E17 of1 mice embryos

Experiments were conducted in accordance with the Swiss Federal Guidelines for Animal Experimentation and were approved by the Cantonal Veterinary Office for Animal Experimentation (Vaud, Switzerland). Primary cultures of cortical neurons were prepared from embryonic day 17 (E17) OF1 mice embryos (Charles River Laboratories) as previously described (Allaman et al., 2010 (link)). Briefly, minced pieces (1–2 mm³) of isolated cerebral cortices were enzymatically digested with papain and gently triturated to a single-cell suspension. Dissociated cells were plated onto poly-L-ornithine-coated cell culture dishes at a density of 4 × 10⁴ cells/cm² and maintained in Neurobasal medium supplemented with B27, GlutaMax, penicillin (50 U/mL), and streptomycin (50 μg/mL) (Invitrogen) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. These culture conditions typically produced 93–95% pure neuronal cultures (Belanger et al., 2011 (link)). Neurons were used for experiments at day in vitro 11.

Primary cultures of cortical neurons were prepared from embryonic day 17 (E17) OF1 mice embryos (Charles River Laboratories, L’Arbresle, France) as previously described^{6 (link)}. Neurons were plated on 25 mM poly-L-Ornithine (Sigma) coated coverslips at an average density of 4 × 10⁴ cells/cm² and maintained in Neurobasal medium (Gibco, containing 25 mM Glucose) supplemented with B27 (Gibco), GlutaMAX, penicillin (50 U/mL) and streptomycin (50 µg/mL) (Invitrogen, Basel, Switzerland) at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air and were used at day in vitro (DIV) 17 to 22. These culture conditions typically produced 93% pure neuronal cultures, as assessed by microtubule-associated protein 2 (MAP2, neuronal marker) and glial fibrillary acidic protein (GFAP, astrocytic marker) co-immunostaining^{66 (link)}.