Primary hippocampal neuron cultures

Briefly, a 22 mm diameter glass coverslip (0.09 mm thickness) was silicone-sealant-fastened to the bottom of a 35 mm culture dish with a bored hole of 20 mm in diameter and sterilized as we previously described (Lin et al. 2004). Coverslips were coated with poly-D-lysine. Hippocampi were dissected from CO₂-anaesthetized neonatal Sprague–Dawley timed-pregnancy rats (Envigo, Indianapolis, IN, USA) at 0–24 h of life. Rats were fed a diet of regular chow, ad libitum. Hippocampi were enzymatically digested in Earle’s balance salt solution (EBSS) supplemented with 1% glucose and cysteine-activated papain. Digestion was blocked with dilute DNase, and cells were rinsed in fresh EBSS and plated in plating medium (minimal essential medium with Earle’s salts, 10% fetal bovine serum, 5% horse serum, 2 mM glutamine, 10 mM sodium pyruvate, 0.6% glucose, 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin) at 1.0 × 10⁶ cells/dish. After 18 h, cell adherence was established. Cells were then grown in neurobasal medium (NbActiv1; BrainBits LLC, Springfield, IL, USA) and incubated at 37°C in a 5% CO₂ biological incubator.