Fig. 1

Expression of NMDA receptors in the human primary brain endothelial cells and its activation induces Ca²⁺ influx. A and B Expression of the human NMDA receptor subunit 1 (NR1) in the primary human brain endothelial cells were confirmed using western blot (A) and immunofluorescence assay (B). Scale bar indicates 25 μm. C and D (C) Confirmation of NR1 expression on the cell surface. Primary human brain endothelial cells were stained with anti-NR1 (Red, 1:500), wheat germ agglutin (WGA, Green, 1:1000). Arrow indicates where surface localization of NR1 is observed. D Image from (C) was 3D reconstructed by LAS X 3D visualization software. E Expression of NR1 on the brain endothelial cells in vivo. Brain endothelial cells were stained with CD31 (Red), NR1 with anti-NR1 (Red, 1:100). Scale bar indicates 5 μm. F and G (F) Application of NMDA induces influx of Ca²⁺ in the brain endothelial cells (upper panel) that is blocked with application of MK801, a specific blocker of NMDA receptor (bottom panel) that was measured by real time confocal microscopy. Scale bar indicates 75 μm. G Relative intensity of Fluo 4 was quantified and was presented as a graph (n = 4, two-tailed unpaired student t-test, ** indicates where p value is less than 0.01)