Primary neuronal culture and drug treatment

Before culture, 20-mm glass coverslips were coated with 1 mg/mL poly-D-lysine at room temperature for 20 minutes; coated coverslips were then placed into 12-well culture plates. Mouse neonates at postnatal day 0 were sacrificed and their cerebral cortices were excised under a dissection microscope. Cortical tissues were triturated for disaggregation and plated on poly-D-lysine-coated glass coverslips in Neurobasal medium containing B27 serum-free supplement and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). Cultures were incubated at 37°C in an 95% air, 5% CO₂, and 90% relative humidity. Half of the growth medium was replaced every two days. Primary neurons were cultured for 15 to 19 days prior to immunostaining. For repetitive stimulation with KCl ⁵⁵, neurons (15 days in vitro) were treated repetitively with 90 mM KCl for 3 min and a spaced recovery for 10 min. Neuronal cells were harvested or fixed 30 min after the last stimulation.