G-Protein Coupled Receptor-Evoked Glutamate Exocytosis from Astrocytes: Role of Prostaglandins
Figure 2
Analysis of VGLUT1-pHluorin vesicles that colocalize with markers of early or recycling endosomes. (a)–(d) Estimation of the size of vesicles expressing VGLUT1-pHluorin. Analysis of individual vesicle was performed in confocal images of VGLUT1-pHluorin-expressing astrocytes by plotting fluorescence intensity of pHluorin spots against distance from the centre of the spot (black curve ± SD). Such an analysis provided an estimation of the average fluorescence profile otherwise called “radial sweep” [23]. The fluorescence intensity values obtained from the radial sweep were well fitted by a one-dimensional Gaussian function (red curve). Such a curve represents the average radial sweep value obtained from 20 vesicles. Note that the half maximum value of pHluorin-expressing vesicle positive for EAA1 ((b), marker of early endosomes, nm) is similar to that of 200 nm fluorescent beads ((a), nm) and the half maximum value of pHluorin-expressing vesicle that do not express EAA1 ((d), nm) is similar to that of 40 nm fluorescent beads ((c), nm). (e), (f) Temporal distribution of VGLUT1-pHluorin and Alexa-Tf 568 fusion events evoked by DHPG application. (e) Each individual histogram represents the number (mean ± SD) of fusion events detected from VGLUT1-pHluorin vesicles in a 50 ms-long frame ( cells). (f) Fusion events (mean ± SD) detected from VGLUT1-pHluorin and Alexa-Tf568 double positive vesicles in the same cells as in (e). Each histogram represents the number of fusion events detected in a 50 ms-long frame ( cells).
