Yano et al crobiota at birth(postnatal day(P)O), weaningP21), or early adulthood (P42)and then evaluated at P56 for levels of 5-HT and expression of 5-HT-related genes GF mice conventionalized at each age with an SPF microbiota exhibit restored serum( Figure 1A)and colon(Figure 1B)5-HT levels, with more pronounced effects seen at earlier ages of olonization. Colonic expression of TPHI and SLC6A4 is similarly corrected by postnatal conventionalization of GF mice( Figures IC and lD), with more substantial changes from PO conventionalization. Increases in 5-HT are localized to colonic ECs(Figure 2). These findings indicate that postnatal reconstitution of the gut microbiota can correct the 5-HT deficiency seen in GF mice and further suggest that gut microbes exert a continuous effect on 5-HT synthesis by modulating EC function. Overall, we demonstrate that microbiota- mediated elevation of host 5-HT is postnatally inducible, persistent from the time of conventionalization and not dependent on the timing of host development To assess the reversibility of microbial effects on host 5-HT metabolism, we depleted the 9 gut microbiota in SPF mice via bi-daily antibiotic treatment beginning on PO, P2l or P42 and until P56. Treatment of P42 SPF mice with a cocktail of ampicillin, vancomycin, neomycin and metronidazole(reikvam et al., 2011)sufficiently recapitulates GF-associated deficits in serum and colon 5-HT and alterations in host colonic TPHI and SlC6A4 expression(Figures I and 2). Interestingly, PO and P21 antibiotic treatment also induces GF related deficits in colonic 5-HT, but the effects on serum 5-HT are more pronounced when dministered at P42, compared to PO and P21(Figure 1), suggesting potential confounding effects of early life or prolonged antibiotic treatment on microbiota-mediated modulation of peripheral 5-HT. Antibiotics can elicit several direct effects on host cells( Shimizu et al 2003; Westphal et al., 1994), which may underlie differences between PO treatment and gF status. That P42 antibiotic treatment of SPF mice results in 5-HT phenotypes analogous to those seen in GF mice demonstrates that microbiota effects on host 5-HT can be abrogated postnatally and further supports the plasticity of 5-HT modulation by indigenous gut microbes. Altogether, these data indicate that the gut microbiota plays a key role in raising levels of colon and serum 5-HT, by promoting 5-HT in colonic ECs in an inducible and Indigenous Spore-Forming Microbes Promote Host Serotonin Biosynthesis In light of our finding that 5-HT levels are decreased in colons but not small intestines of GF mice compared to SPF controls, we hypothesized that specific subsets of gut microbes are esponsible for affecting host 5-HT pathways. Mice monocolonized with Bacteroides fragilis or Segmented Filamentous Bacteria(SFB) display deficits in serum 5-HT that are omparable to those seen in GF mice(Figure 3A). Moreover, postnatal colonization(P42) with Bacteroides uniformis, altered Schaedler flora(ASF), an eight-microbe consortium known to correct gross intestinal pathology in GF mice(Dewhirst et al., 1999), or with cultured Bacteroides spp. from the SPF mouse microbiota, has no significant effect on the 5- hT deficiency seen in GF mice(Figures 3A and 3B). Interestingly, GF mice colonized at P42 with indigenous spore-forming microbes from the (Sp), known to be dominated by Clostridial species(Atarashi et al Stefka et al 2014)(Figure S7 and Table $4), exhibit complete restoration of serum and colon 5-HTto levels observed in SPF mice(Figures 3A and 3B). Consistent with this, Sp colonization of Cell. Author manuscript; available in PMC 2016 April 09microbiota at birth (postnatal day (P) 0), weaning (P21), or early adulthood (P42) and then evaluated at P56 for levels of 5-HT and expression of 5-HT-related genes. GF mice conventionalized at each age with an SPF microbiota exhibit restored serum (Figure 1A) and colon (Figure 1B) 5-HT levels, with more pronounced effects seen at earlier ages of colonization. Colonic expression of TPH1 and SLC6A4 is similarly corrected by postnatal conventionalization of GF mice (Figures 1C and 1D), with more substantial changes from P0 conventionalization. Increases in 5-HT are localized to colonic ECs (Figure 2). These findings indicate that postnatal reconstitution of the gut microbiota can correct the 5-HT deficiency seen in GF mice and further suggest that gut microbes exert a continuous effect on 5-HT synthesis by modulating EC function. Overall, we demonstrate that microbiota￾mediated elevation of host 5-HT is postnatally inducible, persistent from the time of conventionalization and not dependent on the timing of host development. To assess the reversibility of microbial effects on host 5-HT metabolism, we depleted the gut microbiota in SPF mice via bi-daily antibiotic treatment beginning on P0, P21 or P42 and until P56. Treatment of P42 SPF mice with a cocktail of ampicillin, vancomycin, neomycin and metronidazole (Reikvam et al., 2011) sufficiently recapitulates GF-associated deficits in serum and colon 5-HT and alterations in host colonic TPH1 and SLC6A4 expression (Figures 1 and 2). Interestingly, P0 and P21 antibiotic treatment also induces GF￾related deficits in colonic 5-HT, but the effects on serum 5-HT are more pronounced when administered at P42, compared to P0 and P21 (Figure 1), suggesting potential confounding effects of early life or prolonged antibiotic treatment on microbiota-mediated modulation of peripheral 5-HT. Antibiotics can elicit several direct effects on host cells (Shimizu et al., 2003; Westphal et al., 1994), which may underlie differences between P0 treatment and GF status. That P42 antibiotic treatment of SPF mice results in 5-HT phenotypes analogous to those seen in GF mice demonstrates that microbiota effects on host 5-HT can be abrogated postnatally and further supports the plasticity of 5-HT modulation by indigenous gut microbes. Altogether, these data indicate that the gut microbiota plays a key role in raising levels of colon and serum 5-HT, by promoting 5-HT in colonic ECs in an inducible and reversible manner. Indigenous Spore-Forming Microbes Promote Host Serotonin Biosynthesis In light of our finding that 5-HT levels are decreased in colons but not small intestines of GF mice compared to SPF controls, we hypothesized that specific subsets of gut microbes are responsible for affecting host 5-HT pathways. Mice monocolonized with Bacteroides fragilis or Segmented Filamentous Bacteria (SFB) display deficits in serum 5-HT that are comparable to those seen in GF mice (Figure 3A). Moreover, postnatal colonization (P42) with Bacteroides uniformis, altered Schaedler flora (ASF), an eight-microbe consortium known to correct gross intestinal pathology in GF mice (Dewhirst et al., 1999), or with cultured Bacteroides spp. from the SPF mouse microbiota, has no significant effect on the 5- HT deficiency seen in GF mice (Figures 3A and 3B). Interestingly, however, GF mice colonized at P42 with indigenous spore-forming microbes from the mouse SPF microbiota (Sp), known to be dominated by Clostridial species (Atarashi et al., 2013; Stefka et al., 2014) (Figure S7 and Table S4), exhibit complete restoration of serum and colon 5-HT to levels observed in SPF mice (Figures 3A and 3B). Consistent with this, Sp colonization of Yano et al. Page 4 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript