Borosilicate glass capillary tubes

Spiking HEK-293 cells were placed in the same bath solution as that used for QPI at room temperature (25 °C). Glass micropipettes were pulled from borosilicate glass capillary tubes (Warner Instruments) using a PC-10 pipette puller (Narishige) and were loaded with internal solution containing (in mM) 125 potassium gluconate, 8 NaCl, 0.6 MgCl₂, 0.1 CaCl₂, 1 EGTA, 10 HEPES, 4 Mg-ATP, and 0.4 Na-GTP (pH 7.3, adjusted with NaOH; 295 mOsm, adjusted with sucrose). The resistance of the pipettes filled with internal solution varied between 2 and 3 MΩ. After setting up the whole cell configuration, the membrane potential during the spontaneous action potentials of spiking HEK cells was monitored with a Multiclamp 700B amplifier (Molecular Devices) under the current clamp mode.

Whole cell patch-clamp recordings from isolated bladder sensory neurons were obtained with the perforated technique using Amphotericin B. Current-clamp recordings were performed at room temperature with an Axopatch 200B patch-clamp amplifier (Molecular Devices) and data were captured with a Digidata 1440 A acquisition system and pClamp 10 (Molecular Devices). Signals were low-pass filtered at 1 kHz (four-pole Bessel filter) and digitized at 5 kHz. Micropipettes were pulled from borosilicate glass capillary tubes (Warner Instruments) with a PP-830 puller (Narishige). Fire-polished micropipettes with a tip resistance of 1.5–3 mΩ were used for patch-clamp recordings. The pipette filling solution contained (in mM): 145 KCl, 1 MgCl₂, 0.1 CaCl₂, 1 EGTA, and 10 HEPES (pH 7.2). Amphotericin B was added to the pipette solution to a final concentration of 120 μg/ml. The extracellular bath solution contained (in mM): 135 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 glucose and 10 HEPES pH 7.4. A gravity-feed perfusion system (Automate Scientific) was used to exchange solutions.

Whole-cell patch-clamp recordings were performed in the voltage-clamp mode using an EPC-10/2 amplifier (HEKA, Lambrecht/Pfalz, Germany). The recording pipettes were pulled from borosilicate glass capillary tubes (Warner Instruments, Hamden, CT) and had a resistance of 3–5 MΩ; only whole-cell patches with series resistances <15 MΩ were used for recording, and the membrane potential was held at −70 mV. The pipette solution consisted of 130 mM K-gluconate, 1 mM EGTA, 5 mM Na-phosphocreatine, 2 mM Mg-ATP, 0.3 mM Na-GTP, 5 mM QX-314, and 10 mM HEPES, pH 7.3. The bath solution consisted of 25 mM HEPES, pH 7.3, 128 mM NaCl, 30 mM glucose, 5 mM KCl, 5 mM CaCl₂, 1 mM MgCl₂, plus 50 μM D-AP5 and 20 μM bicuculline. The D-AP5, bicuculline, and QX-314 were from TOCRIS Bioscience, and the other chemicals were from Sigma-Aldrich (St. Louis, MO). For mEPSC recordings, 1 μM tetrodotoxin (TOCRIS Bioscience, Bristol, United Kingdom) was added to the bath solution. For evoke-EPSC trains, presynaptic inputs were stimulated with a CBAEC75 concentric bipolar electrode (FHC, Bowdoinham, ME) that was placed near (between 100 and 120 μm from) the recording neuron.