Whole-cell patch-clamp recording from the cultured hippocampal neurons

Based on the procedures of Wang, et al. [36], the whole-cell patch-clamp technique was performed to record APs, I_Na and K_v currents at 22–25 °C. The recording pipettes were pulled using a multistage micropipette puller (P-97, Sutter Instruments, Novato, CA, USA) and a borosilicate capillary glass. The tip resistance of the pipettes was 3–5 MΩ after being filled with the intracellular solution. The hippocampal neurons were then incubated with extracellular solution. We randomly selected hippocampal neurons with a smooth and bright appearance and no visible organelles for recording under an inverted microscope (BX51W1, Olympus, Japan). Signals were filtered, amplified, and digitized using a Multiclamp 700 B amplifier (Molecular Devices, Sunnyvale, CA, USA) and a DigiData 1440A digitizer (Molecular Devices). The data were recorded and analyzed using the pClamp 10.1 software (Molecular Devices). The series resistance was compensated for 85–90%. Recordings were discarded if the series resistance was over 20 MΩ or changed by over 20% during the experiments.

For recording the APs, the intracellular solution contained 130 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂·6H₂O, 10 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP·3H₂O (pH 7.3 with KOH); the extracellular solution contained 130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂·6H₂O, 10 mM HEPES, 10 mM glucose (pH 7.4 with NaOH).

For recording I_Na, the intracellular solution contained 130 mM CsCl, 1 mM MgCl₂·6H₂O, 10 mM EGTA, 20 mM TEA-Cl, 10 mM HEPES, and 3 mM Na₂ATP·3H₂O (pH 7.3 with CsOH); the extracellular solution contained 125 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂·6H₂O, 10 mM HEPES, 10 mM glucose, 0.2 mM CdCl₂, 4 mM 4-AP, and 20 mM TEA-Cl (pH 7.4 with NaOH).

For recording K_v currents, the intracellular solution contained 140 mM KCl, 1 mM MgCl₂·6H₂O, 10 mM EGTA, 10 mM HEPES, and 4 mM Na₂ATP·3H₂O (pH 7.3 with KOH); the extracellular solution contained 145 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂·6H₂O, 10 mM HEPES, 10 mM glucose, 0.2 mM CdCl₂, and 0.001 mM tetrodotoxin (pH 7.4 with NaOH). In addition, 20 mM TEA-Cl and 4 mM 4-AP were used to block I_DR and I_A, respectively.

To eliminate the influence of neuronal size, we normalized the currents to the cell membrane capacitance to calculate current densities (pA/pF).