Measuring GABA-A Receptor Currents

To initiate a GABA_A receptor current neurons were voltage-clamped at a holding potential of -100 mV, and 2 mM of GABA were applied for 1–2 seconds [26 ]. These changes resulted in large outward GABA_A- receptor currents that were easily detected and differentiated from other currents. From the GABA_A receptor currents produced, we measured the peak amplitude, baseline holding current, decay time as the 90%-to-10% decay time, and the area under the curve as the integrated area between the measured current and the baseline using Clampfit software (Molecular Devices, Sunnyvale, CA, USA). Sheets were then perfused with oxygenated aCSF and 1 μM Alfaxalone for 15 min, and the GABA_A receptor current decay time, integrated area under the curve, peak amplitude, and baseline holding current were measured again. This procedure was repeated utilizing gabazine (25 μM), a GABA_A receptor antagonist, rather than Alfaxalone, to see if the currents could be blocked to confirm we were recording GABA_A receptor currents [26 ]. Additionally, Alfaxalone-free stock Alfaxalone solution (vehicle, a generous gift from Jurox; Rutherford, NSW, Australia) had no significant impact when applied alone (S1 Fig and S1 Dataset) (n = 4).
To construct a dose-response curve, GABA_A receptor current measurements following acute Alfaxalone treatment were normalized to pre-treatment values to determine the relative change in decay time, area under the curve, and peak amplitude. The same process to determine relative changes in decay time, area under the curve, and peak amplitude was repeated for tissue sheets perfused with oxygenated aCSF and 0.1 μM, 0.5 μM, 1 μM or 1.5 μM Alfaxalone for 15 min (S2 Fig and S1 Dataset). Data was fit to the 4-parameter Hill equation (log-logistic equation) to construct a dose response curve for each of the parameters [28 (link)].