Taurine Effect on Stem Cells and Synaptogenesis Figure 5. Effects of taurine treatment on cell differentiation. EdU labeling and differentiated cell markers (DCX labels neurons and GFAP labels astrocytes) were used to assess the differentiation and fate choice of newly generated cells in NPCs and in DG of cultured hippocampal slices. Representative images of cultured P5 hippocampal stem/progenitor cells treated with taurine (100 mM) and allowed to differentiate for 5 days before fixation for immunohistochemical processing (A). Quantitative analysis of cells positive for EdU and DCX or GFAP indicating that the percentage of DCX or GFAP was not altered by taurine treatment (B). Representative images showing cells positive for EdU and DCX or GFAP in DG of hippocampal slices (C and D). Quantitative analysis of cells positive for EdU and DCX or GFAP in the dentate gyrus indicating that the percentage of DCX or GFAP was not altered by taurine treatment (E). All quantitative data are expressed as mean 6 SEM. Scale bar = 20 mm. doi:10.1371/journal.pone.0042935.g005 factor for neurogenesis and neuronal development. Whittle et al. [44] recently reported that fetal Down syndrome brains showed reductions in the levels of serotonin, taurine, and dopamine in the frontal cortex. Juveniles passerine birds fed taurine-rich diets as neonates matures into much larger risk takers and more adept at spatial learning tasks This also explains the importance of taurine in brain development. Taurine’s effects on the neurons suggest their functions in enhancing synaptic plasticity and enhancing learning and memory. It is well known that hippocampal neurogenesis and neuritogenesis is accompanied with a better spatial learning performance. Thus, this study provides evidence that increased proliferation and neuritogenesis in the hippocampus may be a part of a basis for the beneficial effect of taurine on behavioral performance. Materials and Methods Neural Progenitor Cultures NPC isolation was performed according to established protocols [45,46]. Briefly, 5 days old C57/BL6 mice were sacrificed and then the hippocampus, as a whole, was gently separated from the corpus collosum. The tissue samples were diced with a scalpel blade in Hanks’ Balanced Salt Solution (HBSS) (Invitrogen) and centrifuged at 2000 rpm for 5 min. After removing the superna- tant, tissue samples were enzymatically digested with Papain (Worthington) containing DNase I 250 U/ml and Dispase II 1 U/ ml at 37uC for 30 min. Tissues were then dissociated into single cells and centrifuged to obtain cell pellet. The pellet was resuspended into Dulbecco’s Modified Eagle Medium (DMEM)/ nutrient mixture F-12 (Invitrogen), supplemented by 1% N2 supplement (Invitrogen), 1% penicillin/streptomycin and 1 mM l- glutamine. Finally, cells were washed and resuspended in culture media supplemented with FGF-2 (R&D Systems) and EGF (Merck), both at 20 ng/ml in 5 mg/ml heparin. Primary hippocampal cells were incubated for 7 days to allow neurosphere formation. The neurospheres were then dissociated using accutase and NPCs were cultured as monolayer on laminin (10 mg/ml) coated plates. The NPCs are characterized by staining for proliferation and stem cell markers (Figure 1). Cells are harvested and seeded at a density of 20,000/cm2 per well on 12 mm coverslips precoated with laminin. The cells were treated with taurine for 48 hrs. To measure whether taurine supplementation promotes NPC proliferation, 10 uM of thymidine analogue, 5 ethynyl-29-deoxyuridine (EdU) (Invitrogen) was added after 45 hrs for 3 hrs and fixed with paraformaldehyde (PFA). The cells were stained for EdU using EdU staining kit according to manufactur- er’s instructions (Invitrogen). EdU positive cells were then counted using cell profiler software. Figure 1B shows that all DAPI positive cells are nestin positive and the proliferating cells are labeled with EdU. Data were analyzed by one-way ANOVA with Dunnet’s post hoc test. P,0.05 is considered as significant. To determine whether ERK1/2 signaling pathway mediates the action of taurine on cell proliferation, P5 hippocampal stem/ progenitor cells were exposed to the ERK1/2 inhibitor PD98059 (10 mM) followed by taurine treatment. The data were analysed by one-way ANOVA followed by Dunnett’s Post-hoc test. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42935