Figure 1. Mitochondrial distribution in cortical neurons during the development of dendritic arbors. A, A P7.5 cortical section showing the distribution of EGFP-labeled neurons. The dotted line indicates the boundary between layers 1 and 2. The numbers show cortical layers. B–I, Pyramidal neurons expressing EGFP and mito-RFP in E18.5 (B, C), P7.5 (D, E), P14.5 (F, G), and P21.5 (H, I) cortices. Plasmids encoding fluorescent proteins were electroporated in utero into ventricular zone cells at E13.5–13.75 to visualize the neuronal morphology and mitochondria. Dendritic mito-RFP signals are located mostly in the proximal portion at E18.5 and P7.5, but almost evenly distributed throughout the dendritic arbors at P14.5 and P21.5. The red dotted lines indicate the boundary between the marginal zone and the upper cortical plate (B, C) or between layers 1 and 2 (D–I). The regions indicated by the red brackets in D, E, H, and I are enlarged in D′–E″ and H′–I″. D′–E″, H′–I″, Enlarged images of the distal and proximal portions of the apical dendrite at P7.5 and P21.5. Mito-RFP signals (red) are more densely distributed in the proximal portion than in the distal portion of the apical dendrite at P7.5 (D′–E″), while they are approximately evenly distributed in both portions at P21.5 (H′–I″). Arrowheads point to branch points. J, K, Analysis of the developmental changes in the dendritic morphology: (J) number of branch points, (K) length of dendrites including all branches. Dendrites grow extensively from E18.5 to P14.5, but not between P14.5 and P21.5; n = 39 cells, two brains for E18.5; 21 cells, three brains for P3.5; 20 cells, three brains for P7.5; 9 cells, two brains for P14.5; and 15 cells, three brains for P21.5. L, Analysis of the dendritic distribution of the mito-RFP signals. Dendrites were divided into consecutive 10 μm segments based on the distance from the soma, and the percentage of mito-RFP pixels in each segment to the total number of pixels in the soma-dendrite compartment was calculated. Clear peaks from the mito-RFP signal are seen in the proximal portion of dendrites in E18.5 and P7.5 neurons, but not in P14.5 and P21.5 neurons. M, Normalized distribution of mito-RFP signals to dendritic size. The values obtained in L were further divided by the proportion of the area of each segment to the total area of the soma-dendrite compartment. Normalized signals are concentrated in the proximal portion of the dendrites at E18.5 and P7.5; n = 35 cells, two brains for E18.5; 21 cells, three brains for P3.5; 15 cells, three brains for P7.5; 4 cells, two brains for P14.5; and 7 cells, three brains for P21.5. A.U., arbitrary units. Scale bars: A, 250 μm; B, C, 10 μm; D, E, 20 μm; D′–E″, H′–I″, 10 μm; F–I, 40 μm. Error bars indicate SEM; **p < 0.01, ***p < 0.001, Mann–Whitney U test with Bonferroni correction (3 tests).