Borosilicate micropipettes

For thalamic recordings, the skull was exposed and a craniotomy (0.5–1 mm in diameter) was made above the VPM of the thalamus (3 mm posterior and 2.7–3.2 mm lateral to bregma) and a portion of the dura mater was carefully removed. Extracellular single-unit recordings were performed with high resistance (30–40 MΩ) borosilicate micropipettes (o.d., 1.5 mm; i.d., 0.86 mm; Sutter Instruments, Novato, CA, USA), pulled with a P-97 micropipette puller (Sutter Instruments) and filled with 2 M K-acetate. Intracellular “Blind” sharp recordings were obtained using high resistance (60–100 MΩ) borosilicate micropipettes (o.d., 1.5 mm; i.d., 0.86 mm; Sutter Instruments, Novato, CA, USA), pulled with a P-97 micropipette puller (Sutter Instruments) and filled with 2 M K-acetate. To assess the synaptic inputs of VPM cells QX-314 (30–50 mM) was added to the intracellular solution to prevent action potentials. Micropipettes were vertically advanced to the VPM and recordings were made at a depth of 4.3–5.2 mm. To protect the brain from drying, we covered the craniotomy with a few drops of artificial cerebrospinal fluid (ACSF). Signals were amplified using Axoclamp-2B (Axon Instruments, Foster City, CA, USA) and digitized at 20 kHz.

Electrophysiological experiments on different cell lines (A549, WI-38, and HLF) were performed using the whole-cell configuration of the patch-clamp technique. Cells were placed (passaged) on sterile 4 × 4-mm coverslips in 35-mm Petri dishes 1–2 days before the experiment. Ion current records were acquired with the Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA), the analog–digital interface Digidata 1550A and PC running the Clampex software (Molecular Devices). Borosilicate micropipettes (BF‐150‐110‐10, Sutter Instruments, Novato, CA, USA) were pulled at a P-97 puller (Sutter Instrument) to a resistance of 3–6 MΩ when filled with a relevant intracellular solution containing (in mM) 140 K-Aspartate, 5 NaCl, 1 MgCl₂, 2 EGTA/KOH, 20 HEPES/TRIS, and 0.176 CaCl₂ to establish free ionized calcium concentration [Ca²⁺]i at 0.01 µM. Typical bath solution (in the chamber) contained (in mM) 145 NaCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES/TRIS (pH 7.4). For bath extracellular solution with pH 5.5, HEPES/TRIS was replaced by MES. Gap-free ion currents were recorded at holding membrane potential -50 mV. Data were low-pass filtered at 200 Hz and analyzed using Axon pClamp 10.5 software suite (Molecular Devices).