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).