Figure 3
From: Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior
Mechanisms underlying endothelial Gabrb3's actions. (A) Co-labeled image of isolectin B4, KCC2/NKCC1 and DAPI in periventricular endothelial cells. (B) The endothelial GABAA receptor on periventricular endothelial cells is functional. Focal application of muscimol (30 μM) evoked an inward current consistently in whole-cell voltage-clamp recording of periventricular endothelial cells held at −70 mV (92.5 ± 16.3 pA, n = 8). (C) Traces showed an inward current of 100 pA induced by muscimol (30 μM) that was blocked by BMI (10 μM). (D) Muscimol application (30 μM) produced no current response in Gabrb3ECKO periventricular endothelial cells. (E, F) Traces showed inward currents of 100 pA induced by muscimol (30 μM) and blocked by BMI (10 μM) in Gabrb3fl/fl(E) and Gabrb3ECKO(F) cortical neuronal cells. (G, H) Increase of intracellular calcium upon muscimol treatment (30 μM) was significantly retarded in Gabrb3ECKO periventricular endothelial cells (H) when compared to the control. (I) Calcium imaging data were quantified by normalizing the values after muscimol application to that before muscimol application; Data represent mean ± SD (n = 7, *P < 0.05, Student's t-test). (J-L) With or without muscimol application, Gabrb3fl/fl and Gabrb3ECKO periventricular endothelial cells were exposed to BrdU (1 mM BrdU per ml medium) for 1 h followed by Isolectin B4/BrdU double labeling. Muscimol application significantly increased cell proliferation in Gabrb3fl/fl periventricular endothelial cells, but Gabrb3ECKO periventricular endothelial cells showed no change. BrdU-labeling indices were quantified in L; Data represent mean ± SD (n = 7, *P < 0.05; Student's t-test; 'M': muscimol). (M) Co-labeling with isolectin B4 and GABA antibodies showed that GABA expression was significantly downregulated in Gabrb3ECKO periventricular endothelial cells when compared to Gabrb3fl/fl endothelial cells. (N) As a result, GABA secretion from E15 Gabrb3ECKO periventricular endothelial cells measured by ELISA was significantly decreased when compared to Gabrb3fl/fl endothelial cells; Data represent mean ± SD (n = 6, *P < 0.05, Student's t-test). (O) A diagrammatic illustration of how endothelial cell-secreted GABA influences critical events during brain development. Wild-type embryonic telencephalon with normal periventricular vascular network (red lattice pattern) and normal endothelial GABA signaling pathway (orange yellow hue) promotes tangential GABAergic neuronal migration (green circles) from the ventral telencephalon where they originate (big green circle). In Gabrb3ECKO telencephalon, there is a partial loss of endothelial GABA secretion (light yellowish hue). This affects periventricular angiogenesis (dotted red pattern) and GABAergic neuronal tangential migration with reduction in GABAergic neurons in the developing neocortex. Scale bars: A, 15 μm; G, 100 μm; (applies to H), J, 50 μm; (applies to K, M).
