For this study and to address our questions, we optimized a technique to use state-of-the-art methods and directly correlate physiology with ultrastructure of synapses. We performed high-pressure freezing (HPF) of live acute brain slices with optogenetic stimulation immediately prior to freezing, with millisecond precision, to capture presynaptic changes after action potential evoked synaptic transmission under physiological conditions. We were able to: (1) express the light-activated channel channelrhodopsin specifically in granule cells in hippocampus dentate gyrus; (2) characterize light-evoked responses in both granule cells and CA3 pyramidal neurons with electrophysiology, matching those evoked traditionally with electrical stimulation; (3) successfully freeze acute slices with a new HPF system still hardly used by many groups in the world; (4) stimulate mossy fibers in the HPF chamber with light and successfully evoke synaptic transmission; (5) apply mild and strong stimulation paradigms, with evidence of depletion of docked vesicles with strong stimulus, as well as evidence of ultrafast endocytosis.
Such depletion of docked vesicles suggests that docked vesicles represent the releasable vesicle pool and therefore that docked vesicles can be used as a structural correlate for the readily-relesable pool (RRP) of vesicles. We however noticed that after this depleting-protocol, some vesicles remained docked. We interpret this result in two fold. Firstly, this could indicate that not all docked vesicles are part of the RRP, although the majority of them are. However this could also indicate that at some point during the stimulus, rapid recruitment and replenishment of the docked vesicle pool started to happen. Thus, this method now also allows for future studies to investigate kinetics of vesicle recruitment and replenishment at the active zone, by looking at different time points after evoked synaptic transmission, with different levels of activity.
