Fig. 5: Simultaneous tracking of the reserve and recycling pool of SVs in live SynTKO hippocampal neurons.
a Representative epifluorescence image of a neuronal segment of a Synapsin triple knockout (SynTKO) hippocampal neuron, transfected with Synaptotagmin1-pHluorin (Syt1pH) acquired before addition of anti-green fluorescent protein (GFP) Atto565-tagged nanobodies (At565Nb). The dashed boxes in panel ‘a’ highlight a (ii) presynaptic compartment and (iii) peri-synaptic axonal segment. b Merged maximum intensity projection of Syt1pH-bound anti-GFP Atto647N-tagged nanobodies (Syt1pH/At647Nb; reserve pool; 48 h chase) and Syt1pH/At565Nb (recycling pool; 10 min chase). c Trajectory map of tracked reserve SVs containing Syt1pH/At565Nb in the (ii) presynapse and (iii) axonal segment. d Trajectory map of tracked recycling SVs containing Syt1pH/At647Nb in the (ii) presynapse and (iii) axonal segment. (e) Merged trajectory maps. f, h Average mean square displacement (MSD; µm2) of resting reserve SVs (black), reserve SVs after stimulation (red) and recycling SVs (green) within the (f) presynapses and (h) axons. g, i Area under the MSD curve (AUC; µm2s) for (g) presynapses and (i) axons. Data are displayed as mean ± SEM. Values were obtained from n = 24 presynapses (Resting RP), n = 15 presynapses (Stimulated RP) and n = 16 presynapses (Recycling Pool) in (f, g), from n = 10 axons (Resting RP), n = 7 axons (Stimulated RP) and n = 5 axons (Recycling Pool) in (h, i). Data were obtained from three biological replicates. Statistical comparisons were performed using the one-way ANOVA and Tukey’s multiple comparisons test in (g) and (i). Scale bar 1 µm (a, b) and 200 nm (c–e). Source data are provided as a Source Data file.
