Isolation of Hippocampal Neurons from Mice

Hippocampal neurons from E17-E19 C57BL/6 mice brains were prepared according to standard procedures^{57 (link)}. Briefly, hippocampal neurons were incubated for 10 min at 37 °C in trypsin-EDTA solution (0.025%/0.01% w/v; Biochrom AG) in 1 × PBS, and tissues were mechanically dissociated at RT with a fire polished Pasteur pipette. Cell suspension was harvested and centrifuged at 600 × g for 5 min. The pellet was resuspended in MEM (Biochrom AG) containing 5% fetal bovine serum, 5% horse serum, 2 mM glutamine, 1% penicillin-streptomycin antibiotic mixture (Sigma-Aldrich), and 25 mM glucose. The cells were plated on poly-l-lysine (0.1 mg mL⁻¹; Sigma-Aldrich) pre-coated wells. After 72 h, medium was replaced again with glutamine-free medium and experiments were performed at 12–14 days in vitro (DIV).