Panel A includes line traces and dot plots. The line traces show the average fluorescence of SypHy upon 20 Hertz stimulation with 200 action potentials (APs) in Control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST (blue), and CAST/ELKS-cDKO with ELKS (green) neurons. The x-axis represents time in seconds, and the y-axis represents normalized fluorescence. The dot plots show the peak of 200 APs and the decay constant (tau) after stimulation. Panel B includes line graphs and dot plots. The line graphs show the total recycling pool size in Control (black) and CAST/ELKS-cDKO (red) neurons upon 20 Hertz stimulation with 600 APs. The x-axis represents time in seconds, and the y-axis represents normalized fluorescence (delta F/ delta F NH4Cl). The dot plots show the peak of 600 APs and the rise constant (tau) during stimulation. Panel C includes line graphs and dot plots. The line graphs show the average for estimating the surface fraction of SypHy and vesicular pH in Control (black) and CAST/ELKS-cDKO (red) neurons. The x-axis represents time in seconds, and the y-axis represents normalized fluorescence (delta F/ delta F NH4Cl). The dot plots show the quantification of the surface fraction ratio of SypHy and vesicular pHs.
Measurement of the SV recycling process in CAST/ELKS-cDKO hippocampal neurons. (A) SV release and endocytosis kinetics in CAST/ELKS cDKO neurons. Activity-dependent SypHy fluorescence response in control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST (blue), or CAST/ELKS-cDKO with ELKS (green) in mouse hippocampal cultured neurons. Left traces show average fluorescence of individual SypHy upon 20 Hz stimulation with 200 APs. Results are mean ± SEM. n = 10 images, each. Right dot plots show the peak of 200 APs (left) and the decay constant (tau) after stimulation (right). P values were obtained by comparison with cDKO neurons (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, *P < 0.05, ***P < 0.001). (B) TRP size and SV replenishment kinetics in CAST/ELKS-cDKO neurons. Left traces show the TRP size in control (black) or CAST/ELKS-cDKO (red) neurons. Results are mean ± SEM. n = 15 images, each. Right dot plots show the peak of 600 APs (left) and the rise constant (tau) during stimulation (right). P values were obtained by nonparametric Mann–Whitney test, *P < 0.05, ****P < 0.0001. (C) Surface ratio of SypHy and vesicular pH of SypHy-bearing vesicles in the CAST/ELKS cDKO neurons. Left traces show the average for estimating the surface fraction of SypHy and vesicular pH in control (black) or CAST/ELKS-cDKO (red) neurons. Results are mean ± SEM. n = 10 images, each. Right dot plots show quantification of the surface fraction ratio of SypHy (right) and vesicular pHs (left). P values were obtained by nonparametric Mann–Whitney test, **P < 0.01.