In this study, we sorted adult rat TG neurons into groups on the basis of soma diameter [small-sized (<30 μm) and medium-sized (30 to 45 μm)] and restricted the electrophysiological recordings to small-sized neurons, most of which are nociceptors (22, 23, 38). Whole-cell recordings were carried out at room temperature (22° to 24°C) as previously described (39, 62, 64). Coverslips plated with TG neurons were transferred to a Warner recording chamber (Warner Instruments) and perfused with external solution for T-type channel current recording containing 5 mM BaCl2, 140 mM TEA-Cl, 5 mM CsCl, 0.5 mM MgCl2, 5.5 mM d-glucose, 10 mM Hepes (pH 7.3) (with TEA-OH), and 305 mOsm. The electrodes were pulled from borosilicate glass (Sutter Instruments) and had 3 to 4 megohm resistance when filled with the internal solution composed of 110 mM CsCl, 4 mM Mg–adenosine 5′-triphosphate (ATP), 0.3 mM Na2–guanosine 5′-triphosphate (GTP), 25 mM Hepes, 10 mM EGTA (pH 7.4) (with CsOH), and 295 mOsm. The whole-cell recordings were conducted using a MultiClamp 700B patch-clamp amplifier (Molecular Devices), and the output was digitized with a Digidata 1322A converter (Molecular Devices). Data were low-pass–filtered at 2 kHz, digitized at 20 kHz, and stored on a computer equipped with pClamp 10.2 (Molecular Devices). The series resistance was adequately compensated by at least 80%. To enable recordings of isolated T-currents, recordings were carried out in extracellular bath solution containing 5 μM nifedipine (L-type channel blocker) and 0.2 μM ω-conotoxin MVIIC (N-type and P/Q-type channel blocker). In experiments for whole-cell voltage-gated potassium channel (Kv) current recordings, the extracellular solution contained 140 mM choline-Cl, 1 mM MgCl2, 5 mM KCl, 0.03 mM CaCl2, 10 mM glucose, 10 mM Hepes (pH 7.4) (with KOH), and 310 mOsm. The intracellular pipette solution contained 140 mM KCl, 0.5 mM CaCl2, 0.3 mM Na2-GTP, 3 mM Mg-ATP, 1 mM MgCl2, 5 mM EGTA, 10 mM Hepes (pH 7.4) (with KOH), and 295 mOsm. The two kinetically different Kv currents in small-sized TG neurons were separated by the two-step voltage protocol as previously described (62, 65). In current clamp and whole-cell Nav current recording experiments, the extracellular solution contained 125 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM d-glucose, 25 mM Hepes (pH 7.4) (with NaOH), and 305 mOsm. The intracellular pipette solution contained 110 mM KCl, 10 mM NaCl, 25 mM Hepes, 0.3 mM Na2-GTP, 4 mM Mg-ATP, 2 mM EGTA (pH 7.3) (with KOH), and 295 mOsm. To perform current clamp recordings, we added 5 mM TEA (the delayed rectifier K+ channel blocker), 0.2 μM ω-conotoxin-MVIIC, and 5 μM nifedipine to the external solution. Using a pressure-pulsed microinjector (Picopump PV820, World Precision Instruments), BDNF was puff-applied through a glass pipette, the tip of which was placed 15 to 25 μm from the soma of TG neurons. In cells dialyzed with compounds, the resistance of the patch electrodes ranged from 2 to 3.5 megohms for intracellular delivery of agents, and recordings were started at least 5 min after breaking into the whole-cell configuration.

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