Panel A shows a western blot analysis of CAST, ELKS, and Tubulin protein levels in mouse hippocampal neurons from CAST-flox/ELKS-flox mice. The blot compares Control (Cre-mutant), CAST/ELKS-cDKO (Cre), CAST/ELKS-cDKO with CAST (Cre with CAST), and CAST/ELKS-cDKO with ELKS (Cre with ELKS). Panel B presents line traces of activity-dependent Syn-GCaMP8s fluorescence response in Control (black) and CAST/ELKS-cDKO (red) neurons upon a 10 Hertz, 10 AP stimulation. The right dot plots show the fluorescence response at the 1st AP and 10th AP. Panel C displays line traces of activity-dependent iGluSnFR3 fluorescence response in Control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST (blue), and CAST/ELKS-cDKO with ELKS (green) neurons upon a 10 Hertz, 10 AP stimulation. The right dot plots show the fluorescence response at the 1st AP and 10th AP. Panel D shows a line graph of sucrose solution-mediated iGluSnFR3 fluorescence response in Control (black) and CAST/ELKS-cDKO (red) neurons upon 250 mM sucrose solution stimulation. The right dot plot shows the increase in fluorescence of iGluSnFR3 in response to 250 mM sucrose solution for each image.
Measurement of calcium concentration at presynaptic terminal and glutamate release in CAST/ELKS-cDKO hippocampal neurons. (A) CAST/ELKS protein expression level in mouse hippocampal neurons from CAST-flox/ELKS-flox mouse. CAST, ELKS, and tubulin were detected by the western blot analysis in control (Cre-mutant), CAST/ELKS-cDKO (Cre), CAST/ELKS-cDKO with CAST (Cre with the short CaMKII promoter, Ca0.3-derived CAST), and CAST/ELKS-cDKO with ELKS (Cre with Ca0.3-derived ELKS). (B) Calcium concentration at presynaptic terminal in CAST/ELKS cDKO neurons. Activity-dependent Syn-GCaMP8s (synaptophysin-GCaMP8s) fluorescence response in control (black) or CAST/ELKS-cDKO (red) mouse hippocampal cultured neurons. The left traces show the average fluorescence of individual Syn-GCaMP8s upon a 10 Hz, 10 AP stimulation. Results are mean ± SEM. n = 15 images, each. The points for the 1st AP and 10th AP are indicated by arrows and arrowheads, respectively. The right dot plots show the 1st AP (right) and the 10th AP data (left). P values were obtained by nonparametric Mann–Whitney test. (C) Released glutamate in CAST/ELKS-cDKO neurons. Activity-dependent iGluSnFR3 fluorescence response in control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST (blue), or CAST/ELKS-cDKO with ELKS (green) in hippocampal neurons. The left traces show average fluorescence of individual iGluSnFR3 upon the 10th AP stimulation (10 Hz). Results are mean ± SEM. n = 15 images, each. The points for the 1st AP and 10th AP are indicated by arrows and arrowheads, respectively. The right dot plots show results from the 1st AP (right) and 10th AP (left). P values were obtained by comparison with cDKO neurons (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, *P < 0.05, **P < 0.01, ****P < 0.0001). (D) RRP size in CAST/ELKS cDKO neurons. The sucrose solution mediated iGluSnFR3 fluorescence response in control (black), CAST/ELKS-cDKO (red) in the hippocampal neurons. The left trace shows average fluorescence of individual iGluSnFR3 upon 250 mM sucrose solution stimulation. Results are mean ± SEM. n = 10 images, each. The right dot plot shows the increase in fluorescence of iGluSnFR3 in response to 250 mM sucrose solution for each image. P values were obtained by nonparametric Mann–Whitney test, **P < 0.01. Source data are available for this figure: SourceData F1.