Bw51wi microscope

Primary CNs from WT and Ifnb^–/– were cultured as described above. PIAS2 was overexpressed using AAV6 pAAV-SYN1–mCherry-mouse Pias2 or pAAV-SYN1–mCherry (Vector Builder) by CaCl2 method on day 1 of culture or knocked down by siRNA with the Accell SMART pool system (Thermo Scientific) for the last 72 h after 9–10 days in culture. SHSY5Y cells were kept in artificial cerebrospinal fluid, containing (in mM) 125 NaCl, 2.5 KCl, 26 NaHCO₃, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, and 25 glucose, in 95% O₂ and 5% CO₂ at room temperature. Glass pipettes (borosilicate, resistance 5–10 MΩ, Sutter Instruments, US) were filled with (in mM): 122 K-gluconate, 2.5 MgCl₂, 0.3 CaCl₂, 5.6 Mg-gluconate, 5 K-HEPES, 5 H-HEPES, 5 Na₂ATP, 1 EGTA, 10 biocytine, Alexa Fluor^®488 (10 µM, Sigma), and KOH to adjust the pH to 7.4.
Cells were visualized under a BW51WI microscope (Olympus, Japan). Whole-cell recordings were made in voltage clamp mode with a Multiclamp 700B amplifier (Molecular Devices, US). Recordings were sampled at 20 kHz with a 16-bit analog-to-digital converter (DIGIDATA 1440; Molecular Devices, US) and displayed with Clampex 10.2 (Molecular Devices, US). Fast transient sodium inward currents [34 ] were evoked by depolarizing steps of 10-mV increments, applied from −90 mV. The inward current amplitude (calculated as the difference between the peak and baseline) was maximal at 0 or +10 mV.

Visually guided patch-clamp recordings were performed in whole-cell configuration with a Multiclamp 700B amplifier (Molecular Devices, USA) in voltage-clamp mode. Cells were visualized by means of a BW51WI microscope (Olympus, Japan) equipped with an oblique illumination condenser or with a differential interference contrast system. The pipette solution (in mM: 122 K-gluconate, 2.5 MgCl₂, 5.6 Mg-gluconate, 5 K-HEPES, 5 H-HEPES, 5 Na₂ATP, 1 EGTA, 2.5 biocytine, HCl to adjust the pH to 7.4) contained the fluorescent dye Alexa 488 (250 μM, Sigma-Aldrich). Electrodes had an input resistance ranging from 4 to 8 MΩ. Recordings were sampled at 10–20 kHz and treated offline with a Bessel filter with a cutoff frequency set at 400 Hz. Data were sampled with a 16-bit analog-to-digital converter (DIGIDATA 1440; Molecular Devices, USA) and displayed by means of Clampex 10.2 software (Molecular Devices, USA). Data are presented without liquid-junction potential correction unless specified.