Figure 4.

Activity is not necessary during PMA treatment for redistribution of vesicles between pools. A, Separate sets of synapses were treated with PMA or 4α-PMA either during loading with 47 mm K⁺ or after 5 min of dye washout. After washout of dye and residual PMA for an additional 5 min, synapses were imaged during a 15 s application of 500 mm sucrose, followed by multiple rounds of 90 mm K⁺ solution for maximal destaining. B, Sample responses of synapses from a single coverslip during 15 s of 500 mm sucrose application. Synapses treated with PMA showed faster kinetics of dye loss compared with synapses treated with 4α-PMA as a control. C, Total vesicle pool size as measured by the dye fluorescence trapped in synapses (ΔF) was not significantly different between the different loading paradigms (n = 6-7 coverslips each), although there was a tendency toward a smaller pool size in cultures treated with PMA after dye loading and washout compared with 4α-PMA, suggesting some spontaneous dye loss after PMA treatment (PMA treatment after load, 75 ± 11% of 4α-PMA-treated synapses; p = 0.12). D, The ratio of dye loss during sucrose application to the total pool size, labeled Δsuc/ΔF, showed a significant increase in synapses treated with PMA either during or after dye loading (n = 6-7 coverslips each). ^*p < 0.05; ^**p < 0.02. Error bars indicate SE.