Fig 1.
Generation and characterization of hiPSC derived neuron and astrocyte co- culture.
(A) Workflow and timeline to produce mature neurons from WTC11 neurogenin 2 inducible hiPSCs (B) Co- culture stained for mature neuronal marker MAP2 (green), astrocyte marker (red) and DAPI (blue).
Fig 2.
Co-culture electrophysiological maturation.
24-well quantification of (A) Mean firing rate (Hz), (B) Number of active electrodes, (C) Network burst frequency (Hz), (D) Network burst duration (s), (E) Network interburst interval coefficient of variation, and (F) Synchrony index on DIV 11, 17, 26, and 33. (G) Representative raster plots. Statistical analysis was performed with repeated measures ANOVA and Bonferroni corrections Error bar: SEM. N = 24 independent wells. p-value < 0.05 *, p-value < 0.01 **, p-value < 0.001 ***.
Fig 3.
Glutamate receptor inhibitor treatment.
Wells containing cultures DIV 33 were treated with either 10 μM CNQX, 10 μM AP5, both 10 μM CNQX and 10 μM AP5, or 0.1% DMSO (n = 4 per group). Measurements are shown at baseline (BL), 2 hours of treatment, 24 hours of treatment, 4 hours after a 50% media change at 24 hours after BL (wash 1) and 4 hours after a second 50% media change at 48 hours after BL (wash 2). Significant differences were seen in the mean firing rate (A) and percent change of the mean firing rate (B), the number of network bursts per 5-minute recording (C) and respective percent change (D), and the synchrony index (E) and respective percent change (F). Representative raster plots and histograms are shown in G. Repeated measures ANOVAs were significant (p < 0.05) for all parameters shown. A-priori post-hoc comparisons between experimental treatments and DMSO were performed, and p-values were Bonferroni corrected (*p < 0.05 and both CNQX and AP5+CNQX differed from DMSO #p < 0.05 and AP5+CNQX differed from DMSO). Error bars represent SEM.
Fig 4.
GABA receptor inhibitor treatment.
Wells containing cultures DIV 35 were treated with either 50 μM bicuculline or 50 μM picrotoxin (n = 5 per group). There were no significant differences in the parameters of mean firing rate (A), percent change of mean firing rate (B), network burst frequency (C), percent change in network burst frequency (D), synchrony index (E), and percent change in synchrony index (F) at 2 hours and 4 hours after treatment following baseline (BL) or after a 50% media change 24 hours later (wash). Error bars represent SEM.
Fig 5.
(A) Representative raster plots and histograms of relative population firing rates for baseline, 24 hours after treatment, and after 2 washouts. (B) Mean firing rate (Hz) (C) Number of active electrodes (D) Network burst frequency (Hz) (E) Network burst percentage (%) (F) Synchrony Index (G) Network Interburst Interval Coefficient of Variation. Statistical analysis was performed with repeated measures ANOVA and Bonferroni corrections. Error bar: SEM. N = 4 independent wells. p-value < 0.05 *, p-value < 0.01 **, p- value < 0.001 ***.