Figure 5.

Increased dye labeling and faster activity-dependent destaining kinetics after PMA treatment during spontaneous dye uptake. A, Synapses were treated with 1 μm PMA or 4α-PMA either during spontaneous FM2-10 dye loading (in 4 mm K⁺/2 mm Ca²⁺ and 1 μm TTX) or after 5 min of dye washout for 10 min. After an additional 5 min of dye washout, synapses were imaged during multiple rounds of 90 mm K⁺ application. B, Plot showing that the average median size of the dye-labeled pool increases when PMA is present during dye loading (n = 5-9 coverslips for each condition). ^***p < 0.001 for PMA during dye load compared with all other loading conditions. C, Plot showing the average kinetics of dye destaining in response to high K⁺ application in different loading conditions. Dye destaining in synapses in which PMA was present during the loading protocol show biphasic release typical of dye release from the evoked pool. Dye release in all other conditions shows monoexponential slow dye loss typical of high K⁺ destaining of the spontaneous vesicle pool. D, The fast time constant of release in a double-exponential fit of dye release under 90 mm K⁺ application is faster in synapses loaded in the presence of PMA compared with other loading paradigms. Here, we should note that double-exponential fits were used for all traces to facilitate quantification and comparison. Traces from individual experiments could be more reliably described with two exponentials (albeit slow ones for all conditions except for “PMA during”), although the average traces shown in C (again except for “PMA during”) may not seem to justify double-exponential fits. E, The slow time constant in a double-exponential fit of the release kinetics was not altered by PMA. F, The ratio of the amplitude of the fast-to-slow component of dye release was significantly larger in synapses that were loaded in the presence of PMA. ^**p < 0.005 between PMA during load and all other loading conditions. Error bars indicate SE.