Fig. 2

Synaptic expression of SynaptoZip and activity-dependent uptake of SB. a Presynaptic boutons expressing GZ (green) in acute hippocampal slices (Schaffer collateral, CA1). Slices were bathed with SB (red; SB 5 nM, 5 min, 24 °C, 30 mM isosmotic KCl). b–d Super-resolution microscopy of cultured CA3-CA1 hippocampal synapses. b Presynaptic boutons expressing GZ (green), with superimposed labeled synaptic vesicles, after spontaneous uptake of SB (hot red LUT; SB 5 nM; 60 min incubation, 37 °C). c Magnified view of synaptic vesicles loaded with SB. d Analysis of vesicles diameter following spontaneous uptake of SB (N = 428 SB-labeled vesicles from 24 synapses; 41.2 ± 16.2, mean ± SD). e, f Time-lapse imaging of SB uptake (SB 5 nM, 24 °C; color map, LUT at the bottom) in a group of boutons along a single axon (cultured hippocampal CA3–CA1 neurons), before and during trains of presynaptic action potentials (APs). In f, green lines are normalized SB uptake time series for N = 18 individual boutons (same experiment as in e; thick black line is the average SB uptake trace; blue diamonds correspond to time frames shown in e). The red bar refers to the presence of SB 5 nM in the bath, the blue bar indicates the stimulation epochs (trains of 10 AP at 10 Hz inter-leaved with 4 s pauses). Pale blue areas signal the occurrence of stimulation when SB is present. On top of graph, expanded traces to illustrate SB uptake (black lines; scale bar: 0.05 a.u. vs. 2 min) from 4 out of the 18 boutons (indicated by white arrowheads in e, GZ), and its rate (green lines, time derivative; scale bar: 0.05 a.u. h⁻¹ vs. 2 min), during the second stimulation epoch. Notice that, despite the common trend, each synapse shows its own SB uptake dynamics. Scale bars: 4.11 µm (a, e), 400 nM (b), 40 nM (c)