Two-Electrode Voltage Clamp Recording

Defolliculated Xenopus laevis oocytes (stage V–VI) were obtained from Ecocyte BioScience (Austin, TX) and injected with cRNAs encoding GluN1 and GluN2 at a 1:2 ratio. The cRNA was diluted with RNase-free water to give responses with amplitudes ranging between 200 and 2000 nA (0.2–10 ng total cRNA). Following cRNA injection, the oocytes were stored at 15–19°C in Barth’s solution that contained (in mM) 88 NaCl, 2.4 NaHCO₃, 1 KCl, 0.33 Ca(NO₃)₂, 0.41 CaCl₂, 0.82 MgSO₄, and 5 Tris-HCl (pH 7.4 with NaOH), supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA), and 100 μg/ml gentamicin (Fisher Scientific, Pittsburg, PA). Recordings were performed 2–5 days following cRNA microinjection at room temperature (23°C) using a two-electrode voltage-clamp amplifier (OC725; Warner Instrument, Hamilton, CT) to measure current responses to 100 μM glutamate and 30 μM glycine, unless otherwise stated. The signal was low-pass filtered at 10–20 Hz (4-pole, −3 dB Bessel) and digitized at the Nyquist rate using PCI-6025E or USB-6212 BNC data acquisition boards (National Instruments, Austin, TX). Oocytes were placed in a custom-made chamber and continuously perfused (2.5 ml/min) with oocyte recording solution containing (in millimolar concentrations) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl₂, and 0.01 EDTA (pH 7.4 with NaOH). Solutions were applied by gravity, and solution exchange was controlled through a rotary valve (Hamilton, Reno, NV). Recording electrodes were filled with 0.3–3.0 M KCl, and current responses were recorded at a holding potential of −40 mV. Data acquisition, voltage, and solution exchange were controlled by custom software. Concentration-effect curves were expressed as a percentage of the response in the absence of test ligand and fitted by

where N is the Hill slope and maximum is the maximal response predicted for saturating concentration of potentiator.