Pattern of Innervation by Axons expressing Alpha-Synuclein
Throughout the stomach and proximal duodenum, axons immunoreactive for alpha-synuclein were so densely distributed that the myenteric plexus was clearly delineated (). Large numbers of labeled fibers filled the interganglionic connectives, including the secondary and tertiary interganglionic connectives of the primary plexus. Individual axons were identifiable within the densely stained plexus, and were typically smooth in appearance when running within the connectives. Upon entering the ganglia, however, the same axons contained numerous axonal swellings along their length (). Within a ganglion, axons immunoreactive for alpha-synuclein formed an elaborate network encircling the myenteric neurons with tightly apposed strings of varicosities. These rings of varicosities occurred around both alpha-synuclein-negative () and alpha-synuclein-positive neurons ().
Panels A and B are both photomontages illustrating the innervation of the myenteric plexus by alpha-synuclein-positive fibers and varicosities in the stomach (A) and proximal duodenum (B) of control rats. Scale bar in A = 50 µm for A,B.
Figure 2 Alpha-synuclein-positive varicosities were observed throughout the myenteric ganglia of the stomach (A,B) and the duodenum (C–E) of control rats. Alpha-synuclein-positive varicosities encircle both darkly stained alpha-synuclein-positive neurons (more ...)
In addition to projecting to and through the myenteric plexus, axons immunoreactive for alpha-synuclein innervated the longitudinal smooth muscle of the forestomach (), and blood vessels in the stomach () and duodenum. Alpha-synuclein-positive axons in the longitudinal smooth muscle of the forestomach were in close apposition to unstained intramuscular interstitial cells of Cajal whose processes and somas were silhouetted by the stained axons.
Enteric Neurons expressing Alpha-Synuclein
A subset of myenteric neurons expresses alpha-synuclein in both the nucleus and the cytoplasm of the somata. The density of myenteric neuronal somata immunoreactive for alpha-synuclein was low in the stomach, but became progressively greater in the small intestine as we sampled from sites more distal to the pyloric sphincter ().
Figure 3 Mean ± S.E.M. density of neurons in the myenteric plexus immunoreactive for alpha-synuclein. The density of neurons positive for alpha-synuclein was greater in the small intestine compared to the stomach, and within the small intestine the density (more ...)
Distribution of Alpha-synuclein-positive Myenteric Neurons Co-reactive for either NOS, Calbindin, or Calretinin
Since nitrergic neurons (expressing NOS) and cholinergic neurons (expressing calbindin or calretinin) are the two mutually exclusive chemical phenotypes of enteric neurons that collectively make up the myenteric neuronal population and since hypothetically alpha-synuclein expression might be correlated with one of the phenotypes, the patterns of co-localization were examined quantitatively.
In the survey of stomach whole mounts (ventral; n = 18), 715 myenteric neurons immunoreactive for alpha-synuclein were examined for co-reactivity with either NOS, calbindin, or calretinin (6 whole mounts/antibody). There was an average of 39.7 alpha-synuclein-positive neurons surveyed per ventral whole mount, with positive staining occurring in the nucleus and cytoplasm of both strongly- and weakly-stained cells. The neurons immunoreactive for alpha-synuclein were spread throughout the stomach with 64% located within the boundaries of the nonglandular forestomach, and the remaining 36% contained within the corpus-antrum. In the stomach whole mounts double labeled for alpha-synuclein and NOS, 58% of the alpha-synuclein-positive neurons overall were immunoreactive for NOS. When whole mounts were double labeled for alpha-synuclein and calbindin, 35% of the alpha-synuclein-positive neurons overall were immunoreactive for calbindin. Similarly, in whole mounts double labeled for alpha-synuclein and calretinin, 35% of the alpha-synuclein-positive neurons overall were co-reactive for calretinin.
Expressing co-localization for the stomach as overall averages masked a dramatic and specific pattern of co-expression. By instead plotting the regional organization of double labeled cells in the stomach, we determined that 90% of the alpha-synuclein neurons in the forestomach were co-reactive for NOS, while 92% of the alpha-synuclein neurons in the corpus-antrum were co-reactive for either calbindin or calretinin (). This pattern of regional specificity for double labeled cells could not easily be explained away by the distribution of the individual markers because neurons immunoreactive for either NOS, calbindin, or calretinin were distributed throughout the myenteric plexus of the forestomach, corpus, and antrum, and were not localized within one single defined region of the stomach ().
Figure 4 Plotting the distribution of myenteric neurons within the ventral stomach wall that were co-reactive for alpha-synuclein and either NOS, calbindin, or calretinin revealed regional specificity for the different chemical phenotypes. Neurons double labeled (more ...)
Figure 5 The regional specificity of neurons co-reactive for alpha-synuclein and either NOS, calbindin, or calretinin (illustrated in ) cannot be explained by the distribution of the three phenotypes throughout the stomach and duodenum. Neurons positive (more ...)
For the duodenum, a total of 631 neurons immunoreactive for alpha-synuclein were sampled from the 18 whole mounts double labeled for either NOS, calbindin, or calretinin (6 whole mounts/antibody). Similar to stained neurons in the stomach, the nucleus and the cytoplasm of the cells were positive for alpha-synulcein. In whole mounts processed for both alpha-synuclein and NOS, 40% the alpha-synuclein cells were co-reactive for NOS. When whole mounts were double labeled for alpha-synuclein and calbindin, 13% of the alpha-synuclein-positive neurons were immunoreactive for calbindin. Finally, in whole mounts double labeled for alpha-synuclein and calretinin, 38% of the alpha-synuclein-positive neurons were co-reactive for calretinin. Myenteric neurons immunoreactive for each of the three antibodies (i.e., NOS, calbindin, and calretinin) were distributed throughout the ganglia of the duodenal whole mounts (), and no regional pattern (such as seen in the stomach) was apparent.
Expression of Alpha-Synuclein within Vagal Efferent but not Vagal Afferent Terminals
Bilateral injections of Dextran-Texas Red into either the dmnX or the nodose ganglia resulted in the selective labeling of, respectively, either vagal efferent or afferent terminals. Within the GI tract, the Dextran-Texas Red labeled vagal efferent fibers were located within the connectives and ganglia of the myenteric plexus, and, upon entering a ganglion, produced endings containing dense terminal-like varicosities around one or more neurons. Immunohistochemistry combined with anterograde labeling revealed that vagal preganglionic axons express a high level of alpha-synuclein. In fact, every Dextran-Texas Red labeled varicosity was unmistakably positive for alpha-synuclein.
The presence, however, of alpha-synuclein-positive, Dextran-Texas Red negative neural elements highlights the fact that a) not all vagal efferents are labeled following typical injections of tracers into the dmnX, and b) the non-vagal origin of some of the alpha-synuclein axons and terminals.
In addition, and importantly in terms of potential pathways for an alpha-synuclein-mediated retrograde transfer of a pathogenic stimulus (as postulated by Braak and co-workers), alpha-synuclein-positive neurons in the myenteric plexus were consistently found to be either completely or partially encircled by dense, varicose vagal efferent endings that were positive for alpha-synuclein (). Not all neural elements positive for alpha-synuclein, however, were vagal efferents.
Figure 6 Varicosities along the vagal efferent axons innervating the myenteric ganglia were positive for alpha-synuclein, and in close proximity to myenteric neurons that were also positive for alpha-synuclein. Vagal efferent innervation (red) of the myenteric (more ...)
Following injection of Dextran-Texas Red into the nodose ganglia, the myenteric plexus of the stomach and small intestine and the longitudinal smooth muscle of the forestomach were both innervated by their respective specialized vagal afferent terminals (Phillips and Powley, 2000
). Specifically, the myenteric plexus was innervated by intraganglionic laminar endings (IGLEs) consisting of numerous plates of puncta that form a fine laminar covering that encapsulates one or more neurons within a ganglion; whereas, the longitudinal smooth muscle was innervated by intramuscular arrays (IMAs) consisting of a parent axon which typically, upon entering the muscle layer, bifurcates multiple times, creating an array of parallel terminal elements that terminate within one or more adjacent muscle sheets. The circular muscle layer was removed to facilitate access of the primary antibodies to the myenteric plexus, so observations on the innervation of the circular muscle by IMAs were not possible. Anterograde labeling of vagal afferent terminals combined with immunohistochemical labeling of alpha-synuclein did not find any expression of the antigen within the two types of afferent terminals.
IGLEs were, however, in close registration with alpha-synuclein-positive varicosities within the ganglia of the myenteric plexus (), and in some cases the laminae of IGLEs were either superficial or deep to alpha-synuclein-positive neurons (). Similarly, IMAs terminating within the longitudinal smooth muscle were intertwined with alpha-synuclein-positive fibers and unstained interstitial cells within the same muscle sheets, but were not themselves positive for alpha-synuclein.
Figure 7 Vagal afferent terminals in the myenteric plexus were not co-localized with alpha-synuclein, though some afferent terminals associated with alpha-synuclein-positive enteric neurons. Vagal afferent innervation (red) of the gastric myenteric plexus was (more ...)
Verification of Vagotomies with Retrograde Transport of FluoroGold
Since vagotomy is the most definitive means of eliminating vagal efferents and thus gauging which subgroups of alpha-synuclein-positive neurites in the GI tract are vagal efferents (see above) and which are intrinsic (or, of course, extrinsic sympathetic efferents or afferents), we evaluated the completeness of vagotomy of our operated animals at the time of euthanasia. Six rats satisfied the stringent criteria used to establish that the subdiaphragmatic vagotomy had eliminated all of the vagal abdominal branches: 1) compared to shams (n = 5) with all vagal branches intact and their corresponding columns in the dmnX heavily labeled with FluoroGold, completely vagotomized rats had very few FluoroGold-labeled motor neurons distributed through the dmnX, and 2) none of the rats judged to have complete vagotomies had concentrations of preganglionic neurons in either the gastric or celiac columns of the left and right dmnX ().
Figure 8 The vagal innervation of the GI tract was eliminated by cutting and cauterizing the left and the right trunks of the nerve at the level of the diaphragm. The medial and lateral columns of the dmnX are both clearly labeled with FluoroGold in the sham rats (more ...)
Pattern of Innervation by Alpha-Synuclein in Vagotomized Rats
Elimination of the vagal innervation to the GI tract resulted in a dramatic reduction in the density of the innervation of the myenteric plexus by axons and varicosities immunoreactive for alpha-synuclein (). Whereas in the sham rats the myenteric plexus was well defined by the alpha-synuclein innervation (cf. ), the myenteric plexus of vagotomized rats was only discernible because of residual background staining. Ganglia and fiber tracts which were richly innervated in sham rats, consisted of only scattered varicosities and stray fibers, respectively, in the vagotomized rats.
Figure 9 Alpha-synuclein-positive neurons (A), varicosities (D) and axons (F) were present in the myenteric plexus of the stomach of sham rats with their vagal efferent innervation intact. Vagotomy did not eliminate the presence of alpha-synuclein-positive neurons (more ...)
Following vagotomy, myenteric neurons immunoreactive for alpha-synuclein remained. These alpha-synuclein neurons, which in sham operated controls were typically encircled by closely apposed and varicose alpha-synuclein-positive terminals, were frequently found to be bare of any varicose alpha-synuclein-positive contacts in the vagotomized rats. In addition, elimination of the vagal innervation unmasked the axonal processes of the more strongly labeled alpha-synuclein-positive enteric neurons. These axons of enteric neurons, which had been obscured by the dense networks of vagal neurites positive for alpha-synuclein in the control and sham rats, could be followed for long distances as they tapered off within other ganglia or the smooth muscle layers (). In addition to intrinsic neurons immunoreactive for alpha-synuclein, the innervation of the longitudinal smooth muscle layer of the forestomach and the blood vessels by fibers immunoreactive for alpha-synuclein remained after vagotomy.
Vagal Efferent Preganglionic Contact with Immunohistochemically Identified Subsets of Neurons
Double-label immunohistochemistry was done in conjunction with anterograde labeling of vagal preganglionics to identify the types of alpha-synuclein-positive myenteric neurons contacted by vagal efferent terminals. NOS-, calbindin-, and calretinin-immunoreactive neurons were common throughout the myenteric plexus of the stomach and duodenum, however calbindin and calretinin neurons were not as abundant as neurons immunoreactive for NOS. The highly varicose vagal efferent fibers either partially or completely encircled alpha-synuclein-positive neurons in the stomach and duodenum co-reactive for either NOS, calbindin, or calretinin (). Neurons immunoreactive for either NOS, calbindin, or calretinin but not alpha-synuclein were also routinely observed in close association to vagal efferent varicosities.
Figure 10 Vagal efferent varicosities positive for alpha-synuclein were in close proximity to myenteric neurons co-reactive for alpha-synuclein and either NOS (A), calbindin (B) or calretinin (C). Dextran-Texas Red tracing of vagal efferents (red) was combined (more ...)
Extrinsic and Intrinsic Origin of Alpha-Synuclein Immunoreactive Inclusions
A distinct axonopathy represented by markedly swollen alpha-synuclein immunoreactive axons and terminals was observed, albeit infrequently, in the stomach and small intestine of adult rats. Such swollen alpha-synuclein immunoreactive axons could be followed for several millimeters as they ran within the connectives and through various ganglia before terminating in close proximity to myenteric neurons (). These dystrophic swellings were in some cases as large as the smaller neuronal somata (), and appeared to be packed with unstained or lightly stained organelles, presumably mitochondria. We were able to determine, by anterograde labeling of the vagal efferent innervation of the myenteric plexus with Dextran-Texas Red in combination with immunohistochemical labeling of alpha-synuclein, that some of the markedly swollen varicosities immunoreactive for alpha-synuclein were vagal efferent axons and terminals. These markedly swollen alpha-synuclein-positive vagal efferent terminals were observed encircling myenteric neurons that were also immunoreactive for alpha-synuclein (). Not all of the dystrophic axons immunoreactive for alpha-synuclein, however, were vagal efferents because some remained following complete subdiaphragmatic vagotomy ().
Figure 11 Markedly swollen axons, indicative of dystrophy or degeneration, were positive for alpha-synuclein (brown DAB precipitate) and found to be in close proximity to myenteric neurons (light blue cells labeled with Cuprolinic Blue) in the stomach of a 10 month (more ...)
Figure 12 Alpha-synuclein-positive axons were observed with dystrophic swellings along their length within the myenteric plexus of the small intestine (A). Similar alpha-synuclein-positive axonopathies were observed in the small intestine of vagotomized rats confirming (more ...)