Terry L. Powley

The fiber composition of the abdominal vagus of the rat.

SummaryThe 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 >10% of them were evaluated morphometrically.The means of unmyelinated axon diameters for each of the five branches were similar (0.75–0.83 μm). 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 <2.6 μm in diameter and had sheath widths between 0.1 and 0.5 μm. The gastric branches, however, also contained a few larger myelinated fibers with sheath widths as great as 0.85 μm. Whole mounts revealed fibers which were not of supradiaphragmatic orgin 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.

Longitudinal columnar organization within the dorsal motor nucleus represents separate branches of the abdominal vagus

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.

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. J. Comp. Neurol. 421:302–324, 2000.

Aging of the myenteric plexus: neuronal loss is specific to cholinergic neurons

Neuron loss occurs in the myenteric plexus of the aged rat. The myenteric plexus is composed of two mutually exclusive neuronal subpopulations expressing, respectively, nitrergic and cholinergic phenotypes. The goal of the present study, therefore, was to determine if neuron loss is specific to one phenotype, or occurs in both. Ad libitum fed virgin male Fischer 344 rats of 3 and 24 months of age were used in each of two neuronal staining protocols (n=10/age/neuron stain). The stomach, duodenum, jejunum, ileum, colon, and rectum were prepared as whole mounts and processed with either NADPHd or Cuprolinic Blue to stain, respectively, the nitrergic subpopulation or the entire population of myenteric neurons. Neuron numbers and sizes were determined for each preparation. Neuron counts from 24-month-old rats were corrected for changes in tissue area resulting from growth. There was no age-related loss of NADPHd-positive neurons for any of the regions sampled, whereas significant losses of Cuprolinic Blue-labeled neurons occurred in the small and large intestines of 24-month-old rats. At the two ages, the average neuron sizes were similar in the stomach and small intestine for both stains, but neurons in the large intestine were significantly larger at 24 months. In addition, numerous swollen NADPHd-positive axons were found in the large intestine at 24 months. These findings support the hypothesis that age-related cell loss in the small and large intestines occurs exclusively in the cholinergic subpopulation. It appears, however, from the somatic hypertrophy and the presence of swollen axons that the nitrergic neurons are not completely spared from the effects of age.

ALPHA-SYNUCLEIN-IMMUNOPOSITIVE MYENTERIC NEURONS AND VAGAL PREGANGLIONIC TERMINALS: AUTONOMIC PATHWAY IMPLICATED IN PARKINSON’S DISEASE?

The protein alpha-synuclein is implicated in the development of Parkinsons disease. The molecule forms Lewy body aggregates that are hallmarks of the disease, has been associated with the spread of neuropathology from the peripheral to the CNS, and appears to be involved with the autonomic disorders responsible for the gastrointestinal (GI) symptoms of individuals afflicted with Parkinsons. To characterize the normative expression of alpha-synuclein in the innervation of the GI tract, we examined both the postganglionic neurons and the preganglionic projections by which the disease is postulated to retrogradely invade the CNS. Specifically, in Fischer 344 and Sprague-Dawley rats, immunohistochemistry in conjunction with injections of the tracer Dextran-Texas Red was used to determine, respectively, the expression of alpha-synuclein in the myenteric plexus and in the vagal terminals. Alpha-synuclein is expressed in a subpopulation of myenteric neurons, with the proportion of positive somata increasing from the stomach (approximately 3%) through duodenum (proximal, approximately 6%; distal, approximately 13%) to jejunum (approximately 22%). Alpha-synuclein is co-expressed with the nitrergic enzyme nitric oxide synthase (NOS) or the cholinergic markers calbindin and calretinin in regionally specific patterns: approximately 90% of forestomach neurons positive for alpha-synuclein express NOS, whereas approximately 92% of corpus-antrum neurons positive for alpha-synuclein express cholinergic markers. Vagal afferent endings in the myenteric plexus and the GI smooth muscle do not express alpha-synuclein, whereas, virtually all vagal preganglionic projections to the gut express alpha-synuclein, both in axons and in terminal varicosities in apposition with myenteric neurons. Vagotomy eliminates most, but not all, alpha-synuclein-positive neurites in the plexus. Some vagal preganglionic efferents expressing alpha-synuclein form varicose terminal rings around myenteric plexus neurons that are also positive for the protein, thus providing a candidate alpha-synuclein-expressing pathway for the retrograde transport of putative Parkinsons pathogens or toxins from the ENS to the CNS.

Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers

Accurate estimates of the total number of neurons located in the wall of the gut are essential for studies of the enteric nervous system (ENS). Though several stains and antibodies are used routinely as pan-neuronal markers, controversies of relative sensitivity and completeness have been difficult to resolve, at least in part because comparisons often must be made across experiments and laboratories. Therefore, we evaluated the efficacy of four putative pan-neuronal markers for the ENS, under comparable conditions. Neurons in the myenteric plexus of wholemounts taken from the small intestines of Fischer 344 rats were stained using Cuprolinic Blue, anti-HuC/D, anti-protein gene product 9.5, or FluoroGold injections followed by permanent labeling with an antibody to the FluoroGold molecule. All four markers had useful features, but both protein gene product 9.5 and FluoroGold were found to be problematic for obtaining reliable counts. As a result, only neurons labeled with either Cuprolinic Blue or anti-HuC/D were compared quantitatively. Based on counts from permanently labeled tissue, Cuprolinic Blue and HuC/D were similarly effective in labeling all neurons. Because the two protocols have different strengths and weaknesses, Cuprolinic Blue and HuC/D provide a complementary set of labels to study the total neuronal population of the ENS.

Characterization of vagal innervation to the rat celiac, suprarenal and mesenteric ganglia

In order to shed light on the controversial issue of vagal innervation of the solar plexus ganglia, vagal efferent preganglionic fibers were anterogradely labeled by injecting the fluorescent carbocyanine dye Dil into the dorsal motor nucleus (dmnX). Additionally, Fluorogold was used to label the ganglia in toto, providing a counterstain and the possibility of UV light-guided dissection of the various ganglia. Using optical sectioning of whole mounted intact ganglia by means of laser scanning confocal microscopy, a considerable number of Dil-labeled vagal terminal-like structures were found in the major ganglia (celiac, superior mesenteric and suprarenal). Additionally, vagal efferent terminals were regularly found in microganglia associated with the periarterial plexuses of the celiac and superior mesenteric arteries, and in a few cases in small ganglia of the intermesenteric and renal plexuses. By using animals with prior selective vagal branch vagotomies, leaving only one (or a pair) of the three major abdominal divisions intact, it was concluded that the two celiac branches contribute the bulk of this vagal innervation, with the two gastric and the unpaired hepatic branch providing a small contribution mostly limited to the celiac ganglia. From control experiments, which involved Dil injections (1) into the dmnX in animals whose visceral afferents had been previously destroyed by capsaicin; (2) into the nodose ganglia, in order to anterogradely label vagal afferents; and (3) into the cervical vagus nerve as a control for uptake by fibers of passage, it was concluded that the identified terminal-like structures were vagal efferents and not inadvertently labeled afferents. We suggest that these vagal terminals have to be regarded either as ectopic parasympathetic junctions, or as part of a vagal mechanism for gating of sympathetic ganglionic transmission. Functionally, the parasympathetic innervation of the solar plexus may provide not only the classic vagal influence on gastrointestinal targets, but also vagal control of the adrenal glands and possibly other abdominal organs that have not been traditionally regarded as vagal targets.

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. J. Comp. Neurol. 428:558–576, 2000.

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 μl 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. J. Comp. Neurol. 434:358–377, 2001.

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. J. Comp. Neurol. 381:81‐100, 1997.