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Figure 4 | Molecular Brain

Figure 4

From: Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis

Figure 4

Best1-mediated glutamate release from cultured astrocytes by using glutamate-sensitive FRET sensor, GluSFnR. A) Schematic diagram of principles of FRET-based glutamate sensor and imaging. Released glutamate from astrocyte binds to glutamate binding motif of GluSnFR (glutamate FRET sensor), resulting in decrease of FRET between CFP and YFP or increased CFP/YFP ratio. B) Representative FRET images from GluSnFR-expressing cultured astrocytes processed by CFP/YFP emission ratio. The white bar shown at 0 s image indicates the puff of TFLLR (500 μM, 100 msec). C) Astrocytic GluSFnR responses (CFP/YFP ratio change) by respective puff-treated extracellular glutamate concentration at the time point indicated by an arrow. D) The graph showing the relationship between glutamate concentration and peak value of relativeCFP/YFP ratio. The calculated EC50 was ~ 25.57 μM. E) The graph shows the averaged relative CFP/YFP ratio values from time-lapse imaging using GluSFnR expressing-cultured astrocytes. Scrambled shRNA-expressing astrocytes (Sc-shRNA); Best1-shRNA-expressing astrocytes (B1-shRNA); naïve astrocytes pretreated with BAPTA-AM (BAPTA; 30 μM); naïve astrocytes pretreated with niflumic acid (NFA; 100 μM); naïve astrocytes treated with recording buffer puff (Buffer puff). F) Bar graph representing averaged relative CFP/YFP ratio (analyzed during the period indicated by the gray box in Figure 1F). *p < 0.05, **p < 0.01 vs. Sc-shRNA-expressing group. Numbers on each bar indicate number of cells from at least three independent culture batches.

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