(A) CAG promoter (pCAG)-TurboRFP, TRETight2 promoter (pTRE-Tight2)-Kir2.1EGFP, and pCAG-tTA2s were transfected. (B) Doxycycline (Dox) was administered from P6. The blue bars indicate the period of Dox treatment. (C) Kir2.1EGFP (Kir-GFP) and TurboRFP (RFP) signals in P6, P10, and P15 cortical sections. Right panels show three representative neurons (in columns) with Kir-GFP and RFP signals (in rows) at each age. At P6 (before Dox treatment), the signals of Kir-GFP (stained with anti-GFP antibody) were primarily observed in the periphery of the soma and along dendrites, implying that Kir-GFP was transported to the cellular membrane. Note that RFP signals were localized in the central part of the cell body. At P10 and P15 (4 days and 9 days after Dox treatment), Kir-GFP signals were not preferentially observed in the periphery of the soma and along dendrites anymore. Low-level signals in the central part of the cell body may suggest low-level expression of Kir-GFP even after Dox treatment. Alternatively, these may reflect the contaminated RFP signals in the acquisition of the GFP channel. (D) Average signal intensity of Kir-GFP (x-axis) and RFP (y-axis) in each cell is displayed at P6, P8, P10, P12, and P15. (E) Each dot represents KirGFP/RFP ratio of a cell at each age. Note that KirGFP/RFP ratio decreases after Dox administration at P6. Scale bar in the left panel of (C), 50 μm; right panel, 10 μm. P6, n=10 sections from three mice. P8, n=7 sections from two mice. P10, n=10 sections from three mice. P12, n=10 sections from three mice. P15, n=10 sections from three mice.