Note the high degree of match between EGFP-Synapsin1a and FM4-64 in both merged images. Bar, 10 μm. (B) Bar graphs quantifying the percent colocalization between EGFP-Synapsin and FM4-64 puncta in control cultures (EGFPSyn) or those lacking Piccolo (EGFPSynPclo28; > 800 puncta per condition, two experiments). (C) Bar graph of FM4-64 fluorescence intensity at EGFP-Synapsin1a puncta comparing the relative sizes of the TRP of SVs at control synapses (EGFPSyn) and those lacking Piccolo (EGFPSynPclo28). FM intensity values at EGFPSyn-expressing boutons were normalized against those from neighboring uninfected cells to enable cross-coverslip comparison. Mean normalized FM intensity from control boutons was set to 100; that from Pclo28 boutons was ratioed against this value ( > 800 puncta, two experiments). (D and E) Destaining kinetics of the TRP at 10 Hz (D) and 5 Hz (E) comparing synapses with (EGFPSyn) and without (EGFPSynPclo28) Piccolo ( = 5 experiments per condition). (F) Bar graph comparing the size of the RRP of SVs in boutons with (EGFPSyn) or without (EGFPSynPclo28) Piccolo, as determined by either the application of 500 mM sucrose (left) or 2-Hz, 30-s stimulation (right). No significant differences were found ( test, P > 0.5). Sucrose experiments were performed twice for each condition and stimulation experiments three times. (G) Destaining kinetics of the RRP during 2-Hz, 30-s stimulation for control (EGFPSyn) boutons and those lacking Piccolo (EGFPSynPclo28; = 5 experiments per condition). Error bars indicate SEM.Copyright information:Taken from "Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis"The Journal of Cell Biology 2008;181(5):831-846.Published online 2 Jun 2008PMCID:PMC2396795.