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. 2017 May 18;2(10):e88609. doi: 10.1172/jci.insight.88609

Figure 7. Axonal branching was altered in the brains of the mouse model.

Figure 7

(A) Axonal branching of the GFP-positive cells in the contralateral hemisphere in P10 mouse brains that had been transfected with a GFP expression plasmid at E15.0. In the control brains (Control), the GFP-labeled axons branched extensively in the superficial layers (I and II/III). However, in the occluded brains (Occluded), extensive axonal branching was also observed in the deeper layers (layers V and VI). Scale bar: 200 μm. (B) Fluorescence intensity of GFP in each layer was quantified to analyze the contralateral axonal branching. The GFP intensity was significantly reduced in layers I and II/III and increased in layer V in the occluded brains as compared with the control brains (n = 6, respectively). *P < 0.05, **P < 0.01, ***P < 0.001, repeated-measures ANOVA followed by Bonferroni post-hoc test. (C) GFP-positive axons from the contralateral neocortex branched around the abnormally distributed BrdU-positive cells in layer V of P10 occluded mouse brains (arrows). Scale bar: 20 μm. (D) Schematic representation showing the procedure of in utero electroporation of the right and left neocortices. To visualize the axonal projections from the right neocortex (contralateral hemisphere), a tdTomato expression plasmid was transfected into the right neocortex at E16.5 and a GFP expression plasmid was immediately transfected into the left neocortex, followed by a sham operation (Control) or maternal uterine artery occlusion (Occluded). (E) The brains were analyzed at P17.5. Sections were counterstained with DAPI. Some of the GFP-positive cells (green) were distributed in the deeper layers and the white matter in the occluded brains. While the tdTomato-labeled axons (magenta) from the contralateral hemisphere made extensive branching in the superficial layers of the control brains, abundant axonal branching was also observed in the deeper layers of the occluded brains (bottom, indicated by arrows). (F–N) The boxes in E are shown at high magnification in F, I, and L, respectively. High-magnification images of the boxes in F, I, and L are shown in G, J, and M, respectively. In F and G, 25 confocal planes are merged. In I and J, 22 confocal planes are merged. In L and M, 21 confocal planes are merged. Single confocal sections of G, J, and M are shown in H, K, and N, respectively. Arrows in G, J, and M indicate contacts between the tdTomato-labeled axons and the GFP-labeled dendrites. Scale bar: 10 μm (F, I, and L); 5 μm (G, H, J, K, M, and N).