This finding is broadly consistent with a landmark study in dissociated hippocampal cultured neurons looking at a different functional pool—the readily releasable pool—which characterized the tendency for vesicles to occupy positions close to the active zone (Schikorski and Stevens, 2001). In theory, our total recycling pool could include a subset of preferentially reused vesicles (Ertunc et al., 2007; Pyle et al., 2000) and the spatial bias we observe here could be indicative of a fast mode of recycling (Gandhi and Stevens, 2003; Park et al., 2012; Zhang et al.,

2009); further work will be find more needed to test the relevance of these ideas in native terminals. To explore the generality of our findings, we also used a modified form of our FM dye photoconversion method to characterize the nanoscale appearance of functional vesicle pools in vivo, in this case, specifically recruited by activity driven by defined sensory input. This report establishes an experimental strategy for delineating function-ultrastructure characteristics of synapses from intact brain. Notably, selleck chemical our findings regarding functional pool organization in visual cortex were highly consistent with those in hippocampal slices: functional vesicles were preferentially located near the active zone, suggesting that this is a shared feature among different types of small central

synapses. We investigated a possible role for the cytoskeletal element actin as a candidate in contributing to spatial segregation. We showed that stabilizing actin with jasplakinolide these disrupted the preferential distribution of recycling vesicles, indicating that remodeling actin is important

in facilitating the repositioning of recycling vesicles toward the active zone after endocytosis. These findings are broadly compatible with the current model for actin function in the presynaptic terminal as a scaffolding element, guiding vesicle-associated components to their destination during repeated cycles of activity (Sankaranarayanan et al., 2003; Shupliakov et al., 2002) (also see Pechstein and Shupliakov, 2010). Importantly, we show that actin stabilization, and by association the abolition of preferential recycling pool distribution, does not prevent vesicle turnover but does affect the rate of release; experiments measuring FM dye loss show clear stimulation-evoked destaining but notably the timecourse of exocytosis is significantly slower compared to controls. Given that a clear direct role for actin in driving synaptic vesicle exocytosis has not been established (Sankaranarayanan et al., 2003), the effects we observe most likely result from disruption of the recycling pool distribution. We suggest that the preferential spatial positioning of functional vesicles might contribute to efficient vesicle release during sustained activity. Interestingly, the segregation of recycling vesicles toward release sites is not a universal property of presynaptic terminals.