Fig. 4: Spontaneous and evoked activity in neural networks in Hi-Q brain organoids.

A Fluorescence intensity image of an organoid loaded with the calcium indicator OGB-1. B Exemplary traces of single cells revealing spontaneous calcium signaling in day 40 and day 150 old brain organoids. Asterisks highlight occasional synchronized activity. Wash-in of tetrodotoxin (TTX; 1 µM) strongly dampens spontaneous calcium signaling. C Pie charts illustrating active and inactive cells in brain organoids across all age groups (days 30, 40, 50, and 150). D Box plots showing median (line), mean (square), interquartile range (box), and standard deviation (whiskers) of the frequency of spontaneous calcium activity under control conditions and in the presence of TTX on day 40, 50, and 150 old brain organoids. Data are from three independent batches of the organoids in each age group, each containing at least four organoids (n = 4). Gray diamonds represent single data points/cells analyzed. E Intracellular calcium transients evoked by bath application of 1 mM glutamate (upper row) or 1 mM GABA (lower row) under control conditions and in the presence of ionotropic glutamate receptor inhibitors (APV for NMDARs and NBQX for AMPARs) or the presence of inhibitors of voltage- ion channels (TTX for Nav and NiCl2 for Cav) in day 50 brain organoids. Gray traces show individual cellular responses in one particular experiment; black traces are averages of the individual traces. F Box plots showing median (line), mean (square), interquartile range (box), and standard deviation (whiskers) glutamate- or GABA-induced calcium responses of day 50 brain organoids in control conditions and in the presence of indicated receptor/channel blockers. Data are from three independent batches of the organoids, each containing at least five organoids (n = 5). Gray diamonds represent single data points/cells analyzed. Gray diamonds are single data points/cells analyzed. For (D, E), data are presented in Tukey box-and-whisker plots indicating median (line), mean (square), interquartile range (IQR; box), and standard deviation (whiskers). In addition, all individual data points are shown in gray underneath the Tukey plots. Data were statistically analyzed using one-way ANOVA followed by a post hoc Bonferroni test. The following symbols are used to illustrate the results of statistical tests in the figures: *: 0.01 ≤p < 0.05; **: 0.001 ≤p < 0.01; ***: p < 0.001. “n” represents the number of cells analyzed; “N” means the number of individual experiments/brain organoids. Each series of experiments was performed on at least three different organoids.