Presynaptic calcium currents and synaptic vesicle release in RIM-BP2 KO mice

(A) The terminal was depolarized from -80 mV to +10 mV in WT (black) and RIM-BP2 KO (gray) hippocampal mossy fiber boutons. Ca2+ currents (ICa) and membrane capacitance (Cm) in response to a 10 ms pulse (Vm) are shown. (B) (top) The peak Ca2+ currents are plotted against the pulse duration. WT vs KO, p = 0.034 (ANOVA). (bottom) The capacitance jumps are plotted against the pulse duration. WT vs KO, p = 0.23 (ANOVA). Black filled circles and gray open circles represent the data from WT (n = 9 from 8 animals) and RIM-BP2 KO (n = 8 from 6 animals), respectively. Each data point represents mean ± SEM. (C) Experimental protocol and representative traces for presynaptic Ca2+ current measurements in WT (black) and RIM-BP2 KO (gray) boutons. Terminals were sequentially depolarized for 5 ms with 2 ms intervals from -80 mV to +70 mV by 10 mV steps. (D) Current-voltage relationships of peak Ca2+ currents in WT (black filled circle; n = 4-6 from 4 animals) and RIM-BP2 KO (gray open circle; n = 4-5 from 5 animals). ICas were elicited by 5 ms depolarizations. Two curves were significantly different (p < 0.01 from mixed model). Each data point represents mean ± SEM. Numerical values of plots are provided in Figure 1-source data 1.

Individual value plot of Fig. 1B

Individual values of ICas (top) and Cms (bottom) in WT (pink) and RIM-BP2 KO (green) terminals are plotted against the pulse duration. Numerical values of plots are provided in Figure 1-source data 1.

The effects of high extracellular calcium concentration on Ca2+ currents and capacitance changes.

(A) The ICas (top) and the Cms (bottom) are plotted against the pulse duration. Black filled squares and gray open squares represent the data from WT (n = 6-8 from 6 animals) and RIM-BP2 KO (n = 3-4 from 2 animals) at 4 mM [Ca2+]ext, respectively. For comparison, the WT data in 2 mM [Ca2+]ext are superimposed (black filled circle; n = 9) (the same data sets as Fig. 1B). Each data point represents mean ± SEM. ICas were significantly different between WT and KO in 4 mM [Ca2+]ext (p < 0.01 from mixed model). The WT ICas in 2 mM [Ca2+]ext and the KO ICas in 4 mM [Ca2+]ext were significantly different (p < 0.02 from mixed model), but Cm were not significantly different (p = 0.57 from mixed model). (B) Average ICas (top) and Cms (bottom) in response to a 100 ms pulse in WT (black bars) and RIM-BP2 KO (gray bars) terminals. Extracellular Ca2+ concentration was 2 mM (left) or 4 mM (right). Error bars show SEM. Circles indicate individual values. Numerical values of plots are provided in Figure 2-source data 1.

Individual value plot of Fig. 2A

Individual values of ICas (top) and Cms (bottom) recorded at 4 mM [Ca2+]ext in WT (pink) and RIM-BP2 KO (green) terminals are plotted against the pulse duration. Numerical values of plots are provided in Figure 2-source data 1.

Calcium-dependence of the release kinetics and the RRP size

(A) (top) The relationship between peak Ca2+ currents and pulse durations at different [Ca2+]exts. (bottom) The relationship between capacitance jumps and pulse durations at different [Ca2+]exts. Gray open circles, black diamonds and black triangles represent the data from RIM-BP2 KO at 2 mM [Ca2+]ext (n = 8) (the same data sets as Fig. 1B), WT at 1 mM [Ca2+]ext (n = 3-5 from 4 animals) and WT at 1.5 mM [Ca2+]ext (n = 5-7 from 6 animals), respectively. Each data point represents mean ± SEM. (B) Capacitance jumps at various [Ca2+]exts are plotted against calcium current amplitudes. Pulses were 100 ms depolarization from -80 mV to +10 mV. Each data point represents mean ± SEM. Data points were fitted with a Hill equation with n = 3. (C) (left) Average ICas at indicated [Ca2+]exts were normalized to the response at 2 mM [Ca2+]ext in each genotype. (right) Average Cms at indicated [Ca2+]exts were normalized to the 2 mM [Ca2+]ext response in each genotype. Numerical values of plots are provided in Figure 3-source data 1.

Individual value plot of Fig. 3A

Individual values of ICas (top) and Cms (bottom) recorded at 1 mM (green) or 1.5 mM (pink) [Ca2+]ext in WT are plotted against the pulse duration. Numerical values of plots are provided in Figure 3-source data 1.

The relationship between synaptic vesicle release and total Ca2+ charge

Capacitance jumps elicited by various pulse durations are plotted against total Ca2+ charge. The total Ca2+ charge was calculated by multiplying a peak Ca2+ current amplitude by pulse duration. Each symbol represents a data point obtained from WT (black filled symbols) and RIM-BP2 KO (gray open symbols) mice at various [Ca2+]ext. Data points were fitted by a single exponential function. Numerical values of plots are provided in Figure 3-figure supplement 2-source data 1.

The effects of high EGTA on calcium currents and synaptic vesicle exocytosis

(A) Example traces in response to a 5 ms depolarizing pulse to +10 mV in WT (left) and RIM-BP2 KO (right) boutons. Ca2+ current (ICa) and membrane capacitance (Cm) recorded with 0.5 mM EGTA (black) or 5 mM EGTA (red) in the patch pipette are shown. Note that each trace was obtained from different terminals but the traces were superimposed. (B) The ICas (top) and Cms (bottom) are plotted against the pulse duration. The patch pipette contained 5 mM EGTA. Black filled diamonds and gray open diamonds represent the data from WT (n = 4-5 from 4 animals) and RIM-BP2 KO (n = 4 from 3 animals), respectively. Each data point indicates mean ± SEM. (C, D) Average Cms (C) and ICas (D) elicited by a 20 ms pulse in WT (filled bars) and RIM-BP2 KO (hatched bars). The extracellular Ca2+ concentration was 1 mM (from 5 animals) or 2 mM. The intracellular solution contained either 0.5 mM EGTA (black) or 5 mM EGTA (red). Error bars show SEM. Circles and diamonds indicate individual values. Numerical values of plots are provided in Figure 4-source data 1.

Individual value plot of Fig. 4B

Individual values of ICas (top) and Cms (bottom) recorded in the presence of 5 mM EGTA in WT (green) and KO (pink) were plotted against the pulse duration. Numerical values of plots are provided in Figure 4-source data 1.

The evoked EPSC amplitudes in WT and KO, and their sensitivity to the extracellular Ca2+

(A) The mossy fiber-evoked EPSCs measured at CA3 pyramidal cells. The fibers were stimulated twice with an interval of 20 ms. The responses of WT in 1.5 mM and 2 mM [Ca2+]ext (n = 7 cells from 5 animals), as well as those of KO in 2 mM and 4 mM [Ca2+]ext (n = 7 cells from 6 animals) are shown. The stimulus artifacts were blanked. (B) The amplitude ratios of WT EPSCs (2 mM / 1.5 mM [Ca2+]ext) and KO EPSCs (4 mM / 2 mM [Ca2+]ext) are shown. The concentrations were chosen to set the Ca2+ influx similar between WT and KO from Fig. 1-Fig. 3. (C) The paired pulse ratios under 4 conditions (WT in 1.5 mM and 2 mM [Ca2+]ext, KO in 2 mM and 4 mM [Ca2+]ext). Numerical values of plots are provided in Figure 5-source data 1.

The time course of synaptic facilitation/depression in WT and KO

(A) Mossy fibers were stimulated at 50 Hz (25 times) and the evoked EPSCs were measured in 1.5 mM (n = 5 cells from 4 animals) and 2 mM [Ca2+]ext (n = 7 cells from 5 animals) in WT. The stimulus artifacts were blanked. (B) Mossy fibers were stimulated at 50 Hz (26 times in this particular example), and the evoked EPSCs were measured in 2 mM (n = 7 cells from 6 animals) and 4 mM [Ca2+]ext (n = 8 cells from 6 animals). (C) The time course of EPSC amplitudes during a 50 Hz train. The data were obtained from WT and KO in 2 mM [Ca2+]ext. The time courses were significantly different (p < 0.01 from ANOVA). (D) The same as (C), but the data were obtained from WT in 1.5 mM [Ca2+]ext and KO in 2 mM [Ca2+]ext. The time courses were significantly different (p < 0.05 from ANOVA). (E) The same as (C), but the data were obtained from WT in 2 mM [Ca2+]ext and KO in 4 mM [Ca2+]ext. Numerical values of plots are provided in Figure 6-source data 1. The time courses were not significantly different (p = 0.12 from ANOVA). Numerical values of plots are provided in Figure 6-source data 1.

The EPSC amplitudes plotted against the stimulus number

The same data sets as Fig. 6C-Fig. 6E, but the absolute EPSC amplitudes are plotted instead of the normalized amplitudes. Numerical values of plots are provided in Figure 6-source data 1. Raw data were summarized. In Fig. 5 and Fig. 6, the effect of holding potential was corrected so that the values were adjusted to those at -70 mV. Numerical values of plots are provided in Figure 6-source data 1.

The individual data of Fig. 6

WT in 2mM (A) and 1.5 mM (B) [Ca2+]ext, and KO in 2 mM (C) and 4 mM [Ca2+]ext (D) are shown. Numerical values of plots are provided in Figure 6-source data 1.

RIM-BP2 deletion alters the signal intensity of Cav2.1 clusters within the AZ

(A) Confocal (top) and STED (bottom) images of Munc13-1 (magenta) and Cav2.1 (yellow) clusters at hippocampal mossy fiber terminals of WT (left) and RIM-BP2 KO (right) mice. (Scale bar: 500 nm.) (B) Confocal images of PSD95 (cyan) and VGLUT1 (white) to identify glutamatergic synapses in CA3 stratum lucidum: mossy fiber-CA3 synapses. (Scale bar: 500 nm.) The image was taken from same region shown in (A), (left). (C) Histograms (top) and cumulative distribution plots (bottom) of the total signal intensity of Cav2.1 (left), Cav2.2, and Munc13-1 (right) at AZs in WT (black) and RIM-BP2 KO (red) mice. (D) The average nearest neighbor distance between Cav2.1 and Munc13-1 clusters in WT (black; n = 4 animals) and RIM-BP2 KO (red; n = 4 animals) mice. Several hundreds of AZs per image were analyzed. Data show the average value of distance per animal and error bars represent SEM. Each data point indicates individual values. Numerical values of plots are provided in Figure 7-source data 1.

STED microscopy confirmed loss of RIM-BP2 proteins in KO terminals

Confocal (top) and STED (middle) images of RIM-BP2 (yellow) in WT (left) and RIM-BP2 KO (right) hippocampal mossy fiber terminals identified by PSD95 (cyan) and VGLUT1 (white) (bottom). (Scale bar: 500 nm.) The bottom images show the same region as top and middle.

STED imaging of Cav2.2 at hippocampal mossy fiber terminals

Confocal (top) and STED (middle) images of Munc13-1 (magenta) and Cav2.2 (yellow) clusters in WT (left) and RIM-BP2 KO (right) mice. Confocal images of PSD95 (cyan) and VGLUT1 (white) in the same region as top and middle are also shown (bottom). (Scale bar: 500 nm.)

Immunohistochemical characterization of large VGLUT1 positive terminals in the CA3 stratum lucidum

(A) Confocal images of VGLUT1 (cyan) and mGluR1α (yellow) were taken with STED images of Munc13-1 (magenta) in the CA3 stratum lucidum. Arrows indicate representative large MF terminals identified by VGLUT1 signals (Scale bar: 5 µm.) The right graph shows the proportion of large VGLUT1 positive terminals contacting mGluR1α positive processes. (B) Same as (A) but for GluA4 instead of mGluR1α. (Scale bar: 5 µm.)

The area, length, width of Cav2.1 cluster

In A, distribution plots of area (left), length (middle) and width (right) are shown. In B, cumulative distribution plots are shown. The same samples as Fig. 7. Numerical values of plots are provided in Figure 7-figure supplement 4-source data 1.

Quantification of the signal intensity using confocal images

The signal intensity of Munc13-1 (left), Cav2.1 (middle), and Cav2.2 (right) at AZs in WT (black) and RIM-BP2 KO (red) mice were quantified from confocal images. The position of AZ was determined from the Munc13-1 STED data, and the signal intensity of Munc13-1, Cav2.1 and Cav2.2 was measured. Histograms (A) and cumulative distribution plots (B) are shown. The same samples as Fig. 7. Numerical values of plots are provided in Figure 7-figure supplement 5-source data 1.