E17 rat cortices were isolated in cold dissection buffer [1× Hanks’ balanced salt solution, 2.5 mM Hepes (pH 7.4), 30 mM d-glucose, 1 mM CaCl2, 1 mM MgSO4, and 4 mM NaHCO3] and digested in digestion buffer [1 mM Hepes (pH 7.4), 20 mM glucose, 82 mM Na2SO4, 30 mM K2SO4, 6 mM MgCl2, 0.25 mM CaCl2, 0.001% phenol red, and 0.126 mM NaOH] plus 200 U of papain activated with cysteine-HCl and neutralized to pH 7. Cortices were washed, dissociated, and plated in plating media [minimum essential medium with 10% fetal bovine serum (FBS), 5% horse serum, and 1 mM glutamine]. Cells were plated on 10 cm Nunc plates coated with poly-d-lysine (20 μg/ml) and laminin (2 μg/ml) at 12.5 million per dish and kept at 37°C and 5% CO2. After 1 day in culture, media were replaced with supplemented neurobasal [neurobasal with 1× B27, 1 mM glutamine, 1× penicillin-streptomycin, and 10 μM 5-flouro-2′-deoxyuridine (FdU)]. Before stimulation, cells were starved for 16 hours by replacing two-thirds of media with NB without B27 (1 mM glutamine, 1× penicillin-streptomycin, and 10 μM FdU). E15.5 mouse cortices were isolated and digested as for rat cortices. Cells were plated on coverslips in 24-well plates coated with poly-d-lysine (40 μg/ml) and laminin (2 μg/ml) at 0.35 million per well and kept at 37°C and 5% CO2.

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