Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon - PubMed (original) (raw)

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon

Maurizio Mazzoni et al. Cell Tissue Res. 2024 Jan.

Abstract

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19-22% and 13-17%, respectively, P < 0.001-0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62-85%), whereas 18-38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33-36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

Keywords: Cholinergic and nitrergic transmission; Excitatory motor neurons; Inhibitory motor neurons; Interneurons; Secretomotor neurons.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Graphs showing the density of neurons containing HuCD/SP-immunoreactivity (IR) (a, b), HuCD/ChAT/SP-IR (c, d), or HuCD/SP/nNOS-IR (e, f) in the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP) of the ascending and descending colon. Neuronal density is expressed as numbers of neurons/mm2 (a, c, d) and % of neurons visualized with HuCD-IR (b, d, f) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between different plexuses within the same colonic region) (#P < 0.05; ##P < 0.01; ####P < 0.0001 between the same plexus in different colonic regions)

Fig. 2

High-resolution confocal images of the submucosal (a–c, d–f) and myenteric (g–i) plexus of the porcine colon. a Immunofluorescence staining obtained with the pan neuronal marker HuC/D in the inner submucosal plexus of the descending colon. b Substance P (SP)-immunoreactive (-IR) neurons that exhibit an irregular, ovoidal soma profile (arrows) some of which with thick dendrites. SP-IR fibers in the ganglia show varicosities (arrowheads) and often form baskets around HuC/D neurons. c Overlap staining of HuC/D-IR and SP-IR. d–f Representative images of the outer submucosal plexus of the ascending colon. d HuC/D-IR neurons, e SP-IR neurons, and f merge staining. Arrows in d–f indicate examples of HuC/D/SP-IR neurons exhibiting rounded or oval profile. g–i Myenteric plexus of the ascending colon. Arrows point to neurons co-expressing HuC/D- (g) and SP-IR (h) (i, respective overlapping). SP-IR neurons displayed a variable size and morphology. SP-IR was detectable in thinner and thicker bundles of fibers (both exhibited varicosities) running around HuC/D-IR neurons (arrowheads)

Fig. 3

High-resolution confocal images. Double labeling of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivities (IR) in ganglion cells in the outer submucosal plexus in the ascending colon (a–c) and in the descending colon myenteric plexus (d–f, g–i). a–c Arrowheads point to some ChAT-IR neurons containing SP-IR, whereas arrows point to examples of ChAT-IR neurons lacking SP-IR. d–f SP/ChAT-IR neurons (arrowheads), ChAT-IR neurons lacking SP-IR (arrows) as an example of a SP-IR neuron that does not contain ChAT-IR (asterisks). Blue arrowheads (d–f and g–i) indicate SP-IR (f): varicose fibers. g–i Some neurons co-expressing SP- and ChAT-IR (arrowheads) and a ChAT- IR neuron that does not contain SP-IR (arrows) and is surrounded by SP-IR fibers with varicosities. Blue arrowheads also point to prominent varicose fibers

Fig. 4

High-resolution confocal images showing staining for neuronal nitric oxide synthase (nNOS) (a, d, g) and SP (b, e, h) and merging of both staining (c, f, i) in the outer submucosal (a–c and g–i) and myenteric (d–f) plexus of the ascending colon. Arrows point to nNOS immunoreactive (-IR) neurons co-expressing SP-IR, while arrowheads indicate examples of nNOS-IR neurons lacking SP-IR and blue arrowheads point to neurons that do not contain SP-IR. Some nNOS-IR neurons showed cellular projections (dendrites or axons) emerging from the cell body and size and morphology variability

Fig. 5

Graphs showing the density of neurons containing SP/ChAT/nNOS-IR (a) in the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP) of the ascending and descending colon expressed as numbers of neurons/mm2. The graph in b shows the % of SP-IR neurons that contain both ChAT- and nNOS-IR (*P < 0.05; **P < 0.01; ****P < 0.0001 between different plexuses within the same colonic region) (###P < 0.001; ####P < 0.0001 between the same plexus in different colonic regions)

Fig. 6

High-resolution confocal images showing colocalization of SP- (a), nNOS- (b), and ChAT-immunoreactivity (IR) (c). d The overlay of SP/nNOS/ChAT-IRs. Double arrowheads point to SP/nNOS/ChAT-IR neurons, arrows point to nNOS/ChAT-IR lacking SP-IR, whereas asterisks indicate a SP-IR neuron positive for ChAT-IR but negative for nNOS-IR

References

1. Bassols A, Costa C, Eckersall PD, Osada J, Sabrià J, Tibau J. The pig as an animal model for human pathologies: a proteomics perspective. Proteomics Clin Appl. 2014;8:715–731. doi: 10.1002/prca.201300099. -DOI -PubMed
1. Blumenthal JA, Onorato JJ, Carey HV. Muscarinic inhibition of substance P induced ion secretion in piglet jejunum. Can J Physiol Pharmacol. 1998;76:169–175. doi: 10.1139/y98-009. -DOI -PubMed
1. Bornstein JC, Furness JB, Costa M. An electrophysiological comparison of substance P-immunoreactive neurons with other neurons in the guinea-pig submucous plexus. J Auton Nerv Syst. 1989;26:113–120. doi: 10.1016/0165-1838(89)90159-8. -DOI -PubMed
1. Brehmer A, Croner R, Dimmler A, Papadopoulos T, Schrödl F, Neuhuber W. Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine. Auton Neurosci. 2004;112:49–59. doi: 10.1016/j.autneu.2004.03.005. -DOI -PubMed
1. Brookes SGH. Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec. 2001;262:58–70. doi: 10.1002/1097-0185(20010101)262:1<58::AID-AR1011>3.0.CO;2-V. -DOI -PubMed

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon - PubMed (original) (raw)