Auditory Neuroscience, 1995, Vol 1, pp. 139-150 Reprints available directly from the publisher Photocopying permitted by license only

1995 Harwood Academic Publishers GmbH Printed in the United States of America

Salicylate Ototoxicity: The Effects on Basilar Membrane Displacement, Cochlear Microphonics, and Neural Responses in

the Basal Turn of the Guinea Pig Cochlea

E. MURUGASU*+ and I. J. RUSSELL*

*School o f Biological Sciences, University o f Sussex, Brighton, BN1 9QG, United Kingdom, and tENT Department, Royal Sussex County Hospital, Brighton, BN2 5BE, United Kingdom

(Received August 26,1994; accepted November 17,1994)

The self-mixing effect of a laser diode was used to measure frequency tuning of basilar membrane displacements in the 15 kHz region of the guinea pig cochlea while perfusing the scala tympani with either artificial perilymph or millimolar salicylate solutions. Following perfusion with either 2.5 or 5 mM sodium salicylate, the tips of the tuning curves became reversibly desensitized by up to 45 dB sound pressure level, 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. The changes in the tip of the tuning curve could be explained if sali­cylate caused a reduction in feedback from the outer hair cells (OHCs). An increase in the compliance of the cochlear partition, possibly by reducing the turgidity of the OHCs, would also cause a desensi­tization of the tip and a shift to lower frequencies. In addition, it would account for the sensitization of the tail of the tuning curve. In some instances, sensitization of the tail and high-frequency shoul­der of the tuning curve occurred only after the sal­icylate was washed out of the scala tympani. These changes could be accounted for if salicylate also caused a second, opposing effect, such as an in­crease in the basolateral conductances of the OHCs, with a slower time course than that of the proposed increase in basilar membrane compliance. From measurements made at the round window at fre­quencies between 1 and 25 kHz, it was concluded that cochlear microphonics and the compound ac­

*Corresponding author: I. J. Russell, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG United Kingdom.

tion potential were poor indicators of the action of salicylate on basilar membrane displacement and that salicylate had an additional action on the re­sponse properties of the auditory nerve. The find­ing that salicylate-induced changes in the mechanical responses of the basilar membrane could recover before cochlear microphonics was taken to indicate that the OHCs may continuously adjust their operating conditions to optimize the sensitivity and frequency selectivity of the cochlea.

Key words: Cochlear mechanics, basilar membrane dis­placement, salicylate, cochlear microphonics, eighth nerve action potential, adaptation

THE ADMINISTRATION OF MODERATELY high doses of aspirin can induce reversible hearing loss and severe tinnitus (recently reviewed by Jastreboff and Sasaki, 1994). Severe, flat, and high-frequency (HF) hearing losses of up to 40 dB can be induced by taking aspirin at levels often prescribed for the treatment of rheumatoid arthritis (4 to 8 g/day). The mechanism of salicylate-induced hearing loss is not well understood and some of the observations and measurements ob­tained to date are ambiguous and contradictory. Depending on the route of administration, the animal species, and the level and frequency of the auditory stimulus, salicylate can either have no effect, enhance or reduce the cochlear microphonics (CM), (Silverstein et al., 1967; McPherson and Miller, 1974; Puel et al., 1990; Stypulkowski, 1990; Fitzgerald et al., 1993) reduce or en­hance acoustic distortion, (McFadden and Plattsmier, 1984; Long and Tubis, 1988; Penner, 1989; Stypulkowski, 1990; Brown et al., 1993; Fitzgerald et al., 1993) increase or reduce spontaneous afferent activity in the auditory nerve, (Evans and Borerwe, 1982; Meikle and Charnell,1994) decrease the amplitude of the VUIth nerve com­

139

140 E. MURUGASU and I. J. RUSSELL

pound action potential (CAP), or elevate the threshold of the CAP, (Puel et al., 1989,1990; Stypulkowski, 1990; Fitzgerald et al., 1993). In experiments on isolated outer hair cells (OHCs), salicylate increases the conductance and reversibly decreases the turgidity, capacitance, and electromotility of OHCs, (Shehata et al., 1990, 1991; Tunstall et al., 1994). Thus, it is apparent that salicylate probably acts at several sites along the sensory process­ing pathway in the cochlea, including the OHCs and inner hair cell (IHC) afferent synapses. To date, the ac­tion of salicylate on the mechanical properties of the cochlear partition has been inferred from measurements of the CM and from changes in the acoustic distortion, (Stypulkowski, 1990; Fitzgerald et al., 1993). The mea­surement of CM, while relatively easily achieved, has provided apparently contradictory and ambiguous re­sults. This may indicate that salicylate alters the electri­cal impedance of the cochlear partition in addition to its more specific actions on the electrical and mechanical properties of the OHCs. In this article we have investi­gated the action of salicylate on the mechanical proper­ties of the cochlear partition. We used a laser-diode interferometer to measure basilar membrane (BM) dis­placements in situ in the basal, HF turn of the guinea pig cochlea with signals reflected directly from the BM while perfusing the scala tympani of the basal turn with arti­ficial perilymph and with solutions containing salicy­late.

MATERIALS AND METHODS

Animal Preparation and Electrophysiological Recording

Twenty-seven young pigmented guinea pigs weighing 180 to 300 g were used in this study. The animals were anesthetized with sodium pentobarbital (30 mg/mL), phenoperidine (lmg/mL) and droperidol (4 mg/mL), (Evans, 1979) and a tracheal cannula was inserted. The body temperature of the guinea pig was kept at 38°C with a heating blanket. The heart rate was monitored through a pair of skin electrodes placed on either side of the thorax. The right cochlea was exposed through a lateral opening in the temporal bone and was back illu­minated by a fiber optic light guide inserted through a hole made in the basal wall of the bulla. The BM was ex­posed through a small fenestra approximately 1 by 0.5 mm made in the bony wall of the basal turn of the cochlea. The CAP of the cochlear nerve and the CM were recorded with an electrode placed on the round win­dow. The silver-silver chloride reference electrode was inserted in the neck muscles. A fine cannula (less than 100 urn diameter) was inserted through a small incision in the round window and HEPES-buffered, Hank's bal­anced salt solution (HBSS, pH 7.4, osmolality 320 m Osm/L) and solutions of sodium salicylate in HBSS ad­

justed to pH 7.4 with 0.1 M sodium hydroxide were per­fused, at room temperature (22° to 24°C) through the basal turn of the cochlea at a rate of 2.5 (iL/minute with a syringe pump. In common with Puel et al., 1990, and Fitzgerald et al., 1993, no attempt was made to warm the solution to body temperature because perfusion with artificial perilymph did not cause perceptible changes in either the CAP audiogram or BM displacement re­sponses to tones. The solution flowed out through the fenestra in the basal turn, where it was collected by a dental point that acted as a fine, absorbent wick. The wick was positioned so that fluid did not escape into the middle ear, and the shape of the meniscus of the peri­lymph in the fenestra was kept constant to provide a view of the BM. Thus, only regions basal to the 10 kHz region of the cochlea were fully perfused with HBSS or salicylate solution. In this series of experiments the cri­terion for a sensitive preparation was one in which the change in the pure tone CAP threshold audiogram over the range from 1 to 25 kHz following exposure of the BM was less than 10 dB.

Measurement of Basilar Membrane Displacement

Displacements of the BM were measured by the self-mix­ing effect of a diode laser. The technique is similar to that using gas lasers, (O'Neil and Beardon, 1993) but the in­terferometer is more compact and easier to implement for in vivo measurements. The self-mixing in a laser diode depends on reflecting back a small proportion of the light emitted by the laser into the laser cavity, (Koelink et al., 1992). Interference between light within the cavity and light reflected back into the cavity from the target mod­ulates the intensity of the laser. As the target is displaced along the beam axis, the intensity of the laser varies si­nusoidally with a period corresponding to a displace­ment ofX/2, where A. is the wavelength of the laser light, 670 nm. For the case of a target oscillating by an amount p about a fixed position Po:

P(t) I Po + psin(oot) (1)

where P(t) is the target position. Providing that the inter­ferometer is operating about the quadrature point, that is, it is operating about a position where Po is an integer function of X/2, the method underestimates p by less than 3% for changes in p of ± 45 nm about the quadrature point. The displacement-dependent effect was measured in the signal of the photodiode, which is located behind the laser crystal in the diode laser housing. The bandwidth of the displacement-dependent signal was 200 kHz. Calibration of the signal was achieved by displacing the interferom­eter a known amount by a piezoelectric driver that moved 12.5 nm/V. The interferometer was continuously ad­justed for maximum signal and calibrated during the ex­periment to compensate for any slight variations in reflectivity from the BM that might occur during perfu­

SALICYLATE AND BASILAR MEMBRANE DISPLACEMENT 141

sion of the scala tympani. The piezoelectric driver was also fed by an amplified signal from the photodiode, which was low-pass filtered with a comer frequency of 100 Hz and a direct current reference voltage for setting the quadrature point of the interferometer. This system provided a negative feedback system that enabled the in­terferometer to track gross movements of the prepara­tion, such as those caused by respiratory movements and heart beat, while preserving sensitivity for the HF, tone- evoked displacements of the BM. Although the experi­ments were performed on a vibration isolation table (Newport), the system could not cope with gross relative movements between the diode and the BM more than 10 |J.m. If strong extraneous movements caused the operat­ing point of the interferometer to move away from the quadrature point, then there was a pronounced reduc­tion in the sensitivity and sometimes a reversal in phase of the apparent motion of the BM. In most instances when this happened the measurements were repeated; in oth­ers, it was sometimes possible to correct for the change in gain and phase reversal after the event. The high-pass characteristics of the displacement-measuring system prevented the recording of tonic displacements of the BM that were presumably represented, together with the larger, gross displacements of the preparation, in the feed­back signal to the piezoelectric transducer.

The beam from the laser diode was focused to form a 10 Jim diameter spot in the middle of the basal turn of the exposed basilar membrane. Only light from the plane of focus was reflected back into the laser cavity. The most reflective structures in this region of the BM are the bases of the outer pillar cells and Deiters' cells and it is likely that the measurements of BM displace­ment reported here are based on reflections from these structures. In these experiments it was relatively easy to direct the beam to any position on the BM that was vis­ible through the opening in the wall of the basal turn of the cochlea and to compare vibrations of the BM with those of a fixed structure, such as the spiral lamina, that did not produce tuned displacements to tones presented at the tympanic membrane. The frequency range of the BM that was exposed by the opening was 14 to 19 kHz, but most measurements reported here were made close to 15 kHz.

Signal AnalysisVoltage signals from the interferometer were amplified and directed to a two-channel lock-in amplifier set in quadrature (model 5210, EG & G, Princeton). The time constant of the lock-in amplifier was 10 ms and the ref­erence signal was the driving voltage to the HF sound delivery system. Signals from the in-phase and quadra­ture channels of the lock-in amplifiers were sampled at 0.1 to 0.5 ms intervals and the magnitude of the phasic response of the voltage signal from the interferometer was computed either on-line or from data stored on disk

by the microcomputer. Each measurement of magnitude is the mean of eight samples.

Acoustic Stimulation

Sound was delivered to the tympanic membrane by a closed acoustic system that was calibrated in situ before and during the experiment following major procedures, including the opening of the middle ear, fenestration of the basal turn of the cochlea, and perfusion of the basal turn of the cochlea. HF tones were delivered through a Bruel & Kjaer 3134 1/2 inch condenser microphone. A Bruel & Kjaer 3134 1/2 inch probe microphone, which was calibrated in the closed sound system against a Bruel & Kjaer 3135 1/4 inch microphone located at the plane of the tympanic membrane, was used to calibrate the acoustic system in situ. Continuous tones and tone bursts were presented at known sound pressure level (SPL), expressed in this article in decibels (re 2 x 10~5 Pa).

RESULTS

Frequency and Level Dependence of Basilar Membrane Displacement

BM displacement at the 15 kHz region as a function of level for different frequencies is shown in Figure 1 A. For frequencies close to the best or characteristic frequency (CF) of the measurement site, BM displacement in­creases with level with a slope close to 1 for SPLs below 50 dB. For levels above this, the BM displacement is very compressive, although it continues to grow with a slope of about 0.12 to 30 nm rms with increasing level between 50 and 100 dB SPL. For frequencies above the CF, the in-

dB (SPL) dB (SPL)

FIGURE 1 (A) Basilar membrane (BM) displacement as a func­tion of level for a single preparation at 1 ,1 3 ,16 (characteristic frequency [CF]) and 19 kHz. Each point is the mean of eight mea­surements (B). BM displacement as a function of level for another preparation at 16 (CF) and 19 kHz. Each point is the mean ± the standard deviation of 10 successive measurements, each of which is the on-line computed mean of eight measurements. SPL = sound pressure level.

142 E. MURUGASU and I J. RUSSELL

crease in BM displacement with level is also very com­pressive but at these frequencies, such as 19 kHz (Fig. 1A), the whole relationship is less sensitive and the BM displacements saturate when they approach about 8 nm. In fact, BM displacement level functions start to satu­rate at increasingly lower magnitudes of BM displace­ment with increasing frequency for frequencies just below the CF. For frequencies more than about one-half octave below CF, BM displacements do not saturate for levels below 90 dB SPL. Thus, the level and frequency- dependent properties of BM displacements in response to pure tones reported here are in accord with earlier measurements (Sellick et al., 1982; Cooper and Rhode,1992) and closely resemble the phasic voltage responses of OHCs recorded from the basal turn of the guinea pig cochlea in situ, (Russell et al., 1995).

As an estimation of the amount of variation in the measurements, the means and standard deviations of

10 successive groups of measurements of BM displace­ment at two different frequencies are shown for a sin­gle preparation in Figure IB. In general, twice the standard deviation of the measurements was less than 10% of the mean. This technique effectively smooths the data and removes notches and apparent nonmonotonic changes that sometimes appear in the data when only single measurements are made (Fig. 2B, low frequency [LF], and 3B, LF) Unfortunately this process was too time consuming to be used in the experiments described and all curves are shown as the on-line computed average of eight measurements.

Effect of Salicylate on Basilar Membrane Displacement

There was no observable effect on either tone-evoked re­sponses of the BM or the CAP audiogram during perfu­

B1

"e

Stimulus level (dB SPL)

FIGURE 2 BM displacement as a function of level in three different preparations (A, B, C) at the characteristic frequency (CF, 15 kHz), at 1 kHz (low frequency [LF]) and at a frequency above the CF (high fre­quency [HF]). Open circles, during control perfusions of the scala tympani with artificial perilymph, solid circles, during perfusion with 2.5 mM sal­icylate; and open diamonds after washout with artificial perilymph. SPL = sound pressure level.

SALICYLATE AND BASILAR MEMBRANE DISPLACEMENT 143

sion of the scala tympani in the basal turn of the cochlea with artificial perilymph. However, perfusion with mil- limolar concentrations of salicylate in artificial peri­lymph caused reversible changes in the mechanical responses of the BM to tones. For frequencies around the best or CF of the recording site (14 to 19 kHz), perfusion with concentrations of either 2.5 or 5 mM salicylate re­duced the magnitude of tone-evoked BM displacements and shifted the displacement-stimulus level functions along the level axis in the direction of higher stimulus levels by up to 40 dB (Figs. 2A,B,C,3A,B, CF). The sali­cylate-induced reduction in sensitivity of the level-in- tensity functions for frequencies around the tip was reversible and recovery to the original control conditions was achieved within 20 to 30 minutes when the salicy­late perfusion was replaced by artificial perilymph. Perfusion with 2.5 mM salicylate produced reversible de­sensitization of between 20 and 40 dB of tone-evoked re­sponses to HFs above CF which are similar to those seen at the CF (Fig. 2A,B,C, HF). Perfusion with 5 mM con­centrations produced more variable results. In four of five cases, it caused a reversible decrease of about 15 dB in the sensitivity of BM displacement (Fig. 3B,HF). In one case (Fig. 3A, HF), perfusion with 5 mM salicylate had little apparent effect on the sensitivity of the BM re­

sponse, but the response became sensitized by about 20 dB following washout with artificial perilymph, and this effect was not reversible within the 50-minute time scale of the experiment. For LFs well below the CF, that is, be­tween 1 and 4 kHz, perfusion with 2.5 mM salicylate caused a 10 to 25 dB sensitization that usually (Fig. 2B,C, LF), but not always (Fig. 2A,LF), recovered following washout with artificial perilymph. In all but one of five preparations perfusion with 5 mM salicylate caused a desensitization that changed to sensitization following washout (Fig. 3B,LF). In one preparation (Fig. 3A,LF), perfusion with 5 mM salicylate had only a slight effect on the 1 kHz BM responses. However, following washout with artificial perilymph, they became more sensitive by about 10 dB for levels below 70 dB SPL.

Effect of Salicylate on Basilar Membrane Tuning

Iso-response, frequency-tuning curves were derived from the displacement-level functions. A displacement of 1 nm was chosen as the criterion for the iso-response tuning curve because this closely corresponded to the threshold of neural excitation in the cochlea. In the ex­amples shown in Figure 4, it can be seen that the net ef­fect of the salicylate perfusion is to cause a 25 to 45 dB

Stimulus Level (dB SPL)

FIGURE 3 Basilar membrane (BM) displacement as a function of level in two differ­ent preparations (A, B) at the characteristic frequency (CF, 15 kHz), at 1 kHz (low fre­quency [LF]) and at a frequency above the CF (high frequency [HF]) Open circles, during control perfusions of the scala tympani with artificial perilymph; solid circles, during per­fusion with 5 mM salicylate; and open diamonds, after washout with artificial perilymph. SPL = sound pressure level.

144 E. MURUGASU and I. J. RUSSELL

Frequency (kHz) Frequency (kHz)

FIGURE 4 One nanometer isothreshold basilar membrane (BM) displacement tuning curves in four preparations (A, B, C, D). Open circles, during control perfusions of the scala tympani with artificial perilymph; solid circles, during perfusion with ei­ther 2.5 mM salicylate (A, B) or 5 mM salicylate (C, D); and open diamonds, after washout with artificial perilymph. SPL = sound pressure level.

desensitization of the tip of the tuning curve and a shift of the tip to lower frequencies. The changes in the tip region are complicated and the desensitization of the tip, in most cases, is complemented by an increased sen­sitivity to frequencies one-half octave below the tip. Following the subsequent washout of the salicylate with artificial perilymph, the original tip may be almost com­pletely restored but the secondary tip may remain (Fig. 4B) or, indeed, only appear after the washout (Fig. 4C). In Figure 4B, the action of salicylate resulted in only a moderate change of about 10 dB in the sensitivity of the tip of the tuning curve, but it caused profound changes to the shape of the tuning curve, largely through the ap­pearance of a notch in the tip of the curve and through the increased sensitivity of the BM response one-half of an octave below the tip. The LF tail of the tuning curve was reversibly sensitized when the scala tympani was perfused with 2.5 mM salicylate (Fig. 4A,B) but perfu­sion with 5 mM salicylate caused either no apparent change (Fig. 4C) or an increase in the threshold of the LF tail (Fig. 4D). In both cases, the tail became sensitized following washout with artificial perilymph.

A summary of the effects of perfusing the scala tym­pani of the basal coil with a 2.5 mM salicylate on the fre­

quency selectivity of the BM in the 15 kHz region in seven different cochleas where it was possible to obtain full tuning curves before, during, and after scala tym­pani perfusion with salicylate is shown in Table I. The HF and LF slopes of the tuning curves under the con­trol conditions were similar to those reported previ­ously (Sellick et al., 1982) The Q]0dBs were somewhat sharper, and the tip to shoulder measurements were similar to those of OHC receptor potential tuning curves, (Kossl and Russell, 1992) Following salicylate perfusion, the mean desensitization of the tips was al­most 30 dB, the mean downward shift in the CF was 2 kHz, the Q10c|b was reduced to about a third and the sen­sitivity of the tail (1 nm threshold at 2 kHz) became sen­sitized by almost 10 dB.

Effects of Salicylate on Cochlear Microphonics Measured at the Round Window

Salicylate perfusion of the scala tympani caused changes in the magnitude of the CM recorded at the round window, which were not tightly correlated with salicylate-induced changes in the BM responses mea­sured simultaneously from the same preparation (com­

SALICYLATE AND BASILAR MEMBRANE DISPLACEMENT 145

TABLE IComparison of Frequency Tuning at the 15 kHz Place Before and After Perfusion _______________ with 2.5 mM Salicylate

Artificial Perilymph SalicylateNumber of preparations 1 nm threshold at tip Characteristic frequency of tip 1 nm threshold at 2 kHz Tip to shoulder (mean level

between 1 and 8 kHz) Low-frequency slope High-frequency slopeQlOdB

726.4 ± 10.6 dB SPL*15.2 ± 0.4 kHz68.3 ± 5.2 dB SPL

46.7 ± 9.2 dB SPL115.2 ± 32.9 dB/octave217.3 ± 26.1 dB/octave

10.6 ± 0.9

754.8 ± 5.5 dB SPL

13 ±1.1 kHz59.4 ± 4.4 dB SPL

23.5 ± 11.5 dB SPL 33.2 ± 12.4 dB/octave

140.8 ± 56.6 dB/octave 3.6 ±1.4

* SPL = sound pressure level

pare figs. 2 and 3 with figs. 5 and 6). The CM recorded at 15 kHz, which corresponds to the CF of the BM mea­surements, maybe enhanced (Fig. 5 A) or reduced (Figs. 5B, 6A,B) by perfusion with either 2.5 mM (Fig. 5) or 5 mM (Fig. 6) salicylate. Following washout with artificial perilymph, the magnitude of the CM may be increased (Fig. 5A,B) or decreased (Fig. 6A,B) when compared to preperfusion controls. These observations for the CM were not shared by the BM responses at 15 kHz (CF), which were always reduced by perfusion with salicy­late and recovered when the perfusate was restored to artificial perilymph (Figs. 2,3). CM responses recorded to 1 kHz tones are believed to reflect the electrical and mechanical responses of OHCs in the basal turn of the cochlea, (Patuzzi et al., 1989). However, salicylate-in­duced changes in CM response to 1 kHz tones were not correlated with salicylate-induced changes in BM re­sponses to tones at either the CF (15 kHz) or to 1 kHz tones. In general, perfusion with salicylate caused a re­duction in the magnitude of the CM at 1 kHz, whereas washout with artificial perilymph usually caused an in­crease in magnitude of the CM relative to preperfusion levels (Fig. 5A,B) for tone levels above 70 dB (Figs. 5A, 6A) and 90 dB (Fig. 5B). For levels below this, the effect of salicylate was completely reversible. In Figure 6A, perfusion with 5 mM salicylate caused irreversible changes in the 15 kHz CM recorded at the round win­dow but reversible changes in the mechanical responses at the CF (Fig. 3A,C,F).

Time Course o f R esponse to and Recovery from Salicylate Perfusion

In this set of experiments, instead of perfusing the BM continuously with either artificial perilymph or salicy­late solutions, the scala tympani was perfused for 2 min­utes with either a 5 or 10 mM solution of salicylate at a rate of 2.5 |0.L/ minute without subsequent washout with artificial perilymph, and the time course of the effects of this perfusion on the responses of the BM, CM, and CAP

were recorded to tones at the CF of the BM measure­ments and at 1 kHz. The time course of response to, and recovery from, 2 minutes of salicylate perfusion of both the CAP and the 1 nm BM response thresholds in one preparation is shown in Figure 7. In this and four simi­lar experiments, the time courses for recovery of the CAP and BM response thresholds were different. In all of the five experiments the CAP threshold did not re­cover within 30 to 40 minutes, which was the time course

Stimulus level (dB SPL)

FIGURE 5 Cochlear microphonics (CM) at 1 and 15 kHz as a function of level in two preparations (A, B). Open circles, dur­ing control perfusions of the scala tympani with artificial peri­lymph; solid circles, during perfusion with 2.5 mM salicylate; and open diamonds, after washout with artificial perilymph. SPL = sound pressure level.

146 E. MURUGASU and P J. RUSSELL

Stimulus level (dB SPL)FIGURE 6 Cochlear microphonics (CM) at 1 and 15 kHz as a function of level in two preparations (A, B). Open circles, dur­ing control perfusions of the scala tympani with artificial peri­lymph; solid circles, during perfusion with 5 mM salicylate; and open diamonds, after washout with artificial perilymph. SPL = sound pressure level.

Time (minutes)FIGURE 7 Time courses of response to and recovery from 2- minute perfusion of 10 mM salicylate of the scala tympani (in­dicated by open rectangle) beginning 12 minutes after gathering baseline measurements of the 1 nM basilar membrane (BM) dis­placement threshold and compound action potential (CAP) de­tection threshold to tones at 19.5 kHz. SPL = sound pressure level

for total recovery of the BM response threshold and sometimes did not recover within 2 hours after the ex­periment was terminated. Magnitude level functions for CM at 1 and 15 kHz and BM displacement at 15 and 16 kHz (the CF was between these two frequencies) for a single preparation taken at intervals before, during, and following a 2-minute perfusion of 5 mM salicylate are shown in Figure 8. Salicylate perfusion had no appar­ent effect on the 1 kHz CM response for levels below about 80 dB SPL and suppressed the CM above this level but without full recovery within the duration of the ex­periment. The salicylate perfusion augmented the 15 kHz CM at all levels below about 95 dB SPL and recov­ery was complete within 15 minutes for levels below 60 dB SPL, but there was no full recovery within 40 min­utes for levels above this. In contrast, salicylate perfu­sion caused a very strong reduction (30 to 45 dB compared with 8 dB for the CM) in BM displacement to15 and 16 kHz tones, which are frequencies close to the tip of the tuning curve (Fig. 9). These responses fully re­covered within 35 minutes (Figs. 7,8) and were notice­ably augmented to moderate and high level tones above about 60 dB SPL (Fig. 8), although there was no im­provement in detection threshold (Fig. 9).

DISCUSSION

Perfusion of millimolar concentrations of salicylate into the scala tympani causes reversible changes to tone- evoked displacements of the BM. These include a de­crease in the sensitivity of the tip of the tuning curve by up to 40 dB, displacement of the tip to lower frequen­cies, and sensitization by up to 20 dB of the mechanical responses of the BM to frequencies one-half octave below the tip and in the tail of the tuning curve.

Salicylate has been implicated in blocking the volt­age-dependent capacitance and motility of OHCs, (Ashmore, 1989; Shehata et al., 1990,1991; Tunstall et al.,1994). A reduction in electromechanical feedback to the cochlear partition could account for the changes we have observed close to the tip of the tuning curve. Indeed, de­sensitizing and broadening of the tip, a shift of the tip to lower frequencies, and sensitization of the BM re­sponses to frequencies one-half octave below the CF would all be consistent with a reduction in the gain of the OHC feedback, as has been postulated from models of cochlear frequency tuning mechanism with active feedback, (e.g. Neely, 1993). In this model the gain and the phase of the OHC feedback are crucial for IHC ex­citation. The gain of the feedback directly influences the magnitude of the displacement of the OHC hair bun­dles and their effect on the relative shear displacement between the tectorial membrane and the cuticular plate in the region of the IHC bundles. The phase of the OHC feedback determines whether the shear displacements of the IHC hair bundles are attenuated or amplified. As first proposed by Mountain et al., 1983, and shown by

SALICYLATE AND BASILAR MEMBRANE DISPLACEMENT 147

30 40 50 60 70 80 90 100 30 40 50 60 70 80 90 100

Stimulus Intonsity (dB SPL) stimulus Intensity (dB SPL)

FIGURE 8 The magnitude of the cochlear microphonics (CM) at 1 and 15 kHz (upper panels) and basilar membrane (BM) dis­placement at 15 and 16 kHz (lower panels) recorded from a sin­gle preparation at intervals (indicated by symbols) following a 2-minute perfusion of the scala tympani with 5 mM salicylate. SPL = sound pressure level.

direct measurements of OHC receptor potentials made in situ (Kossl and Russell, 1992), the phase of the recep­tor potential at CF lags the BM displacement by 90°. This is 180° out of phase with BM damping and hence pro­vides "negative damping" (amplification) to the cochlear partition (Kim et al., 1980). For frequencies on the LF tail of the tuning curve, the phase of the OHC feedback is in phase with BM displacement and shear displacement between the tectorial membrane and the cuticular plate is attenuated in the region of the IHC hair bundles. For frequencies well below the CF, IHCs are only excited at levels when the OHC receptor potentials are beginning to saturate and the effective gain of the OHC feedback is at a minimum (Kossl and Russell, 1992). This finding is in accordance with Neely's 1993 model because a reduction in gain would increase the relative shear between the tympanic membrane and cu­ticular plate. Such a reduction in the gain of OHC feed­back could account for the increased sensitivity that has been observed in the tail of IHC and neural tuning curves (Russell and Sellick, 1978; Salvi et al., 1983; Robertson, 1982; Liberman and Dodds, 1984; Versnel et

Stimulus Frequency (kHz)

FIGURE 9 One nanometer isothreshold tuning curve from same preparation as in Figure 8. The solid circles indicate the thresholds to 15 and 16 kHz tones 5 minutes after the start of the 2-minute perfusion of 5 mM salicylate into the scala tympani and the stars indicate measurements taken 35 minutes after the start of the perfusion. (Characteristic frequency, 15.5 kHz; Q10dB = 15.)

al., 1992) following mechanical damage to, and noise trauma of, the cochlea.

This explanation, alone, would not account for the increased sensitivity of the LF (1 to 8 kHz) BM responses reported here unless a reduction in the gain of the OHC feedback also increased the compliance of the cochlear partition. This would increase the transverse motion of the BM for frequencies in the tail region of the tuning curve. The consequences of increased compliance on BM tuning have been modelled by Allen 1990 where the predicted changes are similar to the salicylate-induced changes reported here. That is, a reduction in the stiff­ness of the cochlear partition causes a downward shift in the CF of the tuning curve, a broadening and desen­sitization of the tip, and a corresponding sensitization of the tail region of the tuning curve. Another reported action of salicylate perfusion on isolated OHCs is a re­versible reduction in turgor pressure (Shehata et al., 1990). This might be expected to lead to a change in the axial stiffness of the OHCs and this has been shown to be the case. In a recent study, both the axial stiffness and voltage-dependent motility of isolated OHCs was re- versibly reduced by up to 50% following perfusion with 5 mM salicylate. (Russell and, Schauz, in preparation). Such a reduction in stiffness of major elements in the cochlear partition has been proposed to account for a downward shift in the frequency of the distortion peak of acoustic emissions recorded from human subjects fol­lowing aspirin administration (Brown et al., 1993). It

148 E. MURUGASU and I. J. RUSSELL

could also account for the changes observed here, al­though it is not known if salicylate also acts to change the mechanical properties of other elements of the cochlear partition.

In summary, a reduction in both the feedback and the axial stiffness of OHCs would account for the effects of salicylate on BM tuning reported here. Interestingly, sal­icylate has been reported to reduce both OHC motility and axial stiffness.

Cochlear Microphonics as a Monitor of the Outer Hair Cell Transducer Current and Basilar Membrane Mechanics

It is generally conceded that the CM is generated by the OHCs (Dallos and Cheatham, 1976) and, although sim­ple to record from the round window, the CM is very difficult to interpret. This is because the CM is a field potential that is generated by many sources distributed along the length of the BM whose output is subject to phase cancellation and smearing due to the electrical length constant of the cochlear partition (Kohlloffel, 1970; Honrubia et al., 1973). Interpretation of the round window CM was compounded in the experiments re­ported here because only the scala tympani of the basal turn of the cochlea was perfused. Thus, the level-de- pendent effects of salicylate on the CM to 1 kHz tones were probably because the 1 kHz CM recorded at the round window originates from a wide region of the cochlea and not just the basal turn (see Fitzgerald et al.,1993). The CM is a voltage measured across one resis­tive arm of a complex electrical network often repre­sented as one variable resistance (the transducer channel) in series and in parallel with other components, including the basolateral membranes of the OHCs and the summed electrical impedance of the cochlear parti­tion (Davis, 1965; Honrubia and Ward, 1969; Schmiedt and Zwislocki, 1977). However, these other elements should not be considered passive, (see Corey and Hudspeth, 1983) and their effective electrical imped­ance can be modified by voltage, ion- and ligand-gated channels (see Oesterle and Dallos, 1990; Kolston and Ashmore, 1992; Dulon, 1994). Also, the action of salicy­late on these other elements is unknown. In fact, the sal­icylate-induced increases and decreases in the magnitude of the CM observed in the experiments re­ported here and elsewhere could be attributed to changes in the current flowing in each of the different arms of the complex resistive network from which the CM is measured. In the majority of cases reported here the information provided by the CM gives a false rep­resentation of the action of salicylate on the mechanical tuning of the BM. For example, in the preparation shown in Figure 4C, perfusion with a sequence of perilymph, 5 mM salicylate, then perilymph caused a reversible loss of BM tuning. However, according to the CM measure­ments, the same sequence caused a progressive and ir­

recoverable loss of CM (Fig. 6B). One could interpret this finding as indicating that salicylate had irreversibly blocked the OHC transducer conductance, although there is direct evidence to the contrary (Kimitsuki et al.,1994). It is more likely that the perfusion effectively shunted current from the segment of the network across which CM is measured. Small changes in the relative impedance of different elements of the network might have profound consequences. For example, the large augmentation of the CM observed by Fitzgerald et al. 1993 (Fig. 5A) during salicylate perfusion might be ac­counted for by slight changes in the balance of the net­work, which increases the current flow in the CM arm of the network. These increases in CM are much larger than can be accounted for by an increase in the potas­sium conductances of the OHC basolateral membranes (Stypulkowski, 1990; Fitzgerald et al., 1993).

In conclusion, although round-window CM is easy to measure, it is too complex a signal to be used as a monitor of cochlear mechanics and OHC activity.

Salicylate Has Opposing Effects on Outer Hair Cell Voltage Responses and Basilar Membrane Mechanics

The discrepancies between laboratories on the attrib­uted action of salicylate on CM and the considerable variation in level and time-dependent effects on both the CM and BM displacement reported here may be be­cause salicylate has at least two opposing effects on OHCs. By comparison with studies on isolated OHCs, these effects may be a rapidly acting, reversible reduc­tion in electromotility, voltage-dependent capacitance, and stiffness of the OHCs (Shehata et al., 1990; Tunstall et al., 1993; Schauz and Russell, in preparation) and a slowly acting irreversible increase in the basolateral membrane conductances (Shehata et al., 1990). A de­crease in both the OHC feedback and stiffness might be expected to cause a decrease in the CM and BM dis­placement at the CF, whereas an increase in basolateral conductance might be expected to increase the magni­tude of the CM and possibly BM displacement if the in­creased conductance resulted in an increased driving voltage for the OHC motility. Under certain conditions, the two effects may cancel each other so that salicylate perfusion has no apparent effect on BM mechanics. However, an augmented response was then observed following washout with artificial perilymph (Figs. 3A, 4C) which may be due to a slower recovery of the con­ductance increase. A maintained conductance increase may also account for observations that salicylate-in- duced suppression of CM is followed by CM augmen­tation after washout (fig. 5A, 5B). Whether one observes a salicylate-induced augmentation (Fig. 2C,LF) or a suppression (Fig. 2C, HF) of BM displacement or CM may depend on a fine balance in the action of salicylate on the electrical and mechanical properties of the OHCs.

SALICYLATE AND BASILAR MEMBRANE DISPLACEMENT 149

Adaptation in Outer Hair Cells

The time course of the change in CM, CAP, and BM me­chanics from the onset of salicylate perfusion is about 6 minutes, which is similar to the time course of the sali­cylate-induced reduction in motility, turgor pressure, and axial stiffness of OHCs in vitro (Shehata et al., 1990• Schauz and Russell, in preparation). However, the du­ration of the salicylate-induced changes in the CM and CAP far outlast those seen in the BM mechanics. The lack of correlation between the time course of the action of salicylate on the CAP and BM mechanics is not sur­prising because it is known that salicylate has an inde­pendent action on transmitter release at the afferent synapse (Evans and Borerwe, 1982; Meikle and Chamell, 1994). The long-term changes in CM represent changes in the current flow through the OHC transducer conductance may be a result of conductance changes in elements both in series and possibly in parallel with the OHC transducer conductance. If this is so, then the find­ing that the BM mechanics can recover while the net flow of current through the transducer conductance remains altered may indicate that the OHCs are capable of adapt­ing to minor changes in their electrical environments. That is, it is proposed that in response to long-term changes in membrane potential the OHCs undergo me­chanical changes that adjust the operating point of the hair cell transducer to optimize their sensitivity to BM displacement. In this respect it has been suggested that basal turn OHCs may be continuously adjusting to op­timize the sensitivity and frequency selectivity of the cochlea (Russell et al., 1986; Russell and Kossl, 1991;1992). Current injection into OHCs alters the direct current component and gain of the OHC receptor po­tential by shifting the operating point (Russell and Kossl, 1991). Furthermore, it has recently been shown that fol­lowing a reduction of endocochlear potential, and hence a reduction in current flow through the OHC transducer conductance, acoustic distortion products recover be­fore the endocochlear potential is fully restored (Mills et a l, 1993). It seems that there is increasing evidence that the operating point of the OHC transducer is con­tinuously adjusted to optimize the cochlea's perfor­mance.

ACKNOWLEDGEMENTS

We thank James Hartley for designing and building electronic ap­paratus and Wally Barnett for the mechanical construction of the interferometer. For comments on the manuscript, we thank Ann Brown, Gerhard Frank, Manfred Kossl, and Guy Richardson. This research was supported by the Medical Research Council, Hearing Research Trust, Royal Society. E. Murugasu is supported by a Lee Kuan Yew Scholarship.

REFERENCES

Allen, J. B., Dallos, P., Geisler, C. D., Mathews, J. W., Ruggero, M.

A., & Steele, C. R. (eds). Modelling the noise damaged cochlea. In: The Mechanics and Biophysics of Hearing. Berlin: Springer-Verlag, pp. 324-332.

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.

Brown, A. M., Williams, D. M., & Gaskill, S. A. (1993). The effects of aspirin on cochlear mechanical tuning. / Acoust Soc Am, 93,3298-3307.

Cooper, N., Rhode, W. S. (1992). Basilar membrane mechanics in the hook region of the cat and guinea pig cochlea. Sharp tun­ing and nonlinearity in the absence of basilar membrane po­sition shifts. Hear Res, 63 ,163-190.

Corey, D. P. & Hudspeth, A. J. (1983). Analysis of the microphonic potential of the bullfrog's sacculus. J Neurosci, 3 ,942-961.

Dallos, P. & Cheatham, M. A. (1976). Production of cochlear po­tentials by inner and outer hair cells. / Acoust Soc Am, 60, 510-512.

Davis, H. (1965). A model for transducer action in the cochlea. Cold Spring Harbor Symp. Quant. Biol., 30 ,181-190.

Dulon, D. (1994). Calcium signals and mechanical properties of isolated Deiters cells of the organ of Corti. In: Flock A, Ulfendahl M, (eds): Active Hearing (in press).

Evans, E. F. (1979). Neuroleptanaesthesia for the guinea pig: an ideal anaesthetic procedure for long term physiological stud­ies of the cochlea. Arch Otolaryngol, 105,185-186.

Evans, E. F. & Borerwe, T. A. (1982). Ototoxic effects of salicylates on the responses of single cochlear nerve fibres and cochlear potentials. Br J Audiol, 16,101-108.

Fitzgerald, J. J., Robertson, D., & Johnstone, BM. (1993). Effects of intra-cochlear perfusion of salicylates on cochlear micro- phonic and other auditory responses in the guinea pig. Hear Res, 67 ,147-156.

Honrubia, V. & Ward, P. H. (1969). Dependence of the cochlear microphonics and the summating potential on the endo­cochlear potential. J Acoust Soc Am, 46,388-392.

Honrubia, V., Strelioff, D., & Ward, P. H. (1973). A quantitative study of cochlear potentials along the scala media of the guinea pig. J Acoust Soc Am, 54,600-609.

Jastreboff, P. J. & Sasaki, C. T. (1994). An animal model of tinni­tus: a decade of development. Am. J Otol, 15,19-27.

Kim, D. O., Neely, S. T., Molnar, C. E., & Mathews, J. W. (1980). An active cochlear model with negative damping in the cochlear partition: comparison with Rhode's ante- and post­mortem results. Scand Audiol Suppl, 9, 63-81.

Kimitsuki, T., Nakagawa, T. G., Hisashi, K , Komune, S., & Uemura, T. (1994). The effects of ototoxic drugs on mecha- noelectric transduction in chick cochlear hair cells. Euro Arch Otorhinolaryngol, 251,53-56.

Koelink, M. H., Slot, M., de Mul, F. F., Greve, J., Graaff, R., Dassel, A. C. M., & Aardnoudse, J. G. (1992). Laser Doppler ve- locimeter based on the self-mixing effect in a fibre-coupled semiconductor laser: theory. Appl Optics, 18, 3401-3408.

Kohlloffel, L. V. E. (1990). Longitudinal amplitude and phase dis­tribution of the cochlear microphonics (guinea pig) and spa­tial filtering. J Sound Vib, 11,325-334.

Kolston, P. J. & Ashmore, J. F. (1992). Action of ATP on isolated

150 E. MURUGASU and I. J. RUSSELL

Hensen's cells of guinea pig cochlea investigated by calcium imaging and whole-cell recording. J Physiol (Lond), 446,389P.

Kossl, M. & Russell, I. J. (1992). The phase and magnitude of hair cell receptor potentials and frequency tuning in the guinea pig cochlea. J Neurosci, 12,1575-1586.

Liberman, M. C. & Dodds, L. W. (1984). Single neuron labelling and chronic cochlear pathology. Ill Stereocilia damage and alterations of threshold tuning curves. Hear Res, 16,55-74.

Long, G. R. & Tubis, A. (1988). Modification of spontaneous and evoked acoustic emissions and associated psychoacoustic microstructure by aspirin consumption. / Acoust Soc Am, 84, 1343-1353.

McFadden, D. & Plattsmier, H. S. (1984) Aspirin abolishes spon­taneous otoacoustic emissions. J Acoust Soc Am, 76,443-448.

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

Meikle, M. B. & Chamell, M. G. (1994). Salicylate-induced changes in asynchronous neural activity recorded from the round window. Assoc Res Otolaryngol Abstr, 17,394.

Mills, D. M., Norton, S. J., & Rubel, E. W. (1993). Vulnerability and adaptation of distortion product otoacoustic emissions to en- docochlear potential variation. J Acoust Soc Am, 94, 2108-2122.

Mountain, D. C., Hubbard, A. E., & McMullen, T. A. (1988). Electromechanical processes in the cochlea. In: de Boer, E., Viergever, M. A. (eds): Mechanics o f Hearing. Delft, The Netherlands Delft University Press, pp. 119-126.

Neely, S. T. (1993). A model of cochlear mechanics with outer hair cell motility. J Acoust Soc Am, 94 ,137-146.

O'Neil, M. P., Beardon, A. (1993). The amplitude and phase of basilar membrane motion in the turtle measured with laser- feedback interferometry. In: Duifhuis, H., Horst, J. W., van Dijk, P., van Netter, S. M., (eds): Biophysics of Hair Cell Sensory Systems. Singapore: World Scientific, pp. 398-405.

Oesterle, E. C. & Dallos, P. (1990). Intracellular recordings from supporting cells in the guinea pig cochlea: DC potentials. / Neurophys, 64, 617-636.

Patuzzi, R., Yates, G. K., & Johnstone, B. M. (1989). Outer hair cell receptor currents and sensorineural hearing loss. Hear Res, 42,47-72.

Penner, M. J. (1989). Aspirin abolishes tinnitus caused by sponta­neous otoacoustic emissions: a case study. Arch Otolaryngol Head Neck Surg, 115,871-875.

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

Robertson, D. (1982). Effects of acoustic trauma on stereocilia

structure and spiral ganglion cell properties in the guinea pig cochlea. Hear Res, 7,55-74.

Russell, I. J., Cody, A. R., & 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.

Russell, I. J. & Kossl, M. (1991). The voltage responses of hair cells in the basal turn of the guinea pig cochlea. J Physiol (Lond), 235,493-511.

Russell, I. J. & Kossl, M. (1992). Sensory transduction and fre­quency selectivity in the basal turn of the guinea pig cochlea. Phil Trans R Soc Lond B, 336,317-324.

Russell, I. J., Kossl, M., & Murugasu, E. (1995). A comparison be­tween tone-evoked voltage responses of hair cells and basi­lar membrane displacements recorded in the basal turn of the guinea pig cochlea. In: Advances in Hearing Research: Proceedings of the Xth International Symposium on Hearing, Manley, G. A., Fasti, H., Klump, G., Koppl, C., Oeckinghaus, H., (eds) Singapore: World Scientific, (in press).

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

Salvi, R., Hamemik, R. P., & Henderson, D. (1983). Response pat­terns of auditory nerve fibres during temporary threshold shift. Hear Res, 10,37-67.

Schmiedt, R. A. & Zwislocki, J. J. (1977). Comparison of sound- transmission and cochlear-microphonic characteristics in Mongolian gerbil and guinea pig. J Acoust Soc Am, 61, 133-149.

Sellick, P. M., Patuzzi, R., & Johnstone, B. M. (1982). Measurements of basilar membrane motion in the guinea pig using the Mossbauer technique. J Acoust Soc Am, 72,131-141.

Silverstein, H., Bernstein, J. M., & Davies, D. G. (1967). Salicylate ototoxicity: A biochemical and electrophysiological study. Ann Otol Rhinol Laryngol, 76,118-128.

Shehata, W. E., Brownell, W. E., Cousillas, H., & Imredy, J. P.(1990). Salicylate alters membrane conductance of outer hair cells and diminishes rapid electromotile responses. Assoc Res Otolaryngol Abstr, 13, 252.

Shehata, W. E., Brownell, W. E., & Dieler, R. (1991). Effects of sal­icylate on shape, electromotility and membrane characteris­tics of isolated outer hair cells from guinea pig cochlea. Acta Otolaryngol (Stockh), 111, 707-718.

Stypulkowski, P. H. (1990). Mechanisms of salicylate ototoxicity. Hear Res, 46 ,113-146.

Tunstall, M. J., Gale, J. E., 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, 75P-76P.

Versnel, H., Schoonhoven, R., & Prijs, V. S. (1992). A single-fibre and whole nerve responses to clicks as a function of inten­sity in the guinea pig. Hear Res, 5 9 ,138-156.