8 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 0.7 mM NaH2PO4, 10 mM HEPES, 5.6 mM glucose, 2 mM pyruvate, and 2 mM creatine. Both stria and spiral ganglia were peeled from the organ of Corti and the remaining epithelium was placed into a glass-bottomed recording chamber. The tectorial membrane was removed and the organ of Corti held in place with single strands of dental floss. Apamin was included at 100 nM to block small conductance calcium-activated potassium currents. Fire-polished borosilicate patch

electrodes of resistance 3–5 M Ω were used for all recordings. Unless otherwise stated, the internal solution for turtle contained 110 mM CsCl, 5 mM MgATP, 5 mM creatine phosphate, 1 mM ethylene glycol-bis (β-amino ethyl ether)- N,N,N′nN ′-tetraacetic acid (EGTA), 10 mM HEPES, 2 mM ascorbate (pH see more 7.2). Osmolality was maintained Ulixertinib at 255 mosmls by adjusting CsCl levels; pH was 7.2. For perforated-patch recordings the internal solution contained 110 mM Cs aspartate, 15 mM CsCl, 3 mM NaATP, 3 mM MgCl2, 1 mM BAPTA, and 10 mM HEPES. Amphotericin dissolved in dry DMSO was used as the perforating agent. In several experiments Alexa 488 was included in the recoding pipette to verify the whole-cell mode was not obtained. For rat hair cells the internal solution contained

90 mM Cs methylsulfonate, 20 mM TEA, 1 mM EGTA, 5 mM MgATP, 5 mM creatine phosphate, 3 mM ascorbate, 3 mM MgCl2, and 10 mM HEPES. Stimulus protocols were applied 10 min after achieving whole-cell mode to allow equilibration of internal solution and run up of ICa (Schnee and Ricci, 2003). Hair cells were voltage clamped with a lock-in amplifier (Cairn) allowing for capacitance measurements as initially described by Neher and Marty (1982) and later used for hair cell recordings (Johnson et al., 2002 and Schnee et al., 2005). A ± 40 mV sine wave at 1.5 kHz was imposed onto the

membrane holding potential, blanked during depolarization that elicited ICa, and resumed so that capacitance measurements before and after stimulus were obtained. Capacitance data Rebamipide were amplified and filtered at 100 Hz offline. This amplifier was also used initially for validation of the two-sine wave method (see below). The multiclamp amplifier (Axon Instruments) was also used for capacitance measurements. All data were sampled with a Daq/3000 (IOtech) driven by jClamp software (Scisoft). Vesicle release was determined by measuring membrane capacitance correlates of surface area change. Capacitance was measured with a dual sinusoidal, FFT-based method (Santos-Sacchi, 2004 and Santos-Sacchi et al., 1998) relying on component solutions of a simple model of the patched cell (electrode resistance, Rs, in series with a parallel combination of membrane capacitance, Cm, and membrane resistance, Rm; see Figure 1 in Santos-Sacchi, 2004).