Outer hair cell electromotility: The sensitivity and vulnerability of the DC component

Heurrng Research, 52 (1991) 28X-304 B.V. 037%5955/91/$03.50

288

1 1991 Elsevier Science Publishers HEARES

01532

Outer hair cell electromotility: The sensitivity of the DC component Burt N. Evans, Richard

Hallworth

Auditor?; Physiology Laborator?, (The Hugh Knowles Center) and Department Ewn.~ton, U. S. A. (Received

5 September

1990: accepted

and vulnerability

and Peter Dallos

oJ Neurohiolog): und Physlologv, Northwestern

25 October

Uni~ew~v.

1990)

A technique was devised in order to study the fast electromechamcal length changes of outer hair cells at low stimulus levels. Solitary outer hair cells were drawn into a glass microchamber. Length changes were evoked by the application of transcellular potentials and were detected with a photodiode. The method is non-invasive to the cell and offers superior sensitivity and stability for the recording of cell length changes. The function relating command voltage and cell length (V-6’L) change was determined. A nonlinearity. consisting of a DC component superimposed on the AC response. was shown to he present at the lowest stimulus levels measured. The nonlinearity was sensitive to imposed electrical bias as well as vulnerable to overstimulation. The observations are interpreted in reference to the V-6L function. A parallel is suggested between the nonlinearity seen in the mechanical response and that observed in the responses of the intact cochlea.

Hair cell, outer:

Motility:

Nonlinearity

Introduction One possible problem in assigning a functional role to the fast motility of isolated outer hair cells (Holley and (OHCs) is its very robustness Ashmore, 1988; Patuzzi et al., 1989). Motile responses (length changes) are usually studied in vitro following the trauma of enzymatic or mechanical dissociation. Fast length changes apparently require no messenger substances or metabolic substrates and seem impervious to most drugs or toxins (Kachar et al., 1986; Holley and Ashmore. 1988). In contrast, basilar membrane vibration and auditory nerve responses are well known to be vulnerable to numerous forms of accidental or deliberate manipulations (Robertson, 1974; Rhode, 1978; Salvi et al., 1983; Russell and Cowfey, 1983).

Correspondence to: Burt N. Evans. Laboratory. Northwestern University, Evanston. IL 60208, U.S.A.

Auditory Physiology 2299 Sheridan Road,

It has been previously shown that sinusoidal voltage commands evoke length changes in OHCs which are profoundly nonlinear (Evans, 1988; Evans et al., 1989: Santos-Sacchi, 1989; Evans, 1990). The nonlinearity is manifested as a DC component of the length change in either the contraction or extension direction. As nonlinearities are extremely prominent in almost any measure taken in vivo, it is attractive to consider what role OHC motility may play in the generation of cochlear nonlinearities. The available evidence suggests that the nonlinearities of cochlear responses are more sensitive to manipulation than are the responses themselves. For example, manipulation of the endolymphatic potential (EP) has a profound effect on the summating potential and distortion components as well as on tuning, but affects the AC receptor potentials to a much lesser extent (Honrubia and Ward, 1969; Dallos et al., 1969; Durrant and Dallos, 1972; Sewell, 1984). Similarly, biasing of the cochlear partition by low-frequency sound has small effects on microphonic potentials but

289

dramatically alters summating potentials and tuning of auditory nerve fiber responses (Durrant and Dallos, 1974; Klis and Smoorenburg, 1985; Klis et al., 1988). Hip-intensity acoustic stimulation produces a similar reduction in amplitude and hnearization of both inner and outer hair cell receptor potentials (Cody and Russell, 1988). If the nonlinearity of OHC motility plays a part in the generation of cochlear nonlinearities, then it is reasonable to expect that the nonlinearity of motility would likewise be selectively sensitive to manipulation as well as vulnerable to overstimulation. A novel approach has been developed in order to study OHC motility in vitro (Evans et al., 1989). The method gives superior sensitivity and mechanical stability during recording and enables us to simulate an in vivo-like electrical and ionic environment across the cell. Using this method, we have extended our previous findings to show that the nonlinearity of motility is explicable by the length change function with respect to stimulus voltage. We further show that the nonlinearity is both sensitive to electrical polarization and vulnerable to overstimulation. A number of possible physiological implications of the nonlinear nature of the response are then discussed.

mented by 15 mM HEPES and 0.1% papain (Sigma) and adjusted to pH 6.9-7.0, osmolarity 320 mOsm. Cells were selected for experiments if they showed no obvious signs of damage or deterioration, such as granularity or swelling, and if the diameter was constant and not much larger than the diameter of the nucleus. The cells used in this study ranged from 45 to 80 pm in length. The experiments described here were conducted at room temperature. Microchamber

Isolated OHCs were gently drawn partway into a ~cropipette (Fig. 1) which resembled, in principle, the suction pipette employed by Baylor et al. (1979) for the study of isolated retinal rods. The

Methods Preparation

of cells

Outer hair cells were isolated from whole cochleas of young albino guinea pigs (Charles River) by enzymatic digestion and gentle trituration as previously described (Evans, 1990). A suspension of cells was plated in aliquots into small chambers made from plastic cover slips and maintained near 4°C in a container designed to prevent evaporation. The normal experimental medium was Leibovitz’s L-15 (Gibco), supplemented with 15 mM HEPES and 0.35 mg/ 100 ml BSA (Sigma), and adjusted to pH 7.35, 320 mOsm. In some experichloride ments, 15 mM tetraethylammonium (TEACI, Calbiochem) and 2 mM cobalt chloride were added to the medium and the osmolarity and pH re-adjusted. The enzymatic digestion medium was prepared from a published Leibovitz’s L-15 formula (Gibco), omitting everything except the monovalent ion salts. The solution was supple-

Fig. 1. Photomicrograph of a solitary OHC (a) 50% inserted and (b) fully inserted in a ~cr~hamber.

290

micropipette differed in detail from that used by Baylor and co-workers in that it was much wider away from the aperture so that the cell was free to extend and contract. A superficially similar but narrower suction pipette has previously been used by Gitter et al. (1988) in a study of the passive mechanical properties of OHCs. The suction pipettes in which the experiments were performed will hereinafter be referred to as ‘microchambers’, in part to distinguish them from the two other kinds of micropipettes used in this study. Microchambers were fabricated from pipettes made of borosilicate thin-wall glass tubing (2 mm o.d.) pulled to a long tapered shank on a vertical electrode puller (Narashige). The pipette was scored with a diamond knife and broken off under microscopic observation at a diameter of about 60 pm. The tip was then brought close to a glasscoated platinum filament (A-M Systems) and polished under direct observation to a final inner diameter of 8 pm. The pipette was then bent to 45” at a point approximately 5 mm from the tip and coated with a silane compound (hexamethyldisilane, Sigma) which served to improve the seal and to reduce the likelihood of damage to the cell. Experimental chamber, cell transfer and cell manipulation The microchamber was mounted in an experimental bath on the stage of an inverted microscope (Zeiss). The experimental bath (Fig. 2a) was equipped with a Ag/AgCl ground electrode and with inlet and outlet ports to permit solution changes. The microchamber was mounted in a suction electrode holder and was positioned so that the distal 5 mm lay parallel to and close to the bottom of the experimental bath. The electrode holder contained a Ag/AgCl wire which was connected to the external voltage command generation and current recording circuitry. The suction port of the electrode holder was connected to a micrometer-driven syringe to provide the controlled suction and pressure needed to insert and expel the cell. A separate holding compartment containing dispersed cochlear epithelium was placed in a recess adjacent to the experimental bath. Individual cells were selected from the compartment and transferred to the bath by means of a wide-bore

a

b

R r _

*_

1

‘C-

r

Fig. 2. (a) Diagram of the stimulus generation and current recording apparatus. illustrating the experimental bath, the microchamber and current-to-voltage converter. The inset shows a microchamber in cross-section with an isolated OHC fully inserted. (h) Diagram of the optical length change measurement system (see text for explanation).

suction pipette (about 100 pm diameter) mounted on a triaxial mechanical manipulator. The transfer pipette was maintained air-filled by positive pressure except for a small column of fluid (approximately 100 pl) near its tip. Another pipette was used in the experimental bath to facilitate the manipulation of cells and for maneuvering them into the aperture of the microchamber. This pipette (4 pm i.d.) was fabricated from 1.5 mm borosilicate electrode glass (A.M. Systems. type 6030) pulled on an electrode puller (David Kopf, Model 700) using a two-stage pull similar to that described by Corey and Stevens (1983). The pipette was mounted in a suction electrode holder on a triaxial pneumatic micromanipulator (de Fonbrune).

291

Lmgth changeme~urements Fig. 2b is a diagram of the length change measurement system. The preparation was illuminated from a battery-driven 200W incandescent bulb, the output of which was brought to the microscope condenser C via a light pipe. Light from the condenser passed through the microchamber M and the microscope objective 0. The image of the cell was split along two paths at the binocular head of the microscope. One path was led via an eyepiece E directly to a video camera MC (Panasonic) where it was used to monitor the entire field. The other path was used for measurement of length changes. The image of a cell edge (usually the nuclear pole) passed through an eyepiece E, the optical lever L (see below) and the rectangular slit S. The slit was rotatable so that it could be aligned with the movement axis of the cell. A beam splitter was used to allow part of the light passing through the slit to fall on the photodiode P (PIN-IOD, United Detector Technology). The remainder of the light was passed to a second video camera SC (Javelin) which was used to monitor the position of the cell in the slit. Both video images were simult~eously displayed in a split screen format, and the combined image was recorded on videotape. The entire apparatus was mounted on a vibration isolation table (TMC) and shielded by a Faraday cage. Length changes were measured by the change in the current of a photodiode when the cell image was displaced in a rectangular slit. The output of photodiode P was detected by a current-to-voltage converter A (20 Mf2 feedback resistor). The offset due to the total flux was nulled manually by voltage V, and the result was amplified (10~ x >. The photodiode detector and amplifier, together, had a corner frequency of 600 Hz and a rolloff of 6 dB per octave. With averaging, movement amplitudes as low as 10 nm could be detected routinely. The photodiode output signal was calibrated at the beginning of each run by an optical lever method (Clark et al., 1990). Briefly, motion of the cell was simulated by means of a glass plane which was, at rest, perpendicular to the light path and in front of and parallel to the slit. Rotation of the plane about an axis perpendicular to the slit

translated the cell image along the long axis of the slit by a known amount. The rotation was driven via a stepper motor (Berger-Lahr) under computer control.

Stimulus generation and protocols Length changes were evoked by voltage commands applied between the microchamber and the experimental bath. Voltage commands were generated by means of a programmable stimulus generator (Qua Tech) and applied via a current-to-voltage converter (Yale Mk. V, 100 Mf2 feedback resistor). All voltage command levels are expressed in mV. In our convention, positive voltage commands made the experimental bath positive with respect to the microchamber, implying a depolarization of the basolateral cell membrane inside the microchamber. Both sinusoidal and square-wave stimuli were employed in this study. Sinusoidal stimuli consisted of bursts (100 Hz), presented at approximately 5/s. The square-wave stimulus was a pair of pulses of opposite polarity, 3 ms in duration. Two closely-related pulse paradigms which were also used are described in the legend of Fig. 5. Electrical signals corresponding to length changes and to evoked currents were digitized by a Metrabyte DASH-16F data acquisition board in an IBM compatible computer. The photodiode output signal was low-pass filtered at 600 Hz for the sinusoidal stimuli and 300 Hz for the squarewave stimuli (&pole Bessel, Frequency Devices). Analog data were simultaneously recorded on video tape via a two-channel PCM interface (Instrutech). Responses to sinusoidal stimuli were averaged using a procedure which limited the number of command presentations required to obtain a pre-determined signal-to-noise ratio (B. Clark, in preparation). Therefore, the number of presentations required for each of the response waveforms presented here varied from 32 to a maximum of 128. The microchamber resistance as well as the preparation resistance (resistance of the microchamber with a cell inserted) were measured from the current evoked by 5 mV step commands. Microchamber resistances were in the range of 50% 700 kf2; preparation resistances were 5 to 20 MO.

292

r

Fig. 3. Equivalent circuit of the microchamber with a fully inserted cell. R,,, and R,,, represent the external and mternai membrane resibtances respectively, R,,, the shunt resistance across the sea1 between she microchamber and the cell and R, the series resistance of the microchamber. C,,, and C,,,, represent the external and internal cell membrane capacitances, C{ the applied command potential and y,,, the potential drop evoked by V, across the resistance R,,,,.

Equivulent circuit and model From published studies. it is reasonable to assume that the length change is driven by the tra~smembrane potential change of the cell (Santos-Sacchi and Dilger, 1988). We will refer to the mechanism which controls cell length as the V-M. (Voltage-to-Movement) converter. Fig. 3 shows the equivalent circuit of a cell when inserted in the microchamber. The voltage command V,. is applied across the external and internal membrane resistances (R,,, and R,,,) of the OHC in series, which together form a resistive voltage divider. The effective voltage command to the basolateral membrane, v,,,, is therefore given by Y. R;,,,/( R,,, + R,,,, j. The cell is shunted by the resistive seal between the ~cro~hamber and the cell, R,,?. The total resistance seen by an applied voltage command, the preparation resistance, therefore includes the cell resistance, the shunt resistance, and the series resistance of the microchamber, R , . However, the preparation resistance is lo-30 times the series resistance of the microchamber, R v, hence R, can be neglected, No series resistance compensation was therefore performed. R,,, and R,,,, are both calculated to be larger than the measured preparation resistance (Dallas, 1983), so it is reasonable to conclude that

the preparation resistance is almost entirely that of the seal resistance *, which is in parallel to the series combination of R,.,, and R,,f,. The membrane capacitances C,,,, and C,,, modify the temporal properties of the motile response, but have not been found to be significant in interpreting the results presented here. When the cell is fully inserted into the microchamber, R,,, za R,,,, and the voltage divider ratio approximates the ratio of apical and basal resistances in vivo. R,/R,,, otherwise known as the shape factor, cy (Dallos, 1983). The voltage divider effect of R,,, and R,,,, ensures that only a small fraction of the voltage command is dropped across the basolateral membrane. We compute that the maximum sinusoidal command voltage used in this study (125 mV) results in a relativeIy modest potential drop across the basolateral mem-

* The input resistance of isolated outer hair cells is approximately 25-40 MC? (Ashmore and Meech, 1986). Since this value is the parallel combination of R, and R,, the latter must he larger individually than the input resistance of the ceil.

293

brane *. If the cell is only partly inserted, the applicable voltage divider ratio increases in favor of R,,,. This permits the delivery of larger voltage

commands to the basolateral membrane possible for fully inserted cells.

than is

Results

* The voltage drop across the interior membrane, y:,,, may be computed as a function of the command voltage, V,, and the ratio of interior and exterior membrane resistances. When the cell is fully inserted, the interior and exterior resistances are equal to the in viva apicai and basal resistances R, and R,. If the shape factor a is given by a = RJR, then the potential given by

drop across the interior

resistance,

v,,,, is

The maximum amplitude of sinusoidal command voltage used in this study was 125 mV peak. Assuming a range for a from 0.05 to 0.15 (depending on cell length), the computed maximum driving voltage ranged from 6.0 to 16.3 mV. Around detection threshold for motility (V, = 3.9 mV). the computed driving voltage ranged from 0.19 mV to 0.56 mV.

a

The voltage to length change (V-&L) function of OHC motility As described in the Methods section, the voltage divider formed by the external and internal membranes is a function of the extent to which the cell is inserted into the ~crochamber. In the special case when the cell is inserted 50%, the areas of the external and internal portions of the membrane are equal and the voltage divider ratio is approximately 0.5 (provided the specific resistance of the membrane is constant for everywhere on the cell and LYis small). Thus the stimulus delivered to the basolateral membrane can be calculated with more certainty than if the cell were partially or fully inserted. We took advantage of the above configuration to examine the voltage to length change (VGL)

b

r

0

1

I

1

10 Time

,

20

I

I

30

I

,

40

(ms)

Fig. 4. (a) Superimposed length change responses to square wave pulses at the nuclear pole from a cell inserted 50%. Command voltages were in six equal steps from 40 mV to 240 mV. The waveforms have been smoothed once, using forward and reverse passes of an unwei~t~ 3-point smoother. The scale bar represents 100 nm. The cell length was 67 pm. (b) The peak step responses of the inserted half of the cell plotted as a function of the command potentiai (same cell as in (a)). The fitted curve was obtained to equation 1 by an adaptive procedure using a modified pattern search algorithm (Hooke and Jeeves, 1961). The parameters for the fitted curve are given in Table I.

294

function of OHCs. Isolated cells were examined via step commands and the movements of the nuclear pole were monitored. In order to study the V-M converter isolated from any contribution by nonlinearities due to time-and-voltage dependent conductances in the cell membrane, a series of experiments were performed in L-15 medium containing TEACl and cobalt chloride (Ashmore and Meech, 1986; Santos-Sac&i and Dilger, 1988) (see Methods). An example of the length changes evoked by voltage pulse commands is shown in Fig. 4a. The movements are asymmetric, in that positive commands evoked much larger movements in the contraction direction than did negative commands in the extension direction. The peak step responses for this cell are plotted in Fig. 4b as a function of the command voltage. The rising phase of the cell’s length change is faster than the measurement system can properly resolve, so the length changes measured are likely to be an underestimate of the instantaneous responses. However, in separate experiments (not shown) we have confirmed that the V-SL function determined in this manner can be used to predict the peak amplitudes of responses to sinusoidal stimuli (100 Hz). Fig. 5 shows three examples of V-6L functions obtained from other cells by the same or similar methods. The curves fitted to the data in Figs. 4 and 5 are second-order Boltzmann functions of the form 6L

=

k, 1

+

,hwr~,)[l

+ ,4o-l~“)]

-

The values of the coefficients required to fit the responses in Fig. 5 are given in Table I. In general, we find the values of a to be 3-6 times b, [lo and D, to be both positive and ug to be greater than u,. The V-&L function is nonlinear with a hard saturation in the contraction direction at about + 160 mV and a more gradual saturation in the

k,

where 6L is the evoked length change of the inserted half of the cell, 0 is the command potential, and u, 6, u,), vi, k,, and k, are constants. TABLE

I

THE VALUES OF THE COEFFICIENTS TO EQUATION THE CELLS PRESENTED IN FIGS. 4 AND 5

Curve

Length

a

4 5. a 5. b 5. c

67 75 55 72

U(l

h

WV)

(w0 Fig. Fig Fig Fig

Fig. 5. Three representative examples of V-SL functions obtained by the same or closely similar methods as in Fig. 4. Plot (a) was obtained as for Fig. 4 (pulse durations, sampling rates and filter settings are given in the text). Plot (b) was obtained from the initial peak amplitudes of the responses to 50 ms pulses presented in steps of 12.5 mV (sampling rate 2000 Hz, low-pass filter cut-off frequency 600 Hz). The pulses were presented for each polarity separately, in order of increasing size. Plot (c) was obtained using a series of 5 ms pulses of alternating polarity and increasing stimulus amphtude, from 40 mV to 280 mV. The filter low-pass cutoff was 600 Hz. Plot (b) was also obtained in media containing TEA and cobalt. The data in this figure were obtained from waveforms which were not smoothed. The coefficients of the fitted curves and the cell lengths are given in Table I.

0.049 0.035 0.039 0.021

142.08 102.23 75.65 160.65

0.014 0.010 0.011 0.010

1 WHICH

WERE

REQUIRED

TO FIT THE V-6L FUNCTIONS

1’1

k,

k,

Slope

@VI

(nm)

(nm)

(nm/mV

12.61 14.56 77.94 62.24

328.36 1900.71 611.14 1430.23

240.43 1309.87 425.20 963.42

0.92 4.14 1.73 3.43

TEA/Co

No No Yes No

OF

--

295

contraction direction. As shown in Fig. 6b, both the AC and DC components grow approximately linearly with stimulus amplitude. For a fully inserted cell, the voltage commands applied represent effective stimuli to the cell membrane which are within the physiological range. Assuming a shape factor (Y of 0.05 for a fully inserted cell, the highest level used in this study (V, = 125 mV) would be expected to evoke a potential of 6.0 mV peak across the interior membrane. We commonly find a contraction asymmetry at the lowest stimulus levels employed. Fig. 7 shows the low-level responses for the same cell reptotted on a different scale. At the lowest level used, the stimulus applied transcellularly was 3.9 mV. If a is 0.05,then this stimulus corresponds to 0.19 mV across the inserted membrane. From in vivo measurements in third turn hair cells one can estimate that the peak-to-peak receptor potential at 0 dB SPL is about 0.1 mV at best frequency (Dallos, 1986). Consequently, our lowest command voltages are clearly in the physiological range and are estimated to evoke potentials across the inserted portion of the cell which correspond to near-threshold receptor potentials.

extension direction. The function does not completely saturate in the extension direction, even for applied commands of -200 mV (estimated to be approximately - 100 mV across the internal membrane). The origin or set-point of the function is closer to the extension saturation than to the contraction saturation. A similar form of the function has been inferred from previous experiments (Evans, 1990), and is consistent with our previously-reported observations (Evans et al., 1989). The V-&L function in media containing TEA and cobalt (an example of which is given in Fig. 5b) showed no sig~fic~t differences implying that the function is representative of the V-M (voltageto-movement) converter, and is independent of membrane nonlinearities. The nonlinearity of OHC sinusoidal responses

Fig. 6a shows an example of the length change responses to sinusoidal voltage commands, obtained for a fully inserted cell. The response consists of a phasic, or AC, component in which the positive phase of the command results in cell contraction. Supe~mposed on the sinusoidal responses is a tonic, or DC, component in the

a

b 1000

r

Stimulus Amplitude (mV)

t

-10

r

f

1

1

0

Time

(ms)

I 80

-100

-

1000

Fig. 6. (a) Average length change response waveforms from a 70 pm cell to sinusoidal commands at levels from 125 mV peak decreasing in 6 dB steps. The effective stimulus amplitudes to the cell are calculated to range from 0.19 to 5.95 mV peak, assuming a = 0.05. The scale bar represents 400 nm. (b) Magnitude of the AC and DC response components for the cell in (a).

296

I

’

1

Sensitivity of the DC component to biasing The microchamber method also facilitates the simulation of an electrical bias to the OHC by the addition of a DC pedestal upon which the AC stimulus is superimposed. The effects of bias potentials on the nonlinearity of the length change response were examined for both low and highamplitude commands. It was found that the DC component was selectively sensitive to bias. The direction of the effects depended, however, on the amplitude of the sinusoid as well as on the magnitude and direction of the bias command. Fig. 8a shows the effect of small (90 mV) positive and negative biases on the length change response waveforms for a fully inserted cell. The unbiased sinusoidal stimulus command amplitude was 125 mV. The positive bias resulted in a large increase of the asymmetry in the contraction direction, with a smaller increase in the AC component of the response. A bias of opposite polarity induced a small reduction of the AC response component but resulted in a more severe reduction of the DC component. Fig. 8b shows the

I

’

0

Time

(ms)

Fig. 7. The average responses of the same cell as in Fig. 6 at levels 3.9, 7.8 and 15.6 mV peak, replotted at a larger scale to demonstrate the asymmetry at low stimulus levels. Scale bar represents 50 nm. The waveforms have been smoothed once, using forward and reverse passes of an unweighted 3-point smoother.

a

b Ee1

/ -100

1

la

-60 Step

90

mV

/

j

-20

11

/

20

Command

100

(mV)

, I I / I I , / -100 -60 -20 1 20 4

1,

60

/

60

I

/

100

80

0 Time

(ms)

Fig. 8. (a) Effect of 90 mV (bottom) and -90 mV (top) step bias command potentials on the response of a fully inserted ceil to a 125 mV peak sinusoidal burst. The response to the unbiased stimulus is plotted in the center. The sinusoidal stimulus waveform, sampling rate and filter cutoff frequency were as for Fig. 6. The scale bar represents 50 nm. The cell length was 45 pm. (b) AC and DC components of the cell in (a), for biases from - 90 mV to 90 mV.

291

-140

mV

1 140

0

mV

80

Time

(ms)

Fig. 9. Effects of 140 mV (bottom) and - 140 mV (top) applied bias commands on the responses of a cell to a 125 mV peak sinusoidal command. This cell was inserted 50% and the experimental medium contained TEA and cobalt. The scale bar represents 400 nm. The cell length was 77 pm.

effects of a range of transcellular bias commands from -90 mV to + 90 mV (in 6 dB steps) on the sinusoidal responses of the same cell as in Fig. 8a. The DC component increased by greater than a factor of 3 (10.2 dB) from -90 mV to + 90 mV while the AC response increased by less than 5 dB over the same range. The calculated amplitude of the effective sinusoidal stimulus to the cell membrane was 6.0 to 16.3 mV peak, depending on the assumed value of (Y (0.05 to 0.15). On the same assumptions, the calculated effective amplitude of the 90 mV bias command ranged from 4.3 to 11.7 mV. When the unbiased response is similar in size to the cell’s dynamic range of motility, the length change responses exhibit different properties. Fig. 9 shows the effect of large positive and negative applied bias commands (140 mv) on the responses to large sinusoidal commands (125 mV). In this case, the cell was inserted 50%, so the effective stimulus was much larger than in the previous experiment. In addition, the experimental medium contained TEA and cobalt to block active conductances. A contraction asymmetry was obtained in the unbiased condition. As shown, positive biases reversed the contraction asymmetry towards extension, while negative biases served to increase

the contraction asymmetry. Similar results are obtained in the normal medium without TEA and cobalt. Assuming, as before, that the voltage divider ratio was 0.5, the effective sinusoidal stimulus was 62.5 mV peak and the effective bias voltages were 70 mV. Vulnerability of the DC component to overstimulation It was shown in the previous section that the DC component of the motile response is more sensitive than the AC component to manipulation by electrical biasing. We also find that repetitive stimulation at moderate levels can modify the response. As observed with electrical biasing, the effects of overstimulation are more apparent in the DC component of the response than in the AC component. The result from a typical experiment is shown in Fig. 10. In this experiment, the cell was fully inserted into the microchamber and the motile responses were measured in response to repeated sequences of sinusoidal bursts (62.5 mV peak). The data points represent the AC or DC responses to a sequence of 13 groups of 32 stimuli. Consecutive presentations of the stimulus resulted in a clear loss of the DC response component (nine sequences were required in this case). The AC and DC responses were examined four additional times at intervals up to 2400 seconds after the overstimulation was initiated. As shown, the DC component did not recover over time but continued to lose magnitude. At the end of the monitoring period, the DC component, initially in the con-

AC c

Time

(seconds)

Fig. 10. The AC and DC responses to repetitive presentations of a 62.5 mV peak sinusoidal command. The cell was fully inserted. The cell length was 65 pm.

298

traction direction, reversed to extension. During the same period, the AC component remained relatively unaffected. The resting length of the cell was monitored over the entire period of the experiment and did not change by more than 1 pm (or approximately 1.5% of cell length). The input level used to elicit the loss of the DC component is unlikely to represent a large stimuIus to the basolateral membrane. Using the assumptions discussed in the Methods section, such a stimulus represents a peak amplitude of 3.0 to 8.2 mV membrane potential change, which is within the range seen in outer hair cells at low frequencies (Dallas, 1986; Cody and Russell, 1987). Discussion

The microchamber method The microchamber method, combined with the other procedures, offers several advantages when compared to other methods used to study OHC motility. The microchamber provides a stable platform and locates the cell in the optimum transverse orientation for the measurement of length changes. Performing the experiment in a separate ~omp~tment from the cell suspension results in improved optical clarity and isolation from noise contributed by the Brownian motion of small debris in the cell isolate. The design of the experimental bath permits the exchange of the external solution without unnecessarily subjecting other cells to the experimental treatments. When the bath solution is artificial endolymph and the microchamber is filled with artificial perilymph, the in vivo electrolyte configuration about the cell is simulated. The axial current flow from the bath to the ~cr~hamber also simulates in viva conditions better than either free-field or whole-cell patch stimulation. Further, it becomes possible to simulate the in vivo standing current flow through the cell by polarization, in lieu of an endocochlear potential. Control of the stimulus to the basoIateral membrane, while not as precise as for the patch method, is far more interpretable than for free field in vitro stimulation. More importantly, the stimulus levels can be made unambiguously physiological in magnitude. Finally, it is known that patch clamping depressurizes OH&, which

are naturally turgid (Ashmore, 1987; Holley and Ashmore, 1988) and that reducing the internal pressure can diminish or even abolish the motile response (Holley and Ashmore, 1988). The microchamber method does not puncture the cell, thus preserving the motility mechanism as close to intact as possible. Against these advantages, it must be noted that control of the OHC basolateral membrane potential is not as precise as is sometimes desirable, nor is the exact resting membrane potential of the cell known. It is likely that isolated cells are often depolarized compared to their in vivo counterparts. In fact, Ashmore (1988) and Santos-Sac&i and Dilger (1988) have both pointed to observations consistent with isolated cells being depolarized. However, the consistency of our data, and our repeated observation of a contraction nonlinearity over a large stimulus intensity range suggest that the cells in our preparation are not significantly compromised by depola~zation. It is generally apparent from the cellular responses when this is not the case.

The voltage to iength change (V-&L) function An underlying assumption in these studies is that cell length change is controlled by the transmembrane potential. Previous experiments by Santos-Sac&i and Dilger (1988) demonstrate convincingly that OHC length changes are not controlled by the whole cell current. However, their data do not rule out the possibility that the length change is controlled by a component of evoked current which was left unblocked by their pharmacological regime. The most persuasive evidence that the length changes are in fact controlled by voltage rather than by current is the lack of a reversal potential for the length change observed by us or by other workers (Ashmore, 1987; Santos-Sac&i and Dilger, 1988). Plots describing the length change as a function of membrane potential have been presented elsewhere (Ashmore, 1987; Santos-Sac&i, 1989). Ashmore (1987) showed a curve which was similar to those presented here. However, his curve was symmetrical and the voltage range is approximately double what we have observed. More recently, Santos-Sac&i (1989) presented nine plots of individual cell responses which, in contrast to

299

Ashmore, were asymmetric though essentially linear on both sides of the origin. No saturation in the contraction direction was clearly evident in the plots (although the extension asymmetry response to a sinusoidal burst command at a depolarized holding potential shown for a cell elsewhere in the paper clearly implies that a contraction saturation exists). Also, the extension direction saturation reported by Santos-Sac&i was more abrupt than we have observed. If the Ashmore curve is corrected for series resistance as suggested by Santos-Sac&i, it becomes asymmetrical and more closely resembles our curve. However, unlike our findings, the saturations at either extreme of the Ashmore curve would still be similar to each other. In contrast, while the asymmetry of the SantosSacchi curve corresponds closely to ours, we find it difficult to reconcile the apparent linearity of his curve with our data. At low voltage command levels, the relatively insensitive measurement technique used may account for the failure to observe the curvature of the V-6L function. The slope gains of the functions at the origin for the cells in Figs. 4 and 5 are presented in Table I. They were calculated from the fitted curves, and range from 0.9 to 4.8 nm/mV. These values are directly comparable to the value of 2 nm/mV presented by Santos-Sac&i (1989) since, although they are calculated for the inserted half of the cell only, the command voltage delivered to the membrane is expected to be only half of the applied command. As shown in Table I, we find that the slope can vary significantly. By inspection, much of the variation could be accounted for by differences in cell length. It is not yet clear to what extent the remaining variability may be accounted for by individual cell variability or by damage due to isolation. In the Results section we showed that a second-order Boltzmann equation could be used to fit the V-6L function. The data presented here could also be fitted by fourth or fifth order polynomial equations. However, the polynomial fits did not adequately describe the saturation of the function at extreme voltages. The Boltzmann equation was obtained from a model that describes the microphonic measured from the frog sacculus as a function of otolith displacement (Corey and Hudspeth, 1983), and the relationship

also describes the receptor conductance in turtle hair cells as a function of cilia deflection (Crawford et al., 1989). The underlying theory is that receptor conductance channels exist normally in one of three states, one of which is open and the other two are closed. Energy delivered via cilia stimulation opens receptor conductance channels by modifying two energy barriers which govern the rates of transition between the states. It is tempting to speculate that the resemblance between the two systems may have a functional basis. Elsewhere (Dallos et al., 1991; Hallworth et al., 1990) we present evidence that the mechanism of OHC motility may be considered to consist of a large number of small, independent generators of cell length change, whose contributions add. It is entirely possible that each of these generators may fluctuate between three states. For example, two of the states may be extended (‘long’) and one may be contracted (‘short’). If energy barriers exist between these states, and if the membrane potential were capable of modifying the energy barriers, then a V-6L function similar to what we have observed would very likely be obtained.

The V-6L function indicates cell condition In many cells the voltage to length change function obtained was less asymmetrical than those presented here, that is, the origin was closer to the midpoint of the function. If the mechanism of length change is voltage-controlled, then the origin, or set point, should reflect the resting membrane potential of the cell. It can then be inferred that symmetrical functions are most likely to come from depolarized cells. It can also be inferred that the data discussed in this study do not come from depolarized cells. In the data recently presented by Santos-Sac&i (1989) the contraction saturation voltage under voltage clamp, when observed, appears to occur at about +20 mV membrane potential. As remarked in the Results section, we observe the contraction saturation for commands of about + 160 mV (corresponding to effective voltage commands across the inserted half of the cell of + 80 mv). Thus the resting membrane potential of the cell in Fig. 4 was probably approximately - 60 mV. We will argue later in this discussion that, for the conclusions reached in this

300

paper, an exact knowledge potential is not required.

of the cell’s membrane

OHC motility bus an essential nonlinearity Fig. lla shows an example of a V-6L function (top) closely resembling the ones presented in the

Results section, but with arbitrary units on both the abscissa and ordinate. Underneath is plotted the curvature (second derivative with respect to voltage) of the function. Three points on the function are of special interest. The origin, 0, or set point of the curve, can presumably be equated

b

a

Membrane

1

Potential

d

Fig. 11. (a) Top: hypothetical V-&L function of the V-M converter. Point 0 is the set-point and point H is the mid-point of the function, halfway between the extension and contraction saturations. The point marked M is the point of maximum slope. Bottom: second derivative of the function. The maximum slope occurs where the second derivative is zero. (b) A contraction asymmetry at all stimulus levels is predicted by the function when the set-point is at its normal position. (c) The asymmetry of a small amplitude response is predicted by the curvature around the set-point. The curve represents a small section of the V-8L function around the normal set-point 0. (d) The effect of large biases on large amplitude responses is predicted by the location of the set point with respect to the mid-point of the function.

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with the membrane potential of the cell. We will attempt to show in this discussion that changes in the set point due to biasing or overstimulation can be explained, to a first approximation, by changes in the resting membrane potential. In other words, the simplest explanation of the changes in response waveform is that the function remains unchanged but that the set-point 0 changes. The point marked H represents the approximate midpoint between extension and contraction saturations. The point labelled M represents the point of maximum slope (first derivative) of the transfer function, which is therefore the point of maximum small-signal gain. Note that the maximum slope occurs at a more positive membrane potential than either the mid-point H or the normal set-point 0. We estimate that the point of maximum slope occurs at a point close to 0 mV resting potential. Thus motility apparently does not operate at its maximum gain under normal physiological conditions. It is easy to show that the magnitude and sign of the curvature of the V-6L function uniquely determine the magnitude and direction of the asymmetry for the responses to sinusoidal stimuli. As Fig. lla shows, the curvature is negative around the normal set point, therefore it should not be surprising that a contraction asymmetry was observed in Fig. 7, even for the lowest amplitude stimuli employed. The nonlinearity of OHC electromotility is thus an essential nonlinearity, that is, it is present at the lowest response levels. This conclusion does not require the cell to be accurately maintained at a particular membrane potential; it applies at any set point within the physiological range, since the curvature is non-zero and negative throughout this range. This is demonstrated more clearly in Fig. llb, in which the responses to large-amplitude and small-amplitude stimuli at the normal set-point are compared. Note that the large amplitude asymmetry is also determined by the form of the function, rather than the slope. This will be discussed later in this section. It is also predictable that, around the normal set-point, the DC component of the small-amplitude response is more affected by biasing than is the AC component, since the second derivative of the function is changing faster than the gain. Fig.

llc shows that the effect of biasing on small amplitude sinusoidal responses is predicted by the steadily changing curvature around the normal set point. Positive biases increase the slope and consequently the amplitude and curvature, thus leading to a greater contraction asymmetry, as demonstrated by point 1. Similarly, negative biases decrease both the curvature and the slope, thus decreasing both the amplitude and the asymmetry, as seen by the response at point 2. These waveforms are comparable to the data in Fig. 8a. Note that small amplitude extension asymmetries are not possible around the normal set-point. Small amplitude extension asymmetries are possible only if the cell becomes depolarized past the point of maximum slope. In contrast, the asymmetry of large amplitude responses is determined not by the local curvature but rather by the position of the set point in relation to the midpoint of the function. This is demonstrated in Fig. lld. At set points more negative than the mid-point, large amplitude responses are always contraction asymmetries whereas at points more positive, the responses always exhibit extension asymmetries. This explains the effects of large biases on large amplitude sinusoidal responses, as demonstrated in Fig. 9. It may also explain the effects of overstimulation as observed in Fig. 10. Overstimulation may simply cause depolarization due to trauma, which is then expressed as a change of set-point to a position more positive than the mid-point. Note that while low-level responses for setpoints negative with respect to the point of maximum slope M are always contraction asymmetries, there is a range of possible set-points between H and M where the low level responses would show contraction asymmetries, but the high level responses would result in extension asymmetries. This may explain the high-amplitude extension asymmetry described in Evans et al. (1989) and Evans (1990). In these examples, the cell may have been already depolarized or may have come to that point due to overstimulation, as in Fig. 10. In either case, it is unlikely that the high-amplitude extension asymmetry would occur under normal physiological conditions. Since the voltage to length change function was not measured in these cells, and since some morphological changes de-

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monstrably did occur (at least in Evans, 1990) it remains plausible that the V-&L function in these cells was itself modified in some way. The no~~~~ear~t~of UHC rn~t~~~t~may be an important source of cochlear no~I~~earity While it has become commonplace to consider OHC motility as a possible substrate of the cochlear amplifier (e.g. Ashmore, 1987), this notion has been challenged for a variety of biophysical objections, such as its presumed linearity and low sensitivity (e.g., Mountain and Hubbard, 1989; Hudspeth, 1989). For example, one recent theoretical discussion sought to explain a variety of nonlinear cochlear phenomena and pathologies by assigning the fundamental nonlinearity solely to the OHC transduction process (Fatuzzi et al., 1989). This position was reached by elimination: motility was dismissed as unimportant at low stimulus levels because the responses were assumed to be both linear and unaffected by trauma, based on previously-reported voltage-clamp studies. In fact, neither of these assumptions is correct, It is demonstrated in this paper that motility is nonlinear at stimulus levels corresponding closely to the lowest detectable sounds in vivo. Furthermore, we have demonstrated that the no~nearity is sensitive to biasing as well as vulnerable to overstimulation. Small depolarizations can have significant impact on the DC component of the motile response, even when its AC magnitude is little affected. Whereas the relationship of the nonlinea~ty of motility and cochlear nonlinearities are presently unknown, it is now clearly inappropriate to rule out a potential contribution of motility to cochlear nonlinearities on the basis that it is either linear or invulnerable. It is interesting to note that the apparent labile nature of the motile response has not been reported from voltage clamp studies (Ashmore, 1987; Santos-Sac&i, 1989). It may be the case that the vulnerability of the motile response does not lie in the V-M converter itself, but is a consequence of cell depol~tion. As the voltage clamp imposes its own polarization on the cell, it might therefore be predicted to obscure any tendency for such changes to occur.

The relationship of motility to cochleur function The observation that the DC component of motility is both sensitive to bias and vulnerable to oversiimulation leads to a comparison with the cochlear amplifier, which appears to have similar properties. It is plausible that experimental manipulations which compromise the nonlinearity and sensitivity of the cochlea do so by modifying the resting membrane potential of OHCs, thereby adjusting the set point of the V-SL function. For example, loss of sensitivity and tuning in IHC receptor potentials due to acoustic overstimulation has been shown to occur concomitantly with depolarization of OHCs, by a few mV (Cody and Russell, 1985). The membrane potential change in OHCs observed by Cody and Russell is similar in magnitude (3-4 mV) to what would be expected to occur after loss of the EP (Dallos, 1983), which also causes a decrease in sensitivity and tuning in primary afferent discharges (Sewelf, 1984). There is a clear analogy between loss of a 90 mV positive transcellular bias (the endolymphatic potential) in vivo and the application of a 90 mV negative transcellular bias in our experiments, as in Fig. 8. In either case, the effect on the nonlinearity of motility is much more prominent than on the amplitude of the motile response. Similarly, other manipulations which cause a decrease in cochlear sensitivity and tuning (efferent stimulation and acoustic biasing) might also be reasonably expected to affect the membrane potential of OHCs, and therefore the nonlinea~ty of the response, The need for caution in motility studies One final note is that future studies should pay close attention to the possibility of inducing overstimulation in vitro, The stimuli used in the vulnerability experiments (Fig. IO) evoked peak length changes of only a few hundred nm. This size of length change is visually undetectable at the magnifications typically employed for motility studies. Yet, even at these amplitudes, profound and irreversible changes were observed in response to repetitive stimulation. It thus appears n~essary to study OHC electromotility at response levels commensurate with estimated in vivo displacements of the organ of Corti. This way, it is more

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likely that the full expression of the response may be preserved. Acknowledgements

We thank King-wai Yau, William Brownell, and Brian Clark for helpful discussions, Brian Clark for programming, and Albert Farbman for the glass knives. We acknowledge the thorough and perceptive comments of one of our anonymous reviewers. This work has been supported by NIH grants DCOO089, DC~708 and NS07223, and the American Hearing Research Foundation. References Ashmore, J.F. (1987) A fast motile response in guinea-pig outer hair cells: The cellular basis of the cochlear amplifier. J. Physiol. 388, 323-347. Ashmore, J.F. (1988) Ionic mechanisms in hair cells of the mammalian cochlea. In: W. Hamann and A. Iggo (Eds.), Progress in Brain Research. Vol. 74. Elseviet, Amsterdam, pp 3-8. Ashmore. J.F. and Meech, R.W. (1986) Ionic basis of the resting potential in outer hair cells isolated from the guinea pig cochlea. Nature 322, 368-371. Baylor, D.A., Lamb, T.D. and Yau, K.-W. (1979) The membrane current of single rod outer segments. J. Physiol. 288, 589-611. Clark, B.A., Hallworth, R. and Evans, B.N. (1990) Calibration of photodiode measurements of cell motion by a transmission optical lever method. Pfltigers Arch. 415, 490-493. Cody, A.R. and Russell, I.J. (1985) Outer hair cells in mammalian cochlea and noise-induced hearing loss. Nature 315, 662-665. Cody, A.R. and Russell, I.J. (1987) The responses of hair cells in the basal turn of the guinea-pig cochlea to tones. J. Physiol. 383, 551-569. Cody, A.R. and Russell, I.J. (1988) Acoustically induced hearing loss: Intracellular studies in the guinea pig cochlea. Hear. Res. 35, 59-70. Corey, D.P. and Hudspeth, A.J. (1983) Kinetics of the receptor current in bullfrog saccular hair cells. J. Neurosci. 3, 962916. Crawford, A.C., Evans, M.G. and Fettiplace, R. (1989) Activation and adaptation of transducer currents in turtle hair cells. J. Physiol. 419, 405-434. Dallas. P. (1983) Some electrical circuit properties of the organ of Corti: I. Analysis without reactive elements. Hear. Res. 12, 89-119. Dallas, P. (1986) Neurobiology of cochlear inner and outer hair cells: intracellular recordings. Hear. Res. 22, 185-198. Dallos. P., Evans, B.N. and Hallworth, R.J. (1990) Nature of

the motor element in el~tro~netic shape changes of cochlear outer hair cells. Nature (in press). Dallas, P., Schoeny, Z.G., Worthington, D.W. and Cheatham, M.A. (1969) Co&ear distortion: effect of direct-current polarization. Science 164, 449-451. Durrant, J.D. and Dallos, P. (1972) Influence of DC polarization of the cochlear partition on the summating potentials. J. Acoust. Sot. Am. 52, 542-552. Durrant, J.D. and Dallos, P. (1974) Modification of DIF summating potential components by stimulus biasing. J. Acoust. Sot. Am. 56, 562-570. Evans, B.N. (1988) Motile response patterns and ultrastructural observations of the isolated outer hair cell. Thesis, University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences, Houston, Texas. Evans, B.N. (1990) Fatal Contraction: Ultrastructural and electromechanical changes in outer hair cells following transmembraneous electrical stimulation. Hear. Res. 45, 265-282. Evans, B.N., Dallos, P. and Hallworth, R.J. (1989) Asymmetries in motile responses of outer hair cells in simulated in uioo conditions. In: D. Kemp and J.P. Wilson (Eds.) Mechanics of Hearing, Plenum, New York, pp 205-206. Gitter, A.H., Rudert, M. and Zenner, H.-P. (1988) Cell clamp technique to measure the compliance of mammalian cochlear outer hair cells in vitro. Pfliigers Arch. 412, R 75 Suppl. 1. Hallworth. R., Evans, B.N. and Dallas, P. (1990) Outer hair cell electromotility: Localization and dist~bution of the force-generating mechanism. Sot. Neurosci. Abstr. 16,1078. Holley, M.C and Ashmore, J.F. (1988) On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea. Proc. R. Sot. B232, 413-429. Honrubia, V. and Ward, P.H. (1969) Mechanism of production of cochlear microphonics. J. Acoust. Sot. Am. 47, 498-503. Hooke, R. and Jeeves, T.A. (1961) Direct search solution of numerical and statistical problems. J. Assoc. Comp. Mach. 8, 212-229. Hudspeth, A.J. (1989) How the ear’s works work. Nature 341. 397-404. Kachar, B., Brownell, W.E., Altschuler, R. and Fex, J. (1986) Electrokinetic shape changes of cochlear outer hair cells, Nature 322, 365-368. Klis, J.F.L. and Smoorenburg, G.F. (1985) Modulation at the guinea pig round window of summating potentials and compound action potentials by low frequency sound. Hear. Res. 20, 1523. Klis, J.F.L., Prijs, V.F., Latour, J.B., and Smoorenburg, G.F. (1988) Modulation of cochlear tuning by low-frequency sound. Hear. Res. 36, 163-174. Mountain, D.C. and Hubbard, A.E. (1989) Rapid force production in the cochlea. Hear. Res. 42, 195-202. Patuzzi, R.B,, Yates, G.K.. and Johnstone, B.M. (1989) Outer hair cell receptor current and sensorineural hearing loss. Hear. Res. 42. 47-72. Rhode, W.S. (1978) Some obse~ations on cochlear mechanics. J. Acoust. Sot. Am. 64, 158-176.

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