2.3. Patch-Clamp Recordings

Whole-cell currents from cultured neurons or HEK293 cells were recorded using a MultiClamp 700B patch-clamp amplifier with Digidata 1440A controlled by pClamp v10.2 software (Molecular Devices). Recordings were low-pass-filtered at 100 Hz. Perfusing solution exchange was performed by BPS-8 fast-perfusion system (ALA Science Inc., Farmingdale, NY, USA) with the tip of the manifold placed 100–200 µm from the recorded cell. The external bathing solution contained (in mM): 140 NaCl; 2.8 KCl; 1.0 CaCl₂; 10 HEPES, at pH 7.2–7.4, 310 mOsm. The pipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, 300 mOsm, pH 7.4. Patch-pipettes of 4−6 MΩ were pulled from Sutter BF150–89–10 capillaries. Experiments were performed at 23–25 °C. Both neurons and HEK293 cells were voltage-clamped at −70 mV (holding voltage, V_h). The liquid junction potential was ~12 mV between the Na⁺-containing bathing solution and the Cs⁺-containing pipette solution. Wherever V_h is shown, this value is indicated without a correction for the liquid junction potential. To activate NMDARs, both N-methyl-d-aspartate (NMDA) or l-homocysteine (HCY) were always co-applied with 30 µM glycine as a co-agonist.