Fig. 3: Communication via Interluminescence in co-cultured populations depends on intact synaptic connections.

a Schematic of experimental design and constructs used for separate nucleofections of cortical and hippocampal or striatal primary neurons. Cortical neurons were nucleofected with the luciferase construct (hPOMC1-26-sbGLuc-P2A-dTomato) and plated in the upper compartment of a 2-chamber silicon divider (group of red spheres covering the upper half of MEA electrodes), and their natural synaptic targets, hippocampal or striatal neurons, were nucleofected with the excitatory opsin construct (ChR2(C128S)-EYFP) and plated in the lower compartment (group of green spheres covering the lower half of MEA electrodes). The next day neurons had attached and the divider was removed (fluorescent image; ×5 magnification; scale bar: 216 µm) showing the expression of luciferase and opsin in respective neuronal populations. Cultures matured over the next 3 weeks, with processes from cortical neurons growing deep into the hippocampal or striatal areas (fluorescent image; ×20 magnification; scale bar: 54 µm) showing the processes from the cortical population (dTomato) contacting the hippocampal neurons (EYFP). b Illustrations showing the layout of electrodes (light gray circles) in 1-well MEAs with a co-culture (left panel) when the inter-population connections are intact (un-cut: upper left) or severed (cut: lower left) by making a cut between the two populations (fluorescent images; 20x magnification; scale bar: 54 µm). Recordings from one electrode within the postsynaptic population (right panels) when treated with blue light (blue solid triangle), presynaptic electrical stimulation (red bolt), and CTZ (orange pipette tip) with connecting processes intact (right upper) versus cut (right lower). c Schematic showing the color code (left panel) used for the ladder plots for both un-cut (peach) and cut (gray) co-cultures in the right panel. Ladder plots (right panel) showing change in number of spikes of postsynaptic neurons 5 s before and after each treatment (blue light; electrical stimulation; CTZ) for both the un-cut (peach) and cut (gray) conditions (blue light, un-cut n = 63, p < 0.0001, cut n = 58, p < 0.0001; electrical stimulation, un-cut n = 54, p < 0.0001, cut n = 32, p = 0.0965; CTZ, un-cut n = 51, p < 0.0001, cut n = 38, p = 0.7388; 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 CTZ). ns, not significant; ****p < 0.0001.