Our findings suggest that a small recycling pool supports neurotransmission in native central synapses and that the physical position of recycling vesicles in the terminal is an important factor in Erastin their favored stimulus-driven fusion. To label functional vesicle pools in native hippocampal tissue, we prepared acute slices from rat brain and activated CA3 axons while the styryl dye FM1-43 (Betz and Bewick, 1992; Gaffield and Betz, 2006; Ryan et al., 1993) was applied to a target region in CA1 (Zakharenko et al., 2001) (Figure 1A). Confocal imaging revealed clear punctate

fluorescent staining (Figure 1B), the intensity of which was stimulus dependent (0–2,400 action potentials [APs]), consistent with the loading of synaptic vesicles in presynaptic terminals (Figure 1C). Labeling intensity reached saturation when electrical stimulation exceeded 600 APs and this

maximal load was not significantly different from the intensity of synapses labeled with hyperkalemic stimulation (Figure 1C, bottom, see figure legend for statistics). Next, we tested whether labeled terminals were release competent by monitoring fluorescence intensity during a further round of stimulation. Synapses readily underwent activity-evoked destaining consistent with exocytosis and dye loss (Figures 1D and 1E). Across the synaptic population, the timecourse selleckchem of destaining became faster as the stimulation frequency increased but was highly variable between terminals (Figures 1E and 1F), reflecting substantial heterogeneity in individual synaptic release properties similar to previous findings in cultured hippocampal neurons (Branco et al., 2008; Murthy et al., 1997; Waters and Smith, 2002; Welzel et al., 2011). To establish that the recycling pool accessed during these destaining experiments had the same composition as the pool that was dye marked

during the loading protocol—in other words that it was preferentially ADP ribosylation factor reused—we compared our experimental dye loss profiles to simulated destaining curves based on the reuse of varying fractions (0%–100%) of the recycling pool (see Experimental Procedures). The experimental data were best described by the simulated destaining profile corresponding to ∼90% vesicle reuse (see Experimental Procedures), implying that the recycling pool was essentially immutable over the timecourse of our experiments. These results demonstrate the robust stimulus-driven FM dye labeling and subsequent reuse of functionally recycling synaptic vesicles in native hippocampal slice. Next, we used an experimental approach that allows dye-labeled functional vesicle pools to be visualized at ultrastructural level.