Fig. 2: Modulation of postsynaptic neural activity by Interluminescence in mixed cultures of pre- and postsynaptic neurons.

a Schematic: Interluminescence via an Optical Synapse. Luciferases (blue colored enzyme inside the gray circles) are released from presynaptic vesicles and, in the presence of the luciferin, emit light (bioluminescence: light bluish glow) that activates postsynaptic opsins (magenta; downward black arrows indicating ion movement through open channels). b Schematic of experimental design and constructs used for separate nucleofections of cortical neurons with either hPOMC1-26-sbGLuc-P2A-dTomato or one of the opsins, ChR2(C128S)-EYFP (excitatory) and hGtACR2-EYFP (inhibitory); neurons were then mixed (red and green spheres), plated on MEAs and maintained for 2–3 weeks until recording. c Illustrations (upper panels) and corresponding representative traces from individual electrodes of MEAs (middle and lower panels) showing response of postsynaptic neurons to blue light (blue solid triangle), CTZ-induced bioluminescence (orange pipette tip) and vehicle (gray pipette tip) expressing the excitatory opsin ChR2(C128S) (middle panels) and the inhibitory opsin hGtACR2 (lower panels). d Schematic showing the time windows for analyzing the number of spikes for each treatment (upper panel) and ladder plots from multiple experiments (middle panels for excitatory and lower panels for inhibitory opsin-expressing postsynaptic neurons as depicted for individual traces in (c). ChR2(C128S), blue light, n = 62, p < 0.0001, CTZ, n = 62, p < 0.0001, vehicle, n = 24, p = 0.6498; hGtACR2, blue light, n = 49, p < 0.0001, CTZ, n = 49, p < 0.0001, vehicle, n = 49, p = 0.5594; Wilcoxon matched-pairs signed rank test. The artifacts due to addition of reagents in MEAs are overlaid by a vertical white bar in all MEA recording traces (the white gap right after addition of either CTZ or vehicle). ns, not significant; ****p < 0.0001.