Figure 8

Postnatal consequences of loss of endothelial cell-derived GABA. (A, B) Significantly affected regions in Vgat^ECKO brain at P30 were cingulate, motor, somatosensory and piriform cortex in which reductions in vessel density were observed. Images depict somatosensory cortex. Vgat^ECKO data were normalized to Vgat^fl/fl data; Data represent mean ± SD (n = 6, ^*P < 0.05, Student's t-test). (C, D) Extravascular IgG staining was observed in P30 Vgat^ECKO cerebral cortex (yellow arrows, C) and IgGs formed halos around microvessels (D). (E-G) Co-labeling with isolectin B4 revealed that while IgGs are localized to vessels in Vgat^fl/fl cortex (E), IgG leakage and uptake by neurons were observed at various time points after status epilepticus (F, G; n = 8). (H, I) Concurrent reduction in GABA cells was observed in the cortical regions examined. The GABA cell distribution was very abnormal in Vgat^ECKO with several cells clustered in layer IV-V and few to none in upper layers (red asterisks) indicative of cortical asynchrony. Vgat^ECKO data normalized to Vgat^fl/fl data (I); Data represent mean ± SD (n = 6, ^*P < 0.05, Student's t-test). (J) Numbers of calretinin⁺, somatostatin⁺, neuropeptide Y⁺ and parvalbumin⁺ subclasses in somatosensory cortex from P30 old mice. Vgat^ECKO data normalized to Vgat^fl/fl data (J); Data represent mean ± SD (n = 10, ^*P < 0.05, Student's t-test). (K, L) Parvalbumin immunoreactive cells in the Vgat^ECKO somatosensory cortex (L) showed a similar abnormal profile as GABA immunoreactive cells (H). Yellow asterisks in (L) point to significant reduction of parvalbumin⁺ cells in layers II/III and yellow arrow points to cells abnormally clustered in layer V. (M) Quantification of parvalbumin⁺ cells in Vgat^fl/fl and Vgat^ECKO somatosensory cortical layers; Data represent mean ± SD (n = 10, ^*P < 0.05, Student's t-test). (N-Q) Representative images of the basket cells in the Vgat^fl/fl(N) and Vgat^ECKO(P) somatosensory cortex. Basket cells sampled (red boxes; N, P) were mainly located at the layer II-III close to neighboring pyramidal cells (red asterisks) of the somatosensory cortex. Higher magnification of the basket cell morphology was illustrated in O and Q. Compared to Vgat^fl/fl cortex (O), Vgat^ECKO cortex showed a significant retraction of dendritic trees (red arrows in Q). (R) Comparison of dendritic length of Vgat^fl/fl and Vgat^ECKO basket cells. There was a 41% reduction of dendritic lengths of basket cells of the Vgat^ECKO group when compared to the Vgat^fl/fl group (n = 9, ^*P < 0.05, ANOVA). (S) Comparison of frequency of dendritic intersections × 30-μm interval from the soma of basket cells between Vgat^fl/fl and Vgat^ECKO group. There was a significant reduction in the frequency of intersections at a distance of 60-120-μm from the soma of basket cells of the Vgat^ECKO group (n = 9, ^*P < 0.05, ANOVA and post hoc tests). Scale bars: A, 100 μm (applies to C, H, K, L, N, P), E, 50 μm (applies to F,G), D, 25 μm (applies to O, Q).