Sodium cyanate

EC, butyl carbamate (BC) as an internal standard, sodium cyanate, and 2-aminobenzoic acid were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA). Chloroform was obtained from Samchun Pure Chemical (Pyeongtaek, Korea), and ethanol was obtained from Honeywell Burdick & Jackson (Ulsan, Korea). Sodium hydroxide and citric acid were purchased from Junsei Chemicals (Tokyo, Japan). Hydrochloric acid was supplied by Daejung Chemicals and Metals (Siheung, Korea). All chemicals used in this study were of analytical grade. Water was deionized using an ultra-pure and pure all-in-one water purification system (Dongwon Scientific Co., Seoul, Korea).

Newly postnatal C57bl/6j mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center, Beijing. Anaesthetized mice were sacrificed by cervical dislocation. All tissues were maintained in d-Hank's solution (KCl 0.4 g, KH₂PO₄ 0.06 g, NaCl 8.0 g, NaHCO₃ 0.35 g, Na₂HPO₄•12H₂O 0.132 g, d-Glucose 1.0 g in 1 L dddH₂O, pH 7.4) and chilled on ice. The dissected medial prefrontal cortex was centrifuged (1000 r/min, 7 min, room temperature) after dispersed by trituration and 0.25% trypsin digestion (Hyclone. 5 min, 37 °C). Culture medium was DMEM (Gibco) supplemented to 10% FBS (Gibco) for the first 4 h, then changed to neurobasal (Gibco) with 2% B27 (Gibco) and 0.5 mM l-glutamine (Sigma). Cells were plated on 0.1% poly-l-lysine (Sigma, for urea stimulation) or poly-ornithine (Sigma, for cyanate, because cyanate is fast reacted with residue of lysine) coated 10 cm dishes or 6-well plates at 1 × 10⁶ cells/ml of medium, and maintained in an incubator at 37 °C and 5% CO₂. urea (Sigma), mannitol (Sigma), sodium cyanate (Sigma) and sodium chloride (Sigma) were dissolved into the medium for stimulation. Neurons after cultured for 15 days was subjected to subsequent experiments.