Endothelial Cdk5 deletion causes progressive astrogliosis and decreased astrocytic glutamate uptake. (A and B) Representative Western blot of GFAP (A) and quantification of GFAP expression (B) in the cortex (CTX) and hippocampus (HP) in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 16 wk (n = 3 mice per group; **, P < 0.01; unpaired two-tailed Student’s t test). (C–F) Representative stitched images of immunostaining for GFAP (green; C) and S100β (red; E) in the hippocampus in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. DAPI staining is shown in blue. Quantification of the area occupied by GFAP+ cells (D) and the density of S100β and GFAP double-positive cells (F) in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at the ages of 4, 8, 16, and 24 wk (n = 3 mice per group; *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test). Bars: C, 500 µm; E: main images, 100 µm; insets, 50 µm. (G) Intrinsic membrane properties, including Rin, τm (Tau), Cm, and RMP recorded in hippocampal astrocytes from brain slices in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk (n = 5 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). (H and I) Glutamate uptake by astrocytes was assessed after puff application of 100 µM glutamate for 500 ms before and after the application of 100 µM TBOA (H) or 200 µM DHK (I) in brain slices in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk. Quantification of transport currents associated with TBOA-sensitive glutamate uptake (H, right) and DHK-sensitive glutamate uptake (I, right) in brain slices in Cdh5-Cre;Cdk5f/f and control mice at 4 wk (n = 5 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). (J and K) Representative Western blots of GLT1 and GLAST in astrocytes (J) and quantification of GLT1 and GLAST expression (K) in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice treated with saline or CEF at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (L) PTZ-induced seizure onset latency and duration in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice treated with saline or CEF at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (M) Representative traces of AP responses to positive current injection treatments at 150 pA with saline or CEF in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk. (N) Quantification across 0–300-pA current injections in 10-pA steps (n = 5 mice per group; ***, P < 0.001; two-way ANOVA followed by Tukey’s multiple comparisons test). (O) Representative GLT1 transport currents following DHK incubation in the hippocampus from 4-wk-old Cdh5-Cre;Cdk5f/f and Cdk5f/f mice with or without CEF. (P) Quantification of the effect of CEF administration on GLT1 transport currents (n = 5 mice per group; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). The numbers inside the bars represent the numbers of cells from five mice. The bars with error bars represent means ± SEM; n.s., not significant.
