Optogenetic stimulation and recording of human neurons. (A) A 30 mm distance (d) from the MEA electrodes provided an optimal power density of ∼0.29 mW/mm² from a 470 nm LED, while limiting noise on the MEA measurement system. (B) Non-optogenetic iNeurons (i.e., GCaMP6) did not respond to blue light stimulation (denoted as +) on MEAs (n = 18–24 electrodes each). (C) Raster plots of OptoNeuron monocultures in response to increasing optical stimulation frequency (1–40 Hz) at pulse widths varying from 1 to 5 ms. Below 3 ms, there was minimal detectable pacing of cells in response to light. A 5 ms pulse width was deemed optimal for subsequent experiments. Each horizontal row represents one electrode (pre-threshold; n = 20 electrodes per group). (D) As there was no detectable correlation between spontaneous and stimulated (1–40 Hz) firing rates, we determined a minimum threshold cutoff of 0.1 Hz for both spontaneous and stimulated firing rates. This threshold was applied across monoculture and coculture groups. Dotted lines indicate linear regression fits (top). Inset boxes (bottom) show detail from x = [0,0.3]Hz and y = [0,0.8]Hz. (E and F) Spontaneous versus light-induced firing rates (n = 7–41 electrodes each, after threshold) of day-21 OptoNeuron spheres on MEAs (E) and temporal attachment to Matrigel (F; n = 8–9 spheres each time point) under different media conditions (see Fig. 2 G). (G) Viability (n = 5–10; measured by a CellTiter-Glo 3D Assay and normalized to sphere area) of day-12 and day-18 spheres in NM+Dox, in the absence or presence of chronic BDNF and NT3 treatment. Results are shown as mean ± SEM. In B, D and E, dots represent individual spheres or electrodes. For B, E, and G, significance was determined using two-tailed paired t tests, with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.