Yano et al Page 6 then tested for GI transit and colonic neuronal activation at P56. Sp colonization ameliorates GF-associated abnormalities in GI motility, significantly decreasing total transit time and increasing the rate of fecal output in a Tph-dependent manner(Figures 4A and 4B). Similar ffects are seen in SLC6A4+/-and--mice, where Sp colonization of antibiotic-treated mice restores Gi transit time toward levels seen in SPF SLC644 +/and -/-controls Consistent with deficits in GI motility, steady-state activation of 5-HT receptor subtype 4 (S5HT4-expressing neurons in the colonic submucosa and muscularis externa is decreased GF mice compared to SPF controls, as measured by colocalized expression of 5HT4 with the immediate early gene, c-fos(Figure 4C, 4D and 4E). Colonization of GF mice with Sp increases 5HT4+ c-fost staining to levels seen in SPF mice, and this effect is dependent on colonic Tph activity(Figures 4C, 4D and 4E), which aligns well with the understanding that Sp-induced elevations in colonic 5-HT promote Gl motility by activation of 5HT4+ enteric 9 neurons(Mawe and Hoffman, 2013). In addition, colonic activation of intrinsic afferent primary neurons(IPANs)of the myenteric plexus is decreased in GF mice(Mc Vey Neufeld et al., 2013)and improved by colonization with Sp, as measured by colocalization of c-fos and the IPAN marker, calretinin( Calb2)(Figure 4F). Inhibiting Tph activity with PCPA decreases IPAN activation in Sp-colonized mice, suggesting that some IPAN responses to Sp depend on host 5-HT synthesis(Figures 4F). Altogether, these findings indicate that S mediated increases in colonic 5-HT biosynthesis are important for gut sensorimotor function Microbiota-Mediated Regulation of Host Serotonin Modulates Platelet Function Platelets uptake gut-derived 5-HT and release it at sites of vessel injury to promote blood coagulation. To determine if microbiota-dependent modulation of colon(Figures I and 3) and plasma(Figure SlA)5-HT impacts platelet function, we colonized p42 mice with Sp and then examined blood clotting, platelet activation and platelet aggregation at P56. In a tail bleed assay (Liu et al., 2012), GF mice exhibit trending increases in time to cessation of bleeding compared to SPF mice, suggesting impaired blood coagulation( Figure 5A) Colonization of GF mice with Sp ameliorates abnormalities in bleeding time to levels seen in SPF controls, and this effect is attenuated by intrarectal administration of PCPA(Figure 5A), indicating that Sp-mediated improvements in coagulation may be dependent on colonic Tph activity. Notably, the impact of acute colonic PCPA treatment on reducing 5-HT content and 5-HT-related functions in platelets may be tempered by the fact that mouse platelets have a lifespan of-4 days(Odell and Mc, 1961). There were no significant differences between treatment groups in total platelet counts( Figure S5A) In light of inherent limitations of the tail bleed assay (Liu et al., 2012), we focused subsequent experiments particularly on platelet activity. Platelets isolated from GF mice display decreased activation in response to in vitro type I fibrillar collagen stimulation, as measured by reduced surface expression of the activation markers granulophysin( CD63), P- (Ziu et al., 2012). Sp colonization of GF mice leads to partial restoration in the expression of platelet activation markers, and this effect depends on colonic Tph activity(Figures 5D, 5E and 5F).Moreover, Cell. Author manuscript; available in PMC 2016 April 09then tested for GI transit and colonic neuronal activation at P56. Sp colonization ameliorates GF-associated abnormalities in GI motility, significantly decreasing total transit time and increasing the rate of fecal output in a Tph-dependent manner (Figures 4A and 4B). Similar effects are seen in SLC6A4 +/− and −/− mice, where Sp colonization of antibiotic-treated mice restores GI transit time toward levels seen in SPF SLC6A4 +/− and −/− controls (Figure S4E). Consistent with deficits in GI motility, steady-state activation of 5-HT receptor subtype 4 (5HT4)-expressing neurons in the colonic submucosa and muscularis externa is decreased in GF mice compared to SPF controls, as measured by colocalized expression of 5HT4 with the immediate early gene, c-fos (Figure 4C, 4D and 4E). Colonization of GF mice with Sp increases 5HT4+ c-fos+ staining to levels seen in SPF mice, and this effect is dependent on colonic Tph activity (Figures 4C, 4D and 4E), which aligns well with the understanding that Sp-induced elevations in colonic 5-HT promote GI motility by activation of 5HT4+ enteric neurons (Mawe and Hoffman, 2013). In addition, colonic activation of intrinsic afferent primary neurons (IPANs) of the myenteric plexus is decreased in GF mice (McVey Neufeld et al., 2013) and improved by colonization with Sp, as measured by colocalization of c-fos and the IPAN marker, calretinin (Calb2) (Figure 4F). Inhibiting Tph activity with PCPA decreases IPAN activation in Sp-colonized mice, suggesting that some IPAN responses to Sp depend on host 5-HT synthesis (Figures 4F). Altogether, these findings indicate that Sp￾mediated increases in colonic 5-HT biosynthesis are important for gut sensorimotor function. Microbiota-Mediated Regulation of Host Serotonin Modulates Platelet Function Platelets uptake gut-derived 5-HT and release it at sites of vessel injury to promote blood coagulation. To determine if microbiota-dependent modulation of colon (Figures 1 and 3) and plasma (Figure S1A) 5-HT impacts platelet function, we colonized P42 mice with Sp and then examined blood clotting, platelet activation and platelet aggregation at P56. In a tail bleed assay (Liu et al., 2012), GF mice exhibit trending increases in time to cessation of bleeding compared to SPF mice, suggesting impaired blood coagulation (Figure 5A). Colonization of GF mice with Sp ameliorates abnormalities in bleeding time to levels seen in SPF controls, and this effect is attenuated by intrarectal administration of PCPA (Figure 5A), indicating that Sp-mediated improvements in coagulation may be dependent on colonic Tph activity. Notably, the impact of acute colonic PCPA treatment on reducing 5-HT content and 5-HT-related functions in platelets may be tempered by the fact that mouse platelets have a lifespan of ~4 days (Odell and Mc, 1961). There were no significant differences between treatment groups in total platelet counts (Figure S5A). In light of inherent limitations of the tail bleed assay (Liu et al., 2012), we focused subsequent experiments particularly on platelet activity. Platelets isolated from GF mice display decreased activation in response to in vitro type I fibrillar collagen stimulation, as measured by reduced surface expression of the activation markers granulophysin (CD63), P￾selectin and JON/A (integrin αIIbβ3) (Figures 5D, 5E and 5F) (Ziu et al., 2012). Sp colonization of GF mice leads to partial restoration in the expression of platelet activation markers, and this effect depends on colonic Tph activity (Figures 5D, 5E and 5F). Moreover, Yano et al. Page 6 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript