Characterization of shDrp1-induced astrocyte clustering. (A) Overview of PALE combined with EdU labeling. (B) Representative images of V1 cortices with astrocytes expressing mCherry-CAAX shRNA (red) against Drp1 (shDrp1) or scrambled control (shControl) and EdU labeling (magenta) at P4 and P14. Scale bar, 200 µm. (C) Quantification of Edu+ cells in shControl or shDrp1 cortices at P4, P8, P10, P12, and P14. N = 3–4 male and female mice/condition, three cortical section images/mouse. Data points are time point averages ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test. (D) Representative images of V1 cortices with astrocytes expressing mCherry-CAAX shRNA (magenta) against Drp1 (shDrp1) or scrambled control (shControl) at 2 and 6 mo. Scale bar, 200 µm. (E) Quantification of astrocyte cell or cluster area per field of view at 2 and 6 mo. N = 3 male and female mice/condition (large circles), three cortical section images/mouse (small gray dots). Data points are mean ± SEM. Nested t test. (F) Representative images of V1 astrocytes from three mice per condition expressing mCherry-CAAX and shControl (top two rows) and shDrp1 (bottom two rows) stained with an antibody against Cx30 (cyan) at P21. Scale bars, 20 µm.