Two electrode voltage clamp recordings from Xenopus oocytes

Healthy-looking, defolliculated stage V-VI Xenopus laevis oocytes (EcoCyte Bioscience) were injected with cRNA for GluN1 and GluN2 in a 1:2 ratio (5–10 ng total in 20–50 nl water). Experiments utilized human GluN1/GluN2A and GluN1/GluN2B, rat GluN1/GluN2A_C1/GluN2A_C2 [33], and rat GluN1/GluN2C and GluN1/GluN2D cRNA. Following injection, oocytes were maintained at 15°C in Barth’s culture medium 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 7.4 with NaOH), 1 U/mL penicillin, 0.1 mg/mL gentamicin sulfate, and 1 μg/mL streptomycin. Recordings were performed 2–7 days after injection at 23°C. Oocytes were transferred to a recording chamber and continuously perfused with extracellular recording solution containing (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5–1 BaCl₂, 0.01 EDTA and brought to pH 7.4 with NaOH. Microelectrodes were fabricated from borosilicate glass (World Precision Instruments catalog no. TW150F-4; Sarasota, FL, USA) and voltage clamp and current monitoring were achieved with a two-electrode voltage clamp amplifier (Warner Instruments model OC-725C; Hamden, CT, USA). Currents were low-pass filtered at 10 Hz and digitized at 20 Hz using custom acquisition software written in LabWindows/CVI (National Instruments, Austin, TX, USA), which also controlled solution exchange. Oocytes were held at -40 mV unless otherwise stated. The NMDAR open probability was estimated as previously described using MTSEA to lock open NMDA receptors expressing mutant GluN1(A652C)[62]. Open probability was calculated as (γ_MTSEA/γ_CONTROL)/potentiation where γ_MTSEA and γ_CONTROL are chord conductances estimated from control and MTSEA-modified GluN1/GluN2A receptors [62] and potentiation is the ratio of current observed after MTSEA modification to control.