Sutter p97

Slices were transferred to a recording chamber and were continuously perfused with oxygenated Tyrode’s solution at 32°C - 34°C which included synaptic blockers SR95531 (gabazine, 10μM), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10μM), and (R)-CPP (10μM). All drugs were purchased from Tocris (Bristol, United Kingdom). Glass patch pipettes (Sutter Instruments BF150–86-7.5) were pulled using a Sutter P97 (Sutter Instrument, Novato, CA, USA) to produce tip resistances of 2 – 4 MΩ and were filled with a K-gluconate intracellular solution (290 mOsm, pH 7.37) consisting of 132 K-gluconate, 4.4 Na-gluconate, 4.4 NaCl, 2.2 MgCl₂, 1.1 EGTA, 11 HEPES, 22 sucrose, 14 TRIS creatine phosphate, 4 Mg-ATP, 0.3 and TRIS-GTP. Stable whole-cell recordings were acquired at 100 kHz in pClamp 10.3 and pClamp 10.7 (Molecular Devices, San Jose, CA, USA) via an Axon 700B multiclamp amplifier and Digidata 1440 digitizer. Cells were visually identified and included for recording based on an access resistance below 20MΩ, and the demonstration of a robust fast inward sodium current in voltage clamp and overshooting action potentials in current clamp.

Patch pipettes were manufactured from soda lime capillary glass (Thermo Fisher Scientific) using a Sutter P-97 (Sutter Instrument) puller. When filled with standard recording solutions, pipettes had a tip resistance of 1–3 MΩ. Recordings were filtered at 5 kHz and sampled at 10 kHz, with manual capacitance compensation and series resistance compensation at 80%, and stored directly on a computer hard drive using an Axopatch 200B amplifier, Digidata 1440 digitizer, and Clampex 10 software (Molecular Devices). Zero current levels are denoted by dashed lines in representative current panels. The external (bath) solution had the following composition (in mM): 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, and 10 HEPES, adjusted to pH 7.3 with NaOH. The internal (pipette) solution had the following composition (in mM): 135 KCl, 5 EGTA, and 10 HEPES, adjusted to pH 7.2 using KOH. Chemicals were purchased from Sigma-Aldrich or Thermo Fisher Scientific. Patch-clamp experiments were conducted at room temperature (22 ± 1°C).