Whole-cell patch clamp Electrophysiology
These experiments were performed as described earlier (Benerjee et al.,
2017; 2020). Briefly, resected hippocampal and non-seizure control
tumour periphery tissues were placed in ice-cold, carbogenated (95%
O2, 5% CO2) artificial cerebrospinal
fluid (ACSF; NaCl, 125 mM; KCl, 2.5 mM; CaCl2, 2 mM;
NaHCO3, 25 mM; NaH2PO4,
1.25 mM; MgCl2, 1 mM; and Glucose,25 mM.) and brought to
the laboratory, followed by 350µm thick slices were prepared in a
vibratome. Slices were prepared by making tangential cuts to the outer
surface of the cortical specimens and were incubated at room temperature
for 30 mins and transferred to a recording chamber which was perfused
(2ml/min) with carbogenated ACSF. Normal appearing pyramidal neurons
with thick pyramid like soma and single tapering dendrite were
morphologically identified visually using Infrared-assisted
video-microscopy with differential interference contrast (IR-DIC). Cells
on the surface slice preparations were usually dead, so we used the
pale-looking pyramidal neurons from layer III or IV for our studies.
Patch pipettes with resistance of 3-5 MΩ were filled with internal
solution containing HEPES, 10 mM; MgCl2, 2 mM,
Cs-methanesulfonate, 130 mM; EGTA, 10 mM; CsCl, 10 mM. Passive membrane
properties of neurons were determined by “membrane test” function of
pCLAMP 10.0 software (Molecular Devices, USA). Whole cell patch clamp
recordings were performed from those pyramidal neurons using an
amplifier (Axopatch 200B, Molecular Devices, USA). Spontaneous
excitatory postsynaptic currents (EPSCs) were recorded at -70mV holding
potential and spontaneous inhibitory postsynaptic currents (IPSCs) at
0mV respectively. The access resistance were between 15-20 MΩ and change
of more than 20% from this range was not reliable and data of those
neurons were discarded. The leak current during recording was between
50-150 pA. If it was more than 200 pA, the data became unreliable. All
recordings were performed at room temperature (22-24°C)
Data were analysed in pCLAMP 10.0 software. Frequency, amplitude, rise
time (10-90%) and decay time constant (τd) of the
sEPSCs and sIPSCs were measured. The amplitude threshold for sEPSCs and
sIPSCs were -5pA and +10 pA respectively. All recordings were visually
inspected to select events which show a steep rising and exponential
decay phase for kinetic analysis of EPSCs and IPSCs. Events that did not
show the above-mentioned rise and decay phase were not typical synaptic
waveform and these were rejected manually. Events that showed multiple
peaks were excluded for kinetic analysis (rise time and decay time
constant) but included for frequency calculation as multiple events.
Cumulative distribution of inter-event interval and amplitude in
non-seizure control vs MTLE-HS groups were compared using
Kolmogorov-Smirnov test (K-S test).