Substance-P (SP) is a commonly used marker of nociceptive afferent axons, and it plays an important role in a variety of physiological functions including the regulation of motility, gut secretion, and vascular flow. Previously, we found that SP-immunoreactive (SP-IR) axons densely innervated the pyloric antrum of the flat-mount of the mouse whole stomach muscular layer. However, the regional distribution and morphology of SP-IR axons in the submucosa and mucosa were not well documented. In this study, the mouse antrum-pylorus-duodenum (APD) were transversely and longitudinally sectioned. A Zeiss M2 imager was used to scan the serial sections of each APD (each section montage consisted of 50–100 all-in-focus maximal projection images). To determine the detailed structures of SP-IR axons and terminals, we used the confocal microscope to scan the regions of interest. We found that 1) SP-IR axons innervated the muscular, submucosal, and mucosal layers. 2) In the muscular layer, SP-IR varicose axons densely innervated the muscles and formed varicose terminals which encircled myenteric neurons. 3) In the submucosa, SP-IR axons innervated blood vessels and submucosal ganglia and formed a network in Brunner’s glands. 4) In the mucosa, SP-IR axons innervated the muscularis mucosae. Some SP-IR axons entered the lamina propria. 5) The muscular layer of the antrum and duodenum showed a higher SP-IR axon density than the pyloric sphincter. 6) SP-IR axons were from extrinsic and intrinsic origins. This work provided a comprehensive view of the distribution and morphology of SP-IR axons in the APD at single cell/axon/varicosity scale. This data will be used to create a 3D scaffold of the SP-IR axon innervation of the APD.

P物质（SP）是伤害性传入轴突的常用标志物，在调节运动、肠道分泌和血管流量等多种生理功能中发挥着重要作用。此前，我们发现SP-免疫反应性（SP-IR）轴突密集地支配小鼠全胃肌肉层平坦的幽门窦。然而，粘膜下层和粘膜中 SP-IR 轴突的区域分布和形态尚未得到很好的记录。在这项研究中，对小鼠胃窦-幽门​​-十二指肠（APD）进行了横向和纵向切片。使用蔡司 M2 成像仪扫描每个 APD 的连续切片（每个切片蒙太奇由 50-100 个全焦点最大投影图像组成）。为了确定 SP-IR 轴突和末端的详细结构，我们使用共焦显微镜扫描感兴趣的区域。我们发现 1) SP-IR 轴突支配肌肉层、粘膜下层和粘膜层。2）在肌肉层，SP-IR曲张轴突密集地支配肌肉并形成包围肌间神经元的曲张末梢。3）在粘膜下层，SP-IR轴突支配血管和粘膜下神经节，并在布伦纳腺中形成网络。4)在粘膜中，SP-IR轴突支配粘膜肌层。一些 SP-IR 轴突进入固有层。5)胃窦和十二指肠的肌层显示出比幽门括约肌更高的SP-IR轴突密度。6) SP-IR 轴突有外在和内在起源。这项工作提供了 APD 中 SP-IR 轴突在单细胞/轴突/静脉曲张尺度上的分布和形态的全面视图。该数据将用于创建 APD 的 SP-IR 轴突神经支配的 3D 支架。