Mice purchased from Janvier were anesthetized with isoflurane (Abbott Laboratories) in an oxygen carrier before the administration of temgesic (Schering-Plough). Endotoxin-free plasmids were injected into lateral ventricles of E14.5 mouse embryo forebrains using a FemtoJet microinjector (Eppendorf) with 0.1% fast green for visualization. Injection was followed by electroporation (5 pulses of 24 mV at 50-ms intervals for 950 ms) using platinum electrodes (Sonidel, catalog no. CUY650P3) connected to an electroporator (ECM 830, BTX). Embryos were coelectroporated with tamoxifen-inducible CreERT2 expressing plasmid along with two Cre-dependent constructs expressing DsRed and shAtat1/sh-scrambled and with LAMP1-Emerald–expressing plasmid. After electroporation, embryos were placed back in the abdominal cavity. For injection, 4OHT and progesterone were dissolved in ethanol 100% at concentrations of 20 and 10 mg/ml, respectively, and then diluted with nine volumes of corn oil (Sigma-Aldrich). Diluted tamoxifen solution (2 mg/ml, 100 μl per mouse) was intraperitoneally injected three times at E17, E18, and P1, and pups brains were dissected at P2. Brains were embedded in agarose 4% HBSS solution and cut into coronal sections (300 μm) using a vibratome (Leica VT1000S, Leica Microsystems). The slices were placed on Matrigel-coated (Corning) MatTek glass-bottom dishes and covered with half-diluted Matrigel with supplemented Neurobasal culture medium. Sections were incubated for 30 min at 37°C before imaging. Time-lapse imaging was performed on a Zeiss Super Resolution LSM 880 Airyscan Elyra S1 (×63 magnification) at a 500-ms interval for 60 s at the corpus callosum midline. The recoding microscope chamber was heated at 37°C and supplied with 5% CO2.

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