visual cortex. Pyramidal cell number is reduced and neurons show poor arborization in taurine deficient kitten [19,20]. These studies further demonstrate the importance of taurine in the developing brain. Taurine is also reported to increase or restore cell proliferation of human fetal neurons [21] and has been postulated to influence neurotransmission [22]. Altogether, these findings point to taurine being essential for optimal proliferation, develop- ment and maturation of brain cells. The main objective of the present work is to determine the potential effect of taurine on neural stem/progenitor cell proliferation and neurogenesis in the developing brain, using cultured neural progenitor cells, and cultured hippocampal slices representing in vitro/ex-vivo models of early post natal neurogenesis and also in embryonic hippocampus of taurine supplemented embryos. In addition, we investigated the mechanism of the taurine effect on neural stem/progenitor cell proliferation. We also studied the effect of taurine on neurite growth and synaptogenesis in cultured embryonic hippocampal primary neurons. Results Taurine Enhances Proliferation of Neural Stem/ progenitor Cells (NPC) The capacity for self-renewal and continuous proliferation is one of the key characteristics of stem/progenitor cells in vitro [23]. To evaluate the effect of taurine on NPCs, we isolated NPCs from hippocampus of 5 days old mice. NPCs were characterized based on their ability of self-renewal, expression of NPC markers as well as their ability to differentiate to neuronal and glial populations. All the cells express the standard NPC markers such as Nestin, Sox2, BLBP and Vimentin. They also express the proliferation marker Ki67 and their expression is constant regardless of the passage number (Fig. 1A). In order to determine the effects of taurine on NPC proliferation, we treated the cells with different concentrations of taurine (10 mM, 100 mM, 500 mM, 2.5 mM and 5 mM) and labeled the dividing cells with 5-ethyl-29-deoxyuridine (EdU). All cells on the coverslips are positive for DAPI and Nestin, and the dividing cells are labeled with EdU (Fig. 1B). The number of EdU positive cells in 100 mM taurine -treated group increased significantly by 28% (P,0.05) as compared to the control. A 20% increase was also observed when treated with 500 mM or 2.5 mM taurine. (Fig. 2A–B, P,0.05), indicating that taurine at appropriate concentrations could stimulate the proliferation of P5 NPCs. However, taurine at a lower concentration of 10 mM did not induce any significant effect on NPC proliferation and at higher concentration (5 mM), the number of NPCs decreased (P,0.01) as compared to control. EdU labeling was also used to determine the proliferation of cells in the dentate gyrus of cultured hippocampal slices. The total number of EdU positive cells in taurine (400 mM) treated group was significantly more (by 38%) as compared to the control group (Fig. 3A–B, P,0.05), indicating that taurine stimulates the proliferation of neural stem cells in the dentate gyrus of the developing brain. Targeting of nutritional compounds to the embryonic brains during the prenatal developmental periods could lead to a more complete understanding of the effect of these compounds on the developing brain in vivo. Therefore, we evaluated the effects of taurine on stem/neural progenitor cell proliferation in the embryonic hippocampus in vivo via in utero microinjection. In these experiments, taurine was injected directly into ventricles of the embryonic stage 13 (E13) fetal mice brains. The embryonic stage E13 was selected because it is just before the expected peak of cell division and migration in the hippocampus. Taurine Effect on Stem Cells and Synaptogenesis Dividing cells were labeled with EdU, and these EdU positive cells in the developing hippocampus were analyzed by quantifying the fluorescence intensity of EdU in hippocampal slices from pups that were sacrificed and fixed at E17. We found that taurine stimulated a 31% increase (P.0.05) in the EdU intensity as compared to control (Fig. 4A, B). The Effect of Taurine on the Differentiation of Cultured P5 Stem Cells/Neural Progenitor Cells to Specific Lineages Next, we determined if taurine affects the differentiation of hippocampal stem/progenitor cells in vitro. NPCs were induced to differentiate in the differentiation media for 5 days that contains a lower concentration of FGF-2 and no EGF as published previously [24]. The low concentration of FGF-2 is essential for survival of our P5 NPCs and does not impair the differentiation efficacy. After 5 days of differentiation, the number of Ki67 and Nestin positive cells decrease dramatically from 100% to less than 8% (data not shown) and these P5 NPCs can be differentiated into doublecortin- (DCX) expressing neurons and glial fibrillary acid protein- (GFAP) expressing astrocytes (Figure 5A). Taurine treatment showed no significant effect on the percentages of cells that were positive for the neuronal marker, DCX or the glial marker, GFAP (Fig. 5B). To determine if taurine treatment can affect the fate of newborn cells in organotypic brain slices, EdU-labeled cells were analyzed 6 days after EdU was added to hippocampal slices treated with taurine or vehicle. This length of time is known to be sufficient for newly proliferated EdU-labeled cells to differentiate into their different lineages. The differentiation of EdU-labeled cells was identified through immunohistochemistry using specific antibodies against the neuronal marker, DCX, or the glial marker, GFAP (Fig. 5C–E). Analysis of co-localization of EdU with DCX or GFAP indicated that neither the percentages of DCX nor GFAP expressing cells were significantly altered by taurine treatment. These ex vivo findings are consistent with our results from the earlier in vitro studies showing no significant effects of taurine on the differentiation of P5 NPCs (Fig. 5B). Taurine-induced Proliferation is Dependent on the Phosphorylation of p44/42 MAPK The p44/42 MAPK (ERK1/2) growth signaling pathway has been shown to be involved in many different cellular processes and is essential for proliferation of NPCs [25]. To determine if taurine activates this ERK1/2 signaling pathways in P5 hippocampal stem/progenitor cells, phosphorylation of ERK1/2 was evaluated by western blot analysis following taurine treatment. Taurine treatment dramatically elevated the level of phosphorylated ERK1/2 (P,0.01, Fig. 6A and B) and a modest increase in the total ERK1/2 (P,0.05, Fig. 6C and D). Pretreatment with ERK1/2 inhibitor, PD98059 inhibited the taurine-stimulated increase in phosphorylation of ERK1/2. We normalized the levels of phosphorylated ERK1/2 by levels of total ERK1/2 proteins (Fig 6 E) and our results showed that pretreatment with PD98059 specifically inhibited taurine-stimulated increase in phosphoryla- tion of ERK1/2. These results suggest that ERK1/2 signaling pathways are activated by taurine in hippocampal progenitor cells. To further determine whether activation of the 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 pretreatment with the ERK1/2 inhibitor PD98059 diminished the taurine-induced effect on proliferation (Fig. 7A–B). These results further suggest that activation of PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42935