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).