Spinal afferent innervation in flat-mounts of the rat stomach: anterograde tracing.

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 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 neurons and the circular muscle, 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.

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

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 or vesicular acetylcholine transporter 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.

Mapping the Organization and Morphology of Calcitonin Gene-Related Peptide (CGRP)-IR Axons in the Whole Mouse Stomach.

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.

Topographical distribution and morphology of SP-IR axons in the antrum, pylorus, and duodenum of mice.

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.

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

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 SP-immunoreactive (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: antrum-pylorus, 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.