Two-Electrode Voltage-Clamp Recordings from X. laevis.

X. laevis stage VI oocytes (Ecocyte Biosciences, Austin, TX) were isolated as previously described (Hansen et al., 2013) and injected with 50 nl of water containing 5–10 ng of cRNA encoding the GluN1 and GluN2 NMDAR subunits (3:7 ratio). Oocytes were stored at 15°C in media containing (in mM) 88 NaCl, 2.4 NaHCO₃, 1 KCl, 0.33 Ca(NO₃)₂, 0.41 CaCl₂, 0.82 MgSO₄, 5 Tris-HCl (pH was adjusted to 7.4 with NaOH), 1 U/ml penicillin, 0.1 mg/ml gentamicin sulfate, and 1 μg/ml streptomycin. Two to seven days after injection, two-electrode voltage-clamp recordings were performed at room temperature. All extracellular solutions contained (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl₂, and 0.01 EDTA (pH 7.4 with NaOH). Gravity-applied solutions (4 to 5 ml/min) were exchanged by an eight-port Modular Valve Positioner (Hamilton Company, Reno, NV) and controlled by custom software. Voltage and current electrodes were filled with 0.3 M and 3.0 M KCl, respectively. Oocyte currents were recorded at a holding potential of −40 mV and recorded by a two-electrode voltage-clamp amplifier (OC-725B or C; Warner Instruments, Hamden, CT).

(+)-CIQ and related analog stocks were prepared as a 20 mM solution in dimethylsulfoxide (DMSO), and working solutions were prepared during rapid stirring immediately before the experiment. For concentration-response curve recordings, 1-5 mM 2-(hydroxypropyl)-β-cyclodextrin was used to increase the solubility of modulators and subsequently added to all agonist solutions to control for any direct effects. Unless indicated otherwise, current responses were elicited by application of 100 μM glutamate plus 30 μM glycine. Currents from the GluA1 and GluK2 receptors were evoked with 100 μM glutamate; oocytes expressing GluK2 were soaked in 10 μM concanavalin-A for 5 minute prior to recording. Currents were evoked for the following receptors using the agonist concentrations indicated: GABA_C ρ1 (2 μM GABA), GABA_A α1β2γ2 (20 μM GABA), glycine α1 (50 μM glycine), 5-HT3A (15 μM serotonin), nicotinic acetylcholine α1β1δγ (20 μM acetylcholine), α4β2 (1 μM acetylcholine), α7 (100 or 300 μM acetylcholine), and the P2X2 receptor (9 μM ATP).