The time series of fi(t) were analyzed with NETCAL (36) to infer the timing of neuronal activations using the Schmitt trigger method. This method scans the fluorescence traces for events that first pass a high threshold and then remain elevated above a second lower threshold for at least a certain minimum duration. Shorter events are discarded. In our analysis, we used +3 SDs of the mean of the baseline noise as the high threshold, +1.5 SD as the low threshold, and 200 ms as the minimum event length. Schmitt spike inference was contrasted with the time-derivative thresholding method (14). The overall results were consistent across methodologies.

Network bursts provided a quick insight into the collective dynamics in cultures and were defined as those activity episodes in which more than 75% of the neurons fired simultaneously for more than 500 ms. Experiments with less than 10 network bursts were discarded in the analysis of dynamical richness and effective connectivity.

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