Expression of 5-HT3 receptors in the rat gastrointestinal tract Jörg Glatzle, Catia Sternini, Carla Robin, Tilman T. Zittel, Helen Wong, Joseph R. Reeve, Helen E. Raybould  Gastroenterology  Volume 123, Issue 1, Pages 217-226 (July 2002) DOI: 10.1053/gast.2002.34245 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Schematic view of the 5-HT3R, a ligand gated ion channel, containing a large extracellular domain, 4 transmembrane regions, and large intracellular loop. In rats, 2 splice variants exist; the long-form 5-HT3L with a 6-amino acid insertion (marked with the 2-way arrow) and the short-form 5-HT3S. The antibody region for the present c-terminal antibody is marked accordingly. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Myenteric neurons immunopositive for the 5-HT3R in cryostat sections (A) and whole mounts (C) of the ileum as well as submucosal plexus neurons in cryostat sections of the ileum (D). The 5-HT3R immunostaining is predominantly located on the plasma membrane of the neurons (C, arrows left to right and insert); however, in some myenteric neurons, the 5-HT3R immunoreactivity is located in the cytoplasm (C, arrowhead) and some neurons do not express 5-HT3R immunoreactivity (C, double arrow). The insertion in (C) is a high magnification of a myenteric neuron showing 5-HT3R immunostaining on the plasma membrane. Many fibers in the myenteric plexus are immunopositive for the 5-HT3R (C, large arrows, right to left). The distribution and pattern of the 5-HT3R tissue staining is similar throughout the GI tract. Preabsorption with the synthetic peptide abolished the 5-HT3R staining demonstrating the specificity of the antibody to the tissue staining in cryostat sections (B) as well as in whole mounts (E). Images are captured at a magnification of ×60; the insert in (C) is enlarged with a magnification zoom of 2.5. LM, longitudinal muscle; CM, circular muscle. Scale bars = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 5-HT3R immunopositive fibers in the duodenal mucosa running the length of the villi (A, arrow, section perpendicular to the lumen) and in the mucosa encircling the intestinal crypts (B, arrow, section tangential to the lumen). Scale bars = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) 5-HT3R– and 5-HT–immunopositive endocrine cell in the duodenum (arrow). In the duodenum, many endocrine cells were immunopositive for 5-HT, but a minority expressed both 5-HT and 5-HT3R immunoreactivities. (B) 5-HT–immunopositive endocrine cell in the duodenum in close anatomical relation to a 5-HT3R–immunopositive fiber. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 5-HT3R immunoreactivity (green), SOM immunoreactivity (red), and their colocalization in myenteric plexus neurons in cryostat sections of the ileum. Some enteric neurons expressed both SOM and 5-HT3R immunoreactivities (right panels); however, the 5-HT3R immunostaining was predominantly located on the plasma membrane (double arrows), whereas the SOM immunoreactivity was only observed in the cytoplasm (single arrow). Some enteric neurons immunopositive for only SOM and not 5-HT3R were also identified throughout the entire intestine (left panels). Scale bar = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 5-HT3R immunoreactivities (green), VAChT (red), SP (red), and VIP (red) immunoreactivities and their colocalization (yellow) in myenteric plexus neurons in cryostat sections of the ileum. Many enteric neurons and fibers were double labeled for VAChT and 5-HT3R immunoreactivities throughout the entire intestine. Some 5-HT3R–immunopositive enteric neurons and fibers also contained either SP or VIP and 5-HT3R immunoreactivities throughout the entire intestine. Scale bar = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Myenteric plexus neurons immunopositive for 5-HT3R and VAChT (upper panels) or immunopositive for 5-HT3R and VIP (lower panels) in whole mounts of the rat ileum. Scale bar = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 ICCs immunoreactive for the 5-HT3R (A and B) and immunoreactive for the c-kit receptor (C) in whole mounts of the rat ileum. ICCs often form a dense network in the level of the myenteric plexus (A). ICCs, immunopositive for the c-kit receptor can be identified in the level of the deep muscle plexus (DMP) and the level of the myenteric plexus (MP) in cryostat sections of the rat ileum (D). The tissue staining for c-kit receptor was abolished by preabsorption with the synthetic peptide, demonstrating the specificity of the tissue staining (E). LM, longitudinal muscle; CM, circular muscle. Scale bars = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 5-HT3R immunoreactivity (A, green), c-kit immunoreactivity (A, red) and their colocalization (C, yellow) on ICC-MP in the rat ileum of whole mounts. Many ICCs were seen expressing both 5-HT3R and c-kit immunoreactivites both in the level of the deep muscle plexus (DMP) and the myenteric plexus (MP). Some ICCs, however, were only immunopositive for the c-kit receptor and did not express 5-HT3R immunoreactivity (not shown). Scale bar = 10 μm. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 5-HT3R–positive fibers in the mucosa of the duodenum of (A) sham-operated rats, (B) vagotomized rats, and (C) perineural capsaicin-treated rats. Vagotomy and functional vagal differentiation by capsaicin markedly reduced the number of 5-HT3R immunoreactive fibers in the duodenum. Gastroenterology 2002 123, 217-226DOI: (10.1053/gast.2002.34245) Copyright © 2002 American Gastroenterological Association Terms and Conditions