(a) Inhibitor toxicity measured in HEK293T cells using a standard dead cell apoptosis assay. Healthy cells are not fluorescent (green), dead cells are propidium iodide (PI) and annexin V (AV) positive (red). Apoptotic cells are only AV positive (grey), while necrotic cells are only PI positive (black). Hoescht nuclear counterstain was to determine the total number of cells. Values are mean +/- SEM, n = 7–8 for each condition from three independent trials. (b) Inhibitor toxicity measured in primary cultures of rat cortical neurons using PI (Dead) and Hoechst (Total) stains. Values are mean +/- SEM, n = 10 fro each condition from two independent trials. (c) Representative anti-GFP immunoblots showing time-course of cleavage for DNV and PRV washout with the modified EDVVCC/QMSY model. PRV has the fastest apparent dissociation from the protease, as the model is almost completely cleaved within 1 hr following washout. (d) Representative, individual ROI (black) and average (green) iGluSnFR traces showing the Ca2+-dependence of release in WT rat hippocampal neurons transduced with iGluSnFR. The examples shown were from a single FOV for each condition. (e) Quantification of miniature glutamate transients (mGT) in WT rat hippocampal neurons at 15 DIV using iGluSnFR (Marvin et al., 2013) in 2 mM extracellular Ca2+. One µM TTX was used to block action potentials where indicated; in some experiments, forskolin (10 µM) was also included to increase mGT frequency. Addition of PRV drastically increased the rate of spontaneous glutamate release events; TTX blocked this increase. A low dose of PRV (0.5 µM) did not influence spontaneous release. Two separate trials were conducted; bars represent means and individual dots represent events/s from a FOV (n) imaged for one minute, n = 4 to 10. (f) Normalized, average traces (n = 5 to 6) of evoked glutamate release from a single action potential in WT neurons, and WT neurons treated with 0.5, 1, or 3 µM PRV, in 1 mM extracellular Ca2+. (g) Representative confocal images of 20 DIV rat hippocampal neurons stained with anti-PSD95, anti-vGLUT1, and anti-MAP2. Neurons grown in vehicle, 0.5 µM, or 5.0 µM PRV. Quantitation of synapse number to the right, control (grey) 0.5 µM (green), or 5.0 µM PRV (blue).