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).