Axon 700b amplifier

Bam8-22-responding vagal sensory neurons were first identified by calcium imaging before they were patched for current clamp recording. Patch pipettes had resistances of 2–4 MΩ. Action potential measurements were performed with an Axon 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). Neurons were perfused with 2uM Bam8-22 for 20s. All experiments were performed at room temperature.

Whole-cell patch-clamp recordings were performed at room temperature (22–24°C) using an Axon 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) on the stage of an inverted phase-contrast microscope equipped with a filter set for eYFP visualization (Nikon Instruments Inc., Melville, NY, USA)^{14 (link)}. Pipettes pulled from borosilicate glass (BF 150–86-10; Sutter Instrument, Novato, CA, USA) with a Sutter P-1000 pipette puller had resistances of 2–4 MΩ for whole-cell patch-clamp recordings when filled with pipette solution containing in 10⁻³M concentrations: KCl 140, MgCl₂ 1, Mg-ATP 3, EGTA 1, sucrose 10 and HEPES 10 with pH 7.3 and 315 mOsm/l osmolarity. Cells were continuously perfused with standard extracellular solution containing, in 10⁻³ M concentrations: NaCl 140, KCl 5, EGTA 0.5, MgCl₂ 1, CaCl₂ 2, D-Glucose 10, and HEPES 10 (pH 7.4 and 340 mOsm/l osmolarity). Whole-cell membrane currents were recorded using gap-free recording at holding potential of −60 mV. Data were acquired using Clampex 10.4 software (Molecular Devices, Novato, CA, USA). Currents were filtered at 2 kHz and digitized at 10 kHz. Data were analyzed and plotted using Clampfit 10 (Molecular Devices, Novato, CA, USA).

The coverslip was transferred into a perfusion chamber with extracellular solution (in mM: 145 TEA-Cl, 5 CaCl₂, 0.8 MgCl₂, 10 HEPES, 5 glucose; pH adjusted to 7.39 with TEA-OH, and osmolarity adjusted to 320 mOsm with sucrose). GFP-expressing neurons with cell body diameters between 22 and 25 μm were recorded in the whole-cell voltage-clamp configuration. The intracellular pipette solution contained (in mM): 135 CsCl, 1 CaCl₂, 2 MgCl₂, 1.5 MgATP, 0.3 Na₂GTP, 11 EGTA, 10 HEPES, with pH of 7.3 (adjusted by CsCl) and osmolarity of 310 mOsm (adjusted by sucrose). Electrodes were pulled (Model pp-830, Narishige International USA, Inc. Long Island, NY) from borosilicate glass (WPI, Inc., Sarasota, FL) and had a resistance of 2–4 MΩ. Whole-cell currents were measured with an Axon 700B amplifier and the pCLAMP 9.2 software package (Molecular Devices, Sunnyvale, CA). Current traces were sampled at 10 kHz and low-pass filtered at 2 kHz. Low-voltage-activated (LVA) I_ca was evoked at −40 mV (20 ms) from a holding potential of −80 mV, and HVA I_ca was evoked at 10 mV (20 ms) from a holding potential of −60 mV. All experiments were performed at room temperature (~25°C). The liquid junction potential, cell membrane capacitance, and series resistance were electronically compensated. An experimenter blind to genotype and/or drug treatment conditions performed all electrophysiologic recordings.