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. 2021 Jul 16;10:e63756. doi: 10.7554/eLife.63756

Figure 9. Action potential trains activate Rac1 in presynaptic terminals.

(A) Experimental design in organotypic hippocampal slices. (B) Schematic of Rac1 sensor. Activation of Rac1 leads to its association with the GTPase-binding domain of Pak2R71C,S78A (PBD2), increasing FRET between GFP and mCherry. This is measured as a decrease in fluorescence lifetime, or an increase in binding fraction. (C) Representative 2pFLIM images of a bouton before and after stimulation for 2 s at 50 Hz. Scale bar, 1 μm. (D) Mean time course of the change in binding fraction of the Rac1 sensor in aCSF (cyan, n=15 boutons/5 slices) with (E) quantification; one-way repeated measures ANOVA (F6,84=3.89, p=0.0018) with Dunnett’s multiple comparisons test vs the baseline (−30–0 s): 0–30 s (p=0.0005), 30–60 s (p=0.0102), 60–90 s (p=0.0142), 90–120 s (p=0.2881), 120–150 s (p=0.6807). (F) Mean time course of Rac1 sensor in TTX (black, n=12/4) with (G) quantification; one-way repeated measures ANOVA (F6,66=0.8539, p=0.5334) with Dunnett’s multiple comparisons test vs the baseline (−30–0 s): 0–30 s (p=0.9930), 30–60 s (p=0.9839), 60–90 s (p=0.6430), 90–120 s (p=0.7654), 120–150 s (p=0.6548). (H) Mean time course of Rac1 sensor in Cd2+ (green, n=13/4) with (I) quantification; one-way repeated measures ANOVA (F6,72=0.2728, p=0.9479) with Dunnett’s multiple comparisons test vs the baseline (−30–0 s): 0–30 s (p>0.9999), 30–60 s (p=0.9996), 60–90 s (p=0.9996), 90–120 s (p=0.9997), 120–150 s (p=0.8896). All data are mean ± SEM. *p<0.05, ***p<0.001, n.s. not significant.

Figure 9.

Figure 9—figure supplement 1. Presynaptic boutons in organotypic slices used for 2pFLIM contain Synapsin.

Figure 9—figure supplement 1.

(A) Representative image of an organotypic hippocampal slice microinjected in CA3 with AAV-mEGFP-Rac1 and AAV-mCherry-PBD2-mCherry on DIV10 and fixed and stained on DIV20 for DAPI (blue), GFP (green), mCherry (magenta), and Synapsin1 (cyan). Scale bar, 300 μm. (B) Representative image of axons in CA1 expressing the Rac1 activity sensor. Green arrow points to a Synapsin1+ bouton, and magenta arrow points to a bouton without Synapsin1 staining. Scale bar, 15 μm. (C) Quantification of percent of GFP+ mCherry+ boutons with Synapsin1 staining (n=142 boutons/8 slices). All data are mean ± SEM.