Nteed by setting both intracellular and extracellular chloride concentrations equal. All chemicals used were purchased from Sigma (St. Louis, MO).Cell capacitanceAn Axon Instruments (Foster City, CA) 200B amplifier was used for wholecell recording. To measure both AC capacitance and integrated charge movement, a simple voltage protocol was designed that included both step stimulation and dual sine stimulation at a range of dual-sine interrogation frequencies (Fig. 1, A and B). For AC admittance analysis, real and imaginary components of currents were corrected for the recording-system frequency response (19). No averaging was used with this protocol. Membrane capacitance (Cm) was measured using a continuous dual-frequency (discrete sinusoidal frequencies at f1 and f2, where f2 ?2 ?f1) voltage-stimulus protocol (19,21). Simultaneous AC Cm sampling resolutions (5.12, 2.56, 1.28, and 0.064 ms) were achieved by stimulating with a summed multisine voltage (the multi-dual-sine approach) whose phases were the same. Primary frequencies (f1) were 195.3, 390.6, 781.3, and 1562.5 Hz; all frequencies (10 mV peak) were superimposed onto steps from ?60 to ?00 mV for a duration of 700 ms at a clock sample period of 10 ms. We limited our voltage delivery to ?60 to 100 mV because with this protocol, larger voltages caused cell recordings to be lost or unstable. On return from each step to a sinusoidal-free holding XAV-939 web potential of 0 mV for at least 40 ms (see Fig. 1, where only a portion of the protocol is plotted), voltage-sensor displacement currents are extractable. Thus, this approach provided both mutlifrequency AC capacitance and step-induced charge movement measures within one protocol, an important approach that ensures that the preparation is quasistationary in time. After gigohm seal formation, stray capacitance was cancelled with amplifier order DM-3189 compensation controls, as is usually done. Since stray capacitance is frequency dependent, it is important to ensure that it is cancelled out at each recording frequency. The existence of stray capacitance causes an apparent frequency dependence of linear capacitance, which should not be frequency dependent. Measures of Rs are similarly affected. Consequently, in the whole-cell configuration, residual stray capacitance at each of the recording frequencies was cancelled with further manipulations of amplifier compensation by ensuring that cell linear capacitance (or, equivalently, Rs) was constant across frequency. This was done at very positive voltages, where OHC capacitance is dominated by linear capacitance. Removal of stray capacitance is necessary to meet Cm estimation algorithm requirements (19). We have used this compensation approach to ensure accurate measures of hair cell synaptic vesicle release at high interrogating dual-sine frequencies (20,22). The Supporting Material Appendix expands on our approach. For each cell, capacitance data were fit to the first derivative of a twostate Boltzmann function with an additional component describing theMATERIALS AND METHODSWhole-cell patch-clamp recordings were made from single isolated OHCs from the organs of Corti of Hartley albino guinea pigs. After animals were overdosed with isoflurane, the temporal bones were excised and the top turns of the cochleae dissected free. Enzyme treatment (1 mg/mL Dispase I for 10 min) preceded trituration, and isolated OHCs were placed in a glass-bottom recording chamber. An inverted Nikon (Tokyo, Japan) Eclipse TI-2000 micro.Nteed by setting both intracellular and extracellular chloride concentrations equal. All chemicals used were purchased from Sigma (St. Louis, MO).Cell capacitanceAn Axon Instruments (Foster City, CA) 200B amplifier was used for wholecell recording. To measure both AC capacitance and integrated charge movement, a simple voltage protocol was designed that included both step stimulation and dual sine stimulation at a range of dual-sine interrogation frequencies (Fig. 1, A and B). For AC admittance analysis, real and imaginary components of currents were corrected for the recording-system frequency response (19). No averaging was used with this protocol. Membrane capacitance (Cm) was measured using a continuous dual-frequency (discrete sinusoidal frequencies at f1 and f2, where f2 ?2 ?f1) voltage-stimulus protocol (19,21). Simultaneous AC Cm sampling resolutions (5.12, 2.56, 1.28, and 0.064 ms) were achieved by stimulating with a summed multisine voltage (the multi-dual-sine approach) whose phases were the same. Primary frequencies (f1) were 195.3, 390.6, 781.3, and 1562.5 Hz; all frequencies (10 mV peak) were superimposed onto steps from ?60 to ?00 mV for a duration of 700 ms at a clock sample period of 10 ms. We limited our voltage delivery to ?60 to 100 mV because with this protocol, larger voltages caused cell recordings to be lost or unstable. On return from each step to a sinusoidal-free holding potential of 0 mV for at least 40 ms (see Fig. 1, where only a portion of the protocol is plotted), voltage-sensor displacement currents are extractable. Thus, this approach provided both mutlifrequency AC capacitance and step-induced charge movement measures within one protocol, an important approach that ensures that the preparation is quasistationary in time. After gigohm seal formation, stray capacitance was cancelled with amplifier compensation controls, as is usually done. Since stray capacitance is frequency dependent, it is important to ensure that it is cancelled out at each recording frequency. The existence of stray capacitance causes an apparent frequency dependence of linear capacitance, which should not be frequency dependent. Measures of Rs are similarly affected. Consequently, in the whole-cell configuration, residual stray capacitance at each of the recording frequencies was cancelled with further manipulations of amplifier compensation by ensuring that cell linear capacitance (or, equivalently, Rs) was constant across frequency. This was done at very positive voltages, where OHC capacitance is dominated by linear capacitance. Removal of stray capacitance is necessary to meet Cm estimation algorithm requirements (19). We have used this compensation approach to ensure accurate measures of hair cell synaptic vesicle release at high interrogating dual-sine frequencies (20,22). The Supporting Material Appendix expands on our approach. For each cell, capacitance data were fit to the first derivative of a twostate Boltzmann function with an additional component describing theMATERIALS AND METHODSWhole-cell patch-clamp recordings were made from single isolated OHCs from the organs of Corti of Hartley albino guinea pigs. After animals were overdosed with isoflurane, the temporal bones were excised and the top turns of the cochleae dissected free. Enzyme treatment (1 mg/mL Dispase I for 10 min) preceded trituration, and isolated OHCs were placed in a glass-bottom recording chamber. An inverted Nikon (Tokyo, Japan) Eclipse TI-2000 micro.
