Auditory Neuroscience, 1995, Vol 1, pp. 309-319 Reprints available directly from the publisher Photocopying permitted by license only 1995 Harwood Academic Publishers GmbH

Printed in Singapore

Salicylate Ototoxicity: Effects on the Stiffness and Electromotility of Outer Hair Cells Isolated from the Guinea Pig Cochlea

IAN RUSSELL and CORNELIA SCHAUZ

School o f Biological Sciences, University of Sussex, Falmer, Brighton, BN19QG, United Kingdom.

(Received November 12 ,1994, accepted February 23,1995)

The axial stiffness and the forces produced by the voltage-dependent motility of outer hair cells iso­lated from the guinea pig cochlea were determined by measuring nanometer displacements of a fine glass fiber of known stiffness in a bathing solution of Eagle's minimal essential medium with Hank's salts. For outer hair calls with a mean length of 60 (im, the compressive stiffness along the long axis of the cell was 0.755 mN/m. An inverse relationship was found between outer hair cell length and axial stiffness; the shorter outer hair cells were stiffer. Outer hair cells that were partially sucked into the tip of a micropipette underwent sinusoidal length changes when a transcellular sinusoidal voltage was applied between the pipette and bath solu­tions. The electromotile forces generated by the outer hair cells with a mean length of 59 (im were estimated to be 4.1 pN/jiV. A direct correlation was found between outer hair cell length and the forces they could develop. Both the axial stiffness and the electromotile forces were reduced by about 65% when the outer hair cells were perfused with 5 mM salicylate solution that was adjusted for pH. It was concluded that salicylate-induced changes in the axial stiffness and electromotile force generation of the outer hair cells could account for the salicylate- induced desensitization and frequency shift of the tip of the tuning curve and the enhanced sensitiv­ity of the tail of displacement tuning curves of the basilar membrane, which have been measured in situ.

Key words: Cochlea, outer hair cell, salicylate, electro- motilty, mechanics

Corresponding author: Prof. Ian Russell, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN19 QG, United Kingdom.

THE INGESTION of acetyl salicylic acid or aspirin, can induce reversible reductions in auditory sensitivity and selectivity, changes in otoacoustic emissions, and tinni­tus. There are several routes through which aspirin might interfere with audition. The effects on tinnitus are thought to be due to interaction with afferent transmis­sion at the inner hair cell (IHC) synapse and more cen­trally (see Jastreboff and Sasaki, 1994 for a recent review.) The reversible induction of deafness and alterations in acoustic distortion indicate outer hair cell (OHC) in­volvement and the results from animal experimentation reveal that salicylate acts at several sites within the cochlea, including the OHCs, with conflicting actions on the tone-evoked responses. Administration of salicylate, either intravenously or through scala tympani perfu­sion, increases spontaneous rates in Vlllth nerve fibres (Evans and Borerwe, 1982) or reduces asynchronous neural activity recorded at the round window (Meikle and Chamell, 1994), decreases the amplitude of the Vlllth nerve compound action potential (CAP) and ele­vates the threshold of the CAP (Puel et al., 1989,1990; Stypulkowski, 1990; Fitzgerald et al., 1993). Reports of the action of salicylate on the cochlear microphonic (CM) are conflicting. Depending on the route of administra­tion, the animal species and the level and frequency of the auditory stimulus, salicylate can either have no ef­fect or reduce the CM (Silverstein et al., 1967; McPherson and Miller 1974; Puel et al, 1990) or enhance CM (Stypulkowski, 1990; Fitzgerald et al., 1993). Similarly, both salicylate and aspirin administration can either en­hance or decrease acoustic distortion (Stypulkowski, 1990; Kossl, 1992; Fitzgerald et al, 1993; Brown et al,1993). Perfusion of the scala tympani with either 2.5 or 5 mM salicylate causes a reversible broadening, down­ward frequency shift, and decrease in the sensitivity of the tip of basilar membrane (BM) displacement iso-re sponse tuning curves by up to 40 dB, which is accom­panied by a 10 dB sensitization of the low-frequency tail of the tuning curve (Murugasu and Russell, 1995). The effects of salicylate on the tip of the BM tuning curve are compatible with salicylate, causing a decrease in posi­tive mechanical feedback to the cochlear partition and point to the OHC motor as the site of action. Negative feedback from the OHCs has been proposed to mini­

309

310 I. RUSSELL and C. SCHAUZ

mize the shear displacement between the tectorial mem­brane and the cuticular plate in the region of the IHC stereocilia for frequencies less than one-half octave below the tip of the tuning curve (Mountain et al., 1983; Kossl and Russell, 1992; Neely, 1993). A decrease in OHC negative feedback would account for the sensitization of the tails of neural tuning curves following selective damage to the OHCs (Liberman and Dodds, 1984). In experiments on OHCs isolated from the mammalian cochlea, salicylate has been known to cause reversible reductions in the voltage-dependent capacitance and motility of the OHCs (Ashmore, 1989; Shehata et al., 1990, 1991; Tunstall et al., 1994). If, as has been proposed, the voltage-dependent motility of OHCs forms the basis of the positive feedback to the cochlea (Brownell et al., 1985; Ashmore, 1987), then the action of salicylate on OHCs would account for the desensitization of the tip of the BM tuning curve but not necessarily the sensitization of the low-frequency tail of the mechanical tuning curve unless salicylate also caused an increase in compliance of the cochlear partition. Indeed, salicylate has been shown to cause a reversible reduction in the turgidity of OHCs (Shehata et al, 1990,1991). In the experiments re­ported here we have investigated the action of salicylate on the axial stiffness of the OHCs and the forces gener­ated by the OHC motor to see if changes in these two parameters might account for the salicylate-induced changes in the mechanical properties of the cochlear par­tition, which have been measured in situ.

MATERIALS AND METHODS

Preparation and Experimental Procedure

Adult pigmented guinea pigs (200-300 g) were killed by an overdose of sodium pentobarbitol. After cervical dis­location their temporal bones were rapidly removed. The bulla was opened, the cochlea was relieved of its bony shell, and the stria vascularis was removed to ex­pose the organ of Corti. The organ of Corti from the first2.5 turns (0.035-4 kHz; Greenwood, 1990) was carefully detached from the BM, and the coils of the organ ware transferred with a pipette to a small Perspex chamber. The cells were isolated nonenzymatically by trituration through an Eppendorf pipette tip (10-100|il). The dis­section and the experiments were carried out in Eagle's minimal essential medium with Hanks' salts (pH 7.5 os­molality 322 m osmol H). The osmolality of the 5 mM sodium salicylate in Hank's balanced salt solution (HBSS) was 322 m • osmol • I-1 and the pH was adjusted to 7.5 with NaOH. The dissection yielded large numbers of birefringent cells. Experiments were carried out only on OHC's that appeared undamaged. That is, they did not undergo reversible length changes, were relatively constant in diameter, the nucleus was not displaced and the organelles did not jitter about. The lengths of the OHCs used in this experiment were between 41 and 84

Hm and they were 9 to 11 (am in diameter. The volume of the fluid in the chamber (500 (il), and the osmolality of the fluid was kept constant by addition of small amounts (10 jal) of distiled water to compensate for water loss through evaporation in early experiments. The chamber was continuously perfused with airated solutions at 250 |il/ min in later experiments. The vol­ume of fluid in the chamber could be replaced in about 10 s. The experiment was carried out at room tempera­ture (22°C) in a sound-attenuated booth and all mea­surements reported in this article were carried out within 4 hours of the beginning of the experiment. The cells were viewed at approximately 600 times magnifi­cation through a top focusing Zeiss ACM microscope equipped with Nomarski optics and with a 40 water im­mersion objective. Measurements of the axial stiffness and displacement responses to applied electrical stim­ulation were made from single OHCs selected from the disaggregated organ of Corti. The data presented in this article were obtained from 36 cells.

M echanical Stimulation and Recording

The axial stiffness of OHCs and the forces they devel­oped during voltage-dependent displacements were measured from OHCs held at their basal ends by a large patch pipette filled with HBSS, which was pulled from1.5 mm thin-walled tubing on a laboratory-designed puller. For measurements of axial stiffness, the OHCs were gently affixed to a pipette by means of a manome-

FIGURE 1 Experimental arrangement showing an outer hair cell (OHC) held in a pipette the inside tip diameter of which is sim ilar to the diameter of the OHC. A proportion q of the total length of the OHC protrudes from the tip of the pipette. The fiber of known stiffness rests only on the cuticular plate and not the hair bundle of the OHC. A voltage Vt is applied between the elec­trolytes in the pipette and in the bath solution via a current to voltage converter (solid triangle). The image of the cuticular plate is projected by the microscope optics to lie between the photodi­ode pair of the displacement measuring system.

SALICYLATES AND OUTER HAIR CELL MECHANICS 311

ter attached to the pipette. The inside diameter of the pipette (8 to 9 |im) was such that only the base of the OHC could be drawn into the pipette. For measure­ments of the OHC voltage-sensitive motility the inside diameter of the pipette tips were slightly larger so that the OHCs could be drawn into the pipettes. The exact proportion of the OHC drawn into the pipette could be controlled within a micrometer by adjusting the height of the fluid column of the manometer. For stiffness mea­surements, fluid column of the manometer tended to re­sist any forces applied to the apical surface of the OHC. This was confirmed by using the optical lever technique (see later) to show that displacements of the basal sur­face were negligible relative to those of the apical sur­face when forces were applied to the apical surface.

The axial stiffness was determined from measuring nanometer displacements of the tip of a fine glass fiber (0.5 to 1 |i.m diameter) of known stiffness fused to a glass stub that was attached to a piezo electric bimorph with insect wax and resin. The fiber was formed by dipping the tip of the stub in molten soda glass and then rapidly withdrawing the stub by means of a powerful solenoid. The stiffness of the fibers was calibrated after a tech­nique introduced by Howard and Ashmore (1986) (Kossl et al., 1989). Several acrylic beads (Howmedica dental acrylic) were attached to the fiber tip and the de­flection of the tip measured by means of a horizontally mounted microscope with a graticule eyepiece. After the beads were removed by a gentle air jet it was possible to determine the stiffness of the probe tips as a relation­ship between the recorded displacement and gravita­tional forces exerted by the beads. The density of the acrylic beads was 1.15 x 103 kg/m3 and their diameter was between 3 and 120 p.m. The measured stiffness of the probes used in these experiments varied slightly about 0.3 mN/m.

The mechanical combination of fiber, glass stub, and piezoelectric bimorph had a resonant frequency of above 3 kHz. The bimorph was driven by eight cycles of computer-generated 100 Hz sinusoidal voltage sig­nals delivered at 2 S"1. The voltage signals were attenu­ated so that the range of the free movements of the tip of the glass fiber was restricted to between 1 and 100 nm. The axial stiffness (ka) was measured by comparing the free movement of the tip of the fiber (Lf) of known stiff­ness (kf) when it was moved in a plane parallel to the long axis of the OHC with the deflection of the tip when it was rested against the surface of the cuticular plate (Ln) (see figs. 1, 2), where:

ka = k f(Lf - L h)/Lh (1)

The displacements were measured by projecting a 1000 x magnified image of the fiber onto a pair of pho­todiodes (Crawford and Fettiplace, 1985), and the pho­tocurrent (converted to a voltage) was proportional to the displacement of the fiber. The measurements were

calibrated by centering the image of the fiber on the pho­todiode array and then moving the measuring photo­diodes sinusoidally over a range of ± 13 (im, which corresponded to a ± 13 nm displacement of the fiber. The linear range of the displacement measurements was ex­amined by displacing the photodiode assembly in 10 (xm increments and repeating the calibration procedure. In this way it was discovered that the system was linear for displacements of ± 275 nm about the mean position.

Transcellular voltage signals were delivered to the OHCs between the bath solution and the pipette solu­tion by means of a laboratory-constructed current to voltage converter. This method of electrically stimulat­ing the OHCs is according to the microchamber tech­nique devised by Evans et al., (1989), where a resistive seal is formed at the opening at the tip of the pipette and the OHC membrane, which separates the electrolytes within the pipette from those in the surrounding bath solution. The interior of the cell is equipotential so that when the inside of the pipette is negative the segment of membrane within the pipette is depolarized and that exposed to the bath solution is hyperpolarized. According to Dallos et al. (1993) and with their simpli­fying assumptions, including that the lateral membrane of the OHC has uniform resistivity, the OHC diameter is constant, the series resistance of the pipette is zero, and that both the shunt resistance at the seal between the pipette opening and the OHC membrane and the re­sistance of the apical membrane is infinite, then the OHC and the pipette behave as a voltage divider, dividing the transcellular voltage Vt as:

dV[ = Vr(l — q) (2)

and

dV2 = -V Tq (3)

where q is the proportion of the cell excluded from the pipette dVj is the voltage drop across the excluded por­tion of the OHC, and dV2 is the voltage drop across the portion of the OHC inserted within the pipette.

OHC displacements in response to transcellular elec­trical stimulation were recorded by projecting a 1000 times magnified image of either the cuticular plate or the base of the cell onto the pair of photodiodes.

The phasic component of the fiber and OHC dis­placements was measured by feeding the voltage out­put of the photodiode measuring system into the inputs of a pair of lock-in amplifiers (Ortec Brookdeal 9503-SC), which were set in quadrature phase and with integra­tion times of 10 ms. The output of the two lock-in am­plifiers and the photodiode system were each filtered at 800 Hz by eight pole Bessel filters and digitized at 4.4 kHz. The modulus and phase of the phasic component of displacement were computed on-line and these sig­nals, together with the filtered, unprocessed, output

312 I. RUSSELL and C. SCHAUZ

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Displacement of free fibre (nm)FIGURE 2 (A) The displacement of the tip of the fiber (stiffness 0.305 mN/m) when in contact with the cuticular plate of an outer hair cell (OHC, 83 |im long, 11 im diameter) as a function of the free motion of the fiber when driven by the piezoelectric bimorph at 100 Hz. Open circles: measurement in Hank's balanced solid solution (HBSS); solid triangles: measurement in 5 mM salicylate. Each point is the mean and standard deviation of five measurements. Sample records of measurements of the displacement of the tip of the calibrated fiber are inset in the figure. Each record is the mean of eight samples. (B) The displacement of the tip of the calibrated fiber when in con­tact with the cuticular plate of an OHC (73|xm long, 9 (im diameter) as a function of the free motion of the fiber when driven by the piezo­electric bimorph at 100 Hz. Open circles: measurement in HBSS; solid triangles: measurement in 5 mM salicylate; open diamonds: measurement following washout of the salicylate by HBSS.

from the photodiodes were stored on disc for later com­puter analysis.

RESULTS

Effect o f Salicylate on Axial Stiffness o f Outer Hair Cells

The axial stiffness of OHCs held at their basal pole by a micropipette (Fig. 1) was obtained by measuring the nanometer deflections of the tip of a fine glass fiber of known stiffness when the tip of the fiber was unimpeded and when it was pushed against the cuticular plate (Fig.2). From the relationship between these two measure­

ments (see equation 1) the axial stiffness of the OHCs was computed. For the 22 OHCs of length 59.73 ± 11.32 |j.m (mean ± sd) for which this parameter was measured, the axial stiffness was 0.755 ± 0.742 mN/m. In Figure 3, the axial stiffness is plotted as a function of OHC length for all 22 OHCs in this particular study. The OHCs formed naturally into two populations, stiff OHCs de­noted by open and closed circles and relatively compli­ant hair cells denoted by open triangles. For the 11 stiffest OHCs in Figure 3 of mean length 61.93 ± 11.41 |im, the axial stiffness was 1.157 ± 0.306 mN/m. A linear rela­tionship exists between the inverse of axial stiffness, axial compliance, and OHC length (see the insert of Fig.3) with a correlation coefficient greater than 0.99 for the

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SALICYLATES AND OUTER HAIR CELL MECHANICS 313

OHC length (pm)

FIGURE 3 The stiffness of the outer hair cell (OHC) body meausred in the plane of the long axis of the cell body as a func­tion of cell length. The stiffest cells in the population are shown by open and closed circles, the more compliant cells are shown by open triangles. The regression lines are based on the inverse of the linear regression of the compliance of the OHC body as a function of cell length for the same data shown in the inset. The regression lines through each set of data in the inset are given by, x = 0.0202L - 0.331 (r = 0.99) for the stiff OHCs, and T = 0.0616L - 0.412 (r = 0.56) for the compliant OHCs, where x = compliance and L = cell length (|im).

stiff OHCs. The inverse of the linear correlations be­tween OHC length and axial compliance was fitted to the data from both the stiff and the compliant popula­tions of OHCs (Fig. 3), where it can be seen that axial stiffness increases as OHC length decreases. It is possi­ble that the relationship between OHC length and axial stiffness is apparent rattier than real because the banana­shaped longer OHCs may buckle when axial forces are applied. To minimize any buckling, the forces were kept to a minimum within the constraints of the sensitivity of the displacement measuring system.

The effect of salicylate on axial stiffness was mea­sured for 16 of the OHCs (those denoted by open sym­bols) shown in Figure 3. When the HBSS bathing the OHCs was replaced by sodium salicylate, the axial stiff­ness was reduced. Despite of the large variation in the measurements of axial stiffness (0.69 ± 0.48 mN/m,

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FIGURE 4 The axial stiffness of the outer hair cells (OHC) de­noted by the open symbols in fig. 3, measured in HBSS (k hbss) as a function of the same stiffness when measured after the OHCs had been perfused for 2 minutes in 5 mM sodium sali­cylate (ks). The regression line is given by 1% = 0.756Hgss - 0.082. HBSS = Hank's balanced salt solution.

mean ± SD) there was a good correlation (shown in Fig.4) between the axial stiffness of the OHCs measured in HBSS and after 2 minutes of perfusion with salicylate perfusate where it can be seen that the stiffness is re­duced by about 30%. The reduction in stiffness was re­versible (Fig. 2C). W ith longer perfusions, the OHC axial stiffness declined by about 65% during 6 minutes of per­fusion with 5 mM salicylate and recovered to control levels after 15 minutes following washout with HBSS, (Fig. 5). The effect was usually not reversible if the pe­riod of salicylate perfusion extended beyond about 6 minutes.

Effect o f Salicylate on Electromotility o f Outer Hair Cells

OHCs that were partially sucked into the tip of a mi­cropipettes underwent sinusoidal length changes when a sinusoidal voltage was applied between the solution inside the pipette and the bath solution (Fig. 6). The mag­nitude of the movement increased with the voltage and with the length of the excluded portion of the OHC, and the direction of the movement was determined by the polarity of the bath solution (Dallos et al., 1993). The forces exerted by the voltage-dependent movements of the OHCs against the calibrated glass fiber placed on the cuticular plate were measured and expressed as pico- Newton per millivolt potential difference across the

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FIGURE 5 Time course of changes in the axial stiffness of an outer hair cell OHC, (84 |im long, 10.5 (im diameter) following replacement of Hank's balanced salt solution (HBSS) in the bath with 5 mM salicylate and after washout of the salicylate with HBSS.

membrane of the excluded portion of the OHC. The po­tential difference across the excluded portion of the OHC was computed according to Dallos et al., 1993, (see equation 2), where the OHC in the pipette is considered as a potential divider. On the basis of this calculation, and with the assumptions that are made in this calcula­tion (Dallos et al, 1993), the mean forces of 10 OHCs (mean length, 59.4 ± 8.2 mm) was 4.1 ± 2.6 pN/mV. A correlation was found between OHC length and the forces they could develop (Fig. 7). When perfused with5 mM salicylate for periods of 4 to 6 minutes, the forces exerted by the electromotility were reversibly attenu­ated by about 65% to 1.4 ± 1.2 pN/mV (n = 10) (Fig. 6). The correlation between the forces developed by OHCs in HBSS and those developed in salicylate (Fig. 8) was only 0.67. This correlation is much weaker than the cor­relation of 0.99 for the axial compliance of OHCs mea­sured in HBSS and salicylate (Fig. 4), although the standard deviations from the mean of the two parame­ters (stiffness and force) as functions of OHC length were similar in the two cases (compare Figs. 3 and 7).

DISCUSSION

When perfused with a 5 mM solution of salicylate, the axial stiffness, electromotility, and the forces generated by the electromotile process of OHCs are reversibly de­creased. These findings extend and confirm earlier re­ports by Shehata et al. (1990, 1991) that millimolar

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FIGURE 6 Voltage-dependent displacements of an outer hair cell OHC; (84 |im long, 10 |im diameter) held in a pipette in re­sponse to 100 Hz transcellular voltage signals of 36 mV when 26.5 nm of the OHC protruded from the tip of the pipette dur­ing perfusion with Hank's balanced salt solution (HBSS), per­fusion with salicylate for 2 minutes, and following 5 minutes washout with HBSS.

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FIGURE 7 The force generated by the outer hair cell (OHC) motor as a function of OHC length measured in Hank's bal­anced salt solution. The regression lines are given by F = 0.297L -14.726, where F is the axial force in pN/mV and L is the OHC length in |im.

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SALICYLATES AND OUTER HAIR CELL MECHANICS 315

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FIGURE 8 The force generated by the outer hair cell (OHC) motor measured in HBSS (FHBSS) as a function of the force gen­erated by the OHC after 2 minutes of perfusion in 5 mM sodium salicylate (Fs). The regression line is given by Fs = 0.303FHBSS + 0.144.

concentrations of salicylate block the motor and reduce the turgidity of OHCs. The main finding reported here is that salicylate reduces both the axial stiffness and the forces generated by the electromotility in similar pro­portion. This proportionality between the stiffness of the OHC and the force it develops is predicted in a piezo­electric model of OHC function (Mountain and Hubbard, 1994) where a decrease in cell stiffness would decrease the force acting on an external load, since the membrane compliance will shunt the load. It is not pos­sible, on the basis of these experiments to say if salicy­late has two independent effects, one of which reduces the cell turgor and hence the forces exerted by the OHC motor, and another on the motor itself (Ashmore, 1989; Tunstall et a l, 1994). Recent studies by Tunstall et al,(1994) support a model in which salicylate partitions into the membrane in an undissociated form and binds to the motor. It is possible that these two effects are dif­ferent expressions of the same action of salicylate on pro­teins associated with the OHC plasma membrane. For example, it may be that the OHC motors have a tonic, "corseting" action in the plasma membrane that helps maintain OHC turgor and that the salicylate blockade of the voltage-dependent capacitance results in a re­duction of both the motility and the turgor. In fact, Holley and Ashmore (1988) have already demonstrated that the expression of electromotility and cell turgor are inextricably linked. Dieler et al., (1991) have shown a cor­respondence between the reversible, salicylate-induced reduction in electromotility and ultrastructural changes

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in OHCs associated with the lateral cistemae. This ob­servation, and a corresponding finding in hair cells of the avian cochlea (Dieler et al, 1994) indicate either that salicylate has additional sites of action in OHCs other than the motor or that the fenestration of the subsynap- tic cistemae is due to an indirect action of salicylate.

Salicylate has been shown to have a variety of effects on different animal tissues. For example, salicylate may alter the membrane conductances of mammary cells to non selective cationic channels, potassium and calcium (Shennan, 1992). Salicylate has also been reported to mo­bilize internal calcium stores in mouse pancreatic cells, which has led to the activation of calcium-dependent potassium conductances (Drews et al, 1992), and acetyl- salicylate probably acting as salicylate has been shown to induce mitochondrial dysfunction in liver cells, which is potentiated by a rise in intracellular calcium (Tomoda et al, 1994). Irreversible, salicylate-induced conductance changes in OHCs were observed by Shehata et ah ', (1991) but not by Tunstall et al, (1994). It remains to be seen if the reduction in axial stiffness observed here is due to reversible fenestration of the lateral cistemae observed by Dieler et al. (1991) or if it is a consequence of the re­versible blockade of the OHC motor.

A hint that the action of salicylate on OHC stiffness may involve mechanisms different from those responsi­ble for salicylate-induced changes in the voltage-depen­dent force generated by the OHCs, might be drawn from a comparison between Figures 4 and 8. It is evident from Figure 4 that a very strong, linear proportionality exists between the axial stiffness of OHCs measured in HBSS and of the same OHCs measured in salicylate. This pro­portionality is much weaker for the measurement of the voltage-dependent force shown in Figure 8, which might indicate that several factors act either directly or indirectly on the manifestation of voltage-induced force production by the OHCs. For example, a breakdown in the propor­tionality might be expected if salicylate also induced a conductance change in the excluded portion of the OHC. A change in conductance would alter the voltage divi­sion of the transcellular stimulating voltage and hence the voltage-dependent forces generated by the OHC.

In summary, the observations reported here confirm earlier findings that salicylate blocks OHC electro- motilty and reduces OHC turgidity and hence the axial stiffness that is essential for directing the forces exerted by the OHC motility.

Comparison with In Vivo Basilar Membrane Measurements

Measurements of the effects of salicylate on the mechan­ical and electromotile properties of the OHCs were prompted by measurements made in situ of the effects of salicylate perfusion the scala tympani on BM displace­ments to tones (Murugasu and Russell, 1995). Following perfusion of the scala tympani with either 2.5 or 5 mM

316 1 RUSSELL and C. SCHAUZ

sodium salicylate, the tips of the mechanical tuning curves became reversibly desensitized by up to 45 dB sound pressure level (SPL) the best frequency of the tip shifted to lower frequencies by about 2 kHz, the tip be­came broader, and the tail of the tuning curve became sen­sitized by about 10 dB. In accordance with a model of the noise-damaged cochlea proposed by Allen (1990), the changes in the mechanical responses of the BM could be explained if salicylate causes an increase in the compli­ance of the cochlear partition, possibly by reducing the axial stiffness of the OHCs by up to 50%, which is simi­lar to the salicylate-induced reduction in axial stiffness measured in OHCs. It is possible that the stiffness of other components in the cochlear partition are also changed by salicylate but the mechanical properties of most other major components, with the exception of the OHCs and perhaps Hensen's cells, are probably determined by their impressive cytoskeletons of actin and tubulin, and it is unlikely that these are altered either directly or indirectly through exposure to salicylate within the time scale of the experiment.

Comparison w ith Similar M echanical Measurements on Isolated Outer Hair Cells

The mean stiffness of 0.775 mN/m (0.268 to 1.785 mN/m) for OHCs with a mean length of 60 (xm reported here compares with 0.544 mN/m (0.15 to 1.1 mN/m) for cells between 53 and 75 (Am in length first measured by Holley and Ashmore (1988), 3 mN/m of Zenner et al., (1992), 0.2 mN/m of Gitter et al., (1993), and with measurements of the axial compliance (inverse of stiff­ness) of OHCs made by Hallworth (1995). In accordance with his findings, we have also demonstrated a linear relationship between the axial compliance and OHC length (Fig. 3, inset) which indicates that OHCs behave as simple springs whose compliance is proportional to their length. OHCs are cylindrical cells with diameters that remain remarkably constant along the length of the cochlea (Pujol et al., 1990). The linear relationship be­tween OHC length and axial compliance reported by Hallworth (1995), and here, might be expected if, at least in the apical turns of the cochlea, the structural proper­ties of the OHC body is relatively homogeneous along its long axis. Thus, the axial stiffness of the OHCs is matched to their location in the cochlear partition. The smallest OHCs are located in the stiff, high-frequency regions of the cochlea and the outermost and smallest hair cells at any one position along the BM are located over the relatively stiff region of the BM close to the spi­ral ligament. By comparison, the tallest OHCs are lo­cated close to the most compliant region of the BM (Olson and Mountain, 1993).

Our findings that the electromotile forces generated by the OHCs with a mean length of 59 |im in our study had a mean value of 4.1 pN/mV correspond closely to Hallworth's (1995) measurements of between 2 and 20

pN/mV and an estimate of 14 pN/mV by Zenner et al. (1992) for the same parameter. These measurements are two to three orders of magnitude less than estimates made by Iwasa and Chadwick (1992) and similar ones from in vivo measurements of basilar membrane motion by Xue et al. (1993) of 0.2 to 1.24 nN/mV for the voltage- dependent OHC force generation that should be exerted by OHCs to produce electrically-induced displacements of the BM.

In accordance with Hallworth, we have shown a di­rect relationship between the electromotile force per mil­livolt generated by the OHCs and cell length. The relationship seems surprising because the gain of the cochlear amplifier increases with frequency from about6 dB of amplification for frequencies around 1 kHz to more than 40 dB for frequencies around 18 kHz in the guinea pig cochlea (see Dallos [1992] for a review). Furthermore, the stiff, high-frequency region of the BM requires greater forces to displace it a given amount than does the low-frequency region of the cochlea (von Bekesy, 1960). The unexpected results obtained here may be due to variations in the mechanical loading of the OHCs by the flexible fiber. Hallworth (1995) has shown that the force generated by an OHC motor can be substantially increased when the OHC is compressed by very small amounts by the flexible fiber used to mon­itor the force. The amount of force generated by the OHC motor is increased in inverse proportion to the mem­brane compliance (Holley and Ashmore, 1988) and hence shunting of the load can be effectively decreased by increasing the distributed load on the OHC mem­brane. Thus, to some extent the results presented here and by Hallworth (1995) are all the more surprising be­cause the longer OHCs are more compliant than are the shorter OHCs and should, presumably shunt a greater proportion of any applied external load. In situ, it would be necessary for any shunting of the applied load to be kept to a minimum possibly as a result of maintained turgor pressure and through interaction with other structural elements in the organ of Corti so that OHCs in the basal turn can exert forces that are up to three or­ders of magnitude greater than those that have been recorded from isolated OHCs.

The problem of OHC electromotility at very high fre­quencies is compounded because OHC length changes are proportional to the transmembrane potential (Santos-Sacchi and Dilger, 1988) and it has long been es­tablished that the phasic voltage responses of IHCs and OHCs are limited by the hair cell membrane time con­stants (Russell and Sellick, 1978,1983). As a consequence, OHC voltage responses to tones at the frequency of their place in the high-frequency region appear to be insuffi­cient to drive OHC motility by between one and two or­ders of magnitude (Russell and Kossl, 1992). High-frequency, tuned, BM responses can be elicited through high-frequency transorgan of Corti stimulation (Nuttall and Dollan, 1993; Xue et al., 1993), which has

SALICYLATES AND OUTER HAIR CELL MECHANICS 317

been taken to indicate that the electromotility of OHCs is engaged during active feedback in the cochlea. However, for OHCs to generate the large forces neces­sary to displace the basilar membrane of the basal turn, Xue et al., (1993) have estimated that OHCs are required to generate membrane potentials that are three orders of magnitude larger than those that have been recorded from basal turn OHCs in situ in response to characteris­tic frequency tones close to the neural threshold (Russell et al., 1986). It now remains to be seen if the apparent in­constancies between the measured performance of OHCs and what they are required to do in situ can be reconciled if the responses of OHCs to electrical stimu­lation and, indeed, to acoustic stimulation, are a re­sponse to transepithelial rather than to transmembrane voltage in the organ of Corti (Dallos and Evans, 1995).

Do Changes in the Axial Stiffness o f Outer Hair Cells Alter the Stiffness o f the Basilar Membrane

As we have reported here, perfusion with salicylate re­duces OHC axial stiffness and electromotility by about 65%. This change will have significant effect on both the stiffness and motion of the BM if the axial stiffness of the OHCs is a major component of the total stiffness of the cochlear partition. Support for this idea comes from the finding that voltage-dependent movements of apical turn OHCs in an isolated preparation of the cochlea have sufficient force to move both the basilar membrane and cuticular plate and that these movements are blocked by salicylate (Mammano and Ashmore, 1993). At present, there is an apparent mismatch between estimates of OHC stiffness (0.8 N/m, Olson and Mountain, 1993) based on point measurements of BM stiffness in the basal turn of the cochlea in situ (1.5 to 15 N/m) (Gummer et al., 1981; Miller, 1985; Olson and Mountain, 1994) and measurements of the axial stiffness of isolated OHCs (0.1 to 10 mN/m). If the stiffness of OHCs in situ is similar to that measured for isolated OHCs, then salicylate-in­duced changes in OHC stiffness and electromotility will have little impact on the stiffness and motion of the cochlear partition. In the light of current evidence this seems unlikely. The apparent discrepancy between BM stiffness (measured in the basal turn) and the axial stiff­ness of isolated OHCs may be largely because isolated OHCs are usually harvested from the apical coils where they are more robust and easier to isolate than those from more basal coils. There are, as yet, no comprehensive measurements of BM stiffness from the apical coils of the cochlea. However, estimates of the stiffness of the BM in this region can be made by combining. Greenwood's (1990) frequency map of the guinea pig cochlea with mea­surements of the stiffness in the arcuate region of the guinea pig cochlea by Gummer et al. (1981) (see Mammano and Nobili, 1993) to provide estimates of the stiffness of the BM in the 1 to 4 kHz region of the guinea pig BM of between 2 and 10 mN/m. These estimates are

similar to the measured axial stiffness of OHCs isolated from this region of the cochlea. According to the rela­tionship shown in Figure 3 for the stiff OHCs, OHCs with lengths of about 20 |im, similar in dimension to those found in the basal, high-frequency turn of the cochlea, might be expected to have an axial stiffness of about 14 mN/m, which is an order of magnitude less than the in situ estimates of OHC stiffness made in the basal turn of the gerbil cochlea (Olsen and Mountain, 1993). However, slight changes in the constants of the relationship shown in Figure 3 can have dramatic effects on the estimated stiffness of OHCs. For example, according to this rela­tionship OHCs 17 nms in length or OHCs 20 (im in length but with a diameter about 1 (im less than those in the api­cal two turns of the cochlea would have an axial stiffness that would meet the requirements of Olsen and Mountain's in situ estimates. In conclusion, it seems that salicylate-induced changes in the displacement tuning curves of the BM can be accounted for by changes in the axial stiffness and electromotility of OHCs.

ACKNOWLEDGMENTWe thank James Hartley for designing and making electronic equipment and Drs. Ann Brown, Manfred Kossl, Come Kros, and Guy Richardson for helpful comments on the manuscript. The work was supported by grants from the Manual Research Council, Royal Society, and Hearing Research.

REFERENCES

Allen, J. B. (1990). Modelling the noise damaged cochlea. In: The Mechanics and Biophysics o f Hearing, Dallos, P., Geisler, C. D , Mathews, J. W., Ruggero, M. A., and Steele, C. R., eds. Springer-Verlag, Berlin, pp. 324-332.

Ashmore, J. F. (1987). A fast motile response in guinea pig outer hair cells: The cellular basis of the cochlear amplifier. J. Physiol. (Lond.) 388,323-347.

Ashmore, J. F. (1989). Effects of salicylate on a rapid charge move­ment in outer hair cells isolated from the guinea pig cochlea J. Physiol. (Lond.) 412,46P.

Bekesy, G. von., (1960). Experiments in Hearing. McGraw-Hill, New York.

Brown, A. M., Williams, D. M. and Gaskill, S. A. (1993). The ef­fects of aspirin on cochlear mechanical tuning. J. Acoust. Soc. Am. 9 3 ,3298-3307.

Brownell, W. E., Bader, C. R., Bertrand, D., and Ribaupirre, Y.(1985). Evoked mechanical responses of isolated cochlear outer hair cells. Science 227,194-196.

Crawford, A. C., and Fettiplace, R. (1985). The mechanical prop­erties of ciliary bundles of turtle cochlear hair cells. J. Physiol. (Lond.) 364, 359-379.

Dallos, P. (1992). The active cochlea. J. Neurosci. 12 ,4575-4585.

Dallos, P., Hallworth, R. and Evans, B. N. (1993). Theory of elec­trically-driven shape changes of cochlear outer hair cells. /. Nettrophys. 70 ,299-323.

Dallos, P., and Evans, B. N. (1995). High frequency motility of

318 I. RUSSELL and C. SCHAUZ

outer hair cells and the cochlear amplifier. Science 267, 2006-2009.

Dieler, R., Shehata-Dieler, W. E., and Brownell, W. E. (1991). Concomitant salicylate-induced alterations of outer hair cell subsynaptic cistemae and electromotility. /. Neurocytol. 20, 637-653.

Dieler, R., Shehata-Dieler, W. E., Richter, C.-P., and Klinke, R.(1994). Effects of endolymphatic application of salicylate in the pigeon. II: Fine structure of auditory hair cells. Hear. Res. 74, 85-98.

Drews, G., Garrino, M. G., and Henquin, J. C. (1992). Arachidonic- acid metabolism in mouse pancreatic B cells. Diabetes, 41, 620-626.

Evans, B. N., Dallos, P., and Hallworth, R. (1989). Asymmetries in motile responses of outer hair cells in simulated in vivo con­ditions. In: Cochlear Mechanisms, Wilson, J. P., Kemp, D. T., eds., Plenum, New York, pp. 205-206.

Evans, E. F and Borerwe, T. A. (1982). Ototoxic effects of salicy­lates on the responses of single cochlear nerve fibres and cochlear potentials. Br. J. Audiol., 1 6 ,101-108.

Fitzgerald, J. J., Robertson, D., and Johnstone, B. M. (1993). Effects of intra-cochlear perfusion of salicylates on cochlear micro- phonic and other auditory responses in the guinea pig. Hear. Res. 6 7 ,147-156.

Gitter, A. H., Rudert, M., and Zenner, H-P. (1993). Forces involved in length changes of cochlear outer hair cells. Pflugers Arch. 424 ,9-14.

Greenwood, D. D. (1990). A cochlear-position function for several species— 29 years later. J. Acoust. Soc. Am. 87, 2592-2605.

Gummer, A. W., Johnstone, B. M., and Armstrong, N. J. (1981). Direct measurements of basilar membrane stiffness in the guinea pig. J. Acoust. Soc. Am. 7 0 ,1298-1309.

Hallworth, R. (1995). The biomechanics of outer hair cell motility. In Active Hearing, A. Flock, D. Ottoson and M. Ulfendahl (eds.) Elsevier, Cambridge, in press.

Holley, M. C., and Ashmore, J. F. (1988). A cytoskeletal spring in cochlear outer hair cells. Nature 335 ,635-637.

Howard, J., and Ashmore, J. F. (1986). Stiffness of sensory hair cells in the sacculus of the frog. Hear. Res. 23,93-104.

Iwasa, K. H., and Chadwick, R. S. (1992). Elasticity and force gen­eration of cochlear outer hair cells. J. Acoust. Soc. Am. 92, 3169-3173.

Jastreboff, P. J., and Sasaki, C. T. (1994). An animal model of tin­nitus: A decade of development. Am. J. Otol. 1 5 ,19-27.

Kossl, M. (1992). High frequency distortion products from the ears of two bat species, Megaderma lyra and Carollia perspicil- lata. Hear. Res. 5 6 ,156-164.

Kossl, M., Richardson, G. P., and Russell, I. J. (1989). Stereocilia bundle stiffness: effects of neomycin sulphate, A23187 and Concanavalin A. Hear. Res. 44, 217-230.

Kossl, M., and Russell, I. J. (1992). The phase and magnitude of hair cell receptor potentials and frequency tuning in the guinea pig cochlea. f. Neurosci. 1 2 ,1575-1586.

Liberman, M. C., and Dodds, L. W. (1984). Single neuron la­belling and chronic cochlear pathology. Ill Stereocilia dam­age and alterations of threshold tuning curves. Hear. Res. 26 ,55-74.

McPherson, D. L., and Miller, J. M. (1974). Choline salicylate. Effects on cochlear function. Arch. Otolaryngol. 99,304-308.

Mammano, F., and Ashmore, J. F. (1993). Reverse transduction measured in the isolated cochlea by laser Michelson Interferometry. Nature 365,838-841.

Mammano, F., and Nobili, R. (1993). Biophysics of the cochlea: Linear approximation. J. Acoust. Soc. Am. 93, 3320-3332.

Meikle, M. B., and Charnell, M. G. (1994). Salicylate-induced changes in asynchronous neural activity recorded from the round window. Abstr 17th Midwinter Meeting of the Association of Research in Otolaryngology, Clearwater, Florida, 394.

Miller, C. E. (1985). Structural implications of basilar membrane compliance measurements. J. Acoust. Soc. Am. 77,1465-1474.

Mountain, D. C., and Hubbard, A. E. (1994). A piezoelectric model of outer hair cell function. J. Acoust. Soc. Am. 95 ,350-354.

Mountain, D. C., Hubbard, A. E., and McMullen, T. A. (1983). Electromechanical processes in the cochlea. In: Mechanics o f Hearing, de Boer, E., and Viergever, M. A., eds., Delft U. P., The Netherlands, pp. 119-126.

Murugasu, E. and Russell, I. J. (1995). Salicylate ototoxicity: The effects on basilar membrane displacement cochlear micro­phonics and neural responses in the basal turn of the guinea pig cochlea. Auditory Neurosci. 1 ,139-150.

Neely, S. T. (1993). A model of cochlear mechanics with outer hair cell motility. /. Acoust. Soc. Am. 9 4 ,137-146.

Nuttall, A. L., and Dollan, D. F. (1993). Basilar membrane veloc­ity responses to acoustic and intracochlea electric stimuli. In Biophysics o f Hair Cell Sensory Systems Duifhuis, H., Horst, J. W., van Dijk, P., and van Netten, S. M., eds., Singapore: World Scientific, pp. 288-295.

Olson, E. S., and Mountain, D. C. (1993). Probing the cochlea partition's properties with measurements of radial and longitudinal stiffness variations. In Biophysics o f Hair Cell Sensory Systems, Duifhuis, H., Horst, J. W., van Dijk, P., and van Netten, S. M., eds. Singapore: World Scientific, pp. 288-295.

Olson, E. S., and Mountain, D. C. (1994). Mapping the cochlear partition's stiffness to its cellular architecture. J. Acoust. Soc. Am. 9 5 ,395-400.

Puel, J-L., Bledsoe, S. C., Bobbin, R. P., Caesar, G., and Fallon, M.(1989). Comparative actions of salicylate on the amphibian lateral line and guinea pig cochlea. Comp. Biochem. Physiol. 93, 73-80.

Puel, J-L., Bobbin, R. P. and Fallon, M. (1990). Salicylate, mefena- mate, meclofenate and quinine on cochlear potentials. Otolaryngol. Head Neck Surg. 102,66-73.

Pujol, R., Lencir, M., Ladrech, S. Tribillac, F., and Rebillard, G. (1991). Correlation between the length of the outer hair cells and frequency coding in the cochlea. In: Auditory Physiology and Perception. Cazals, Y., Homer, K , and Demany, L., eds. Oxford: Pergammon, pp. 44-51.

Russell, I. J., and Sellick, P. M. (1978). Intracellular studies of hair cells in the mammalian cochlea. J. Physiol. (Lond.) 284, 261-290.

Russell, I. J., and Sellick, (1983). Low frequency characteristics of intracellularly recorded receptor potentials in mammalian hair cells. /. Physiol. (Lond.) 435, 493-511.

SALICYLATES AND OUTER HAIR CELL MECHANICS 319

Russell, I. J., and Kossl, M. (1992). Voltage responses to tones of outer hair cells in the basal turn of the guinea pig cochlea: significance for electromotility and desensitization. Proc R Soc. Lond. B. 247,97-105.

Russell, I. J., Cody, A. R., and Richardson, G. P. (1986). The re­sponses of inner and outer hair cells in the basal turn of the guinea pig cochlea and in the mouse cochlea grown in vitro Hear. Res. 2 2 ,199-216.

Santos-Sacchi, J., and Dilger, J. P. (1988). Whole cell currents and mechanical responses of isolated outer hair cells. Tissue Cell Res. 229 ,467-481.

Silverstein, H., Bernstein, J. M., and Davies, D. G. (1967). Salicylate ototoxicity: A biochemical and electrophysiological study. Ann. Otol. Rhinol. Laryngol. 7 6 ,118-128.

Shenata, W. E., Brownell, W. E., Cousillas, H., and Imredy, J. P.(1990). Salicylate alters membrane conductance of outer hair cells and diminishes rapid electromotile responses. (Abstr.) 13th Midwinter Meeting of the Association of Research in Otolaryngology, Clearwater, Florida, 252.

Shehata, W. E., Brownell, W. E. and Dieler, R. (1991). Effects of salicylate on shape, electromotility and membrane charac­teristics of isolated outer hair cells from guinea pig cochlea. Acta Otolaryngol. (Stockh.) I l l , 707-718.

Shennan, D. B. (1992). Salicylate induced cation fluxes across bi­ological membranes—a study of the underlying mecha­nisms. Biochem. Pharmacol. 44, 645-650.

Stypulkowski, P. H. (1990). Mechanisms of salicylate ototoxicity. Hear. Res. 4 6 ,113-146

Tomoda, T., Takeda, K., Kurashiga, T., Enzan, H., and Migahara, M. (1994). Acetylsalicylate-induced mitochondrial dysfunc­tion and its potentiation by calcium. Liver 14 ,103-108.

Tunstall, M. J., Gale, J. E., and Ashmore, J. F. (1994). The effect of salicylate on the voltage-dependent capacitance of outer hair cells isolated from the guinea pig cochlea. ]. Physiol. (Lond.) 476. P 75P-76P.

Xue, S., Mountain, D. C., and Hubbard, A. E. (1993). Direct mea­surement of electrically-evoked basilar membrane motion. In Biophysics o f Hair Cell Sensory Systems Duifhuis, H., Horst, J. W., van Dijk, P., and van Netten, S. M., eds. Singapore: World Scientific, pp. 361-369.

Zenner, H-P., Gitter, A. H., and Ernst, A. (1992). Stiffness, com­pliance, elasticity and force generation of outer hair cells. Acta Otolaryngol. (Stockh.) 112,248-253.