Figure 2. BzATP-evoked Ca2+ responses in hiPSC-derived neurons. (a) Average time series showing response of hiPSC line 1-derived neurons to the application of BzATP (300 μM) in the absence (black trace) and presence (green trace) of AFC-5128 (30 nM) (Mann-Whitney U test, ****p < 0.0001, n = 76). (b) Individual peak ΔF/F0 amplitude during BzATP or BzATP and AFC-5128 co-application (Mann-Whitney U test, ****p < 0.0001, n = 73) and (c) AUC during BzATP or BzATP and AFC-5128 co-application (Mann-Whitney U test, ****p < 0.0001, n = 73).(d) Response to BzATP application in hiPSC line 1-derived neurons in the absence (black trace) and presence (purple trace) of JNJ-47965567. (Mann-Whitney U test, ****p < 0.0001, n = 86).(e) Individual peak ΔF/F0 amplitude during BzATP or BzATP and JNJ-47965567 co-application (Mann-Whitney U test, ****p < 0.0001, n = 86) and (f) AUC during BzATP or BzATP and JNJ-47965567 co-application (Mann-Whitney U test, ****p < 0.0001, n = 86). (g) Average time series showing response of hiPSC line 2-derived neurons to the application of BzATP (300 μM) in the absence (black trace) or presence (green trace) of AFC-5128 (30 nM) (Mann-Whitney U test, ***p < 0.0001, n = 57). (h)Individual peak ΔF/F0 amplitude during BzATP or BzATP and AFC-5128 co-application (Mann-Whitney U test, ***p = 0.0111, n = 52) and(i) AUC during BzATP or BzATP and AFC-5128 co-application (Mann-Whitney U test, ***p = 0.0007, n = 52). (j) Response to BzATP application in hiPSC line 2-derived neurons in the absence (black trace) and presence (purple trace) of JNJ-47965567. (Mann-Whitney U test, ****p = 0.0002, n = 26). (k) Individual peak ΔF/F0 amplitude during BzATP or BzATP and JNJ-47965567 co-application (Mann-Whitney U test, ****p = 0.0002, n = 26) and (l) AUC during BzATP or BzATP and JNJ-47965567 co-application (Mann-Whitney U test, ****p = 0.0002, n = 26).
Figure 3. hiPSC-derived neuronal PTX model of epileptiform-like events in vitro. (a) Schematic view of experimental design for acute epileptiform-like activity model. After loose-patch recording in baseline conditions, sustained epileptiform-like activity was induced by 10 min exposure to 100 µM PTX. Loose patch-clamp recordings were continued in PTX during sustained epileptiform-like activity. (b) Exemplary traces of loose-patch recording showing PTX-induced bursting activity (red). (c)Effect of PTX (100 µM) on burst frequency (Mann-Whitney U test, *p = 0.0358 , n = 8). (d) Synchronization matrix based upon the peaks of the different fluorescence traces in the presence of physiological saline, PTX or TTX (n = 60). (e) Raster plot of calcium transients in the presence of physiological saline, PTX or TTX (n = 60).
Figure 4 Effect of AFC-5128 on burst parameters in acute or chronic epileptiform-like activity model. (a) Schematic showing experimental design for acute epileptiform-like activity model. Loose-patch recordings were performed in the presence of physiological saline, PTX or PTX and P2X7 antagonists AFC-5128 or JNJ-47965567. (b-e) Effect of AFC-5128 on burst parameters in acute PTX model of acute epileptiform-like activity. Burst frequency: baseline vs. PTX, p = 0.3074; PTX vs. PTX and AFC-5128, p = 0.4591; baseline vs. PTX and AFC-5128, p > 0.9999; number of spikes in burst: baseline vs. PTX, p > 0.9999; PTX vs. PTX and AFC-5128, p = 0.3074; baseline vs. PTX and AFC-5128, p = 0.4591; burst duration: baseline vs. PTX, p > 0.9999; PTX vs. PTX and AFC-5128, p > 0.9999; baseline vs. PTX and AFC-5128, p > 0.9999; interburst interval: baseline vs. PTX, p = 0.8593; PTX vs. PTX and AFC-5128, p = 0.0990; baseline vs. PTX and AFC-5128, p = 0.8593 (n = 12). Friedman’s ANOVA with Dunn’s multiple comparisons test. (f-i) Effect of JNJ-47965567 on burst parameters in acute PTX model of epileptiform-like activity. Burst frequency: baseline vs. PTX, p > 0.9999; PTX vs. PTX and JNJ-47965567, p = 0.7446; baseline vs. PTX and JNJ-47965567,p = 0.4467; number of spikes in burst: baseline vs. PTX, p= 0.9999; PTX vs. PTX and JNJ-47965567, p = 0.0975; baseline vs. PTX and JNJ-47965567, p = 0.1841; burst duration: baseline vs. PTX, p = 0.1841; PTX vs. PTX and JNJ-47965567, p = 0.0975; baseline vs. PTX and JNJ-47965567, p > 0.9999; interburst interval: baseline vs. PTX, p > 0.9999; PTX vs. PTX and JNJ-47965567, p = 0.5443; baseline vs. PTX and JNJ-47965567, p = 0.1841 (n = 7). Friedman’s ANOVA with Dunn’s multiple comparisons test. (j) Schematic showing experimental design for chronic epileptiform-like activity model. Neuronal networks were treated with PTX for 2 days followed by treatment with PTX and neuroinflammatory agents for 7-12 days before loose-patch recordings were performed. (k-n) Effect of AFC-5128 on burst frequency, spikes per burst, burst duration and IBI in chronic model of epileptiform-like activity. Burst frequency: PTX vs. PTX and AFC-5128, *p = 0.0342; number of spikes in burst: PTX vs. PTX and AFC-5128, p = 0.3054; burst duration: PTX vs. PTX and AFC-5128, p = 0.5879; interburst interval: PTX vs. PTX and AFC-5128, *p = 0.0479 (n = 13), Wilcoxon matched-pairs signed rank test. (o-p) Histogram of IBI distribution using log transformed data overlaid with PDF in the presence of PTX or PTX and AFC-5128. Note the appearance of a population of bursts with longer IBI in the presence of AFC-5128 (indicated by arrow). (q) KDE estimation indicating the appearance of a peak (indicated by arrow) at longer IBI in the presence of PTX and AFC-5128. (r-s) Histograms of IBI distribution in the presence of PTX or PTX and AFC-5128 fitted with a gamma function. (t) Cumulative frequency distribution of IBI upon PTX or PTX and AFC-5128 treatment. (Kolmogorov–Smirnov test, **p = 0.0198, n = 12).
Figure 5 Effect of AFC-5128 on epileptiform-like activity in CBZ-resistant model. (a) Schematic showing experimental design for CBZ-resistant epileptiform-like activity model. Neuronal networks were exposed to 100 µM PTX for 2 days, followed by PTX and neuroinflammatory agents for 7-12 days before loose-patch-clamp recordings were performed in the presence of PTX or PTX and CBZ or PTX, CBZ and AFC-5128. (b) Effect of burst frequency in the presence of CBZ (n = 16). Neurons that failed to reduce burst frequency by 70% or more upon CBZ exposure were classified as CBZ-resistant. (c)Chart showing percentage of CBZ-sensitive or resistant neurons (n = 16).(d) Exemplary traces showing bursting activity in the presence of PTX (top), PTX and CBZ (middle) or PTX, CBZ and AFC 5128 (bottom) at indicated concentrations. (e-h) Effect of co-application of CBZ and AFC-5128 on burst parameters Burst frequency: PTX vs. PTX and CBZ: p = 0.8666; PTX and CBZ vs. PTX, CBZ and AFC-5128: *p = 0.0442; spikes in burst: PTX vs. PTX and CBZ: *p = 0.0173; PTX vs. PTX and CBZ and AFC-5128: **p = 0.0017; burst duration: PTX vs. PTX and CBZ: p = 0.1390; PTX and CBZ vs. PTX, CBZ and AFC-5128:p = 0.0585; PTX vs. PTX, CBZ and AFC-5128: **p = 0.0098; IBI: PTX vs. PTX and CBZ: p = 0.5748; PTX and CBZ vs. PTX, CBZ and AFC, p = 0.5526, n = 12, Friedman’s ANOVA with Dunn’s multiple comparisons test. (i-k) Histogram of IBI distribution using log transformed data overlaid with PDF in the presence of PTX or PTX and CBZ or PTX, CBZ and AFC-5128. Note the appearance of a population of bursts with longer IBI in the presence of AFC-5128 (n = 12). (l) KDE estimation indicating lower density in the prese of PTX, CBZ and AFC-5128 (n = 12). (m-o) IBI distribution in the presence of PTX or PTX and CBZ or PTX, CBZ and AFC-5128 fitted with a gamma function (n = 12). (p) Cumulative frequency distribution of IBI upon PTX and CBZ or PTX, CBZ and AFC-5128 treatment. (Kolmogorov-Smirnov test, **p = 0.008, n = 12).