Figure 7.

P-null SV2A does not rescue Syt1–pHluorin retrieval after shRNA knockdown. A, Images of cultures transfected with shRNA–Syt1–pHluorin vectors that are either empty or contain oligonucleotides against SV2A (shRNA1). Grayscale panels highlight transfected neurons (GFP), whereas false color panels display endogenous SV2A revealed by immunofluorescence staining. Arrows highlight nerve terminals. Scale bar, 1 μm. B, Neurons transfected with WT Syt1–pHluorin and either empty (blue) or SV2A shRNA (shRNA1, red) were stimulated with a train of 300 action potentials (10 Hz). Graph displays the mean ΔF/F₀ time course for Syt1–pHluorin ± SEM normalized to the peak of stimulation (significantly different between time points 96–140 s; p < 0.05; n = 7 empty, n = 5 shRNA1). C, Neurons transfected with a pSUPER vector expressing both Syt1–pHluorin and shRNA1 were coexpressed with either mCer empty vector (mCer), WT mCer–SV2A, or T84A mCer–SV2A. Neurons were stimulated as in B, and the graph displays the mean ΔF/F₀ time course for Syt1–pHluorin ± SEM normalized to the peak of stimulation (mCer and WT traces significantly different between 60 and 200 s; p < 0.05, T84A and WT traces significantly different from 68–200 s; p < 0.05; n = 5 mCer, n = 6 WT, n = 7 T84A). D, Surface expression of Syt1WT–pHluorin in neurons expressing a combination of empty or shRNA1 pSUPER vector and mCer, WT, or T84A mCer–SV2A displayed as a percentage of total pHluorin ± SEM (*p < 0.05, ***p < 0.001 to empty/mCer; ^###p < 0.001 to shRNA1/mCer; ⁺p < 0.05 to shRNA1/WT; n = 7 for all except n = 5 for shRNA1/T84A). E, Neurons transfected with WT synaptophysin–pHluorin (Syp–pHluorin) and either empty (blue) or SV2A shRNA (shRNA1, red) were stimulated as in B. Graph displays the mean ΔF/F₀ time course for Syp–pHluorin ± SEM normalized to the peak of stimulation (n = 9 empty, n = 6 shRNA1, p > 0.05). In B, C, and E, the bar indicates the period of stimulation.