(A) Application of NBQX and muscimol silence network activity in a neuronal culture. Example traces show current clamp recordings of two cultured pyramidal cells first in Mg2+ free Tyrode’s solution and then in Mg2+ free Tyrode’s solution containing 5 µM NBQX plus 5 µM muscimol (green bar). Upon perfusion of NBQX and muscimol, spontaneously arising action potentials are silenced and the voltage fluctuations of each cell driven by excitatory synaptic network activity are silenced. (B) NBQX and muscimol allow reliable stimulation, and prevent reverberatory excitatory network activity by taking advantage of the predominant somatodendritic expression of GABAA receptors. Example traces show Fluo-4 AM fluorescence (reporting bulk Ca2+ changes, rather than synapse specific Ca2+ as monitored by GCaMP6f-PSD95) from 2.5 µm circular ROIs placed on the cell soma or a proximal, isolated dendritic process for two cells. Cells are first imaged in Mg2+ free Tyrode’s solution and stimulated 10 times with 30 mA from a 3 mm spaced parallel bipolar electrode at 0.1 Hz (red marks denote stimulus). During the first train of stimulations, network activity generates multi AP responses that are visible as complex, multi-peak signals in the dendrite which are consistent with network bursting activity (black traces). At the cell soma, a smoothed version of the same signal is visible where each stimulus generates a longer Ca2+ signal. Cells are washed for 1 min with Mg2+ free Tyrode’s solution plus NBQX and muscimol and again stimulated 10 times at 0.1 Hz (green traces). During the second stimulation, each stimulus generates a unitary response with a single peak in the dendrite. At the cell soma, only small Ca2+ signals are visible in the presence of NBQX and muscimol which is consistent with GABAA currents shunting excitatory inputs from the dendrite. The lack of signal at the cell soma compared to the dendrite confirms that Ca2+ signals seen after NBQX and muscimol application are driven by synaptic activity elicited by the stimulus rather than a stimulus artifact which would be visible in both the somatic and dendritic compartments. (C) Spontaneous event amplitude coefficient of variation does not show a population of ROIs with multiple synaptic inputs. Coefficient of variation was calculated for each ROI with two or more spontaneous events during the experiment in Figure 2. Each mark represents a single ROI. If a non-negligible population of ROIs received fluorescent signals from multiple synapses we would expect to see a population of ROIs with both high spontaneous response rate and a high coefficient of variation among amplitudes. We do not detect a population of ROIs where this is the case. (D) The analysis in C. was repeated for evoked signals, and again we did not detect a population of high variance and high Rp ROIs that would indicate multiple inputs per ROI.