Volume 131, Issue 1, Pages 97-107 (July 2006) Ligand-Induced 5-HT3 Receptor Internalization in Enteric Neurons in Rat Ileum  Samara L. Freeman, Jorg Glatzle, Carla S. Robin, Melissa Valdellon, Catia Sternini, James W. Sharp, Helen E. Raybould  Gastroenterology  Volume 131, Issue 1, Pages 97-107 (July 2006) DOI: 10.1053/j.gastro.2006.04.013 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Photomicrographs showing immunolocalization of 5-HT3R immunoreactivity in myenteric neurons of the rat ileum from (A) control, (B) 5-HT–treated rats, (C) or rats in which the intestine was perfused with vehicle or with (D) 990 mmol/L glucose. Confocal images (.5-μm sections; magnification, 150×) of myenteric neurons were analyzed by Scion Image software for subcellular distribution of the 5-HT3R. 5-HT3R immunoreactive pixels on the membrane and in the cytoplasm were determined by an observer unaware of the treatment. Scale bar represents 10 μm. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 5-HT3R immunoreactivity is decreased in the membrane of ileal myenteric neurons by acute administration of exogenous 5-HT or by acute release of endogenous 5-HT. 5-HT was increased by (A) injection of exogenous 5-HT (100 μg intraperitoneally) or by (B) release from endogenous stores after infusion of hypertonic glucose (180 mg) into the duodenum. Both procedures resulted in a significant decrease of 5-HT3R immunoreactivity at the membrane of myenteric neurons. (A) □, Vehicle; ■, 5HT. (B) □, Vehicle; ■, glucose. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 5-HT3 receptor immunoreactivity in the cytoplasm of myenteric neurons colocalizes with clathrin, an early endosome marker. Confocal images (.5-μm sections; magnification, 150×) of myenteric plexus neurons were acquired. (A) Image showing 5-HT3 receptor immunoreactivity visualized using donkey-anti-rabbit IgG conjugated with AlexaFluor 488 (green). (B) Clathrin immunoreactivity visualized using a donkey-anti-mouse IgG conjugated with Rhodamine (red). (C) Merged images from A and B. Colocalization of 5-HT3R immunoreactivity and clathrin is indicated by yellow areas on the image. (D, E) Enlarged sections (blue box) of the merged image showing yellow vesicles in the cytoplasm of myenteric neurons that represents colocalization of 5-HT3 receptor immunoreactivity and immunoreactivity for clathrin. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 5-HT3R immunoreactivity is decreased in the membrane of ileal myenteric neurons in an in vitro preparation of the ileum. (A) Administration of the 5-HT3R agonist, 2-Me-5-HT, resulted in internalization of the receptor after 2 minutes, which returned to control levels by 60 minutes. *P < .01, saline vs 2-Me-5-HT n = 63 and 48 neurons, respectively. , Control at 60 minutes; ▧, 2-Me-5-HT; ■, control at 120 minutes; ▤, 2-Me-5-HT + 60 minutes. (B) 5-HT3R immunoreactivity is decreased in the membrane of ileal myenteric neurons, which is blocked by the presence of the specific receptor antagonist, ondansetron. *P < .001; 2-Me-5-HT vs ondansetron + 2-Me-5-HT, n = 64 and 18 neurons, respectively). In addition, ondansetron alone increased the amount of immunoreactivity at the membrane of myenteric neurons (#P < .05, saline vs ondansetron, n = 50 and 18 neurons, respectively). , Control; ▧, 2-Me-5-HT; ■, ondansetron; ▤, ondansetron + 2-Me-5-HT. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Reproduction of Western blot showing immunoreactive product with a molecular weight of 60 kilodaltons. c, control; f, fluoxetine treatment, 6 days 20 mg/kg orally. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Intestinal secretion in a segment of ileum, measured in anesthetized rats, is dose-dependently increased by exogenous administration of the 5-HT3R agonist, 2-Methyl 5-HT. *P < .05 vehicle vs 2-Methyl 5-HT; **P < .01 vehicle vs 2-Methyl 5-HT. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Intestinal secretion in a segment of ileum in response to exogenous administration of 5-HT (A) or in response to luminal glucose (B) is attenuated in rats chronically treated with fluoxetine. N = 6–8 in each group; P < .01 vehicle vs 2-Me-5-HT or glucose. (A) □, Vehicle; ■, 2-Me-5-HTt. (B) □, Vehicle; ■, glucose. Gastroenterology 2006 131, 97-107DOI: (10.1053/j.gastro.2006.04.013) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions