Brain slice preparation and slice-whole cell patch current-clamp patch recording

After passive avoidance test, brains were quickly removed and immersed in an oxygenated (95% O₂/5% CO₂), ice-cold artificial cerebrospinal fluid (ACSF) solution containing the following composition (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 1.3 MgSO₄, 2.4 CaCl₂, 26 NaHCO₃, and 8 _D-glucose. Coronal mPFC slices (300-µm) were cut using a vibratome (5,100 mz-22, Campden Instrument, England). During 15 min, brain slices were incubated with warm (30°C) oxygenated (95% O₂/5% CO₂) N-Methyl-D-glucamine (NMDG) ACSF containing the following (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 10 MgSO₄, 0.5 CaCl₂, 26 NaHCO₃, 25 _D-glucose, 110 NMDG, and 80.05 HCl. Then, slices were incubated with oxygenated (95% O₂/5% CO₂) ACSF for >1 h at room temperature. Recordings were performed in a submersion-type chamber (2~4 ml/min perfusion of oxygenated ACSF at 33~35°C) mounted on an upright microscope (Nikon Eclipse FN1, Tokyo, Japan). Recorded action potential (AP) spikes in the mPFC region using a borosilicate glass capillary electrode with internal solution connected to a Multi-Clamp Amplifier (Molecular Devices, San Jose, CA, USA). Recording pipettes were filled with intracellular solution (in mM): 120 K-MeSO₃, 10 Hepes, 0.25 EGTA, 2 MgCl₂, 0.1 CaCl₂, 10 Na₂-Phosphocreatine, 4 Mg-ATP, and 0.3 Na-GTP (pH 7.4). Patch pipettes (3~5 MΩ) were pulled on a Narishige electrode puller PC-10 (Narishige, Tokyo, Japan) from Kimax borosilicate glass capillaries (TW150-4, World precision Instruments, Inc., Sarasota, FL, USA) with an inner diameter of 1.12 mm and an outer diameter of 1.5 mm. The analog signal was digitized by an A/D converter (Digidataolecular Devices) and collected with pClamp10.7 software (Molecular Devices). Currents were Bessel-filtered at a cut-off frequency of 5 kHz and digitally sampled at 10 kHz. Including measuring resting membrane potential, all recording was performed in nACSF.