mSCaTs measured by GCaMP6f imaging reflect NMDAR activation at individual synapses following spontaneous single vesicle release. mSCaTs measured by GCaMP6f imaging reflect NMDAR activation at individual synapses following spontaneous single vesicle release. A, Cultured hippocampal neuron infected with AAV-GCaMP6f. Left, GCaMP6f average (green); middle, ΔF calculated by subtracting GCaMP6f average from GCaMP6f maximum projection (magenta); right, merge of GCaMP6f average (green) and ΔF (magenta). Bottom, Zoom in of boxed spine from cell in top left. First panel is GCaMP6f average, the 2nd through 4th panels are individual frames showing a single mSCaT at 0, 400, and 800 ms, respectively. Red circles indicate ROIs for data traces shown in B. B, ΔF/F traces from spine and dendrite regions circled in red in A. C, Frequency histogram of mSCaT amplitude for individual synapses across 923 spines from 8 cells. D, Frequency histogram of mSCaT frequency for individual synapses across 923 spines from 8 cells. E, Representative GCaMP6f traces demonstrating that treatment with ryanodine, thapsigargin, DNQX, and nifedipine (blockers) did not alter mSCaT amplitude compared with vehicle treatment. F, Quantification of effect of blockers on mSCaT amplitude compared with vehicle treatment revealed that blockade of non-NMDAR sources of Ca2+ did not impact mSCaT amplitude. G, Representative GCaMP6f traces demonstrating that treatment with APV eliminated mSCaTs compared with vehicle treatment. H, Treatment with APV eliminates 94% of events. I, Raising extracellular Ca2+ causes increased mSCaT amplitude, whereas application of 30 μm, 100 μm, and 1 mm Mg2+ reduce mSCaT amplitude. For example traces, see Extended Data Figure 1-1. ns, not significant; ****p < 0.0001. Sarah R. Metzbower et al. eNeuro 2019;6:ENEURO.0419-18.2019 ©2019 by Society for Neuroscience