Electrical properties of frog saccular hair cells: distortion by enzymatic dissociation

Abstract

Although it is widely accepted that the electrical resonance seen in many types of auditory and vestibular hair cells contributes to frequency selectivity in these sensory systems, unexplained discrepancies in the frequency (f) and sharpness (Q) of tuning have raised serious questions. For example, enzymatically dissociated hair cells from bullfrog (Rana catesbeiana) sacculus resonate at frequencies well above the range of auditory and seismic stimuli to which the sacculus is most responsive. Such disparities, in addition to others, have led to the proposal that electrical resonance alone cannot account for frequency tuning. Using grassfrog (Rana pipiens) saccular hair cells, we show that the reported discrepancies in f and Q in this organ can be explained by the deleterious effects of enzyme (papain) exposure during cell dissociation. In patch-clamp studies of hair cells in a semi-intact epithelial preparation, we observed a variety of voltage behaviors with frequencies of 35-75 Hz. This range is well below the range of resonant frequencies observed in enzymatically dissociated hair cells and more in tune with the frequency range of natural stimuli to which the sacculus is maximally responsive. The sharpness of tuning also agreed with previous studies using natural stimuli. In contrast to results from enzymatically dissociated hair cells, both a calcium-activated K+ (KCa) current and a voltage-dependent K+ (KV) current contributed to the oscillatory responses of hair cells in the semi-intact preparation. The properties of the KCa and the Ca2+ current were altered by enzymatic dissociation. KV and a small-conductance calcium-activated K+ current were apparently eliminated.

Fig. 1.

Scanning electron micrograph illustrating the semi-intact epithelial preparation. A beveled piece of hair was used to plow a furrow through the saccular epithelium (A), revealing basolateral aspects of hair cells (B). Scale bars: A, 50 μm;B, 5 μm.

Fig. 2.

Current-clamp recordings showing stereotypical electrical resonance from an enzymatically dissociated hair cell (A) and the variations seen in the semi-intact epithelial preparation (B–D), evoked by depolarizing current steps (top trace) of amplitudeI_stim. A, Damped sinusoidal voltage oscillations (I_stim = 225 pA;f = 185 Hz; Q = 15.3;V_rest = −86 mV). B, Damped oscillations (I_stim = 100 pA;f = 59 Hz; Q = 2.9;V_rest = −91 mV). C, Oscillations that gave way to spike-like behavior (I_stim = 125 pA; f = 53 Hz; V_rest = −86 mV). D, Sawtooth (arrow) at the onset of the voltage response (I_stim = 175 pA; f = 45 Hz; V_rest = −65 mV). Resting potentials (V_rest) were measured immediately before the step. All recordings were made using perforated patch.

Fig. 3.

Average current-clamp behaviors of enzymatically dissociated cells (□; n = 40) and hair cells in the semi-intact preparation (•; n = 50).A, Frequency–current relationships. Not all cells oscillated at every current amplitude tested. B, Voltage–current relationships. Membrane potential was measured either as the steady-state voltage at the end of the current step or, when the oscillations lasted for the duration of the step, as the average potential around which the cell oscillated. Both panels show mean ± SD.

Fig. 4.

Effects of 1 mm 4-AP and 6 mm TEA on current-clamp recordings from enzymatically dissociated hair cells. A, Resonance recorded in normal extracellular solution (control:I_stim = 200 pA; f = 225 Hz; V_rest = −72 mV) was not affected by 4-AP. B, TEA reversibly eliminated resonance (control, solid line: I_stim = 200 pA; f = 222 Hz;V_rest = −52 mV). Oscillations recovered fully when TEA was removed (recovery, dotted line). Both recordings were made using perforated patch. The records inA are the average of five presentations.

Fig. 5.

Effects of 1 mm 4-AP and 6 mm TEA on current-clamp recordings from hair cells in the semi-intact epithelial preparation. A, Voltage oscillations recorded in normal extracellular solution (control, solid line: I_stim = 225 pA;f = 82 Hz; V_rest = −70 mV) were eliminated by 4-AP and partially restored when 4-AP was removed (recovery, dotted line). B, TEA transformed the damped sinusoidal oscillations found in this cell (control, solid line: I_stim = 200 pA; f = 67 Hz; V_rest= −94 mV) into spike-like oscillations. This effect was partially reversed when TEA was removed (recovery, dotted line). Recordings from both cells were made using perforated patch. The traces in A are the average of five presentations.

Fig. 6.

Effects of TEA, Ibtx, and 4-AP on outward currents in enzymatically dissociated hair cells. Essentially all of the outward current recorded in normal extracellular solution (control) was eliminated by 6 mm TEA (A) or 2 μm Ibtx (B). Application of 1 mm 4-AP (C) had no effect. The panels to the right show the I–Vrelationship for each cell before (□) and during (•) drug application. Currents for I–V plots were calculated at the end of each 50 msec step. All recordings were made using a whole-cell configuration and from a holding potential of −70 mV. For each family of traces, the largest amplitude voltage step (corrected for series resistance errors) is indicated next to the step protocol. For clarity, some of the data traces used to construct theI–V plots are not shown.

Fig. 7.

Effects of TEA, Ibtx, and 4-AP on outward currents from hair cells in the semi-intact epithelial preparation. Application of 6 mm TEA (A) or 1 μmIbtx (B) blocked a fast component of the outward current, leaving a more slowly activating component. 1 mm4-AP (C) blocked a slow component of the outward current, leaving a rapidly activating outward current that partially inactivated. Nearly all outward current was eliminated by applying both 1 mm 4-AP and 6 mm TEA (D). The panels to the right show the I–V relationship for each cell before (□) and during (•) drug application (see Fig. 6 legend). The recordings in B were made using perforated patch; the others (A, C, D) were made in the whole-cell configuration.

Fig. 8.

TEA-, 4-AP-, and Ibtx-sensitive currents (dark traces) in hair cells in the semi-intact epithelial preparation obtained by subtracting the currents after drug application (smaller-amplitude light traces) from the currents in control conditions (larger-amplitude light traces). A, Current blocked by 6 mmTEA activated rapidly and partially inactivated. B, One micromolar Ibtx blocked a similar component of the current.C, One millimolar 4-AP blocked a slowly activating current. Because of series resistance errors in the step potential it was necessary to estimate the current at −20 mV by linear interpolation of currents from adjacent voltage steps. These are the same cells as in Figure 8A–C.

Fig. 9.

Papain transforms voltage oscillations and outward currents in the semi-intact epithelium in situ. Traces from continuous perforated patch recordings taken before (control) and after perfusion of papain solution onto the epithelium. A, After 15 min in papain, the frequency of the voltage oscillations recorded in current-clamp increased from 36 Hz to 164 Hz. (I_stim = 200 pA; V_rest = −72 mV in control and −91 mV after papain treatment). Traces are the average of 10 presentations.B, In another cell, the papain solution transformed the outward currents from those typical of hair cells in the semi-intact epithelial preparation (control) into currents typical of enzymatically dissociated hair cells (after 21 min in papain). The time constant for decay of the tail current after voltage steps to −36 mV decreased from 6.5 msec before enzyme treatment to 4.4 msec. The largest amplitude voltage step (corrected for series resistance errors) is indicated next to the step protocol.

Fig. 10.

Isolation of Ca²⁺currents. All recordings, except for C, were made with Cs⁺ internal solution. A, A sag in the inward current was evident in hair cells in the semi-intact preparation but not in enzymatically dissociated hair cells (B). C, NMG internal solution blocked the sag. The corresponding voltage steps (corrected for series resistance errors) are indicated by each trace (A–C). D, Peak Ca²⁺ current I–Vrelationships from enzymatically dissociated hair cells (□,n = 12) and hair cells in the semi-intact preparation (•, n = 10). E, Ca²⁺ current activation kinetics (τ_m, see text for definition) versus membrane potential (symbols are the same as in D: □,n = 11; •, n = 7). All recordings were made in the whole-cell configuration with 10 mm extracellular Cs⁺ to eliminate the activation of a K_IR current during the leak pulses. Values in D and E are mean ± SEM.