Fig 2: cPAF enhances presynaptic vesicle exocytosis. (A–D) The Syn-pHluorin fluorescence (F) at single boutons (from primary hippocampal cultures transfected with Syn-pHluorin) increases after each 100 pulse stimuli at 10 Hz as a portion of labeled vesicles are exocytosed and then returns to baseline as synaptic vesicles are internalized. Treatment with NH4Cl raises the pH of all internal vesicle and results in maximal pHluorin fluorescence representative of the total vesicle pool. All Syn-pHluorin measurements are normalized to the ?F with NH4Cl. (A) Representative fluorescence images of single boutons treated with 1 µM cPAF or vehicle. Images are 4.3 µm × 4.3 µm. (B) Examples of individual traces (?F/?FNH4Cl) from representative boutons treated with 1 µM cPAF. (C) Traces show the average Syn-pHluorin ?F/?FNH4Cl over time for all boutons treated with 1 µM cPAF or vehicle. Vehicle, 20 min: 263 boutons from seven coverslips; cPAF, 20 min: 120 boutons from five coverslips. (D) Average ?F/?FNH4Cl response to the two before treatment 100 pulse stimuli shown overlapping the average response to the two 100 pulse stimuli given after 1 µM cPAF or vehicle treatment. (E) Scatter plot of the average peak amplitude of ?F/?FNH4Cl induced by 100 pulse stimuli before and after 20 min treatment with vehicle or 1 µM cPAF. (F) Non-linear regression of decay part of ?F/?FNH4Cl trace (in D) was used to quantitate the endocytic decay constant (k) followed by testing the null hypothesis of one rate for all data sets (p = 0.807). (G) Average peak amplitude of ?F/?FNH4Cl induced by 100 pulse stimuli before and after 20 min treatment with vehicle or 1 µM cPAF. Statistical analysis was performed using two-way ANOVA with repeated measures followed by Sidak’s multiple comparison tests. (H) Quantification of the % change in peak amplitude due to 20 min treatment of vehicle or 1 µM cPAF. Statistical analysis used paired Student’s t-test. (I) Quantification of the % change in peak amplitude due to 2 min treatment of vehicle or 1 µM cPAF. Some samples were also pretreated 30 min with BN52021 (a PAFR inhibitor) or PKCi (a PKC inhibitor). Statistical analysis used one-way ANOVA with Sidak’s multiple comparison tests. For all graphs statistical significance is indicated by the following markings: *p < 0.1; **p < 0.01; ***p < 0.001; ns = not significant. Error bars are ±SEM.