Preparation of organotypic rhinal-hippocampal slice cultures
Organotypic rhinal-hippocampal slice cultures were prepared from Sprague-Dawley rats (6 to 7 days old) as described previously (Dyhrfjeld-Johnsen et al., 2010; Magalhães et al., 2018; Valente et al., 2021) with some modifications. This method allows organotypic slices to develop spontaneous epileptiform pyramidal cell discharges. After decapitation, the brains were removed and briefly placed in a cold Gey’s balanced salt solution (GBSS, Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 25 mM D-(+)-glucose (Sigma, St. Louis, MO, USA) under sterile conditions. The hippocampus, together with the entorhinal cortex and perirhinal cortex, was meticulously separated and sliced (350 µm thick) transversely using a McIlwain tissue chopper. Four slices were then transferred to porous insert membranes (0.4 µm) (PICM 03050, Millipore, Bedford, MA) placed in each well of six-well culture trays (Corning Costar, Corning, NY). Each well contained 1 mL of culture media made up of 50% Opti-MEM I Reduced Serum Medium, 25% Hank’s Balanced Salt Solution (HBSS), 25% heat-inactivated horse serum (HS) (Thermo Fisher, Waltham, Massachusetts, USA), 25 mM D-(+)-glucose (45% in water, Sigma), 30 µg/mL Gentamycin solution (50 mg/mL, Thermo Fisher). The slices were then kept at 37 °C in 5% CO2 and 95% O2 for 2 weeks. The culture medium was changed every 2–3 days with Opti-MEM medium pre-heated at 37 °C. The day before the experiments, the culture medium was changed to serum-free Neurobasal A (NBA) medium supplemented with 2% B27, 1mM L-glutamine, 30 µg/mL gentamycin (all from Thermo Fisher).