05) were determined by one-way ANOVA with a Newman-Keuls test or Student’s t test. All measurements were made at room temperature. Introduction of plasmid DNA into the neuroepithelial cells of mouse embryonic neocortex in utero was performed as described elsewhere (Tabata and Nakajima, 2001), with
minor modifications. In brief, the uterine horns were exposed at E14.5, and ∼1 μl DNA solution www.selleck.co.jp/products/MDV3100.html (0.2–5 μg/μl of each plasmid, depending on the construct) was injected into the lateral ventricle of each littermate. Embryos were then electroporated with an electroporator CUY21EDIT (BEX, 0.5 cm puddle type electrode, 33–35 V, 50 ms duration, four to eight pulses). After electroporation, the uterine horns were returned to the abdominal AT13387 clinical trial cavity to allow the embryos to continue development. For Leu incorporation (Figure 6D), the embryos were harvested 4 days after electroporation, and the brains were then subjected to the imaging analysis. For Cmn incorporation (Figures 6E–6H), the uterine horns were exposed again at E16.5, and Cmn (500 mM, 2–5 μl) was injected to the electroporated side or both sides of the lateral ventricle. The uterine horns were placed
back into the abdominal cavity again. Twelve to forty-eight hours after Cmn injection, the embryos were harvested, and the brains were then subjected to the imaging analysis or electrophysiology as described later. For imaging analysis, the brains were fixed with 4% paraformaldehyde in PBS at 4°C for 2–4 hr. After equilibration with 30% (w/v) sucrose in PBS, the fixed brains were embedded in optimal cutting temperature compound (Sakura) and frozen. Coronal sections (10 μm thick) were prepared by cutting the frozen brains with a cryostat CM3050S (Leica), and the fluorescence of GFP and mCherry was detected using microscopies.
DAPI (Sigma) was used to counterstain nuclei. After in utero electroporation and Cmn delivery, E17.5–E18.5 mice embryos were harvested, and sagittal slices (200 μm) from their Suplatast tosilate neocortices were prepared in ice-cold artificial cerebral spinal fluid (ACSF) (119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl2, 2.5 mM CaCl2, 1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose, pH 7.3) continuously bubbled with 95%/5% O2/CO2. Vibratome slices were warmed to 33°C and incubated for 42 min in ACSF supplemented with 3 mM myo-inositol, 0.4 mM ascorbic acid, and 2 mM sodium pyruvate and then transferred to the recording chamber superfused with ACSF (2 ml/min). Neurons were visualized with a Hamamatsu digital camera (Model C8484) on an Olympus microscope (BX51WI), and whole-cell patch-clamp recordings (Axopatch 200B) were made from neurons in the neocortex. PIRK-expressing neurons were identified by GFP and mCherry fluorescence. The internal solution contained 130 mM potassium gluconate, 4 mM MgCl2, 5 mM HEPES, 1.1 mM EGTA, 3.4 mM Na2ATP, 10 mM sodium creatine phosphate, and 0.1 mM Na3GTP at pH 7.3 with KOH. BaCl2 (0.5 mM) was diluted into ACSF and applied directly on to the slice.