Topographical organization and morphology of substance P (SP)‐immunoreactive axons in the whole stomach of mice

Abstract

Nociceptive afferents innervate the stomach and send signals centrally to the brain and locally to stomach tissues. Nociceptive afferents can be detected with a variety of different markers. In particular, substance P (SP) is a neuropeptide and is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. However, the topographical distribution and morphological structure of SPimmunoreactive (SP-IR) axons and terminals in the whole stomach have not yet been fully determined. In this study, we labeled SP-IR axons and terminals in flat mounts of the ventral and dorsal halves of the stomach of mice. Flat-mount stomachs, including the longitudinal and circular muscular layers and the myenteric ganglionic plexus, were processed with SP primary antibody followed by fluorescent secondary antibody and then scanned using confocal microscopy. We found that (1) SP-IR axons and terminals formed an extensive network of fibers in the muscular layers and within the ganglia of the myenteric plexus of the whole stomach. (2) Many axons that ran in parallel with the long axes of the longitudinal and circular muscles were also immunoreactive for the vesicular acetylcholine transporter (VAChT). (3) SP-IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus; many of these were VAChT immunoreactive. (4) The regional density of SP-IR axons and terminals in the muscle and myenteric plexus varied in the following order from high to low: antrum-pylorus, corpus, fundus, and cardia. (5) In only the longitudinal and circular muscles, the regional density of SP-IR axon innervation from high to low were: antrumpylorus, corpus, cardia, and fundus. ( 6) The innervation patterns of SP-IR axons and terminals in the ventral and dorsal stomach were comparable. Collectively, our data provide for the first time a map of the distribution and morphology of SP-IR axons and terminals in the whole stomach with single-cell/axon/synapse resolution. This work will establish an anatomical foundation for functional mapping of the SP-IR axon innervation of the stomach and its pathological remodeling in gastrointestinal diseases.

Organization and morphology of calcitonin gene‐related peptide‐immunoreactive axons in the whole mouse stomach

“…Following previously established protocols for whole mount processing (Li et al, 2014;Ma et al, 2023;Rysevaite et al, 2011), samples were washed 6 × 5 min in 0.1 M PBS (pH = 7.4) and immersed in blocking solution (0.1 M PBS containing 2% bovine serum albumin, 10% normal donkey serum, 2% Triton X-100, and 0.08% sodium azide) for 5 days.…”

“…How to cite this article: Ma, J., Nguyen, D., Madas, J., Bizanti, A., Mistareehi, A., Kwiat, A. M., Chen, J., Lin, M., Christie, R., Hunter, P., Heal, M., Baldwin, S., Tappan, S., Furness, J. B., Powley, T. L., & Cheng, Z. J. (2023).…”

“…Substance P (SP), calcitonin gene‐related peptide (CGRP), and transient receptor potential cation channel subfamily V member 1 (TRPV1) have been commonly used as three major nociceptive makers. Recently, we provided a comprehensive map of the SP‐IR axon innervation in flat mounts of the whole mouse stomach muscular layers (Ma et al., 2023). Earlier studies (Ekblad et al., 1985; Furness et al., 1991; Green & Dockray, 1988) showed that many SP‐IR nerve fibers are of intrinsic origin, and our study supported this (Ma et al., 2023).…”

“…Recently, we provided a comprehensive map of the SP‐IR axon innervation in flat mounts of the whole mouse stomach muscular layers (Ma et al., 2023). Earlier studies (Ekblad et al., 1985; Furness et al., 1991; Green & Dockray, 1988) showed that many SP‐IR nerve fibers are of intrinsic origin, and our study supported this (Ma et al., 2023). Using the same methodology for the identification of morphology and distribution of nociceptive nerves, we applied immunohistochemical (IHC) labeling of CGRP to determine the distribution and morphology of CGRP‐IR axon innervation in the whole stomach muscular layers of mice, as well as to determine the origin(s) of these axons.…”

“…Given that a comprehensive view of the innervation pattern of CGRP‐IR axons and terminals is important for the understanding of nociceptive processes, we used flat‐mount preparations of the whole stomach muscle layers which retained the complete axon network. In addition to the holistic view, we also focused on specific gastric targets, such as myenteric ganglia, longitudinal and circular muscles, and blood vessels in different regions (Ma et al., 2023; Sharrad et al., 2015). Previously, we have studied the distribution pattern and morphology of CGRP‐IR and SP‐IR axons in flat mounts of the atria of the heart as well as SP‐IR axons in flat mounts of the whole stomach muscular layers (Li et al., 2014; Ma et al., 2023).…”