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.

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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.

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin-biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in the flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: 1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. 2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. 3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric ganglia and circular muscles, exhibiting polymorphic terminal structures. 4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.