Cochlear resonance in the mustached bat: Behavioral adaptations

Hearing Research, 50 (1990) 259-274 Elsevier

HEARES

259

01478

Cochlear resonance in the ~ustachcd O.W. Henson ‘, P.A. Koplas

2, A.W. Keating

bat: Behavioral adaptations I, R.F. Huffman

’ and M.M. Henson

’

’ Department oJ Cell B~olou and Anatomy, The Unroersity of North Curolinn, Chapel Hill, ’ Currtculum tn h’eurohtologr, The Uniuersity of North Carolina, Chapel Hill and ’ Drcwon of Otolaryngokogy/Head und h’eck Surgeg~. Deportment The Uniuersity of North Carolma, Chapel Hill, North Carolmcr. U.S. A. (Received

22 November;

accepted

of Sur,yerv.

30 June 1990)

Mustached bats. Pteronotus p. parnefiii, use complex, multiharmonic biosonar signals with prominent approx. 60 kHz (CF) components. The sense of hearing is especially acute to sounds near 60 kHz and the cochlea shows a number of specializations m the 60 kHz region. Foremost is a remarkable degree of cochlear resonance. In this study it is shown that: 1) any sounds near the resonance frequency elicit a pronounced resonance that continues after the stimulus terminates; 2) Doppler-shifted echoes of the bat’s own cries may cause resonance; 3) continuous resonance can be produced by stimulating the ear with broadband noise but such resonance does not interfere with the bat’s ability to Doppler-shift compensate during simulated flight; 4) significant changes in the resonance frequency of the cochlea occur during and after flight; 5) the changes in resonance can be dependent or independent of body temperature changes; and 6) mustached bats continuously adJust the CF component of their pulses to keep the second harmonic echoes in a constant frequency band near the resonance frequency. Thus. mustached bats not only compensate for Doppler-shifts imposed by their movements relative to that of a target. but they cochlear resonance compensate to deal with small changes in the micromechanical properties of the cochlea. Cochlear

potentials:

Bats; Cochlear

resonance;

Cochlear

emissions;

Mustached bats, Pteronotus parneilii, use complex biosonar signals to detect, identify and capture insects in complex echoic environments (Goldman and Henson, 1977; Henson et al., 1987). Each emitted pulse contains a long constant frequency (CF) component that is usually preceded and followed by frequency modulated (FM) components. The initial FM, terminal FM and CF components are represented in a series of at least four harmonics (Fig. 1). When mustached bats are not flying the emitted second harmonic CF component is maintained in a relatively narrow (approx. 200-400 Hz) frequency band: the center of this band is near 61 kHz

Correspondence to: O.W. Henson, Department of Cell Biology and Anatomy, The University of North Carolina, Taylor Building, CB 7090, Chapel Hill, NC 27599, U.S.A. FAX (919) 966-1856.

037~-5955~~/$03.50

‘G 1990 Elsevier Science

Publishers

Echolocation

and is called the resting frequency (Schnitzier, 1970a,b). When flying or moving, the bats systematically adjust the frequency of the CF component in such a way that the echo frequency is maintained in a narrow band called the reference frequency. This echolocative behavior is known as Doppler-shift compensation (Schnitzler, 1970a,b). There is no question that accurate Doppler-shift compensation requires an auditory system that can resolve very small changes in frequency. Indeed, it has become firmly established that the frequency resolving power of the ear of the mustached bat is phenomenal. The cochlea and neural centers throughout the auditory system have specific parts that show exceptionally sharp tuning, and neurons tuned to the approx. 61 kHz, second harmonic of the biosonar signals comprise about one third of each acoustic center (Pollak and Bodenhamer, 1981; Suga and Jen, 1977: Pollak and Casseday, 1989). The cochlea of mustached bats has several unique properties that are undoubtedly associated

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with sharp tuning (Henson et al., 1985; Henson and Henson, 1988; KBssl and Vater 1985a, 1985b; Vater, 1988). Foremost of these is a degree of mechanical resonance that far surpasses that seen in the cochlea of any other mammal. Resonance can easily be observed from recordings of co&fear emissions or the cochlear microphonic (CM) output of hair cells. It is most pronounced when the ear is stimulated with sound at (Fig. 2B) or near (Fig. 2C) the resonance frequency (Henson et al., 1985, K&s1 and Vater, 1985a; Suga et al., 1975; Suga and Jen, 1977); when the stimulus frequency is far removed from the resonance frequency, little or no resonance is seen (Fig. 2A). Sharp tuning at the hair cell level was first described by Poilak et

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Fig. 2. The demonstration of cochlear resonance in CM potentials recorded from the ear of the mustached bat. When a 4.5 ms. 45 kHz tone pulse with a fast (0.1 ms) rise-decay time and rectangular envelope stimulates the ear (A) the CM response (CM) appears rectangular and the output of a RMS/DC converter resembles a square wave;no evidence of resonance exists. When the stimulus is at the resonance frequency of the ear (B), the rise and fall times of the CM enveIope are long compared to the stimulus and the ear rings for a considerable time after the end of the stimulus. When the stimulus frequency is higher or lower than the resonance frequency of the cochlea (C), the CM envelope and the RMS/DC converter output show prominent beats during the time that the stimulus and resonance interact; after the end of the stimulus the CM evoked by resonanCe alone exists and the resonance shows a slow decay.

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Fig. 1. Frequency characteristics of the biosonar signals of the mustached bat, Pteronotur p. parneifii. This frequency vs time display is composed of a series of spectral slices, each covering sequential, 0.5 ms. overlapping segments of a 17 ms time window. Note the initial upward sweeping, frequency modulated component, the long constant frequency component and the terminal downward sweeping, FM component. Fach of these components is represented in a series of four harmonics. Each tic mark on the time axis represents 1.0 ms.

al. (1972) and was attributed to resonance by Suga et al. (1975) and Suga and Jen (1977). Although cochlear resonance can easily be produced with electronically generated stimuli with fast rise-decay times, there is Iittle information on whether this resonance occurs under natural conditions, i.e. in relation to the intense emitted pulses or the returning echoes which are actively held near the resonance frequency. If resonance does

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occur, one must ask how stable it is, and how such a sensitive detection and fine frequency resolving system can operate effectively when the system is continuously resonating at or near the same frequency as the echoes it must detect and analyze. The main purpose of this report is to show that: 1) resonance may occur under natural conditions while the animals are actively echolocating; 2) continuous resonance does not interfere with Doppler-shift compensation; 3) the cochlear resonance frequency is not stable during and after flight, and 4) the bats adjust the frequency content of their pulses, not only for Doppler-shift compensation but also for changes in the resonance frequency of their ears. Methods Animals studied The animals used for these studies were mustached bats, Pteronotus p parnellii, from Jamaica, W.I. The electrophysiological results are based on the study of six unanesthetized animals with chronically implanted electrodes, but the majority of the data were obtained from three animals whose cochlear microphonic (CM) potentials and degree of cochlear resonance were relatively stable over periods ranging from four to nine weeks. After surgery and electrode implantation, these bats continued to be strong, fast fliers and they were healthy throughout the time periods tested. Experiments were conducted almost daily on these three individuals. Resting frequency determinations Resting frequency values were determined for each bat studied. As previously noted, this is the frequency of the second harmonic, CF component emitted when the animals were not flying. The bats were placed in a small cylindrical cage (21.6 cm diameter; 21.6 cm long) and the emitted pulses were detected with a 6.25 mm Brtiel and Kjaer microphone (Model 4135) and associated measuring amplifier system (Model 2608). The biosonar signals detected by the microphone were passed through a narrow bandpass filter to eliminate the higher and lower harmonics (TTE, Inc. Model K18 E3007, 3 dB points at 59.65 and 64.0 kHz).

To decrease the required sampling rate and increase the frequency resolution, the filtered signals were heterodyned with a 59 kHz signal from a crystal oscillator and low pass filtered (- 3 dB point was 3 kHz) to eliminate the sum frequency. The heterodyned signals and associated trigger pulses, generated by a custom made phaselocked-loop pulse detector, were connected to a 12 bit, 1 MHz FFT spectrum analyzer/digital oscilloscope (Rapid Systems, Inc., Model R350). This system was configured to perform a rectangular windowed 1024 point FFT on 16 consecutive pulses, and then display a spectral average. A cursor was placed at the maximum peak of the spectral display to determine the average pulse frequency for the 16 pulses. The sampling frequency used to digitize the heterodyned pulses was 10 kHz which resulted in an FFT binwidth of approximately 10 Hz. Chronic electrodes for recording CM potentials For electrode implantation, the bats were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Inc). A posterior skin incision was made over the dorsal part of the skull and the underlying part of the temporalis muscle was removed bilaterally. The bone was then cleaned and coated with a thin layer of cyanoacrylic adhesive (Loctite, Superbonder 409 gel) mixed with glass microspheres. An uninsulated tungsten ground electrode was implanted in the lateral lobe of the cerebellum. For recording CM potentials, a hole was drilled near the lambdoid ridge and a tefloncoated tungsten electrode was advanced toward the cochlear aqueduct. When high amplitude potentials were evident in response to broad band acoustic stimuli, the electrode was glued in place. Care was taken not to penetrate the aqueduct. After the electrodes were implanted, a small section of a square, hollow, brass rod was glued to the skull. When recording from the unanesthetized animal, the head was stabilized in a set position by clamping onto this rod. CM potentials were amplified with an EG&G (Princeton Applied Research, Model 1130) preamplifier (10,000 X gain)with bandpass filter settings of 1 kHz and 300 kHz. The potentials were additionally filtered with a custom made tracking filter or a narrow bandpass filter (TTE, Inc., Model K18, E3007)

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Fig. 3. CM amplitude vs frequency curves. When acoustic stimuli are systematically changed in frequency and SPL, the CM amplitude reaches a maximum at a specific frequency point. This point. represented by vertical lines. is constant (62.4 kHz in this preparation) when the SPL is low (lowest three traces, 30.40 and 45 dB) but it becomes progressively lower as the SPL is increased ahove 50 dB. In this study. changes in the position of the CM amplitude peak for a given SPL were used to measure changes in the resonance frequency (see Fig. 7).

which ratio.

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Acoustic stimulation Pure tone acoustic stimuli were delivered to the ear with an electrostatic transducer (Polaroid Corp.) placed 15 cm in front of the bat’s ear. The speaker was oriented to give a maximum CM response to a frequency close to the resonance frequency of the ear. An accurate (50 ppM) 12 MHz synthesized function generator (Wavetek. Model 23) was used to produce the signals. Stimulus frequency, duration, rise-decay time, SPL, interstimulus intervals and frequency steps were computer controlled with custom-designed hardware and software. CM amplitude us frequency curves Prior to most experiments, the response properties of the ear were assessed by plotting CM amplitude vs. frequency curves for a series of different SPLs (Fig. 3). Low level stimulus response curves (30-50 dB SPL) were always run first because higher level stimuli often caused shifts in the position of the peak amplitude of subsequent curves. For plotting CM amplitude vs. frequency curves in the sharply tuned region of the ear, frequency steps of lo-25 Hz were used.

The CM was fed to a custom designed KMS-DC’ converter which was interfaced with an A/D converter board of a DEC micro PDP-I l,/ 23 minicomputer. The average CM amplitude was taken as the voltage output of the ear during a 5.0 ms interval that began 3 ms after the beginning of the stimulus. In this way, amplitude modulations (beats) present in the initial segment of the rcsponse were not included in the averaged part of the CM. By using relatively low level stimuli. the middle ear muscle reflex contractions were avoided. For any point in the response curve. the CM amplitude represented the average value for eight stimulus presentations. These curves not only proved useful for determining changing properties of the ear but also for examining the time course of changes (see Fig. 5). Resonance /requeng measurements Two methods were used to measure the resonance frequency of the ear. One method involved the use of CM amplitude vs frequency curves and the other used FFT spectral analysis techniques. Both methods provided similar values for the resonance frequency or changes in this frequency. Descriptions of the CM amplitude vs frequency curves for the mustached bat can be found in the reports of Henson et al. (1985) and K&s1 and Vater (1985) and some specific details will be reviewed in order to understand the methods employed in the present study. A more detailed account of these curves and their changing properties will be the subject of another paper. When acoustic signals with SPLs (generally below 50 dB) are used for plotting CM amplitude vs frequency curves, a distinct amplitude peak occurs at the resonance frequency of the ear. When the resonance frequency changes. the position of the amplitude maximum shifts and the amount of shift can be accurately measured from graphic displays. When the SPL of the stimulus is progressively elevated between 50-75 dB the CM maximum gradually shifts to lower frequencies such that the position of the peak does not correspond to the resonance frequency determined for low stimulus levels. Nevertheless, the shift in the position of the CM amplitude peak for a given SPL provides accurate values for resonance frequency shifts regardless of the SPL used.

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binwidth of approx. 10 Hz. The actual resonance frequency was then determined by adding the beat frequency value displayed by the computer to the stimulus frequency. Because of the high accuracy (10 ppM) of our signal generating system. any changes in the resonance system could be attributed to the changing properties of the ear and not to drift in the frequency of our signals. When recording the CM from active animals we usually used a high-Q filter. To demonstrate that the filter was not ringing and causing the beats, the CM was recorded simultaneously on two different channels of the tape recorder: one channel received the output of the high Q (60663 BP, -3 dB points) filter and the other received the output from the broad-band (20--120 kHz) filter. Similar beat patterns were seen in oscillographic displays from both channels. Also, the fact that the resonance frequency was physiologically vulnerable (see below) indicates that the observed resonance was not an artifact. Fig 4. Measurement of spectral energy in heterodyned signals as a technique for rapid, (+lO Hz) resonance frequency determinations. A shows the digitized CM response to a 4.5 ms, 61 kHz tone pulse. The amplitude modulations in the response are beats due to the interaction of the 61 kHz CM with the CM generated by resonance of the cochlea. When the CM is fed to an RMS/DC converter, a sinusoidal output is displayed and the frequency of this output is the difference frequency between the signal generated CM and the resonance generated CM. B shows the spectral energy displayed for the digitized window in A: a cursor marks the tip of the peak. The frequency of this peak (0.7227 kHz) is displayed on the screen and the actual resonance frequency (61.722 kHz) was determined by adding the displayed value (0.722 kHz) to the 61.0 kHz stimulus frequency. The peaks to the left of the main peak are the result of off-set voltages in the RMS/DC output.

The most accurate and rapid method of recording the resonance frequency was to present a 6-10 ms stimulus about 1000 Hz lower than the predicted resonance frequency. When the rise-decay time was fast (0.1 ms) the spectral energy associated with fast rise-time signals caused the ear to resonate strongly for about 5 ms and the CM evoked by the resonance interacted with the CM produced by the stimulus to create marked beats. The beats in the CM envelope were preserved as a sinusoidal waveform with an RMS/DC converter (Fig. 4). An unwindowed 1024 point FFT, with a sampling rate of 10 kHz, provided a frequency

Recording cochlear potentials during simulated flight The recording of cochlear potentials from echolocating bats was accomplished by mounting the bats on a pendulum and swinging them toward and away from fixed objects. Under these conditions the bats emitted streams of ultrasonic pulses and they actively Doppler-shift compensated. Details concerning the design of this system and the associated electronic components can be found in the reports of Henson et al. (1982) and Kobler et al. (1985). To obtain data on spectral peaks in the CM generated by emitted pulses, echoes and resonance, the CM during pendulum swings was recorded on magnetic tape (Racal, Store 7D). The taped records were then slowed down, digitized and analyzed; computer software programs provided time vs spectral peak data or a display of energy peaks for the entire period sampled (see Fig. 5). In addition, studies of Doppler-shift compensation performance relied on measurements of pulse frequency adjustments during different velocity points in pendulum swings. The frequency of the second harmonic CF component of the emitted pulses was measured throughout the acceleration and deceleration phases of the forward swing.

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Fig. 5. Demonstration of co&tear resonance in a bat actively involved in echolocation during simulated flight on a pendulum. A shows a digitized version of an emitted pulse (I’) detected by a microphone beneath the animal’s mouth. After the end of the pulse there is a faint echo (E). The accompanying higheat spectral peaks vs time display shows that most of the spectral peeks occur in relation to the CF component of the pulse (approx. 60.5 kHz). A spectrogram of the entire digitized window (B) shows that most of the energy is centered near 60.5 kHz. C shows the digitized CM potentials ehcited by the same pulse and by echoes and co&ear resonance. Strong beats due to interactions of the emitted pulse with a Doppler-shifted e&o are evident ~ou~out the period of p&e-echo overlap. Less pronounced amplitude peaks (arrows) probably represent beats created by the ~teraction of co&ear resonance with the initial part of the pulse (two arrows), and later, the returning echo (three arrows). The accompanying highest spectral peak display shows a scattering of echo energy between 61 and 61.5 kHz and a clear series of peaks near 62 kHz and close to the measured resonance frequency of this animal’s cochlea. D shows a spectral display for the CM during the window shown in C; only a small peak corresponding to the pulse CF can be seen, while the peaks corresponding to the echo CF and co&ear resonance are high in amplitude.

Noise stimulation In some experiments we used broadband noise to stimulate the ear and create continuous co&ear resonance. The signal was produced by a random noise generator (General Radio, type 13903) and this was fed to an electrostatic loudspeaker (Polaroid Corp.). Spectral analysis of the output showed a relatively flat frequency response in the 40-90 kHz band with a maximum peak at 48 kHz. Temperature measurements Body temperature measurements were made in one of two ways. In one method, a temperature probe was chronically implanted between the tem-

poral& muscle and the skull. The tip of the probe was over the thin membrane covering the paraflocculus of the cerebellum. In the second method, the tip of the probe was placed in the midsagittal line in a deep skin fold between the occiput and back. Since simultaneous measurements from both points yielded similar values in terms of body temperature changes, the noninvasive skin fold measurements were used in all but two of the experiments. ‘when measurements were made from the skin fold it was important to have the probe spring-mounted so that the horizontal shaft of the probe was pressed into the deepest part of the fold. The temperature probes used (Sensortek, Inc.,

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Model IT-18 or IT-21) and the recording device (Sensortek, Inc., Model BAT-12) allowed temperature measurements with a resolution of 0.1” C. Results

Cochleor ~es5n~nce during ec~olocfftion When CM potentials elicited by natural biosonar signals were recorded from bats swinging on a pendulum, pronounced amplitude modulations (beats) were commonly seen in the CM envelope during periods of pulse-echo overlap (Fig. 5C). This has previously been noted by Henson et al. (1982; 1987) and the beats in these instances could be traced to the interaction of emitted signals with Doppler-sifted echoes; they were not related to cochlear resonance. In addition to these beats, however, the CM envelopes often showed repetitive amplitude modulations both prior to pulseecho overlap and after the end of a pulse (Fig. 5A). The beats in the initial segments of the signals can be explained best by the simultaneous presence of some of the initial, slow FM, and/or the beginning of the CF, of an emitted signal and cochlear resonance. Beats which occurred in relation to echoes that did not overlap the pulse can be accounted for by interactions of the echo CF with cochlear resonance. The beat frequencies observed were at or close to those predicted from knowledge of the difference frequency between an animal’s cochlear resonance frequency and the frequency of the biosonar signal CF components. It is also important to note that beats in the CM were sometimes observed when the pendulum was not moving and therefore when no Doppler-shifted signals were reaching the ear. Thus, these beats cannot be attributed to interactions between multiple Doppler-shifted echoes and CF pulse components. To demonstrate when resonance was occurring, we processed 32 ms windows containing pulseand echo-evoked, CM potentials. A computer program was used that identified high amplitude spectral peaks in 0.512 ms, overlapping segments. Fig. 5 shows the highest spectral peaks detected in relation to a digitized emitted pulse with a Doppler-shifted echo (A) and the digitized CM response of the ear to the same emitted pulse and echo (C). To the right of each frequency vs time

display are spectral displays for the entire digitized period. In A, there is a series of dots that represent spectral peaks which correspond in time to the approx. 60.5 kHz, CF component of the emitted pulse and the Doppler-shifted (61.5 kHz) CF component of the echo. The spectral peak for the pulse CF is pronounced in B while the echo energy is small. The spectral data for the CM (C and D) were very different from the pulse data: spectral peaks for the pulse energy at 60.5 were absent (C) or very small (D), but the echo-related peaks were prominent. In addition, C and D show spectral peaks near the known resonance frequency of this animal’s cochlea (62 kHz). The peaks which correspond to resonance in C appear to be especially prevalent after the end of the emitted puise. In D, the height of the spectral peak suggests that resonance occurred over a substantial part of the digitized time period. The amplitude of the resonance-evoked spectral peak in Fig. SD is one of the most marked of any of our records. In other cases the spectral peak recorded under these conditions was less pronounced, the third peak being half the value of the CF echo peak; in many cases no peak could be seen even when beats in the CM response suggested the presence of resonance. We interpret this as a limitation of our peak detection software which scaled the response in proportion to the much more prominent peaks produced by the echo-evoked CM. Also, it should be noted that we were only able to record separate peaks like those shown in Fig. 5 when the frequencies of the peaks were well separated. In many of our preparations the echo frequency and resonance frequency were so close that the detection and measurement of the resonance could not be made. It should be emphasized that the high amplitude of the spectral peak potentially attributed to resonance in Fig. 5D, may have been produced by a strong brief period of resonance, a long period of low level resonance or a combination of the two. Creation of co~~j~u~~ resonance with broad-hind noise From a physical point of view it is obvious that any sound energy near the resonance frequency of the cochlea should cause the cochlea to resonate. Indeed, when broad-band noise was presented to the cochlea of the mustached bat, continuous reso-

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Fig. 6. Demonstration of ccchlear resonant: produced by stimulating the ear with low level (55 dB) broadband noise (A) and the absence of any change in Doppler-shift compensation when the noise stimulus was presented during pendulum swings (B). The curves show the changing frequency of the CF pulse component during acceleration (falling frquency) and deceleration (rising frequency) phases of two forward swings. The curve with the open circles was obtained when no noise was present. The curve with the solid shows a curve obtain& while both ears were being stimulated with 70 dB random noise.

nance was easily demonstrated. We have examined this phenomenon in six animals to date and in every case continuous resonance was indicated from FFT displays; these showed a single high amplitude peak that corresponded to the known resonanCe frequency of the bat’s ear (Fig. 6A). In addition, when the noise was presented in combination with a pure tone, continuous beating occurred at the frequency difference between the

resonance frequency and the tone. When the noise was putsed, resonance after the termination of each pulse. like that in Fig. 2B, was seen. The creation of continuous resonance hy noise stimulation gave us the opportunity to determine if Doppler-shift compensation was affected when the ears were ringing. Noise. which was shown to produce continuous resonance, was presented by a pair of loudspeakers mounted on the pendulum, one beamed into each ear. The bats were first swung with no noise and then with progressively higher noise in the approx. 55-75 dB SPL, range. Under these circumstances there was no consistent difference between Doppler-shift compensation in the noise and no noise conditions (Fig. 6B). Resonance ffequenq changes associafed with f&h and sip& emissions In some cases we noted that the co&ear resonance frequency during echolocation on the pendulum was slightly higher in frequency than that measured when the animal was stationary. In order to determine if there were consistent and systematic changes in the resonance frequency. we measured the resonance frequency prior to and i~~iate~y after the animal flew for 15 min. Two methods were used, one to show that the entire CM amplitude vs frequency curves shifted to higher frequencies, and the other to determine the time course of resonance frequency changes. The CM amplitude vs frequency curves in Fig. 7A show the characteristic type of shift observed between pre-flight and post-flight recordings. It can be seen that two superimposed pre-flight curves are identical and the peak amplitude is centered at 62.15 kHz. The two post-flight curves had a peak amplitude at about 62.40 kHz and they were not as stable as the pre-flight curves. in this case, the shift in the position of the CM peak was 250 Hz. In almost ail cases, the peak amplitudes and shapes of the pre-flight and post-flight curves were similar; the only change was in the position of the peak. Fig. 7B shows the same two sets of curves as in 7A, as well as additional curves numbered 3, 4. 5, 7. and 8. No. 1 is the first curve recorded after the animal was mounted in the recording system (approx. one minute after the end of a 15 min flight). The other numbers refer to the curves obtained in

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associated with flight might be body temperature dependent. A correlation of resonance frequency and temperature can be demonstrated by anesthetizing the animal or cooling the tissue near the ear with a subsequent fall in body temperature. Simultaneous measurements of the body temperature and resonance frequency during pre- and post-flight periods, however, did not show consistent, parallel changes. Fig. 8, for example, shows a case where there was a progressive decrease in the resonance frequency prior to flight and during the time that the body temperature was rising. After flight both the temperature and resonance frequency decreased in a somewhat parallel fash-

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FIN. 7. CM amplitude vs frequency curves prior to and after flight. A shows two supe~mpos~ pre-flight curves and the first two curves recorded immediately after 15 minutes of flight: an approx. 300 Hz shift of the amplitude peak is evident. B shows additional curves in the sequential order (by number) that they were obtained after the end of the flight. This series reveals that the amplitude peak gradually shifted to lower frequencies and approached the pre-flight curves. Note that the maximum amplitude of the peak does not change as the curve shifts.

-400 succession; each curve required about 30 s to acquire. With time, it is clear that the peak of the shifted curves returned nearly to the pre-flight position. Using the previously described spectral estimate technique for measuring the resonance frequency, we found additional evidence of resonance frequency shifts prior to and after flight. Averaged spectral values for the resonance frequency were obtained every 15 s. Plots of the frequency changes as a function of time after flight (Figs. 8 and 9) revealed an average rate of change of about 26 Hz/min over periods of 6 to 10 min. The rate, however, was often much steeper during the initial minute of measurements than in later ones; this is evident from the initial slopes of the post-flight curves in Fig. 8. Since body temperature is known to affect the resonance frequency (K&s1 and Vater, 1985b) it was anticipated that the resonance frequency shifts

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lar rate. In all instances the highest resonance frequencies for a given animal were observed immediately after flight, and there was always a decline with time. Resonance frequency shifts in range of 150-300 Hz were routinely recorded. In Figs. 8 and 9 it should be noted that postflight temperature and resonance frequency data were not obtained for the first one or two minutes of the post-flight period because of the time required to mount the animal in the recording chamber and to hook up the electrodes. Thus, the total change was probably greater than that shown in the graphs. Comparison of resting frequency and resonance frequency Since the ear of the mustached bat resonates at a higher frequency after flight than before, we measured changes in the resting frequency of the

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Fig. 9. Comparison of post-flight, resonance frequency and body temperature data after two successive flights. In both cases the resonance frequency was high when the first measurements were made and rapidly decreased while the animal was resting. Body temperature, by contrast, steadily increased after the first flight and decreased after the second. These data, like those in Fig. 7, show that there is no consistent relationship between small body temperature changes and resonance frequency changes.

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Note, however, that during the post-flight period, after about 200 s, the temperature was relatively stable between 40.5 and 40.6 degrees, but the resonance frequency continued to decline steadily. Fig. 9 shows curves for two successive post-flight measurements which even more dramatically indicate a lack of correspondence between changes in the resonance frequency and the temperature of the tissue near the ear. In one case the temperature after flight was rising and in the other case the temperature was falling, but resonance was decreasing in both cases at a simi-

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Fig. 10. Changes in resting frequency prior to and after four successive periods of flight and echolocation. (A) shows individual data points as a function of time. In (B) the recorded values have been placed in 5 min bins. The error bars equal I SD of all values for five minutes; 3-5 values were obtained per minute, and each value was determined from the displayed peak for 16 consecutive FFTs (see Fig. 4). Note that even at the end of 15 min the resting frequency is higher than that measured prior to flight.

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emitted pulses prior to and after flight to determine if the CF component frequency characteristics changed in relation to the changing properties of the ear. Resting and resonance frequency measurements could not be made at the same time but it was established that ~~ed~ately after flight the resting frequency, like the resonance frequency, continuously declined in small steps and at a rate that was similar to that demonstrated for the resonance frequency. Also, like the changes in the resonance frequency, the change in the resting frequency was generally in the 150-300 Hz range (50 to 450 Hz total range). Fig. 1UA shows the repeatability of the resting frequency changes after four successive flights in a single bat and Fig. 10B shows histograms for the data placed in five minute bins. Tn addition to examining the resting frequency we also examined the reference frequency (the frequency where the CF echo is stabilized) during the middle portions of pendulum flight; the final deceleration portion of each swing was not analyzed because the bats consistently showed a change in compensation behavior during the final part of the deceleration phase. The data from five to six pendulum swings were analyzed for each bat and from 25-75 pulses were analyzed for each ~nd~v~du~_ As shown in Table I, the post-flight reference frequency was elevated in five of the six animals tested and the difference ranged from 59 to 450 Hz. Here too, there was always a significant time delay (approx. 1 min) between the end of each flight and when the first frequency measurements could be made. Thus, the values may be

TABLE

I

COMPARISON OF PRE-FLIGHT AND POST-FLIGHT BODY TEMPERATURES AND MEAN RESTING FREQUENCIES RECORDED DURING PENDULUM SWINGS BAT No.

1 2 3 4 5

Pre-flight

Post-flight

Temp

Ref. freq

Temp

Ref. freq

35.6 37.2 37.6 35.1 37.1

60.9 62.04 61.95 61.17 61.2

35.9 35.7 36.8 38.3 38.1

60.82 62.49 62.09 61.32 61.8

* P value 5 0.005.

Change

-wHz* +450 Hz +.59 Hz + 1.50 Hz -cl80 Hz

* * * *

low. Temperature measurements were made with a rectal probe. Note also the lack of correlation between frequency changes and body temperatures. Discussion Evidence has been presented that the cochlea of the mustached bat resonates during, and for a period of time after, the ear is stimulated with natural and/or electronically generated ultrasonic signals near 61 kHz. In the case of natural biosonar signals emitted by the animals, we found beats in the CM indicating an interaction (interference) between two frequencies which appeared to be the echo frequency and the resonance frequency. There is no question about the identification of the echo but the identification of resonance is less certain. One line of evidence in favor of resonance is that the beats were present after the end of the emitted pulses and at the time when the Doppler-shifted echo would be expected to both produce, and interact with, the cochlear resonance. A second point is that the spectral peak corresponded in frequency to the known resonance frequency of the cochlea. Although we believe that the high amphtude spectral peaks in the CM potential (Fig. 5D) were the result of the ear being forced into resonance by the energy in the approx. 61.5 kHz echo, it is possible that there may have been double Doppler-shifts, whereby the primary echo reverberated from the pendulum back to the primary target and then back to the bat. This double shift would have placed the second echo CF close to the resonance. From a purely physical point of view it would seem most unusual if the ear did not resonate whenever the ear was stimulated with sounds at or near the resonance frequency; we know that the ear of the mustached bat is relatively undamped and in every animal studied to date we have seen pronounced resonance when the ear is stimulated by a wide variety of sounds (cticks, noise and both CF signals and FM signals). In addition, previous studies have shown that when the ear is stimulated with slow sweeping, low level signals, there are pronounced cochlear emissions and there is a destructive interference with the incoming sounds when the stimulus frequency is near the resonance

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frequency. These strong cochlear emissions are especially indicative of resonance evoked by acoustic signals. (Henson et. al.. 1985: K&l and Vater. 1985b). Since many different types of stimuli can cause resonance it seems likely that this resonance must occur in a variety of natural circumstances and most notable in caves where mustached bats live in large colonies; echoes from the beating wings of insects would also be expected to add resonance at a time when critical signal detection and analysis would be required. With this in mind, it is not surprising that a bat’s ability to Doppler-shift compensate is not noticeably affected by continuous resonance (Fig. 6B). If one accepts the idea of a nearly continuous resonance during echolocation one must question its functional significance. Is it a simple by-product of a sharply tuned system or does it have a clear functional value? Do the bats perceive the resonance? How can a Doppler-shift compensation system operate effectively when the receiver is ringing continuously?. Kesonance of the cochlea may be interpreted as a by-product of the mechanical properties of the inner ear and/or part of a mechanism that has evolved for very sharp tuning. Cochlear resonance seems, at least partially, to fit some of the models proposed for sharp neural tuning in other mammals but it is impossible to satisfy the criteria for any one model. The publications of Kbssl and Vater (1985a,b; 1990) and Vater (1988) are especially interesting in terms of putative cochlear mechanisms. An emerging theme is that the most sensitive (lowest threshold) units are at the resonance frequency while the most sharply tuned (highest Q) units have best frequencies that are slightly lower and thus in a more apical position in the cochlea. If this is true, then the pronounced resonance could be responsible for establishing the very sharp tuning which is obviously required for Doppler-shift compensation. In the mustached bat the sharply tuned cochlear nucleus units have Q-rOdBswhich range from about 50 to more than 300. A unit with a best frequency of 61 kHz, a symmetrical response area, and a Q- IodBof 244 could be responsive to resonance but non-responsive to an echo 200 Hz higher in frequency and 15 dB louder. Since the high frequency slope is often much greater than the low

frequency slope of a tuning curve, even smaller frequency shifts and greater SPL differences could account for selective responses to resonance vs echo vs pulse emission frequencies. If one advocates the presence of different pools of neurons devoted to resonance processing vs Dopplershifted echo processing then the ability of the system to function effectively during continuous resonance can be explained. With the knowledge of the presence of very sharp tuning in the mustached bat, several studies have attempted to determine the exact relationship between the resting frequency of the emitted pulses. the frequency of resonance and the narrow frequency band to which many units are tuned (Pollak and Bodenhamer. 1981; Henson et al.. 1982; K&l and Vater, 1985a). The interrelationships of these frequencies have not been satisfactorily established because in all cases the data are flawed by the fact that the conditions under which the different frequencies were recorded were not specified. From the present study it is obvious that the resting frequency will vary according to what the animal was doing immediately before the recordings were made. Based on other experiments in our laboratory, we have determined that after surgery and implantation of an electrode. the resonance frequency of the ear may not reach a normal range until after five or six days: the values may be 500 or more Hz lower than those determined from healthy, fully recovered animals. In future experiments involving the resting frequency. attention must be given to these details. Although previous studies have not recognized the changing resonance. or changes in resting frequencies based on prior activity, the data strongly suggest that the emitted CF and the returning echoes are held at frequencies below resonance. This is certainly evident in Fig. 5 where the resonance and echo (reference) frequency were measured simultaneously. If cochlear resonance and related micromechanical events set the stage for the exquisite tuning seen in auditory units, it is not surprising that the bats adjust the physical characteristics of their sonar signals to match both the echo characteristics and the changing micromechanical properties of the ear. It is interesting in this respect that vocal compensation for changes in cochlear reso-

271

nance seems to be as sensitive and finely adjusted as Doppler-shift compensation. This fine adjustment is evident from the data in Figs. 7 and 8. A prime question that arises is how single unit properties are affected by shifts in the resonance frequency. Experiments are currently being undertaken in our laboratory to address this question. Resonance frequency compensation provides good evidence that the resonance of the ears is perceived, although we cannot be certain that it is at a conscious level. In humans it is well known that the ears may ring and produce acoustic emissions (Kemp, 1978). These emissions are usually at such low levels that they are not heard and thus they are much less pronounced than the up to 70 dB SPL emissions recorded from the ears of mustached bats. In some humans, audible emissions occur (tinnitis) and they are not only heard (Kemp, 1979; Wilson, 1980; Wit and Ritsma, 1979) but may be so distracting that they have driven patients to seek special medical attention. In animals, the CM potential is generally a good indicator of the responsiveness of the ear and hearing as a whole. The prominence of the CM potentials produced by resonance in Pteronotus parnellii is thus indicative of perception. Even more convincing are neurophysiological correlates of resonance perception from single-unit, multiunit and evoked potential studies (Suga and Jen, 1977; Suga et al., 1975; K&s1 and Vater, 1990). Tonic unit activity has been observed after the end of a pure tone stimulus and the duration of the tonic activity is similar to the duration of resonance usually produced by such stimuli. Prominent multi-unit activity and evoked neural potentials are easily observed in response to the onset of resonance at the end of a stimulus, This ‘off response’ is tuned to the same frequency as the resonance frequency and is physiologically vulnerable in the same way that the sharply tuned resonance peak of the CM is vulnerable (Pollak et al., 1979). Furthermore, single unit responses capable of encoding the beat frequency produced by the interaction of resonance with the resonance-eliciting stimuli have been recorded (Kbssl and Vater, 1990). There are several ways that cochlear resonance could be beneficial to echo perception and analysis other than its association with sharp tuning. It

might provide a reference frequency which could be valuable for fine frequency analysis, especially if beat detection is important. It is often argued that beats can not be heard by bats because the frequency is so low, but there is ample evidence that each beat can be encoded as if it were a single stimulus (Suga et al., 1975; Suga and Jen, 1977). In most cases resonance demonstrated by CM responses appears linked to the emission of sounds from the ears (cochlear emissions). It seems inevitable that these approx. 70 dB SPL emissions produce a complex array of interference patterns with returning echoes. If these patterns are perceived in any way, it would allow for more sophisticated imaging than would otherwise be possible. In humans, for example, it has been reported that a tone produced by a loudspeaker placed near the ear enhances sound field imaging (see High Technology Business. Feb., 1988, p. 11). The 150-300 Hz changes in the resonance frequency and resting frequencies recorded prior to and after flight are not impressive in terms of the total, approx. 110 kHz hearing range in Pteronotus parneflii. However, in terms of Doppler-shift compensation, which operates over about a 1000 Hz frequency band, and in terms of the approx. 300-500 Hz band occupied by numerous, sharply tuned neurons. it is a substantial percentage. One of the intriguing aspects of cochlear resonance in the mustached bat is its susceptibility to change under certain conditions. Some of these conditions have been explored and well documented by Kossl and Vater (1985b). Kossl and Vater noted that the resonance (cochlear emission) frequency decreased as the SPL of a resonanceeliciting stimulus increased. The nature of the changes they observed appear to be illustrated by the shifting position of the amplitude peak in CM amplitude vs frequency curves (see Fig. 3). They also showed that stimulation of the ear with tones in the lo-60 kHz range (70-80 dB SPL for one minute) caused a temporary increase in the emission frequency; within a 5-20 min time span the frequency gradually returned to normal. The emission amplitude under these conditions was not observed to change. The observed frequency shift (without a change in emission amplitude) and the associated recovery time, seem to correlate with

272

the CM resonance data reported here (Fig. 7). Thus, during flight and echolocation, the almost continuous bombardment of the ears by the biosonar signals may be the cause of the resonance frequency change. It should be noted, however, that after flight, when the bats are ‘resting’, they often emit nearly continuous streams of pulses during the time that the resonance frequency is falling. We initially anticipated that body temperature would rise in the flying bat and fail during periods of rest and that these temperature changes would explain the postflight changes in resonance. This prediction was based on experiments by K&l and Vater (1985b) and on experiments in our laboratory (unpublished, Koplas and Henson) which clearly showed that the body temperature and cochlear resonance frequency of anesthetized bats progressively decreases with time. The often observed increases in body temperature during postflight periods (Fig. 9) may be explained by two mechanisms: first by an initial lowering of the temperature during flight, as a result of evaporative cooling from the expanded wing surfaces; and secondly, by a rise in temperature when these cooling surfaces were folded and the animal and its wings were placed in a small Styrofoam chamber where air circulation was minimal. There may be other interwoven factors, none of which have been adequately investigated. to explain the changes in resonance frequency. Possibilities include changes in cochlear blood flow or tissue substances, such as prostaglandins. The latter occurs in high concentrations in the ear (Escoubet et al., 1985) and has the capacity to affect a variety of contractile tissues, including perhaps the tension fibroblasts in the lateral wall of the spiral ligament (Henson et al., 1984, 1985: Henson and Henson, 1988). Another likely candidate for producing micromechanical changes in the cochlea is the system of medial efferent neurons which innervate the outer hair cells. These are well developed in the mustached bat and each hair cell has a single, large efferent terminal (Bishop and Henson, 1988). Many studies on other mammals have suggested that the activity of these efferent nerves causes micromechanical changes and alters the physiological properties of the cochlea (Brown, 1988; LePage, 1989: Mott et al.,

1989). The 5-10 min time frame of the changing resonance frequencies, however, is long compared to that which might be brought about by either increasing or decreasing efferent nerve activity and subsequent hair cell motility. LePagr (1989). for example, has demonstrated two types of baseline movements of the basilar membrane: the time courses of these changes can be measured in milliseconds and seconds, compared to the much longer time intervals required for resonance changes in the mustached bat. Mott et al. (1989) have also examined changes in the frequency of cochlear emissions and the time frame is small compared with the resonance frequency changes observed in this study. Thus, evidence that the resonance shifts in Pteronotw were the result of efferent nerve stimulation is not strongly supported at this time. Acknowledgements

This work was supported by PHS grant NS 12445 from the National Institute of Neurological and Communicative Disorders and Stroke and grant DC 00114 from the National Institute on Deafness and Other Communicative Disorders. References Bishop, A.L. and Henson, O.W., Jr. (1988) The efferent auditory system in Doppler-shift compensating bats. In: P.E. Nachtigall and P.W.B. Moore (Eds.), Animal Sonar. Processes and Performance, NATO AS1 series, Series A: Life Sciences, Vol. 156. Plenum Press, pp. 307.-310. Brown. A.M. (1988) Continuous low level sound alters cochlear mechanics: an efferent effect? Hear. Res. 34, 27-38. Escoubet, B., Amsallem, P.. Ferrary, E. and Tran Ba Huy, P. (1985) Prostaglandin synthesis by the cochlea of the guinea pig. Influence of aspirin, gentamicin and acoustic stimulation. Prostaglandins 29, 589-599. Goldman, L.J. and Henson, O.W.. Jr. (1977) Prey recognition and selection by the constant frequency bat, Preronorus 9. pameliii. Behav. Ecol. Sociobiol. 2. 411-419. Henson. M.M.. Henson, O.W., Jr. and Jenkins. D.B. (1984) The attachment of the spiral ligament to the cochlear wall: anchoring cells and the creation of tension. Hear. Res. 16. 231-242. Henson. MM. and Henson, O.W.. Jr. (1988) Tension fibroblasts and the connective tissue matrix of the spiral ligament. Hear. Res. 35, 237-258. Henson, M.M., Burridge, K., Fitzpatrick, D.. Jenkins, D.B. Pillsbury. H.C. and Henson, O.W.. Jr. (1985) Immunocytochemical localization of contractile and contraction associated proteins in the spiral ligament of the cochlea. Hear. Res. 20, 207-214.

213 Henson, O.W., Jr., Bishop, A., Keating, A., Kobler, J., Henson. M., Wilson, B. and Hansen, R. (1987) Biosonar imaging of insects by Pteronotus parnellii, the mustached bat. Nat. Geographic Res. 3, 82-101. Henson. O.W., Jr. and Henson, M.M. (1988) Morphometric analysis of cochlear structures in the mustached bat, Pteronouf purnel~~i pamellii. In: P.E. Nachtig~l and P.W.B. Moore (Eds.), Animal Sonar Processes and Performance, NATO AS1 series, Series A: Life Sciences, Vol. 156. Plenum Press, pp. 301-305. Henson, O.W., Jr., Pollak, G.D., Kobler, J.B., Henson, M.M. and Goldman, L.J. (1982) Cochlear microphonic potentials elicited by biosonar signals in flying bats, Pteronotw p, pamellii. Hear. Res. 7, 127-147. Henson, O.W.. Schuller, G. and Vater, M. (1985) A comparative study of the physiolo~cal properties of the inner ear in Doppler-shift compensating bats (~hinoloph~ rouxi and Pteronotus pameiiii). J. Comp. Physiol. A. 157, 587-597. Kemp, D.T. (1978) Stimulated acoustic emissions from human auditory system. J. Acoust. Sot. Am. 64. 1386-1391. Kemp, D.T. (1979) Evidence of mechanical nonlinearity and frequency selective wave amplification in the cochlea. Arch. Otol. Rhinol. Laryngol. 224, 37-45. Kobler, J.B., Wilson, B.S., Henson, O.W., Jr. and Bishop, A.L. (1985) Echo intensity compensation by echolocating bats. Hear. Res. 20, 99-108. K&l. M. and Vater, M. (1985a) The cochlear frequency map of the mustached bat, Pteronotus p. pamellii. J. Comp. Physiol. A. 157, 687-697. Kiissl, M. and Vater. M. (1985b) Evoked acoustic emissions and cochlear microphonics in the mustached bat, Pteronotuspamellii. Hear. Res. 19, 157-170. Kiissl, M. and Vater, M. (1990) Resonance phenomena in the cochlea of the mustached bat and their cont~bution to neuronal response characteristics in the cochlear nucleus. J. Comp. Physiol. A. 166, 711-720. LePage, E.L. (1989) Functional role of the olivo-cochlear bundle: A motor unit control system in the mammalian cochlea. Hear. Res. 38, 177-198. Mott, J.B., Norton, S.J., Neely, ST. and Warr, W.B. (1989)

Changes in spontaneous otoacoustic emissions produced by acoustic stimulation of the contralateral ear. Hear. Res. 38. 229-242. Pollak, G.D. and Bodenhamer, R.D. (1981) Specialized characteristics of single units in the inferior coiliculus of mustache bats. Frequency representation, tuning and discharge patterns. J. Neurophysiol. 46, 605-620. Pollak, G.D., Henson, O.W., Jr, and Novick, A. (1972) Cochlear microphonic audiograms in the ‘pure tone’ bat, 0nlonyc~eri.s parnellii. Science, 176, 66-68. Pollak, G.D and Casseday, J.H. (1989) The neural basis of echolocation in bats. Springer-Verlag, Berlin, p. 143. Pollak, G.D., Henson, O.W., Jr. and Johnson. R. (1979) Multiple specializations in the peripheral auditory system of the CF-FM bat, Pteronotus pameflii. J. Comp. Physiol. A. 131. 255-266. Schnitzler, H.-U. (1970a) Echoortung bei der Fledermaus. Chilonycreris ruhiginosa. 2. Vet-@. Physiol. 68, 25-38. Schnitzler, H.-U. (1970b) Comparison of echolocation behavior in Rhinolophus ferrum-equinum and Chilon.vcterrs ruhiginosa. Bijdr. Dierk 40, 77-80. Suga, N., Simmons, J.A. and Jen, P.H-S. (1975) Peripheral specializations for fine frequency analysis of Dopplershifted echoes in the auditory system of the ‘CF-FM’ bat. Pteronotur parnellii. J. Exp. Biol. 63. 161-192. Suga, N. and Jen, P.H-S. (1977) Further studies on the peripheral auditory system of the CF-FM bats specialized for fine frequency analysis of Doppler-shifted echoes. J. Exp. Biol. 69, 201-232. Vater, M. (1988) Cochlear physiology and anatomy in bats. In: P.E. Nachtigall and P.W.B. Moore (Eds.), Processes and Performance, NATO AS1 Series, Series A: Life Sciences, Vol. 156. Plenum Press, pp. 301-305 Wilson, J.P. (1980) Evidence for a cochlear origin for acoustic re-emissions, threshold fine-structure and tonal tinnitis. Hear. Res. 2, 233-252. Wit, H.P. and Risma, R.J. (1979) Evoked acoustical responses from the human ear. Stimulated acoustic emissions from the human ear. J. Acoust. Sot. Am. 66. 91 l-913.