Negative pole disc type electrodes

In utero electroporation targeting the prefrontal cortex (PFC) region was performed by our published protocol with some modifications.^{20 (link)} Pregnant C57/BL6 mice (Charles River) were anesthetized at embryonic day 14.5 (E14.5) by intraperitoneal administration of a mixed solution of Ketamine HCl (100 mg/kg), Xylazine HCl (7.5 mg/kg), and Buprenorphine HCl (0.05 mg/kg). After the uterine horn was exposed by laparotomy, inducible shRNA to DISC1 (2 µg/µl) and CALNL-GFP (1 µg/µl) together with CAG-ER^T2CreER^T2 (1 µg/µl) (molar ratio, approximately 3:1:1) were injected into the bilateral ventricles with a glass micropipette made from a microcapillary tube (GD-1; Narishige). The embryo’s head in the uterus was held between the custom-made electrode, consisting of one positive and two negative pole disc-type electrodes (Nepagene). Electrode pulses (33V; 50 ms) were charged four times at intervals of 950 ms with an electroporator (CUY21EDIT; Nepagene). After electroporation, the uterine horn was replaced in the abdominal cavity to allow the embryos to continue to develop. Brains extracted from DISC1 knockdown and control animals were assessed to confirm GFP expression in proper PFC regions after behavioral characterization. All experiments were performed in accordance with the institutional guidelines for animal experiments.