Fig 2: Coupling distance between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin.(A) Example scan in ZnT3-positive area of the CA3 stratum lucidum in 100-µm-thick hippocampal slices: confocal scan (top), raw gSTED scan (middle), and deconvolved gSTED scan (bottom). Staining for Cav2.1 (green), Munc13-1 (magenta), and Homer1 (cyan). (B) Example of an analyzed synapse: The distance between Cav2.1 (green) and Munc13-1 (magenta) was measured only if they were in close proximity to a Homer1-positive spot (cyan). Line profiles were plotted at the dotted line (top), drawn through the intensity maxima of the Cav2.1 and Munc13-1 signals (arrowheads). The distance was calculated between the intensity maxima of the Cav2.1 and Munc13-1 signals, shown in the example normalized intensity plots for control (middle) and forskolin-treated (bottom). (C) The distribution of measured distances between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin-treated (p = 0.81, 2-sample Kolmogorov–Smirnov test). Frequency distribution (left y-axis, bars) and cumulative frequency (right y-axis, lines) with a bin size of 20 nm, for control (blue) and forskolin-treated (red). (D) The mean distance between Cav2.1 and Munc13-1 in the CA3 stratum lucidum is unchanged in control versus forskolin-treated. Scatter plot from all measured synapses: distances (nm) for CA3 control in blue (n = 584 synapses from 11 animals) and CA3 forskolin-treated in red (n = 525 synapses from 11 animals). Bar graphs show mean values ± SEM. Significance tested with Mann–Whitney U test (p = 0.62). CS, cumulative frequency; CTRL, control; FD, frequency distribution; FSK, forskolin; gSTED, time-gated stimulated emission depletion; KS, Kolmogorov–Smirnov test; MW, Mann–Whitney U test. The data underlying this figure can be found at doi: 10.5281/zenodo.4498214.

Fig 3: EPAC and PKCe act on vesicle docking, synaptic release, and Rab3-RIM1-Munc13 complex.(A) Representative EM (23,000×) of PF-PC synapses of WT and Rapgef3/4-dKO mice. Scale bars: 200 nm. The inserts show docked vesicles. Unpaired t test. ****p<0.0001. (B) Representative EM of PF-PC synapses of Prkcef/f and Prkce-cKO mice. Scale bars: 200 nm. Unpaired t test. ****p<0.0001. (C) Example PC mEPSCs in Atoh1Cre and Rapgef3;Rapgef4-cKO mice. Lower: statistics of inter-event interval and amplitude. Grey dots indicate individual data points. Frequency: 2.0±0.2 Hz (Atoh1Cre) and 1.4±0.2 Hz (Rapgef3;Rapgef4-cKO; p=0.0036). Amplitude: 18.3±1.3 pA (Atoh1Cre) and 18.5±1.3 pA (Rapgef3;Rapgef4-cKO; p=0.46). Unpaired t test. n=for all groups. **p<0.01. (D) Example PC mEPSCs from Prkcef/f and Prkce-cKO mice. Frequency: 1.9±0.1 Hz (Prkcef/f; n=19) and 1.3±0.1 Hz (Prkce-cKO; n=20; p=0.00059). Amplitude: 17.9±1.2 pA (Prkcef/f; n=19) and 17.5±1.1 pA (Prkce-cKO; n=20; p=0.39). Unpaired t test. ***p<0.001. (E) Representative responses of Atoh1Cre and Rapgef3;Rapgef4-cKO PCs to 100 Hz PF stimulation. The artifacts were truncated and each EPSC were aligned to its initial rising point. RRP was defined as the y-intercept of linear portion of cumulative amplitude curve. For RRP (inset), Atoh1Cre: 861±113; Rapgef3;Rapgef4-cKO: 790±101; p=0.31, unpaired t test. For cumulative amplitude, Atoh1Cre: 5815±360 pA; Rapgef3;Rapgef4-cKO: 3848±66 pA; p<0.001, unpaired t test. For Pr, Atoh1Cre: 0.17±0.03; Rapgef3;Rapgef4-cKO: 0.11±0.01; p=0.043, unpaired t test. n=7 for both groups. *p<0.05. ***p<0.001. (F) Representative responses of Prkcef/f and Prkce-cKO PCs to 100 Hz PF stimulation. The artifacts were truncated and each EPSC was aligned to its initial rising point. For RRP, Prkcef/f, 764±100; Prkce-cKO, 728±106, p=0.40, unpaired t test. For cumulative amplitude, Prkcef/f, 5940±337 pA; Prkce-cKO, 3755±181 pA; p<0.001, unpaired t test. For Pr, Prkcef/f, 0.19±0.04; Prkce-cKO, 0.12±0.01; p=0.034, unpaired t test. n=7 for both groups. *p<0.05. ***p<0.001. (G) Cerebellar synaptosomes from WT and Rapgef3/4-dKO mice were immunoprecipitated by anti-RIM1 antibody, and the immunoprecipitates were probed with antibodies to Munc13-1, Rab3A, and RIM1. Rabbit IgG was negative control. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to WT. Munc13-1: 100 ± 6% (WT) and 62 ± 8% (Rapgef3/4-dKO; p=0.0081, n=4). Rab3A: 100 ± 5% (WT) and 63 ± 10% (Rapgef3/4-dKO; p=0.019, n=4). Total Rab3A and RIM1 were normalized to WT. Munc13-1: 100 ± 2% (WT) and 98 ± 4% (Rapgef3/4-dKO; p=0.73, n=6). Rab3A: 100 ± 5% (WT) and 98 ± 4% (Rapgef3/4-dKO; p=0.77, n=6). Unpaired t test. *p<0.05. **p<0.01. (H) Immunoprecipitation of Munc13-1 and Rab3A with RIM1 in cerebellar synaptosomes from Prkcef/f and Prkce-cKO mice. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to WT. Munc13-1: 100 ± 2% (Prkcef/f) and 70 ± 8% (Prkce-cKO; p=0.0030). Rab3A: 100 ± 2% (Prkcef/f) and 89 ± 4% (Prkce-cKO; p=0.019). Total Rab3A and RIM1 were normalized to Prkcef/f. Munc13-1: 100 ± 3% (Prkcef/f) and 96 ± 5% (Prkce-cKO; p=0.46). Rab3A: 100 ± 7% (Prkcef/f) and 106 ± 5% (Prkce-cKO; p=0.52). n=6 for all groups. Unpaired t test. *p<0.05. **p<0.01. (I) Cerebellar synaptosomes (WT) mice were incubated in control buffer or 8-pCPT (20 µM, 30 min) and eV1-2 (5 µM, 30 min) and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to control. Munc13-1: 100 ± 8% (Ctrl); 138 ± 12% (8-pCPT; p=0.041 vs. Ctrl); 96 ± 12% (8-pCPT+eV1-2; p=0.97 vs. Ctrl; p=0.029 vs 8-pCPT). Rab3A: 100 ± 5% (Ctrl); 168 ± 12% (8-pCPT; p=0.0011 vs. Ctrl); 133 ± 12% (8-pCPT+eV1-2; p=0.069 vs. Ctrl; p=0.046 vs 8-pCPT). One-way ANOVA test. n=4 for all groups. *p<0.05. **p<0.01. (J) Cerebellar synaptosomes (WT) were treated with control buffer or FR236924 (FR) (200 nM, 30 min) and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to Ctrl. Munc13-1: 100 ± 4% (Ctrl) and 144 ± 16% (FR; p=0.041). Rab3A: 100 ± 4% (Ctrl) and 175 ± 13% (FR; p=0.0016). Unpaired t test. n=4 for all groups. *p<0.05. **p<0.01. (K) Cerebellar synaptosomes (Prkce-cKO) were treated with control buffer or FR236924 and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to Prkce-cKO. Munc13-1: 100 ± 3% (Prkce-cKO; n=4) and 100 ± 12% (Prkce-cKO +FR; p=0.99; n=4). Rab3A: 100 ± 2% (Prkce-cKO; n=8) and 108 ± 9% (Prkce-cKO +FR; p=0.37; n=8). Unpaired t test. See the online Figure 2—source data 1 file for source data of western blots in this figure. Figure 2—source data 1.The uncut gel of western blots in Figure 2.