Supplementary Figure 5: SynOptopatch in hiPSC-derived neurons.

a, Schematic of synOptopatch in hiPSC-derived neurons. b, Schematic of excitatory cortical neuron differentiation, gene transduction, maturation and measurement. c, Blue light stimulation induced single-trial PSPs and action potentials (red: before; black: after NBQX addition). Due to lower CheRiff expression than in primary neurons, stimulation required higher blue light intensities (400 mW cm^–2 versus 20–120 mW cm^–2 for primary neurons) to evoke presynaptic action potentials (n = 12 neurons; representative data are shown). d, PSPs were stable for 1 h (red: before; black: 1 h without perturbation). Right: quantification of AUC before and after 1 h (AUC, A.U., 0.58 ± 0.16 versus 0.50 ± 0.11, n = 7 neurons, P = 0.4, two-sided paired-sample t-test). e, A synaptic blocker (25 μM NBQX) largely eliminated the PSPs. The residual transient in the presence of NBQX occurred concurrent with the blue light stimulation, indicating that this signal arose from blue light crosstalk, a consequence of the higher blue stimulation power needed (also visible in d and f). Right: quantification of AUC before and after addition of NBQX (AUC, A.U., 0.97 ± 0.1 versus 0.53 ± 0.07, n = 12 neurons, ***P = 2 × 10⁻⁵, two-sided paired-sample t-test). f, A positive allosteric modulator of AMPARs and kainate receptors (CTZ, 50 μM) increased the PSP amplitude. Right: quantification of AUC before and after addition of CTZ (AUC, A.U., 0.63 ± 0.26 versus 1.4 ± 0.48, n = 6 neurons, *P = 0.055, two-sided paired-sample t-test). g, Exclusive expression of QuasAr2 and CheRiff in hiPSC-derived neurons. Green: presynaptic cells expressing CheRiff-CFP. Red: postsynaptic cells expressing QuasAr2-Citrine, showing Citrine fluorescence. All scale bars, 10 μm (n = 3 culture dishes; representative data are shown) All shaded error bars and error bars, s.e.m. All statistics are mean ± s.e.m.