Electrophysiology

Whole-cell patch clamp recording was performed between 1 and 3 d after plating HEK cells for coculture. We recorded from both neurons and double-fluorescent HEK cells in the same cultures. An Axopatch 200B (Molecular Devices) amplifier controlled by pClamp 10 (Molecular Devices) was used to acquire one to four 1-min gap-free recordings. Coverslips with cocultured cells were transferred to 35-mm dishes containing extracellular solution consisting of (in mM) 140 NaCl, 5 KCl, 5 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4 at room temperature). Note that 5 mM of calcium increases mEPSC frequency and facilitates their study (Scanziani et al., 1992; Bekkers and Stevens, 1995; Chiang et al., 2018). NMDA receptors, γ-aminobutyric acid receptors, and action potentials were blocked with 50 µM amino phosphonovaleric acid, 10 µM SR95531, and 0.5 µM tetrodotoxin, respectively. Aniracetam was added to the extracellular solution for the indicated experiments. Patch pipettes (3–7 MΩ when filled) were pulled with Narishige PC-10 puller. After coating with Sylgard, the pipettes were filled with intracellular solution consisting of (in mM) 120 Cs-gluconate, 8 CsCl, 2 NaCl, 10 EGTA, 5 phosphocreatine, 5 HEPES, 2 Mg-ATP, and 0.3 Na-GTP. Membrane potential was held at −65 mV throughout recordings. Series resistance was compensated >75%, and cells with series resistance >20 MΩ before compensation were discarded.