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.

Nociceptive afferents from spinal dorsal root ganglia (DRG) and vagal nodose ganglia innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. In our other two posters (Mistareehi et al. 2022, Nguyen et al. 2022), nociceptive afferents are detected with SP and CGRP markers. To specifically study spinal afferent innervation of the stomach, we injected tracer Dextran Biotin (DB) into the left DRG (T7‐T11; 1.5 µl/each ganglion). 14 days after tracer injection, rats were perfused through the left ventricle with 40℃ PBS (1M phosphate‐buffered saline, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1 M PBS (4℃, pH 7.4). After the perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was removed. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser and greater curvatures, thus yielding two flat‐mounts per animal. The external muscle wall of the stomach was then isolated as a whole mount by removing the gastric mucosa and submucosa. Ventral stomach flat‐mounts were examined using the Neurolucida system. We found that Tracer DB‐labeled axons formed serval categories of the terminal structures. The simple type: long varicose axons without branching; The branching type: axons with multiple branches running in parallel with either circular or longitudinal muscles; The complex type: single axons with multiple branches running in various directions in both circular and longitudinal muscle layers; The ganglionic type:axons innervating myenteric ganglia; The blood vessel type: axons innervating blood vessels. Our work will provide a foundation for specific labeling of spinal afferent innervation of the stomach. In the future, we will use tracer injection of DRG and nociceptive markers (e.g., SP, CGRP, TRPV1) to examine topographical innervation of spinal nociceptive afferents in the stomach. The first two authors contributed equally to this work.

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Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers [e.g., substance P (SP) and calcitonin gene-related peptide (CGRP)]. We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: 1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP-IR axons densely innervated the blood vessels. 3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. 4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. 5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. 6) CGRP-IR axons did not colocalize with tyrosine hydroxylase (TH) or vesicular acetylcholine transporter (VAChT) axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. 7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

This protocol describes the process of identifying the distribution and morphology of substance P (SP) immuno-reactive axons and terminals in the antrum, pylorus, and duodenum of mice. The antrum-pylorus-duodenum (APD) region was sectioned and immunohistochemically labeled for SP. To determine the distribution and morphology of SP-IR axons and calculate their density in different layers of APD, a Zeiss M2 Imager was used to scan each serial section. Each section was presented as a montage of approximately 50 all-in-focus maximum projection images. To determine the detailed structures of SP-IR axons and terminals, the confocal microscope was used to scan the regions of interest.