Potassium currents in auditory hair cells of the frog basilar papilla

Hearing Research 132 (1999) 117^130

Potassium currents in auditory hair cells of the frog basilar papilla Michael S. Smotherman *, Peter M. Narins Department of Physiological Sciences, University of California, 405 Hilgard Ave., Los Angeles, CA 90095-1527, USA Received 5 November 1998; received in revised form 24 February 1999; accepted 3 March 1999

Abstract The whole-cell patch-clamp technique was used to identify and characterize ionic currents in isolated hair cells of the leopard frog basilar papilla (BP). This end organ is responsible for encoding the upper limits of a frog's spectral sensitivity (1.25^2.0 kHz in the leopard frog). Isolated BP hair cells are the smallest hair cells in the frog auditory system, with spherical cell bodies typically less than 20 Wm in diameter and exhibiting whole-cell capacitances of 4^7 pF. Hair cell zero-current resting potentials (Vz ) varied around a mean of 365 mV. All hair cells possessed a non-inactivating, voltage-dependent calcium current (ICa ) that activates above a threshold of 355 mV. Similarly all hair cells possessed a rapidly activating, outward, calcium-dependent potassium current (IK…Ca† ). Most hair cells also possessed a slowly activating, outward, voltage-dependent potassium current (IK ), which is W80% inactive at the hair cell Vz , and a fast-activating, inward-rectifying potassium current (IK1 ) which actively contributes to setting Vz . In a small subset of cells IK was replaced by a fast-inactivating, voltage-dependent potassium current (IA ), which strongly resembled the A-current observed in hair cells of the frog sacculus and amphibian papilla. Most cells have very similar ionic currents, suggesting that the BP consists largely of one homogeneous population of hair cells. The kinetic properties of the ionic currents present (in particular the very slow IK ) argue against electrical tuning, a specialized spectral filtering mechanism reported in the hair cells of birds, reptiles, and amphibians, as a contributor to frequency selectivity of this organ. Instead BP hair cells reflect a generalized strategy for the encoding of high-frequency auditory information in a primitive, mechanically tuned, terrestrial vertebrate auditory organ. ß 1999 Elsevier Science B.V. All rights reserved. Key words: Frog; Hearing; Hair cell; Basilar papilla

1. Introduction The frog inner ear contains three auditory organs: the sacculus, amphibian papilla, and basilar papilla. Of these, the basilar papilla is responsible for encoding the upper limits of the animal's spectral sensitivity (Feng et al., 1975). It responds to airborne sounds of 1.2^2.0 kHz in the leopard frog (Ronken, 1990). The anatomy of the frog basilar papilla (BP) is unique (Wever, 1973; Wilczynski and Capranica, 1984): the BP is a doughnut-shaped organ, with approximately 50^100 hair cells ¢xed within a rigid cartilaginous base along one-half of the inner wall (Frishkopf and Flock, 1974 ; Lewis and Li, 1975). An overlying tectorial membrane covers all but the most peripheral hair cells. How tun-

* Corresponding author. Tel.: +1 (310) 206-8407; Fax: +1 (310) 206-3987; E-mail: [email protected]

ing is achieved by this organ is poorly understood, but it appears unlikely that hair cells contribute via electrical tuning in this frequency range (Smotherman and Narins, 1999a,b). Thus, the frog BP o¡ers us the opportunity to study the electrophysiological properties of hair cells in a high-frequency end organ that has neither a basilar membrane (or analogous structure to support a traveling wave) nor electrical tuning. The physiological response properties of auditory afferent ¢bers innervating the frog BP have been described by several investigators (Frishkopf and Goldstein, 1963 ; Feng et al., 1975 ; Megela and Capranica, 1981 ; Lewis et al., 1982 ; Megela, 1984 ; Ronken, 1990, 1991 ; Yamada, 1997), with a surprisingly uni¢ed picture emerging. For a given animal, a¡erent ¢bers exhibit nearly identical response properties, including characteristic frequencies and consistently poor sharpness of tuning : for example, for the leopard frog the 10 dB bandwidth is nearly an octave wide centered around

0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 9 ) 0 0 0 4 7 - 7

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1.6 kHz. The whole organ appears tuned to a constant bandwidth presumably pertaining to sounds of behavioral signi¢cance, which varies from species to species. Given the homogeneity of BP a¡erent ¢ber responses, it appears that very little spectrographic analysis occurs within the BP. The electrical properties and ionic currents of frog auditory hair cells have been described for the sacculus (Hudspeth and Lewis, 1988; Holt and Eatock, 1995) and amphibian papilla (Smotherman and Narins, 1998). This paper describes the electrical properties and ionic currents of high-frequency auditory hair cells in the leopard frog basilar papilla. Its completion makes possible a comparison of hair cell biophysics across the entire auditory range of the frog. Within the basolateral membrane of BP hair cells, we have identi¢ed one calcium current and four potassium currents, three of which are common to nearly every BP cell examined. Each of the ionic currents identi¢ed here has been described in detail in other frog auditory hair cells; only the combination of ionic currents observed in BP hair cells is unique to this organ. 2. Methods 2.1. Dissociation of hair cells Basilar papillae were dissected out of pithed and decapitated adult northern leopard frogs (Rana pipiens pipiens) and were treated for 20 min at room temperature with 500 mg/l papain (Calbiochem, San Diego, CA) and dissolved in a dissociation solution containing (in mM): NaCl 120, KCl 5, CaCl2 0.1, D-glucose 3, HEPES 10; pH 7.2. Papillae were then transferred to a dissociation solution with bovine serum albumin (BSA, 500 mg/ml) replacing papain for 30^45 min at less than 10³C, after which they were transferred to a 0.3 ml recording dish containing dissociation solution alone, and hair cells were gently scraped free with a tungsten needle. A typical dissociation produced three

to ¢ve uncompromised hair cells, which settled to the base of the recording chamber. It has been shown that the use of papain as a dissociative agent may alter the electrical properties of frog saccular hair cells (Armstrong and Roberts, 1998), however we have uncovered no consistent evidence of enzymatic degradation in isolated frog auditory hair cells (Smotherman and Narins, 1999b). While papain was reported to remove a voltagedependent potassium current (IK ) in frog saccular hair cells (Armstrong and Roberts, 1998), we were able to record and characterize a very similar IK in papain-dissociated BP hair cells. We would not argue that BP hair cells are una¡ected by the enzymatic dissociation procedure used here, but the di¤culty of isolating BP hair cells even with papain prohibited us from more thoroughly addressing this issue. Following hair cell dissociation the recording chamber was placed on the stage of an inverted microscope (Nikon Diaphot, Japan), and the dissociation solution was replaced via a slow continuous perfusion system with a control external recording solution (see Table 1). After W10 min of exposure to the standard external solution, hair cells adhered more ¢rmly to the chamber £oor, allowing the perfusion rate to be raised to approximately 1 ml/min. A series of ionic and pharmacological agents was incorporated into the recording solution (Table 1). Recording solution exchange was achieved via continuous gravity perfusion of the entire recording chamber. The solution was allowed to equilibrate for at least 2 min before experiments continued and was maintained until no further change was observed in the ionic currents. Recordings were stable and consistent enough to allow several solution exchanges and reversals. 2.2. 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 2-stage vertical pipette puller (Narishige, Japan) to tip diameters of approximately

Table 1 Composition of solutions (in mM) CsCl Internal CsCl internal Reduced Ca2‡ Control BaCl2 (U = BaCl2 ) CdCl2 (U = CdCl2 ) 4-AP (U = 4-AP) TEA (U = TEA)

^ 110 ^ ^ ^ ^ ^ ^

NaCl

KCl

CaCl2

MgCl2

K-asp.

Glucose

HEPES

EGTA

^ 114 110 110 110 110U 110U

4 ^ 5 5 5 5 5 5

0.1 0.1 0.1 4 4U 4U 4 4

2 2 ^ ^ ^ ^ ^ ^

106 ^ ^ ^ ^ ^ ^ ^

3 3 3 3 3 3 3 3

10 10 10 10 10 10 10 10

2 2 0.5 ^ ^ ^ ^ ^

Solutions adjusted to pH 7.2 with NaOH. Changes in external KCl or CaCl2 concentrations described in text were balanced by an equimolar change in NaCl. Solutions were stored for up to 3 days at 5³C. Internal solutions contained 5 mM Na2 -ATP. For BaCl2 , CdCl2 , 4-AP and TEA, U represents the concentrations listed in the text.

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1 Wm. Electrode resistances typically ranged from 4 to 10 M6. Series resistances during recordings ranged from 6 to 30 M6 and were compensated 60^95% during voltage-clamp recordings using the compensation circuitry of the ampli¢er. Cell capacitances ranged from 4 to 7 pF. Cell capacitances and uncompensated series resistances produced theoretical voltage-clamp time constants below 84 Ws, although minor compensationinduced transients limited most experiments to greater than 50 Ws settling times. Cell capacitances and series resistances were taken to be the values read from the ampli¢er's compensation dials, however these values were found to be within 5% of those calculated by ¢tting a single exponential function to a capacity transient and estimating Cm from the area under the curve (Cm = Q/vV). Series resistance and cell capacitance compensation were updated continuously throughout all experiments. Electrode tip junction potentials (13 mV, pipette negative) were subtracted as in Fenwick et al., 1982. Leak subtraction and correction for voltage errors were performed o¡-line during data analysis. In some cases, records were averaged to improve the signal-to-noise ratio. We used the Axopatch 200A (Axon Instruments, Foster City, CA) for all current- 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. Voltage and current waveforms were low-pass ¢ltered by the ampli¢er at 2 kHz cuto¡ frequency. Experiments were performed at room temperature (19^20³C). 2.3. Data analysis Current- and voltage-clamp data were stored digitally and later analyzed o¡-line using the Pclamp 5.5 Clamp¢t program (Axon Instruments). For determining activation and relaxation time constants, exponential curves were ¢t using a least-squares algorithm in Pclamp. Boltzmann curves of the general form I=I max ˆ 1=f1 ‡ exp……V m ÿ V 1=2 †=k†g where Vm is membrane potential, V1=2 is the potential at which activation (or inactivation, with the appropriate modi¢cations) is half-maximal, and k is a constant re£ecting the steepness of the voltage dependence. They were ¢t using the program SigmaPlot 4.0 (Jandel Scienti¢c, Corte Madera, CA), using the Marquardt-Levenberg algorithm. Tail current amplitudes at step o¡set were estimated by ¢tting a single exponential function to the tail current and extrapolating back to the time at which the step turned o¡. Steady-state current ampli-

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tudes were determined by averaging the last 50 ms of a 125 ms voltage step. Peak currents were determined by eye during computer analysis. In some instances conductance amplitudes are presented along with current amplitudes. The potassium conductance, gK , was calculated to take into account di¡erences in ionic driving forces (resulting from di¡erent [K‡ ]ext ) using the relationship gK = I/Vhold -VK , where VK is the potassium equilibrium potential. Results are presented as mean þ S.D. unless otherwise noted. 3. Results 3.1. Static electrical properties Hair cells isolated from the basilar papilla are the smallest hair cells in the frog auditory system, primarily because they exhibit the shortest cell-body lengths (W20^30 Wm). Although they are probably more columnar in shape in situ, they typically appear spherical upon dissociation. Whole-cell capacitances, measured at the onset of the experiment in voltage-clamp mode, ranged from 4 to 7 pF around a mean of 5.6 þ 1.0 pF (n = 60). Capacitances tended to increase slightly with recording time, in conjunction with a mild visible swelling of the basolateral membrane. We attribute this in part to a shift in the osmotic gradient caused by the replacement of the cytoplasm with pipette solution, and in part to the calcium-dependent fusion of synaptic vesicles with the plasma membrane due to the repeated depolarization of the hair cell as part of the experimental protocol (Parson et al., 1994). Zero-current membrane potentials were measured under current- and voltage-clamp mode shortly after beginning each experiment (both producing similar values for a given cell). Zero-current potentials (Vz ) ranged from 350 to 380 mV, with a mean of 364.9 þ 8.0 mV. This resting potential is consistent with the presence of an inward rectifying potassium current, which actively contributes to the resting potential as an outward current above EK . In both the frog sacculus (Holt and Eatock, 1995) and amphibian papilla (Smotherman and Narins, 1999a) hair cells possessing the inward recti¢er IK1 were found to have resting potentials around 370 mV, while those lacking IK1 rested around 355 mV. As described below, most BP hair cells possess IK1 , although the current density (pA/pF) is typically less in the BP than in the AP or sacculus (Smotherman and Narins, 1999a). 3.2. The voltage-dependent calcium current, ICa A voltage-dependent calcium current could be ex-

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Fig. 1. The voltage-dependent calcium current. A: The voltage-dependent inward current evoked by a series of depolarizing steps in membrane potential from a holding potential of 373 mV, recorded with CsCl replacing KCl in the pipette. B: Steady-state (open circles) currents plotted versus holding potential and peak tail currents (¢lled circles) at 373 mV following steps to the same set of potentials. This steady-state I-V curve is typical of L-type calcium currents. The tail current I-V curve is ¢t by a Boltzmann function (solid line) of the form I = Imax / {1+exp[(Vm 3V1=2 )/k]} using a least-squares criterion. I is the peak amplitude at a given potential, Imax is the peak amplitude with all the calcium channels open, Vm is the holding potential, V1=2 is the holding potential at which half the peak amplitude is evoked, and k is a slope factor describing the voltage dependence of activation. For the curve shown Imax = 3157 pA, V1=2 = 349 mV, and k = 6.2. Hair cell speci¢cations: spherical cell, 4.5 pF whole-cell capacitance, 2.2 M6 uncompensated series resistance (usr), 20 pA leak current (not subtracted) at an interim Vhold of 373 mV.

posed through the external application of 10 mM TEA+5 mM 4-AP, or through the infusion of Cs‡ into the cell from the pipette recording solution. Under these conditions the properties of ICa were studied at potentials positive to the activation range (330 mV) of the inward recti¢er, thus eliminating any potential contributions of IK1 to the net (steady-state) inward current. In 4 mM [Ca2‡ ]ext the exposed inward current was typically less than 100 pA and complete run-down occurred within 5 min, making it very di¤cult to perform a detailed pharmacological assessment of this current. Barium could substitute for calcium as the primary charge carrier, and barium currents were recorded in a subset of cells (5 mM Ba2‡ , n = 4), however, these were not substantially larger than the calcium currents recorded, and run-down occurred just as rapidly. Fig. 1 displays the largest inward calcium current recorded in

a BP hair cell. The peak inward current evoked during a depolarizing step was typically achieved between 323 and 328 mV, similar to that reported for calcium currents of the frog sacculus (Hudspeth and Lewis, 1988; Armstrong and Roberts, 1998) and amphibian papilla (Smotherman and Narins, 1999a). The mean peak steady-state current was 61 þ 33 pA (n = 14). The reversal potential for the steady-state calcium current typically occurred between +5 and +20 mV (Fig. 1), which is well below the calculated ECa of approximately +80 mV, but not substantially di¡erent from values reported in the literature under similar conditions (Ohmori, 1984 ; Hudspeth and Lewis, 1988; Fuchs and Evans, 1990 ; Smotherman and Narins, 1998). The inward current was found to be highly sensitive to external calcium concentrations, increasing by nearly 4-fold when [Ca‡2 ]ext was increased to 20 mM (n = 3).

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Fig. 2. Three outward potassium currents. Depolarizing a BP hair cell from holding potentials positive to 360 mV evokes a non-inactivating outward current in all three examples shown. A hyperpolarizing prepulse potential of 3120 mV for 2 s or longer usually evoked one of two di¡erent inactivating outward currents during the succeeding depolarizing step; either the fast-inactivating IA (A), or the more slowly activating IK (B). Only occasionally both IA and IK were observed in the same cell (C). Protocol shown in A is the same for all three sets of recordings. A: 4 pF, 5 M6 usr, Vz = 357 mV. B: 6.5 pF, 3.6 M6 usr, Vz = 355 mV. C: 5 pF, 4.6 M6 usr, Vz = 373 mV. D: Histogram of the frequency of occurrence of di¡erent activation time constants (dact ) for BP hair cells. Using the same protocol as described above, the non-inactivating component of the outward current evoked at 0 mV was digitally subtracted from the inactivating component, and a single exponential curve was ¢t to the initial rising phase of the waveform. Time constants tended to fall within one of two populations: those currents identi¢ed as Acurrents tended to have activation times of less than 5 ms, while those identi¢ed as K-currents had longer activation times, typically around 10^12 ms. The distribution of di¡erent activation time constants is consistent with the presence of two populations within the BP. Data from hair cells exhibiting both IA and IK were not included in this histogram. The IK shown in B was one of the slowest examples observed, exhibiting an activation time constant of 23 ms.

Additionally, it was found that an inward current remained when calcium was replaced by barium in the external solution (n = 4). When added to the control solution, low concentrations (500 WM to 2 mM) of external cadmium rapidly eliminated the outward, calcium-dependent potassium current (n = 7), and no inward currents were recorded in the presence of cadmium. The threshold for activation of the inward current, as determined by eye from the steady-state I-V curves, typically occurred between 355 and 360 mV; the midpoint of activation (V1=2 determined from Boltzmann ¢ts to tail current amplitudes) was 343.0 þ 2.1 mV (n = 5). Activation was very fast, and was best ¢t by a single exponential curve. The mean time constant of activation, measured at the potential producing the peak steady-state current, was 0.68 þ 0.20 ms (n = 14).

The time course of inward tail current deactivation was best ¢t by a double exponential curve. The mean dfast upon return to a holding potential of 373 mV was 0.35 þ 0.11 ms (n = 14); the dslow was 2.73 þ 0.68 ms (n = 14). The inward current was not observed to inactivate under any conditions. The properties of this ionic current are consistent with those of an L-type calcium current, such as those described in hair cells of the frog sacculus (Hudspeth and Lewis, 1988; Roberts et al., 1990), frog amphibian papilla (Smotherman and Narins, 1999a), and basilar papilla of the turtle (Art and Fettiplace, 1987) and chick (Fuchs and Evans, 1990). This inward current passes calcium and barium ions, and is blocked by cadmium ions. It activates rapidly and exhibits a voltage dependence identical to the calcium current reported in

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other frog hair cells, and it does not exhibit inactivation. The depreciated quality of this current under our recording conditions has prevented us from performing a more detailed pharmacological analysis at this time. However, the evidence presented here is only intended to su¤ciently establish that an inward calcium current is present in these hair cells. We conclude that BP hair cells possess an L-type calcium current which is primarily responsible activating an outward, calcium-dependent potassium current and initiating the fusion of synaptic vesicles at the hair cell basolateral membrane. 3.3. Three di¡erent outward potassium currents in BP hair cells In all hair cells tested, the outward current evoked during a depolarizing step was dominated by a noninactivating calcium-dependent potassium current. Preconditioning the hair cell with a 2^8 s hyperpolarization prior to depolarization usually resulted in the addition of one out of two di¡erent inactivating, voltage-dependent potassium currents. The inactivating currents were separated into two populations based upon their kinetics of activation and inactivation: a fast-inactivating current similar to the A-current described in hair cells of the frog sacculus and amphibian papilla, IA (Hudspeth and Lewis, 1988; Smotherman and Narins, 1999a), and a much more slowly activating and inactivating outward current similar in appearance to the voltage-dependent IK , observed in hair cells of the frog sacculus (Armstrong and Roberts, 1998) and chick and turtle basilar papilla (Fuchs and Evans, 1990; Goodman and Art, 1996a). A small population of hair cells exhibited IA (8/46; the 8 coming from six di¡erent animals) (Fig. 2A). Most hair cells (32/46) were found to possess the slowly activating IK (Fig. 2B). In the rare exception (2/46; two di¡erent animals) we found evidence of both fast and slow inactivating currents in the same cell (Fig. 2C). In six cases we found no evidence of either inactivating current. Fig. 2D quanti¢es the distribution of the two inactivating potassium currents based upon their time course of activation. A histogram of the activation time constant produces a clearly bimodal distribution, and suggests that the observed variation re£ects two kinetically distinct populations, rather than one highly variable population. 3.3.1. The calcium-dependent potassium current, IK…Ca† For the purposes of isolating and characterizing IK…Ca† a 2 s pre-pulse potential of 360 mV or higher could be relied upon to remove all IA , but not all of IK . Four second prepulses, or sustained interim holding potentials as high as 340 mV, would inactivate more than 90% of IK . But in most cases we could not achieve

100% inactivation of IK . This does not hinder the pharmacological characterization of IK…Ca† so long as this fact is taken into consideration during analysis. Fig. 3A illustrates the net current elicited by a series of depolarizing steps in control external solution. From a holding potential of 360 mV, most of IK is inactive, and IK…Ca† dominates the outward current. Yet with increasing depolarization, IK will slowly overcome its inactivation during prolonged depolarizations, adding a slowly activating component to the net outward current. In Fig. 3B, the addition of cadmium to the external recording medium eliminates more than 50% of the outward current evoked during depolarizing steps from a Vh of 360 mV. Cadmium blocks the inward calcium current, and has been shown to rapidly and completely eliminate the calcium-dependent potassium current (Smotherman and Narins, 1999a). The outward current appearing in the presence of cadmium is IK . Cadmium has been observed to cause a positive shift of up to 40 mV in the inactivation range of IA in frog saccular hair cells (M.S. Smotherman, personal observation), and here we found the same e¡ect on IK . Hence, IK now appears activated from a more depolarized holding potential. The kinetics of the outward current recorded in cadmium are considerably slower than those seen in the control solution, supporting the claim that the evoked current is IK . IK…Ca† was found to be sensitive to the external calcium concentration, and IK could be revealed in a zero-calcium recording solution. The sensitivity to external cadmium and calcium concentrations suggests that the non-inactivating outward current is a calcium-dependent potassium current similar to the one identi¢ed on other hair cell preparations. To further test the pharmacological identity of IK…Ca† , the e¡ect of TEA (Sigma, St. Louis, MO) was assessed on the non-inactivating component of the outward current. We found that 1.1 mM TEA routinely eliminated the non-inactivating component (n = 8), but left intact the inactivating outward currents. In contrast, 10 mM 4-AP had no signi¢cant e¡ects on the amplitude or time course of the non-inactivating outward current. The scorpion toxin charybdotoxin (ChTx) is regarded as a selective blocker of the BK-type (and not SK-type) calcium-dependent potassium channels, and has been shown to do so in frog auditory hair cells (Smotherman and Narins, 1999a). We applied 1 WM ChTx to BP hair cells, and found it rapidly blocked the non-inactivating component of the outward current, but not the inactivating component. ChTx has been reported to e¡ect some voltage-dependent potassium currents, however in this preparation as in the frog AP (Smotherman and Narins, 1999a) we observed no e¡ect of ChTx on any of the voltage-dependent potassium currents. At this relatively high concentration ChTx is expected to completely eliminate any contribution of the BK-type

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Fig. 3. The outward current is sensitive to cadmium. The addition of 2 mM cadmium to the external recording solution blocks the inward calcium current and therefore selectively blocks the calcium-dependent potassium current IK…Ca† . In A, a slowly activating outward current can be seen to develop on top of the non-inactivating outward current with increasing depolarization, suggesting that IK inactivation can be overcome by either greater or more prolonged depolarizing steps. In the presence of cadmium (B), approximately half the outward current appears to be removed, revealing an outward current with an initial time course of activation which is much slower than in A. The appearance of IK from this holding potential is an illusion, since it was found that cadmium causes a dramatic depolarizing shift in the activation and inactivation voltage range for this current. C: Initial and peak outward current-voltage relationships are plotted for the data shown in A, and peak outward currents for B, following the addition of cadmium. Initial values from A are presumed to re£ect only IK…Ca† , while peak values incorporate IK . 6 pF, 4.6 M6 usr, Vh = 363 mV between trials. D: Histogram of the frequency of occurrence of gK…Ca† amplitudes for BP hair cells. Conductance amplitudes shown here were calculated from the non-inactivating steady-state current evoked at 320 mV from a (continuous) interim holding potential of 353 mV.

IK…Ca† , and yet even from a holding potential of 350 mV (well above the range of resting potentials observed), a small outward current remains. This may be attributed to either an incomplete block by ChTx or to a small percentage of activated IK . The preferential sensitivity of the non-inactivating outward current to low concentrations of TEA but not 4-AP, its block by ChTx, and its sensitivity to calcium concentration and external cadmium support the conclusion that BP hair cells possess the BK-type calcium-dependent potassium current, IK…Ca† . The kinetics and activation range of IK…Ca† were similar to those reported elsewhere (Hudspeth and Lewis, 1988 ; Smotherman and Narins, 1998, 1999a). The threshold and voltage dependence of activation (Fig.

3C) essentially mirrors that of the inward calcium current. The time course of activation is swift following a brief lag, during which internal calcium levels begin to rise. For BP hair cells, the time to half maximum (T1=2 ) of the outward current varied from 3.0 to 0.5 ms between 350 and 0 mV, and was generally consistent between hair cells. The time course of tail current deactivation could be well ¢t by a single exponential curve. Tail current time constants varied between 0.7 and 4.0 ms (mean 2.4 þ 1.6 ms). Tail currents were frequently obscured by the much slower IK deactivation. It was usually possible to minimize the contribution of IK by preconditioning the cell to potentials more positive than 360 mV, or in some cases IK…Ca† tail currents were analyzed following the digital subtraction of IK during

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Fig. 4. Voltage dependence and kinetics of IA . A: IA was exposed through the application of 5Wg/ml charybdotoxin, a potent IK…Ca† channel blocker, to the external recording solution. The inactivating current shown here is typical of the fast A-current observed in other BP hair cells. A hyperpolarizing prepulse of 2 s at 3120 mV was su¤cient to remove all inactivation prior to the voltage-dependent activation of the current on succeeding depolarizing steps ranging here from 350 to 0 mV. B: Tail currents were collected at a range of membrane potentials increasing from 370 to 335 mV in steps of 5 mV following the large outward current evoked by brief depolarizations to 320 mV from a 3120 mV holding potential. C: Inactivation was assessed by varying the amplitude of the 2 s prepulse potential prior to a 300 ms step to 0 mV. The amplitude of the current evoked is expressed as a percent of the maximum (Imax ) and plotted against the prepulse potential (open circles). Activation was assessed by measuring the peak amplitude of the tail current at 360 mV following brief depolarizing steps (20 ms) from a holding potential of 3120 mV. Activation (¢lled circles), also expressed as a percent of the maximum, is plotted against amplitude of the voltage step. Boltzmann functions were ¢t to both activation (solid line) and inactivation (dotted line) data points: for activation, V1=2 = 334.7 mV, k = 11.6; for inactivation V1=2 = 390.3 mV, k = 6.6. D: A single exponential curve was ¢t to the time course of activation, and the time constants derived from the curve were plotted against step potential. Tail current deactivation was also ¢t with a single exponential curve, and the time constant (dd ) is plotted against membrane potential. 4 pF, 3.7 M6 usr, Vz = 370 mV.

analysis o¡-line. Steady-state current amplitudes, averaged over the second 25 ms of a depolarizing step to 320 mV, varied between 150 and 1500 pA, with means of 564 þ 342 pA in 5 mM [K‡ ]ext , and 631 þ 173 pA in 2 mM [K‡ ]ext . The mean conductance (which allows for a comparison that corrects for di¡erences in VK ) at 320 mV was 7.9 þ 4.0 nS (n = 46; 8.2 þ 5.3 nS in 5 mM [K‡ ]ext , and 7.5 þ 2.1 nS in 2 mM [K‡ ]ext ). The distribution of conductance amplitudes observed in BP hair cells is illustrated in Fig. 3D. 3.3.2. The fast-inactivating potassium current, IA A fast-inactivating outward current identi¢ed as IA has been described in hair cells of the frog sacculus (Hudspeth and Lewis, 1988; Smotherman and Narins, 1999a) and in the low- to mid-frequency region of the leopard frog amphibian papilla (Smotherman and Nar-

ins, 1999a). A thorough pharmacological assessment of this current in the present study was impractical because of its infrequent occurrence in BP hair cells. The identi¢cation of IA in BP hair cells is primarily based upon its activation and inactivation kinetics, which closely match those reported for the frog sacculus and amphibian papilla. Pharmacologically, this current was relatively insensitive to TEA, being exposed rather than blocked by 10 mM TEA (n = 2). It was sensitive to low concentrations of 4-AP: 1 mM 4-AP rapidly removed the fast-inactivating component of the outward current in each cell tested (n = 3). A greater sensitivity to 4-AP than TEA has commonly been used to identify A-currents in hair cells (Hudspeth and Lewis, 1988; Goodman and Art, 1996a). Fig. 4 displays IA exposed through the application of ChTx, with an analysis of its kinetics and ionic sensitivity. The

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Fig. 5. Kinetic and pharmacological analysis of IK . Di¡erent pharmacological blockers of IK…Ca† left intact an outward potassium current with consistent kinetic properties. In the presence of (A) 5 Wg/ml ChTx and (B) 1.1 mM TEA, IK was evoked by depolarizing steps in membrane potential (increasing in 10 mV steps) following a prepulse potential of at least 2 s at 3120 mV. C: Activation (open circles) and inactivation (¢lled circles) were assessed for the hair cell shown in A as described in Fig. 4, except that a 4 s prepulse was used for assessing inactivation. Solid lines represent Boltzmann curves ¢t to each set of data points. For activation, V1=2 = 347 mV, k = 4.7; for inactivation, V1=2 = 379.8 mV, k = 31.4. D: The time course of activation was ¢t with a single exponential curve and the time constant (dact ) is plotted versus step potential. In this ¢gure, a range of time constants is provided for IK isolated in ChTx, TEA, and as determined by digital subtraction of IK…Ca† from the net outward current in the control solution (outward currents evoked from a holding potential of 350 mV were subtracted from the net outward currents evoked from a holding potential of 3120 mV). In each case the approximate range of time constants is similar over the activation range of IK . Each set of data points was best ¢t by an exponential function (solid lines). A: 6.5 pF, 3.6 M6 usr. B: 7 pF, 4.0 M6 usr. C: Same cell as in A. D: ChTx and TEA data from cells shown in A and B; control cell, 5 pF, 6 M6 usr.

threshold of activation varied between 360 and 370 mV, and depolarizing steps to 320 mV or higher resulted in maximum tail current amplitudes. The rapid activation of IA follows a single exponential time course, with a voltage-dependent time constant that varies between 5.5 and 1.0 ms (between 370 and 320 mV, respectively). The activation and inactivation ranges overlap around the Vz of the cell. Inactivation was steep below Vz , with a typical V1=2 of W380 mV, and was completely removed by a v1 s conditioning

step to 3120 mV. In each case, inactivation appeared to follow a single exponential decline, suggesting that for this current a single process accounts for inactivation. Inactivation time constants ranged between 100 and 150 ms at 320 mV. The time course for tail current deactivation was ¢t to a single exponential curve. Deactivation time constants ranged between cells from 20 to 50 ms at the mean Vz of 365 mV. Peak tail current amplitudes reversed at 376 mV (n = 4), which is close enough to EK (384 mV) to support the assumption that

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Fig. 6. Comparing inactivation of IA and IK . Preconditioning steps to 3120 mV of 2^8 s were used prior to depolarization to evoke the inactivating outward currents in addition to IK…Ca† . A and B compare the inactivation rates for IA and IK on proportionally equal time scales. Single exponential curves (solid lines shown for IK ) ¢t well (c = 21 pA) to the time course of inactivation. Inactivation is approximately 10 times slower for IK than IA . By stretching out the time scale for IA to match that of IK (C and D), it appears that these two currents exhibit a very similar process of inactivation.

this conductance passes potassium ions primarily. The peak amplitude of IA , as determined by subtracting the non-inactivating current from the inactivating current evoked from a holding potential of 3120 mV, varied between 50 and 600 pA, with a mean of 238 þ 142 pA (Vh = 0 mV). 3.3.3. The slowly activating potassium current, IK In the majority of hair cells tested, a slowly activating, slowly inactivating, voltage-dependent potassium current could be elicited. Fig. 5 displays IK isolated under two di¡erent conditions, ChTx and TEA. In neither example was there evidence of IA . IK could also be exposed by adding external cadmium. As in the case for IA , cadmium was found to shift both the inactivation and activation voltage ranges of IK by approximately 40 mV positive. In the presence of cadmium IK is not inactivated at a Vh V360 mV, and its threshold of activation is near 330 mV. A hyperpolarizing preconditioning potential is required to evoke a sizable IA or IK in ChTx or TEA, but in the presence of cadmium, a

shift in the inactivation range allows activation from more depolarized potentials. The kinetics of activation appear similar under either condition, which are in general substantially slower than the rates of activation observed for both IK…Ca† and IA . Inactivation was examined with preconditioning voltage steps of 2^8 s. Steps longer than 2 s resulted in smaller k values and slightly depolarized V1=2 s. Inactivation typically reached a plateau within 4 s at 0 mV. Similarly, removal of inactivation appeared complete after approximately 4 s at 3120 mV. The inactivation V1=2 following 4 s preconditioning steps was 378 þ 3 mV (n = 10), which was not observed to change signi¢cantly with longer preconditioning steps (n = 4 at 8 s, and n = 1 examined with 11 s preconditioning steps). Inactivation was voltage-dependent, becoming faster with increasing depolarization. Activation occurred above a threshold of W365 mV, and tail current amplitudes peaked following steps to v330 mV. Activation time constants varied between cells, ranging between 10 and 50 ms at 320 mV. The time course of activation was highly voltage-dependent (Fig.

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Fig. 7. The fast inward-rectifying potassium current, IK1 . A rapidly activating inward current was evoked by hyperpolarizing the cell below EK during a series of holding potentials increasing from 3120 to 370 mV in 10 mV steps (Vh = 360 mV). In A, the current can be observed to exhibit mild inactivation with increasing hyperpolarization. B: Peak and steady-state (the mean of the last 50 ms of pulse) current amplitudes are plotted versus holding potential. 6 pF, 8 M6, usr. In this hair cell, IK1 was studied in the presence of 2 mM cadmium from a holding potential of 360 mV in order to minimize the contributions of other ionic currents. C: Histogram of the distribution of peak current amplitudes of IK1 for BP hair cells recorded during a 200 ms voltage step to 3120 mV in 5 and 2 mM [K‡ ]ext .

5D), but was generally similar whether recorded in control solution or pharmacologically isolated. Around Vz the activation can be expected to occur over a period of hundreds of milliseconds. Deactivation time constants varied between 20 and 60 ms at a holding potential of 360 mV. Tail current amplitudes for IK reversed polarity at a mean potential of 380 þ 4 mV (n = 3), supporting the assumption that IK is a potassium current. In 5 mM [K‡ ]ext , the peak amplitude of IK , taken from the ¢rst 100 ms of a depolarizing step to 0 mV, varied between 100 and 400 pA, with a mean of 185 þ 94 pA. Comparing IK and IA reveals that these two potassium currents are essentially identical in their voltage dependence, ionic selectivity, and pharmacological sensitivity. Only in their kinetics are they di¡erent : IK is approximately 10 times slower than IA in both its activation and inactivation times. Fig. 6 compares the time course of inactivation for examples of IA and IK . Despite di¡erences in their speeds, the shape of the inacti-

vation is very similar (Fig. 6C,D), suggesting a similar underlying mechanism. Thus, it appears reasonable to predict that the underlying ion channels are closely related, and perhaps represent RNA splice variants of the same channel. Indeed, given the inactivation range of IK , this current might be more appropriately named a slow IA . 3.4. The inward-rectifying potassium current, IK1 A fast-activating inward current was evoked upon hyperpolarization to potentials below EK in most BP hair cells (Fig. 7). This current strongly resembles the inward-rectifying potassium current IK1 in the leopard frog sacculus (Holt and Eatock, 1995) and amphibian papilla (Smotherman and Narins, 1998b), and KIR in the turtle BP (Goodman and Art, 1996b). We believe the KIR in turtle BP to be essentially the same as IK1 described in frog sacculus and amphibian papilla. IK1 is

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relatively insensitive to high concentrations of TEA, 4AP and cadmium, but is rapidly blocked by low concentrations of external barium (500 WM). The inward current was larger in 5 mM [K‡ ]ext than in 2 mM [K‡ ]ext , where it was observed in 26 of 28 cells. In 2 mM [K‡ ]ext the current was readily separable from leak current in 11 of 18 hair cells. The steady-state current reversed polarity at 378 and 398 mV in 5 and 2 mM [K‡ ]ext respectively. This current activates below a threshold of W330; mV, and is outward between 330 mV and EK . Tail current amplitudes peak following steps 93100 mV. Activation is typically very fast ( 6 2.5 ms at 3100 mV) and shows little variation between hair cells. IK1 exhibits mild inactivation with increasing hyperpolarization (Fig. 7B), which is a characteristic common to the inward recti¢er IK1 , identi¢ed in saccular hair cells (Holt and Eatock, 1995). Deactivation was well described by a double exponential function, with an initial fast time constant (dfast ) of approximately 0.35 ms, and a larger, slower component (dslow ) of typically 2.3^2.7 ms at Vh = 360 mV. The amplitude of IK1 varied between 50 and 780 pA (Vh = 3120 mV), although most cells possessed currents of 150^350 pA (Fig. 7C). The mean conductance for cells in 2 or 5 mM [K‡ ]ext was 8.3 þ 4.0 nS (n = 11) and 7.0 þ 3.8 nS (n = 26), respectively. 4. Discussion The frog BP is probably the simplest hearing organ among terrestrial vertebrates, and yet the mechanisms underlying its physiology are far from understood. The BP is located in a tubular outpocket of the sacculus, ending in a thin contact membrane separating endolymph from perilymph. The sensory epithelium includes 50^100 hair cells, aligned halfway around the inner surface. Overlying the hair cells is a tectorial curtain which spans approximately half the BP chamber. This organ encodes a bandwidth of frequencies that may begin just above, or for some species several kHz above, the highest frequencies encoded by the amphibian papilla. The frequency selectivity of this organ changes systematically with growth (Shofner and Feng, 1981) and between conspeci¢cs of di¡erent size (Ronken, 1990), and between species of di¡erent size (Megela and Capranica, 1981 ; Zelick and Narins, 1985; Narins and Wagner, 1989; Ronken, 1990), which strongly suggests a mechanical origin for its frequency selectivity. BP a¡erent ¢bers from the same animal all exhibit nearly identical tuning curves. Characteristic frequencies (CFs) vary slightly between animals (Megela and Capranica, 1981 ; Ronken, 1990; Yamada, 1997). Thresholds at CF may span a wide range within the same animal (Ronken, 1990). Unlike the low-frequency

units of the AP, BP a¡erents are not suppressible (Feng et al., 1975). Moreover, BP ¢ber CFs are insensitive to temperature changes (Stiebler and Narins, 1990 ; Van Dijk et al., 1990). The anatomy and physiology of this organ suggest that it owes its frequency selectivity to the gross mechanical properties of the inner ear structures, prior to the stimulation of hair cells, and that very little signal conditioning occurs within the BP sensory epithelium. The nature of the in situ receptor potential is unknown. Electrical tuning, such as that reported in frog saccular and amphibian papillar hair cells, and in reptilian and avian hair cells, is not suspected to contribute to the spectral sensitivity of the BP. Given the lack of specialization within the BP, hair cells of the frog basilar papilla should re£ect the most primitive electrical properties of any terrestrial vertebrate auditory transduction system. The innervation pattern of the BP again re£ects this organ's generalized architecture. Up to ¢ve times as many a¡erent ¢bers as hair cells are present in the sensory epithelium of the BP (Frishkopf and Flock, 1974; Lewis et al., 1982 ; Simmons et al., 1992), resulting in a high degree of divergence. However, a single a¡erent nerve ¢ber may synapse onto as many as seven di¡erent hair cells (Simmons et al., 1992), which means there must be a high degree of overlap between a¡erent ¢bers, with each of 300^500 nerve ¢bers potentially receiving input from up to 10% of the BP's 50 or so hair cells. Within this framework it is not surprising that all BP tuning curves resemble each other. The role of the hair cell in such a system must be limited to mechanoelectrical transduction, which provides us with ideal circumstances for elucidating the roles of ionic currents in simple auditory hair cells. Most BP hair cells possessed nearly identical ionic current compositions: a small inward calcium current coupled to a rapidly activating calcium-dependent potassium current (IK…Ca† ), a slow, voltage-gated potassium current that was mostly inactive at the cells' resting potential (IK ), and an inward-rectifying potassium current (IK1 ) that was active at rest and second only to IK…Ca† in amplitude. The kinetics of ICa and IK1 were identical to their counterparts in the sacculus and amphibian papilla. The kinetics of IK…Ca† were fast, but not as fast as hair cells of the caudalmost AP (Smotherman and Narins, 1999a), suggesting that temporal resolution is not the most critical function of BP hair cells. BP a¡erents are incapable of phase-locking to the ¢ber's CF (Narins and Wagner, 1989 ; Stiebler and Narins, 1990 ; Ronken, 1990), and our data demonstrate that none of the outward currents could track a waveform of 1 kHz or higher. In light of this limitation, it seems likely that receptor potentials in BP hair cells must be dominated by a DC component. BP hair cells are likely to substantially ¢lter out the higher frequency AC com-

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ponents of auditory signals in several places, with lowpass ¢ltering arising from hair bundle dynamics, the membrane time constant, and from delays in synaptic transmission (Weiss and Rose, 1988). The conclusion that BP hair cell electrical properties are not designed for speed is also supported by the presence of IK , the slowest conductance reported for any frog auditory hair cell. The IK identi¢ed here is very similar in both its kinetics and voltage dependence to the IK identi¢ed in frog saccular hair cells (Armstrong and Roberts, 1998), and its role in the sacculus has been compared to that of the IK observed in low-frequency hair cells of the turtle basilar papilla (Goodman and Art, 1996a). Unlike the frog hair cell IK , the IK in turtle BP hair cells is not inactivated at the hair cell's resting potential. Its presence in BP hair cells casts doubt on a frequencyrelated distribution of this ion channel in frogs. It may prove that the presence of IK re£ects an adaptation of frog hair cells within mechanically tuned auditory structures. Due to its inactivating property, potential roles for IK in BP hair cells depend greatly on the in vivo resting potential, which remains unknown at present. IK1 has been shown to hyperpolarize a hair cell's zero-current membrane potential (Holt and Eatock, 1995 ; Smotherman and Narins, 1998, 1999a) and may contribute to the initial depolarization via positive feedback with ICa (Goodman and Art, 1996b). In situ resting potentials well below the threshold for calcium current activation (W355 mV) are supported by the very low levels of spontaneous activity in BP a¡erent ¢bers (Zelick and Narins, 1985; Ronken, 1990). In the frog AP, there is a strong correlation between the presence of IK1 in hair cells and a low spontaneous ¢ring rate in their associated a¡erent ¢bers (Smotherman and Narins, 1999a). It appears that this correlation is maintained in the BP. In vivo recordings of both resting potentials and acoustically evoked receptor potentials are needed before an accurate analysis of the behavior of BP hair cells can be considered complete. The presence of IA is not surprising, since it has been found in all three frog auditory organs. Assigning a functional signi¢cance for IA in hair cells becomes dif¢cult however, when the distribution of this current is considered within the other two auditory organs. IA is found in most saccular hair cells (M.S. Smotherman, personal observation), but it is restricted to the lowest frequency hair cells in the amphibian papilla (Smotherman and Narins, 1999a). Thus in the sacculus and AP, a role for IA in low-frequency electrical band-pass ¢ltering being performed by the hair cell's basolateral membrane is conceivable (as described for IK in turtle hair cells by Goodman and Art, 1996a), but a similar role in BP hair cells would appear unlikely, or at best ine¤cient, for the high frequencies encoded by the BP. If the role of IA in frog auditory hair cells is consistent across

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all three organs, then the distribution of IA is either unrelated or indirectly related to stimulus frequency ; otherwise a changing role of IA must be assumed for any model of hair cell biophysics, which appears less likely given the many general similarities between most frog auditory hair cells (for example stereovillar bundle architectures, and the presence of ICa and IK…Ca† as the predominant ionic currents). This argument applies equally to IK , which has now been reported in frog saccular hair cells (Armstrong and Roberts, 1998). Both IA and IK have been reported in chick cochlear hair cells (Murrow and Fuchs, 1990; Murrow, 1994) and IK has been found in low-frequency hair cells of the turtle cochlea (Goodman and Art, 1996a). The IK in frog BP hair cells is similar to the IK in chick and turtle in all but its inactivation range. The IK inactivation range in the frog is hyperpolarized by as much as 30 mV in comparison to the turtle and chick, and more importantly it is mostly inactive at the hair cells' in vitro resting potential. In this regard IK is similar to IA , which leaves open the possibility that the IK reported here is a modi¢ed form of IA . In the turtle cochlea, IK was selectively distributed across the sensory epithelium (Goodman and Art, 1996a), and in the chick, fast- and slow-inactivating potassium currents are spatially separated across the cochlear sensory epithelium (Murrow and Fuchs, 1990 ; Griguer and Fuchs, 1996). Thus in amphibia, reptiles and birds, fast and slow voltage-dependent potassium currents are selectively, spatially distributed within the epithelium. In conclusion, our investigation into the electrical properties of BP hair cells has revealed what is most likely the general design for primitive auditory hair cells in the terrestrial environment. BP hair cells are the smallest hair cells in the frog auditory system, and possess ionic currents identical to those found in the frog sacculus and amphibian papilla. The ionic current composition of BP hair cells is di¡erent from those of the sacculus and AP, but does not appear specialized in any way to the high-frequency input these cells must encode. It remains to be seen how this collection of ionic currents mediate acoustic transduction and neurotransmitter release in situ. Acknowledgements We thank Drs. D.D. Simmons, C. Bertolotto, A. Purgue, W.M. Yamada and M.L. Garant for their technical assistance and many helpful discussions. We would also like to thank the reviewers for their helpful comments. This work was supported by the National Institute of Deafness and Other Communications Disorders Grant DC00222 to P.M. Narins and a Hyde Fellowship from the UCLA Department of Physiolog-

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ical Science to M.S. Smotherman. All experiments comply with the NIH Principles of Animal Care, Publication 86-23, and all current U.S. laws. References Armstrong, C.E., Roberts, W.M., 1998. Electrical properties of frog saccular hair cells: distortion by enzymatic dissociation. J. Neurosci. 18, 2962^2974. Art, J.J., Fettiplace, R., 1987. Variation of membrane properties in hair cells isolated from the turtle cochlea. J. Physiol. 385, 207^242. Feng, A.S., Narins, P.M., Capranica, R.R., 1975. Three populations of primary auditory ¢bers in the bullfrog (Rana catesbeiana): Their peripheral origins and frequency sensitivities. J. Comp. Physiol. 100, 221^229. Fenwick, E.M., Marty, A., Neher, E., 1982. A patch-clamp study of bovine chroma¤n cells and of their sensitivity to acetylcholine. J. Physiol. 331, 577^597. Frishkopf, L.S., Flock, A., 1974. Ultrastructure of the basilar papilla, an auditory organ in the bullfrog. Acta Otolaryngol. 77, 176^184. Frishkopf, L.S., Goldstein, M.H., 1963. Responses to acoustic stimuli from single units in the eighth nerve of the bullfrog. J. Acoust. Soc. Am. 35, 1219^1228. Fuchs, P.A., Evans, M.G., 1990. Potassium currents in hair cells isolated from the cochlea of the chick. J. Physiol. 429, 529^551. Goodman, M.B., Art, J.J., 1996a. Variations in the ensemble of potassium currents underlying resonance in turtle hair cells. J. Physiol. 497, 395^412. Goodman, M.B., Art, J.J., 1996b. Positive feedback by a potassiumselective inward recti¢er enhances tuning in vertebrate hair cells. Biophys. J. 71, 430^442. Griguer, C., Fuchs, P.A., 1996. Voltage-dependent potassium currents in cochlear hair cells of the embryonic chick. J. Neurophysiol. 75, 508^513. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. P£ugers Arch. 391, 85^100. Holt, J.R., Eatock, R.A., 1995. Inwardly rectifying currents of saccular hair cells from the leopard frog. J. Neurophysiol. 73, 1484^ 1501. Hudspeth, A.J., Lewis, R.S., 1988. Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bullfrog, Rana catesbeiana. J. Physiol. 400, 237^274. Lewis, E.R., Li, C.W., 1975. Hair cell types and distributions in the otolithic and auditory organs of the bullfrog. Brain Res. 83, 35^50. Lewis, E.R., Baird, R.A., Leverenz, E.L., Koyama, H., 1982. Inner ear: Dye injections reveal peripheral origins of speci¢c sensitivities. Science 215, 1641^1643. Megela, A.L., 1984. Diversity of adaptation patterns in responses of eight nerve ¢bers in the bullfrog, Rana catesbeiana. J. Acoust. Soc. Am. 75, 1155^1162. Megela, A.L., Capranica, R.R., 1981. Response patterns to tone bursts in peripheral auditory system of anurans. J. Physiol. 46, 465^478.

Murrow, B.W., 1994. Position-dependent expression of potassium currents by chick cochlear hair cells. J. Physiol. 480, 247^259. Murrow, B.W., Fuchs, P.A., 1990. Preferential expression of transient potassium current (IA ) by `short' hair cells of the chick's cochlea. Proc. R. Soc. Lond. B 242, 189^195. Narins, P.M., Wagner, I., 1989. Noise susceptibility and immunity of phase locking in amphibian auditory-nerve ¢bers. J. Acoust. Soc. Am. 85, 1255^1265. Ohmori, H., 1984. Studies of ionic currents in the isolated vestibular hair cell of the chick. J. Physiol. 350, 561^581. Parsons, T.D., Lenzi, D., Almers, W., Roberts, W.M., 1994. Calciumtriggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells. Neuron 13, 875^883. Roberts, W.M., Jacobs, R.A., Mudspeth, A.J., 1990. Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. 10, 3664^3684. Ronken, D.A., 1990. Basic properties of auditory-nerve responses from a `simple' ear: The basilar papilla of the frog. Hear. Res. 47, 63^82. Ronken, D.A., 1991. Spike discharge properties that are related to the characteristic frequency of single units in the frog auditory nerve. J. Acoust. Soc. Am. 90, 2428^2440. Shofner, W.P., Feng, A.S., 1981. Post-metamorphic development of the frequency selectivities and sensitivities of the peripheral auditory system of the bullfrog, Rana catesbeiana. J. Exp. Biol. 93, 181^196. Simmons, D.D., Bertolotto, C., Narins, P.M., 1992. Innervation of the amphibian and basilar papillae in the leopard frog: Reconstructions of single labeled ¢bers. J. Comp. Neurol. 322, 191^200. Smotherman, M.S., Narins, P.M., 1998. E¡ect of temperature on electrical resonance in leopard frog saccular hair cells. J. Neurophysiol. 79, 312^321. Smotherman, M.S., Narins, P.M., 1999a. Two populations of auditory hair cells in the frog amphibian papilla (submitted). Smotherman, M.S., Narins, P.M., 1999b. Electrical tuning in the frog amphibian papilla (submitted). Stiebler, I.B., Narins, P.M., 1990. Temperature dependence of auditory nerve response properties in the frog. Hear. Res. 46, 63^82. Van Dijk, P., Lewis, E.R., Wit, H.P., 1990. Temperature e¡ects on auditory nerve ¢ber response in the american bullfrog. Hear. Res. 44, 231^240. Weiss, T.F., Rose, C., 1988. Stages of degradation of timing information in the cochlea: A comparison of hair-cell and nerve-¢ber responses in the alligator lizard. Hear. Res. 33, 167^174. Wever, E.G., 1973. The ear and hearing in the frog, Rana pipiens. J. Morphol. 141, 461^478. Wilczynski, W., Capranica, R.R., 1984. The auditory system of anuran amphibians. Prog. Neurobiol. 22, 1^38. Yamada, W.M. III, 1997. Second-order Weiner Kernel Analysis of Auditory A¡erent Axons of the North American Bullfrog and Mongolian Gerbil Responding to Noise. Ph.D. Thesis, University of California, Berkeley, CA. Zelick, R., Narins, P.M., 1985. Temporary threshold shift, adaptation, and recovery characteristics of frog auditory nerve ¢bers. Hear. Res. 17, 161^176.

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