Brief light pulses evoked network-wide spiking responses, only in cultures transduced by AAV. Responses were quantified over 60 repetitions by computing the peri-stimulus histogram of spike times (PSTH) (A), detected over 5 ms bins across all microelectrodes (Fig. 1A). In all experiments, the time course of the PSTH was stereotyped and consisted of an early and late phase, similarly to what was described for extracellular electrical stimulation. While intensity was fixed, the duration of light pulses was changed systematically and it was unexpectedly found to significantly modulate the late (C,D) but not the early phase of the PSTH (B). Longer pulses significantly (p < 0.001) delayed (C) and weakened (D) the late response peak, without affecting the early phase (circles in A). Evaluating the non-parametric Kendall’s rank coefficient, a significant correlation (p < 0.0001) was only found between pulse duration and late-peak latency (0.47) or between pulse duration and late-peak amplitude (−0.36). Panels B-D summarize the results over 39 MEAs, while the insets show averages across several MEAs, over five distinct sets of sister cultures, remarking the consistency of the findings across different biological preparations.