Nociceptive afferent axons of the stomach send signals centrally to the brain and locally to stomach tissues. Nociceptive axons can be detected with a variety of different markers. In particular, the neuropeptide substance P (SP) is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. In addition, the Local release of SP contributes to tissue protection and repair in response to noxious stimuli. However, the topographical distribution and morphological structure of SP-immunoreactive (SP-IR) axons and terminals in the whole stomach have not yet been well determined. In this study, we labeled SP-IR axons and terminals in flat-mounts and serial sections of the antrum, pyloric sphincter, and duodenum (APD) of the mouse stomach. The flat-mount stomachs included the external muscles (longitudinal and circular layers) and the myenteric ganglionic plexus, whereas APD serial sections also included the submucosa and mucosa. Tissue samples were scanned using confocal and Zeiss M2 imager microscopy to acquire detailed high-resolution images of SP innervation and produce montages of the whole stomach flat-mount and APD serial sections. Additionally, we double-labeled the samples with vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), and injected the stomach walls with DiI-retrograde tracer to determine the connectivity map between SP-IR axons and their possible extrinsic origin. We found that: 1) SP-IR axons innervated all layers including the external muscles, myenteric ganglia, submucosa, submucosal ganglia, and mucosa. Many axons were immunoreactive for VAChT but not TH. 2) SP-IR axons and terminals formed an extensive network in the muscular layers and ran in parallel with the long axis of the external muscles. 3) SP-IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus 4) In the submucosa, SP-IR axons innervated blood vessels and submucosal ganglia and formed a nerve network around duodenal Brunner's glands. 5) In the mucosa, SP-IR varicose axons were organized into bundles in the muscularis mucosa and lamina propria at the base of the mucosa. Some SP-IR axons were found in the gastric subepithelial level and duodenal villi. 6) SP-IR axon density in the muscles and myenteric plexus was much higher than in the submucosa and mucosa. 7) The density of SP-IR axons and terminals in the flat-mounts varied in different stomach regions in the following order from high to low: antrum-pylorus, corpus, fundus, and cardia. 8) In the APD area, the muscular wall of the antrum and duodenum showed a higher SP-IR axon density than in the pyloric sphincter. 9) The extrinsic origin of SP-IR axons in the stomach derived mainly from the spinal T7-T11 dorsal root ganglia with a lesser contribution from the vagal nodose ganglia, but not from the celiac ganglia, the dorsal motor nucleus of vagus, or the nucleus tractus solitarius. Collectively, our data provide for the first time a map of the topographical 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 nociceptive afferent innervation of the stomach and its pathological remodeling in gastrointestinal diseases.


If this is your thesis or dissertation, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu

Graduation Date





Cheng, Zixi


Doctor of Philosophy (Ph.D.)


College of Medicine


Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences




CFE0009656; DP0027583





Release Date

February 2028

Length of Campus-only Access

5 years

Access Status

Doctoral Dissertation (Campus-only Access)

Restricted to the UCF community until February 2028; it will then be open access.