All the experimental protocols were approved by the Italian Ministry of Health. Primary cultures were obtained from hippocampi of mice (C57BL6J, Charles River, Milano, Italy) at embryonic day 18 (E18). Embryos were removed and dissected under sterile conditions. Hippocampi were dissociated by enzymatic digestion in trypsin (0.125% for 20 min at 37°C). Trypsin activity was blocked by adding complete media (Neurobasal2, Gibco, Milano, Italy) supplemented by B27 (2%, Gibco, Milano, Italy), alanyl glutamine (2 mM, Gibco, Milano, Italy), and penicillin/streptomycin (both 1 mM, Sigma, Milano, Italy) containing 10% fetal bovine serum (FBS, Gibco, Milano, Italy). After trypsinization, tissues were rinsed in complete media without FBS and dissociated with a plastic pipette. Neurons were plated at a concentration of on glass-bottom Petri dishes (P35G-0-14-C, MaTek Corporation, Ashland), whose surface had been treated with poly-d-lysine (0.1%, Sigma, Milano, Italy) to allow cell attachment. These plated neurons were kept in the incubator for 2 h, allowing them to attach, and thereafter, the dishes were filled with 3 ml of serum-free medium. Experiments were carried out with neurons at 2 DIV.