Yano et al Page 1 I ontribute to its effects on Gl motility. Notably, deconjugated bile salts are reported to alter gut sensorimotor activity(Appleby and Walters, 2014), which supports our hypothesis that Sp-induced increases in deoxycholate, among other metabolites, contribute to its ability to elevate colonic 5-HT and decrease intestinal transit time While we demonstrate that Sp-mediated induction of colonic and blood 5-HT regulates Gl motility and platelet function in mice, further research is needed to explore additional mplications of microbially induced 5-HT on host health and disease(OMahony et al 2014). Peripheral 5-HT modulates several cellular processes, including osteoblast differentiation, erythropoiesis and immunity. Moreover, gross abnormalities in brain structure are observed in Tph1+/-embryos from Tph1-/-mothers( Cote et al., 2007 indicating that maternal peripheral 5-HT is important for offspring neurodevelopment. Placentally-derived 5-HT also influences neurodevelopment, influencing thalamocortical axon guidance(Bonnin et al., 2011). Interestingly, the indigenous microbiota also modulates 9 hippocampal levels of 5-HT(Clarke et al., 2013), revealing a role for the microbiota egulating the brain serotonergic system. Overall, our findings provide a mechanism by which select microbes and their metabolic products can be used to promote endogenous, localized 5-HT biosynthesis and further alter host physiol EXPERIMENTAL PROCEDURES See Supplemental information for additional details and references PCPA Treatment At 2 weeks post-bacterial treatment, mice were anesthetized with isoflurane, and PCPA (90 mg/kg)(Liu et al., 2008)was administered intrarectally every 12 hours for 3 days using a sterile 3.5 Fr silicone catheter inserted 4 cm into the rectum. Mice were suspended by tail for 30 s before return to the home cage For mock treatment, mice were anesthetized and intrarectally injected with sterile water as vehicle Serotonin Measurements Serotonin levels were detected in sera and supernatant of tissue homogenates by ELISA according to the manufacturer's instructions(Eagle Biosciences ). Readings from tissue samples were normalized to total protein content as detected by bCa assay (Thermo Pierce). Data compiled across multiple experiments are expressed as 5-HT concentrations normalized to SPF controls within each experiment RIN14B In Vitro Culture Experiments RIN14B cells(ATCC)were seeded at 105 cells/cm2 and cultured according to methods described in(Nozawa et al., 2009). Total colonic luminal contents were collected from adult SPF, GF and gF mice colonized with spore-forming bacteria, suspended at 120 ul mg in HBSS supplemented with 0. 1% BSA and 2 uM fluoxetine, and centrifuged at 12,000 xg for 10 min Supernatants were passed through 0. 2 um pore syringe filters. Cultured RIN14B cells were incubated with colonic luminal filtrate for 1 hr at 37oC Cell. Author manuscript; available in PMC 2016 April 09contribute to its effects on GI motility. Notably, deconjugated bile salts are reported to alter gut sensorimotor activity (Appleby and Walters, 2014), which supports our hypothesis that Sp-induced increases in deoxycholate, among other metabolites, contribute to its ability to elevate colonic 5-HT and decrease intestinal transit time. While we demonstrate that Sp-mediated induction of colonic and blood 5-HT regulates GI motility and platelet function in mice, further research is needed to explore additional implications of microbially induced 5-HT on host health and disease (O'Mahony et al., 2014). Peripheral 5-HT modulates several cellular processes, including osteoblast differentiation, erythropoiesis and immunity. Moreover, gross abnormalities in brain structure are observed in Tph1+/− embryos from Tph1−/− mothers (Cote et al., 2007), indicating that maternal peripheral 5-HT is important for offspring neurodevelopment. Placentally-derived 5-HT also influences neurodevelopment, influencing thalamocortical axon guidance (Bonnin et al., 2011). Interestingly, the indigenous microbiota also modulates hippocampal levels of 5-HT (Clarke et al., 2013), revealing a role for the microbiota in regulating the brain serotonergic system. Overall, our findings provide a mechanism by which select microbes and their metabolic products can be used to promote endogenous, localized 5-HT biosynthesis and further alter host physiology. EXPERIMENTAL PROCEDURES See Supplemental information for additional details and references PCPA Treatment At 2 weeks post-bacterial treatment, mice were anesthetized with isoflurane, and PCPA (90 mg/kg) (Liu et al., 2008) was administered intrarectally every 12 hours for 3 days using a sterile 3.5 Fr silicone catheter inserted 4 cm into the rectum. Mice were suspended by tail for 30 s before return to the home cage. For mock treatment, mice were anesthetized and intrarectally injected with sterile water as vehicle. Serotonin Measurements Serotonin levels were detected in sera and supernatant of tissue homogenates by ELISA according to the manufacturer’s instructions (Eagle Biosciences). Readings from tissue samples were normalized to total protein content as detected by BCA assay (Thermo Pierce). Data compiled across multiple experiments are expressed as 5-HT concentrations normalized to SPF controls within each experiment. RIN14B In Vitro Culture Experiments RIN14B cells (ATCC) were seeded at 105 cells/cm2 and cultured according to methods described in (Nozawa et al., 2009). Total colonic luminal contents were collected from adult SPF, GF and GF mice colonized with spore-forming bacteria, suspended at 120 ul/mg in HBSS supplemented with 0.1% BSA and 2 uM fluoxetine, and centrifuged at 12,000 ×g for 10 min. Supernatants were passed through 0.2 um pore syringe filters. Cultured RIN14B cells were incubated with colonic luminal filtrate for 1 hr at 37°C. Yano et al. Page 11 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript