Fig. 4: Optogenetics inhibition and synchronicity of burst activity between organoids.

A Optogenetic setup for inhibiting the synaptic interaction between left and right organoids through axon bundle. (i) A microfluidic device for optogenetic control consisting of fiber guides (thin channels) for optical fiber insertion. (ii) ArchT was expressed in the connected organoid by AAV. The light at 565 nm waveband illuminated the axon bundle in microfluidic device. (iii) An optical fiber was connected to 565 nm LED and a pulse generator (Arduino). An optical fiber was positioned perpendicular to the axon bundle with a 100 μm gap. Light exposure timing and a representative channel from MEA amplifier were recorded in TTL logger to synchronize TTL signal and recording. B An axon bundle and an optical fiber. Curved structure serves as a PDMS lens which helps the light to be focused on the axon bundle. C LFP and raster plot from left (pink) and right (blue) organoids of a connected organoid with or without light illumination (orange bar: TTL). D Synchronized burst frequency was around 0.65 Hz in the absence of light illumination and decreased to zero during light on. Then, synchronized burst frequency immediately increased and recovered after stopping the light. E Wavelet coherence by wavelet transformation revealed that slow wave oscillation disappeared during light illumination, indicating the vanishment of any correlative activity during light exposure. F Light illumination completely suppressed these synchronized burst activities. n = 8 from 3 organoids. P = 1.5e−8. G Inter-regional synchronicity measured by delta phase-delta phase coupling showed significant decrease during light illumination. n = 5 from 3 organoids. P = 0.0008 (first light off/light on); 0.0001 (second light off/light on). H Total numbers of single spikes were calculated in a 5 min time slot. Light-on significantly induced the increase in number of spikes. *p < 0.05; Paired-test (two-sided) for F and one-way ANOVA with Tukey’s multiple comparison for G. Data are presented as mean values ± SD.