Sound-evoked e¡erent e¡ects on cochlear mechanics ofthe mustached bat

Markus Drexl a;b;�, Manfred Ko«ssl b

a Department Biologie II der Ludwig-Maximilians-Universita«t Mu«nchen, Luisenstrasse 14, D-80333 Munich, Germanyb Zoologisches Institut der Johann-Wolfgang-Goethe Universita«t Frankfurt, Frankfurt, Germany

Received 5 February 2003; accepted 22 July 2003

Abstract

The influence of the crossed medial efferent system on cochlear mechanics of the mustached bat was tested by measuring delayedevoked otoacoustic emissions (DEOAEs), cochlear microphonics, distortion product otoacoustic emissions (DPOAEs) andstimulus frequency otoacoustic emissions. Contralaterally delivered sinusoids, broadband noise and bat echolocation calls wereused for acoustic stimulation of the efferent system. With all four measures we found a level-dependent suppression understimulation with both broadband noise and echolocation calls. In addition, the sharply tuned cochlear resonance of the mustachedbat which is involved in processing echolocation signals at 61 kHz shifted upward in frequency by several 100 Hz. Presentation ofsinusoids did not have any significant effect. DEOAEs and DPOAEs were in some cases enhanced during contralateralpresentation of the bat calls at moderate intensities. The most important function of the efferent system in the mustached batmight be the control of the extraordinarily fine-tuned resonator of this species, which is close to instability as evident from the verypronounced evoked otoacoustic emissions which sometimes convert into spontaneous otoacoustic emissions of high level.8 2003 Elsevier B.V. All rights reserved.

Key words: Mustached bat; Crossed medial e¡erent; Cochlear mechanics; Otoacoustic emission; Cochlear microphonics

1. Introduction

Acoustic stimulation of one ear signi¢cantly in£uen-ces the response properties of the contralateral ear.These alterations have been shown in studies of thecochlear microphonics (CM) (Fex, 1959), compound

action potentials (Kiang et al., 1970; Liberman, 1989)and otoacoustic emissions (OAEs) (e.g. Collet et al.,1990) and are mainly of a suppressive nature. Theyare mediated by the medial e¡erent ¢bers, which syn-apse on outer hair cells (OHCs) and blocking (Avan etal., 1996; Yoshida et al., 1999) or sectioning (Kujawaand Liberman, 1997) of these ¢bers eliminates the ob-served e¡ects. Several studies have shown that activityof these e¡erent ¢bers might in£uence the motility ofOHCs by altering their sti¡ness and membrane poten-tial (e.g. Dallos et al., 1997), which could result in achange of the cochlear ampli¢er’s activity and thereforein the amount of energy fed back into the travellingwave (Roddy et al., 1994; Guinan, 1996). Several au-thors suggest a protective e¡ect of the e¡erent system,which is thought to prevent acoustic overstimulationand consequent temporary or permanent thresholdshifts of the ear (e.g. Patuzzi and Thompson, 1991;Maison et al., 2002). The rather small e¡erent e¡ectson CM are usually hard to detect in non-specialized

0378-5955 / 03 / $ ^ see front matter 8 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0378-5955(03)00235-1

* Corresponding author. Tel. : +49 (89) 5902 202;Fax: +49 (89) 5902 450.

E-mail address: markus.drexl@stud.uni-muenchen.de (M. Drexl).

Abbreviations: CEC, contralaterally presented echolocation call ;CF, constant frequency; CF2, constant frequency of the secondharmonic of the echolocation call ; CLN, contralaterally presentedbroadband noise; CM, cochlear microphonics; CRF, cochlearresonance frequency; DEOAE, delayed otoacoustic emission;DMPO, dorsomedial periolivary nucleus; DPOAE, distortionproduct otoacoustic emission; DT, decay time; FFT, fast Fouriertransform; LOC, lateral olivocochlear system; MEM, middle earmuscles; MOC, medial olivocochlear system; OAE, otoacousticemissions; OHC, outer hair cell ; SFOAE, stimulus frequencyotoacoustic emission

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mammals. The mustached bat (Pteronotus parnellii par-nellii) appears a perfect model for the investigation ofe¡erent systems, because its highly specialized inner earproduces extraordinarily loud OAEs. Delayed OAE(DEOAE) (Ko«ssl and Vater, 1985a) and CM potentialsshow a long prominent ringing after o¡set of a shorttone burst (Suga and Jen, 1977; Henson et al., 1985).These acoustic and electric cochlear products are highlysensitive to stimulation of the e¡erent system (Hensonet al., 1995).

The echolocation calls of the mustached bat consistof a long constant frequency (CF) part ending with ashort downward modulation. The CF component is anadaptation for hunting insects in dense vegetation. Thebats are able to detect slight frequency and amplitudemodulations in the CF echo, so-called glints, caused bythe prey’s wing beat (review: Neuweiler, 1990). Relative

movements between bat and prey during echolocationcause Doppler shifts in echo frequency, which are com-pensated by lowering the frequency of the emitted call(review: Neuweiler, 1990). The basal turn of the cochleaof the mustached bat contains an acoustic fovea, whichconsists of a spatial expansion of the representation offrequencies between about 54 and about 70 kHz (Ko«ssland Vater, 1985b). Within this fovea, a densely inner-vated region processes the dominant echolocation fre-quency at 61 kHz. The a¡erent neurons innervating theinner hair cells of this region show a high sensitivityand are ¢nely tuned with Q10 dB values of up to 400(Suga and Jen, 1977). The enhanced tuning is generatedby hydromechanical specializations that involve mor-phological discontinuities of the basilar and tectorialmembrane and result in a mechanical resonance. Thisresonance is also responsible for the extraordinarily

Fig. 1. (a) Example of SFOAE evoked with a frequency sweep from 60 to 63 kHz at a sound pressure level of 35 dB SPL. Note the suddendip in the amplitude (solid line) coinciding with a phase change of approx. 180‡ (dashed line) at 61.5 kHz. The cochlear resonance frequencywas de¢ned as the frequency between amplitude maximum and minimum where maximal phase change occurred (vertical dotted line). (b) Ex-ample of DEOAE evoked with a stimulus of 60.95 kHz at 35 dB SPL (duration 2 ms, rise/fall time 0.2 ms, indicated with a horizontal bar).The DEOAE is evident in the long-lasting ringing after stimulus o¡set. (c) Envelope of DEOAE recording. Decay time was de¢ned as the timeneeded for the ringing to decrease to 37% of the initial value (the same calculations were performed on the ringing evident in CM recordings).(d) Example of DPOAE during stimulation with 62.5 kHz (f1) and 63.2 kHz (f2). The stimulus levels were at 60 and 50 dB SPL, respectively.Note the most prominent cubic distortion product 2f13f2.

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loud OAEs and long ringing of DEOAE and CM (Sugaand Jen, 1977; Henson et al., 1985; review: Ko«ssl andVater, 1995; see also Fig. 1).

The e¡erent system of the mustached bat consistsbasically of the same elements as found in non-echolo-cating mammals (Bishop and Henson, 1987). It can bedivided into a medial (MOC) and lateral (LOC) olivo-cochlear system. The MOC neurons compose the dor-somedial periolivary nucleus (DMPO) and innervate thesomata of the OHCs of the ipsi- and contralateral co-chlea with a majority of crossed ¢bers. The neurons ofthe LOC are situated in the interstitial nucleus andcontact a¡erent endings of type I spiral ganglion neu-rons in the ipsilateral cochlea. The crossed olivoco-chlear bundle consists mainly of ¢bers from theMOC. The DMPO receives bilateral input mainlyfrom the cochlear nucleus of the ascending auditorypathway (Bishop and Henson, 1987). Unlike commonlaboratory mammals, the OHCs of the mustached batreceive only one synaptic contact from medial e¡erent¢bers, and there is a gradient in decreasing size of thee¡erent terminals from the ¢rst to the third row ofOHCs. The largest e¡erent terminals can be found inthe ¢rst row of the densely innervated region of thecochlea. One e¡erent neuron innervates a maximalnumber of six OHCs, and this number decreases inthe acoustic fovea to one OHC. In this region, theremight be a ¢ne e¡erent control of cochlear mechanics(Xie et al., 1993). The main transmitter of the mediale¡erent system appears to be, as in other mammals,acetylcholine (Bishop and Henson, 1987).

Henson et al. (1995) showed that contralateral acous-tic stimulation shortens the ringing time of CM re-corded in the ipsilateral ear, this e¡ect being mediatedby MOC ¢bers. In this study, we will extend these re-sults and will show a stimulus-dependent suppression orfacilitation of CM and OAEs.

2. Materials and methods

2.1. Acoustic measurements

Since the experiments were non-invasive, acoustic dis-tortion products were measured in fully awake, non-sedated mustached bats (Pteronotus parnellii parnellii,see Silva Taboada, 1979) from Cuba (n=21, averagebody weight 12 g). For each bat, the constant frequencyof the dominant second harmonic of the echolocationcall (CF2) was determined from fast Fourier transforms(FFT) of 20^25 calls. The CF2 frequency can shift forseveral hundred Hz in dependence on body temperatureand prior £ight activity (Henson et al., 1995). For thisreason, the experiments took place in a sound-attenu-ated, electrically shielded and heated chamber (temper-

ature 30‡C) and between the recording sessions, thebats were kept in cages of 60U50U40 cm to preventextensive £ight activity. For the measurements, the ani-mals were restrained in a styrofoam device that pre-vented extensive movements. The head was ¢xed witha mouth holder consisting of a dental impression platemade of dental acrylic. A closed coupler system incor-porating two 1/2Q BpK 4133 microphone capsules driv-en as loudspeakers (see Fig. 2 for FFT of the loud-speaker’s frequency response) and a microphone (1/4QBpK 4135) was positioned close to the eardrum withina distance of 0.3^1 mm under visual control. A third1/2Q microphone capsule with a small plastic tube at-tached was inserted into the contralateral meatus. Thecoupler’s loudspeakers delivered the acoustic stimuli toevoke the OAEs, whereas the contralateral loudspeakerserved to stimulate the e¡erent system. Stimulus gener-ation and data acquisition were controlled by programswritten in Testpoint (Keithley). The three stimuli weregenerated by D/A conversion using two Microstar DAPdigital signal processing boards, fed into three GPIB(General Purpose Interface Bus)-controlled attenuators(design by Jim Hartley, University of Sussex, Brighton,UK), and sent to the loudspeakers after ampli¢cation.To ensure de¢ned stimulus levels (dB re 20 WPa), theipsilateral speakers were separately calibrated in situ forfrequencies between 1 and 100 kHz before and duringthe experiment, in cases where the coupler positionshifted due to movements of the bat. The contralateralspeaker was calibrated o¥ine. The validity of the SPLcalibration was veri¢ed frequently by using referencedvoltage values of the setup’s output obtained with acalibrator (BpK 4230) generating a sinusoid of 1 kHzat 94 dB SPL. The recorded OAEs were fed via themicrophone and the measuring ampli¢er (BpK 2610)into an A/D input of the DAP boards. All input andoutput channels of the boards were sampled synchro-nously at 333 kHz per channel. A detailed descriptionof the acoustic coupler and measurement procedures is

Fig. 2. FFT of the frequency response of the BpK 4133 1/2Q micro-phone capsule.

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given by Ko«ssl (1994). For contralateral stimuli of up toabout 75 dB SPL there was no crosstalk above theipsilaterally measured noise level of 316T 4 dB SPL.

2.2. Stimulus frequency OAEs (SFOAEs) and DEOAEs

To evoke SFOAEs, we used a continuous tone at aconstant level swept upward in frequency. The sweepwas centered at the CF2 with a sweep range of usually2 kHz. At the frequency of the cochlear resonance, theingoing tone stimulus interferes with the outgoingSFOAE to produce a characteristic pattern of a soundpressure minimum and maximum associated with phasechanges in the frequency response. Usually the stimuluslevel was adjusted below 40 dB SPL. The use of louderstimuli than the optimal one of usually 35 dB SPLresulted in a decrease of the emission since the SFOAEinterference pattern became less pronounced at higherlevels. The transition between level minimum and max-imum, where maximum phase change occurred (see Fig.1a), was used to de¢ne the SFOAE frequency, whichranged from 59.88 to 62.28 kHz (n=10).

To elicit DEOAEs, a short pure tone burst (duration2 ms, rise/fall time 0.2 ms) was delivered to the ipsi-lateral ear and the resulting response at the tympanumwas recorded (see Fig. 1b). In the mustached bat,DEOAEs show a long ringing after stimulus cessation(see Fig. 1b). The frequency and duration of this ring-ing was investigated at various levels of contralateralstimulation. For this purpose, an envelope was ¢ttedto the response (polynomial ¢t). From this envelope,we calculated the decay time (DT), i.e. the time thecurve takes to decrease to 1/e = 37% of the initial value,measured at 0.1 ms after the o¡set of the ipsilateralstimulus (see Fig. 1c). With this decay time, the Q valuewas computed according to the formula Q=DTUZUCRF (Henson et al., 1995) with CRF= cochlear res-onance frequency as derived from FFTs calculated forthe time span of ringing.

2.3. Distortion product otoacoustic emissionmeasurements (DPOAEs)

DPOAEs are most probably generated at the zone ofoverlap of the two stimulus traveling waves close to thef2 frequency place (Brown and Kemp, 1984; Martin etal., 1987; Kummer et al., 1995) and should thereforere£ect OHC activity at this place. The cubic distortion2f13f2 proved to be most prominent (see Fig. 1d). Weoptimized the DPOAE level by choosing an f1 fre-quency that, at low stimulus levels, produces maximumDPOAE levels for given f2 frequencies of about 30 kHzand 60 kHz (at the constant frequency of the ¢rst andsecond harmonic of the bat’s echolocation call). Thecorresponding frequency ratio f2/f1 is de¢ned as the

best ratio, which was between 1.001 and 1.1. The levelof f2 (L2) was set relative to the level of f1 (L1) accord-ing to L1 =L2310, a stimulus condition that produceshigh DPOAE levels in mammals (Probst et al., 1991;Ko«ssl, 1994). After adjusting f1 according to the bestratio, growth functions of 2f13f2 under control condi-tions (no contralateral stimulation) and contralateralstimulation were recorded during a stepwise increaseof the ipsilateral stimulus levels and measuring the levelof the DPOAE.

2.4. CM measurements

For the surgery necessary to introduce the recordingelectrode for CM potentials the bats were anesthetized(2 mg pentobarbital plus 1 mg ketamine/100 g bodyweight). The CM surgery was done according to Hen-son and Pollak’s (1972) method: after making an inci-sion along the skull’s midline and removal of the adja-cent skin and muscles, a metal rod was glued onto thefrontal part of the skull with cyanoacrylic glue anddental acrylic. A small hole was drilled at the occipitalridge of the skull and a tungsten electrode (impedance0.5 M6) was advanced through the cerebellum in sucha way that the tip of the electrode just reached the co-chlear aqueduct. During insertion of the electrode,acoustic stimuli around the individual bat’s CF fre-quency were delivered and the optimal position of theelectrode was judged from observing increases in CMamplitude. An Ag/AgCl electrode for grounding pur-poses was placed in the forebrain. After positioning,the electrodes were glued in place with cyanoacrylicglue and dental acrylic. After surgery, the bats wereallowed to recover for 2^3 days.

CM and DEOAEs were recorded simultaneously inresponse to the same acoustic stimuli in fully awake,non-sedated bats. For this purpose, the bat was re-strained in a styrofoam device and the head was stabi-lized with the metal rod. To record CM, the implantedelectrodes were connected to a custom-built (Jim Hart-ley, University of Sussex, Brighton, UK), battery-pow-ered measuring ampli¢er (gain 10 000U), band-pass ¢l-tered (50 Hz to 120 kHz) and further ampli¢ed. Thesignal was fed into the A/D input of the DAP boardand treated the same way as the DEOAE, i.e. changesin amplitude and the Q value of the CM ringing wereanalyzed.

In all experiments, stimulus trains were delivered at arepetition period of 25 ms. These trains were designedin such a way that the ipsilateral stimulus was presented5 ms after the onset of the contralateral stimulus (du-ration 25 ms and therefore continuous). We used con-tralateral broadband noise (CLN), sinusoids, and thebat’s individually recorded echolocation call (CEC)for contralateral stimulation.

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Statistical analysis was carried out using SPSS 11.0(SPSS) and involved one-sided Wilcoxon and t-tests.

The animal experiments complied with the ‘Principlesof animal care’, publication No. 86-23, revised 1985 ofthe National Institute of Health and also with Germanfederal regulations (approved by the Regierung vonOberbayern, 211-2531-31/97 and 211-2531-37/98).

3. Results

At the beginning of each recording session, the CF2frequency of the bat call was measured and the CRF of

every animal was determined from SFOAE measure-ments. Among the 21 animals used, the CF2 rangedfrom 59.6 to 61.7 kHz and the CRF spanned a rangefrom 59.8 to 62.2 kHz. The CRF was on average 380 Hzhigher than the individual CF2. The CF2 of femaleswas on average 300 Hz higher than that of males (seealso Ko«ssl and Vater, 1985a). During the recording ofthe calls, the individual bats kept their calls constantwithin a maximum standard deviation of 160 Hz.

3.1. SFOAE measurements

Contralateral stimulation with broadband noise

Fig. 3. (a) Decrease of the amplitude and increase of frequency of the SFOAEs evoked with a frequency sweep from 59.5 to 60.1 kHz at 30dB SPL during contralateral presentation of broadband noise (1^100 kHz) in dependence on the level of contralateral stimulation (as indicatednext to the corresponding graph). (b) Box plot of positive frequency shifts (n=7) of SFOAE frequency in dependence on the level of contralat-erally presented broadband noise (CLN, 1^100 kHz) in comparison to control measurements without CLN stimulation, sampled from eightbats. Asterisks indicate statistically signi¢cant di¡erences (Wilcoxon test, P6 0.05). Explanations of box percentile values are given next to theright outermost box. (c) No change of SFOAE frequency and amplitude occurs when a pure tone of 40 kHz at various sound pressure levels(indicated next to the corresponding graph) was used as contralateral stimulus. The SFOAE was evoked with a frequency sweep of 59.5 to60.1 kHz at 25 dB SPL.

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(CLN, 1^100 kHz) at levels ranging from 16 to 76 dBSPL resulted in a progressive decrease of emission am-plitude linked with progressive upward shifts of theSFOAE frequency. Fig. 3a shows an example ofSFOAEs evoked with a stimulus sweep of 30 dB SPLduring contralateral stimulation with levels rangingfrom 16 to 76 dB SPL. They clearly illustrate the abovementioned e¡ects, which were observed in eight out ofnine animals. The positive frequency changes (n=7) incomparison to control measurements of these eight an-imals range from 20 to 200 Hz (Fig. 3b) and show astatistically signi¢cant (Wilcoxon test, P6 0.05) in-crease with increasing CLN level in comparison to con-trol measurements without contralateral stimulation.The contralateral presentation of pure tones (between10 and 90 kHz) at the same sound pressure levels didnot elicit any e¡ect (see Fig. 3c for representative exam-ple).

CLN-induced frequency shifts persist after stimuluscessation and then slowly return to the initial value (seeFig. 4). The recovery time di¡ered among individuals,but never exceeded 29 min in nine specimens examined(data not shown). In the animal shown in Fig. 4, thesuppressive e¡ects on SFOAE amplitude tend to bemore con¢ned to the duration of contralateral stimula-tion than e¡ects on SFOAE frequency. However, this isa single observation and we do not have enough datafrom other individuals to give strong evidence for thispossibility.

3.2. DEOAE measurements

Contralateral stimulation with CLN caused a level-

dependent decrease in the amplitude and duration ofthe DEOAE ringing (Fig. 5a), whereas sinusoids didnot show any e¡ect. In addition, the decay time de-creased, which can be described as a decrease of theQ value of the underlying resonance. DEOAEs wereevoked with frequencies set to the CRF of the individ-ual bat (between 59.5 and 62 kHz) at 45 dB SPL. InFig. 5b the reduction of DEOAE Q values at a CLNlevel of 58 dB SPL in comparison to the control mea-surement with no contralateral stimulation is summa-

Fig. 4. Time course of frequency shifts of SFOAEs elicited with afrequency sweep from 60.5 to 61.2 kHz at 35 dB SPL after contra-lateral presentation of broadband noise (1^100 kHz, 60 dB SPL,5 min). The control measurement was recorded before contralateralstimulation; the following measurements were recorded several min-utes after contralateral stimulation as indicated next to the trace.Vertical lines represent the limits within the SFOAE frequency shifts(i.e. the control value and the maximal shift right after CLN pre-sentation).

Fig. 5. (a) Example of a decrease in the ringing amplitude of a DEOAE evoked with a stimulus of 61 kHz at 45 dB SPL during presentationof CLN (1^100 kHz) at di¡erent sound pressure levels. Note the decrease of the amplitude and decay time with increasing CLN levels. The in-set shows the construction of the graph: the ¢rst 8 ms of every trace which contain the stimulus (horizontal bar) have been removed for clarity.(b) Box plot of DEOAE Q values of 95 measurements sampled from 14 specimens with (CLN) and without (control) presentation of CLN (1^100 kHz) at a level of 58 dB SPL. DEOAEs were evoked with frequencies between 59.5 and 62 kHz at 45 dB SPL, corresponding to the bat’sCRF. The Q values during contralateral stimulation are statistically lower than the control values (Wilcoxon test, P6 0.001). Explanations ofbox percentile values are given next to the right outermost box.

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rized for 95 measurements of 14 bats (highly signi¢cantwith Wilcoxon test, P6 0.001). At this CLN level, themaximal observed changes in the Q values (and there-fore decay time) amounted to 54%, and the amplitudeof the DEOAE ringing, measured 2 ms after ipsilateral

stimulus o¡set, decreased by the maximal value of8.7 dB (data not shown). During CLN presentation,suppressive e¡ects were not accompanied by frequencyshifts.

In control measurements at an ipsilateral stimulus

Fig. 6. (a) Means and standard deviations (n=6) of DPOAE growth functions evoked with f1 and f2 frequencies between 60.8 and 62.8 kHz(as indicated next to the corresponding graph) before (solid line) and during CLN (dashed line) stimulation of 58 dB SPL for three bats (as in-dicated by the bat number at the bottom of each graph). The top two panels are two separate sets of data taken from the same bat and aretherefore obtained with the same frequencies of the primary tones. The dotted line represents the noise level. (b) Example of the dependence ofDPOAE amplitude on the level of CLN: growth functions are shown (control conditions: solid line; CLN stimulation at 38 (dotted line) and58 dB SPL (dashed line)). (c) Averaged growth function, sampled from nine bats (six pairs of growth functions) with (dashed line) and withoutCLN stimulation (solid line) at a constant level of 58 dB SPL. Arrows indicate L2 levels where signi¢cant di¡erences (t-test, P6 0.05) between2f13f2 levels measured during control and CLN conditions occurred. Error bars indicate standard deviations.

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frequency of 50 kHz (a frequency signi¢cantly belowthe CRF) there were no signi¢cant changes of theacoustic response measured at the tympanum, even athigh contralateral stimulus intensities (76 dB SPL, datanot shown). This suggests that middle ear muscles(MEM) were not involved in the observed amplitudereduction of OAEs evoked with frequencies aroundthe cochlear resonance.

3.3. DPOAE measurements

Six pairs of growth functions of the most prominent2f13f2 DPOAE were obtained with and without CLNat 58 dB SPL, from each of nine specimens. ForDPOAEs evoked with a f2 of close to 60 kHz, the pre-sentation of CLN of 58 dB SPL elicits suppressive ef-fects of up to 14 dB, which grow with increasing ipsi-lateral stimulus levels (Fig. 6a shows data from threebats). The slope of the DPOAE growth functions isreduced during contralateral stimulation. Suppressivee¡ects are most prominent at a medium to high primarylevel (Fig. 6c) with the e¡ects decreasing at L2 levelsfrom 60 dB SPL onwards (as shown in Fig. 6b). Wedid not use data obtained with L2 levels of more than60 dB SPL for the calculation of averaged growth func-tions, because such intensities potentially activate theMEM. The suppressive e¡ects are positively correlatedwith the level of CLN (Fig. 6b). Averaged growth func-tions from nine bats show a signi¢cant di¡erence (t-test,P6 0.05) between data sampled with and without con-tralateral presentation of CLN of 58 dB SPL at an f2level from 30 dB SPL onwards (Fig. 6c). For DPOAEgrowth functions evoked at ipsilateral stimulus frequen-cies of about 30 kHz (which is close to the constantfrequency of the ¢rst harmonic of the bat’s echoloca-tion call) with and without contralateral stimulationwith broadband noise of 58 dB SPL, there are no de-tectable changes: Fig. 7 shows two sets of mean growthfunctions (six pairs of growth functions) and their stan-dard deviations, sampled from two bats. Contralateral

presentation of pure tones from 10 to 70 kHz duringacquisition of DPOAEs evoked with primary tonesclose to 60 kHz does not a¡ect the amplitude of the2f13f2 DPOAE either: Fig. 8 shows the 2f13f2DPOAE levels with and without contralateral presenta-tion of sinusoids at a level of 58 dB SPL for two di¡er-ent primary tone levels (L2 = 25 or 45 dB SPL).

3.4. Facilitation and suppression of OAEs duringcontralateral presentation of the bat’s ownecholocation call

The individual echolocation signals of ¢ve bats wererecorded and used for contralateral stimulation. Thedistribution of control Q values (no contralateral stim-ulation) of 56 DEOAE recordings (evoked with fre-quencies between 59.5 and 62 kHz at 45 dB SPL)from ¢ve bats and their corresponding values duringcontralateral presentation of echolocation calls (CEC)at 58 dB SPL displays a complex pattern (Fig. 9a): the

Fig. 7. Means and standard deviations (n=6) of DPOAE growth functions evoked with f1 and f2 frequencies close to 30 kHz (as indicatednext to the corresponding graph) with (dashed line) and without (solid line) presentation of CLN at 58 dB SPL. Data are sampled from twobats. The dotted line represents the noise level.

Fig. 8. Level of 2f13f2 DPOAE evoked with f1 and f2 frequenciesof 60.6 and 61.2 kHz and a constant primary level L2 of 35 dBSPL (lower trace) and 55 dB SPL (upper trace) with (open symbols)and without contralateral presentation of pure tones of frequenciesbetween 10 and 70 kHz at 58 dB SPL (solid symbols). The dottedline represents the noise level.

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observed range of control Q values can be separatedinto three domains of low, intermediate and high Qvalues. Corresponding to these domains, di¡erent qual-itative changes of Q values during contralateral stimu-lation with echolocation calls can be observed (Fig.9a,b): the low Q value domain shows enhancement ofQ values (statistically signi¢cant with Wilcoxon test,P6 0.05), for the intermediate domain there is no sta-tistically signi¢cant change of the Q values, and thehigh Q value domain shows suppression (statisticallysigni¢cant with Wilcoxon test, P6 0.05) during pre-sentation of echolocation calls. The slopes of regressionlines of control values and contralateral stimulationvalues di¡er, whereas the CEC regression line is lesssteep. To rule out that the observed e¡ect is an artifactcreated by the plotting method, Fig. 9c,d show a con-trol plot which was created in just the same way as Fig.9a,b, i.e. the two data sets (control 1 and control 2) areseparated in time by just the same extent (30 s) as the

data shown in Fig. 9a,b. In this control plot no contra-lateral stimulation was used. Since the pattern shown inthis control plot is di¡erent from the one shown in Fig.9a,b and since there is no statistical di¡erence betweenthese two control traces, we are con¢dent that the e¡ectshown in Fig. 9a,b is due to e¡erent activity and not anartifact caused by the plotting method.

Enhanced Q values did not return to the initial valuewithin several hours, while suppressed Q values re-turned to the initial value right after cessation of con-tralateral stimulation, suggesting that suppressive e¡ectshad a shorter time course than enhancing e¡ects. Butmore data are required to assess this possibility. Resultsfrom contralateral presentation of individually recordedecholocation calls during recording of DPOAE growthfunction at primary frequencies of about 60 kHz di¡erfrom the ones acquired during contralateral presenta-tion of CLN and support partly the results of the lattersection: Fig. 10 shows three sets of mean DPOAE

Fig. 9. (a) Distribution of DEOAE Q values (n=56) obtained from ¢ve bats with (CEC, open symbols) and without contralateral presentationof the echolocation call at 58 dB SPL (control, solid symbols). Solid and dashed lines represent linear regression of control and CEC values, re-spectively. The control values are arranged according to increasing Q values and can be divided into domains of low, intermediate and high Qvalues. DEOAEs were evoked with frequencies between 59.5 and 62 kHz, corresponding to the bat’s individual cochlear resonance frequency,at a level of 45 dB SPL. (b) Box plot of DEOAE Q values with (CEC) and without contralateral presentation of echolocation calls (control)within the three Q value domains de¢ned in panel a. Data are replotted from panel a. Asterisks indicate statistical di¡erence (Wilcoxon test,P6 0.05). Explanations of box percentile values are given next to the right outermost box. Panels c and d correspond to a and b, but showcontrol values, i.e. the data are obtained in just the same way as in a and b, but control 1 and control 2 are obtained without any contralateralstimulation. There is no statistical di¡erence between the two control data sets.

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growth functions (six pairs of growth functions) fromthree bats and their standard deviations during controland CEC conditions. In all three sets, there is a ten-dency of enhancement of the 2f13f2 growth functionduring CEC at 58 dB SPL. In Fig. 10 top, there couldbe a change of the e¡ect of CEC presentation withincreasing L2 from suppression to enhancement. How-ever, the signi¢cance of this e¡ect at low L2 levels isdoubtful, since statistical analysis of the results shownin Fig. 6 (during CLN presentation) revealed that sig-ni¢cant e¡ects only appear with L2 levels of more than30 dB SPL.

At a given 2f13f2 level of 15 dB SPL, the L2 levelduring CLN presentation at 58 dB SPL needed to beraised by on average 9.5 dB SPL in comparison to thecontrol condition, while the L2 could be lowered by on

average 3 dB during presentation of CEC at 58 dB SPLto achieve the same 2f13f2 level.

3.5. Simultaneous recordings of CM and DEOAE duringcontrol and CLN conditions reveal the samecharacteristics

To study if contralateral acoustic stimulation has adi¡erent e¡ect on DEOAEs and on OHC receptor po-tentials, we simultaneously evoked CM and DEOAEwith an ipsilateral tonal stimulus between 60.5 and61.2 kHz at 45 dB SPL (corresponding to the individualbat’s CRF) and sampled data (n=19) with and withoutCLN stimulation from two bats.

The Q values of CM and DEOAE measurementsduring the control condition are statistically not di¡er-ent (Fig. 11). Maximal di¡erences of single valuesamount to 0.4 ms decay time, which corresponds to75 Q value units. Under the same conditions as in theprevious experiments, we evoked CM and DEOAE si-multaneously during presentation of CLN and deter-mined the decay time and Q value of the CM andDEOAE ringing. Again, the Q values from CM andDEOAE are statistically not di¡erent and single valuesdi¡er by not more than 90 Q units. In both measures,the control values and values obtained during CLNpresentation at 58 dB SPL are statistically di¡erent(Wilcoxon text, P6 0.001, Fig. 11). This emphasizesthat in the mustached bat cochlea, there is a ratherdirect correspondence between hair cell excitation and

Fig. 10. Means and standard deviations (n=6) of DPOAE growthfunctions evoked with f1 and f2 frequencies close to 60 kHz (as indi-cated next to the corresponding graph) with (dashed line) and with-out (solid line) CEC of 58 dB SPL. Data are sampled from threebats. The dotted line represents the noise level.

Fig. 11. Boxplot of Q values (n=19, two bats) obtained from con-current measurements of CM and DEOAE evoked with the sameipsilateral stimulus during conditions with no contralateral stimula-tion (CM and DEOAE, respectively) and with contralateral pre-sentation of CLN at 58 dB SPL (DEOAE+CLN and CM+CLN, re-spectively). Note that control values from CM and DEOAEmeasurements and CLN values from CM and DEOAE measure-ments are not statistically di¡erent, whereas corresponding controland CLN values from CM and DEOAE measurements are statisti-cally di¡erent, as indicated by asterisks (Wilcoxon test, P6 0.001).The evoking stimulus was set to the bat’s CRF of 61.5 and 61.2kHz at 45 dB SPL. Explanations of box percentile values are givennext to the right outermost box.

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mechanical activity in the organ of Corti, which leadsto a re-emission of sound energy.

4. Discussion

4.1. Methodical considerations

In this study, putative e¡erent-induced changes ofCM were assessed by measuring OAEs and CM. BothOAE and CM could be in£uenced by MEM activity(e.g. Buki et al., 2000), but we are con¢dent thatMEM e¡ects are excluded by the used stimulus para-digms. Henson et al. (1995) report that for the mus-tached bat sound levels of at least 75 dB SPL are re-quired to elicit MEM contractions. In the present study,OAEs and CM are usually evoked with ipsilateral stim-ulation levels between 25 and 60 dB SPL. Suga and Jen(1975) report an increasing threshold for MEM con-tractions with increasing stimulus frequency, resultingin no signi¢cant attenuation at a stimulus frequencyof 70 kHz with a test tone of 90 dB SPL. It thereforeseems unlikely that the ipsilateral stimulus in thepresent study elicited MEM contractions, since weused exclusively high frequency tones (30 and 60 kHz)at stimulus levels below 75 dB SPL. For SFOAE re-cordings, stimulus levels of not more than 40 dB SPLwere used. In addition, the duration of the ipsilateralstimulus was only 2 ms, which seems to be too short forMEM to cause signi¢cant attenuation (Suga and Jen,1975). Since the MEM re£ex can be evoked bilaterally,the contralateral stimuli we used should also be consid-ered a source of potential ipsilateral MEM activity. Butwe detected putative e¡erent e¡ects with contralateralintensities well below 60 dB SPL (the low frequencyMEM threshold reported by Suga and Jen, 1975) andthus we are con¢dent that the observed e¡ects are notof MEM origin. Therefore, the observed frequencyshifts and amplitude alterations should be due to e¡er-ent activity and not be a result of middle ear activity orother mechanisms, which could a¡ect the amplitude ofthe ipsilateral stimulus. This is supported by the factthat the amplitude of the impulse response evoked byother frequencies than the CRF was not a¡ected bycontralateral stimulation, even at highest sound pres-sure levels. If contralateral stimulation had caused ipsi-lateral MEM contractions during CM recording withfrequencies around the resonance frequency, it shouldhave a¡ected CM evoked with frequencies di¡erentfrom the cochlear resonance as well. Regarding a pos-sible involvement of MEM in CM enhancement, Pilz etal. (1997) showed a shift of the non-linear, bell-shapedCM I/O function to higher input levels, which results inan enhancement of CM amplitude. These e¡ects requirehigh stimulus levels of more than 100 dB SPL and are

most prominent at low frequencies (5 kHz). The max-imal enhancing e¡ect on CM in response to tones of2^3 kHz shown in Nuttall (1974) was elicited by elec-trical stimulation of the tensor tympani, which caused a‘near-maximal contraction’ and might not represent thestate of MEM contraction elicited by moderate acousticstimulation. In addition, it is interesting to note thatnone of the studies on the auditory periphery of bats(Henson, 1965; Suga and Jen, 1975; Henson et al.,1995) reports enhancement of CM amplitude byMEM activity. Therefore, we are con¢dent that the in-crease of OAE amplitudes is due to e¡erent activity andnot a result of MEM contractions.

Finally, one needs to consider that in the mustachedbat, the CM and DEOAE response at resonance is verynon-linear and small input level changes can induce alarge change in the response. To avoid small changes inthe level of the ipsilateral stimuli which could be causedby movement of the awake bats, we frequently recali-brated our recording system. In addition, Q values ofringing of DEOAE and CM seems to be independent ofstimulus level, at least in the range of moderate stimuluslevels we used in our experiments. This is supported byresults of Henson et al. (1995), who found that Q valuesof CM ringing are not dependent on stimulus level.Thus, even if small changes in the ipsilateral stimulusamplitude should occur, they are unlikely to a¡ect thedecay time of ringing in DEOAE and CM measure-ments.

4.2. E¡erent e¡ects on CM

Several studies report suppressive e¡ects on CM dueto contralateral stimulation with broadband noise (e.g.Popelar et al., 1999; Che¤ry-Croze et al., 1993; Micheylet al., 1999; Collet et al., 1992), which is in accordancewith our results. We found a positive correlation be-tween CLN level and a reduction of the decay timesof the ringing of CM and DEOAE, and in addition, adecrease of the SFOAE amplitude with a shift of theemission frequency to higher values. In addition, broad-band noise was the most e¡ective contralateral stimulusto elicit suppressive e¡ects on ipsilateral OAEs andCM, whereas pure tones did not cause any suppression.This ¢nding is comparable to results obtained from ex-periments in humans, where the in£uence of stimulusbandwidth on suppression of OAEs was tested (Berlinet al., 1993; Maison et al., 2000; Velenovsky andGlattke, 2002) and a positive correlation between stim-ulus bandwidth and amount of suppression was found.A possible explanation for the greater e¡ectiveness ofbroadband noise might be that the activity of the MOCbundle is facilitated by spatial integration of their in-puts or due to amplitude £uctuations of the stochasticbroadband noise (Maison et al., 2000). This might also

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be the reason for the lack of suppressive e¡ects in themustached bat while using contralaterally presentedpure tones. In humans, a stimulus level of at least60 dB SPL is needed to produce signi¢cant suppressivee¡ects with pure tones (Maison et al., 2000). Therefore,given that the threshold for pure tone-elicited e¡erente¡ects in the mustached bat can be di¡erent from thatin humans, the stimulus level of pure tones used in ourexperiment might not have been high enough to elicite¡erent-mediated suppression, since we did not usestimulus levels of more than 70 dB SPL to avoidMEM activity.

The changes in DEOAE, CM and SFOAE recordingsfound in the present study support data of Henson etal. (1995) on changes of CM ringing during contralat-eral stimulation with broadband noise. Comparable re-sults were also reported for other species. Mott et al.(1989) showed frequency shifts and amplitude altera-tions of spontaneous OAEs in humans. Manley et al.(1999) demonstrated similar e¡ects in the barn owl:during loud contralateral stimulation they were ableto elicit frequency shifts of spontaneous OAEs of upto 125 Hz and they were able to suppress these emis-sions down to the noise £oor.

A sti¡ening of OHCs and hence the organ of Corti isthe most likely source of positive frequency shifts of thecochlear resonance. Another possibility would be thatfrequency shifts are generated by changes in cochlearplace (Hu¡mann and Henson, 1992). In addition, sincethe resonance shifts to lower frequencies with high stim-ulus levels (Ko«ssl and Vater, 1985a), it is possible thatthe upward shift of the resonance frequency during andafter contralateral stimulation could be caused by ane¡erent-mediated attenuation of e¡ective energy actingon OHCs.

Contralateral stimulation with the bat’s own echolo-cation call elicited two opposing results. Presented atmoderate intensities, such stimulation caused an en-hancement of the OAE. In contrast, CLN usually pro-duced suppression. In humans, Micheyl et al. (1999)found non-monotonic, frequency-dependent e¡ects forthe contralateral suppression of transient evoked oto-acoustic emissions (equivalent to DEOAEs). In addi-tion, the recording of DPOAEs during contralateralacoustic stimulation resulted in suppressive and enhanc-ing e¡ects during di¡erent sessions and under the samestimulus conditions. Similar results were reported bySiegel and Kim (1982) from experiments with electricalstimulation of the e¡erent ¢bers. Dolan et al. (1997)showed decrease and enhancement of basilar membranemovement with di¡erent stimulus paradigms.

E¡erent-mediated enhancement of OAEs might notbe caused by an enhancement of basilar membranemovement, but could be the result of a change in theinterplay of di¡erent OAE sources. If interference and

possible cancellation between di¡erent OAE sources ismodi¢ed by e¡erents, an apparent enhancement of theOAE does not necessarily require an increase in theamplitude of one of the components but it would besu⁄cient if it changes its phase.

Contralateral stimulation with broadband noise andecholocation calls caused tonic e¡ects that are evidentin a sustained frequency shift of SFOAE and a long-lasting increase in the decay time of DEOAE. Xie andHenson (1998) describe a tonic reduction of the CMdecay time during contralateral presentation of tape-recorded echolocation calls and social calls of the mus-tached bat. Within 20 min to 2 h after stimulus o¡set,the decreased CM decay time recovered to its initialvalue. Bats that were just removed from their colonyshowed a decreased CM decay time as well, probablycaused by the acoustic background in the colony. Afterextensive echolocation during £ight the bats againshowed a decreased decay time, most likely caused bytheir own echolocation calls. These results partially con-trast with the present study: contralateral stimulationwith echolocation calls caused not only suppression, butin some cases also an enhancement of cochlear mechan-ics. Xie and Henson (1998) showed that the CM ofmustached bats in their natural acoustical environmentis suppressed. This could provide protection for the¢nely tuned inner ear of this species, which may be ofparticular importance, since these animals live in largecolonies and are therefore exposed to a cacophony ofsocial and echolocation calls, comprising a frequencyrange of 6^120 kHz and reaching sound levels of upto 120 dB SPL. In this study, we did not observe anytonic suppression of CM, but tonic shifts of the CRF. Itis interesting to note that amplitude alterations are notcrucially linked to frequency changes during periods oftonic e¡ects, which suggests that e¡erent stimulationa¡ects sti¡ness of OHCs (which should dominate theCRF) and the cochlear ampli¢er (which should domi-nate amplitude alterations of cochlear products) in adi¡erent way and with di¡erent time constants. E¡erente¡ects manifest on two di¡erent time scales (Cooperand Guinan, 2003), one within milliseconds, the otherwithin seconds, which are usually attributed to changesin the activity of the cochlear ampli¢er (the fast e¡ect)and the sti¡ness of the OHCs (the slow e¡ect). In asingle bat (Fig. 4) we could observe that OAE ampli-tude alterations recovered shortly after contralateralacoustic stimulation while frequency changes did not,which might be linked to the separate fast and slowactivity of the e¡erent system. But more data are re-quired to resolve this issue.

The e¡erent system of the mustached bat seems tobe most e¡ective in a frequency region close to 60 kHz,as evident from DPOAE measurements at di¡erentfrequencies, which is in accordance with the highest

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density of synapses of medial e¡erent ¢bers in this re-gion.

Overall, the most important function of the e¡erentsystem in the mustached bat might be the control of theextraordinarily ¢ne-tuned resonator of this species,which is close to instability as evident from the verypronounced evoked OAEs which sometimes convertinto spontaneous OAEs of high level.

Acknowledgements

We thank the Cuban Ministry of Science, Technol-ogy and Environment (CITMA) for kind permission todo research on and to export mustached bats. We alsothank Marianne Vater and Bernhard Gaese for valua-ble comments on earlier versions of the manuscript.This study was supported by the DFG, Ko 987/6-3.

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