Time-lapse Imaging of Cortical Slices

Cortical slice cultures were prepared and time-lapse imaging was acquired as previously described8 (link)51 (link). About 1 day after in-utero electroporation, embryos were removed and the brain was extracted into ice-cold artificial cerebrospinal fluid containing the following: 125 mM NaCl, 5 mM KCl, 1.25 mM NaH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂, 25 mM NaHCO₃, 20 mM glucose pH 7.4 and 310 mOsm l⁻¹. Brains were embedded in 3% low-melting agarose in artificial cerebrospinal fluid and sectioned at 400 mm using a vibratome (Leica Microsystems). Brain slices were transferred on to a slice culture insert (Millicell) in a glass-bottom Petri dish (MatTek Corporation) with culture medium containing (by volume): 66% Basal Medium Eagle (BME), 25% Hanks, 5% fetal bovine serum, 1% N2, 1% penicillin/streptomycin/glutamine (Invitrogen) and 0.66% D-(1)-glucose (Invitrogen). Cultures were maintained in a humidified incubator at 37 °C with constant 5% CO₂ supply. Twenty-four hours later, Petri dishes with slice cultures were transferred to an inverted microscope FV1000MPE-IX81ZDC (Olympus) and imaged every 20 min for about 2–3 days. Images were analysed by FluoView (Olympus), Imaris (Bitplane) and Photoshop (Adobe Systems).