Fig. 4

Shedding of vesicular contents (exocytosis) from microglia expressing the pore or non-pore-forming P2X7R was visualized by labeling the cells with the membrane permeant dye FM1-43 and measuring the changes in cell surface fluorescence upon P2X7R stimulation. ATP and BzATP were used to stimulate P2X7R. a An activated microglia expressing the pore-forming P2X7R, which was then loaded with FM1-43. Red: FM1-43. Green: P2X7R-EGF expression. Upon ATP stimulation, there appeared to be the presence of vesicular structures arising on the surface of the cell (arrows) which were then released into the extracellular milieu. The images are from 0 to 3.5 min after stimulation with 3 mM of ATP. b The cell membrane fluorescence increased with ATP stimulation (coinciding with fusing of the vesicular membrane with cell surface membrane). This was then followed by a decreased in membrane fluorescence. Arrow indicates time of application of 3-mA ATP. c The change in membrane fluorescence (measured in experiments conducted up to 25 min) in response to ATP (3 mM) was significantly higher in cells expressing P2X7R-EGFP (n = 13 cells from 13 different rats) compared to those expressing P2X7RG345Y-EGFP (n = 12 cells from 12 different rats)