, 2009). Tissue was viewed using a 60× (1.0 NA, Olympus) water-immersion objective through a 1–4× magnifier onto a digital Rolera XR (Qimaging) or analog Pictilisib concentration OLY-150 (Olympus) camera on a BX51 microscope (Olympus). Tissue was dissected and perfused at rates of 0.35–0.5 ml/min with external solution containing (in mM): 140 NaCl, 2 KCl,

2 CaCl2, 2 MgCl2, 10 HEPES, pH = 7.4, at 300–310 mOsm. In addition, an apical perfusion protected the hair bundles from internal solution with rates of 0.07–0.1 ml/min using pipettes with tip sizes 40–200 μm. In all preparations, the tectorial membrane was peeled off the tissue. Experiments occurred on multiple recording stations with comparable capabilities but often-different specific equipment, attesting to the robustness of the data. Whole-cell patch-clamp was achieved on IHCs or first or second row OHCs from middle to apical cochlea turns using an Axon 200A or 200B amplifier (Molecular Devices)

with thick-walled borosilicate click here patch pipettes (<4 MΩ) filled with an intracellular solution containing (in mM): 125 CsCl, 3.5 MgCl2, 5 ATP, 5 creatine phosphate, 10 HEPES, 1 Cesium BAPTA, 3 ascorbate, pH = 7.2, and 280–290 mOsm. For the 10 mM BAPTA solution, removal of an equivalent osmolality of CsCl offset the increased BAPTA concentration. For the EGTA internal (in mM), 1 EGTA replaced cesium BAPTA and ascorbate increased to 4 mM. For Ca2+ imaging, 1 mM Fluo-4 or Fluo-4FF (Invitrogen) and 0.05 mM Alexa 594 hydrazide (Invitrogen) were added to the EGTA internal. 1.4 Ca2+ internal contained (in mM): 121 CsCl, 3.5 MgCl2, 3.5 CaCl2, 3.5 ATP, 5 creatine phosphate, 10 HEPES, 2 ascorbate, pH = 7.2, at 280–290 mOsm. For the 1.4 mM Ca2+ internal, free Ca2+ concentration was measured using

a MI-600 Ca2+ electrode (Microelectrodes) calibrated using Ca2+ buffer standards (CALBUF-2, WPI) and found to be 1.4 mM free Ca2+. Experiments were performed at 18–22°C. Whole cell currents were filtered at 50–100 kHz and sampled at 1 MHz using USB-6356 (National Instruments) see more or Personal DAQ3000 (Iotech) controlled by jClamp (SciSoft). Traces were filtered offline at 30 kHz using Origin 8.6 (OriginLab). Voltages were corrected offline for liquid junction potentials. For inclusion, initial MET currents greater than 600 pA in 2 mM external Ca2+ and Mg2+ were required. For a sample of 80 cells recorded, the clamp speed was 28 ± 6 μs with series resistance compensation, whole cell capacitance was 11 ± 1 pF, and leak currents at −84 mV holding potential were −65 ± 40 pA. Borosilicate pipettes were fire polished to shapes that matched the hair bundle structures for IHCs and OHCs.