Primary culture of hippocampal neurons, siRNA transfection, and drug treatment

Cultures of hippocampal neurons were performed as previously described [72]. In brief, the E18 SD rats were placed in a sealed anesthesia container with cotton balls thoroughly infiltrated with ether. The pregnant rats were sterilized by soaking in 75% ethanol after the corneal reflex and pain reflex disappeared, and the newborn rats were removed by laparotomy. Fetal rats were decapitated, and the hippocampus was isolated and placed in ice-cold Hank’s Balanced salt solution (HBSS), followed by digestion with 3 ml of 0.125% trypsin at 37 °C for 15 min. A high glucose medium containing fetal bovine serum was added to terminate digestion. The cells were centrifuged at 1000 rpm for 4 min, and the supernatant was removed. Then 2 ml medium at 37 °C was added to resuspend the cells. For primary culture, hippocampal neurons (1 × 10⁴ cells/cm²) were planted in 6- or 24-well plates embedded with poly-d-lysine, with or without small round glass slides. The original culture medium was discarded and replaced with the neural basal neuron culture medium containing B27 following 4–6 h of culture. The cells were cultured at 37 °C, 5% CO₂, and 95% air atmosphere. The fresh medium was replaced every 2–3 days.

The siRNA transfection of the primary neurons was performed according to the manufacturer’s protocol (RiboBio, Guangzhou, China). 20 μl of 20 μM siRNA was added to 120 μl 1× riboFECT™ CP buffer, and then 12 μl riboFECT™ CP reagent was added and incubated at room temperature for 15 min; the transfection complex was transferred to a 2 ml neural basal medium containing cultured primary neurons. The final concentration of siRNA was 200 nM. Neurons continued to be cultured at 37 °C in 5% CO₂ and 95% air for 24 h. The protein was extracted to determine the corresponding siRNA transfection efficiency.

For drug treatment of primary cultured neurons, the BDNF recombinant protein (Peprotech, New Jersey, USA) or TrkB receptor blocker ANA-12 (AbMole, Chicago, USA) was added into the culture medium at a final concentration of 50 ng/ml or 10 μM for 24 h, respectively.