Figure 2. Effects of taurine treatment on proliferation of P5 hippocampal stem/progenitor cells. Cells were treated with five different concentrations (10 mM, 100 mM, 500 mM, 2.5 mM and 5 mM) of taurine for 48 h and labeled with EdU (10 mM) in the last 3 h of incubation. Data are presented as percentage of EdU positive cells normalized to control (A). Data are expressed as mean 6 SEM. * P,0.05 and ** P,0.01. Representative microscopic images showing EdU- labeled P5 hippocampal progenitor cells in control (left panel) and taurine treated groups (right panel)) (B). Scale bar = 20 mm. doi:10.1371/journal.pone.0042935.g002 glioblasts is also observed in the visual cortex [15]. Pyramidal cell number is reduced and cells show poor arborization in taurine deficient kitten [19,20]. These studies further demonstrate the importance of taurine in the developing brain. During the perinatal period, progenitors of neurons and glial divide and migrate to reach their final destinations within various brain regions, creating the structures of the brain and setting the stage for brain function or dysfunction later in life. During this period, brain development is sensitive to the supply of essential nutrients. Therefore, our finding that taurine influences stem cell prolifer- ation, could potentially explain that smaller brain weight and abnormal brain morphology of taurine deficient mouse could be due to decreased proliferation of neural progenitor cell in the absence of taurine. Direct delivery of nutritional compounds targeting into embry- onic brain regions with nutritional compounds during prenatal developmental periods allow a more complete understanding of the effect of these compounds on developing brain. In utero microinjection techniques enable the delivery of compounds directly to the embryo brain and examine neurodevelopment during and after embryonic developmental stages. Targeting of specific brain regions with tracers during developmental periods can be combined with immunohistochemical methods and the administration of pharmacological treatments. Although this Taurine Effect on Stem Cells and Synaptogenesis approach has the advantage of providing a better understanding on how taurine may work in vivo at a physiologically relevant environment, this could also be a disadvantage for the interpre- tation of results. The microenvironment and neighboring cells may or may not condition the effects of taurine. Moreover, the delivery of the appropriate concentration of taurine to the target destinations and cells is also a challenge. Nonetheless, this type of study could still potentially lead to a more complete understanding of the cellular and molecular mechanisms that regulate perinatal pharmacology [27]. Therefore, we injected embryonic mouse with taurine to the embryos on day E13, just before the expected peak of proliferation and migration in hippocampus. Taurine injected mouse showed an increased in EdU labeling, suggesting increased proliferation of stem cells. However, the difference between vehicle and taurine treated groups is less than those observed in the in vitro and ex-vivo experiments where taurine is delivered directly to the cells. This could be due to that embryos may already have enough taurine through the mother and further supplement may not cause dramatic changes. It could also be that insufficient amount of taurine from ventricles (site of delivery) may have reached hippocampus. Our study also supports earlier reports that taurine increases or restores cell proliferation of human fetal neurons [21] and embryonic cultured NPCs [28]. While most of the earlier studies emphasized the importance of taurine during embryonic development, the novelty of our studies is that taurine increases the neural progenitor cell proliferation in cultured P5 hippocampal slices, as well as NPCs derived from P5 mice. This highlights the importance of taurine during early postnatal brain development. A significant increase in the number of newborn cells was observed in taurine treated slices indicating increased neural progenitor cell proliferation suggesting that a relatively short exposure, at early life stages, has a potentially lasting impact on neuron production. In the dentate gyrus of mice and rats, neurogenesis mainly takes place in the first two postnatal weeks and then gradually decreases [29]. Using hippocampal slices, we could demonstrate that taurine treatment within a time window of intense granule cell proliferation, during early postnatal development, notably enhanced granule cell formation. The dentate gyrus belongs to the major network of the hippocampal region, the entorhinal–dentate–CA3–CA1–entorhinal system. Signal processing by this network is essential for establishment/ retrieval of long-term memories. Since newborn granule cells are preferentially incorporated into spatial memory networks [30,31], it is expected that the prominent increase in granule cell number induced by early exposure to taurine is accompanied by enhancement of memory functions played by the hippocampal region. Moreover, clinical evidence suggests a specific effect of taurine on cognitive development. Warthon et al. [32], found that low plasma taurine concentrations in preterm and small-for- gestational age neonates are associated with lower scores on the Bayley mental development index at 18 months and the Wechsler Intelligence Scale for Children-revised arithmetic subtest at 7 years. The authors did not specify a mechanism for this selective effect of taurine. However, they noted that taurine’s concentra- tions are differentially distributed across brain tissues, supporting taurine’s selective effect. In this case, the hippocampus is likely, as others have indicated that it is a ‘‘metabolically needy’’ organ because of its high sensitivity to nutrient and energy alterations [33]. MAPK/ERK can be activated by many signals including mitogenic signals, cytokines, and growth factors. The MAPK/ ERK-dependent pathways play a key role in regulating cell proliferation and differentiation [25]. Previous studies have PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42935