Taurine Effect on Stem Cells and Synaptogenesis Figure 7. Taurine-induced proliferation of P5 hippocampal stem/progenitor cells is dependent on the activation of ERK1/2. Cultured hippocampal stem/progenitor cells were incubated with ERK1/2 inhibitor PD98059 for 30 min followed by treatment with taurine for 48 h and labeling with EdU (10 mM) in the last 3 h of incubation. Quantitative data presented as percentage of EdU positive cells normalized to control, indicates pretreatment with ERK1/2 inhibitor attenuates the taurine induced increase in the number of EdU-labeled cells (A). Representative images showing EdU -labeled P5 hippocampal progenitor cells in control (left panel), taurine treated groups (centre), and ERK1/2 inhibitor + taurine (right panel) (B). Scale bar = 20 mm. All data presented here are expressed as mean 6 SEM. * P,0.05. doi:10.1371/journal.pone.0042935.g007 a 95% air/5% CO2 atmosphere. At 2 days in vitro (DIV), hippocampal cultures were changed to neurobasal media (GIBCO) and the compound was added to the media. The cultures were fixed for immunocytochemistry at 5 DIV for neurite outgrowth analysis, day 9 DIV for staining with anti-synapsin 1 and 15 DIV for staining with anti-PSD95. Data on neurite outgrowth were analyzed by one-way ANOVA with Dunnet’s post hoc test and a t-test was used to analyse data on synaptic puncta. P,0.05 is considered as significant. For biochemical analysis, neurons were treated with the anti- mitotic drug cytosine-b-D-arabinofuranoside (Ara C) to eliminate dividing astrocytes and used at 9–15 days after plating. The culture plates were washed with cool PBS and then added 200 ml lysis buffer over ice. Samples were vortexed and centrifuged at 13,000 rpm for 10 min, the supernatant was collected and stored in 280uC. Data on western blot were analyzed by t test. P,0.05 is considered as significant. Preparation of Organotypic Brain Slices Hippocampal slices were prepared from postnatal day 5 (P5) mice and cultured according to the standard interface method described by De Simoni and Yu [48] with minor modifications. The hippocampi were dissected and placed in 4uC Grey’s Balanced Salt Solution supplemented with Glucose, then sliced into 400 mm -thick slices using a McIlwain tissue chopper. The slices with DG were identified and selected under Zeiss Stemi DV4 Stereomicroscope (Carl Zeiss Pte Ltd, Singapore) and transferred onto a porous membrane inserts (Millipore), placing 6 slices/insert and maintained in an incubator at 5% CO2/air at 37uC. The slice culture medium consisted of 50% Minimal Essential Medium with Glutamax (GIBCO), 25% Earle’s balanced salt solution, 25% heat-inactivated horse serum, Penicillin-Streptomycin 50 mg/ml, and glucose (6.5 mg/ml). The medium was changed every second day. On day 7 the culture medium was changed to serum free condition and taurine was added to the medium on day 9 and PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e42935