The data reported here firmly establish a role for syp in facilitating rapid and efficient SV endocytosis in mammalian central neurons. Syp
−/− neurons exhibited defective SV endocytosis both during and after neuronal activity while exocytosis and the size of the total recycling pool of SVs were unaffected. Truncation of the C-terminal tail of syp led to slower endocytosis during neuronal activity, consistent with a previous study in which a tail fragment was injected into the squid giant axon (Daly et al., 2000
). However, the same truncation mutant had no effect on endocytosis after neuronal activity; hence, the two endocytic processes - one during stimulation and another that is employed after stimulation - are controlled by distinct domains of syp.
The observed defects in SV endocytosis in syp
−/− neurons result in functional consequences including the pronounced depletion and slower recovery of the recycling SV pool. The slow time constant of post-stimulus endocytosis might seem to be at odds with the rapid divergence of the synaptic depression time-course between wt and syp
−/− neurons during sustained stimulation (). Such rapid depression observed in syp
−/− neurons prompted us to test the possibility that rapid retrieval, or ‘kiss and run’ endocytosis of vesicles, is affected in the absence of syp. We note that whether kiss-and-run/fast retrieval (within ~ 1 sec) is a common mode of endocytosis in hippocampal synapses remains the subject of debate (Balaji et al., 2008
; Ertunc et al., 2007
; Granseth et al., 2006
; Zhang et al., 2009
). We calculated the rate of vesicle retrieval, that occurs during stimulation, as a fraction of the total recycling pool (as determined by the maximal ΔF values in the Baf traces) (). For wt neurons, only ~1.3 % of total recycling pool appears to undergo endocytosis within 1 s; this result argues against the notion that the rapid retrieval (i.e. ‘kiss-and-run’) predominates during sustained transmission. Hence, these results indicate that the rapid divergence of the synaptic depression time-course between wt and syp
−/− neurons cannot be attributed to loss of putative rapid endocytosis.
An alternative explanation for the pronounced synaptic depression in syp
−/− neurons is that syp might regulate another relatively rapid step, such as the clearance of vesicle release sites (Neher, 2010
). Interactions between SNARE proteins on vesicular and target membranes need to be disrupted after exocytosis to allow vesicle recycling. Syp might facilitate this process by binding to synaptobrevin II and clearing it from active zones. The loss of syp might lead to a ‘traffic jam’ of vesicular components at release sites and thereby contribute to synaptic depression during sustained activity. However, it is not known whether the clearance of release sites is a rate-limiting step in hippocampal synapses. Finally, we note that read-outs from pHluorin imaging experiments and physiological recordings might not be directly comparable with each other due to several technical differences. These include (imaging vs electrophysiology) different methods of stimulating neurons (field stimulation vs local stimulation) and differences in temporal resolution (‘s’ vs ‘ms’). Therefore, there are caveats regarding direct comparison of data from these two experimental approaches.
We consider the following possibilities regarding how SV endocytosis can be affected in the absence of syp: (1) unitary endocytic events become slower, or (2) number of SVs that can be retrieved at the same time, ie. ‘endocytic capacity’ is reduced while endocytosis of individual SVs remains unaffected (Balaji et al., 2008
). Since we only measured the macroscopic time-courses of vesicle retrieval, we cannot completely distinguish between these two possibilities. 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., 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
; 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 function of syp in efficient SV endocytosis might involve a physical interaction with syb II (Deak et al., 2004
; Hosoi et al., 2009
; Wienisch and Klingauf, 2006