Pc running pclamp

Whole cell patch clamp recordings were performed using Axopatch 200B or Multiclamp 700B amplifiers, a Digidata 1440 digitizer, and a PC running pClamp (Molecular Devices). Cultured neurons were patched on DIV 14–18 (7–11 days after AAV transduction; AAVs were produced at Janelia Viral Tools Facility). Neurons were bathed in room temperature Tyrode solution containing 125 mM NaCl, 2 mM KCl, 3 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 30 mM glucose and the synaptic blockers 0.01 mM NBQX and 0.01 mM GABAzine. The Tyrode solution pH was adjusted to 7.3 with NaOH and the osmolarity was adjusted to 300 mOsm with sucrose. Borosilicate glass pipette (Warner Instruments) with an outer diameter of 1.2 mm and a wall thickness of 0.255 mm was pulled to a resistance of 5–10 MΩ with a P-97 Flaming/Brown micropipette puller (Sutter Instruments) and filled with a pipette solution containing 155 mM K-gluconate, 8 mM NaCl, 0.1 mM CaCl₂, 0.6 mM MgCl₂, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM Na-GTP. The pipette solution pH was adjusted to 7.3 with KOH and the osmolarity was adjusted to 298 mOsm with sucrose.

Whole cell patch clamp recordings in culture (for Figure 3 and Figure S1) were made using Axopatch 200B or Multiclamp 700B amplifiers, a Digidata 1440 digitizer, and a PC running pClamp (Molecular Devices). For in vitro current-clamp recordings, neurons were patched 14–18 DIV (7–11 days after AAV transduction) to allow for sodium channel maturation. Neurons were bathed in room temperature Tyrode containing 125 mM NaCl, 2 mM KCl, 3 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 30 mM glucose and the synaptic blockers 0.01 mM NBQX and 0.01 mM GABAzine. The Tyrode pH was adjusted to 7.3 with NaOH and the osmolarity was adjusted to 300 mOsm with sucrose. For in vitro voltage-clamp recordings, neurons were patched 19–21 DIV (17–20 days post-transfection) and were done under similar conditions as current-clamp recordings, except the Tyrode also contained 1 μM tetrodotoxin (TTX, Tocris Bioscience). For recordings, borosilicate glass pipettes (Warner Instruments) with an outer diameter of 1.2 mm and a wall thickness of 0.255 mm were pulled to a resistance of 5–10 MΩ with a P-97 Flaming/Brown micropipette puller (Sutter Instruments) and filled with a solution containing 155 mM K-gluconate, 8 mM NaCl, 0.1 mM CaCl₂, 0.6 mM MgCl₂, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM Na-GTP. The pipette solution pH was adjusted to 7.3 with KOH and the osmolarity was adjusted to 298 mOsm with sucrose.