Figure 5.

Functional EM Analysis Reveals an Increase in the Number of Docked Vesicles during PTP

(A) Left: schematic illustration of “flash and freeze” experiments at hippocampal mossy fiber synapses. Thin hippocampal slices were embedded in a sandwich between two sapphire blades, hippocampal GCs were stimulated by light pulses, and slices were subjected to high-pressure freezing at defined time intervals for subsequent EM analysis (Borges-Merjane et al., 2020).

(B) Example transmission EM (TEM) micrographs of active zones of hippocampal mossy fiber synapses in control slices (left), slices frozen immediately after stimulation with 100 light pulses (center), and slices frozen 20 s after stimulation (right). Images were taken from the mossy fiber tract in the stratum lucidum of the CA3 region, primarily CA3b and CA3c. White bars indicate postsynaptic densities.

(C–E) Scatterplot (C), cumulative distributions (D), and histograms (E) of the number of docked vesicles per 100 nm of active zone profile length for a non-light stimulated control (black), slices frozen immediately after HFS_100APs stimulation at 20 Hz (blue), and slices frozen after HFS_100APs stimulation + 20 s recovery time (red). Bars show mean and standard deviation. Each data point in (C) represents an active-zone profile. Note that the number of docked vesicles increased 20 s after HFS, indicating “overfilling” of the docked vesicle pool.

(F–H) Similar plots as shown in (C)–(E) but for diameter of docked vesicles. The diameter of docked vesicles was slightly but significantly larger in the HFS_100APs stimulation + 20 s recovery time in comparison with control experiments. Each data point in (F) represents a docked synaptic vesicle. Data for the HFS_100APs stimulation (blue) include measurements taken from a previous study (Borges-Merjane et al., 2020).

In (C) and (F), boxes indicate mean values, and error bars denote standard deviation. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001.