Fig. 5: PACS1^(+/R203W) excitatory neurons display prolonged network bursting patterns and decreased synchrony.

a NEUROG2 expression in single-cell RNA sequencing data. Scale is in units of log₁₀(value + 0.1). b Experimental protocol for rapid induction of glutamatergic neurons using a dox-inducible system enabling overexpression of NEUROG2. c MAP2 expression in neurons produced with the NEUROG2 overexpression protocol 65 days after viral induction. Scale bar = 100 µm. d Representative day 46 neurons transfected and traced for Sholl analysis. Scale bar =100 µm. The blue and orange colors are used to represent data from PACS1^(+/+) neurons and PACS1^(+/R203W) neurons respectively in all remaining panels. e Number of neurite intersections with concentric circles of increasing distance from the cell body. Dots are averages from n = 4 PACS1^(+/+) cell lines (one C1, two CRISPR A1, and one CRISPR A2 differentiations) and n = 4 PACS1^(+/R203W) cell lines (one CRISPR R203W, two A1, and one A2 differentiations) from two independent experiments. The smoothed curve shows genotype averages with error bands indicating a 95% confidence interval. p > 0.05 for all micron bins using a two-sided student’s t test. f Average dendrites per neuron. Dots are neuronal metrics from n = 4 PACS1^(+/+) cell lines (one C1, two CRISPR A1, and one CRISPR A2 differentiations) and n = 4 PACS1^(+/R203W) cell lines (one CRISPR R203W, two A1, and one A2 differentiations) from two independent experiments; bar represents the mean ± SEM. See Table S1 for further details regarding which cell lines were used. t_5.6 = 0.193, p = 0.854 was calculated using a Welch two sample two-sided t test. g Maximum dendritic length as determined by the furthest concentric circle from the cell body with more than 1 intersection, to avoid axon-related skewing. Dots are neuronal metrics from n = 4 PACS1^(+/+) cell lines (one C1, two CRISPR A1, and one CRISPR A2 differentiations) and n = 4 PACS1^(+/R203W) cell lines (one CRISPR R203W, two A1, and one A2 differentiations) from two independent experiments; bar represents the mean ± SEM. t_4.7 = 0.180, p = 0.865 was calculated using a Welch two sample two-sided t test. h Dots are number of synapsin⁺ cell bodies visible within the multielectrode array (MEA) electrode field averaged by cell line (n = three C1, three CRISPR A1, one CRISPR A2, three CRISPR R203W, three A1, and two A2 differentiations) for each of three independent experiments; bar represents the mean ± SEM. t₁₃ = 0.243, p = 0.812 using a two-sided t test. i–o MEA metrics for neurons days 30–57 of differentiation. Dots represent averages from each cell line, while the smoothed curve shows genotype averages with error bands indicating a 95% confidence interval. n = 8 PACS1^(+/+) lines (four C1, three CRISPR A1, and one CRISPR A2) and n = 9 PACS1^(+/R203W) lines (four CRISPR R203W, three A1, and two A2) from three independent experiments. p values were calculated by conducting an ANOVA between a linear regression with and without genotype included as a covariate. In instances where p values are reported between specific time points, a Kruskal-Wallis test was run. i Mean firing rate in Hz. Genotype had no effect (F_0,2 = 0.933, p = 0.395). y axis is on a log₂ scale for clarity. j Number of electrodes with ≥1 spikes/minute. Genotype had no effect on progression of active electrodes (F_0,2 = 0.090, p = 0.914). k Number of network bursts over 5 min, as defined by ≥18% electrodes simultaneously active within a 100 msec window. Genotype had a significant effect on number of network bursts collectively (F_0,2 = 7.655, p = 5.63E-04), but no individual time point achieved significance. l Network burst duration. Genotype had a significant effect on network burst duration (F_0,2 = 123.17, p < 2.2E-16). Results by isogenic pair and differentiation are shown in Fig. S15a. m Spikes per burst. Genotype had no effect on spikes per burst (F_0,2 = 1.663, p = 0.192). n Interspike interval within a burst. Genotype had a significant effect on interspike interval (F_0,2 = 8.622, p = 2.35E-04). o Width of a correlogram at ½ height of cross-correlation. Genotype had a significant effect (F_0,2 = 37.918, p = 3.11E-15). Individual time points reached significance at day 41 onwards. Example of a neuron activity raster plot across 16 electrodes in PACS1^(+/+) (p) or PACS1^(+/R203) (q) wells. Above the raster plots is a spike histogram showing the collective density of activity over time. A network burst is highlighted in the light gray panels, which is zoomed in to the right. The pink dash below indicates the approximate location of the subsequent zoom panel. Source data for (e–o) can be found in the Source Data file.