Elizabeth A. Baronowsky

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.

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.

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 μl 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. J. Comp. Neurol. 384:248‐270, 1997.

Selective loss of vagal intramuscular mechanoreceptors in mice mutant for steel factor, the c-Kit receptor ligand

Vagal intramuscular arrays are mechanoreceptors that innervate smooth muscle fibers and intramuscular interstitial cells of Cajal of the proximal GI tract. C-Kit mutant mice that lack intramuscular interstitial cells of Cajal also lack intramuscular arrays. Mice mutant for steel factor, the ligand for the c-Kit receptor, were studied to extend and validate these previous findings and to characterize associated changes in food intake. Injections of wheat germ agglutinin-horseradish peroxidase and of dextran into the nodose ganglion were employed to label intramuscular arrays and intraganglionic laminar endings, the other vagal mechanoreceptors found in the gut wall. These two receptor types were inventoried in wholemounts of the stomach and duodenum using a standardized sampling and quantification regime. Steel mutants exhibited a paucity of normal intramuscular arrays and lacked intramuscular interstitial cells of Cajal in the forestomach, whereas their intraganglionic laminar endings appeared normal in number, distribution, and morphology. These observations suggest that intramuscular array losses in steel and c-Kit mutants are specific and result from the elimination of the intramuscular interstitial cells of Cajal, the effect common to both mutations, not from interactions peculiar to background strains or non-specific effects. Double-labeling analyses of intramuscular arrays and intramuscular interstitial cells of Cajal reinforced the hypothesis based on previous findings in the c-Kit mice that these interstitial cells have a trophic effect on intramuscular array development and/or maintenance. Finally, meal pattern analyses revealed decreased meal size and increased meal frequency in steel mutants, with normal daily intake. These alterations suggest short-term feeding controls are affected by the loss of intramuscular arrays and/or intramuscular interstitial cells of Cajal, though long-term controls are unimpaired.

C-Kit mutant mice have a selective loss of vagal intramuscular mechanoreceptors in the forestomach.

Intramuscular arrays are one of two major classes of vagal afferent mechanoreceptors that innervate the smooth muscle wall of the proximal gastrointestinal tract. They consist of rectilinear telodendria that distribute in the muscle sheets, parallel to the long axes of muscle fibers. Intramuscular arrays appear to make direct contact with the muscle fibers, but they also course on, and form appositions with, intramuscular interstitial cells of Cajal. These complexes formed by intramuscular arrays and intramuscular interstitial cells of Cajal suggest that intramuscular arrays might require either structural or trophic support of the interstitial cells of Cajal for normal differentiation and/or maintenance. To evaluate this hypothesis, we have examined the morphology and distribution of vagal afferent endings in the c-Kit mutant mouse that lacks intramuscular interstitial cells of Cajal. Vagal afferents were labeled by nodose ganglion injection of either wheat germ agglutinin-horseradish peroxidase conjugate or a tagged dextran, and the labeled afferent terminals in the stomach were mapped using a standardized quantitative sampling scheme. Intramuscular arrays were dramatically reduced (in circular muscle by 63%; in longitudinal muscle by 78%) in the c-Kit mutant mice relative to their wild-type littermates. Additionally, a substantial number of the surviving axons and terminals in the mutant stomachs were morphologically aberrant. Moreover, the loss of intramuscular arrays in mutants appeared to be selective: the structure, distribution and density of intraganglionic laminar endings, i.e., the other vagal mechanoreceptors in smooth muscle, were not significantly altered. Finally, the conspicuous decrease in intramuscular array density in mutants was associated with a non-significant trend toward loss of nodose ganglion neurons. Collectively these findings suggest that interstitial cells are required for the normal development or maintenance of vagal intramuscular arrays. Therefore, the c-Kit mutant mouse will be valuable for determining the role(s) of interstitial cells in intramuscular array development as well as for providing an animal model with the intramuscular array class of vagal afferents selectively ablated.

Long-term regeneration of abdominal vagus: Efferents fail while afferents succeed

Vagal afferents regenerate, by 18 weeks after subdiaphragmatic transection, to reinnervate the gut and to differentiate into the two types of terminals normally found in the smooth muscle wall of the gastrointestinal (GI) tract (Phillips et al. [2000] J Comp Neurol. 421:325–346). Regeneration, however, is neither complete nor entirely accurate by 18 weeks. Moreover, the capacity of the vagal efferents to reinnervate the GI tract under comparable conditions has not been evaluated. Therefore, to determine whether a more extended postaxotomy survival interval would (1) result in more extensive reinnervation of smooth muscle, (2) facilitate correction of the inaccuracies of the regenerated axons and terminals, and (3) yield motor as well as sensory reinnervation of GI targets, Sprague‐Dawley rats received either complete subdiaphragmatic vagotomies (n = 18) or sham surgeries (n = 12). Physiological endpoints that might normalize as vagal elements regenerated, including body weight, daily food intake, size of first daily meal, and metabolic efficiency, were monitored. At 45 weeks after the vagotomies, the animals were randomly assigned to afferent (wheat germ agglutinin–horseradish peroxidase) or efferent (cholera toxin subunit B–horseradish peroxidase) mapping conditions, and labeled axons and terminals in the stomach and first 8 cm of the small intestine were inventoried in whole‐mounts. Afferent regeneration was more extensive at 45 weeks than previously observed at 18 weeks after surgery; however, the amount of GI innervation was still not comparable to the intact pattern of the sham rats. Furthermore, abnormal patterns of sensory organization occurred throughout the reinnervated field, with small bundles of axons forming complex tangles and some individual axons terminating in ectopic locations. The presence of growth cone profiles suggested that vagal reorganization was ongoing even 45 weeks after surgery. In contrast to this relatively extensive, albeit incomplete, sensory reinnervation of the gut, motor fibers had failed to reinnervate the GI tract. Thus, dramatic differences exist in the regenerative capacities of the sensory and motor arms of the vagus under the same surgical and maintenance conditions. Furthermore, the functional measures of disordered energy regulation did not normalize over the 45 weeks during which afferent but not efferent innervation was restored. J. Comp. Neurol. 455:222–237, 2003.

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 μl 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. J. Comp. Neurol. 421:325–346, 2000.

Versatile, high-resolution anterograde labeling of vagal efferent projections with dextran amines

None of the anterograde tracers used to label and investigate vagal preganglionic neurons projecting to the viscera has proved optimal for routine and extensive labeling of autonomic terminal fields. To identify an alternative tracer protocol, the present experiment evaluated whether dextran conjugates, which have produced superior results in the CNS, might yield widespread and effective labeling of long, fine-caliber vagal efferents in the peripheral nervous system. The dextran conjugates that were evaluated proved reliable and versatile for labeling the motor neuron pool in its entirety, for single- and multiple-labeling protocols, for both conventional and confocal fluorescence microscopy, and for permanent labeling protocols for brightfield microscopy of the projections to the gastrointestinal (GI) tract. Using a standard ABC kit followed by visualization with DAB as the chromagen, Golgi-like labeling of the vagal efferent terminal fields in the GI wall was achieved with the biotinylated dextrans. The definition of individual terminal varicosities was so sharp and detailed that it was routinely practical to examine the relationship of putative vagal efferent contacts (by the criteria of high magnification light microscopy) with the dendritic and somatic architecture of counterstained neurons in the myenteric plexus. Overall, dextran conjugates provide high-definition labeling of an extensive vagal motor pool in the GI tract, and offer considerable versatility when multiple-staining protocols are needed to elucidate the complexities of the innervation of the gut.

Vagal afferent innervation of the lower esophageal sphincter.

To supply a fuller morphological characterization of the vagal afferents innervating the lower esophageal sphincter (LES), specifically to label vagal terminals in the tissues forming the LES in the gastroesophageal junction, the present experiment employed injections of dextran biotin into the nodose ganglia of rats. Four types of vagal afferents innervated the LES. Clasp and sling muscle fibers were directly and prominently innervated by intramuscular arrays (IMAs). Individual IMA terminals subtended about 16° of arc of the esophageal circumference, and, collectively, the terminal fields were distributed within the muscle ring to establish a 360° annulus of mechanoreceptors in the sphincter wall. 3D morphometry of the terminals established that, compared to sling muscle IMAs, clasp muscle IMAs had more extensive arbors and larger receptive fields. In addition, at the cardia, local myenteric ganglia between smooth muscle sheets and striated muscle bundles were innervated by intraganglionic laminar endings (IGLEs), in a pattern similar to the innervation of the myenteric plexus throughout the stomach and esophagus. Finally, as previously described, the principle bundle of sling muscle fibers that links LES sphincter tissue to the antropyloric region of the lesser curvature was innervated by exceptionally long IMAs as well as by unique web ending specializations at the distal attachment of the bundle. Overall, the specialized varieties of densely distributed vagal afferents innervating the LES underscore the conclusion that these sensory projections are critically involved in generating LES reflexes and may be promising targets for managing esophageal dysfunctions.

Vagal Intramuscular Arrays: The Specialized Mechanoreceptor Arbors That Innervate the Smooth Muscle Layers of the Stomach Examined in the Rat

The fundamental roles that the stomach plays in ingestion and digestion notwithstanding, little morphological information is available on vagal intramuscular arrays (IMAs), the afferents that innervate gastric smooth muscle. To characterize IMAs better, rats were given injections of dextran biotin in the nodose ganglia, and, after tracer transport, stomach whole mounts were collected. Specimens were processed for avidin–biotin permanent labeling, and subsets of the whole mounts were immunohistochemically processed for c‐Kit or stained with cuprolinic blue. IMAs (n = 184) were digitized for morphometry and mapping. Throughout the gastric muscle wall, IMAs possessed common phenotypic features. Each IMA was generated by a parent neurite arborizing extensively, forming an array of multiple (mean = 212) branches averaging 193 µm in length. These branches paralleled, and coursed in apposition with, bundles of muscle fibers and interstitial cells of Cajal. Individual arrays averaged 4.3 mm in length and innervated volumes of muscle sheet, presumptive receptive fields, averaging 0.1 mm3. Evaluated by region and by muscle sheet, IMAs displayed architectural adaptations to the different loci. A subset (32%) of circular muscle IMAs issued specialized polymorphic collaterals to myenteric ganglia, and a subset (41%) of antral longitudinal muscle IMAs formed specialized net endings associated with the serosal boundary. IMAs were concentrated in regional patterns that correlated with the unique biomechanical adaptations of the stomach, specifically proximal stomach reservoir functions and antral emptying operations. Overall, the structural adaptations and distributions of the IMAs were consonant with the hypothesized stretch receptor roles of the afferents. J. Comp. Neurol. 524:713–737, 2016.