Movements of the tectorial membrane were measured by imaging 3 μm diameter silica beads (Polyscience) that VE-821 mw were applied at low density on top of the membrane. For assaying hair bundle motion, responses to 25 to 50 presentations were averaged at each stimulus level. Mechanical and electrical stimuli were generated by automated protocols from a Cambridge Electronic Design (CED) Power1401 interface driven by a PC computer, and data were digitized with the interface and analyzed with IGOR Pro v6 (Wavemetrics). Results are presented

as the mean ± 1 standard deviation (SD) and significance assessed by two-tailed Student’s t test. Relationships between the MT current, I, and hair bundle displacement, X, were fit with a Boltzmann equation: I = IMAX/(1 + exp(−(X − XO)/XS)), where IMAX is the maximum current, XO the half saturation Selleckchem MK 2206 displacement, and XS the slope factor; the 10–90 percent working range is given by 4.4·XS. Nonlinear capacitance measurements were performed in an external saline designed to block all voltage-dependent conductances containing (in mM): NaCl, 136; CsCl, 5; CaCl2, 0.5; MgCl2, 2; CoCl2, 2; tetraethylammonium

bromide, 10; 4-aminopyridine, 5; apamin, 0.3 μM; HEPES, 10; glucose 8 (pH 7.4) (321 mOsm/l). The patch-electrode solution was similar to that above with the exception that KCl was replaced with CsCl. For reducing intracellular

chloride, CsCl was isotonically replaced with Cs+ aspartate. A continuous measurement of SHC membrane capacitance was obtained (Santos-Sacchi et al., 1998) by applying a voltage-clamp protocol consisting of a double sine wave (10 mV peak-to-peak at 391 Hz and at 781 Hz) superimposed on a 200 ms voltage ramp from −150 to +150 mV. Voltage commands and data acquisition were controlled with jClamp (www.SciSoftCo.com). SHC capacitances were determined (-)-p-Bromotetramisole Oxalate in the presence and absence of 10 mM Na+ salicylate and the difference capacitance ΔCm was derived. The variation of ΔCm with membrane potential, V, was fit with the first derivative of a two-state Boltzmann function ( Santos-Sacchi et al., 1998): equation(Equation 1) ΔCm=QmaxzekTε(1+ε)2where ε=exp((ze(V−V0.5))kT) Boltzmann parameters were evaluated from the fits: Qmax (maximum nonlinear charge moved), V0.5 (voltage at peak capacitance), and z (valence). In Equation  1, e is the electron charge, k is the Boltzmann constant, and T is temperature; kT/e = 26.4 mV at 33°C. Chickens were killed by decapitation and basilar papillae isolated (five papillae from E21 and four from E16 birds) and used for extraction of total RNA with the Ambion RNAqueous-4PCR kit (Life Technologies). The concentration of RNA for each papilla was ∼19 ng/μl at both ages.