Figure 5.
Figure 5. Endothelial CXCL1 regulates astrogliosis and astrocytic glutamate uptake via CXCR2 in astrocytes. (A) Representative immunostaining images of CXCR2 expression in astrocytes in the CA1 in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk. Bar, 100 µm. (B) Schematic of GFAP-Cre-dependent AAV vectors for CXCR2 silencing. (C) Representative Western blot of CXCR2 and quantification of CXCR2 expression in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with a virus mixture of rAAV-GFAP-Cre and rAAV-mCherry-Con or rAAV-mCherry-shCxcr2 (1:1 mixture) at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (D) Representative images of immunostaining for astrocytic CXCR2 expression in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with a virus mixture (1:1 mixture). Bar, 5 µm. (E) The onset latency and duration of seizures induced by PTZ were assessed in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice after a virus mixture injection (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (F and G) Representative sEPSC (F) and mEPSC (G) traces recorded in pyramidal neurons from the hippocampal CA1 regions of the indicated groups, including the Cdk5f/f + GFAP-Cre/RNAi (Con) group, the Cdh5-Cre;Cdk5f/f + GFAP-Cre/RNAi (Con) group, the Cdk5f/f + GFAP-cre/shCxcr2-RNAi group, and the Cdh5-Cre;Cdk5f/f + GFAP-cre/shCxcr2-RNAi group. (H and I) Cumulative probability plots of amplitudes (H) and inter-event intervals (I) of sEPSC from mice in the indicated groups. The insets depict the average sEPSC amplitudes and frequencies (n = 3–5 mice per group; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). (J and K) Cumulative probability plots of amplitudes (J) and inter-event intervals (K) of mEPC from mice in the indicated groups. The insets depict the average mEPSC amplitudes and frequencies (n = 3–5 mice per group; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). (L) Representative GLT1 transport currents following DHK incubation in the hippocampus from 4-wk-old mice in the indicated groups (left). Quantification of the GLT1 transport currents is shown on the right (n = 3–5 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). The numbers inside the bars represent the numbers of cells from three to five mice. The bars with error bars represent means ± SEM; n.s., not significant.

Endothelial CXCL1 regulates astrogliosis and astrocytic glutamate uptake via CXCR2 in astrocytes. (A) Representative immunostaining images of CXCR2 expression in astrocytes in the CA1 in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk. Bar, 100 µm. (B) Schematic of GFAP-Cre-dependent AAV vectors for CXCR2 silencing. (C) Representative Western blot of CXCR2 and quantification of CXCR2 expression in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with a virus mixture of rAAV-GFAP-Cre and rAAV-mCherry-Con or rAAV-mCherry-shCxcr2 (1:1 mixture) at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (D) Representative images of immunostaining for astrocytic CXCR2 expression in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with a virus mixture (1:1 mixture). Bar, 5 µm. (E) The onset latency and duration of seizures induced by PTZ were assessed in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice after a virus mixture injection (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (F and G) Representative sEPSC (F) and mEPSC (G) traces recorded in pyramidal neurons from the hippocampal CA1 regions of the indicated groups, including the Cdk5f/f + GFAP-Cre/RNAi (Con) group, the Cdh5-Cre;Cdk5f/f + GFAP-Cre/RNAi (Con) group, the Cdk5f/f + GFAP-cre/shCxcr2-RNAi group, and the Cdh5-Cre;Cdk5f/f + GFAP-cre/shCxcr2-RNAi group. (H and I) Cumulative probability plots of amplitudes (H) and inter-event intervals (I) of sEPSC from mice in the indicated groups. The insets depict the average sEPSC amplitudes and frequencies (n = 3–5 mice per group; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). (J and K) Cumulative probability plots of amplitudes (J) and inter-event intervals (K) of mEPC from mice in the indicated groups. The insets depict the average mEPSC amplitudes and frequencies (n = 3–5 mice per group; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). (L) Representative GLT1 transport currents following DHK incubation in the hippocampus from 4-wk-old mice in the indicated groups (left). Quantification of the GLT1 transport currents is shown on the right (n = 3–5 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). The numbers inside the bars represent the numbers of cells from three to five mice. The bars with error bars represent means ± SEM; n.s., not significant.

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