Fig. 6

Calcium dynamics are affected upon POLY(I:C), yet ibuprofen needs iMG to reverse the phenotype. a Example image of iMG-_dissRO immunostained for the neuronal marker MAP2 (grey), the presynaptic marker VGLUT1 (green), and the postsynaptic marker PSD95 (magenta) with zoom-in. Scale bar: 10 µm. b–d Boxplots of synaptic puncta quantification in _dissRO (grey) and iMG-_dissRO (orange) for VGLUT1⁺-puncta per 10 µm MAP2 (a), PSD95⁺-puncta per 10 µm MAP2 (b) and VGLUT1⁺/PSD95⁺-puncta per 10 µm MAP2 (c). Each dot represents one process close to the soma of a single cell. Five independent dissociations from three differentiations. Wilcox-test. e Experimental timeline. At WK17, _dissRO transduced with AAV2-GCAMP6s. preMG added at WK18.5. Four days before calcium imaging, gradual transition to Brain-Phys medium until WK20. f Three example traces of spontaneous calcium transients in iMG-_dissRO. g Example ROI image of iMG-_dissRO expression of the calcium sensor GCAMP6s (green) and tdTomato⁺-iMG (orange). Scale bar: 50 µm. h Spontaneous calcium dynamics during 5 min recording in _dissRO (grey) and iMG-_dissRO (orange). Boxplot of the mean frequency [Hz] (left) and the mean peak amplitude [F/F₀] (right). Wilcoxon rank-sum test. i TTX abolishes calcium transients in _dissRO (left) and iMG-_dissRO (right). Boxplot of mean frequency [Hz] during 150-s baseline recording (baseline) following TTX application for another 150 s (+ TTX). One-sample Wilcoxon signed rank test. j Boxplot of mean frequency [Hz] during 150-s baseline recording (baseline) following exposure to glutamatergic blockers CPP, APB, and NBQX for 150 s in _dissRO (left) and iMG-_dissRO (right). Wilcoxon rank-sum test. k Boxplot of mean frequency [Hz] during 150-s baseline recording (baseline) following Ca2⁺-chelator EGTA application for another 150 s in iMG-_dissRO. One-sample Wilcoxon signed rank test. l, m Spontaneous calcium dynamics during 5 min recording after 24 h of either fresh medium for control (CTRL, grey), POLY(I:C) (magenta), or POLY(I:C) and S(+)-ibuprofen (POLY(I:C) + IBU, blue) exposure in iMG-_dissRO (i) and _dissRO (j). Boxplot of the mean peak amplitude [F/F₀]. Each dot represents an active cell. Recordings from five independent differentiations. Kruskal–Wallis test with post-hoc Dunn’s test. k Spontaneous calcium dynamics in iMG-_dissRO after 24 h POLY(I:C) stimulation and following exposure to glutamatergic blockers CPP, APB, and NBQX. Boxplot of the mean frequency [Hz] (left) and the mean peak amplitude [F/F₀] (right). Each dot represents an active cell from five independent dissociations from three independent differentiations. Wilcoxon rank-sum test.For detailed statistical analysis, see Supplementary Table 4. ***p < 0.001. **p < 0.01. ^nsp > 0.05, not significant. AAV adeno-associated virus, APB 2-aminoethoxydiphenyl borate, CPP 4-(3-phosphonopropyl)piperazine-2-carboxylic acid, CTRL untreated control, _dissRO dissociated retinal organoid cultures without iMG, EGTA Ethylene glycol tetraacetic acid, IBU ibuprofen, iMG microglia-like cells, iMG-_dissRO microglia integrated into dissociated retinal organoid culture, MAP2 microtubule-associated protein 2, NBQX 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline, POLY(I:C) polyinosinic: polycytidylic acid, PSD95 postsynaptic density protein 95, _dissRO dissociated retinal organoid cultures, Sec seconds, TTX tetrodotoxin, VGLUT1 vesicular glutamate transporter 1, WK week after the start of _3DRO differentiation