Axopatch 700b

The electrophysiology was recorded by an Axopatch-700B amplifier and a Digidata 1440 digitizer (Molecular Devices, USA). The MC1R agonist BMS-470539 (BMS, 100 nM, MedChemExpress, China) was used, and the extracellular solution without BMS was defined as control [28 (link)]. The extracellular solution consisted of (in mM) NaCl 150, KCI 5, CaCl₂ 2.5, MgCl₂ 2, HEPES 10, and D-glucose 10 (pH = 7.4 by NaOH, 330 mOsm/L). The intracellular pipette solution contained (in mM) KCl 140, CaCl₂ 1, MgCl₂ 2.5, HEPES 10 and D-glucose 10, EGTA 11, Mg-ATP 5 (pH = 7.2 by NaOH, 310 mOsm/L). The resting membrane potential (RMP) was recorded for 3 min. The currents from -120 pA to 170 pA with an increment of 10 pA were injected into TGNs to evoke action potential (AP) [29 (link)]. The rheobase current was defined as the minimum current required to evoke the first AP. The AP threshold was obtained at dV/dt= 10 mV/ms. The action potential-related parameters including AP latency, the AP amplitude, AP half-width, afterhyperpolarization (AHP) time, and AHP amplitude are measured.

Two Ag/AgCl electrodes were inserted into the electrochemical cell filled with electrolyte (1 M KCl, 10 mM Tris-HCl, pH = 8.0). During the detection of protein molecules, the protein molecules were added to the electrolyte solution, with the glass nanopore fixed to the negative electrode and the positive electrode added outside the nanopore (as shown in Figure 1a). The other ends of the electrodes were connected to the Axopatch 700B patch clamp amplifier (Molecular Devices, San Jose, CA, USA). The signals were digitized using the Axon Digidata 1550A digital-to-analog converter (Molecular Devices, San Jose, CA, USA) and viewed using Clampfit software (Version 10.5.2.6).

HEK293T cells were transfected with pEGFP-ready tagged ANO1 24–36 h before whole-cell patch-clamp recordings of ANO1. The bath solution contained (in mM) 150 NMDG-Cl, 1 MgCl₂, 10 glucose, and 10 HEPES (pH 7.4) and the pipette solution contained (in mM) 150 NMDG-Cl, 10 EGTA, 6.6 CaCl₂, 1 MgCl₂, 3 MgATP, and 5 HEPES (pH 7.2). Pipettes were pulled from borosilicate glass capillaries to have an electrical resistance of 2–3 MΩ after fire polishing. The holding potential was set as −60 mV and ramp pulses were applied from −100 mV to +100 mV in steps of 20 mV over 1 s. The pulse-to-pulse interval was 20 s. All recordings were carried out at room temperature using Axopatch 700B (Molecular Devices, Sunnyvale, CA, USA) and digitalized and analyzed using Digidata 1440A (Molecular Devices) and Clampfit 10.4 (Molecular Devices). Currents were low-pass filtered at 5 kHz and sampled at 10 kHz.

BLA slices were transferred to a submersion-type recording chamber and perfused with room temperature (~25°C) aCSF (2.0 ml/min) for whole-cell voltage clamp recordings similar to previously published reports (Christian et al., 2013 (link)). Data were acquired via Axopatch 700B (Molecular Devices, Foster City, CA) and analyzed offline via pClamp software (Molecular Devices, version 10.5). Inclusion criteria for presumptive principal neurons included high membrane capacitance (>100 pF) and low access resistance in the whole-cell configuration (<20 MΩ, Washburn and Moises, 1992 (link)). Cells in which access resistance or capacitance changed ≥20% during the record or with changes in resting membrane currents ≥100 pA were excluded from analysis. Glutamatergic responses were pharmacologically isolated using 100 μM picrotoxin (a GABA_A antagonist) in the bath aCSF and were recorded with electrodes filled with an internal solution containing (in mM): 145 Cs-gluconate, 10 EGTA, 5 NaCl, 1 MgCl₂, 10 HEPES, 0.4 QX314, 1 CaCl₂-2H₂O, 4 Mg-ATP, and 0.4 Na₃-GTP. Osmolarity of internal solution was corrected to ~285 mOsm with sucrose and pH was adjusted to ~7.25 with D-Gluconic acid. Synaptic responses were electrically evoked using concentric bipolar stimulating electrodes (FHC Inc, Bowdoin, ME, cat. #CBAEB75) placed within the stria terminalis.

Brain neurons were patch-clamped in the whole-cell current-clamp configuration using an Axopatch 700B amplifier (Molecular Devices). Cells in the culture were co-cultured with ZST for 12 h to ensure their effective attachment to neurons. The cells were first washed with our configured bath solution (NaCl 132 mM, KCl 4 mM, MgCl₂ 1.2 mM, CaCl₂ 1.8 mM, HEPES 10 mM, glucose 5.5 mM, pH 7.4) twice to remove the free particles, and then cultured in this bath solution. Borosilicate glass pipettes pulled on a CO₂ laser micropipette puller (Sutter Instruments P-2000) produced 4 MΩ resistances when filled with internal pipette solution (K-Gluconic acid 110 mM, NaCl 10 mM, MgCl₂ 1 mM, EGTA 10 mM, HEPES 30 mM, Mg-ATP 3 mM, Na-GTP 0.3 mM, pH 7.2). Under the current-clamp configuration, the triggered action potentials were recorded upon the illumination of 532 nm laser.

The chemically modified nanopore chip was sandwiched by a custom-built polycarbonate flow cell between two polydimethylsiloxane (PDMS) gaskets to assure that the only path of the ionic current was through the nanopore. The cell was made of two facing Plexiglas chambers filled with filtered 0.02/0.02 M KCl in Fig. 1a. Figure 1c displays the Si₃N₄ chips. Figure 1d–f shows the fluid device used in the experiment. Electrodes (Ag/AgCl) were placed in both chambers and connected to the headstage of a patch clamp amplifier (Axopatch 700B, Molecular Devices Inc., Sunnyvale, CA, USA) which allowed the ionic current to be measured under constant voltage in Fig. 1a. The PS (2.5 ng/mL) was added to the cis side. Signals were acquired at a 100-kHz sampling rate. The amplifier internal low-pass eight-pole Bessel filter was set at 10 kHz. The entire apparatus was placed in a double Faraday cage enclosure on an anti-vibration table. The statistical analysis of the current traces recorded during the PS microsphere translocation experiment was performed with clamp fit 10.3 and Origin 8.0.