2.4. Whole-cell patch-clamp electrophysiology

GABA-activated currents were recorded 24 h after transfection of HEK-293T or GIRK cells. Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from cultured hippocampal neurons at 12–17 days in vitro (DIV). Cells were superfused with a saline solution containing (mM): 140 NaCl, 4.7 KCl, 2.52 CaCl₂, 1.2 MgCl₂, 11 glucose, and 5 HEPES; pH 7.4. sIPSCs from hippocampal culture neurons were recorded in the presence of 2 mM kynurenic acid to block excitatory neurotransmission. For recording K⁺ currents in GIRK cells and neurons, the bath solution was switched to a high K⁺ solution containing 25 mM KCl and reduced NaCl (120 mM), to shift the equilibrium potential for K⁺ from approximately −90 to −47 mV, thus reversing the direction of K⁺ current flux to net inward. GABA_BR-activated K⁺ currents in cultured neurons were recorded in the additional presence of 2 mM kynurenic acid and 20 μM picrotoxin.

Borosilicate glass electrodes (3–5 MΩ) were filled with a K⁺-based internal solution for recording GIRK currents containing (mM): 120 KCl, 2 MgCl₂, 11 EGTA, 30 KOH, 10 HEPES, 1 CaCl₂, 1 GTP, 2 ATP, 14 creatine phosphate, pH 7.0. GABA_AR currents and sIPSCs were recorded with a Cs-based internal solution containing (mM): 120 CsCl, 1 MgCl₂, 11 EGTA, 30 KOH, 10 HEPES, 1 CaCl₂, and 2 K₂ATP; pH 7.2. All internal solutions were adjusted to approximately 305 mOsm/l. Cells were held at −60 mV (sIPSCs) or −60/−70 mV (K⁺ currents). Voltage clamp recordings were undertaken after optimising series resistance (Rs, <10 MΩ) and compensating for the whole-cell membrane capacitance. Membrane currents were filtered at 5 kHz (−3 dB, 6th pole Bessel, 36 dB/octave) and stored for analysis with Clampex 10.

Concentration-response curves were generated by measuring the current (I) for each ligand concentration and normalising to the maximal current response (I_max). Data fitting was performed with a Hill equation:

where I_min defines the pedestal current response, A is the concentration of the agonist, EC₅₀ is the concentration of agonist giving 50% of the maximum response and n is the Hill slope.

For the biphasic curve fits, the following modified Hill equation was used:

Where IC₅₀ defines the concentration of ligand causing a 50% reduction in the maximal current with a Hill slope of m (Halliwell et al., 1999).

sIPSCs were recorded at room temperature (20–23 °C) and detected using WinEDR and WinWCP (Strathclyde Electrophysiology Software UK), and frequency was calculated for 60 s recording epochs. For sIPSC amplitudes, several hundred events were recorded per condition and analysed as an average amplitude per cell.

For kinetic analysis, individual uncontaminated sIPSCs were isolated and the average 10–90% rise time and exponential decay times were measured from the mean sIPSC waveform. Weighted decay times are reported encompassing mono- and bi-exponentially decaying events according to the equation:

where τ₁ and τ₂ are exponential decay time constants, and A₁ and A₂ are the relative amplitude contributions of τ₁ and τ₂, respectively.

For tonic inhibition, to determine the average holding currents, a 30 s continuous holding current recording was sampled every 1 s, discarding epochs that coincided with sIPSCs. Any effect of CGP7930 or bicuculline on the holding current was defined by subtracting the average holding currents in control and during drug application. The baseline root-mean-square current variance (RMS) was calculated before and during drug treatment. This was estimated from a continuous (20 s) current recording, sampled every 100 ms. The median current was calculated every 5 s and values more than twice the standard deviation from the median (usually due to IPSCs) were eliminated. All drugs were applied using a U-tube rapid drug application system, or more slowly via bath perfusion.