Effect of Temperature on Electrical Resonance in Leopard Frog Saccular Hair Cells

M. S. Smotherman and P. M. Narins

Department of Physiological Science, The University of California, Los Angeles, California 90095

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Smotherman, M. S. and P. M. Narins. Effect of temperature on electrical resonance in leopard frog saccular hair cells. J. Neurophysiol. 79: 312-321, 1998. Leopard frog saccular hair cells exhibit an electrical resonance in response to a depolarizing stimulus that has been proposed to contribute to the tuning properties of the frog sacculus by acting as an electrical band-pass filter. With the whole cell patch-clamp technique, we have investigated the effect of temperature on electrical resonances in isolated saccular hair cells, and we have described the effects of temperature on the currents and channel kinetics underlying electrical resonance. A hair cell's onset resonant frequency in response to a constant depolarizing current pulse increases linearly with temperature at a rate of 11 Hz/1°C, exhibiting a mean Q10 of 1.7 between 15 and 35°C. However, offset resonant frequencies continue to double every 10°C, exhibiting a mean Q10 of 2.1. If steady-state voltage during the stimulus is held constant, all oscillatory frequencies increase with a mean Q10 of 2.1. The average level of steady-state depolarization during a +150-pA depolarizing current pulse decreases with increasing temperature (-6 mV from 15 to 25°C). This temperature-dependent reduction of the steady-state membrane potential causes a shift in the voltage-dependent channel kinetics to slower rates, thus reducing the apparent Q10 for onset resonant frequencies. The peak outward tail current and net steady-state outward current, which is the sum of a voltage-dependent inward calcium current (ICa) and an outward calcium-dependent potassium current (IK(Ca)), increase with temperature, exhibiting a mean Q10 of 1.7 between 15 and 25°C. The activation rate (T1/2) of the outward current exhibits a mean Q10 of 2.3 between 15 and 25°C, while the deactivation rate (tau rel) exhibits a mean Q10 of 2.9 over the same temperature range. These results support previous models of the molecular determination of resonant frequency, which have proposed that a combination of IK(Ca) channel kinetics and the overall magnitude of the outward current are primarily responsible for determining the resonant frequency of an isolated hair cell. The robust temperature sensitivity of the hair cell receptor potential contrasts sharply with the temperature-insensitive tuning properties of in vivo saccular nerve fiber recordings. Possible explanations for this discrepancy are discussed.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The frog sacculus is an inner ear organ that responds to primarily substrate-borne vibrations at frequencies extending only to ~160 Hz (Koyama et al. 1982; Lewis 1988). The organ is remarkably sensitive to seismic stimulation (Narins and Lewis 1984), but single nerve fibers are poorly tuned, exhibiting broad pass bands (Lewis 1988). Electrical resonances are exhibited by isolated saccular hair cells and are believed to be the primary mechanism of frequency selectivity for sounds or vibrations entering the sacculus.

The purpose of this study is to quantify the effects of temperature on hair cell electrical resonances and their underlying conductances. The results bear directly on whether or not temperature-dependent changes in the receptor potential can account for the temperature-dependent properties of the intact auditory system. The effect of temperature on single fiber tuning properties has been used to infer the contributions of electrical tuning in the gecko (Eatock and Manley 1981), caiman (Smolders and Klinke 1984), and pigeon (Schermuly and Klinke 1985). In each of these animals, the best excitatory frequency for single auditory nerve fibers increases with a temperature Q10 of 2.0 over its entire acoustical range. By contrast however, in the goldfish sacculus (Fay and Ream 1992) and bullfrog sacculus (Egert and Lewis 1995), single fiber tuning properties are remarkably temperature-insensitive, exhibiting Q10s as low as 1.1. In the frog auditory system, low-frequency amphibian papillar fibers (<1 kHz) exhibit Q10s of ~1.7, whereas high-frequency basilar papillar fibers are temperature insensitive (Stiebler and Narins 1990).

Because most of the animals possessing electrically resonant hair cells are ectothermic, it becomes relevant for our understanding of these animals' sensory ecology to be able to predict how the properties of hair cells change within a physiologically relevant temperature range. The leopard frog (Rana pipiens pipiens) is the most widely distributed amphibian in North America, enduring active body temperature ranges of 8-35°C and known to breed at temperatures <15°C (Zug 1993). Studies of electrical resonance in the turtle basilar papilla have shown that resonant frequency is determined primarily by the kinetics of the calcium-dependent potassium channel (Art et al. 1986), and ion channels are known to be highly temperature sensitive, typically exhibiting Q10s of ~3 (Hodgkin et al. 1952). Using their model of turtle hair cell resonances, Wu et al. (1995) predicted that resonant frequency should exhibit a Q10 of between 2.0 and 2.2. The main conclusion from the present experiments is that frog saccular hair cell electrical resonances are highly temperature sensitive, exhibiting a Q10 of 2.0. The underlying channel kinetics and conductances generally respond to changes in temperature as predicted by Hodgkin et al. (1952).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Dissociation of hair cells

Saccular maculae were dissected from 60 to 80 mm (snout-vent length) pithed and decapitated adult northern leopard frogs(R. pipiens pipiens) and treated for 20 min at room temperature with papain (Calbiochem, San Diego, CA; 500 mg/l) dissolved in a dissociation solution containing (in mM) 120 NaCl, 2 KCl, 0.1 CaCl2, 3 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); pH 7.2. They then were transferred to a dissociation solution with bovine serum albumin (500 mg/l) replacing papain for 15-30 min. Maculae then were transferred to a recording dish with dissociation solution alone, and hair cells were gently flicked free with a tungsten needle. The hair cells settled but did not adhere to the glass bottom of the recording chamber, after which the dissociation solution was replaced via perfusion with a standard external recording solution containing (in mM) 120 NaCl, 2 KCl, 4 CaCl2, 3 D-glucose, and 10 HEPES; pH 7.2. The pipette (internal) solution contained (in mM) 120K-aspartate, 4 KCl, 0.1 CaCl2, 3 D-glucose, 5 HEPES, 2 ethyleneglycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5ATP, and 2 MgCl2; pH 7.2. Under these conditions, both potassium and calcium currents are elevated relative to in vivo estimates. Electrode tip junction potentials were subtracted as in Fenwick et al. (1982). The recording chamber was placed on the stage of an inverted microscope (Nikon Diaphot, Japan) equipped with a ×40 objective with phase contrast optics. Bath temperature was monitored with a thermocouple placed at the center of the bath, and temperature was changed at a rate of 1-2°C/min (a rate comparable with the experiments by Egert and Lewis 1995, although they used microwaves to heat the entire frog) by a temperature controller unit (Sensortek TS-4; Sensortek, Clifton, NJ) attached to the stage.

Whole cell recordings

Currents and voltages were recorded with the conventional whole cell tight-seal patch-clamp technique (Hamill et al. 1981). Borosilicate glass pipettes were pulled with a Narishige two-stage vertical pipette puller (Narishige, Japan) to tip diameters of ~1 µm. Electrode resistances typically ranged from 2 to 10 MOmega . Series resistances during recordings ranged from 6 to 25 MOmega and were compensated 60-95% with the compensation circuitry of the amplifier (Axon Instruments, Foster City, CA). Cell capacitances ranged from 8 to 16 pF, with voltage-clamp time constants ranging from 10 to 160 µs. Steady-state voltage errors due to uncompensated series resistance were corrected post hoc during data analysis. Cell capacitance and series resistances were taken to be the values read from the amplifier's compensation dials, however, these values were found to be within 5% of those calculated by fitting a single exponential function to a capacity transient and estimating Cm from the area under the curve (Cm = Q/Delta V), and RS from Cm and the time constant of the curve (RS = tau /Cm). Some of the current-clamp recordings presented in this study were achieved using the perforated-patch technique (Rae et al. 1991). For perforated-patch recordings, amphotericin B (Sigma, St. Louis, MO) was added to the pipette solution (240 µg/ml), after first being dissolved in dimethyl sulfoxide (60 mg/ml). With this technique, series resistances were typically >20 MOmega , thus limiting their usage for voltage-clamp experiments but providing consistent current-clamp recordings for up to or beyond 1 h. The Axopatch 200A was used for all currrent- (fast current-clamp mode) and voltage-clamp experiments. Stimuli were generated and data were sampled with a 12-bit digital/analog and analog/digital converter (Digidata 1200, Axon Instruments) and controlled by the data acquisition software package pClamp 5.5 (Axon Instruments). Sampling intervals were tailored to the kinetics of the study; in general, however, voltage-clamp data were sampled at 10- or 20-µs intervals, and current-clamp data were sampled at 50- to 100-µs intervals. Voltage and current waveforms were low-pass filtered by the amplifier at 2 kHz cutoff frequency.

Data analysis

Current- and voltage-clamp data were analyzed using the pClamp 5.5 Clampfit program (Axon Instruments). Statistics and figures were produced using the spreadsheet program Excel (Microsoft, Seattle, WA). A hair cells' electrical characteristic frequency was determined to be the frequency exhibited at the onset of a depolarizing current pulse that produces the highest electrical resonance quality factor, Qe (Qe = [(pi fetau )2 + 0.25)]-1/2 (see Crawford and Fettiplace 1981). Typically the resonance with the highest Qe was generated by a +150-pA pulse. Although offset oscillations are regarded as a better measure of a hair cell's natural resonant frequency (Art et al. 1986), onset oscillations are larger and typically last longer in prolonged experiments such as the ones in this study. Saccular hair cells have never been recorded from in vivo, so it is not known whether on- or offset oscillations better represent a saccular hair cell's natural resonant frequency. A +150-pA current pulse provided reliable and measurable oscillations throughout a wide physiological range of temperatures (15-35°C) and for recording times of 30-60 min (depending primarily on series and shunt resistances). In a few cases, a standing current was added in current-clamp mode to raise the cell's membrane potential closer to the lowest potential at which it would oscillate for the purpose of enhancing offset oscillations. However, this current was held constant for the duration of the experiment. No attempt was made to regulate the resting potential during temperature changes.

Values for activation times (T1/2), relaxation constants (tau rel), peak tail currents, and steady-state currents were collected and analyzed by voltage clamping the cell to voltages of -120 to +40 mV in 10-mV steps from holding potentials of -60 or -70 mV, depending on which was closer to the measured cell resting potential. Values for these parameters were collected using a 100-or 500-ms voltage pulse to -30 mV for comparisons between different temperatures. This was typically the minimum voltage at which whole cell currents could be evoked reliably and measured at the lowest temperatures. All data values are given as means ±SD. Q10(temp) values were calculated using the equation(RT2/RT1)[(T2-T1)/10], where R may represent either a rate or an amplitude, and T2 represents the higher temperature and T1 the lower temperature.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Current-clamp recordings

Electrical resonances were recorded under current-clamp conditions in hair cells that were maintained at their resting potential. The average resting potential for all cells recorded at 20°C (n = 62) was -69 ± 9 mV (mean ± SD; voltages corrected for a 13.5 mV junction potential, pipette negative), however, cells were separated easily into two subpopulations, similar to those recognized by Holt and Eatock (1995), of round cells with resting potentials of -62 ± 2 mV (n = 28) and taller hair cells with resting potentials of -80 ± 3 mV (n = 34). Throughout this study no systematic differences were found in the temperature dependence of these two populations; therefore all the results presented here represent both cell types combined. Although resting potentials typically shifted during the course of the experiments, they showed no consistent temperature dependence. The average change in resting potential for hair cells recorded over a range of >= 4°C was -0.1 mV/+1°C ± 1.3 mV (n = 31). Some cells exhibited a reversible temperature-dependent shift in their resting potentials, usually depolarizing with a 10°C increase temperature (+4 ± 1 mV; n = 5), but in two cases, cells reversibly hyperpolarized with increasing temperature (-7 and -9 mV changes). Temperature-dependent shifts in resting potential could not be correlated with the presence or absence of any one or a set of conductances.

Electrical resonances were induced in hair cells by injecting a 500-ms current pulse increasing from -100 to +300 pA in 25-pA steps. The resonant frequency and quality factor (Qe) exhibited voltage dependencies identical to those reported by Hudspeth and Lewis (1988), with resonant frequencies increasing with stimulus amplitude and Qes reaching a peak between -44 and -50 mV (or between +50 and +150 pA of depolarizing current). The voltage waveform (Fig. 1) induced by a current pulse typically exhibits a depolarized onset resonance and may exhibit an offset resonance centered around the resting potential, and both dampen over time with a Qe that can depend on several parameters, including membrane potential, membrane resistance, shunt resistance (or quality of the gigaohm seal), and metabolic state of the cell (which deteriorates during replacement of the cytoplasm with the pipette recording solution). Qe values typically declined rapidly during the course of the experiment and generally confounded an accurate analysis of the effect of temperature on Qe. This effect was compensated partially for either by using the perforated-patch recording technique (Rae et al. 1991) or by decreasing the diameter of the pipette tip to reduce the rate of cell washout. These procedures increased the potential recording time from 20 to >45 min for current-clamp recordings but imposed limitations on the voltage-clamp recordings due to increased series resistance. For this reason most current- and voltage-clamp records were collected separately from different cells. Offset oscillations occurred if the cell's resting potential was within or close to the voltage-activation range of the primary currents. Normally this was not the case, as most cells were resting negative to -60 mV as a result of the low external potassium concentrations (EK = -104 mV). It is likely, based on the presence of spontaneous activity in saccular afferent fibers, that in vivo hair cell resting potentials are close to the threshold of activation for voltage-dependent calcium channels, typically around -55 mV. It is possible to inject a standing current to raise the resting potential to a level conducive to offset oscillations, but we chose instead to record the range of voltage-dependent resonant frequencies with a series of depolarizing current steps (described in a preceding section), which provides the same information while minimizing the metabolic load of the cell during the experiment. After a 150-pA current pulse, offset oscillations were observed in a subset of all recordings (28 of 80) and frequently disappeared within 5 or 10 min of initiating recordings.


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FIG. 1. Electrical resonance at 3 temperatures. Electrical resonances induced by a 150-pA current pulse for 100 ms at 15°C (A), 20°C (B), and 25°C (C), in a spherical saccular hair cell. Initial recordings were made at 15°C; subsequently the temperature was increased at a rate of 1°C/min. Onset resonant frequency, average depolarized membrane potential, and Qe values at 15°C were 125 Hz, -35.5 mV, 15.5; at 20°C were 166 Hz, -37.2 mV, 9.7; and at 25°C were 222 Hz, -39.1 mV, 9.4. Q10 of the onset resonant frequency is 1.8. Offset resonant frequencies at 15 and 20°C were 77 and 111 Hz, respectively. (Zero-current resting potential was -56 mV; series resistance was 12 MOmega ; cell capacitance was 14 pF; shunt resistance was >1.5 GOmega .) Each record represents the average of 10 traces. Recordings corrected for junction potential but not voltage-drop across series resistance.

The effect of temperature on resonant frequency first was investigated by recording the resonant frequency at the initiation of recording from hair cells that had equilibrated at a given temperature for >10 min and by averaging the resonant frequencies of several hair cells recorded at the same temperature. Figure 2 shows the average onset and offset resonant frequencies (collected separately) for cells initially recorded at temperatures ranging from 15 to 35°C in steps of 5°C. The Q10 for averaged onset resonant frequencies between 15 and 35°C is 1.7. Over the entire range, the data appear remarkably well described by a straight line function. A line fit to these points has a slope of 11 Hz/1°C. At 35°C, only 8 of 19 cells exhibited resonant behavior, but it was generally the case that establishing or maintaining a good gigaohm seal was considerably more difficult at this temperature. It is therefore impossible to say if the reduction in resonant behavior is a cellular property or experimental artifact. Gigaohm seals were frequently lost in experiments in which the temperature was raised >30°C. The effect of temperature on offset resonant frequency appears different from onset frequencies in that it is best fit by an exponential curve, exhibiting a consistent Q10 of ~2.1 over the entire range of temperatures included. This difference can be accounted for by a progressive shift in the steady-state voltage response at stimulus onset with temperature change and can be corrected for by comparing resonant frequencies occurring at the same membrane potential but different stimulus intensities at all temperatures measured (see Fig. 3).


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FIG. 2. Average resonant frequency vs. temperature. Cells initially recorded at temperatures between 15 and 35°C were 1st divided into subgroups of 5°C (i.e., 15 ± 2.5°C) and their onset or offset resonant frequencies averaged. Resonant frequencies were recorded during the 1st 5 min of the experiments. Each cell is only represented at 1 temperature. Onset frequencies: for 15°C, n = 15; 20°C, n = 26; 25°C, n = 17; 30°C, n = 8; 35°C, n = 3. Qe values varied between 3 and 45 for this data set. Data were fit to a straight line with a slope of 11 Hz/1°C. Q10 for 15-35°C is 1.7. Considerably fewer cells exhibited resonant behavior >= 30°C (30% of cells recorded) as compared with 20°C (85%). Offset frequencies: for 15°C, n = 11; 20°C, n = 12; 25°C, n = 10; 30°C, n = 8; 35°C, n = 6. Qe values varied between 2 and 18 for this data set. Data were fit to a single exponential by the equation y = 22.4 (100.03X), r2 = 0.99. Q10 for 15-35°C is 2.1. Error bars indicate ±SD.


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FIG. 3. Resonant frequency, membrane potential, and temperature. Resonant frequency plotted as a function of the steady-state membrane-potential induced by depolarizing stimuli of varying amplitude. Current pulses ranging in steps of 25 pA from 50 to 250 pA (15°), 75 to 250 pA (20°), and 125 to 250 pA (25°) were used to induce resonances in an overlapping range of membrane potentials at 3 different temperatures in a single hair cell. As temperature increased, the resulting steady-state membrane potential for a given stimulus is shifted negatively, whereas resonant frequencies increase. Comparing resonant frequencies for a set stimulus amplitude (compare the last point on each curve, 250 pA) fails to take into account the shift in membrane potential and the accompanying effects on the voltage-dependent channel kinetics. Comparing resonant frequencies for a set steady-state membrane-potential results in a similar Q10 value (approx 2.1) for any membrane potential. Data fit with polynomial curves with r2 values of 0.99 or better. Tall cylindrical hair cell, 16.8 pF, 14 MOmega , Vrest = -82 mV, recorded in standard whole cell mode.

Individual hair cell resonant frequencies were recorded over a range of temperatures (Fig. 1) and Q10s calculated for each hair cell. Whole cell recordings were initiated at either 15, 20, or 25°C, and the temperature was raised or lowered at a rate of 0.5°C/min. Current-clamp recordings were made over as wide a temperature range as possible, and at each temperature, a series of increasing current pulses was used to record the voltage-dependent range of resonant frequencies. Figure 3 shows the span of resonant frequencies elicited by a series of depolarizing current pulses for a hair cell at three temperatures. At increasing temperatures, the entire curve of resonant frequencies is shifted both upward and to more negative potentials. The shift to more negative potentials is caused by an increasing net outward current, which reduces the Vss induced by a constant amplitude current pulse. The last data point on each curve represents the resonant frequency in response to a 250-pA current pulse. Estimating a Q10 from this constant stimulus produces a value of 1.6, which underestimates the Q10 of 2.1 calculated using any constant voltage from the overlapping voltage range. Holding voltage constant, the average Q10 value for cells recorded over a span of temperatures within the range of 15-25°C (n = 29) was 2.1 ± 0.3. In all cells in which it was possible to test, the temperature-dependent shift in resonant frequency was reversible (n = 18). Increasing temperature caused the same absolute shift in resonant frequency as decreasing temperature. In five cases where temperature was increased >5°C, the cells were given time to equilibrate at that temperature for a 10-min period and resonant frequency was monitored at 2-min intervals. In every case, the resonant frequency changed by <5% during the 10-min period. Figure 4A shows the resonant frequencies over an extended range of temperatures between 15 and 25°C for five individual hair cells.


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FIG. 4. Current and voltage results from 5 cells. Effect of temperature on resonant frequency (A), steady-state current, Iss (B), activation rate of the outward current, T1/2 (C), and relaxation rate of the outward current, tau rel (D) for 5 hair cells recorded between 15 and 25°C. open circle  (cell 1), 14 pF (cell capacitance), 15 MOmega (series resistance), 80% series resistance compensation (s.r.c); square  (cell 2), 15 pF, 12 MOmega , 95% s.r.c.; diamond  (cell 3), 13 pF, 15 MOmega , 95% s.r.c.; bullet  (cell 4), 8 pF, 20 MOmega , 90% s.r.c.; black-square (cell 5), 11 pF, 25 MOmega , 90% s.r.c. Voltage responses were recorded in response to a 150-pA current pulse while the temperature was raised in cells 2, 3, and 5 or while the temperature was lowered in cells 1 and 4. After a temperature change of ~10°C, the direction of temperature shift was reversed and current-clamp mode was switched to voltage-clamp mode in cells 2, 3, and 5 or voltage- to current-clamp mode in cells 1 and 4. Iss was measured as the average outward current during the last 200 ms of a 500-ms voltage clamp pulse to -30 mV. T1/2 and tau rel were taken from the same voltage-clamp pulses as Iss. Temperature effects were generally independent of the direction of temperature shift.

The damped resonance induced by a constant current stimulus usually decays to a steady-state level of depolarization (Vss). The average Vss for all cells included in the analysis of resonant frequency in response to a 150-pA current pulse at 20°C was 11.6 ± 4.5 mV relative to the average threshold for activation (-55 ± 3.0 mV) of the two primary conductances. This is essentially identical to the value of 11.9 mV predicted using the standard conductance values adopted by Hudspeth and Lewis (1988) of 16.8 nS for gK(Ca) and 4.1 nS for gCa and assuming that Vss represents the product of the input current and the balance of these two conductances [Vss = (Iin)1/(gK(Ca) - gCa)]. Using activation threshold for measuring Vss is necessary for normalizing the steady-state voltage responses because of the considerable variations in resting potentials (nearly 20 mV for both populations), almost all of which were hyperpolarized relative to the activation threshold to a varying extent as a result of the low external potassium concentrations. Nevertheless, activation threshold is the most reasonable point of comparison because it is remarkably consistent between cells, the high-impedence voltage range below the threshold of activation does not contribute significantly to the steady-state response above -55 mV, and the input impedance is determined by the activation-state of these two conductances. In 10 cells recorded from 15 to 25°C, Vss decreased by an average of 4.5 mV ± 2.9 mV; a decrease of 5.0 mV is predicted if both conductances are increased with a Q10 of 1.7, which was the observed amplitude change in whole cell steady-state current (Iss). In 22 of 30 cells observed over a range of temperatures >= 4°C, it was found that Vss reversibly decreased with increasing temperature in 22 of 30 cells by an average rate of -0.6 ± 0.5 mV/°C. In eight cells, there was no change in Vss independent of changes in series resistance, which typically only increased throughout the duration of the experiment.

Voltage-clamp recordings

The electrical resonances observed in auditory hair cells of the fish, frog, and turtle are produced by the voltage-dependent interaction of gCa and gK(Ca) (Art and Fettiplace 1987; Hudspeth and Lewis 1988). On depolarization, the whole cell current is initiated by an inward calcium current but rapidly dominated by an outward potassium current, which is approximately five times greater than the calcium current at a holding potential of -30 mV (Hudspeth and Lewis 1988). The voltage-dependent calcium current's activation and deactivation rates are considerably faster than those for the calcium-dependent potassium current, both being approximately <= 0.3 ms (Hudspeth and Lewis 1988; Roberts et al. 1990) and do not vary appreciably among hair cells. The slower activation and deactivation rates of IK(Ca) make them limiting factors in generating the resonant frequency, although they are not the exclusive determinants. Extensive research in the turtle cochlea (Art and Fettiplace 1987; Art et al. 1986; Wu et al. 1995) has established that the resonant frequency depends on the relative conductance amplitudes and the rates of activation deactivation of the outward current. A similar relationship now is known to exist in the leopard frog amphibian papilla (Smotherman and Narins 1997). In the bullfrog sacculus, a better correlation was observed in a given cell between resonant frequency and the activation rate rather than relaxation rate (Roberts et al. 1986), but attempts to identify these trends are hindered by the very limited range of resonant frequencies observed in the sacculus (typically ~60 Hz, compared with >600 Hz in the turtle and 500 Hz in the amphibian papilla). Present measurements have focused on this set of parameters because they are the most likely ones to be determining the resonant frequency endogenously.

A pharmacological dissection of the currents present in leopard frog saccular hair cells was performed, through which we identified the same set of ionic currents identified in frog saccular hair cells by Hudspeth and Lewis (1988) and Holt and Eatock (1995). On depolarization from a holding potential of -60 mV, the outward current could be blocked completely by 2 mM CdCl2, 2 mM tetraethylammonium (TEA), or 10 µg/ml charybdotoxin, confirming that this current was the large-conductance (BK) calcium-dependent potassium channel. From a holding potential of -120 mV, an additional inactivating voltage-dependent outward current was observed that could be blocked selectively by 4-aminopyridine (4-AP). Analysis of the kinetics and voltage sensitivity suggested that this current closely resembled the A current identified by Hudspeth and Lewis (1988). The addition of TEA and 4-AP revealed a noninactivating inward current that was sensitive to external calcium concentrations and could be blocked by 2 mM CdCl2, demonstrating to our satisfaction that the inward current was the product of an L-type calcium channel. In a subset of hair cells (34 of 62), we observed the presence of a large, fast inward current preferentially activated by hyperpolarizing holding potentials. This inward current was sensitive to external potassium concentrations, growing larger with greater external potassium but was insensitive to CdCl2. This inward potassium current was recognized as the inward rectifier IK1 described in leopard frog hair cells by Holt and Eatock (1995). Hair cells that did not possess this current exhibited another inward rectifier potassium current, IH, which was considerably smaller and slower than IK1 and behaved as described by Holt and Eatock (1995). The potential contributions of the two large voltage-dependent potassium currents IA and IK1 to electrical resonances are not clear. The experimental protocol used for these experiments was designed with an eye toward minimizing their contributions, but no attempts were made (pharmacologically) to specifically exclude these conductances.


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FIG. 5. Temperature dependence of peak tail current amplitudes. Hair cells were voltage clamped to command potentials varying from -70 to +30 mV (increasing by steps of 10 mV) for 500 ms and returned to a resting potential of -60 mV. Data points represent the peak amplitude of the tail current measured at -60 mV after the termination of the command voltage step. Tail current amplitudes typically peaked with command potentials of ~0 mV. To estimate the peak tail current amplitude, tail currents were fit with a single-exponential curve (using the least-squares method for determining best fit) and peak tail current amplitudes were estimated for 0.3 ms after the end of the voltage pulse (0.3 ms is the typical relaxation time constant for the inward calcium current, which imposes a fast but large inward current spike on return to the resting potential). Tail current amplitude changed equally for all voltage steps with a Q10 of ~1.4 in this cell; 15 pF, 12 mOmega , 95% s.r.c. Data were fit with a Boltzman function of the form I = Imax/{1 + exp[(Vm - V1/2)/k]}, where V1/2 is the potential at which the current is half-maximal and k is a constant. For all 3 sets of data, V1/2 is -32 mV and k = 5.5. Changing Imax was sufficient to fit all 3 curves equally well.


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FIG. 6. Temperature-dependent shift in activation rate. Increasing temperature increases both the amplitude and activation rate of the outward current. A: 1st 3.5 ms of a voltage step to -30 mV. Small transient currents were left in to mark the beginning of the pulse. Initial peak of the outward current amplitude exhibits a Q10 of 1.7, whereas the steady-state current (not shown) exhibited a Q10 of 1.5. Q10 for the activation rate, T1/2, was 2.3, decreasing from 2.5 ms at 15°C to 1.1 ms at 25°C. Cell's initial temperature was 15°C: 11 pF, 12 MOmega , 95% s.r.c. B: T1/2 for a typical saccular hair cell recorded between 15 and 25°C. Data were fit to a single exponential by the equation y = 349.4X-1.74, r2 = 0.99. Q10 for 15-25°C is ~2.3.

Peak tail currents were used to estimate changes in gK(Ca). Hudspeth and Lewis (1988) and Roberts et al. (1990) have shown that the calcium current approaches zero shortly after 0.3 ms after the end of a voltage pulse. The peak tail currents were estimated by fitting a single exponential from 0.3 ms after the end of the pulse until the current decayed to baseline and then extrapolating back to the end of the current pulse. Figure 5 shows the relationship between depolarized membrane potentials and the resulting tail current amplitude on return to the resting potential at three temperatures. Amplitude, but not threshold or activation range, changed with temperature, increasing equally across the activation range. Peak tail currents (Imax) increased from 15 to 25°C with an average Q10 of 1.7 ± 0.3 (n = 13).

The whole cell steady-state current (Iss) between -50 and -30 mV is a composite of ICa and IK(Ca). Any temperature effects on Iss would reflect the additive effects of temperature on both conductances. Despite the fact that Iss is the difference between the inward ICa and the outward IK(Ca), any temperature-dependent increase in the calcium conductance will appear as an appropriate increase in the calcium-dependent potassium current. Any changes in Iss must take into account changes in inward conductance, calcium diffusion and buffering, calcium interactions with IK(Ca), and changes in the outward conductance. No attempt was made in this study to separate these factors. Hair cells were voltage clamped from a holding potential of -60 to -30 mV for 100 or 200 ms. Over the temperature range of 15-25°C, Iss exhibited a reversible increase in amplitude with a Q10 of 1.7 ± 0.4 (n = 25). Iss increased in amplitude with increasing temperature in all cells tested, although there was considerable variation in initial current amplitude and temperature sensitivity (Fig. 4B). The magnitude and variations found in hair cell Iss and peak tail current data agree with the values reported for bullfrog saccular hair cells by Hudspeth and Lewis (1988) and Roberts et al. (1990). The temperature-dependent changes in Iss and peak tail currents were reversible and independent of the direction of temperature shift, however, currents generally exhibited mild reduction over time.


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FIG. 7. Temperature-dependent shift in relaxation rate. Increasing temperature increases tail current amplitude and decreases the time constant of relaxation of the outward current. A: relaxation of the outward current at -60 mV after the end of a voltage step to -30 mV. Peak tail current amplitude exhibits a Q10 of 1.7. Tail currents were fit with a single exponential curve to estimate tau rels of 15.7 ms (15°C), 8.4 ms (20°C), and 5.1 ms (24°C). For this cell, the Q10 for the relaxation constant, tau rel, was 3.1. Cell's initial temperature was 15°C: 15 pF, 12 MOmega , 95% s.r.c. B: tau rel for a typical cell recorded between 15 and 25°C. Data were fit to a single exponential by the equation y = 95.7(10-0.05X), r2 = 0.99. Q10 for 15-25°C is ~3.0.

The T1/2 for the outward potassium current was measured from application of the voltage clamp from rest to -30 mV until the current reached a peak value. T1/2 is voltage sensitive, decreasing with increased depolarization, but is remarkably consistent between cells. At 20°C, T1/2 is typically 2.0 ± 0.3 ms (n = 21) and does not appear to vary between cells with respect to offset resonant frequency. Figure 6A shows an example of how increasing temperature causes an increase in the amplitude of the current evoked, a noticeable change in the shape of the activating outward current, and a distinct decrease in the timing of all aspects of inward and outward currents evoked. Figure 6B shows the T1/2 for a typical cell recorded over the range of 15-25°C. The T1/2 decreases as temperature increases, exhibiting a Q10 of ~2.3. Figure 4C shows the T1/2 for five individual cells recorded between 15 and 25°C. The average Q10 calculated for single cells over that same temperature range was 2.3 ± 0.7 (n = 21). The effect of temperature on T1/2 was fully reversible and independent of the direction of temperature shift.

The relaxation time constant (tau rel) of IK(Ca) was fit with a single exponential curve (Hudspeth and Lewis 1988; Roberts et al. 1990) using the least-squares method performed by the Axon Instruments pClamp software package. The tau rel was only measured in experiments in which the recording systems' tau  was not >150 µs throughout the duration of the experiment. Tail current tau rels exhibited a mild dependence on preceding voltage between -50 and +40 mV and, at or above -20 mV, were better fit with a double exponential curve. In some cells, tail currents might have been better fit with a double exponential at higher temperatures but were fit to a single exponential for the purposes of comparison with lower temperatures. Figure 7A shows an example of outward tail currents recorded at 15, 20, and 24°C. Increasing temperature results in larger peak tail currents that decay at a proportionally faster rate. Figure 7B shows the tau rel for a typical cell recorded at multiple temperatures between 15 and 25°C. The Q10 from the data in this curve from 15 to 25°C is ~3.0. The temperature-dependent change in tau rel was best fit with a single exponential curve within this temperature range. The average of individual Q10 values for all cells recorded over a span of temperatures within 15 and 25°C was 2.9 ± 0.3 (n = 15). Figure 4D displays the change in tau rel for five individual cells. The temperature-dependent decrease in tau rel was seen in all cells tested and was always reversible.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Electrical tuning has been proposed as an intrinsic tuning mechanism present in all lower vertebrates (Wu et al. 1995) and therefore is presumed responsible for many of the temperature effects seen in the auditory system. However, the effects of temperature on electrical resonances only have been recorded directly in hair cells of the chick cochlea (Fuchs and Evans 1990) where cells over a wide range of resonant frequencies varied with a Q10 of ~2.0, although it should be noted that in the chick the lowest frequency oscillations were generated with voltage- and not calcium-dependent potassium channels. The model of electrical tuning in the turtle basilar papilla was combined with estimates of temperature-dependent channel kinetics and conductance changes to predict a resonant frequency Q10 of between 2.0 and 2.2 (Wu et al. 1995), which concurs well with our results. Using the equations developed by Art and Fettiplace (1987), we observed changes in the amplitude of the net outward current and in the timing of its underlying channel kinetics that accounted for the temperature-dependent shift in resonant frequency. In the turtle, the relationship between resonant frequency and tau rel is described by an empiric relationship
τ<SUB>rel</SUB> = κ/<IT>F</IT><SUP>2</SUP>
where kappa  is a constant and F is the cell's offset resonant frequency (Art and Fettiplace 1987; Wu et al. 1995). This relationship has not previously been reported for the frog sacculus because of the comparatively restricted range of resonant frequencies observed within the sacculus, however, a similar relationship now has been shown in the leopard frog amphibian papilla (Smotherman and Narins 1997), in which resonant frequencies extend up to >= 500 Hz. Activation and deactivation rates for channels may exhibit Q10s from 2 to 4 (Anderson and Stevens 1973; Fukushima 1982; Hodgkin et al. 1952; Nobile et al. 1990; Papahill and Schlichter 1990), which is a large enough range of temperature sensitivities to allow for a potentially large variation from the predicted resonance Q10. The activation rate of gK(Ca) is a complex process, including the activation of gCa, calcium diffusion, calcium buffering and binding, and gK(Ca) activation. In addition, the gK(Ca) channel must go through multiple activation states and may display more than one conductance state (Art et al. 1995). Our Q10 value of 2.3 reflects all of these factors. The deactivation rate Q10 generally should reflect just the properties of a single channel with changes in temperature. Changes in the tau rel of IK(Ca) reflect changes in the mean open time of this channel but also may reflect the time course of the change in intracellular free calcium (Barrett et al. 1982). However, endogenous calcium diffusion and buffering in hair cells occurs within a few microseconds (Roberts 1993), and it is reasonable to assume that temperature-dependent shifts in these processes will have little impact on the time course of the net outward tail current. Our tau rel Q10 of 2.9 is typical of values for other channels and probably does not reflect a process more complex than the single channel properties of gK(Ca). Amplitude of the outward current also is known to contribute to oscillatory frequency in the bullfrog sacculus (Hudspeth and Lewis 1988) but to a lesser degree than tau rel. Open potassium channel conductances typically exhibit Q10s of 1.4-1.7 (Hille 1992), and the calcium-dependent potassium channel in rat muscle has a Q10 of 1.4 (Barrett et al. 1982). Our value of 1.7 is within the expected range. The effect of increasing outward current on resonant frequency is supported by the Q10 of 2.1, which is a larger change than what would be predicted if resonant frequency was related solely to the square-root of the relaxation constant, ~1.7.

In the turtle (Wu et al. 1995), the caiman (Smolders and Klinke 1984), and pigeon (Schermuly and Klinke 1985), the best frequency for single auditory nerve fibers increased with Q10s of 2.0, supporting the role of electrical tuning in these animals. In the frog sacculus, however, Egert and Lewis (1995) reported remarkably temperature-insensitive tuning properties, with Q10s of <1.1. Similarly, in the goldfish sacculus, which is known to possess electrically resonant hair cells (Sugihara and Furukawa 1989), Fay and Ream (1992) found few systematic temperature-dependent shifts in tuning properties that were consistent with electrical tuning. The temperature insensitivity exhibited by these two examples appears to imply only minimal contributions of electrical tuning. Is the argument against electrical tuning in the bullfrog and goldfish sacculus as strong as the arguments for electrical tuning in the turtle, caiman, and pigeon auditory organs? Without studying the properties of the receptor potential in situ, as has been done only in the turtle, it is impossible to answer this question. Recent work in the toadfish vestibular system (Highstein et al. 1997) has shown that hair cells in situ behave differently than when isolated, exhibiting resonant frequencies that appear to increase by almost an order of magnitude on isolation. We presently know very little about the in situ behavior of auditory hair cells in the frog. The presence of irregular spontaneous activity suggests the hair cell resting potentials are near the calcium channel threshold, about -55 mV, but argues against any rhythmic oscillations around the resting potential (Christensen-Dalsgaard and Jorgensen 1988; Lewis 1988). The only evidence supporting electrical tuning in the frog sacculus is the recording of membrane oscillations in isolated hair cells. Saccular hair cells may behave very differently when their electromotile properties are coupled to the enormous overlying otoconial mass. No evidence presently available precludes saccular hair cells from being electrically matched at a frog's "room" temperature to the most appropriate stimulus range. This study confirms that isolated frog saccular hair cells behave as predicted, but in so doing cannot account for the observed temperature insensitivity of the intact auditory system.

In saccular tuning curves derived from reverse correlation analysis with white noise, Egert and Lewis (1995) examined the temperature sensitivity of several features of the tuning curve worth considering here. Saccular single fiber bandwidths may be as broad as two octaves but typically have Q10dB values between 2 and 10 and frequently exhibit distinct peaks and notches in the tuning curve. Although increasing temperature did not typically shift the entire tuning curve or its peak, it sometimes caused an increase in the high-frequency content of the tuning curve, which resembled the consequences of a low-pass filter increasing its corner frequency. In other cases, they observed the emergence of a second, higher frequency peak with increasing temperatures; they suggested this might be attributable to shifts in the relative contributions of multiple independent natural frequencies. Lewis (1988) has shown previously that saccular tuning curves can be characterized by no less than five natural frequencies, only two of which would be represented by electrical tuning in the hair cell. It is not yet clear what is responsible for tuning curve peaks and notches, but Egert and Lewis (1995) found that when present, the frequencies of these components were as temperature insensitive as the peak of the tuning curve. Taken together, these temperature-sensitive tuning curve properties support a model of saccular tuning that may incorporate electrical tuning as one of several spectral filtering mechanisms in the sacculus, but not the primary one.

Interestingly, both the sacculus and amphibian papilla possess electrically resonant hair cells with overlapping resonant frequencies (Roberts et al. 1986), generated by identical ionic current combinations (Smotherman and Narins 1997), and may respond to overlapping frequency ranges (Feng et al. 1975; Lewis et al. 1982a), yet single fiber recordings exhibit remarkably different tuning properties and temperature sensitivities. In contrast to the broadly tuned, temperature-insensitive sacculus, the amphibian papillar fibers exhibit sharply tuned responses, with temperature Q10s of ~1.7. We believe the different tuning properties must reflect differences in the mechanical processes leading up to stimulation of the hair cell and the concomitant interactions between the hair cells and overlying tectorial structures. The differences in saccular and amphibian papillar fibers (Q10(dB) and Q10(temp)) could be accounted for at least partially if saccular hair cell receptor potentials operated with a significant DC component, as has been described in the lizard (Holton and Weiss 1983) and mammal (Russell and Sellick 1978), while amphibian papillar receptor potentials responded symmetrically around their resting potential. Different receptor potential waveforms for saccular and amphibian papillar hair cells would be reasonable consequences of different modes of stimulation, which would not be surprising considering the entirely different architectures of the two organs. A significant DC component in saccular hair cell receptor potentials could account for both the broad tuning curves and the observed temperature insensitivity of the intact organ. In the electrically tuned hair cells of the turtle cochlea, the acoustically driven receptor potential oscillates around the resting potential in situ (Crawford and Fettiplace 1981), which is necessary if electrical resonances are responsible for high Qe tuning; yet no such recordings have been made in the frog. The amphibian papilla is the only other vertebrate hearing organ in which the hair cells are known to possess a systematic range of resonant frequencies matching that of the turtle (Smotherman and Narins 1997). The sacculus has evolved to be a remarkably sensitive seismic detector with generally poor tuning capacities, whereas the amphibian papilla provides sharp tuning over a frequency range 10 times greater than the sacculus (Yu et al. 1991).

One further possible explanation for the temperature insensitivity of saccular tuning would be its convergent afferent innervation, which through summation of many hair cells onto a single afferent nerve could reduce the effective temperature sensitivity of the fiber. Saccular afferent fibers may receive input from 2 to 30 hair cells (Lewis et al. 1982a). It is not yet clear how convergence contributes to the tuning properties of the sacculus. It typically is assumed that the relative contribution of an individual hair cell to an afferent network remains constant regardless of temperature and that if all cells are affected equally by temperature, then the temperature sensitivity of the afferent fiber essentially would reflect the temperature sensitivity of a single hair cell. It is possible, however, that the relative contributions of individual hair cells might change with temperature. Combining a group of hair cells that encompass a broad range of resonant frequencies and thresholds opens the possibility of a temperature-dependent shift in the set of cells contributing most to the output of the afferent fiber. Saccular hair cell resonant frequencies encompass a range of 100 Hz, saccular fibers have been shown to exhibit an intensity range fractionation, with fiber thresholds varying from <0.005 cm/s2 up to 1.28 cm/s2 (Christensen-Dalsgaard and Jorgensen 1988), and both spike rates and thresholds have been shown to change unpredictably at temperatures >22°C (Egert and Lewis 1995; Stiebler and Narins 1990). The source of these variations in threshold and spike rate are unknown but very likely reflect differences in synaptic architectures within the sacculus. Thus sufficient inherent variations in saccular hair cells, synapses, and fiber types exist to support some unidentified mechanism of temperature accommodation. It must be pointed out, however, that the amphibian papilla is constructed with similar afferent circuitry (Lewis et al. 1982b; Simmons et al. 1992) encompassing an even greater array of frequencies and thresholds and yet maintains a robust temperature sensitivity. Thus unless the details of the innervation patterns prove substantially different between the two organs, it appears unlikely that convergent innervation alone can account for the temperature insensitivity of the sacculus.

In conclusion, the temperature insensitivity of frequency tuning by the frog sacculus cannot be attributed to any unusual properties of its hair cells. The hair cells behave as expected experimentally and presumably maintain their temperature sensitivity in situ. We have focused on the question of how much hair cells contribute to tuning in the sacculus, however, an equally important question remains. How does the sacculus resist the changes observed in the highly temperature-sensitive electrical resonances and provide the frog with a consistent frequency range over considerable daily and seasonal temperature changes? The answer will come when we fully understand the dynamic interactions among this organ's mechanics, hair cells, and their innervationpatterns.

    ACKNOWLEDGEMENTS

  The authors thank Drs. Dwayne Simmons, Cristina Bertolotto, and Francisco Bezanilla for many helpful discussions and for the equipment made available by Dr. Simmons. We also thank the anonymous reviewers for helpful comments.

  This work was supported by National Institute of Deafness and Other Communications Disorders Grant DC-00222 to P. M. Narins. All experiments comply with the National Institutes of Health Principles of Animal Care, Publication 86-23, and all current U.S. laws.

    FOOTNOTES

  Address for reprint requests: P. M. Narins, UCLA Dept. of Physiological Science, 405 Hilgard Ave., Los Angeles, CA 90095-1527.

  Received 25 March 1997; accepted in final form 11 September 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society