Isolation and Culture of Rat Cortical Neurons

Primary rat cortical neurons were obtained and cultured as described previously [13 (link)]. Pregnant SD rats (16–18 days) were used to prepare primary-neuron-enriched cultures. In brief, we first removed the meninges and blood vessels of the brain. The brain tissues were then digested with 0.25% trypsin for 5 min, and the digestion was terminated by washing the tissue three times with PBS. The brain tissue suspension was centrifuged at 500×g for 5 min, and the pellet was then resuspended in neural basal medium (all from GIBCO, Carlsbad, CA, USA). Finally, cells were seeded in 12-well plates and 6-well plates in fresh medium. Afterward, half of the medium was changed every 2 d.