Figure 7.
Astrocytic Drp1 loss dysregulates astrocyte organization and PAP protein expression in the mouse cortex. (A) Diagram of astrocyte organization analysis by nearest and multiple neighbor distance quantification. (B) Violin plot of nearest neighbor average distance between tdTomato+ somas across V1 cortex of Drp1 cWT and cKO mice. N = 4 male and female mice/condition (large circles), ∼200 distances per image, 2–3 cortical images/mouse. Data are mean ± SEM. Nested t test. (C) Distribution of average distances to multiple (9) neighbors. N = 4 male and female mice/condition, ∼200 distances per image, 2–3 images/mouse. Kolmogorov–Smirnov test. (D) Representative images of Drp1 cWT and cKO V1 cortices with tdTomato+ astrocytes (magenta) stained with Cx43 (cyan) at P21. Scale bar, 100 µm. Zoom merge (last column). Scale bar, 20 µm. (E–G) Quantification of (E) individual, (F) variance, and (G) distribution of Cx43 mean gray value per astrocyte in Drp1 cKO and cWT mice. N = 4 male and female mice/condition, n = 15–30 mid-layer astrocytes (L2/3, L4, and L5) per animal from 2 to 3 section images/animal. Nested t test for (E), unpaired, two-tailed t test for (F), and Kolmogorov–Smirnov test for G. (H) Quantification of Cx43 mRNA from isolated cKO and cWT astrocytes. N = 4–6 mice/condition. Data are mean ± SEM. Unpaired two-tailed t test. (I) Representative immunoblot of isolated astrocytes for Cx43 showing its FL form at 40 kDa and its 20 kDa isoform using COX4 as loading control. Immunoblot ran in the same experiment as Fig. 6 B, therefore COX4 loading control image is the same as Fig. 6 B. (J and K) Quantification of FL Cx43 protein and (K) Cx43-20 kDa isoform protein in isolated cKO and cWT astrocytes. N = 3 mice/condition. Data are mean ± SEM. Unpaired, two-tailed t test. (L, N, and P) Representative immunoblots for (L) GLAST, (N) Glut1, and (P) Kir4.1 from immunopurified astrocytes from Drp1 cWT and cKO mice. COX4, a mitochondrial protein, serves as a loading control. (M, O, and Q) Quantification of (M) GLAST, (O) Glut1, and (Q) Kir4.1 protein in isolated cKO and cWT astrocytes. N = 3–4 mice/condition. Data are mean ± SEM. Unpaired, two-tailed t test. Source data are available for this figure: SourceData F7.

Astrocytic Drp1 loss dysregulates astrocyte organization and PAP protein expression in the mouse cortex. (A) Diagram of astrocyte organization analysis by nearest and multiple neighbor distance quantification. (B) Violin plot of nearest neighbor average distance between tdTomato+ somas across V1 cortex of Drp1 cWT and cKO mice. N = 4 male and female mice/condition (large circles), ∼200 distances per image, 2–3 cortical images/mouse. Data are mean ± SEM. Nested t test. (C) Distribution of average distances to multiple (9) neighbors. N = 4 male and female mice/condition, ∼200 distances per image, 2–3 images/mouse. Kolmogorov–Smirnov test. (D) Representative images of Drp1 cWT and cKO V1 cortices with tdTomato+ astrocytes (magenta) stained with Cx43 (cyan) at P21. Scale bar, 100 µm. Zoom merge (last column). Scale bar, 20 µm. (E–G) Quantification of (E) individual, (F) variance, and (G) distribution of Cx43 mean gray value per astrocyte in Drp1 cKO and cWT mice. N = 4 male and female mice/condition, n = 15–30 mid-layer astrocytes (L2/3, L4, and L5) per animal from 2 to 3 section images/animal. Nested t test for (E), unpaired, two-tailed t test for (F), and Kolmogorov–Smirnov test for G. (H) Quantification of Cx43 mRNA from isolated cKO and cWT astrocytes. N = 4–6 mice/condition. Data are mean ± SEM. Unpaired two-tailed t test. (I) Representative immunoblot of isolated astrocytes for Cx43 showing its FL form at 40 kDa and its 20 kDa isoform using COX4 as loading control. Immunoblot ran in the same experiment as Fig. 6 B, therefore COX4 loading control image is the same as Fig. 6 B. (J and K) Quantification of FL Cx43 protein and (K) Cx43-20 kDa isoform protein in isolated cKO and cWT astrocytes. N = 3 mice/condition. Data are mean ± SEM. Unpaired, two-tailed t test. (L, N, and P) Representative immunoblots for (L) GLAST, (N) Glut1, and (P) Kir4.1 from immunopurified astrocytes from Drp1 cWT and cKO mice. COX4, a mitochondrial protein, serves as a loading control. (M, O, and Q) Quantification of (M) GLAST, (O) Glut1, and (Q) Kir4.1 protein in isolated cKO and cWT astrocytes. N = 3–4 mice/condition. Data are mean ± SEM. Unpaired, two-tailed t test. Source data are available for this figure: SourceData F7.

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