Effect of the middle ear reflex on sound transmission to the inner ear of rat

Itmlll ELSEVIER

Hearing Research 105 (1997) 171-182

Effect o f the middle ear reflex on s o u n d transmission to the inner ear o f rat P.K.D. Pilz a,,, j. Ostwald a, A. Kreiter b, H.-U. Schnitzler a a Lehrbereich Tierphysiologie, Universitiit Tiibingen, Morgenstelle 28, D-72076 Tiibingen, Germany b M a x Planck Inslitul fiir Hirnforschung, Frankfurt, Germany Received 30 May 1996; revised 5 November 1996; accepted 15 November 1996

Abstract

The effect of the acoustic middle ear reflex (MER) was quantified using electrodes chronically implanted in the middle ears of rats. Cochlear microphonics (CM) and middle ear muscle EMG were measured under light Ketamin anesthesia after stimulation with tone pulses of 5-20 kHz ranging between 75 and 120 dB SPL. With increasing intensity, the CM measured before the onset of the MER increased to a maximum amplitude and then decreased with higher SPLs. At 10 kHz this maximum was reached at 95 dB SPL, for other stimulus frequencies at higher SPLs. After a latency of 10-20 ms, CM to 10 kHz stimuli of 80-95 dB SPL were decreased by the attenuating action of the MER. The lowest threshold of the MER was also measured at 10 kHz (77 dB SPL in the mean). To stimuli greater than 100 dB SPL after a latency of 6-10 ms, the CM amplitude was increased. That this CM increase to intense stimuli is caused by the action of the MER was confirmed by control experiments such as cutting the tendons of the middle ear muscles. The CM decrease to stimuli below 100 dB SPL, as well as the increase to very intense stimuli, can be explained by sound attenuation caused by the MER, together with the nonlinear dependence of CM amplitude on stimulus level. The observed shift of the maxima of the CM input-output function by the MER to higher stimulus levels probably indicates an increase of the dynamic range of the ear.

Keywords: Cochlear microphonic potential; Acoustic reflex; Middle ear muscle reflex; Stapedius muscle; Electromyography; Rat

1. Introduction

The middle ear reflex (MER) is a contraction of the two muscles in the middle ear elicited by intense acoustic stimuli. Thereby the impedance of the ossicular chain in the middle ear is changed causing a frequency-dependent attenuation of the sound reaching the inner ear. The main function of the M E R seems to be the protection of the inner ear from noise damage (Moller, 1974; Borg, 1971; Borg and Moller, 1967; Borg et al., 1984). However, several factors argue against this theory. First, the latency of the M E R is thought to be too long to protect effectively against impact noise (e.g., Moller, 1974). Secondly, the M E R fatigues if the ear is exposed to loud sound stimuli over * Corresponding author. Tel.: +49 (7071) 297 4835; fax: +49 (7071) 292 618 and 294 634; e-mail: [email protected]

0378-5955/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 6 ) 0 0 2 0 6 - 7

a longer period of time (e.g., Avan et al., 1992; Wilson et al., 1984; but see also Borg and Moller, 1968; Borg and Nilsson, 1984). Finally, in some animals the attenuation found seems to be too small to account for an effective protection (Avan et al., 1992). Different experimental approaches have been used to measure this reflex (MMler, 1984). The M E R has been measured (1) by quantifying the induced change of the impedance in the outer ear channel (Jepsen, 1951; MMler, 1958; Borg and Moller, 1968; Borg, 1972a,b), (2) by measuring its effect on otoacoustic emissions (Whitehead et al., 1991), (3) by measuring the threshold shifts after strong acoustic stimulation with and without intact M E R (Zakrisson, 1975; Henderson et al., 1994; but see also Ryan et al., 1994), (4) by quantifying the electromyograms or the forces which accompany the contraction of the middle ear muscles (Carmel and Starr, 1964; Simmons, 1959; Moller, 1964), and

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(5) by measuring the attenuating effect of the M E R on cochlear microphonic potentials (Wever and Vernon, 1955; G a l a m b o s and Rupert, 1959; Moller, 1964; Murata et al., 1986). The first three methods can be applied in unoperated ears and thus be used in experiments with humans. Only the latter method, that is measuring cochlear microphonics (CM), offers a direct measure of the transfer change to the inner ear by changes in the middle ear. In a given situation (where the electrodes are placed and this place is unchanged) the input-output (I/O) function of CM can be very well reproduced (e.g., Berge et al., 1990). In the following experiment we quantified the effect of the M E R by measuring CM. The major aim of the present study was to measure the transmission change induced by the M E R on the reflex eliciting stimulus itself, over a wide intensity range and with frequencies in the optimal hearing range of the rat. Stimulation of one ear can elicit the M E R in the ipsilateral and in the contralateral ear. Therefore, in the literature either the crossed or the uncrossed M E R was investigated. As Green and Margolis (1984) point out, in most cases the M E R was not measured in the same ear where the reflex was elicited, but most available information on the characteristics of the M E R have been obtained by contralateral measurement. In this type of experiment, the M E R is acoustically elicited in one ear, and its effect measured in the other ear where the researchers are forced to use a second tone as probe stimulus. In the following experiment we measured the M E R in the same ear where this reflex was elicited, thereby quantifying its effect on the transfer of the eliciting stimulus itself. Therefore, we had to use test stimuli with high sound pressure levels (SPL), above the threshold of the MER. This is in contrast to most experiments using test stimuli of moderate intensities ( < 70-80 dB SPL), where the test tone is in the 'log-linear' range of the CM I/O function. Above 70 80 dB SPL CM amplitude no longer increases linearly with stimulus SPL, but reaches a m a x i m u m at about 100 dB SPL (e.g., Davis and Eldredge, 1959; Wever and Vernon, 1955; Dallos and Cheatham, 1976; Berge et al., 1990). In the present paper we show means to quantify transmission changes in the nonlinear range of the CM I/O function. C M are often measured in acute experiments, by recording from the round window of rodents with the bulla open. Open bulla conditions have the disadvantage that the resonance of the bulla is changed. In addition, during acute experiments there may be exudation to the middle ear due to deep and long anesthesia, which changes the impedance. We therefore chronically implanted electrodes to measure CM. We could thus measure CM after recovery of the animals, with the bulla closed, under either light anesthesia or with no anesthesia at all. Finally, in most experiments the M E R is quantified

by measuring its effect on a low-frequency tone. This is done because the M E R has a greater effect on the transmission of stimuli with low-frequency (Moller, 1965). However, we were interested in the effect of the M E R elicited with stimuli in the range where the rat hears best (Kelly and Masterton, 1977; Heffner et al., 1994) and, where the rat has the lowest threshold of another auditory reflex, the acoustic startle response (Pilz et al., 1987). In summary, we quantitatively measured transmission change over a wide range of stimulus intensities and frequencies due to middle ear muscle contraction. Possible other causes for this change were excluded by a series of qualitative experiments. We found a frequencyspecific continuous attenuation of sound transmission to the inner ear. Due to the shape of the C M I/O function this resulted in a decrease or increase (at very high intensities) of CM amplitude.

2. Materials and methods

Subjects were 16 female Sprague-Dawley rats weighing about 220 g. In 13 rats electrodes were chronically implanted bilaterally in the middle ear. Under Ketamin/ Xylazin anesthesia (100 mg/kg Ketamin/4 mg/kg Xylazin) the skin behind the pinna on each ear was laterally incised and the bulla was laid open by cautiously tearing the connective tissue and pulling the muscles in a blunt approach. Holes with a diameter of 1 m m were drilled into the bony walls of the bullae. The uninsulated tip of a teflon-insulated multistrand flexible steel wire electrode was placed at the rim of the round window, close to the bony shelf of the stapedius muscle. The electrodes were fixed and the bullae were closed with dental cement. The skin at the dorsal skull was cut along the midline and the underlying bone was cleared from tissue. A small gold-plated screw in the skull served as indifferent electrode. The electrode wires from the two ears were led subcutaneously to the top of the skull and the three electrodes were connected to a plug socket that was fixed to the skull and the screw with dental cement. All incisions were closed. In the remaining three rats CM were acutely measured with the bulla open. Chronic experiments were performed at least 4 days after surgery, quantitative measurements at least 5 days after surgery. After this time the acoustic startle threshold was measured to control for correct function of the rats hearing ability (Pilz et al., 1987). Due to the position of the electrodes, CM potentials and electromyograms ( E M G ) of the stapedius muscle could be recorded simultaneously. Potentials were amplified (Tectronix power module model T M 501) and the two components separated by high-pass (3 kHz, CM) and low-pass (2 kHz, E M G ) filtering (Krohn Hite filter

P.KD. Pilz et al./Hearing Research 105 (1997) 171-182

model 3550). The signals were stored on two D R channels (CM) and two F M channels (EMGs) of a tape recorder (Racal store 4D) with a bandwidth of 0.l-75 kHz (DR channels) and 0-5 kHz (FM channels). Signals were displayed on a storage oscilloscope (Tektronix model 5113) and analyzed with a frequency analyzer (Medav MOSIP). Examples of CM to 10 kHz stimuli displayed in Figs. 1, 3, 4 and 6 were filtered with a bandpass of 7-14 kHz, and the E M G example (Fig. 6) was filtered with a bandpass of 0.4-2 kHz. The main results described below were first measured qualitatively in awake, freely moving rats. In this case CM varied due to movement and different positions of the rats' ears relative to the loudspeaker. For better control of the stimulus intensity and quantitative evaluation, rats were anaesthetized by Ketamin (100 mg/kg) to a level where they still showed a blink reflex to an air puff to the cornea. If not otherwise indicated, the results are reported from these lightly anaesthetized rats. Stimuli were 100 ms tone pulses with rise and decay times of 0.4 ms shaped by a pulse generator (Pulsar, custom made) and produced by a gated frequency generator (Toellner model 7706). Stimuli were attenuated (Hewlett Packard attenuator model 350D), amplified (Krohn Hite, model DCA-50) and delivered with an interstimulus interval of 2 s by a loudspeaker (Motorola, model KSN 1025) positioned 20 cm in front of the rat's head. Stimulus intensity was measured with a 114" condenser microphone (Bruel and Kjaer, model 4135) located between the rat's ears and displayed with a measuring amplifier (Bruel and Kjaer, model 2606). Stimulus SPL is always given in dB relative to 20 gPa. The rat's body temperature was kept constant at 38°C (Harvard animal blanket control unit). To measure I/O functions, stimuli of 5, 7.05, 10, 14.1 and 20 kHz with intensities increasing from 75 to 120 dB SPL were presented in steps of 5 dB. Each stimulus combination was repeated 5 times. After reaching 120 dB SPL the frequency was changed by random order. There was a 1 min break presented when changing the frequency. For each rat CM amplitude was calculated as the mean for the five stimuli of each block. The effects of cutting the tendons of the middle ear muscles were tested with 7 and 10 kHz stimuli in three acutely prepared rats. For cutting the middle ear muscles rats were anaesthetized with Ketamin (100 mg/kg), Xylacin (4 mg/kg) and Chlorpromazine (5 mg/kg) and one bulla was opened by breaking a hole of several millimeters in diameter. The tendon of the m. tensor tympani was cut with very fine scissors, that of the m. stapedius with the tip of a sharp canula. The electrode was positioned in the same place as in the chronic experiments described above. To minimize influence of exudation in the middle ear, surgery and tests in this open bulla condition were finished as fast as possible. The effects of pentobarbital anesthesia (50

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mg/kg and 0.8 mg/kg Atropine) as well as contralateral stimulation were also tested each in three rats. One rat was stimulated with 50 ms long pulses of broadband noise (bandwidth: 5-20 kHz). The care of the animals and the procedure were approved by the Regierungspraesidium Tuebingen (ZP185).

3. Results 3.1. Action o f the M E R on C M amplitude

The effects of the M E R as judged from the change in the amplitude of CM potentials varies considerably with the intensity of the stimulus used. In the following we will first describe these effects in a typical example and then look for the quantitative aspects of this behavior in more detail. In Fig. 1 the time course of CM oscillograms in one rat is shown for 10 kHz tone pulses between 75 and 120 dB SPL. At 75 dB SPL CM amplitude stayed constant during the stimulus duration. Although there was no change in CM amplitude, often a m . stapedius E M G of low amplitude could be observed (not shown in Fig. 1). At higher stimulus levels, where CM amplitude changed, a m. stapedius E M G always occurred. We will first focus on the changes of CM amplitude shortly following the beginning of the EMG, that means the gross changes after a latency of 6 20 ms. The CM oscillations at 85 and 90 dB SPL, the short peaks at the beginning of CM at stimulus levels above 100 dB SPL, and the short CM dips (very low CM amplitude which with a latency less than 1 ms increased during the first 1.5-3 ms to a plateau) after the initial peak at 120 dB SPL will be treated in more detail later. For stimulus levels between 80 and 100 dB SPL, the CM amplitude was decreased after a latency of 10-20 ms. At higher SPLs, however, this amplitude change was reversed. At 105 120 dB SPL (Fig. 1) all CM envelopes began with a short peak. After this peak, CM amplitude decreased to a plateau for 6-10 ms (e.g., 110 dB SPL; Fig. 1), and then increased to a second, higher plateau. The relation between CM amplitude and stimulus SPL can be described quantitatively by I/O functions. We assumed that the changes in CM amplitude after 620 ms were caused by the MER. Therefore, we measured the CM amplitude of six rats to five stimuli of each frequency and SPL in two different time windows, one before the action of the MER, the other during this action. One I/O function was obtained by measuring the CM amplitude 3 ms after stimulus onset. At this point in time CM amplitude had reached the first plateau for all frequencies and SPLs. The initial peaks at high stimulus

P.K.D. Pilz et al./Hearing Research 105 (1997) 171 182

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levels (e.g., 105-120 dB SPL; Fig. 1) had disappeared and the dips at highest levels (e.g., 120 dB SPL; Fig. 1) h a d almost vanished and thus no longer influenced the first plateau. The following C M decrease (e.g., 80-100 dB SPL) or increase (e.g., 105 120 dB SPL) had a latency which in all cases was longer than 5 ms. Thus, the 3 ms C M amplitude was used as a control value, representing the C M amplitude without being influenced by the M E R . We called the I/O function obtained by measuring the 3 ms C M amplitude the 'control I/O function'. The control I/O functions are shown in Fig. 2A (solid lines). The data shown in Fig. 2 are the grand mean

o f the responses o f the six rats (which each got five stimuli). With increasing stimulus intensity the control I/O functions rose to a frequency-specific maximum, and then decreased with a further increase in stimulus intensity at all frequencies. These courses are bell shaped and rather symmetrical to the m a x i m u m o f the function. The mean o f individual m a x i m a was 95.8 (SD: +2.0) dB SPL at 10 kHz. At all other frequencies tested the m a x i m u m was located at higher intensities: 100.8 ( + 2 . 0 ) dB SPL at 7 kHz, 101.7 ( + 2 . 6 ) dB SPL at 14 kHz, 107.5 ( + 2.7) dB SPL at 5 kHz, and 111.5 ( + 3.4) dB SPL at 20 kHz. The highest m a x i m u m o u t p u t voltage, with a mean o f 594 gV, was found at

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Fig. 2. A : F r e q u e n c y a n d i n t e n s i t y c h a r a c t e r i s t i c s o f C M . F u l l lines: I / O f u n c t i o n s o f C M m e a s u r e d 3 m s a f t e r s t i m u l u s onset. D a s h e d c u r v e s : C M a m p l i t u d e s m e a s u r e d 30 m s a f t e r s t i m u l u s onset. B: A t t e n u a t i o n f u n c t i o n s . T h e a t t e n u a t i o n w a s c a l c u l a t e d f r o m the difference b e t w e e n the t w o c u r v e s in a ( c o r r e s p o n d i n g a r r o w s in A a n d B h a v e the s a m e l e n g t h . F u r t h e r e x p l a n a t i o n see text. M e a n o f 6 rats).

the lowest frequency tested (5 kHz). The amplitude of the m a x i m u m decreased with increasing frequency and fell below 200 pN at 20 kHz. A second I/O function was obtained by measuring CM amplitude 30 ms after stimulus onset. At this point in time the effect of the M E R on C M had fully developed. The 30 ms CM amplitude was located after the beginning of the second plateau. This second plateau was sometimes not constant during stimulus duration. At some stimulus levels and frequencies oscillations occurred (e.g., 85/90 dB SPL; Fig. 1), which influenced the amplitude measured after 30 ms. At higher SPLs the change of C M was not always fully completed after 30 ms, there were still comparatively small and slow C M changes visible after this point in time (e.g., 100 and 120

dB SPL; Fig. 1). Nevertheless the 30 ms CM amplitude was taken as a representative choice because the deviations at that point were relatively small. We called these I/O functions the '30 ms I/O function', and they are shown in Fig. 2A (dashed lines). F o r all frequencies at 75 dB SPL C M amplitude was unchanged by the M E R , so the 30 ms I/O functions were at the same value as the control I/O functions. Above a frequency-dependent SPL, these 30 ms I/O functions separated from the control I/O functions. At 7, 10 and 14 k H z the 30 ms I/O functions also reached a m a x i m u m (115.8 _+3.8 dB SPL, 110.4 _+4.0 dB SPL, and 112.9_+ 7.8 dB SPL, respectively) and decreased again. The m a x i m u m at these frequencies had the same amplitude as in the control I/O functions, but it was reached

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at higher SPLs (an analysis of variances showed a significant influence of rats: F~<27= 143.4, P < 0 . 0 0 1 , and of frequency, F2.27 = 26.2, P < 0.001, but no difference between the m a x i m u m voltage at 3 and 30 ms: FI,27= 1.123, P--0.298). At 5 and 20 kHz the 30 ms I/O functions increased monotonically up to the stimulus level of 120 dB SPL. F r o m the difference between the two I/O functions the attenuating effect of the M E R was calculated. F o r example at 10 kHz, 95 dB SPL the CM amplitude was decreased from 513 to 430 BV. In order to determine the corresponding change in SPL reaching the inner ear, it was necessary to know the stimulus intensity producing the latter CM amplitude (430 BV) under control conditions (Desmedt, 1962; Suga and Jen, 1975). The appropriate SPL, where the M E R does not influence CM, can be taken from the control I/O function measured at 3 ms; in the example a CM amplitude of 430 laV 3 ms after stimulus onset is evoked by 87 dB SPL. CM are therefore attenuated by an equivalent of 95 dB--87 d B = 8 dB. This amount of attenuation is represented by the left arrow in Fig. 2A, with an origin of 95 dB SPL/430 ~tV on the dashed line, and a length in units of the abscissa of 8 dB. At 7, 10 and 14 kHz at the highest SPLs the 30 ms I/O functions exceeded the maximum. In these cases points after the m a x i m u m of the 30 ms I/O function in Fig. 2A have to be compared with points after the m a x i m u m of the control I/O function to calculate the attenuation by the MER. For example, at 10 kHz 115 dB SPL, the amplitude rose from a mean of 191 gV up to 500 gV after 30 ms. 500 gV were elicited by 100 dB SPL stimuli without influence of the MER. Thus, the attenuation in this example is 1 1 5 - 1 0 0 = 15 dB, represented by the right arrow in Fig. 2A. The amount of attenuation by the M E R depended on stimulus intensity and frequency. This relation is described by attenuation functions. With the above-described method for each stimulus the attenuation was calculated, and its dependence on SPL is shown for each frequency in Fig. 2B. For all frequencies tested these attenuation functions showed an almost linear course. There was no change in this behavior when CM I/O functions passed the m a x i m u m C M amplitude

value. The measured decrease of CM amplitude at 20 kHz was very small compared to lower frequencies. Due to the low slope of the I/O function this small change still led to a considerable average attenuation at high SPLs. The linear part of the attenuation functions was extrapolated to an attenuation of zero to calculate the M E R threshold for the particular stimulus frequency for each individual. It was 77.3 ( + 2.5) dB SPL in the mean at 10 kHz, and higher for the other frequencies tested: 82.1 dB SPL (+5.7) at 7 kHz, 84.9 (_+5.2) dB SPL at 5 kHz, 86.7 (_+2.8) dB SPL at 14 kHz, and 104.2 (+10.4) dB SPL at 20 kHz. The steepness of the attenuation functions fell with rising stimulus frequency. The slope (or gain) was - 0 . 6 dB/dB at 5 kHz and fell to - 0 . 2 dB/dB at 20 kHz. The latency of the CM change, as well as the attenuation functions, were monotonic over the intensity range observed. The latency was estimated in some examples with 10 kHz stimulus frequency. The C M decrease at 80 dB SPL began after 18 ms on the average. With increasing stimulus level up to 100 dB SPL this latency shortened continuously to 11 ms. The CM increase had a latency of 10 ms at 100 dB SPL; in some rats at 100 dB SPL 10 kHz the CM increased while in others it decreased. This latency of change decreased to 8 ms at 120 dB SPL. Thus, the latency of the CM change decreased monotonically with increasing stimulus level irrespective of the direction of the CM change. The latency of the m. stapedius E M G in these rats (not shown in Fig. 1) also shortened continuously from 6.5 ( + 1.5) ms at 75 dB SPL to 4.5 (+0.3) ms at 100 dB SPL, and to 4 (+0.3) ms at 120 dB SPL. At the same time the amplitude of the m. stapedius E M G monotonically increased from 84 (_+ 59.6) gV at 75 dB SPL to 210 ( + 79.8) gV at 120 dB SPL. Although reliable m.

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stimuli of 80 90 dB SPL were no longer decreased and to stimuli above 100 dB SPL no longer increased. We also tested the influence of the crossed M E R on the CM change. A contralateral 10 kHz 99 dB SPL stimulus was used to activate an ipsilateral, crossed MER. (In control experiments it was shown that the acoustic crosstalk did not elicit an ipsilateral MER, and that the crossed M E R diminished CM amplitude to subthreshold stimuli of 70 dB SPL.) If a crossed M E R was elicited, the initial plateau of CM to ipsilateral 10 kHz 114 dB SPL tones was higher compared to the case without previously elicited MER. This further suggests that the M E R increases the CM amplitude to intense stimuli by attenuating the incoming sound. 3.2. Noise stimuli

stapedius E M G activity could be observed at 75 dB SPL, there was no transmission change at this intensity. While the attenuation function was linear, the E M G amplitude increased logarithmically with increasing stimulus SPL. We assumed that the above described changes in CM amplitude with a latency of 6-20 ms were caused by the acoustic MER, but cochlear efferents could also be involved. Therefore several tests were performed to prove the contribution of the middle ear muscles. The described changes of CM immediately vanished when the tendons of the middle ear muscles (m. stapedius and m. tensor tympani) were cut. After lesioning, the CM amplitude no longer decreased to stimuli below the maximum of the I/O function (Fig. 3A). At higher SPLs, CM started and stopped with a short peak. Between these two peaks the CM amplitude was constant during the stimulus duration (Fig. 3B), the CM amplitude was no longer increased. Thus, to stimuli with SPLs of 80-125 dB, the CM changes ascribed to the M E R with a latency of 6-20 ms were no longer observed. To get a hint for the relative influence of the two muscles, in one single case CM changes were looked at already after only an m. tensor tympani lesion. Almost no change compared to the unlesioned state was observed; however, in another animal after lesioning only the m. stapedius, the effect of the M E R was strongly diminished. This shows that at least in these cases the influence of the m. stapedius dominated. As shown in Fig. 4, the CM changes also vanished under pentobarbital anesthesia (50 mg/kg). At the beginning of pentobarbital anesthesia the change of CM envelope was still similar to Ketamin anesthesia. After 10 20 min CM envelopes under pentobarbital anesthesia began to differ from those measured with Ketamin. The decrease of CM amplitude to stimuli below the maximum and the increase to supramaximal stimuli became smaller. After about 60 min the effect of the pentobarbital anesthesia was maximal. Now CM to 10 kHz

To exclude influences of acoustic artifacts such as standing waves during sinusoidal stimulation, one rat was stimulated with noise pulses, and the results are shown in Fig. 5. CM envelopes to noise stimuli (band noise: 5-20 kHz) resembled those to tonal stimuli. To 95 dB SPL noise stimuli the CM amplitude was decreased by the M E R (Fig. 5A), and the whole frequency band was diminished (not shown in Fig. 5). At 125 dB SPL, CM first showed a peak followed by an initial plateau, and then an increase by the M E R (Fig. 5B). The frequencies between 7 and 20 kHz were enhanced. Thus, noise stimuli, where the responses should not be influenced by phase cancellation effects, produced CM envelopes and CM changes similar to tonal stimuli.

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Fig. 6. CM potentials and simultaneous electromyogram of the stapedius muscle of the same ear in an awake, freely moving rat. (a) Stimulus (115 dB SPL, 10 kHz; duration: 20 ms including 5 ms rise and decay times). (b) CM. (c) M. stapedius EMG. (d) Calculated C M envelope evoked by this stimulus for a system following passively the control I/O function in Fig. 2A (10 kHz) without reflex activity.

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3.3. Side effects In the following three features of the CM envelopes of Fig. 1 are addressed, which were not explained until now: CM oscillations, initial peaks and short initial dips of CM, which each occurred at certain stimulus intensities. At SPLs slightly above the threshold of the MER, CM amplitude oscillated (e.g., 85 dB SPL; Fig. 1; see also: Murata et al., 1986; Borg, 1971; Suga and Jen, 1975). The frequency of these oscillations shortened with increasing SPL (e.g., 90 dB SPL; Fig. 1). At still higher SPLs these oscillations became smaller and disappeared (e.g., 95/100 dB SPL; Fig. 1). These oscillations were largest at 5 kHz in all rats. They were less marked at higher frequencies and not visible at 20 kHz. Some small irregularities of the 30 ms I/O functions and the attenuation functions resulted from these CM oscillations, which sometimes affected the amplitude measured 30 ms after stimulus offset. This effect mainly influenced the 5 kHz curves (Fig. 2). At SPLs above the maximum of the I/O function, CM envelopes showed a short peak at the beginning (Fig. 1; 105-120 dB SPL; see also Figs. 3 and 5, 125 dB SPL; Fig. 4, 120 dB SPL), which always had almost the same value as the maximum plateau value at this frequency. We assumed that this peak was produced by CM passing the maximum of the I/O function during the short stimulus rise time. To get a better view of this effect, stimuli with prolonged rise time were used. Fig. 6A shows a 10 kHz, 115 dB SPL stimulus with a rise time of 5 ms. For this stimulus an example of a CM envelope and m. stapedius E M G measured in an awake rat are shown (Fig. 6B,C). The CM amplitude increased during the first 0.5 ms, and then decreased, reaching its minimum after 5 ms. Shortly after the begin of activity of the m. stapedius EMG, the CM amplitude increased again. For each point of the stimulus (Fig. 6A) the expected CM amplitude was calculated from the control I/O function in Fig. 2A. This theoretical CM envelope, which would be the output of this system following the stimulus along the control I/O function without any influence of the MER, is shown in Fig. 6D. For example, the maximum of the I/O function (95 dB SPL at 10 kHz) should be reached after 0.5 ms. The CM amplitude should then fall, reaching the initial plateau 5 ms after stimulus onset. Thereafter the CM amplitude should stay constant for the duration of the stimulus plateau. For the first 6 ms the theoretical values (Fig. 6D) agreed well with the actually measured ones (Fig. 6B). After this period measured CM increased. The CM increase shortly followed the start of activity in the simultaneously recorded E M G of the stapedius muscle (Fig. 6C). Therefore we conclude, that the initial part of the CM envelopes (before the influence of the MER) can be explained by a system passively following the

I/O function. (During the short rise times used in the other experiments, the SPL producing the maximum CM output was passed very fast, e.g., during only a quarter of a sine wave at 10 kHz, 120 dB SPL for the maximum SPL + 5 dB. Less than a full sine wave is probably to short for the full production of maximum CM amplitude, which would explain the lower amplitude of the initial peaks in Fig. 1, 110 120 dB SPL, compared with the initial amplitude at 95 dB SPL). It can be seen in Fig. 6D that in a passive system without influence of the M E R two peaks should occur, one at the beginning, the other at the end of the stimulus. The latter could normally not be observed, because the M E R influenced the CM too much (Fig. 6B). But it can be seen if the M E R is eliminated (Fig. 3). If the middle ear muscles are cut, at stimulus levels above the maximum of the I/O function two CM peaks occur: one at the beginning, and the other at the end of the stimulus (125 dB SPL; Fig. 3B). At 7 and 10 kHz, at the highest SPLs used (115 and 120 dB SPL), sometimes the initial peak was followed by a short dip of the amplitude. This dip recovered immediately, CM amplitude reached the initial plateau in general in less than 3 ms (Fig. 1, 120 dB SPL; Fig. 3, 125 dB SPL; Fig. 4, 120 dB SPL). Because the latency is much to short, we conclude that this dip was not caused by the MER.

4. Discussion

We measured changes in sound transmission caused by the M E R depending on stimulus frequency and intensity. Therefore, we quantified CM measured in rats with electrodes chronically implanted in the middle ear. At SPLs above 75 dB SPL, CM were decreased after about 10 20 ms depending on stimulus frequency (example: Fig. 1, 80 100 dB SPL). This decrease of CM amplitude is commonly attributed to the M E R which attenuates the sound reaching the inner ear (e.g., Galambos, 1956; Carmel and Start, 1964; Berge et al., 1990). To very intense stimuli, after 6-10 ms the CM amplitude increased (e.g., Fig. 1, 110 120 dB SPL). We will discuss later that this CM increase is also due to sound attenuation by the MER. The threshold of the M E R was lowest at 10 kHz (77 dB SPL) and rose to lower and higher frequencies. This seems to contradict the results of Murata et al. (1986) and of Berge et al. (1990) who describe the minimum threshold of the M E R in rats as being 3.5 kHz and 2.5 3 kHz, respectively. However, they measured the M E R on open bullae, which shifts the resonance characteristics of the ear to lower frequencies (Nutall, 1974) and changes the gain of the MER (Borg, 1972b). In humans (Moller, 1962a; Jepsen, 1951) the threshold curves of hearing and of the M E R run in parallel. Our data sug-

P.K.D. Pilz et al./Hearing Research 105 (1997) 171 182

gest that the same is true for the rat, where the lowest threshold of the M E R at 10 kHz corresponds with the lowest threshold of hearing and the lowest threshold of the acoustic startle reflex (Kelly and Masterton, 1977; Heffner et al., 1994; Pilz et al., 1987). The amount of sound attenuation by the M E R was calculated as the difference between the SPLs eliciting the respective CM amplitude before and during M E R activity. This procedure was hitherto applied to values below the maximum of the I/O function (Desmedt, 1962; Suga and Jen, 1975), and for our data extended to values above the I/O function maximum, i.e., to the range where the M E R enhanced CM. The resulting attenuation functions are shown in Fig. 2B. The amount of attenuation (gain) of the M E R was highest (0.6 dB/dB) at 5 kHz and fell with rising frequency. This corresponds with the results of other authors (e.g., Zakrisson and Borg, 1974; Pang and Peake, 1986; MMler, 1965; Borg, 1972b). The highest gain of 0.6 dB/dB at 5 kHz was lower than the gain of about 1 dB/dB reported by Wever and Vernon (1955). However, they used a lower frequency of 1 kHz which should result in a higher gain, and they measured on open bullae where the effect of the M E R is overestimated (Borg, 1972b). The CM increase at high stimulus levels is due to attenuation of sound transmission by MER, and a consequence of the physiological basis of CM production. Experimental conditions which are known to diminish the effect of the acoustic M E R also diminished the increase in CM amplitude to intense stimuli. Contralateral stimuli which elicit an ipsilateral M E R (e.g., Moller, 1962a,b; Borg and MMler, 1968; Borg, 1972b, 1973) increased the CM amplitude to intense stimuli. Pentobarbital, which is known to reduce the M E R (Simmons, 1964; Borg and Moller, 1967, 1975), eliminated the increase of CM amplitude to very intense stimuli almost totally. Cutting the tendons of the middle ear muscles eliminated the MER, and thus the increase to very intense stimuli. The absence of a CM increase after cutting the tendons of the middle ear muscles eliminates the possibility that the observed increase of CM was caused by olivocochlear efferents. Olivocochlear efferents can enhance CM (Fex, 1959, 1962), but this enhancement corresponds only to about 2 4 dB (Desmedt, 1962; Desmedt et al., 1971; Gifford and Guinan, 1987). In addition, the enhancement is maximal at low intensities in the range of 15-25 dB SPL (Fex, 1959; Gifford and Guinan, 1987). Olivocochlear efferents may contribute to CM increases at high stimulus levels under some conditions, but they were not responsible for the changes reported here. Therefore, one has to conclude that the observed CM increase to intense stimuli is caused by the MER. Further evidence comes from the continuous dependency of two M E R parameters on SPL. The sound at-

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tenuation increased with the SPL without discontinuity, and the approximate linearity was not disrupted when the change of CM envelope switched from decrease to increase (Fig. 2B). A linear dependence from stimulus level was also observed when studying the crossed M E R (Borg, 1972c), or the amplitude of another auditory reflex, the acoustic startle response (Davis and Wagner, 1968; Pilz and Schnitzler, 1996). The second parameter with a continuous course is the latency of the M E R effect on CM (at 10 kHz from 18 ms at 80 dB SPL, where CM are decreased, to 8 ms at 120 dB SPL, where CM are increased). Thus, sound attenuation by the M E R increased and latency of the M E R decreased continuously with increasing SPL, irrespective of whether they were measured in the range where M E R decreased or increased the CM amplitude. Acoustic artifacts are probably not the reason for the changes of CM amplitude attributed above to the MER. One rationale of the increase of the initial plateau of CM at highest SPLs could have been a decrease of harmonic distortion due to the attenuation of the M E R (Simmons, 1959). This was excluded by frequency analysis (not shown in the results section), where the frequency spectrum of CM was analyzed with short time windows of 2-5 ms at all frequencies and SPLs used. Like Davis and Eldredge (1959) and Hubbard et al. (1979) we observed a decrease of the number and relative intensity of higher harmonics at very high SPLs. The increase of CM amplitude to these stimuli was accompanied by an increase of distortion, not a decrease. Therefore, harmonic distortion should not be responsible for the observed increase of CM amplitude by the M E R at very high stimulus levels. A second rationale could have been standing waves in our experiments. This can be excluded, because the sound pressure measured at the rats ears was perfectly linear during the experiment. Standing waves, e.g., in the outer ear canal, could be excluded by stimulation with a noise band. Noise stimuli of 95 dB SPL were decreased by the MER, while stimuli with 125 dB SPL produced CM with a short peak at the beginning followed by a low initial plateau and then an increase of the amplitude by the M E R (Fig. 5). Our result that not only one frequency but a noise band of 7-20 kHz is enhanced makes standing waves as origin of the form of CM envelope unlikely. (However, this result was obtained from only one rat.) Therefore, the inverted U-shape of the CM I/O function and especially the CM decrease to stimuli above the I/O function maximum must have physiological reasons. Sound is transmitted to the inner ear with a linear I/O function up to at least 130 dB SPL (Guinan and Peake, 1967). Basilar membrane motion increases not linearly, but monotonically with rising SPL (Sellick et al., 1982; Johnstone et al., 1986). Basilar membrane motion is transduced to a receptor potential in outer hair cells (but, other ideas about the function

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of the basilar membrane also exist, see Braun, 1994). Because outer hair cells produce CM (Davis et al., 1958; Dallos et al., 1972) they remain as candidates which cause the decreasing flank of the I/O function at high SPLs. Dallos and Cheatham (1989), measuring intracellular potentials in outer hair cells, describe a decreasing response with increasing input at very high levels. This overstimulation of outer hair cells may cause the overstimulation of CM described in this paper. Other causes could have been standing waves and phase cancellation effects. CM in different turns of the cochlea show phase differences up to 1000 degrees (Tasaki et al., 1952, 1954). Patuzzi (1987) calculated a model which predicted a phase influence on CM saturation - - but of course only for differential recording of CM. Dallos et al. (1971) explain this type of overstimulation by saturation of the local receptors, while distant hair cells contribute to CM more and more; the phase difference increases with distance, and this may lead to an increasing cancellation. But it is not very likely that the arguments for phase cancellation hold against our results with noise stimuli (Fig. 5). As a conclusion, we have to assume that the inverted U-shape of the CM I/O function is not due to acoustic artifacts, but due to the nonlinear characteristics of the outer hair cells, which generate the CM. Both, the decreasing and increasing effects of the M E R on CM are a consequence of the course of the CM I/O function. CM rise to a maximum and fall with further augmentation of stimulus level (Fig. 2; Simmons, 1959; Davis and Eldredge, 1959; Berge et al., 1990). In the ascending limb of the I/O function attenuation leads to a decrease of CM amplitude. Logically, in the descending limb of the I/O function, above the maximum, attenuation of the stimulus leads to an increase of CM amplitude (right arrow in Fig. 2A). Two additional features of the CM envelopes in Fig. 1 deserve comment. First is the short peak at the beginning after stimulation above 100 dB SPL (other examples: Figs. 3 and 4). The peak is due to the fact that the SPL during the stimulus rise time shortly reaches the maximum of the control I/O function. The maximum was passed very fast during the short rise time used in the respective examples. That the CM amplitude indeed follows the I/O function during stimulus onset can be shown with a prolonged rise time (Fig. 6). The second feature is a short dip at the beginning of the CM to the 120 dB SPL stimulus in Fig. 1, and similar dips in Figs. 3 and 4 to the highest intensity in each case. These dips occurred upon very intense stimuli with a short rise time. CM then recovered with a very short latency during about 1.5-3 ms. This effect cannot be explained by the M E R or any other efferent reflex system because the time course is too short. The recovery typically began after less than 1 ms. This seems to be a process in the periphery, such as a change of the working point of the

outer hair cells. The symmetry of outer hair cell potentials and thus their DC potential is intensity dependent (Russel et al., 1986). This asymmetry develops over several cycles of the stimulus. Thus, one can speculate that at high input levels this mechanism may cause the described short dips of CM. The initial peak as well as the short dip can also be seen in the data of K6ssl and Vater (1985) who elicited otoacoustic emissions in the bat Pteronotus. They measured CM to 63 kHz tones where these bats hear very well. While the stimulus SPL at the bats tympanic membrane rises linearly, CM measured 1.5 ms after stimulus onset increase up to 90 dB SPL and decrease with further increasing SPL. Above 90 dB SPL the CM envelopes in Pteronotus peak at the begin and the end of 2 ms stimuli, and they show a short dip after the initial peak at 110 dB SPL. Thus, the described form of CM envelopes obviously is not restricted to rats. Three major functions are in general attributed to the middle ear muscles. First, they prevent desensitization, i.e., overloading of the sensory receptors of the cochlea, thereby maintaining a good level of sensitivity. This is best shown when the middle ear muscles are not activated reflexively by external sound, but by self-generated vocalization. For example, in echolocating bats middle ear muscles begin to contract shortly before the begin of a call and quickly relax after the call, before the comparatively faint echo reaches the ear (Suga and Jen, 1975). Second, in mammals it is well established that the action of the middle ear muscles decreases mainly lower frequencies (e.g., Wever and Vernon, 1955). This action of middle ear muscles has been found to result in an improvement of the discrimination of speech in low-frequency noise (Borg and Zakrisson, 1973). Due to the fact that during sound propagation in natural environment higher frequencies are relatively more attenuated (Michelsen, 1983; Michelsen and Larsen, 1983), it may also be useful for nonvocalizing organisms to diminish the relative energy of low-frequency noise by the M E R for better detection or analysis of high-frequency signals. Third, the middle ear muscles protect the ear from noise damage (Hilding, 1960; Zakrisson and Borg, 1974; Borg and Nilsson, 1984). The three functions of the middle ear muscles are incorporated in the desensitization, interference and injury-preventing theory (DIIP) by Borg et al. (1984). Our findings support these suggestions. First, the MER increases the dynamic range of the ear by attenuating the sound transmission, thereby shifting the maxima of the CM I/O functions to higher intensities. The threshold for the reflex was lowest at the frequency where the rat hears best, at l0 kHz. This may indicate that the MER effectively prevents desensitization. Second, in the rats best hearing range of 5-20 kHz low frequencies are more attenuated than high frequencies. This strengthens the general notion that

P.K.D. Pilz et al./Hearing Research 105 (1997) 171-182

t h e m i d d l e e a r m u s c l e s act as h i g h - p a s s filters, b e c a u s e in o u r s t u d y we m e a s u r e d in a h i g h e r - f r e q u e n c y r a n g e t h a n u s e d in m o s t e a r l i e r r e p o r t s , a n d d e s p i t e the n o t i o n in s o m e p u b l i c a t i o n s t h a t t h e r e l a t i o n o f a t t e n u a t i o n a n d f r e q u e n c y m a y be r e v e r s e d at h i g h f r e q u e n c i e s ( M o l l e r , 1964; N u t a l l , 1974; S i m m o n s , 1959). T h i r d , t h e s t e a d y i n c r e a s e o f a t t e n u a t i o n w i t h i n c r e a s i n g stimu l u s level s h o u l d h e l p to p r o t e c t t h e ear. O n e a r g u m e n t a g a i n s t t h e p r o t e c t i o n h y p o t h e s i s is the s m a l l i n f l u e n c e o f t h e M E R o n i m p a c t n o i s e d u e to t h e reflex l a t e n c y (Pickles, 1988). T h e v e r y s h o r t l a t e n c y f o u n d in o u r s t u d y f o r t h e C M c h a n g e o f o n l y 6 - 1 0 m s at h i g h S P L s c a n n o t p r e v e n t all effects o f i m p a c t n o i s e o n t h e ear, b u t t h e f a c t t h a t this is o n e o f the fastest reflexes in mammals supports the protection hypothesis. I n s u m m a r y , we f o u n d c o n t i n u o u s a t t e n u a t i n g effect o f t h e M E R o n s o u n d t r a n s m i s s i o n to t h e i n n e r e a r in the whole intensity and frequency range studied. The M E R e n h a n c e s t h e C M to v e r y l o u d s t i m u l i b y a t t e n u a t i n g t h e s o u n d r e a c h i n g the i n n e r ear. T h e f o r m o f the C M e n v e l o p e , t h a t is t h e S P L d e p e n d e n t C M d e c r e a s e o r i n c r e a s e as well as the initial p e a k s , c a n be e x p l a i n e d b y t h e n o n l i n e a r I / O c h a r a c t e r i s t i c s o f C M a n d t h e inp u t c h a n g e by t h e M E R . W e c a l c u l a t e d t h e t r a n s m i s sion changes acting on the MER eliciting stimulus. Low frequencies were more attenuated than higher frequencies, b u t s o m e a t t e n u a t i o n w a s still o b s e r v e d at 20 k H z . A t t h e f r e q u e n c y w h e r e t h e rat h e a r s best, at 10 k H z , t h e M E R h a s its l o w e s t t h r e s h o l d .

Acknowledgments S u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B 307). W e t h a n k D r . J f i r g e n H e i t m a n n , D r . U l r i c h E b e r t a n d I n g r i d K a i p f f o r t h e i r h e l p d u r i n g this s t u d y , Prof. Robert N. Leaton for valuable comments on the manuscript and improving the English, and Prof. Axel Michelsen and Dr. Viggo Svane-Knudsen for their time and experimental knowledge spent for a laser-vibrometry s t u d y a c c o m p a n y i n g this s t u d y as well as f o r t h e i r helpful discussion.

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