Patch-clamp Recording of Hippocampal Neurons

Patch-clamp recordings were obtained in acutely prepared coronal hippocampal slices from male rats, as described previously [6 (link)18 (link)]. Briefly, slices were perfused with artificial cerebrospinal fluid (aCSF; in mM: NaCl 126, KCl 2.5, MgSO₄ 1, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 20, ascorbic acid 0.4, CaCl₂ 1, pyruvic acid 2; pH 7.3~7.4; saturated with 95%O₂–5%CO₂) at a ~3 ml/min flow. Recordings were obtained at 32℃ using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). The series resistance was motored throughout the experiments. Neurons localized in the outer half of the granule cell layer were selected to minimize the effects of neurogenesis [19 (link)]. Patch pipettes were filled with a high Cl⁻ containing solution (in mM): KCl 140, HEPES 10, Mg²⁺ATP 5, MgCl₂ 0.9, and EGTA 10. Current output was filtered at 2 kHz and digitized at 10 kHz (Digidata 1322A, pClamp 9 software, Axon Instruments). I_tonic was defined as the difference between the holding current (I_holding) before and after application of the GABA_A receptor blocker bicuculline (20 µM). Drugs were added to the perfusing aCSF solution at known concentrations. All drugs except NO-711 (Tocris, UK) were purchased from Sigma-Aldrich (St. Louis, MO, USA).