Vagal preganglionic axons arborize in the myenteric plexus into two types: nitrergic and non-nitrergic postganglionic motor pools?

Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (∼86%) of vagal efferents were organized into two distinct subtypes. One subtype (∼24% of dextran-labeled efferents, designated "primarily nitrergic") selectively contacted and linked-both within and between ganglia-nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (∼62% of efferents, designated "primarily non-nitrergic") appeared to selectively contact and link-both within and between ganglia-non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (∼14% of labeled preganglionics), appeared to lack "nitrergic selectivity" and to contact both nNOS+ and nNOS- enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes.NEW & NOTEWORTHY The present study highlights the following: 1) one type of vagal efferent axon selectively innervates nitrergic upper gastrointestinal myenteric neurons; 2) a second type of vagal efferent selectively innervates non-nitrergic gastrointestinal myenteric neurons; and 3) the two types of vagal efferents might modulate peristalsis reciprocally and cooperatively.

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.

Vagus nerve stimulation promotes gastric emptying by increasing pyloric opening measured with magnetic resonance imaging.

BACKGROUND: Vagus nerve stimulation (VNS) is an emerging electroceutical therapy for remedying gastric disorders that are poorly managed by pharmacological treatments and/or dietary changes. Such therapy seems promising as the vagovagal neurocircuitry modulates the enteric nervous system to influence gastric functions. METHODS: Here, the modulatory effects of left cervical VNS on gastric emptying in rats were quantified using a (i) feeding protocol in which the animal voluntarily consumed a postfast, gadolinium-labeled meal and (ii) a non-invasive imaging method to measure antral motility, pyloric activity and gastric emptying based on contrast-enhanced magnetic resonance imaging (MRI) and computer-assisted image processing pipelines. KEY RESULTS: Vagus nerve stimulation significantly accelerated gastric emptying (sham vs VNS: 29.1% ± 1.5% vs 40.7% ± 3.9% of meal emptied per 4 hours), caused a greater relaxation of the pyloric sphincter (sham vs VNS: 1.5 ± 0.1 vs 2.6 ± 0.4 mm2 cross-sectional area of lumen), and increased antral contraction amplitude (sham vs VNS: 23.3% ± 3.0% vs 32.5% ± 3.0% occlusion), peristaltic velocity (sham vs VNS: 0.50 ± 0.02 vs 0.67 ± 0.03 mm s-1 ), but not its contraction frequency (sham vs VNS: 6.1 ± 0.2 vs 6.4 ± 0.2 contractions per minute, P = .22). The degree to which VNS relaxed the pylorus was positively correlated with gastric emptying rate (r = .5887, P < .001). CONCLUSIONS & INFERENCES: The MRI protocol employed in this study is expected to enable advanced preclinical studies to understand stomach pathophysiology and its therapeutics. Results from this study suggest an electroceutical treatment approach for gastric emptying disorders using cervical VNS to control the degree of pyloric sphincter relaxation.

Neurotrophin-4 deficient mice have a loss of vagal intraganglionic mechanoreceptors from the small intestine and a disruption of short-term satiety.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs) are the two putative mechanoreceptors that the vagus nerve supplies to gastrointestinal smooth muscle. To examine whether neurotrophin-4 (NT-4)-deficient mice, which have only 45% of the normal number of nodose ganglion neurons, exhibit selective losses of these endings and potentially provide a model for assessing their functional roles, we inventoried IGLEs and IMAs in the gut wall. Vagal afferents were labeled by nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase, and a standardized sampling protocol was used to map the terminals in the stomach, duodenum, and ileum. NT-4 mutants had a substantial organ-specific reduction of IGLEs; whereas the morphologies and densities of both IGLEs and IMAs in the stomach were similar to wild-type patterns, IGLEs were largely absent in the small intestine (90 and 81% losses in duodenum and ileum, respectively). Meal pattern analyses revealed that NT-4 mutants had increased meal durations with solid food and increased meal sizes with liquid food. However, daily total food intake and body weight remained normal because of compensatory changes in other meal parameters. These findings indicate that NT-4 knock-out mice have a selective vagal afferent loss and suggest that intestinal IGLEs (1) may participate in short-term satiety, probably by conveying feedback about intestinal distension or transit to the brain, (2) are not essential for long-term control of feeding and body weight, and (3) play different roles in regulation of solid and liquid diet intake.

Neural pathways involved in the hypothalamic integration of autonomic responses.

Recent mapping studies of hypothalamic and autonomic mechanisms have considerably extended our understanding of the anatomy of this system. The pattern of connections emerging from physiological, anatomical, and histochemical experiments suggests several conclusions about the functional organization of the system as well. Recent evidence supports the idea that the hypothalamic (and other limbic) areas involved in the control of ingestion and metabolism form the rostral pole of a longitudinally and hierarchically organized system that elaborates autonomic responses that influence the energy economy of the animal. Substantially the same pathways are apparently responsible for the modulation of ingestive behavior as well. This circuitry, the "visceromotor system" in Nauta's terminology, seems to weld afferent inputs, particularly those of the gustatory and visceral receptors, into a coordinated integrative control strategy influencing autonomic responses. In addition, the system seems to have unique tissue properties, at least at its two periventricularly located sites of integration with special access to both humorally and ventricularly circulated substrates. These nodes, the basomedial hypothalamus and the vagal complex of the medulla, seem to share similar biochemical specializations reflected in susceptibility to goldthioglucose toxicity, specific insulin binding, and susceptibility to alloxan diabetes.

Organization and distribution of the rat subdiaphragmatic vagus and associated paraganglia.

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.

Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor.

Although the gastric tension receptor has been characterized behaviorally and electrophysiologically quite well, its location and structure remains elusive. Therefore, the vagal afferents to the rat fundus (forestomach or nonglandular stomach) were anterogradely labeled in vivo with injections of the carbocyanine dye Dil into the nodose ganglia, and the nerves and ganglia of the enteric nervous system were labeled in toto with intraperitoneal Fluorogold injection. Dissected layers and cryostat cross sections of the fundic wall were mounted in glycerin and analyzed by means of conventional and laser scanning confocal microscopy. Particularly in the longitudinal, and to a lesser extent in the circular, smooth muscle layers, Dil-labeled fibers and terminals were abundant. These processes, which originated from fibers coursing through the myenteric ganglia and connectives, entered either muscle coat and then ran parallel to the respective muscle fibers, often for several millimeters. They ran in close association with the Fluorogold-labeled network of interstitial cells of Cajal, upon which they appeared to form multiple spiny appositions or varicosities. In the myenteric plexus, two different types of afferent vagal structures were observed. Up to 300 highly arborizing endings forming dense accumulations of small puncta similar to the esophageal intraganglionic laminar endings (Rodrigo et al., '75 Acta Anat. 92:79-100) were found in the fundic wall ipsilateral to the injected nodose ganglion. They often covered small clusters of myenteric neurons or even single isolated ganglion cells (mean = 5.8 neurons) and tended to extend throughout the neuropil of the ganglia. In a second pattern, fine varicose fibers with less profuse arborizations innervated mainly the central regions of myenteric ganglia. Camera lucida analyses established that single vagal afferent fibers had separate collaterals in both a smooth muscle layer and the myenteric ganglia. Finally, Dil-labeled afferent vagal fibers were also found in the submucosa and mucosa. Control experiments in rats with supranodose vagotomy as well as rats with Dil injections directly in the distal cervical vagus ruled out the possibility of colabeling of afferent fibers of passage. In triple labeling experiments, in conjunction with Dil labeling of afferents and Fluorogold labeling of enteric neurons, the carbocyanine dye DiA was injected into the dorsal motor nucleus of the vagus to anterogradely label the efferent vagal fibers and terminals. The different distributions and morphological characteristics of the vagal afferents and efferents could be simultaneously compared. In some instances the same myenteric ganglion was apparently innervated by an afferent laminar ending and an efferent terminal.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleus ambiguus projections to cardiac ganglia of rat atria: an anterograde tracing study.

We injected the anterograde fluorescent tracer 1,1;-dioleyl-3,3,3;, 3;-tetramethylindocarbocyanine methanesulfonate (DiI) into the nucleus ambiguus (NA) and used confocal microscopy to inventory NA fibers and axon terminals in whole-mounts of rat atrial tissues. Both the axons projecting to cardiac ganglia and the innervated principal neurons (PNs) were counted. Rats were injected unilaterally in the NA with DiI, either at four sites (between 600 microm rostral and 600 microm caudal to the obex) or at nine sites (1,600 microm rostral to 1,600 microm caudal). Fluoro-Gold was administered intraperitoneally to retrogradely label neurons of the dorsal motor nucleus of the vagus (DmnX), NA, and cardiac ganglia. To verify that the DiI-labeled fibers examined in the atria originated exclusively from the NA, neurons of the DmnX and the nodose ganglia were surveyed for DiI labeling. Our observations established that (1) NA fibers in the cardiac branches of the vagus numbered in the range of 82-151, left; or 60-122, right. (2) Both left and right NA supplied substantial numbers of fibers to each of the three major cardiac ganglionic plexuses. (3) NA axons terminated in dense basket, or calyx, endings around individual PNs. (4) By issuing divergent collaterals, individual NA fibers supplied numerous PNs with these calyx endings. (5) Labeled axons innervated 2,248 (left vagus) and 1,784 (right), or at least 56% and 45%, of the cardiac PNs. (6) Divergence (i.e., NA axons:PNs innervated) averaged between 1:27 (left vagus) and 1:30 (right vagus). Several features of these NA projections to cardiac ganglia contrasted sharply with those of DmnX projections that we have recently characterized with the same tracing protocol: (1) NA fibers did not innervate small intensely fluorescent cell clusters in cardiac ganglia, whereas DmnX axons did. (2) NA efferent fascicles contained more large fibers (presumably B-type), whereas the DmnX issued more fine caliber fibers (presumably C-type). (3) NA fibers diverged about three times as extensively as did DmnX axons. Taken together, our data strongly suggest that vagal control of the heart involves the convergence and integration of distinct NA and DmnX projections within the cardiac plexuses.

As the gut ages: timetables for aging of innervation vary by organ in the Fischer 344 rat.

To explore the effects of aging on the vagal innervation of the gastrointestinal (GI) tract, male Fischer 344 rats at 3 and 24 months of age were injected in the left nodose ganglion with 3 microl of either 4% wheat germ agglutinin-horseradish peroxidase (to label sensory endings) or 1% cholera toxin subunit B-horseradish peroxidase (to label motor endings). The stomach and duodenum were prepared as wholemounts and processed with tetramethyl benzidine. In addition, to study age-related changes in the myenteric plexus, the stomachs, small intestines, and large intestines from 3-, 12-, 21-, 24- and 27-month-old rats were prepared as wholemounts and processed with Cuprolinic Blue (to stain the neurons). Vagal afferent endings, motor terminal profiles, and myenteric neurons were counted and mapped with a sampling grid. In the stomach, both the vagal and myenteric innervation were stable between the ages of 3 and 24 months; however, a decrease in the number of myenteric neurons in the forestomach was noted at 27 months. In the small and large intestines, myenteric cell loss occurred by 12 months of age, progressed with age, and appeared to be governed by several general principles: (1) the rate of cell loss was organ-specific, with a gradient of increasing severity from proximal to distal in the gut; (2) within organs of the GI tract, the rate of cell loss differed between regions; and (3) for given regions, cell losses progressed linearly with increasing age. The findings suggest that a positive relationship exists between the density of vagal extrinsic innervation and myenteric neuron survival; however, whether this results from the vagal innervation and/or other factor(s) protecting or rescuing myenteric neurons from age-related attrition remains to be determined.

Morphology of identified preganglionic neurons in the dorsal motor nucleus of the vagus.

To determine the degree of variation of neuronal morphology both within and between the subnuclei of the dorsal motor nucleus of the vagus (dmnX), structural features of the preganglionic neurons of each of the five primary subnuclei in the rat dmnX were characterized quantitatively. Each of the columnar subnuclei was separately labeled by application of the retrograde tracer fast blue to its corresponding subdiaphragmatic vagal branch. Fixed brain slices of 100 microns thickness were then prepared in coronal, sagittal, and horizontal orientations. Next, randomly selected fast blue labeled neurons (n = 1,256) were injected with Lucifer yellow, drawn with camera lucida, and digitized. For each cell, three features of the perikaryon and twelve of the dendritic tree were measured. Dorsal motor nucleus neurons with up to eight primary dendrites, 30 dendritic segments, and seventh order dendritic branches were observed. Throughout the dmnX, the dendrites of preganglionic neurons were preferentially oriented in the horizontal plane. Consistent with an organizing role for the columnar subnuclei, most dendrites remained within their column of origin. However, between 5 and 30% of the neurons in each of the columns projected dendrites into adjacent dmnX subnuclei or other brainstem nuclei, including the nucleus of the solitary tract (NTS). The cyto- and dendroarchitectural analyses revealed systematic gradations in morphology, although they did not support the idea that the dmnX was composed of multiple distinct preganglionic types. The most parsimonious interpretation of the data is that dmnX motorneurons are variants of a single prototype, with dendrites varying widely in length and degree of ramification. The extent of an individual preganglionic neuron's dendritic field was predicted by three factors: the cell's rostrocaudal position within the dmnX, its location within a transverse plane (i.e., its coronal position within or ectopic to the dmnX), and its subnucleus of origin. Neurons at rostral and midlongitudinal levels of each column had more extensive dendritic arbors than those at caudal levels. Ectopic neurons had more extensive dendritic fields than similar cells in the corresponding columns; in fact, of all vagal preganglionic neurons, ectopics had the most extensive dendritic fields. Somata and dendrites of celiac column neurons were more extensive than those of hepatic and gastric column cells. These differential regional distributions of vagal preganglionics suggest that their structure and function are correlated.

Topographic inventories of vagal afferents in gastrointestinal muscle.

To inventory and characterize the two types of vagal afferents (both putative mechanoreceptors) in the muscle of the gastrointestinal tract, the authors injected wheat germ agglutinin-horseradish peroxidase into the nodose ganglia of rats that had received unilateral ventral rhizotomies to eliminate efferents. The gut, from the oral esophagus to the distal colon, was divided into wholemounts, processed with tetramethylbenzidine, and surveyed to establish normative topographic maps of afferents. Vagal intraganglionic laminar endings (IGLEs) were ubiquitous, with concentrations varying on a longitudinal gradient (higher rostrally). This overall gradient was punctuated by denser condensations of endings in the oral esophagus, gastric corpus, and distal ileum. In regional specializations, IGLEs were fused into conspicuous, dense networks in the laryngeal esophagus and the antrum. Intramuscular arrays (IMAs) had restricted distributions, including the walls of the stomach and the sphincters throughout the gut. In the forestomach, a singular concentration of orthogonally crossed IMAs was organized into a lattice or "fovea." IMAs displayed variations in morphology, with one specialization consisting of short, terminal processes associated with sphincters and a more widespread form consisting of long, rectilinear processes in the forestomach, along the greater curvature, and in limited intestinal regions. On the basis of their topographic patterns and structural specializations, the two putative mechanoreceptors may have different functions: IGLEs appear situated to integrate intramural tension, and perhaps myenteric neuronal activity, into rhythmical, propagated motor programs, such as swallowing, peristalsis, and emptying. IMAs are distributed strategically and appear to satisfy structural requirements for stretch receptors tuned to tonic or more aperiodic events that may affect central nervous system processing as well as local gastrointestinal coordination.

Dorsal motor nucleus of the vagus neurons: a multivariate taxonomy.

The dorsal motor nucleus of the vagus (DMNX) contains neurons with different projections and discrete functions, but little success has been achieved in distinguishing the cells cytoarchitectonically. The present experiment employed multivariate analytical techniques to evaluate DMNX neuronal morphology. Male Sprague-Dawley rats (n = 77) were perfused, and the brainstems were stained en bloc with a Golgi-Cox protocol. DMNX neurons in each of three planes (coronal, sagittal, and horizontal; total sample = 607) were digitized. Three-dimensional features quantified included dendritic length, number of segments, spine density, number of primary dendrites, dendritic orientation, and soma form factor. Cluster analyses of six independent samples of 100+ neurons and of three composite replicate pools of 200+ neurons consistently identified similar sets of four distinct neuronal profiles. One profile (spinous, limited dendrites, small somata) appears to correspond to the interneuron population of the DMNX. In contrast, the other three distinctive profiles (e.g., one is multipolar, with large dendritic fields and large somata) are different types of preganglionic neurons. Each of the four types of neurons is found throughout the DMNX, suggesting that the individual columnar subnuclei and other postulated vagal motorneuron pools are composed of all types of neurons. Within individual motor pools, ensembles of the different neuronal types must cooperatively organize different functions and project to different effectors within a target organ. By extension, specializations of the preganglionic motor pools are more likely to result from their afferent inputs, peripheral target tissues, neurochemistry, or physiological features rather than from any unique morphological profiles.

Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with dil injected into the dorsal vagal complex in the rat.

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.

Afferent innervation of gastrointestinal tract smooth muscle by the hepatic branch of the vagus.

To survey the vagal hepatic branch afferent projections to and the terminal specializations in the gastrointestinal tract, male Sprague-Dawley rats were given subdiaphragmatic vagotomies, sparing only the common hepatic branch, and were injected with 3 microl of 8% wheat germ agglutinin-horseradish peroxidase in the left nodose ganglion. The nodose ganglia, the stomach, the first 8 cm of duodenum, and the cecum were prepared as wholemounts and were processed with tetramethyl benzidine. Hepatic afferent innervation of the ventral stomach consisted of one or more bundles entering at the lower esophageal sphincter and coursing to the forestomach, where they branched into distinct terminal fields. The only fibers on the dorsal forestomach were distal branches and terminals that wrapped around the greater curvature from the ventral side. Hepatic afferents supplied the forestomach with both intraganglionic laminar endings (IGLEs; putative mechanosensors that coordinate peristalsis) and intramuscular arrays (IMAs; considered tension receptors). IGLEs were located primarily on the ventral wall of the stomach, whereas IMAs were distributed symmetrically. Afferents were also supplied to the distal antrum and the pylorus, with pyloric innervation consisting almost exclusively of IMAs. Innervation of the proximal duodenum was denser in the first 3 cm and decreased progressively caudally, with only meager innervation after 6 cm. Cecal innervation consisted of a few fibers at the ileocecal junction. Duodenal and cecal endings were predominately IGLEs. These results indicate that the hepatic branch carries sensory information from the forestomach, antrum, pylorus, duodenum, and cecum. Furthermore, the different terminals it supplies suggest that the branch mediates a multiplicity of gastrointestinal functions.

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy.

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed "flower-spray" and "end-net" terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes.

Vagal preganglionic projections to the enteric nervous system characterized with Phaseolus vulgaris-leucoagglutinin.

The patterns and extent of vagal preganglionic divergence and convergence within the gastrointestinal tract of the rat were characterized with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Three weeks after tracer was iontophoretically injected into two to four sites within the dorsal motor nucleus of the vagus, wholemounts of perfused gut organs (stomach, duodenum, cecum) were prepared, counterstained with Cuprolinic blue, and processed for PHA-L using the avidin biotin complex with diaminobenzidine. Controls included animals injected with PHA-L after intracranial deafferentations. Well-positioned injections labeled an extremely dense and intricate network of varicose efferent axons throughout the gastric myenteric plexus (including that of the fundus). Individual fibers collateralized extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervated subsets of neurons within ganglia. Most enteric neurons were contacted by varicosities of more than one vagal fiber. The patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resembled the organization in the stomach in many aspects, but the projections in each organ had distinctive characteristics, and label was less dense in the intestines than in the stomach. Vagal preganglionic fibers directly innervated submucosal ganglia, although sparsely. Brainstem injections of PHA-L retrogradely labeled a few myenteric neurons in the corpus, fundus, and duodenum: These "gastrobulbar" and "duodenobulbar" neurons received reciprocal vagal preganglionic innervation. Finally, the PHA-L that spread to the nucleus of the solitary tract occasionally produced transganglionic labeling of afferent intramuscular arrays (gastric fundus). The results of this paper provide strong evidence that the traditional "command neuron" or "mother cell" hypotheses of vagal-enteric organization should be abandoned for an integrative neural network model.

Regenerating vagal afferents reinnervate gastrointestinal tract smooth muscle of the rat.

Peripheral projections of the vagus are known to regenerate after subdiaphragmatic vagotomy, but neither the question of whether the regenerating axons are motor or sensory nor the issue of whether the fibers reinnervate their original targets have been addressed. To determine whether vagal afferents regenerate and whether they differentiate into normal terminal specializations in the reinnervated target organ, male Sprague-Dawley rats underwent complete subdiaphragmatic vagotomies and were injected 18 weeks later with 3 microl of 4% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the left nodose ganglion. To provide a comparison group, an unoperated group (controls) was injected with WGA-HRP in the left nodose ganglion. The esophagus, the entire stomach, the first 8 cm of the duodenum, and the hilus of the liver were prepared as wholemounts and processed with tetramethyl benzidine. Vagal afferents were found to have regenerated and reinnervated the esophagus, stomach, duodenum, and liver. Bundles (two or more axons), individual vagal axons, and terminals in the stomach were counted and mapped with a sampling grid. At 18 weeks postvagotomy, the reinnervated stomach and duodenum contained normal terminals as well as aberrant endings and growth cone profiles. The ingrowing axons reestablished ipsilateral and contralateral projections in the same proportions seen in controls, although the overall density of the different regenerating elements had reached only 7-39% of control values. These findings demonstrate that the gastrointestinal tract and liver can undergo dramatic afferent reinnervation after vagotomy. The presence of differentiated endings at 18 weeks suggests that some afferent function(s) may be restored, and the expression of growth cones suggests that additional regeneration may be ongoing.

Projections of the dorsal motor nucleus of the vagus to cardiac ganglia of rat atria: an anterograde tracing study.

We injected the anterograde fluorescent tracer 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the dorsal motor nucleus of the vagus (DmnX), counterstained the cardiac ganglia with Fluorogold (FG), and used confocal microscopy to examine the distributions and different types of DmnX fibers in wholemounts of the atria. We also quantified the number of DmnX cardiac axons and the number of innervated cardiac principal neurons (PNs). Rats with unilateral DiI injections were used in three different experiments, including unilateral FG soaking of cervical vagal trunks, intracranially rhizotomizing the vagal afferent roots, or contralaterally sectioning the cervical vagus. These manipulations indicated that DiI-labeled cardiac fibers were exclusively from the DmnX. Our observations established that: (1) three major ganglionic plexuses were localized in the epicardium; (2) both sides of the DmnX supplied significant fibers to each of the plexuses; (3) these cardiac efferents formed dense basket terminals around individual PNs; (4) collaterals of individual DmnX fibers diverged, producing calyx endings on multiple PNs; (5) small intensely fluorescent (SIF) cells in the cardiac plexuses were innervated pericellularly; (6) individual axons could innervate both PNs and SIF cells; (7) the total number of DmnX fibers were in the range of [68, 96; left] and [67, 115; right]; (8) these fibers innervated 709 (left) and 494 (right), or at least 18% and 12%, of the PNs, respectively; and (9) vagal preganglionics exhibited a degree of lateralization: Significantly more PNs were contacted by fiber varicosities in the sinoatrial plexus than in the atrioventricular plexus after right DmnX injections. In summary, the present observations suggest that the DmnX plays a significant role(s) in controlling the heart.

Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs), the two putative mechanoreceptors that the vagus nerve supplies to the gastrointestinal smooth muscle, have been characterized almost exclusively in the rat. To provide normative inventories of these afferents for the mouse, the authors examined the endings in the stomach and small intestine of three strains used as backgrounds for gene manipulations (i.e., C57, 129/SvJ, and WBB6). Animals received nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase or dextran-tetramethylrhodamine conjugated to biotin. The horseradish peroxidase tissue was processed with tetramethylbenzidine and was used to map the distributions and densities of the two endings; the dextran material was counterstained with c-Kit immunohistochemistry to assess interactions between intramuscular arrays and interstitial cells of Cajal. IGLEs and IMAs constituted the vagal innervation of mouse gastric and duodenal smooth muscle. IGLE morphology and distributions, with peak densities in the corpus-antrum, were similar in the three strains of mice and comparable to those observed in rats. IMAs varied in complexity from region to region but tended to be simpler (fewer telodendria) in mice than in rats. IMAs were most concentrated in the forestomach and sphincters in mice, as in rats, but the topographic distributions of the endings varied both between strains of mice (subtly) and between species (more dramatically). IMAs appeared to make appositions with both interstitial cells and smooth muscle fibers. This survey should make it practical to assay the effects of genetic (e.g., knockout) and experimental (e.g., regeneration) manipulations affecting visceral afferents and their target tissues.

Diet and cephalic phase insulin responses.

Cephalic phase digestive responses may be particularly critical in determining our various reactions to different diets, since these responses are the first physiological adjustments to food. The potential importance of the cephalic responses is also underscored by the fact that many of the most important food attributes for humans--color, appearance, flavor, aroma, and texture--can influence the individual's gastrointestinal physiology solely by affecting these early metabolic responses. The present survey examines in some detail the data available for one of the responses, the cephalic phase insulin response. Specific shortcomings of the existing analyses are discussed. In addition, given the possible significance of these reflexes, several suggestions for improvements of experimental protocols are considered, and a summary of major experimental questions is provided.