Rat primary cortical neurons were cultured using a method as we described previously (Liu et al., 2007). Briefly, cerebral cortices were removed from the embryos of Sprague–Dawley pregnant rats at 15–18 days gestation (Shouthern Medical University Experimental Animal Center). After removing the meninges, the cortical tissue was minced and incubated with 0.05% trypsin for 30 min at 37°C with gentle trituration. After digestion, the neurons were achieved and suspended in neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine. Before seeding, culture vessels including 96-well plates, 1.2 cm glass slides or 6 cm dishes were coated with poly-L-lysine (PLL; 50 μg/mL, Sigma-Aldrich) at room temperature overnight. Neurons were maintained at 37°C in a humidified 5% CO2 incubator and half of the culture medium was changed every 3 days. The neurons were subjected to experiments 8 days after seeding.