Volving in vivo imaging of enteric neurons with 2PM, although in vivo imaging of enteric neurons with confocal laser endomicroscopy has been recently performed [9]. We detected the formation of newly generated neurons in the thick granulation tissue at the site of anastomosis. Imaging with 2 PM allowed enteric neural imaging several hundred microns deep within the gut of living mouse. In contrast to the brain tissue [7], the structure of the gut tissue is complex, consisting of multiple layers and tissue types, including mucosa, submucosa, circular and longitudinal muscles, blood vessels and crypt glands. Therefore, to enhance visualization of enteric neurons we used Thy1-GFP mice [8] after confirmation of expression of cytoplasmic GFP in enteric neurons in preliminary studies. In the present study, newly formed enteric neurons also expressed cytoplasmic GFP. In future studies, we are planning functional studies of enteric neurons using in vivo imaging with 2PM and genetically encoding calcium indicators [12]. A critical obstacle to overcome in order to obtain clear images of enteric neurons in vivo was to suppress motion disturbance associated with gut motility. Otherwise, Mirin custom synthesis observed images would be blurry and non-interpretable. We found that pinning and intraluminal injection of papaverine eliminated tissue movement and allowed for the acquisition of sharp images.One week after surgical anastomosis, MOS facilitated formation of newly generated enteric neurons in the granulation tissue at the anastomosis. However, even 4 weeks after surgery, only a small number of newborn neurons were identified in the granulation tissue of vehicle-treated control animals. The effects of MOS on neurogenesis were completely antagonized by treatment with a 5HT4 receptor antagonist, indicating that MOS facilitated formation of newborn enteric neurons via 5-HT4-receptor activation. Although the number of newly formed enteric neurons was significantly higher in the MOS-treated mice as compared to antagonist treated and vehicle controls, the distribution pattern of newly formed enteric neurons was similar, i.e., neurons were 69-25-0 chemical information distributed close to the edge of the granulation tissue. This suggested the possibility that neural stem cells were mobilized from the outside of the granulation tissue. Enteric nervous system (ENS) development is relevant to Hirschsprung’s disease (HSCR; congenital aganglionosis of the terminal bowel) and related diseases, which are still imperfectly treated. It is well known that mutations in genes encoding the Ret receptor tyrosine kinase and endothelin receptor type B are 1662274 involved in HSCR pathogenesis [13,14]. We found MOS increased mRNA of c-Ret receptor tyrosine kinase in a rat model and that a 5-HT4 receptor antagonist completely blocked this effect [15]. Therefore, it seems likely that the target molecule of MOS is the Ret receptor tyrosine kinase. Enteric neurogenesis must be strictly controlled, because hyperplasia of enteric neurons due to hypersensitivity for glial cell-derived neurotrophic factor (GDNF)-Ret signaling reversely results in HSCR [16]. Nevertheless, treatment with 5-HT4 receptor agonists such as MOS could be a promising tool to treat HSCR and related disorders. In conclusion, in vivo imaging by 2PM allowed for highresolution deep imaging of the intestines in vivo. Thick granulation tissue at the site of anastomosis, including newly formed ganglionlike structures and nerve fibers, could be studied in the intact mur.Volving in vivo imaging of enteric neurons with 2PM, although in vivo imaging of enteric neurons with confocal laser endomicroscopy has been recently performed [9]. We detected the formation of newly generated neurons in the thick granulation tissue at the site of anastomosis. Imaging with 2 PM allowed enteric neural imaging several hundred microns deep within the gut of living mouse. In contrast to the brain tissue [7], the structure of the gut tissue is complex, consisting of multiple layers and tissue types, including mucosa, submucosa, circular and longitudinal muscles, blood vessels and crypt glands. Therefore, to enhance visualization of enteric neurons we used Thy1-GFP mice [8] after confirmation of expression of cytoplasmic GFP in enteric neurons in preliminary studies. In the present study, newly formed enteric neurons also expressed cytoplasmic GFP. In future studies, we are planning functional studies of enteric neurons using in vivo imaging with 2PM and genetically encoding calcium indicators [12]. A critical obstacle to overcome in order to obtain clear images of enteric neurons in vivo was to suppress motion disturbance associated with gut motility. Otherwise, observed images would be blurry and non-interpretable. We found that pinning and intraluminal injection of papaverine eliminated tissue movement and allowed for the acquisition of sharp images.One week after surgical anastomosis, MOS facilitated formation of newly generated enteric neurons in the granulation tissue at the anastomosis. However, even 4 weeks after surgery, only a small number of newborn neurons were identified in the granulation tissue of vehicle-treated control animals. The effects of MOS on neurogenesis were completely antagonized by treatment with a 5HT4 receptor antagonist, indicating that MOS facilitated formation of newborn enteric neurons via 5-HT4-receptor activation. Although the number of newly formed enteric neurons was significantly higher in the MOS-treated mice as compared to antagonist treated and vehicle controls, the distribution pattern of newly formed enteric neurons was similar, i.e., neurons were distributed close to the edge of the granulation tissue. This suggested the possibility that neural stem cells were mobilized from the outside of the granulation tissue. Enteric nervous system (ENS) development is relevant to Hirschsprung’s disease (HSCR; congenital aganglionosis of the terminal bowel) and related diseases, which are still imperfectly treated. It is well known that mutations in genes encoding the Ret receptor tyrosine kinase and endothelin receptor type B are 1662274 involved in HSCR pathogenesis [13,14]. We found MOS increased mRNA of c-Ret receptor tyrosine kinase in a rat model and that a 5-HT4 receptor antagonist completely blocked this effect [15]. Therefore, it seems likely that the target molecule of MOS is the Ret receptor tyrosine kinase. Enteric neurogenesis must be strictly controlled, because hyperplasia of enteric neurons due to hypersensitivity for glial cell-derived neurotrophic factor (GDNF)-Ret signaling reversely results in HSCR [16]. Nevertheless, treatment with 5-HT4 receptor agonists such as MOS could be a promising tool to treat HSCR and related disorders. In conclusion, in vivo imaging by 2PM allowed for highresolution deep imaging of the intestines in vivo. Thick granulation tissue at the site of anastomosis, including newly formed ganglionlike structures and nerve fibers, could be studied in the intact mur.