2. Methods
2.1. Culture and differentiation of hiPSCs
All hiPSC-related work was approved by the RCSI Research Ethics
Committee (REC202302020). hiPSC line HPSI0114i-eipl_1 (ECACC 77650081;
Culture Collections, Public Health England, UK) was made by
reprogramming with a non-integrating virus from skin fibroblasts from a
healthy individual at passage 32. Cells were maintained under
feeder-free conditions on vitronectin (STEMCELL Technologies, British
Columbia, Canada)-coated 6-well plates in E8 medium (Thermo Fisher
Scientific, Massachusetts, USA). hiPSCs were dissociated by using 0.5 mM
EDTA for 2 min at 37°C, and reseeded at the density of
1 × 104 cells per cm2. For the
neural induction of hiPSCs, approximately 24 h after splitting, culture
medium was switched to Gibco PSC Neural Induction Medium (Thermo Fisher
Scientific, Massachusetts, U.S.A.) containing Neurobasal medium and
Gibco PSC neural induction supplement. Neural induction medium was
changed every other day from day 0 to day 4 of neural induction and
every day thereafter. At day 10 of neural induction, primitive neural
stem cells (pNSCs) were dissociated with Accutase (Thermo Fisher
Scientific, Massachusetts, USA) and plated on Geltrex-coated dishes at a
density of 1 x 105 cells per cm2 in
NSC expansion medium containing 50% Neurobasal medium, 50% Advanced
DMEM/F12, and 1% neural induction supplement (Thermo Fisher Scientific,
Massachusetts, U.S.A.). pNSCs were passaged on the 4thday at a 1:3 split ratio to derive NSCs. For the differentiation of
neurons, NSCs were plated on Geltrex-coated coverslips or on a monolayer
of human primary glia at a density of 5 x 104 cells
per cm2 in neuronal differentiation medium consisting
of neurobasal medium and DMEM-F12 (1:1), with 2% B-27 supplement, 1%
N2 supplement, 1% L-glutamine, 1% nonessential animal acids, 20 ng/ml
brain-derived neurotrophic factor (BDNF), 20 ng/ml glial cell-derived
neurotrophic factor (GDNF) (all from Thermo Fisher Scientific,
Massachusetts, USA), 100 ng/mL cAMP, 100 µM L-ascorbic acid and
penicillin/streptomycin (all from Merck, Missouri, United States). The
culture medium was changed every 2-3 days. Neurons which were
differentiated from hiPSC-derived neural progenitors, hereafter referred
as hiPSC line 2, were obtained from Roche Pharmaceuticals (Lau et al.,
2024).
2.2. Immunocytochemistry
Neurons cultured on coverslips were fixed with a combination of acetic
acid (6.71%) and ethanol (62.5%) for 15 min. After 3 washes in
phosphate buffered saline, cells were permeabilized with 0.1% Triton
for 20 min and blocked in 1% BSA for 30 min. Cells were incubated at
4°C overnight in primary antibody diluted in 1% BSA. Cells were then
incubated in secondary antibody diluted in 1% BSA for 1 h at room
temperature. The following primary antibodies were used: mouse
anti-β-tubulin III as a marker for neuron-specific cytoskeletal proteins
(Biolegend, San Diego, CA, dilution 1:500) and rabbit anti-P2X7R
(Alomone Labs, Jerusalem, Israel, dilution 1:200) or rabbit anti-P2X7R
(Synaptic Systems, Göttingen, Germany, dilution 1:200) as markers for
P2X7R. The respective secondary antibodies were conjugated to Alexa
Fluor-488 or Alexa Fluor-594 (Thermo Fisher Scientific, Massachusetts,
USA) and used at a dilution of 1:1000. Coverslips were mounted and
images were acquired using a Leica DM4000B fluorescence microscope.
2.3. Drug application
Stock solutions of BzATP (Alomone Labs, Jerusalem, Israel), ATP (Merck,
Missouri, United States), AFC-5128 (Beamer et al., 2022) and
JNJ-47965567 (Alomone Labs, Jerusalem, Israel) were diluted and applied
in HEPES-buffered extracellular solution. The drug solutions were
delivered to the recorded cells by a valve-controlled fast multibarrel
superfusion system with a common outlet approximately 350 µm in diameter
(Automate Scientific, California, USA). The application tip was
routinely positioned approximately 1 mm away from and ∼50 µm above the
surface of the recorded cells. A computer connected to Digidata 1550B
controlled the onset and duration of each drug application. The drugs
were used at the following final concentration: BzATP and ATP (300 µM),
AFC-5128 (30 nM) and JNJ-47965567 (100 nM).
2.4. Calcium Imaging
For calcium imaging of BzATP-evoked responses, neurons, differentiated
on Geltrex-coated coverslips without human primary glia, were loaded
with Cal-520 (AAT Bioquest, California, USA) by incubation with the
acetoxymethyl (AM) ester form of the dye at a final concentration of 2
μM in culture media without serum. The dyes were prepared as 5 mM stock
solution in dimethyl sulfoxide (DMSO) and kept frozen at -20°C and
diluted on the day of use. After 45 min, cells were washed several times
with dye-free HEPES-buffered saline solution and transferred to an
imaging chamber on a microscope (Zeiss Axio Examiner, Jena, Germany)
equipped with a Zeiss 40x water immersion objective. Zen Blue imaging
software (Carl Zeiss, Jena, Germany) was used for hardware control and
image acquisition, and image analysis was performed using ImageJ (NIH,
Maryland, USA). All imaging experiments were performed at 34°C in a low
divalent cation-containing bath solution with the composition (in mM):
135 NaCl, 3 KCl, 0.5 CaCl2, 0.1 MgCl2,
10 HEPES and 10 glucose (pH 7.2; osmolality 290-300 mmol/kg). Images
were acquired at 4 Hz. Background fluorescence was measured from the
cell-free area outside the soma of interest in each frame of every time
series. Region of interests (ROIs) were manually drawn around the soma
and baseline fluorescence intensity (F0) was determined by averaging 24
frames preceding the cell’s exposure to BzATP or ATP and the time course
of normalized fractional dye fluorescence [ΔF/F0] was obtained,
where ΔF equals F(t) - F0.
2.5. Induction of acute or chronic epileptiform-like activity
Neurons plated on human primary glia was used for the induction of acute
or chronic epileptiform-like activity. Acute epileptiform-like activity
was induced by exposure to the chemical convulsant picrotoxin (100 µM)
for 10 min. To induce chronic epileptiform-like activity, the cultures
were exposed to picrotoxin (100 µM) for 7 to 12 days where a chronic
epileptiform-like activity with neuroinflammation was induced by
treating the cultures with a cocktail of picrotoxin (100 µM), tumour
necrosis factor-α (TNF-α) (30 ng/ml), IL-1α (3 ng/ml), IL-1β (3 ng/ml)
and complement component 1q (C1q) (400 ng/ml) for 7 to 12 days(Hyvarinen et al., 2019; Stoberl et al., 2023) .
2.6. Burst detection and analysis
Loose patch-clamp experiments were carried out with a Multiclamp 700 B
amplifier (Molecular Devices, California, USA) which was interfaced by
an A/D-converter (Digidata 1550B, Molecular Devices, California, USA) to
a PC running pClamp software (Version 11, Molecular Devices, California,
USA). The signals were low-pass filtered at 2 kHz and sampled at 10 kHz.
Pipette electrodes (G150T-4, Harvard Apparatus, Massachusetts, USA) were
fabricated using a vertical puller (Narishige PC-100, Tokyo, Japan). All
recordings were performed at 34°C in a bath solution containing (in mM):
135 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10
HEPES and 10 glucose (pH 7.2; osmolality 290-300 mmol/kg). Spontaneous
action potential firing and bursts were detected in loose-patch
configuration with patch pipettes filled with the bath solution. A spike
was counted when the signal recorded in loose patch configuration
exceeded a threshold of ±5 σ, where σ was the standard deviation of the
baseline noise during quiescent periods. The following parameters were
used for burst detection: maximum interval to start burst was set to 100
ms, maximum continuing in-burst interval was set to 5000 ms, and minimum
number of spikes in a burst was set to 5.
2.7. Statistical analysis
The manuscript complies with BJP’s recommendations and requirements on
experimental design and analysis. All experiments were designed to
generate groups of equal size, using randomisation and blinded analysis.
All statistical analyses were performed with GraphPad Prism 9 software
(GraphPad Software, San Diego, CA, USA) and are described in Figure
legends. Initially, all datasets were tested for normality using the
Shapiro-Wilk test. For datasets failing normality, Mann-Whitney test
(two-tailed) was performed. For multiple comparisons, repeated-measures
ANOVA and Tukey’s Post hoc tests were performed. Non-parametricKolmogorov-Smirnov test was used to test for one-dimensional
probability distribution. Data are expressed as mean ± SEM.p < 0.05 was considered statistically significant.