The voltage dependence of the mechanoelectrical transducer modifies low frequency outer hair cell electromotility in vitro

Hearing Research 113 (1997) 133^139

The voltage dependence of the mechanoelectrical transducer modi¢es low frequency outer hair cell electromotility in vitro Andrei N. Lukashkin, Ian J. Russell * School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK

Received 21 February 1997; revised 25 June 1997; accepted 18 July 1997

Abstract

The fast outer hair cell (OHC) electromotility is voltage dependent and is driven by changes in the OHC transmembrane potential. Those changes include the receptor potential generated by the variable conductance of the mechanoelectrical transducer (Evans and Dallos, 1993). In the experiments described here, we show that the voltage dependence of the mechanoelectrical transducer influences the low frequency motile responses of OHCs to an external electrical field. OHCs were fully inserted into a glass suction pipette, the microchamber, so that only the cuticular plate and hair bundle were exposed to the bath solution. With this technique, a rectification of the mechanical response, equivalent to an excitatory displacement of the hair bundle, was observed when the command voltage inside the microchamber depolarized the apical membrane. The shape of the response persisted when the OHC voltage-gated conductances were blocked. Following treatment of the hair bundle with BAPTA or dihydrostreptomycin, which are known to impair transduction function (Assad et al., 1991; Kroese et al., 1989), rectification of the motile response disappeared. Keywords :

Cochlea; Outer hair cell; Hair cell transducer; Electromotility; Cochlear ampli¢er

1. Introduction

It is commonly accepted that the sensory function of the cochlea is mediated by the inner hair cells (IHCs), which have multiple a¡erent innervation (Spoendlin, 1988). The motile outer hair cells (OHCs), having rich e¡erent innervation, have been proposed to provide mechanical feedback to the cochlea (Davis, 1983). In this scheme, OHCs in£uence the micromechanics of the cochlea partition to provide both the gain and the compression of cochlear responses which enables the cochlea to function as a sensitive, highly tuned frequency analyzer with an impressive dynamic range. OHCs are capable of changing their length when the potential across the OHC membrane changes (Brownell et al., 1985; Ashmore, 1987; Santos-Sacchi and Dilger, 1988). In situ, the receptor potentials produced by the OHCs in response to acoustic stimulation are believed to control the OHC's voltage-dependent mechanical re* Corresponding author. Tel.: +44 (1273) 678632; Fax: +44 (1273) 678433; E-mail: [email protected]

sponse. OHC electromotility has been observed not only under the action of intra- or extracellularly applied electrical ¢elds but also under mechanical stimulation of the stereocilia (Evans and Dallos, 1993). In Evans and Dallos's experiments, the OHC body was fully inserted into a microchamber so that the basolateral and apical membranes created a voltage divider. In this electrical circuit, the motile response was driven by changes in the intracellular potential, which, in turn, were modulated by the variable mechanoelectrical transducer conductance. Consequently, the intrinsic properties of the mechanoelectrical transduction process may a¡ect OHC electromotility. One of these intrinsic properties is the voltage sensitivity of the mechanoelectrical transducer conductance which has been demonstrated for di¡erent hair cell types including OHCs (Assad et al., 1989; Crawford et al., 1989 ; Kros et al., 1993). We have adopted the microchamber technique, with the OHC fully inserted, to provide a way of observing the dependence of the fast OHC electromotility on the mechanoelectrical transducer. An important aspect of this technique is

0378-5955 / 97 / $17.00 ß 1997 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 7 ) 0 0 1 3 5 - 4

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A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139

134

that the apical and basolateral membrane conductances of the OHC are arranged in series just as they are arranged in vivo (Fig. 1a). This is not the case with the whole-cell technique where the conductance of the basolateral membrane shunts the transducer conductance or in microchamber experiments when the OHCs are only partially inserted into the microchamber. In the experiments described here, we have studied how

the

voltage

sensitivity

of

the

mechanoelectrical

transducer conductance, whereby the open probability of the transducer channels increases with OHC depolarization, may in£uence the low frequency responses of OHCs to an external electrical ¢eld. Measurements of the movement of the synaptic pole of the OHC during sinusoidal voltage changes across the cell were made for two cases. Firstly, for the intact hair bundle and secondly, when the mechanoelectrical transducer conductance was eliminated with blockers.

2. Materials and methods

Guinea pigs were anaesthetized with pentobarbitone. After cervical dislocation their temporal bones were removed. OHCs were isolated non-enzymatically or after enzymatic incubation (1 mg/ml of type IV collagenase, Sigma, Poole, Dorset) by pipetting gently the organ of Corti which had been dissected from the cochlea. OHCs were maintained in an extracellular solution which contained (mM) : 140 NaCl, 0.7 NaH2 PO4 , 5.8 KCl, 1.3 CaCl2 , 0.9 MgCl2 , 5.6

D-glucose,

10 HEPES, pH 7.4.

Vitamins and amino acids for Eagle's Minimal Essential Medium were added from concentrates (Gibco, Paisley, Strathclyde). The osmolality was near 320 mosmol/kg. The modi¢ed extracellular solution which was used to

Fig. 1. a : Elementary electrical circuit of an OHC inserted into the microchamber. Resistance of the cell's apical pole transducer. voltage-

Resistance

and

of

the

time-dependent

basolateral

Ra

includes the

Rb

membrane

Rs

conductances.

is

the

block the OHC voltage-gated conductances contained

ance. The resistance of the microchamber in series with

(mM) : 115 NaCl, 5.8 KCl, 1.3 CaCl2 , 10

is not shown because it is relatively insigni¢cant.

D-glucose,

5

CsCl, 20 TEA-Cl, 1 CdCl2 , 10 HEPES-NaOH, pH 7.4. Vitamins and amino acids were not added to the modi¢ed

solution

to

avoid

precipitation

of

the

cadmium

tive force of the basolateral membrane.

Vc

provide

estimates

of

voltage

sensitivity

Rb

is the electromo-

is the command voltage

of

conductance contained 5 mM BAPTA or 200

ducer

of the BAPTA and the dihydrostreptomycin solutions

resist-

and

The insert shows the equivalent electrical circuit which was used to

Here the resistance of the OHC apical pole

drostreptomycin (Sigma, Poole, Dorset). The osmolality

Ra

applied between the bath solution and the microchamber solution.

compounds. The solution for eliminating the transducer

WM dihy-

E

includes

shunt

resistance

membrane

Rc .

Rtr

in

parallel

with

the

the

Ra

OHC

transducer.

consists of the trans-

resistance

of

the

apical

b : Photodiode response as a function of the displace-

ment of the image of a 5 normalized to the 1

were near 320 mosmol/kg and 300 mosmol/kg respec-

Wm

Wm

diameter glass ¢ber. Responses are

¢ber displacement.

tively and with a pH of 7.4. BAPTA or dihydrostreptomycin were added to the extracellular solution with-

noic acid) (Sigma, Poole, Dorset) to prevent mechanical

out

damage the cells and to improve the resistance of the

vitamins

and

amino

acids.

All

experiments

were

U

6

performed at room temperature (22³C). The cells were

seal (up to 5 M

observed with a top-focusing microscope, using a 40

same extracellular solution which was used to maintain

water immersion objective.

Microchambers for holding the OHCs were pulled

). The microchamber contained the

OHCs, i.e. normal solution or solution with blockers of the

voltage-gated

conductances,

respectively.

The

from borosilicate glass to produce tips with aperture

OHC's bodies were sucked gently into the microcham-

diameters of about 30

ber

then

¢re

polished

to

Wm.

The microchambers were

aperture

diameters

of

8^9

Wm

and coated with linoleic acid (cis-9, cis-12-Octadecadie-

up

to

the

cuticular

plate

so

that

only

the

cell's

apical pole with the hair bundle was in the bath solution (Fig. 1a). Experiments were carried out only on

HEARES 2890 28-11-97

A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139

135

OHCs without any visible sign of trauma or damage of

grabber, Image Nation, Beaverton, OR, USA) before

the hair bundle.

and

during

The

absolute

Transcellular

voltage

signals

were

applied

to

the

the

application

value

of

the

of

an

cell's

electrical length

stimulus.

change

was

OHCs between the bath solution and the microchamber

found by subtracting these two images with a software

solution (Fig. 1a) with a laboratory constructed volt-

package

age-clamp ampli¢er. According to our convention, pos-

quence

itive command voltages corresponded to a positive volt-

the

age

found from the ratio between the photodiode responses

inside

the

microchamber.

This

hyperpolarizes

the

cell's basolateral

the

Rb ,

of

resistance,

believed the

to

apical

be pole

command

much

the basal pole less

(Evans

voltage,

positive

Vb ,

et

than al.,

which

the

resistance,

1991), drops

the

part

across

Ra , of

the

Ra

of the

cell's

and

Rb

values

the of

Seattle,

linearity length

WA,

of

the

changes

USA).

As

photodiode

for

other

a

conse-

response,

stimuli

were

for those stimuli and the calibrated response. Solutions

for

eliminating

the

transduction

process

were applied locally to the hair bundle through a glass pipette with a tip of 50

Wm

diameter. The £ow of the

solution was driven by gravity. Polystyrene latex beads (1.1

Wm

diameter, Sigma, Poole, Dorset) were added to

the solutions to visualize the £ow. The microchamber

WV RR c

Since

(see Fig. 1a) is

basolateral membrane is de¢ned by the ratio

Vb

voltage

membrane.

(Optimas, of

b a

… 1†

with the inserted OHC was ¢lled with solution kept under slight positive pressure to avoid any contamination of the basolateral membranes when BAPTA and

where

Vc

is the command voltage applied between the

dihydrostreptomycin were added to the solution bath-

bath solution and the microchamber solution. The cell's

ing

motile responses were measured using a pair of photo-

OHC

the

apical

surface.

diodes (OSD15-5T, Centronics). The image of the cell's

perfusion pipette from the experimental bath when the

synaptic pole was projected onto one of the photodio-

extracellular

des. The other photodiode was used for di¡erential sub-

OHC responses modi¢ed by dihydrostreptomycin were

traction of light and electrical noise. The ¢ltered output

measured during continuous perfusion of the hair bun-

from the photodiodes was digitized and stored for later

dle with the dihydrostreptomycin containing solution.

responses

were

solution

In

experiments

measured

had

been

after

with

BAPTA,

removal

changed

for

of

the

normal.

computer analysis. The linearity of the photodiode response was con¢rmed by registering the movement of the image of a glass ¢ber driven by a piezo electric

3. Results

bimorph (Fig. 1b). In order to ¢nd the absolute value of cell's length change the image of the cell's synaptic

When fully inserted into microchamber, OHCs dem-

pole was projected simultaneously onto a CCD camera

onstrate a recti¢cation of their extension responses, i.e.

(SSC-M370CE, Sony, Japan) and written (CX frame

with hyperpolarization of the basolateral membrane, as

Fig. 2. Response waveforms of an OHC to a sinusoidal command voltage of 10 Hz before (upper row) and after (lower row) application of BAPTA to the hair bundle. The amplitude of the command voltage is indicated for each column. The response waveform is almost symmetrical for small voltages, but the recti¢cation is clearly apparent for large command voltages in the absence of BAPTA. Cell length : 48 bars : 300 nm and 200 ms.

HEARES 2890 28-11-97

Wm.

Scale

A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139

136

inside the microchamber, hyperpolarization of the basolateral membrane may turn o¡ these conductances. This should lead to an increase of the basolateral membrane resistance

Rb

and to an increase in the cell's mechanical

response. In order to test these two hypotheses we have carried out experiments with blockers of the voltagegated and mechanoelectrical transducer channels. Local perfusion

of

the

hair

bundle

with

BAPTA

solution

(Figs. 2 and 3a) lead to a considerable decrease in the recti¢cation which is associated with positive voltage inside

the

microchamber.

During

these

experiments

similar recti¢cation, and its reduction after BAPTA application, was obtained in solutions containing voltagegated

channels

access

of

blockers

BAPTA

to

(Fig.

the

3b).

apical,

By

restricting

sensory

pole

of

the the

OHCs, the only e¡ect of BAPTA perfusion should be the

irreversible

elimination

of

the

mechanoelectrical

transduction process (Assad et al., 1991 ; Crawford et al., 1991). Similar reduction in the recti¢cation was observed during perfusion of the hair bundle with dihydrostreptomycin cause

a

(Fig.

reversible

4).

Dihydrostreptomycin

blockade

of

the

can

mechanoelectrical

transducer channel (Kroese et al., 1989) and it was possible to observe partial recovery of the recti¢cation after washing out the dihydrostreptomycin (Fig. 4) but not Fig. 3. Mechanical responses for a fully inserted OHC as a function of command voltage. a : Measured in normal extracellular solution, cell length :

35

Wm ;

b:

measured in solution with blockers of the

Wm.

voltage-gated channels, cell length : 40

Open circles represent re-

sponses with intact hair bundle and solid circles represent responses after

application

of

BAPTA.

BAPTA

was

washed

out

with

the

after washing out the BAPTA. Residual recti¢cation of the response was occasionally observed after BAPTA or during dihydrostreptomycin perfusion. This may have occurred

because

mechanoelectrical

we

were

not

transducer

able

eliminate

the

completely.

The probability of the transducer being opened is in-

2‡

bathing solution before measurements were made.

to

conductance

creased in solutions with low Ca

concentration (Ha-

can be seen in Fig. 2. One can see from Eq. (1), when

cohen et al., 1989). However, this e¡ect could not be

Vc

responsible for modifying OHC electromotility in our

is held constant, the voltage drop across the baso-

lateral membrane and, therefore, the amplitude of the

increasing e¡ects.

Rb .

One

Ra

experiments

because

OHC

voltage-dependent

electro-

or

motility was measured only after the extracellular sol-

Two possibilities might account for these

ution containing BAPTA had been replaced with a nor-

cell elongation can be increased with decreasing

could

mechanoelectrical

be

the

voltage

transducer.

brane is depolarized

when

Ra

is hyperpolarized and

the

mal solution. It was sometimes possible to observe a

apical mem-

decrease in the slope of the electromotile response for

dependence

The OHC

of

the basolateral membrane

can be decreased because a

greater proportion of the mechanoelectrical transducer channels will be opened with depolarization of the apical

membrane

(Assad

et

al.,

1989 ;

Crawford

et

al.,

1989 ; Kros et al., 1993). However, it is possible that the observed recti¢cation of the curve when the voltage inside the microchamber is positive, may be generated not only by the voltage dependence of the mechanoelectrical transducer but also by the voltage-gated conductances of the basolateral membrane. For example, the OHCs may be depolarized slightly after the isolation

procedure

voltage-gated

(Evans

channels

et of

al.,

1991)

the

and

basolateral

part

of

the

membrane

(Ashmore and Meech, 1986 ; Santos-Sacchi and Dilger, 1988 ;

Housley

and

Ashmore,

1992 ;

Santos-Sacchi,

1992) may be activated. Therefore, with positive voltage

Fig. 4. E¡ect of dihydrostreptomycin on mechanical responses of a fully inserted OHC. Open circles represent responses with the intact hair bundle and solid circles represent responses during application of

dihydrostreptomycin.

Triangles

show

responses

after

10

min

washout of dihydrostreptomycin with the normal extracellular solution. Cell length : 44

HEARES 2890 28-11-97

Wm.

A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139

137

the largest positive command voltages (Fig. 4). This ef-

been observed for cochlear hair cells, not only for the

fect could be due to weak saturation of the electromo-

steady-state transducer current ^ displacement relation-

tility function. The saturation is most apparent when the

ships, when the voltage dependence can be attributable

elongation

blockers

to the adaptation process, but also for the peak trans-

(Fig. 4). We found it di¤cult to measure a sharp satu-

ducer current ^ displacement relationships which have

ration of the electromotile responses, as has been de-

been measured in the ¢rst few milliseconds of stimulus

scribed earlier for the OHCs in microchamber (Evans

onset, before the start of adaptation (Kros et al., 1993).

et al., 1991 ; Hallworth et al., 1993). This is probably,

In this case, true voltage sensitivity of the mechanoelec-

because only a small fraction of the command voltage is

trical transducer channels may contribute towards the

dropped across the basolateral membrane in our experi-

e¡ect observed in our experiments. Another source of

ments (see Section 4). Of the 34 OHCs tested, only 15 did

voltage dependence, that due to the voltage-dependent

not demonstrate a recti¢cation of the mechanical re-

sti¡ness of guinea pig OHC hair bundles, has been de-

sponse when the inside of the microchamber was made

scribed recently (Zhang et al., 1997). The steady-state

positive. Despite any visible sign of damage, the OHC

position of the hair bundle depends on the vector sum

mechanoelectrical

of

recti¢cation

was

abolished

transducer

appeared

with

to

have

been

compromized by the isolation procedure in these cases.

all

forces

applied

to

the

stereocilia

(Howard

and

Hudspeth, 1988), and consequently, any change in the sti¡ness of the hair bundle will lead to changes in gating spring tension and, in turn, to changes in the number of the open transducer channels.

4. Discussion

The electromotile responses of OHCs under voltage When OHCs are fully inserted into the microcham-

clamp

have

been

¢tted

with

sigmoidal

curves

which

ber (Evans et al., 1989 ; Evans and Dallos, 1993) it is

demonstrate fast saturation in the direction of extension

possible to observe aspects of the fast OHC's electro-

when the cell's membrane potential was held near

motility

mechanoelectrical

mV (Ashmore, 1987 ; Santos-Sacchi, 1989). If the fast

these

OHC

which

transducer.

This

depend is

upon

because,

the

under

conditions,

electromotility

is

controlled

only

by

the

3

70

mem-

the apical and basolateral membrane conductances of

brane potential, as a consequence of the non-linearity

the OHCs are arranged in series, just as they are in the

of the electromotile response, it should be possible to

organ of Corti in vivo (Fig. 1a).

observe some kind of recti¢cation during both elonga-

In the present report, the electromotility of OHCs,

tion and extension, depending upon the value and sign

when fully inserted into the microchamber, became rec-

of the OHC membrane potential. However, this form of

ti¢ed when the command voltage inside the microcham-

recti¢cation is not the basis of that observed in our

ber was positive, i.e. during hyperpolarization of the

experiments.

basolateral membrane and depolarization of the apical

was unknown for the OHCs in our experiments, but

membrane. The recti¢cation survived block of the volt-

it is not essential for understanding the basis of the

age-gated

the

recti¢cation of the electromechanical response described

sensory apical pole of the OHC was exposed to BAPTA

in this paper. This is because the voltage drop across

and dihydrostreptomycin, agents which are known to

the basolateral membrane

interfere with mechanoelectrical transduction (Kroese et

the command voltage for an OHC fully inserted into

al., 1989 ; Assad et al., 1991). Since recti¢cation of the

the microchamber. A value

OHC mechanical response when the inside of the mi-

the ratio of the membrane areas of excluded and in-

crochamber was positive depends on the mechanoelec-

cluded segments of the OHC as a ¢rst approximation

trical transducer, we may conclude that voltage depend-

of the voltage divider

ence

it

of

conductances

the

transducer

but

was

abolished

channels

may

when

modify

the

is

The

estimated

exact

that

resting

Vb

potential

is only a small fraction of

Rb /Ra

Rb /Ra only

membrane

can be found from

(Evans et al., 1991). Thus,

1/23^1/19

of

the

command

electromechanical response of the OHCs in vitro. The

voltage

mechanism of the voltage dependence of the mechanical

for the OHCs shown in Figs. 3 and 4. The recti¢cation

response described in this paper is a matter for specu-

of

lation. Only low frequency electrical signals have been

marked

used in these experiments and the voltage dependence

the potential change across the basolateral membrane

could be due to a number of mechanisms with relatively

is only about 4^5 mV. This change is too small to drive

slow time courses. For example, it is known that adap-

the electromotility into saturation where recti¢cation of

tation of the mechanoelectrical transducer channels in

the extension response would occur. For this to happen,

bullfrog (Hacohen et al., 1989) and turtle (Crawford et

the OHC membrane would have to be depolarized to

al., 1989) hair cells disappears when the apical mem-

about +20 mV (Santos-Sacchi and Dilger, 1988 ; San-

brane is depolarized, thus reducing the driving force

tos-Sacchi, 1989) and it is not possible to achieve this

for the calcium ions. However, an increase in the trans-

value of membrane potential under the conditions of

ducer

our experiments. The ratio

conductance

with

hair

cell

depolarization

has

the

HEARES 2890 28-11-97

is

dropped

elongation for

a

across

the

responses

command

basolateral

of

these

potential

Rb /Ra

of

membrane

OHCs 100

is

quite

mV,

when

can also be calculated

138

A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139

using the data of Kros et al. (1992) for the mechanoelectrical transducer conductance of OHCs of the cultured mouse cochlea (we use a value 8 nS for the total transducer conductance in our calculations) and for the part of the conductance activated at the rest (8% of the maximal value). The speci¢c conductance of the OHC's membrane can be obtained from the data of Housley and Ashmore (1992). In this case the resistance of the cell's apical pole Ra consists of the mechanoelectrical transducer resistance Rtr and the resistance of the apical membrane Rc connected in parallel (see insertion in Fig. 1). These two resistances are connected in series with the basolateral membrane resistance Rb . This way of estimating Rb/Ra provides slightly larger values of 1/ 17^1/15 for the ratio for the OHCs illustrated in Figs. 3 and 4. However, even in this case, the actual voltage change across the basolateral membrane is only about 6^7 mV for a 100 mV command voltage. The electrical circuit in Fig. 1, together with Eq. (1), can be used to provide estimates of the voltage sensitivity of the transducer. Let us assume that, for zero command voltage, the mechanoelectrical transducer is approximated by the resting transducer conductance (Kros et al., 1992) and that elongation of the OHC when the inside of the microchamber is positive is due also to the voltage sensitivity of the mechanoelectrical transducer. Then, for the three OHCs shown in Figs. 3 and 4, the mechanoelectrical transducer conductance would have increased by 0.6, 0.9 and 0.3 nS respectively for a command voltage of 100 mV, which is equivalent to depolarizing the apical membrane by about 5 mV. According to Kros et al. (1992) the mechano-sensitivity of the mechanoelectrical transducer to small displacements of the hair bundle from the resting position is 0.03 nS/ nm. Therefore, a 5 mV depolarization of the apical membrane is equivalent to an excitatory displacement of 0.5^1.5 nm for the hair bundles of the OHCs shown in Figs. 3 and 4.

the OHC electromotility, which were measured in the solution containing 1.3 mM Ca2‡ , may be more pronounced in situ where the Ca2‡ concentration of the endolymph is only 20 WM (Boscher and Warren, 1978). Hair cell depolarization also leads to the displacement of the hair bundles of frog and turtle hair cells (Crawford and Fettiplace, 1985; Assad et al., 1989; Crawford et al., 1989; Assad et al., 1991). This displacement was proposed (Assad et al., 1989) to represent a transition of the hair bundle to another resting position corresponding to an increased number of open transducer channels. However, voltage-dependent displacement of OHC bundles has yet to be observed. Passive displacement of the OHC hair bundles has been observed both in single hair cells and in isolated preparations of the organ of Corti as a result of an active tilting of the hair cell's cuticular plate during exposure to alternating external electrical ¢elds (Zenner et al., 1988; Reuter and Zenner, 1990). Therefore, the angular rotation of the OHCs hair bundles and tension in the gating springs in vivo may be governed by the shear motion of the reticular lamina and tectorial membrane, by the voltage dependence of the OHC mechanoelectrical transducer and, possibly (but see Holley, 1996) by the tilting of the cuticular plate. The co-operative outcome of these e¡ects on the angular rotation of the hair bundle will establish the actual state of the mechanoelectrical transducer and, accordingly, the receptor potential and the electromotile response of the OHC in vivo. The experiments of Russell and Koëssl (1991) can give an idea of complexity of this interaction. These authors observed voltage-dependent shifts of the IHC and OHC mechanoelectrical transducer functions in vivo which cannot be explained either by voltage dependence of the mechanoelectrical transducer (Assad et al., 1989; Crawford et al., 1989; Kros et al., 1993) or by tilting of the cuticular plate (Zenner et al., 1988; Reuter and Zenner, 1990).

4.1. Functional signi¢cance of the voltage dependence in vivo

4.2. Voltage dependence and cochlear non-linearity

The voltage dependence of the mechanoelectrical transducer has been described for di¡erent types of hair cells. Depolarization increased the number of open transducer channels for hair cells in the turtle cochlea (Crawford et al., 1989), the bullfrog vestibular system (Assad et al., 1989) and OHCs of the mouse cochlea (Kros et al., 1993) thus leading to a shift of the mechanoelectrical transducer function to the left along the axes of displacements. Voltage induced shifts of the transducer function depend strongly on the concentration of extracellular Ca2‡ (Hacohen et al., 1989; Crawford et al., 1991) around the apical pole of the hair cell and become more pronounced when the Ca2‡ concentration is lowered. Thus, the voltage dependence of

Patuzzi et al. (1989) and Santos-Sacchi (1993) proposed that the mechanoelectrical transducer function generates a dominant non-linearity which could distort the OHC's electromotility in vivo. Experimental and model investigations have con¢rmed that the non-linearity of the hair cell mechanoelectrical transducer can give rise to non-linear phenomena, such as distortion products and two-tone suppression with patterns similar to those which have been recorded from the peripheral auditory system (Weiss and Leong, 1985; Frank and Koëssl, 1996; Lukashkin and Russell, 1997). It is known that non-linear systems can change their behavior crucially with slight changes in their parameters (e.g. see Andronov et al., 1966). For example, slight changes of the mechanoelectrical transducer operating point, i.e.

HEARES 2890 28-11-97

A.N. Lukashkin, I.J. Russell / Hearing Research 113 (1997) 133^139 shift

the

mechanoelectrical

transducer

function

along

the axes of displacements for several nanometers, could modify the pattern of the distortion products and twotone suppression (Weiss and Leong, 1985 ; Frank and

139

Evans, B.N., Dallos, P., 1993. Stereocilia displacement induced somatic motility of cochlear outer hair cells. Proc. Natl. Acad. Sci. USA 90, 8347^8351. Frank, G., Ko ë ssl, M., 1996. The acoustic two tone distortions 2f1-f2 and f2-f1 and their possible relation to changes in the gain and the

Ko ë ssl, 1996 ; Lukashkin and Russell, 1997). On the oth-

operating point of the cochlear ampli¢er. Hear. Res. 98, 104^115.

er hand, hair cell depolarization a¡ects not only the

Hacohen, N., Assad, J.A., Smith, W.J., Corey, D.P., 1989. Regulation

mechanoelectrical channels are

transducer

open at

rest)

operating

but also

point

other

(more

transducer

parameters (Assad et al., 1989 ; Crawford et al., 1989 ; Kros et al., 1993). Therefore, the voltage dependence of the mechanoelectrical transducer is one of the factors which determine the speci¢c type of non-linearity of the cell's mechanical response and, consequently, the speci¢c type of active non-linear cochlear function.

of tension on hair-cell transduction channels : displacement and calcium dependence. J. Neurosci. 9, 3988^3997. Hallworth, R., Evans, B.N., Dallos, P., 1993. The location and mechanism of electromotility in guinea pig outer hair cells. J. Neurophysiol. 70, 549^558. Holley, M.C., 1996. Outer hair cell motility. In : Dallos, P., Popper, A.N., Fay, R.B. (Eds.), The Cochlea. Springer, New York, p. 421. Housley, G.D., Ashmore, J.F., 1992. Ionic currents of outer hair cells isolated from the guinea-pig cochlea. J. Physiol. (London) 448, 73^98. Howard, J., Hudspeth, A.J., 1988. Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels

Acknowledgments

in the bullfrog's saccular hair cell. Neuron 1, 189^199. Kroese, A.B.A., Das, A., Hudspeth, A.J., 1989. Blockage of the trans-

This work was supported by the MRC. A.N.L. is a Wellcome Prize Research Student. The authors thank

duction channels of hair cells in the bullfrog's sacculus by aminoglycoside antibiotics. Hear. Res. 37, 203^218. Kros,

C.J.,

Ru ë sch,

A.,

Lennan,

G.W.T.,

Richardson,

G.P.,

1993.

James Hartley for designing and constructing the elec-

Voltage dependence of transducer currents in outer hair cells of

tronic

neonatal mice. In : Duifhuis, H., Horst, J.W., van Dijk, P., van

equipment

and

Matthew

Holley

and

Guy

Ri-

chardson for their critical reading of early versions of

Netten, S.M. (Eds.),

Biophysics of

Hair

Cell

Sensory Systems.

World Scienti¢c, Singapore, pp. 141^150.

this manuscript.

Kros, C.J., Ru ë sch, A., Richardson, G.P., 1992. Mechano-electrical transducer currents in hair cells of the cultured neonatal mouse

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