Animal care and experimental procedures were conducted in accordance with the Italian Institute of Technology licensing and the Italian Ministry of Health. The day of mating (limited to 4 h in the morning) was defined as embryonic day zero (E0), and the day of birth was defined as postnatal day zero (P0). E14.5-15.5 (cerebellum) or E17 (hippocampus and cortex) timed-pregnant Sprague Dawley rats (Harlan Italy SrL, Correzzana, Italy) were anesthetized with isoflurane (induction, 3.5 %; surgery, 2.5 %), and the uterine horns were exposed by laparotomy. The DNA (1-2 µg/µl in water) together with the dye Fast Green (0.3 mg/ml; Sigma, St. Louis, MO) was injected (5-6 µl) through the uterine wall into one of the lateral ventricles (hippocampus and cortex) or the 4th ventricle (cerebellum) of each embryos by a 30 gauge needle (Pic indolor, Grandate, Italy). After soaking the uterine horn with a phosphate buffered saline (PBS) solution, the embryo’s head was carefully held between tweezer-type circular electrodes (hippocampus, visual cortex, motor cortex: 10 mm diameter; cerebellum: 5 mm diameter, Nepa Gene, Chiba, Japan), while the third electrode (7x6x1 mm, gold-plated copper) was accurately positioned at different locations, as described in Figure 1. For the electroporation, 5 electrical pulses (hippocampus and cortex: amplitude, 50 V; duration, 50 ms; intervals, 150 ms; cerebellum: amplitude, 35 V; duration, 50 ms; intervals, 150 ms) were delivered with a square-wave electroporation generator (CUY21EDIT, Nepa Gene; ECM 830, BTX, Harvard Apparatus). In a subset of experiments, electroporation of somatosensory and motor cortices, were performed at 20V. For bilateral electroporation experiments, DNA filling in both ventricles was achieved by a single monolateral injection (plus few minutes waiting time for diffusion in the contralateral ventricle) to avoid excessive brain damage. For all animals, the uterine horns were returned into the abdominal cavity after electroporation, and embryos allowed continuing their normal development. For surgery on E14.5-15.5 embryos, illumination was performed with a flexible optic fiber with a cold light source (Olympus KL1550 LCD) from behind the embryo, whereas for surgery on E17 embryos illumination was achieved by two rigid fibers placed above the operating table. In some experiments, a plasmid encoding a red fluorescent protein (Td-Tomato; 1.5 μg/μl) was injected as reporter.