Problem
To distinguish the central cholinergic axons (extrinsic) from enteric cholinergic fibers (intrinsic), and to visualize their spatial configuration in the colonic enteric nervous system (ENS), is fundamental for understanding brain-gut cross-talk via cholinergic pathway, and ultimately efficient neuromodulation. However, the ability to assess their neuroanatomical distinctions is still a challenge due to the lack of specific markers to label central and peripheral cholinergic innervation separately, as well as the lack of a proven approach to visualize the three-dimensional (3D) structure of the ENS.
Solution
Firstly, we established an approach to differentiate central and peripheral cholinergic innervation of the pig colon, a relevant translational model using double immunolabeling. We paired a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody that we developed recently, with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system (CNS). In the whole mount preparation of the colonic myenteric plexus, the dense hpChAT immunoreactive (ir) neurons and fibers, and varicose cChAT-ir fibers, were simultaneously visualized. Notably there were also rare cChAT-ir fibers co-labeled with hpChAT-ir
Fig. 1. A new approach established to differentiate central and peripheral cholinergic innervation of the pig colon. Double immunostaining with antibodies against common type of choline acetyltransferase (cChAT, red, A, D, G) expressed preferentially in the central nervous system, and human peripheral choline acetyltransferase (hpChAT, green, H, I) showed the cChAT labeling of cholinergic neurons/fibers in the cervical vagal bundles (A, C), spinal cord sacral S2 ventral horn (D, F) and in fibers of the whole-mount preparations of the longitudinal muscle-myenteric plexus of the proximal colon (G) while the hpChAT labeling only in the neurons and fibers in myenteric plexus (H, I) and not in the vagus or spinal cord (B, E) of a naïve male pig. C, F, I: merged images of A and B, D and E and G and H respectively. No overlapping of cChAT and hpChAT was found in I. Bar scale in each panel 50 µm.
Secondly, we developed a modified CLARITY tissue-clearing technique to remove the light scattering lipids from colonic muscularis externa, while keeping the proteins and nucleic acids unaffected. This allowed for deep penetration of antibodies and 3D imaging. The 3D images exhibited a clear spatial view of the microstructures of the cholinergic nerve network in the myenteric plexus, showing that intrinsic cholinergic neurons (hpChAT-ir) were closely surrounded by central cholinergic (cChAT-ir) varicose fibers with dot like structures (nerve terminals)
Fig. 2. Spatial configuration of central cholinergic axons and peripheral cholinergic neurons in the pig colonic muscularis externa. 3D images were generated from the pig proximal colonic muscularis externa cleared with a modified CLARITY tissue clearing protocol developed in this study. A: Peripheral cholinergic neurons and fibers were labeled with human peripheral choline acetyltransferase (hpChAT) antibody as green color, while central cholinergic innervation was labeled with common type of ChAT (cChAT) antibody as red color. Bar scale 50 µm. B. High magnification of the insert in A showing that hpChAT immunoreactive (ir) neurons are surrounded by cChA-ir varicose fibers and dot like structures, presumably nerve terminals. Bar scale 20 µm.
This provides neuroanatomical evidence that central efferent cholinergic fibers target myenteric neurons in the pig colon. Thirdly, we developed an approach - adapted from the Imaris 9.7 Surfaces Rendering Technology - for the density measurement of cholinergic innervation in 3D Images of the pig colon
Fig. 3. A computerized automatic measurements of the central cholinergic fibers and peripheral cholinergic fibers + neurons by a developed approach- adapted from the Imaris 9.7 Surfaces Rendering Technology. 3D images with the volume X×Y×Z (axis)=708x708x20-45 (µm3) were acquired from the whole-mount preparations of inner submucosal plexus, outer submucosal plexus and myenteric plexus in the porcine proximal colon. cChAT-ir fibers and hpChAT-ir fibers + somata in the inner submucosal plexus (Fig. 6A), outer submucosal plexus (Fig. 6F) and myenteric plexus (Fig. 6 K) were digitally traced with Imaris SurfaceFunction (Figs. 6B, G, L) respectively and extracted separately (Figs. 6C, H, M and Figs. 6D, I, N). The ganglia containing cholinergic neurons/fibers were demarcated (Figs. 6E, J, O). The volumes of traced nerve fibers and neurons and outlined ganglia were automatically measured with Imaris 9.7 for quantitation of densities of positive cChAT fibers and hpChAT fibers + somata. Bar scale in each panel 50 µm.
This approach allowed us to perform direct, objective and automatic measurements with less biases due to observer/examiner judgment. It was also faster than counting manually via grid-based stereology and histomorphometry, which is both time consuming and labor intensive. This ultimately enabled us to quantify a large number of samples with increased statistical accuracy.
Impact
This study increases our understanding of the brian-gut connection, enhancing our ability to evaluate colonic disorder. It provides strong neuroanatomical evidence of CNS-gut parasympathetic innervation via cholinergic terminals surrounding intrinsic cholinergic neurons, and the consequent ability to regulate colonic function. The quantitative analysis revealed for the first time the differences in density of colonic cholinergic innervation among three colonic segments and plexuses of the central - but not peripheral - cholinergic innervation, with no sex difference. The oral administration of the µ-opioid receptor agonist, loperamide at 3 mg/kg/day for 4 weeks decreased the peripheral cholinergic density in the myenteric plexus of the pig distal colon. These data lay a solid morphological basis for the evaluation of cholinergic alterations of the ENS associated with functional colonic disorders. In addition, such a set of novel approaches can be extended to get insight to the parasympathetic innervation of other parts of the gastrointestinal tract and viscera, in relation with their intrinsic cholinergic system in healthy and disease states.
