Nevertheless, the finding that compensatory vesicle retrieval in syp−/− became slower only after 300 stimuli argues the first scenario. Indeed, the rate of unitary endocytic events is reported to be largely invariant ( Balaji et al., UMI-77 chemical structure 2008). Therefore, we propose that the role of syp is to maintain endocytic capacity in synapses. At the molecular level, syp may recruit or promote the assembly of endocytic components in order to maintain the number

of available “endocytic machines” during and after sustained neuronal activity. It will be of interest to determine whether SV endocytosis is further affected in syp and synaptogyrin double knockout mice with impaired long-term potentiation, a neural substrate for learning and memory (Janz et al., 1999). Future studies will also focus on the molecular mechanisms through which syp interacts with binding partners—e.g., synaptobrevin II, dynamin I, and adaptor protein-I—to control vesicle recycling (Daly and Ziff, 2002, Edelmann et al., 1995, Glyvuk

et al., 2010 and Horikawa et al., 2002). Given that syb II plays a role in vesicle endocytosis and that syp promotes vesicular localization of syb II, it is tempting to speculate that the selleckchem function of syp in efficient SV endocytosis might involve a physical interaction with syb II (Deak et al., 2004, Hosoi et al., 2009 and Wienisch and Klingauf, 2006). Syt1-pH and sypHy constructs were kindly provided by T.A Ryan (New York, NY) and L. Lagnado (Cambridge,

UK), respectively. SV2A-pH is described in Supplemental Experimental Procedures. A syp knock out mouse line was kindly provided by R. Leube (Mainz, Germany) (Eshkind and Leube, 1995). The mouse line was maintained as heterozygous breeding pairs. Primary hippocampal cultures were prepared as described previously in accordance with the guidelines of the National Institutes of Health, as approved by the Animal Care and Use Committee of the University of Wisconsin-Madison (Liu et al., 2009). Viruses were generated in human embryonic kidney 293T cells as previously described (Dong et al., 2006). See Supplemental Experimental Linifanib (ABT-869) Procedures for details. Neurons were continuously perfused with bath solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM Glucose, 50 μM D-AP5, 10 μM CNQX adjusted to 310 mOsm with glucose, pH 7.4) at room temperature during imaging. During resting or washing steps, the 2 mM Ca2+ was replaced with 2 mM Mg2+. For field-stimulation, 1 ms constant voltage pulses (70 V) were delivered digitally using ClampEX 10.0 via two parallel platinum wires spaced by 10 mm in the imaging chamber (Warner Instruments). Time-lapse images taken at 1 s intervals were obtained on an inverted microscope (Nikon TE300) with a 100× oil objective under illumination with a xenon light source (Lambda DG4).