Figure S4. OptoNeuron–astrocyte... | Rockefeller University Press

Figure S4.

$OptoNeuron–astrocyte coculture and dual modulation. (A) Representative confocal Z stack of a single GFAP-positive astrocyte within coculture spheres (ratio of 1:1,000 iAstros:OptoNeurons) after 1 d of coculture (i.e., sphere formation) revealed multiple primary branches immediately extending into the tissue. By day 21, iAstros were extensively elongated and branched. Scale: 100 µm. (B) Synaptophysin (SYP)-positive presynaptic puncta were observed in close proximity to astrocytes by day 21 of coculture. Scale: 50 µm. (C) Spheres containing day-22 OptoNeuron monocultures or OptoNeuron-iAstro cocultures (1:1) demonstrated significantly increased firing rates when exposed to 40 Hz blue light stimulation (+), as measured on MEAs on day 22 of neuronal differentiation with 0.1 Hz cutoff threshold (n = 6–19 electrodes each, after threshold). Shown are mean firing rates after threshold with 40 Hz optical stimulation (left), stimulated response as a percentage of the spontaneous firing rate (middle), and Log2FoldChange (stimulated/spontaneous firing rates; right). Firing rates results are also displayed in Fig. 3 C. OptoNeurons, coculture, and iAstros data are shown left to right in the plots. (D) Oxygen consumption rates (OCR) of iAstro monocultures and OptoNeuron–iAstro cocultures (1:1) at baseline, ATP-linked, maximal, and spare capacity respiration (after treatment with oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin A, respectively), as measured by Seahorse assay (n = 6 spheres each). (E) Control (top) and 6-d CNO–treated (bottom) spheres stained for Synapsin1. Scale: 50 µm. (F) Average ClCasp3 signal as a fraction of total nuclear signal (measured by DAPI) in control and 6-d CNO–treated coculture spheres (n = 7–10 images each). (G) THBS1 protein was significantly increased in astrocyte-conditioned media from monocultures of Astrostim cells stimulated with 2-d CNO treatment compared with controls, as measured by ELISA (n = 3 each). (H) Neither OptoNeurons in monoculture (left) nor cocultured with Astrostim cells (10:1; right) displayed a significant different in either spontaneous or light-induced firing rates with 2-h or 2-d CNO treatment, compared with controls (n = 8–24 electrodes each, after threshold). Results are shown as mean ± SEM. In C and H, dots represent individual spheres or electrodes. For H, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test between spontaneous or light-induced firing rates across groups, with *, P ≤ 0.05.$

OptoNeuron–astrocyte coculture and dual modulation. (A) Representative confocal Z stack of a single GFAP-positive astrocyte within coculture spheres (ratio of 1:1,000 iAstros:OptoNeurons) after 1 d of coculture (i.e., sphere formation) revealed multiple primary branches immediately extending into the tissue. By day 21, iAstros were extensively elongated and branched. Scale: 100 µm. (B) Synaptophysin (SYP)-positive presynaptic puncta were observed in close proximity to astrocytes by day 21 of coculture. Scale: 50 µm. (C) Spheres containing day-22 OptoNeuron monocultures or OptoNeuron-iAstro cocultures (1:1) demonstrated significantly increased firing rates when exposed to 40 Hz blue light stimulation (+), as measured on MEAs on day 22 of neuronal differentiation with 0.1 Hz cutoff threshold (n = 6–19 electrodes each, after threshold). Shown are mean firing rates after threshold with 40 Hz optical stimulation (left), stimulated response as a percentage of the spontaneous firing rate (middle), and Log₂FoldChange (stimulated/spontaneous firing rates; right). Firing rates results are also displayed in Fig. 3 C. OptoNeurons, coculture, and iAstros data are shown left to right in the plots. (D) Oxygen consumption rates (OCR) of iAstro monocultures and OptoNeuron–iAstro cocultures (1:1) at baseline, ATP-linked, maximal, and spare capacity respiration (after treatment with oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin A, respectively), as measured by Seahorse assay (n = 6 spheres each). (E) Control (top) and 6-d CNO–treated (bottom) spheres stained for Synapsin1. Scale: 50 µm. (F) Average ClCasp3 signal as a fraction of total nuclear signal (measured by DAPI) in control and 6-d CNO–treated coculture spheres (n = 7–10 images each). (G) THBS1 protein was significantly increased in astrocyte-conditioned media from monocultures of Astrostim cells stimulated with 2-d CNO treatment compared with controls, as measured by ELISA (n = 3 each). (H) Neither OptoNeurons in monoculture (left) nor cocultured with Astrostim cells (10:1; right) displayed a significant different in either spontaneous or light-induced firing rates with 2-h or 2-d CNO treatment, compared with controls (n = 8–24 electrodes each, after threshold). Results are shown as mean ± SEM. In C and H, dots represent individual spheres or electrodes. For H, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test between spontaneous or light-induced firing rates across groups, with *, P ≤ 0.05.