SV2 Renders Primed Synaptic Vesicles Competent for Ca²⁺-Induced Exocytosis

SV2 deletion selectively impairs Ca²⁺ triggering of release without decreasing the RRP. A, B, Representative traces (A) and mean amplitude and charge transfer (B) of IPSCs evoked by extracellular stimulation at 0.1 Hz frequency in cultured cortical neurons from littermate SV2B KO (BKO) and SV2A/SV2B double-KO mice (DKO). Action potentials were evoked with a focal electrode in a bath solution containing 1 mm Ca²⁺ and 2 mm Mg²⁺. C, D, Ca²⁺ titration of IPSC amplitudes in BKO and DKO neurons plotted in absolute terms (C) or normalized to the maximal Ca²⁺ concentration (D). Recordings were performed in bath solutions containing the indicated Ca²⁺ concentrations with a constant 2 mm Mg²⁺ concentration. E–J, Measurements of the RRP size by application of 0.5 m sucrose in either inhibitory (E–H) or excitatory (I, J) synapses in cultured neurons that were either not treated (E, F, I, J) or treated with 1 μm TTX for 24 h (G, H). Panels show representative traces (E, G, I) and summary graphs depicting the integrated charge transfer during the first 10 s of the response (F, H, J). All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; ***p < 0.001 by Student's t test; in C and D, n = numbers shown above or under data points; the absolute values are significantly different between BKO and DKO neurons at the p < 0.0001 level as tested by 2-way ANOVA).

Effect of ionomycin on release in SV2B KO and SV2A/SV2B double-KO synapses. A, Representative IPSC traces triggered by application of 5 μm ionomycin in SV2B KO (BKO) and SV2A/SV2B double-KO (DKO) neurons in 1 mm Ca²⁺. Excitatory currents were blocked by APV and CNQX. The scale bar applies to both traces. B, C, Total synaptic charge transfer for ionomycin-induced responses (B) and average delays from the point of application of ionomycin to maximum amplitude of inhibitory response (C) in SV2B-deficient and SV2A/SV2B double-deficient neurons. Data shown are means ± SEMs (n = numbers shown in bar diagrams from 2 independent cultures). D, Normalized cumulative charge as a function of time to illustrate the kinetics of ionomycin-induced release in SV2B KO (BKO) and SV2A/SV2B double-KO (DKO) synapses.

SV2 deletion decreases release but increases facilitation during 10 Hz stimulus trains. A, Representative traces of IPSCs induced by a 1 s, 10 Hz stimulus train in cultured neurons from SV2B KO (BKO) and SV2A/SV2B double-KO (DKO) mice (bath solution contains 1 mm Ca²⁺ and 2 mm Mg²⁺). B–D, Summary graphs of the charge transfer during the stimulus train (train release, B), after the train (delayed release, C, a measure of asynchronous release), and over the entire experiment (total charge transfer, D) in BKO and DKO neurons. E, Plot of the normalized amplitude as a function of the stimulus number during the 10 Hz stimulus train. As amplitudes, only the synchronous component of the response is measured. All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; *p < 0.05, **p < 0.01, and ***p < 0.001 by Student's t test; in E, n = 53 for BKO neurons and n = 59 for DKO neurons; values are significantly different between BKO and DKO neurons at the p < 0.0001 level as tested by 2-way ANOVA).

Effect of EGTA-AM on synaptic responses in SV2-deficient neurons. A–C, Representative traces (A) and summary graphs of the amplitudes (B) and synaptic charge transfers (C) of evoked IPSCs in neurons from SV2B KO mice (BKO) and SV2A/B double-KO mice (DKO), recorded before or after pretreatment with 0.1 mm EGTA-AM (>10 min). Data are normalized for the response before the EGTA-AM treatment; no statistically significant difference in the relative effect of EGTA-AM on the response size was detected. D–G, Effect of EGTA-AM on synaptic responses evoked during a 10 Hz stimulus train (1 s). Data shown are representative traces (D) and summary graphs of the train release (E), the delayed release (F), and the total release (G). In E–G, responses are normalized for the naive condition. H, Effect of EGTA-AM on the facilitation induced by deletion of SV2. The graph depicts a plot of the relative amplitude of synaptic responses as a function of the stimulus number during a 10 Hz stimulus train observed in BKO and DKO neurons. As amplitudes, only the synchronous components of the responses are measured. All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; the effect of EGTA-AM is not statistically significantly different between neurons from BKO and DKO mice; in H, n = 29 for BKO synapses; n = 33 for BKO synapses treated with EGTA-AM; n = 26 for DKO synapses; and n = 25 for DKO synapses treated with EGTA-AM; values are significantly different between naive and EGTA-AM-treated neurons from DKO mice at the p < 0.0001 level as tested by 2-way ANOVA, whereas the responses from BKO versus DKO mice are not significantly different after EGTA-AM treatment).

Recovery of synaptic responses in SV2B KO and SV2A/SV2B double-KO synapses after use-dependent depression induced by high-frequency stimulation. A, Representative traces of IPSCs evoked by extracellular stimulation at 0.1 Hz frequency before and after high-frequency stimulation (100 stimuli at 10 Hz frequency) in SV2B-deficient and SV2A/SV2B double-deficient neurons. B, C, Plot of either the absolute IPSC amplitudes (B) or the normalized amplitude (C) as a function of time (10 s) before and after high-frequency stimuli in SV2B KO (BKO) and SV2 double-KO (DKO) synapses. Data shown are means ± SEMs (absolute responses but not the normalized responses are significantly different at the p < 0.001 level as tested by 2-way ANOVA).

Rescue of the phenotype in SV2-deficient neurons by lentiviral expression of EGFP-tagged SV2A. A, Fluorescence (left) and phase-contrast (right) images of neurons infected with lentivirus expressing EGFP-SV2A. B–D, Representative traces (B), mean amplitudes (C), and charge transfers (D) of IPSCs evoked by extracellular stimulation at 0.1 Hz. Responses were monitored in cultured cortical neurons from littermate SV2B KO (BKO) and SV2A/SV2B double-KO mice, the latter either as naive neurons (DKO) or after infection with wild-type EGFP-SV2A-expressing lentivirus (DKO^WT). Action potentials were evoked with a focal electrode in a bath solution containing 1 mm Ca²⁺ and 2 mm Mg²⁺. E–H, Representative traces (E) and summary graphs of the charge transfer during the stimulus train (train release, F), after the train (delayed release, G), and over the entire experiment (total charge transfer, H) in the same neurons described for panel B above. I, Plot of the normalized amplitude as a function of the stimulus number during the 10 Hz stimulus train in BKO and DKO neurons without or with rescue. All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; *p < 0.05 and ***p < 0.001 by Student's t test; in I, n = 35 for BKO neurons; n = 40 for DKO neurons; and n = 32 for DKO^WT neurons; values are significantly different between BKO and DKO neurons at the p < 0.0001 level as tested by 2-way ANOVA).

Structure and mutants of SV2A. A, Schematic diagram of the structure of SV2 proteins and location of mutants analyzed in Figures 7⇓⇓–10. Amino acids residues in the primary sequence of SV2A are indicated by circles, with 12 potential transmembrane regions (TMRs) predicted by sequence analyses. Residues that are conserved in SV2A, SV2B, and SV2C are shown in black, residues that are identical in at least two of the three isoforms in gray, and nonconserved residues in white. Branched lines show positions of N-linked carbohydrates. N and C termini are identified by letters (Janz et al., 1999). The positions of mutations are indicated: DA mutant (D179A/E182A), NQ mutant (N498Q/N548Q/N573Q), KA mutant (K694A), d107 mutant (deletion of residues 1–107). Fluorescent protein EGFP was fused to the N terminus of full-length or SV2A deletion protein as indicated by green arrow. B, C, Immunoblot analysis of the expression of wild-type EGFP-SV2A and of various EGFP-SV2A mutants in lentiviral-infected cultured neurons using antibodies to SV2 (B) and to GFP (C). All blots were additionally blotted for GDI as a loading control (bottom bands shown in the blot, indicated by asterisks); note that depending on expression levels, different amounts of protein were loaded on each lane, as is evident from the loading control.

Rescue of the SV2 KO phenotype by mutant SV2A lacking the N-terminal 107 residues implicated in synaptotagmin binding. A, Fluorescence (left) and phase-contrast (right) images of neurons infected with lentivirus expressing d107 mutant EGFP-SV2A. B, C, Representative traces (B) and summary graphs (C) of IPSCs evoked by isolated action potentials in SV2B KO neurons (BKO) or SV2A/B double-KO neurons without rescue (DKO) or after rescue with wild-type SV2A (DKO^WT) or d107 mutant SV2A (DKO^d107). D–F, Representative traces (D) and summary graphs of train release (E) and total release (F) of BKO neurons and naive and rescued DKO neurons stimulated at 10 Hz for 1 s. G, Plot of the relative amplitude of synaptic responses during a 10 Hz stimulus train as a function of stimulus number in BKO neurons and naive and rescued DKO neurons. All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; *p < 0.05, **p < 0.01, and ***p < 0.001 by Student's t test; in G, n = 13 for BKO synapses; n = 8 for DKO synapses; n = 9 for rescued DKO^WT synapses; and n = 20 for DKO^d107 neurons; values are significantly different between BKO and DKO neurons, but not between BKO and rescued DKO neurons, at the p < 0.0001 level as tested by 2-way ANOVA).

Lack of rescue of the SV2 KO phenotype by DA mutant SV2A containing a double amino acid substitution in TMR1 (D179A/E182A). A, Fluorescence (left) and phase-contrast (right) images of neurons infected with lentivirus expressing DA mutant EGFP-SV2A. B, C, Representative traces (B) and summary graphs (C) of IPSCs evoked by isolated action potentials in SV2B KO neurons (BKO) or SV2A/B double-KO neurons without rescue (DKO) or after rescue with wild-type SV2A (DKO^WT) or DA mutant SV2A (DKO^DA). D–F, Representative traces (D) and summary graphs of train release (E) and total release (F) of BKO neurons and naive and rescued DKO neurons stimulated at 10 Hz for 1 s. G, Plot of the relative amplitude of synaptic responses during a 10 Hz stimulus train as a function of stimulus number in BKO neurons and naive and rescued DKO neurons. All data shown are means ± SEMs (n = numbers shown in bar diagrams from at least 3 independent cultures; *p < 0.05 and **p < 0.01 by Student's t test; in G, n = 16 for BKO neurons; n = 22 for DKO neurons; n = 15 for DKO^WT neurons; and n = 26 for DKO^DA neurons; values are significantly different between BKO and DKO neurons, but not between DKO and DKO^DA rescued neurons, at the p < 0.0001 level as tested by 2-way ANOVA).