Figure 7.

SV2A R383Q cannot rescue Syt1 retrieval kinetics in SV2A-depleted cells. Primary cultures of hippocampal neurons were transfected with a bicistronic vector expressing Syt1-pHluorin (Syt1-pH) and shRNA against SV2A and either mCerulean (mCer), wild-type (WT) SV2A-mCer, or R383Q SV2A-mCer after 7–8 DIV. At 13–15 DIV, neurons were stimulated with a train of 300 action potentials delivered at 10 Hz. Neurons were allowed to recover before being pulsed with NH₄Cl imaging buffer reveal the total Syt1-pH population. A, Representative images are displayed at specific time points indicated, with responsive nerve terminals highlighted using arrowheads. Scale bar = 10 μm. B, Traces display the time course of the average fluorescent Syt1-pHluorin response normalized to the peak of stimulation. Bar indicates the period of stimulation (n = 7 for mCer and n = 11 for both WT and R383Q SV2A-mCer, from four independent preparations). C, Syt1-pHluorin remaining to be retrieved 130 s after termination of stimulation is displayed as F/F₀ ±SEM (one-way ANOVA, *p = 0.0348 WT compared with mCer, p = 0.0473 WT compared with R383Q SV2A-mCer, ns = not significant, mCer compared with R383Q).