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.
The sympathetic nervous system is crucial for controlling multiple cardiac functions. However, a comprehensive, detailed neuroanatomical map of the sympathetic innervation of the heart is unavailable. Here, we used a combination of state-of-the-art techniques, including flat-mount tissue processing, immunohistochemistry for tyrosine hydroxylase (TH, a sympathetic marker), confocal microscopy and Neurolucida 360 software to trace, digitize, and quantitatively map the topographical distribution of the sympathetic postganglionic innervation in whole atria of C57Bl/6 J mice. We found that (1) 4–5 major extrinsic TH-IR nerve bundles entered the atria at the superior vena cava, right atrium (RA), left precaval vein and the root of the pulmonary veins (PVs) in the left atrium (LA). Although these bundles projected to different areas of the atria, their projection fields partially overlapped. (2) TH-IR axon and terminal density varied considerably between different sites of the atria with the greatest density of innervation near the sinoatrial node region (P < 0.05, n = 6). (3) TH-IR axons also innervated blood vessels and adipocytes. (4) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent cells were also strongly TH-IR. Our work provides a comprehensive topographical map of the catecholaminergic efferent axon morphology, innervation, and distribution in the whole atria at single cell/axon/varicosity scale that may be used in future studies to create a cardiac sympathetic-brain atlas.
This protocol describes the process of mapping the topographical organization of tyrosine hydroxylase immune reactive sympathetic postganglionic axons and terminals in the mouse heart. Hearts were removed and separated as whole mounts, then scanned using confocal or zeiss microscopy
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.
The cover image is based on the Research Article Topographical organization and morphology of substance P (SP)‐immunoreactive axons in the whole stomach of mice by Jichao Ma et al., https://doi.org/10.1002/cne.25386.
To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene‐related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague‐Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and ventricles and interventricular septum, and prepared as flat‐mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluro 488 secondary antibody for fluorescence. The flat‐mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP‐IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP‐IR axons entered as large bundles near the superior/inferior vena cava, left pre‐caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP‐IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP‐IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.
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.
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