vagus and nucleus of the solitary tract), and the medial septum. The brain lesions were associated with a chronic increase in the rate of body weight gain and the development of obesity. The lesions are similar to those seen in mice following BPM or gold thioglucose administration, however, the development of the obesity in the rat follows a more protracted time course.

In order to investigate the role of the pituitary in yellow obesity, genetically obese yellow mice (Aya) and normal nonyellow siblings (aa) were either shamoperated or hypophysectomized by a paraphyarngeal method at 45--50 days of age. The operated animals were weighed for 13 wk and their carcasses subsequently were analyzed for fat, water, and lean body mass content. Completeness of hypophysectomy was assessed by microscopic examination of stained serial sections of the brain and attached hypophyseal capsule. Completely hypophysectomized Aya mice still gained more weight than hypophysectomized nonyellow siblings and showed the excessive accumulation of fat characteristic of the yellow obesity. We conclude that the pituitary and the endocrine pathways it controls are not primarily involved in the expression of yellow obesity.

To identify the distribution of central preganglionics associated with each branch of the subdiaphragmatic vagus, the fluorescent tracer True Blue (TB) was administered intraperitoneally to rats with 4 out of 5 branches cauterized, and then, after 72 h, the animals were sacrificed for histological analysis. Each vagal branch contained the axons of a topographically distinct column of cells within the dorsal motor nucleus of the vagus (DMN). The columns representing the 4 branches with the largest numbers of efferents are organized as paired, bilaterally symmetrical, longitudinal distributions on either side of the medulla. Each DMN side contains a column occupying the medial two-thirds or more of the nucleus and corresponding to one of the gastric branches (left DMN, anterior gastric; right DMN, posterior gastric). Also on each side, the lateral pole of the DMN consists of a coherent cell column corresponding to one of the celiac branches (left DMN, accessory celiac; right DMN, celiac). The fifth branch, the hepatic, is represented by a limited number of somata forming a diffuse column largely coextensive with that representing the anterior gastric branch. At some levels of the DMN, the columns overlap. Labeled cells observed in the reticular formation were correlated in number, left-right ratios and response to vagotomy with those in the DMN, which suggests that they are displaced cells of the nucleus. Distributions of labeled cells in the nucleus ambiguus and the retrofacial nucleus were not tightly correlated with those of the DMN. An analysis of cell counts obtained for each of the individual branches suggests that vagal axons do not generally send collaterals through more than one branch.

In order to provide a detailed surgical anatomy of the rat abdominal vagus, we examined pyridine silver-stained tissue from one group of normal animals and a second group that survived 9 months after vagotomy. In the normal sample, as has been established for man, there was considerable variability in the levels at which each of the vagal branches separated from the main trunks. Contrary to reports from dissection studies, most of the branches were not single fiber bundles but rather consisted of two or more separate bundles. At the extreme, the posterior gastric and coeliac branches each consisted of as many as 15 individual bundles. Even the main trunks of the subdiaphragmatic vagus were occasionally observed to have multiple components (anterior trunk, 13% of the cases; posterior, 25%). In addition to the classically recognized hepatic, anterior gastric, coeliac, and posterior gastric branches, we also observed an accessory coeliac branch of the anterior trunk in all animals. This accessory coeliac division originated just caudal to the hepatic branching and extended first laterally and then dorsally while running caudally to exit from the esophagus just before the separation of the coeliac branch from the posterior trunk. The vagi were observed to contain paraganglia consisting of islands of glomus cells, neurons, and extensive capillary beds, all situated within the perineurium. The paraganglia occurred in greatest frequency at the sites where the hepatic and coeliac branches divide from their respective trunks. Paraganglia were also observed peripherally within vagal branches; there they were most numerous within the coeliac branch and least numerous in the accessory coeliac. Other studies yielded evidence that regeneration had occurred after complete vagotomy. First, stumps of the branches distal to the resection scar contained axons. Central to the scar, axons grew out in all directions from the neuroma; some of them appeared to cross the scar and to reinnervate the distal stumps. Secondly, 30% of the animals in which regeneration was thought to be possible increased their insulin secretion in response to electrical stimulation of the cervical vagus. The implications of the above findings for experiments that involve manipulation or recording of the vagus are discussed.

In order to explore the possibility that different-sized vagal motor fibers innervate different abdominal targets as well as to identify efficient stimulation parameters for future experimentation, response characteristics to various stimulus frequencies were determined for 6 visceral responses caused by vagal nerve stimulations. Gastric acid secretion, plasma levels of insulin, glucagon and glucose, as well as heart rate and mean arterial blood pressure were monitored in anesthetized rats subjected to 1-16 Hz square wave electrical stimulation of either the left or right cut cervical vagus. The frequency-response curves evidenced distinctly different profiles for the gastric, pancreatic, and cardiovascular responses respectively: acid secretion was half-maximal at less than 1 Hz, insulin and glucagon responses were half-maximal at approximately 3 Hz, and cardiovascular responses were shifted still more to the right (half maximal frequency approximately 15 Hz). These results suggest but do not prove that the gastric parietal cells may be innervated by small C-fiber caliber axons and the pancreatic islets by axons in the large C-fiber or small B-fiber range. Alternatively, these findings could reflect differences in neuro-effector couplings of the two organs. Furthermore, the present results provide an experimental technique that makes it feasible to select a frequency that will maximize one of the vagally mediated responses or minimize the activation of a second response.

The rat's vagal hepatic branch and associated tissues were studied using light and electron microscopy. Whole mounts, serial sections, and vascular endocasts were used to characterize the tissue from the anterior vagal trunk to the porta hepatis. Fiber number and caliber as well as intraneural organization were analyzed from complete cross-sectional electron micrographic montages of the hepatic branch sampled at its point of separation from the anterior vagal trunk. The hepatic branch ramified from the anterior vagus in one (in 47% of the specimens), two (in 37%) or three (in 16%) bundles. The single bundled hepatic branch contained 2887 +/- 287 unmyelinated fibers, and their size distribution, with a mean diameter of 0.66 +/- 0.02 micron, was Gaussian. Myelinated fibers numbered only 21 +/- 4 per branch and had a complex size distribution ranging from 0.5 to 1.8 micron with a mean of 1.2 +/- 0.03 micron. Forty four +/- 6% of the myelinated fibers were found in a single "subfascicle" in the dorso-medial pole of the nerve. Whole mounts at this level revealed that a distinct bundle, here designated an extrinsic "hepato-gastric bundle", occurred within the hepatic branch and linked the omental hepatic branch and the distal anterior gastric branch, apparently without central vagal connections. In the lesser omentum, between the esophagus and the hepatic artery proper, the hepatic branch formed a plexus which was characterized by numerous nerve divisions, anastomoses and large paraganglia (196-463 glomus cells per paraganglion). This plexiform segment ended with the recombining of the hepatic branch into 5-7 bundles which variously ascended in the porta, descended on the hepatic artery proper, or traversed the portal vein. Through its omental course, the hepatic branch traveled in close apposition to the hepato-esophageal artery and the corresponding vein as well as a prominent lymphatic vessel with associated hemolymph nodes.

The dynamics of gastric acid secretion induced by electrical stimulation of the cervical vagus in the anesthetized rat were investigated using a continuous collection-titration system permitting high temporal resolution. Stimulation with pulse rates of 2 or 4 impulses/s (pps) produced maximal gastric acid responses with small cardiovascular effects. With continuous stimulation, secretion was sustained for at least 1 h. Frequency-response profiles suggested that the parietal cells are innervated predominantly by fine-caliber C-fibers. Continuous stimulation was 3 times as effective as stimulation in bursts of higher frequencies. The minimal latency for the onset of secretion was 2.6 min at 4 pps, however, one- and two-min stimulations still produced proportionate but delayed secretory responses. It is concluded that, with low frequency cervical vagus stimulation, the rat stomach preparation described and employed in the present experiment is a useful model for further studies on the interaction of neural and humoral factors on gastric acid secretion.

A simple and reliable method for labeling the preganglionic neurons which innervate the abdominal viscera is described. Infusion of a True Blue (TB) suspension in several different concentrations directly into the peritoneal cavity consistently labeled the medullary vagal nuclei including the dorsal motor nucleus (DMN), nucleus ambiguus (NA), and retrofacial nucleus (RFN). Quantitative analysis of cell labeling after 30 microliters infusions of a large range of concentrations of TB (0.001-20.0%) showed that: (1) as little as 0.0075 mg of TB was sufficient to label DMN cells distinctly while only 0.075 mg (or larger) doses of TB were adequate for labeling cells of the NA and the RFN, and (2) doses of 3.0 mg (or greater) labeled the maximum number of cells in the DMN, NA, and RFN. Qualitative analysis suggested that medium range doses (0.075-0.75 mg) were optimal for discerning cell size and type throughout each of these nuclei.

The widespread use of new axonal transport tracing techniques in the ANS has resulted in substantially revised and amended descriptions of ANS organization. The present review suggests, however, that at least some of the results on which proposed revisions of ANS anatomy have been based have incorporated artifacts and therefore should be cautiously interpreted. The peripheral nervous system and viscera are composed in part of connective and endothelial tissues that are porous or 'leaky' to solutes with appropriate chemical characteristics, including the major tracer compounds. As a result, several extra-axonal routes for redistribution of label from the application site into other tissues are present. These include (1) diffusion through tissue membranes to enter directly adjacent tissues and (2) leakage into extracellular fluids within the body cavity, vasculature, lymphatics, exocrine ducts, or organ lumens to migrate to more distant tissues. As a consequence of the extreme sensitivity of the methods used, such redistribution of even minute amounts of label can produce false positives. Review of autonomic neuroanatomy suggests additional mechanisms, including tracer uptake by fibers of passage, can produce artifactual staining. Based on these surveys of tissue composition, tracer characteristics and sources of artifact, experimental controls and criteria for identifying and avoiding labeling artifacts are described. Since no single procedure is foolproof for ANS experimentation, the routine application of multiple controls, particularly ones which restrict or prevent tracer diffusion, are needed.

To characterize the lesion produced in the medulla oblongata by gold thioglucose (GTG), the present experiment quantified the medullary damage in C57B1 mice that had become obese after treatment with 800 mg/kg of GTG at 30 days of age. At the rostrocaudal level of the area postrema, the neurotoxin destroyed up to 75% of the neurons in the medial cell column of the dorsal motor nucleus of the vagus (DMX), while sparing the lateral pole of the nucleus. GTG also produced significant tissue loss in the central and commissural subnuclei of the nucleus of the solitary tract (NST). In contrast, the GTG lesion did not affect cell number in the hypoglossal nucleus or reduce the volume of the area postrema. Additional observations indicated that at 48-72 h after GTG administration the affected regions of the medulla already show advanced necrosis including cell loss and gliosis; and when the relative contributions of hypothalamic, DMX, and NST damage to the obesity that develops are evaluated statistically with partial correlational analysis, it appears the the obesity primarily correlates with the hypothalamic lesion produced by GTG.

This experiment analyzed the organization of the rat abdominal vagus. To spare delicate tissues and preserve positional information, untrimmed blocks of the subdiaphragmatic viscera (N = 22) were fixed, impregnated by using a pyridine-silver protocol, and double embedded. Each block was sectioned transversely at 7 micron, and a section every 70 micron from the diaphragm to the cardia was analyzed. The features of the section were traced and digitized for computer reconstruction. Included in the measurements were sizes and locations of bundles, fascicles, and paraganglia. The anterior and posterior vagi were consistently distinctive in size, distribution, cross-sectional shape, and paraganglionic content. In the most common pattern (41% of animals), the anterior trunk coursed longitudinally on the ventral surface of the esophagus, giving off at successively more distal levels the hepatic branch, the accessory coeliac branch and then the bundles of the anterior gastric branch. The posterior trunk separated into a coeliac branch and a posterior gastric branch, each consisting of numerous bundles, in the most distal quarter of the esophagus. Fifty-nine percent of all animals exhibited one or more significant variations in vagal organization (e.g., double primary trunks--41%, supernumerary branches--18%, or atypical branching sequences--9%). Four to 14 vagal paraganglia (mean = 8 +/- 1; equivalent to 32/rat, corrected for sampling) were found in each animal, and no branch was consistently devoid of paraganglia. Ninety-four percent of the paraganglia were located at nerve branch points. Some of the larger paraganglia contained at their central poles one to six neurons with soma diameters ranging from 14 to 22 micron.

To evaluate a hypothesized role of the vagus in the circannual metabolic cycle of hibernators, measures of annual weight gain, weight loss and cycle period were taken for the ground squirrel, Citellus lateralis, maintained in constant environmental conditions before and after subdiaphragmatic vagotomy. The surgery did not alter the peak weights attained, the lowest weights reached, or the period of the circannual cycle.

The role of hyperphagia -n the obesity of the diabetic mouse, C57BL/6J db/db, was investigated. Ingestion patterns and the amount of food for diabetic mice were controlled by yoking their food intake to that of nonobese siblings obtaining their food by bar pressing. Over a period of 6 wk, young (initial ages were 28 days) pair-fed diabetic mice accumulated 42% more body weight and approximately five times more extractable carcass lipid than did their siblings. Weight gain and absolute levels of carcass fat were reduced in food-restricted siabetic mice compared with dbs on unrestricted food intake. However, carcass fat as percentage of wet carcass weight was virtually identical in the restricted and unrestricted dbs (47.6% vs. 49.6%). From these results it is concluded that the heightened adiposity of the diabetic mouse does not require hyperphagia for its expression and thus represents a metabolic obesity.

Genetically obese Zucker "fatty" rats (fa/fa) and nonobese siblings were weighed regularly from 7 to 150 days of age to determine the onset and time course of the excessive weight gain that characterized the fatty rat. At 150 days of age, male and female fatties and their lean littermates were sham operated or hypophysectomized to examine the role of the pituitary in the fatty obesity. The operated animals were then weighed until sacrifice at 220 days of age, when adrenal weights, gonadal weights, uterine weights, and the Lee Index were determined. Fatty rats were significantly heavier than their nonobese littermates even in the 2nd wk of life (day 7-13). Complete hypophysectomy blocked the continued development of the fatty obesity but did not eliminate the adiposity established prior to hypophysectomy. It is concluded that the pituitary and the major endocrine pathways it controls are not the locus of the primary genetic lesion responsible for maintaining the Zucker fatty rat obesity.