, 2010). Whether similar developmental changes also take place at much smaller cortical synapses is unclear. Experiments on acute hippocampal and neocortical slices suggested that short-term plasticity and Pr at small synapses develop similar to the calyx (Bolshakov and Siegelbaum, 1995; Feldmeyer and Radnikow, 2009; Reyes and Sakmann, 1999). In dissociated culture, on the other hand, endocytosis has been reported to be stable over time (Armbruster and Ryan, 2011), and the vesicle retrieval rate at saturating stimulation intensity (Sankaranarayanan and Ryan, 2000), that is, endocytic

capacity, remains low compared to the mature calyx (Renden and von Gersdorff, 2007; Wu et al., 2009). The total number of vesicles increases during development (Mozhayeva et al., S3I-201 solubility dmso 2002), but it is unclear if partitioning into functional and nonfunctional pools is also developmentally regulated. We set out to study the maturation of presynaptic function at Schaffer collateral (SC) synapses

in slice cultures of rat hippocampus, a preparation that closely recapitulates postnatal development (De Simoni et al., 2003). To measure the fraction of released and recycling vesicles, we developed a dual-color release indicator (ratio-sypHy) for use in intact tissue. In immature slice Galunisertib cultures, after 5–7 days in vitro (DIV 5–7), a sizable fraction of vesicles could not be released. In 2–4 week cultures, however, essentially all vesicles were mobilized in response to either high-frequency AP trains or typical CA3 cell place field activity. The rate of vesicle retrieval increased about 7-fold during maturation. Chronic depolarization induced a sizable resting pool at mature SC boutons. Therefore, SC boutons are capable

of reducing their output by removing vesicles from the recycling pool, but this homeostatic mechanism seems to be activated only during periods of pathologically high activity. We conclude that synapses in CA1 undergo a pronounced refinement of vesicle use and recycling during early postnatal development. Our indicator is based on a fusion protein of the pH-sensitive GFP variant superecliptic pHluorin (Miesenböck et al., 1998) with the synaptic vesicle protein synaptophysin I (sypI), a these combination known as sypHy (Granseth et al., 2006). To create a dual emission indicator suitable for ratiometric two-photon microscopy, we fused the extraluminal C terminus of sypHy with the dimeric red fluorescent protein tdimer2 (ratio-sypHy; Figure 1A). The C-terminal tdimer2-tag faces the cytoplasm, providing a red fluorescence signal proportional to the total amount of ratio-sypHy present at a synapse. Because of the fixed stoichiometry, the green-to-red fluorescence ratio of ratio-sypHy was independent of expression level and depth of the synapse in the tissue and could be compared across cells.