Fig. 4: PACS1^(+/R203W) organoids have increased GABAergic synaptic density but no differences in neural activity detected by calcium imaging.

a Experimental workflow for calcium imaging in organoids from two independent differentiations. b Expression of glutamatergic synapse markers in day 79 organoid sections. Co-localization of pre-synaptic marker synapsin 1 (SYN1) and post-synaptic density protein 95 (PSD95) was used to identify glutamatergic synapses. Scale bar = 10 µm in the main panels and 1 µm in the zoomed panel. The median value is indicated by dashed lines and the dotted lines represent quartiles. Data is from n = 16 total sections across two C2, one C3, and one CRISPR A1 PACS1^(+/+) organoids and n = 15 total sections across two CRISPR R203W, one A1, and two A2 PACS1^(+/R203W) organoids from three independent differentiations. See Table S1. U = 118, p = 0.953 using a two-sided Mann-Whitney test. y axis is on a log₁₀ scale. Individual cell line results are shown in Fig. S13a. c Expression of GABAergic synapse markers in day 79 organoid sections. Co-localization of pre-synaptic marker vesicular GABA transporter (vGAT) and post-synaptic marker Gephyrin (GPHN) was used to identify synapses. Scale bar = 10 µm in the main panels and 1 µm in the zoomed panel. The median value is indicated by dashed lines and the dotted lines represent quartiles. Data is from n = 14 sections across two C2, one C3, and one CRISPR A1 PACS1^(+/+) organoids and n = 20 sections across two CRISPR R203W, one A1, and two A2 PACS1^(+/R203W) organoids from two independent differentiations. See Table S1. U = 71, p = 0.015 calculated using a two-sided Mann–Whitney test. y axis is on a log₁₀ scale. Individual cell line results are shown in Fig. S13a. d Representative calcium traces of PACS1^(+/+) (blue) and PACS1^(+/R203W) (red) cells over 300 s of recording. e Bar chart showing mean ± SEM of number of active cells per mm² of an organoid section. Scale is log₁₀ for clarity. There is an effect of day (F_1,23 = 8.576, p_adj = 0.008) but not genotype (F_1,23 = 0.482, p_adj = 0.495) using a two-way ANOVA test with a Tukey HSD correction on log-transformed values. Dots represent sections from n = 5 Day 40 PACS1^(+/+) organoids (three C1 and two CRISPR A1), n = 6 Day 40 PACS1^(+/R203W) organoids (three CRISPR R203W and three A1), n = 8 Day C2 80 PACS1^(+/+) organoids, and n = 7 Day 80 PACS1^(+/R203W) organoids (four CRISPR R203W and three A1) from two independent differentiations for (e–h). See Table S1 for more details on number of replicates per line used in each experiment. Data for each cell line is shown in Fig. S13c. f Bar chart displaying the mean ± SEM of Ca²⁺ event amplitude. There is an effect of day (F_1,23 = 15.481, p_adj = 6.815E-04) but not genotype (F_1,23 = 0.484, p_adj = 0.494) using a two-way ANOVA test with a Tukey HSD correction. Data for each cell line is shown in Fig. S13d. g Bar chart displaying the mean ± SEM of calcium event width. There may be an effect of day (F_1,23 = 3.811, p_adj = 0.064) but not genotype (F_1,23 = 2.648, p_adj = 0.117) using a two-way ANOVA test with a Tukey HSD correction. Data for each cell line is shown in Fig. S13e. h Bar chart showing mean ± SEM of average frequency of calcium events in untreated conditions. There is no effect of genotype (H₁ = 0.111, p_adj = 0.815) or day (H₁ = 0.055, p_adj = 0.815) on event frequency using a one-sided Kruskal-Wallis test for genotype and day followed by a Benjamini Hochberg correction as data was non-parametric (p_shapiro = 9.67E-04). i Plot showing mean ± SEM of the effect of GABA_A antagonist bicuculline (BCU) on calcium event frequency. While BCU has a strong effect on event frequency (t₂₂ = 6.473, p = 1.63E-06 calculated using a one sample two-sided t test), there is no effect of day (F_1,20 = 2.430, p_adj = 0.135) or genotype (F_1,20 = 1.441, p_adj = 0.244) using a two-way ANOVA test with a Tukey HSD correction. Dots represent the difference in event frequency in paired recordings from organoid sections following a 15 min incubation with 50 µM bicuculline. Statistics were calculated from n = 5 Day 40 PACS1^(+/+) organoids (three C1 and two CRISPR A1), n = 6 Day 40 PACS1^(+/R203W) organoids (three CRISPR R203W and three A1), n = 6 Day C2 80 PACS1^(+/+) organoids, and n = 6 Day 80 PACS1^(+/R203W) organoids (three CRISPR R203W and three A1) from two independent differentiations. Data for each cell line is shown in Fig. S13f. Legend also applies to (e–g). Source data for (b–c) and (e–i) can be found in the Source Data file.