Figure 6. Analysis of synaptic vesicle recycling pool size and kinetics of exocytosis using FM dye.

(A) Schematic of the FM1-43 dye loading and unloading protocol used to determine recycling vesicle pool size and destaining kinetics. Synaptic vesicles were loaded with FM1-43 for 2 min using 45 mM KCl, washed for 5 min and then unloaded with 45 mM KCl during image acquisition at 1 second intervals. (B), Quantitation of FM1-43 destaining in autaptic synapses and interneuronal synapses. A baseline of 10 images was collected prior to destaining by addition of 45 mM KCl, indicated by the arrow. (C) Recycling vesicle pool size calculated from the total FM1-43 fluorescence loss from boutons following stimulation for autaptic and interneuronal synapses. (n = 41; 5 different coverslips for autaptic synapses and n = 55; 6 different coverslips for interneuronal synapses). Statistical significance was determined by a Student’s t-test. ***p<0.001. All data shown represent mean ± SEM.