Department of Otology and Laryngology, Harvard Medical School; and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114
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ABSTRACT |
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Kujawa, Sharon G. and M. Charles Liberman. Long-Term Sound Conditioning Enhances Cochlear Sensitivity. J. Neurophysiol. 82: 863-873, 1999. Sound conditioning, by chronic exposure to moderate-level sound, can protect the inner ear (reduce threshold shifts and hair cell damage) from subsequent high-level sound exposure. To investigate the mechanisms underlying this protective effect, the present study focuses on the physiological changes brought on by the conditioning exposure itself. In our guinea-pig model, 6-h daily conditioning exposure to an octave-band noise at 85 dB SPL reduces the permanent threshold shifts (PTSs) from a subsequent 4-h traumatic exposure to the same noise band at 109 dB SPL, as assessed by both compound action potentials (CAPs) and distortion product otoacoustic emissions (DPOAEs). The frequency region of maximum threshold protection is approximately one-half octave above the upper frequency cutoff of the exposure band. Protection is also evident in the magnitude of suprathreshold CAPs and DPOAEs, where effects are more robust and extend to higher frequencies than those evident at or near threshold. The conditioning exposure also enhanced cochlear sensitivity, when evaluated at the same postconditioning time at which the traumatic exposure would be delivered in a protection study. Response enhancements were seen in both threshold and suprathreshold CAPs and DPOAEs. The frequency dependence of the enhancement effects differed, however, by these two metrics. For CAPs, effects were maximum in the same frequency region as those most protected by the conditioning. For DPOAEs, enhancements were shifted to lower frequencies. The conditioning exposure also enhanced both ipsilaterally and contralaterally evoked olivocochlear (OC) reflex strength, as assessed using DPOAEs. The frequency and level dependence of the reflex enhancements were consistent with changes seen in sound-evoked discharge rates in OC fibers after conditioning. However, comparison with the frequency range and magnitude of conditioning-related protection suggests that the protection cannot be completely explained by amplification of the OC reflex and the known protective effects of OC feedback. Rather, the present results suggest that sound conditioning leads to changes in the physiology of the outer hair cells themselves, the peripheral targets of the OC reflex.
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INTRODUCTION |
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Prior exposure to long-duration, moderate-level
sound can protect the ear. Ears that have been "conditioned" in
this way, and subsequently exposed to high-level sound of similar
spectral content, demonstrate smaller permanent threshold shifts (PTSs) than do ears exposed to the high-level sound alone. In the first report
of this phenomenon (Canlon et al. 1988), guinea pigs
conditioned by continuous exposure to a 1-kHz pure tone (81 dB SPL, 24 days) suffered no significant PTS from a subsequent, high-level
exposure (1 kHz; 105 dB SPL, 72 h). In contrast, a control group
that received the high-level exposure alone showed 20-30 dB of PTS at
the same postexposure time (8 wk). Numerous reports of
conditioning-related protection have since emerged. In some, the
conditioning stimulus was presented continuously (e.g., Canlon
and Fransson 1995
; Canlon et al. 1988
,
1992
; Ryan et al. 1994
); in others,
intermittently (e.g., Campo et al. 1991
;
Subramaniam et al. 1993
). In most, moderate-level conditioning took place over days and was followed by exposure to
high-level sound of similar spectral content and shorter duration. "Protection" in such studies is quantified as a reduction in the PTS that otherwise would be associated with exposure to the high-level sound alone.
These studies grew out of earlier work (e.g., Eldredge et al.
1959; Miller et al. 1963
) in which
trauma-producing exposures were delivered intermittently and repeatedly
over days and effects were studied on temporary threshold shifts (TTSs)
measured immediately after each exposure. In these paradigms,
protection is seen as a gradual reduction, as the number of days
increases, in the TTS measured after each day's exposure, i.e., the
ear demonstrates a gradual "toughening" against the high-level
sound (e.g., Boettcher and Schmiedt 1992
, 1995
;
Clark and Bohne 1987
; Sinex et al. 1987
; Subramaniam et al. 1991a
,b
, 1994a
,b
). This
gradually reducing TTS often is coupled to a gradually increasing
residual threshold shift (seen immediately before each daily exposure),
which resolves to a small, but significant PTS and which can be
associated with accumulating hair cell damage/loss (e.g.,
Boettcher and Schmiedt 1992
).
The mechanism(s) underlying conditioning- and toughening-related
threshold protection are not well understood and may not be the same.
In the broadest sense, there are two main possibilities: that
conditioning changes some cochlear elements or that conditioning changes the strength of either the middle-ear muscle (MEM) or the
olivocochlear (OC) reflexes, both of which can protect the ear from
acoustic overexposure. A major role for the MEM reflex has been ruled
out by several lines of evidence (see DISCUSSION for
details). A role for the OC system in these effects remains unresolved
(Brown et al. 1998; Kujawa and Liberman
1997
; Yamasoba and Dolan 1998
; Zheng et
al. 1997a
,b
) and could involve changes in medial (M)OC
responsiveness or changes to the peripheral targets of these neurons,
the OHCs (Canlon and Fransson 1994
; Canlon et al.
1993
; Chen et al. 1995
; Hu and Henderson
1997
), as well as changes in the lateral (L)OC system or its
targets, the terminals of afferent fibers under the inner hair cells.
Despite considerable effort devoted to documenting conditioning-related
protection, detailed consideration of the effects of conditioning, per
se, on cochlear physiology has been largely overlooked. Such
characterization is crucial to understanding the protective mechanism
because overexposure-related PTSs will occur from a new,
postconditioning baseline/state. In some previous work, such
conditioning-related threshold shifts complicated interpretation of the
conditioning-related protection; in other studies, postconditioning testing was either cursory or omitted altogether (see
DISCUSSION). Moreover, this characterization can provide
important clues to the mechanisms underlying the protection. Such a
characterization is the focus of the present report. Data were obtained
as part of an ongoing investigation of a possible OC role in
conditioning-related protection (Brown et al. 1998;
Kujawa and Liberman 1997
). In the present report, we
compare CAP- and DPOAE-based measures of cochlear responsiveness and OC
reflex strength in untreated control versus sound-conditioned animals.
This work was presented, in part, at the Midwinter Meeting of the Association for Research in Otolaryngology, 1996, St. Petersburg Beach, FL.
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METHODS |
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Experimental animals and inclusion criteria
Albino guinea pigs of either sex were used in all experiments. To control for potentially confounding effects of age, animals were accepted into the protocol only if their weights fell within a restricted range (325-350 g), and all animals remained in the protocol for an equal number of days regardless of group assignment (see Fig. 1).
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Animals were required to pass a DPOAE screen (methods detailed later in
the text) designed to rule out preexisting middle ear or cochlear
pathology. Animals were anesthetized (pentobarbital sodium, Nembutal,
25 mg/kg ip) and were tested intact and unrestrained. Low-level
2f1-f2 DPOAEs were recorded
bilaterally in response to nine logarithmically spaced
f2 frequencies across the range 2.78-12.14 kHz.
Animals entered the protocol only if responses to 40 dB SPL primaries
were present (3 dB above the surrounding noise floor) at all nine test
frequencies. Although noise floors vary with frequency, untreated
animals passing such screens invariably demonstrate CAP thresholds
consistent with laboratory norms. Animals recovered from pretest
anesthesia for 5 days before undergoing subsequent treatments. Groups
did not differ (P > 0.05) on the basis of the pretest
measures. All procedures were conducted in accordance with the National
Institutes of Health guidelines and were approved by the Massachusetts
Eye and Ear Infirmary's animal care committee.
Sound exposures
All conditioning and traumatic exposures were delivered to awake
and unrestrained animals in a small, subdivided cage suspended within a
small, reverberant chamber (see Liberman and Gao 1995 for details). The conditioning/traumatic exposure stimulus was a 2- to
4-kHz octave band of noise (OBN) filtered with a 60 dB/octave slope,
amplified and delivered through an exponential horn coupled to the
chamber. Before each exposure, the noise level was measured with a
1/4-in condenser microphone suspended in the cage at the approximate position of the animal's head (without the animal present). Variations in SPL were < 1 dB at any point within
either of the cage's two partitions. At the maximum exposure level
(109 dB SPL), frequency components outside the desired passband were >60 dB below those in the passband.
Experimental groups
Animals that met weight and DPOAE screening requirements were randomly assigned to one of four groups (Fig. 1): Group 1 (Control, n = 18): animals served as untreated, age- (weight-) matched, time controls. After recovering from immediate effects of screening anesthesia, they were returned to a quiet room (ambient SPL ~50 dB) in the animal care facility. Control animals received no additional manipulations and underwent final testing (see following text) on day 32. Group 2 (Condition-only, n = 26): Animals were screened as described in the preceding text. Beginning on day 17, they were sound conditioned by daily exposure to the 2- to 4-kHz OBN (85 dB SPL overall level) on a 6 h on/18 h off schedule for 10 consecutive days. For the next 5 days, animals received no conditioning exposures; final testing took place on the 6th day after the conditioning ended (day 32). Group 3 (Condition-Trauma, n = 24): Beginning on day 6 after screening, animals were conditioned as described in the preceding text. Six days after conditioning (day 21), they were exposed (individually) to the trauma stimulus, the same 2- to 4-kHz OBN, now presented at 109 dB SPL for 4 h. Final testing took place 11 days later (day 32). Group 4 (Trauma-Only, n = 20): Animals received no additional treatments after the DPOAE screening test until undergoing the traumatic exposure on day 21, as above. Final testing occurred 11 days later, on day 32. During all rest periods from exposure, animals in groups 2-4 were housed in the animal care facility with animals from group 1 (Control group).
Final testing
On day 32, each animal was anesthetized (Nembutal, 25 mg/kg ip; Innovar Vet, 0.5 ml/kg im) and surgically prepared for acute physiological measurements. Additional anesthetic (one-third original dose) was administered at 2-h intervals. All procedures were conducted in an acoustically and electrically shielded experimental chamber. Electrocardiograph and rectal temperature were monitored and temperature was maintained near 38°C by heating the air within the chamber and by using a heating pad when necessary. Animals were tracheotomized and usually allowed to breathe unassisted. Rarely, spontaneous middle-ear muscle contractions interfered with DPOAE response recording; in those cases, the animal was paralyzed (curare; 1 mg/kg im) and artificially respirated with room air. The skin and muscles overlying the dorsolateral portions of the skull were reflected and cartilaginous ear canals were severed to allow insertion of the sound delivery systems. The posterior aspects of both bullae were exposed, and a small hole shaved in each to allow placement of a silver wire on the bone ventral to the round window (to record the CAP).
Stimulus generation and response detection
CAPS.
Compound action potential (CAP) thresholds were recorded from all
animals using a calibrated acoustic system consisting of a 1-in
condenser microphone used as a sound source and a 1/4-in microphone coupled to a probe tube (Kiang et al. 1965)
for measurement of sound pressures at the entrance to the bony ear
canal. Tone-pip stimuli (5-ms duration, 0.5-ms rise/fall,
cos2 shaping, delivered 10/s) were presented at
29 logarithmically spaced frequencies from 2.31 to 30.49 kHz. Responses
were recorded via a silver-wire electrode near the round window niche
(e.g., Johnstone et al. 1979
), amplified (10,000 times),
filtered (0.3- to 3-kHz passband), and averaged (16 consecutive stimuli
alternated in polarity to eliminate the cochlear microphonics). At each
frequency, sound pressure was varied (first in 2 dB steps and then in 1 dB steps) to determine the level required to produce a 10-µV
peak-to-peak CAP (CAP threshold functions). As the experimental series
progressed, we decided to examine CAP responses at suprathreshold
stimulus levels. Thus in all later animals, CAP amplitudes were
measured at nine logarithmically spaced stimulus frequencies from 2.78 to 12.14 kHz. At each frequency, stimulus level was increased from 5 to
85 dB SPL in 5-dB steps.
DPOAES. DPOAEs were obtained from all animals. For both screening and final testing purposes, DPOAEs were elicited by equilevel primary stimuli (f1, f2) chosen such that f2 matched certain CAP test frequencies (9 logarithmically spaced f2 frequencies from 2.78 to 12.14 kHz; f2/f1 = 1.2). Primary tones were routed through computer-controlled attenuators to an acoustic probe assembly (Etymotic Research ER10c) coupled to the ear canal. The sensitivity of the probe's microphone (dBV/Pa) was calibrated daily. Ear-canal sound pressure was amplified (gain = 40 dB), filtered (high-pass >1 kHz) and led to a spectrum analyzer (Hewlett-Packard 35660A) for fast Fourier transform (FFT) analysis and display. Under computer control, amplitudes of the 2f1-f2 DPOAE and surrounding noise floor (average of 6 values within 50 Hz of the 2f1-f2 frequency) were extracted from an average of 10 spectra. Responses were obtained as growth functions of increasing primary level (from below "threshold" to 75 dB SPL in 2-dB steps). Distortion at 2f1-f2 was not detectable above the noise floor when measured either in a hard-walled, passive coupler of appropriate volume or in a "dead" ear at primary levels employed in this investigation.
OC REFLEX ASSAYS.
OC reflex strength was measured during the final test in subgroups of
Control and Condition-Only animals. This assay was introduced into the
protocol after the study began; however, after its introduction, all
animals were tested. Contralateral sound-evoked effects and the
pathways thought to subserve them have been reviewed extensively (see,
for example, Kujawa et al. 1994; Warren and
Liberman 1989
). The metric for ipsilaterally evoked OC activity
is based on the rapid, post-onset adaptation of the
2f1-f2 DPOAE described by Liberman et al. (1996)
. To analyze this rapid
adaptation, primary stimuli were presented as 2-s bursts, and DPOAE
amplitude was sampled with time resolution of ~10 ms via a
LabVIEW-based system using linked A-D and D-A boards for stimulus
generation (f1 and f2) and
response analysis. Ear-canal sound pressure was digitized (20-µs
sampling) and broken into contiguous 10.24-ms samples. An FFT was
computed on each waveform sample, and the amplitude of the
2f1-f2 DPOAE extracted. To
eliminate onset transients, the first 10.24 ms of the response waveform
were ignored. To assess contralateral OC reflex strength, a wideband
noise (~70 dB SPL) was introduced to the contralateral ear 1 s
after the onset of primary stimulation. Measurements were obtained
across a range of f2 frequencies (2, 4, 6, 8, 10, 12 kHz; f2/f1 = 1.2). At
each f2 frequency, L1 was
varied in 5-dB steps from 60 to 80 dB SPL, with
L2 = L1
5.
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RESULTS |
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At the final test, amplitude-versus-level functions were obtained
for both CAPs and DPOAEs for each animal from each of the four sound
exposure groups. The relation between the CAPs and the underlying
activity of the auditory nerve fibers that generate them is well
understood (Antoli-Candela and Kiang 1978); thus the CAP
provides a useful metric of the sensitivity of the entire auditory
periphery from middle ear through neural firing. The DPOAEs are useful,
especially at low levels, because of the insight they give into OHC
function in particular (Liberman et al. 1997
; Trautwein et al. 1996
). Thus we chose to combine the two
metrics in hopes of better defining the locus of any functional alterations.
Group means (and standard errors) for each frequency-level
combination are shown in Figs. 2 and
3. To facilitate cross-frequency comparisons as well as comparisons between near-threshold responses and
suprathreshold responses, these mean input-output functions have been
transformed into mean isoresponse contours. Such contours are
illustrated in Fig. 4 for Control
animals. For CAP data (Fig. 4A), the noise floor of our
measurements (given the limited number of responses averaged at each
frequency/level combination) was 1-2 µV, thus a value of 5 µV was
chosen for a near-threshold response value, and response amplitudes 10 and 20 dB greater (15.8 and 50 µV) were chosen for suprathreshold
metrics. For DPOAEs (Fig. 4B), the noise floor was frequency
dependent (see Fig. 3) but was <10 dB SPL at all frequencies. Thus
10 dB was chosen for a near-threshold value and 0 and +10 dB SPL for
suprathreshold values. In control ears, the stimulus level required to
produce near-threshold DPOAEs is roughly 20 dB higher than that
required to produce near-threshold CAPs. However, for both metrics,
roughly a 20-dB increase in stimulus level is required to produce a
20-dB increase in response amplitude.
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Effects of high-level sound: Trauma-Only versus Control groups
Effects of the high-level sound on CAPs and DPOAEs can be seen by
comparing the averaged amplitude-versus-level functions for the
Trauma-Only () and Control (
) groups shown in Figs. 2 and 3. For
both CAP and DPOAE metrics, the overstimulation has shifted the
amplitude-level curves rightward and/or reduced their slopes. Rightward
shifts are maximum in the frequency region of the exposure band and
extending 0.5-1 octave above its upper border; however, decrements in
suprathreshold responses extend to higher frequencies and are apparent
even in regions of normal to near-normal threshold sensitivity.
The magnitude and frequency dependence of noise damage are better seen
in Fig. 5 (), where the data are
converted to isoresponse contours and expressed as permanent
"threshold" shift (PTS) by normalizing Trauma-Only values with
respect to corresponding values from the Control group. For the lowest
isoresponse criteria (Fig. 5, A and C), the
maximum PTS is ~20 or 10 dB when measured via CAPs or DPOAEs,
respectively. For both metrics, the PTS is greater at lower
frequencies. For the highest isoresponse criteria (Fig. 5, B
and D), the maximum PTS grows, its frequency dependence
changes, and the discrepancies between CAP and DPOAE measures increase. For CAP responses, PTS at this high isoresponse criterion shows a
maximum of >40 dB at a distinct peak in the frequency region roughly
0.5 octave above the upper limit of the conditioning exposure band
(Fig. 5B); on the other hand, the DPOAE-based measures do not change greatly from the near-threshold (Fig. 5C) to the
suprathreshold (Fig. 5D) responses. This discrepancy may
reflect a limitation in the dynamic range of the DPOAE metric (see
DISCUSSION).
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Conditioning-related protection: Trauma-Only versus Condition-Trauma groups
The protective effect of the conditioning protocol can be seen in
the amplitude-versus-level functions of Figs. 2 and 3 and in the
comparatively smaller PTSs shown in Condition-Trauma animals (Fig. 5).
For both CAPs and DPOAEs, response "thresholds" are lower (PTSs
are smaller) and suprathreshold amplitudes are greater for
Condition-Trauma animals () than for Trauma-Only animals (
). By
both metrics, protection afforded by this prior conditioning is seen
across a broad range of frequencies with the largest protective effects
appearing at midfrequencies.
The frequency dependence of the protective effect is better illustrated by the contours shown in Fig. 6. To compute "protection," the mean isoresponse contours from Fig. 5 for Condition-Trauma animals were subtracted from Trauma-Only. For both CAP- (Fig. 6A) and DPOAE-based (Fig. 6B) metrics, protection is evident for both near-threshold and suprathreshold response amplitudes; however, the magnitude and frequency dependence of the protection change with response criterion. For the CAP metric, protective effects are largest for frequencies within the exposure band when assessed with near-threshold responses (5-µV isoresponse contours); they demonstrate dramatic growth and show a pronounced peak at frequencies above the exposure band (6-8 kHz) when assessed for suprathreshold levels (15.8, 50 µV) of response. The frequency dependence revealed by the DPOAE-based measures is less clear-cut. However, for both the 0 and +10 dB isoresponse contours, the maximum protection also is seen at test frequencies significantly above the exposure band. The DPOAE-based protective effect does not grow with response criterion as for the CAP. As discussed in the following text, this may reflect a limitation in the dynamic range of the DPOAE metric.
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Conditioning-related response enhancement: Condition-Only versus Control
An important goal in our overall study of conditioning-related protection was to employ a conditioning protocol that "protected" the ear but did not, on its own, cause damage. In the present study, Condition-Only animals were conditioned identically to Condition-Trauma animals. On day 6 postconditioning, when the high-level exposure was delivered to the Condition-Trauma group, the Condition-Only group underwent final testing (Fig. 1).
Comparison of CAP and DPOAE responses from Condition-Only animals to similar data from age-matched controls (without any sound exposures) suggests that the conditioning exposure actually enhances cochlear responses. As seen in Figs. 2 and 3, small, but significant, increases in response amplitudes were seen in the Condition-Only animals, by both CAP and DPOAE metrics (P < 0.01, collapsed across frequency and level). For CAPs, these enhancements were largest for test frequencies from 6 to 10 kHz, i.e., roughly 0.5-1 octave above the exposure band. Indeed, responses at 12 kHz were reduced slightly in the Condition-Only group. For DPOAEs, enhancements were largest at lower frequencies (f2 within the exposure band).
The frequency dependence of the conditioning-related enhancements is better seen in the normalized isoresponse contours of Fig. 5. In these plots, enhancements appear as negative values of PTS. By the CAP metric, enhancement clearly peaks at test frequencies well above the exposure band. For DPOAE-based measures, the enhancement is shifted to lower frequencies. For the CAP metric, the enhancements correspond in frequency to the regions demonstrating maximum protection; for DPOAEs, enhancements appear at frequencies well below those demonstrating the most robust protection.
In contrast to the dramatic growth of CAP-based protection for higher criterion levels of response, CAP-based enhancement is somewhat smaller at the 50- versus the 5-µV amplitude criterion. Greater values of conditioning-related enhancement are seen, however, for DPOAEs at suprathreshold levels (compare Fig. 5, C and D); indeed, DPOAE enhancement was even greater (exceeding 10 dB) at a response criterion of +15 dB SPL (data not shown).
Olivocochlear reflex strength: Condition-Only versus Control
When monitored with fine time resolution,
2f1-f2 DPOAEs demonstrate
rapid onset changes in amplitude (Fig.
7). This onset change can be used to
assay the strength of the ipsilateral (ipsi) OC reflex, i.e., the
magnitude of OC activity elicited by the primary tones, themselves
(Kujawa and Liberman 1998; Liberman et
al. 1996
). Contralateral (contra) OC reflex strength can be assayed in this paradigm by adding a stimulus to the opposite ear and
measuring the associated change in DPOAE amplitude. As described
previously (Kujawa and Liberman 1998
; Liberman et
al. 1996
; Siegel and Kim 1982
), DPOAE amplitudes
can be reduced (Fig. 7A) or enhanced (Fig. 7B) by
OC feedback, depending on the level/frequency combination of primaries.
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This DPOAE-based paradigm was used in the present study to assess the effects of conditioning on OC reflex strength. For the present analysis, the absolute values of sound-evoked OC effects on DPOAE amplitudes were averaged, for each frequency-level combination of primary tones, and compared for Condition-Only versus Control animals (Fig. 8). Several characteristics of OC reflex effects are revealed in these panels. First, for both groups of animals, ipsi and contra effects display the same frequency and level dependence. Over the ranges tested here, effects are largest at higher f2 frequencies (8-10 kHz) and higher primary levels (65-75 dB SPL). This frequency and level dependence was not altered by conditioning. However, the data suggest that conditioning may increase the magnitudes of these sound-evoked OC effects. For most primary levels at f2 values of 8 and 10 kHz, the mean ipsi and contra OC effect magnitudes were higher for Condition-Only than for Control animals; at some frequency and level combinations, averaged effect magnitudes are more than double those recorded for controls. When collapsed across level, these Condition-Only versus Control differences reached significance at f2 = 8-10 kHz for ipsi (P < 0.01) and at f2 = 10 kHz for contra (P < 0.05) measures.
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DISCUSSION |
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Conditioning-related response protection
Conditioning-related protection has been demonstrated in several
species of experimental animals including guinea pig, chinchilla, gerbil, and rabbit (e.g., Campo et al. 1991;
Canlon et al. 1988
, 1992
; Ryan et al.
1994
). Studies conducted in the human (Barrenas et al.
1996
; Miyakita et al. 1992
) have avoided
PTS-producing exposures; however, conditioning-related protection
against TTS has been observed. Across studies, the relationship between
the frequencies of maximal PTS from high-level exposure alone and the
frequencies of maximum conditioning-related protection is fairly
constant. Most studies report ~15-25 dB of threshold protection, maximum in a frequency region within, and to 1 octave above, the exposure stimulus.
Data in the present study show that the magnitude and frequency dependence of the protective effect depend strongly on the criteria, as well as the type of response used to measure "threshold." For the CAP, conditioning-related protection peaked at frequencies within the exposure band when the smallest response criterion (5 µV) was used (Fig. 6A). As the response criterion was increased (to 15.8 and 50 µV), the protection increased dramatically and peaked at higher test frequencies, 0.5-1 octave above the exposure band (Fig. 6A).
For DPOAEs, conditioning-related protection grew slightly with
increasing response criterion and peaked at test frequencies well above
the exposure band (Fig. 6B). In interpreting the small disparity in the peak frequency of protection for CAP versus DPOAE metrics (5.81 and 6.98 kHz in Fig. 6A vs. 6.98 and 8.4 kHz
in Fig. 6B), one must consider the plotting convention
(f2 referent) used for DPOAEs. For the equilevel,
closely spaced primaries used in this investigation, maximum
contributions to the DPOAE amplitude may be closer to the geometric
mean rather than the f2 place (e.g., Martin et al. 1998). Using the geometric mean referent,
the DPOAE-based peak in protection would fall at 6.4 and 7.6 kHz, in
closer agreement with the CAP data.
The striking difference in the growth of the CAP- and DPOAE-based protection metrics with increasing response criterion (Fig. 6, A and B) probably indicates a limitation in the dynamic range of the DPOAE-based assay. As illustrated in Fig. 4, the SPL required to produce our highest criterion (+10 dB SPL) DPOAE response is already >65 dB SPL at certain f2 frequencies even in control ears. At higher primary levels, irreducible animal-generated distortion, as well as system-generated distortion, can produce a robust 2f1-f2 "response" even in a dead ear. This effectively sets the upper limit of useful DPOAE stimulation. Of course, the dynamic range and interpretability of the CAP metric is limited by the spread of excitation with increasing stimulus level to neurons with characteristic frequencies (CFs) above stimulus frequency. Indeed, the apparent shift of the peak protection to higher frequencies for higher criterion levels of response might be related, in part, to such upward spread of activity as stimulus level is increased. Nevertheless, with CAP, the loss of neuronal responsivity in the lesion focus can never be fully compensated by recruitment of "off-CF" neurons and reduction in the CAP amplitude-versus-level functions still can provide information as to lesion severity even at high stimulus levels. Thus the lack of growth of protection shown in Fig. 6B is probably an artifact of the small useful range of input levels for the DPOAE-based metric. Regardless of the criterion level, the limited range of the DPOAE-based assay will tend to underestimate even modest response shifts. This may be one factor contributing to the smaller degree of protection revealed by DPOAE than by CAP measures.
Other factors could contribute to the differences in CAP- and
DPOAE-based measures. Although even near-total loss of IHCs can leave
the DPOAE at 2f1-f2
essentially unaltered (Liberman et al. 1997;
Trautwein et al. 1996
), the normal CAP response requires contributions from both OHCs and IHCs. Thus if the noise-induced pathology in these ears involves a combination of IHC and OHC damage,
as often is seen (Clark and Pickles 1996
;
Engstrom 1983
; Liberman and Kiang 1978
),
one might expect larger PTS measured by CAPs than by DPOAEs. In
support, the type of amplitude-versus-level abnormalities seen in the
CAP in the present study are reminiscent of those seen in cases of
selective IHC damage (Liberman et al. 1997
).
Specifically, such studies have shown that IHC damage decreases the
slope of the amplitude-versus-level functions, thereby causing the PTS
to grow rapidly as the isoresponse criterion is increased (Fig. 5).
Involvement of LOC neurons also could contribute to differences in CAP-
and DPOAE-based reflections of the protection. Because LOC function is
understood so poorly and because no assay exists for LOC activity, an
LOC-mediated contribution to conditioning-related protection cannot be
ruled out. Indeed, data from chronic OCB lesion experiments
(Kujawa and Liberman 1997) suggest a role for LOC
neurons in protection from acoustic injury: complete section of OC
inputs (LOC + MOC) increases PTSs from subsequent exposure whereas
removal of MOC inputs only does not. A possible role for LOC neurons
has been discussed in detail in a previous report (Kujawa and
Liberman 1997
).
Conditioning-related response enhancement
An unexpected finding of these experiments is that conditioning, by itself, enhanced cochlear responsiveness as reflected in CAP and DPOAE amplitudes. When analyzed as isoresponse contours, these changes corresponded to threshold enhancements of ~6 dB (Fig. 5). These enhancements were visible throughout the stimulus level range such that both near-threshold (Fig. 5 A and C) and suprathreshold (Fig. 5, B and D) isoresponse contours were affected. At higher criterion levels, DPOAEs show even greater enhancements from control values.
In the present study, the frequency region of maximal enhancement
effects was different when assayed by CAP- versus DPOAE-based metrics.
For the isoresponse criteria employed here, CAP enhancements were
greatest in the frequency region 0.5-1 octave above the conditioning exposure band; for the DPOAE isoresponse criteria, maximum enhancements were observed at lower frequencies. These results are consistent with
findings for CAPs in another series of experiments from this laboratory
(Brown et al. 1998).
Conditioning-related enhancements of the type described here have not
been reported previously. However, few other experiments have been
structured to screen carefully for such effects. In some
investigations, testing was not done in a condition-only state, except
at very short postexposure times when TTS was clearly evident (e.g.,
Skellett et al. 1996). In others, the methods would not
have revealed the relatively small alterations reported here (e.g.,
Canlon et al. 1988
). Recently Hu and Henderson
(1997)
studied DPOAE growth in four chinchillas conditioned (10 days) and then allowed to recover (5 days) as reported here. No
threshold or suprathreshold DPOAE amplitude enhancements are evident in
those data (their Fig. 3). However, their published plots are
restricted to primary frequencies above the upper limit of their
0.5-kHz octave band conditioning stimulus, whereas our
conditioning-related enhancements were maximal for primaries within the
2- to 4-kHz conditioning band (Figs. 3 and 5, C and
D). Boettcher and Schmiedt (1995)
observed,
in gerbil, that DPOAE amplitudes were unaffected by 12 daily 6-h
exposures to an octave band centered at 4 kHz (80 dB SPL). Of course,
this type of "toughening" paradigm may not lead to the type of
response enhancements seen in the "conditioning" paradigm used in
the present study.
Relation between enhancement and protection and their underlying mechanisms
Conditioning-related enhancement and protection may share a common mechanism or may be completely unrelated. In a trivial sense, the conditioning-related enhancement of cochlear responses must contribute to the observed protective effects. The enhancements represent a readjustment of the preexposure baseline in the Trauma-Only versus Condition-Trauma animals, such that, if the traumatic exposure itself caused an identical decrement in cochlear responsiveness in the two groups, a protective effect of the conditioning would be observed. However, this preexposure enhancement cannot account for all of the protective effect: when viewed either as averaged amplitude-versus-level functions or as averaged isoresponse contours, the conditioning-related decreases in trauma-induced response decrements are much larger in magnitude than the conditioning-related enhancements (see Fig. 5).
Mechanisms underlying conditioning-related protection are poorly
understood. In principle, either could arise from changes in cochlear
physiology, per se, or from alterations in the actions of the two
feedback pathways to the auditory periphery. One of these pathways
involves middle ear muscle activity evoked by high-level sound.
Activation of the middle ear muscles can attenuate transmission of
sound through the middle ear, reducing the effective level of sound
reaching the cochlea. Many of the conditioning/toughening exposure
paradigms described to date have employed stimuli in the frequency
region maximally effective in eliciting such activity (at least in
rabbit and human) (Møller 1984). Three studies have examined a role for the MEM reflex in mediating these protective effects; either by sectioning the MEM tendons (Henderson et al. 1994
; Ryan et al. 1994
) or by muscle paralysis
(Dagli and Canlon 1995
). Each of these studies failed to
identify a significant role for the MEMs in conditioning-related protection.
The OC efferent pathway also is known to play a role in protecting the
ear from acoustic overstimulation. Under certain conditions of
stimulation, threshold losses from acute, high-level sound exposure can
be reduced by manipulations known to raise activity levels in efferent
pathways; for example, by direct electrical activation of MOC fibers
(e.g., Rajan 1988a,b
; Reiter and Liberman 1995
) or by sound stimulation of the contralateral ear (e.g., Cody and Johnstone 1982
; Rajan and Johnstone
1983
). Whether the conditioning-mediated protection is also
OC-mediated is unresolved. If sound conditioning amplifies the strength
of the OC sound-evoked reflex or alters the response of the cochlea to
sound-evoked OC activity, decreased vulnerability to acoustic injury
could result.
We investigated possible conditioning-related changes in OC reflex
strength using a DPOAE-based assay. In Control and Condition-Only ears,
OC reflex strength (ipsi and contra) was maximum at higher f2 frequencies (8-10 kHz) and higher levels
(65-75 dB SPL) of primary tone stimulation. This frequency and level
dependence is consistent with the known longitudinal distribution of
MOC fibers in the guinea pig cochlea (Liberman and Brown
1986; Liberman and Gao 1995
; Robertson
and Gummer 1985
), the sound-evoked response properties of MOC
neurons (Brown 1989
; Liberman 1988
), and
the level dependence of MOC influences on cochlear responsiveness (Gifford and Guinan 1987
; Guinan and Stankovic
1996
). The sound conditioning led to a slight increase in OC
reflex strength (Fig. 8); however, these differences reached
significance only for f2 = 8-10 kHz. Similar
results were found in a parallel study (Brown et al.
1998
) that directly compared sound-evoked discharge rates in
single MOC fibers from Condition-Only versus Control animals. For
high-level, binaural sound, a stimulus condition appropriate to the
study of MOC reflex contributions to protection, firing rates of MOC
neurons with CFs above the conditioning band (6-12 kHz) were increased
significantly in Condition-Only animals relative to Controls.
Interestingly, this CF band corresponds to the frequency band in which
conditioning-related protection was greatest.
It is argued elsewhere (Brown et al. 1998) that
conditioning-related enhancements of MOC reflex strength cannot be
solely responsible for the phenomenon of conditioning-mediated
protection. The key argument is that significant protection is observed
in frequency regions where MOC reflex strength is small and little affected by the conditioning. For somewhat different reasons, it is
also difficult to explain the conditioning-related enhancements of CAP
and DPOAEs based on amplification of the MOC reflex. First, the brief
tone pips used to generate CAPs do not elicit significant MOC activity
(Liberman and Brown 1986
); thus CAP amplitudes
should be unaffected. Second, the frequency dependence of effects on the DPOAE-based metrics are not the same: For DPOAEs, where the stimuli
themselves clearly can produce MOC reflex activity (Liberman et
al. 1996
), DPOAE threshold and suprathreshold response
enhancements were shifted to frequencies substantially lower than those
for which the MOC reflex was amplified.
Alternatively, both conditioning-related response enhancements and
conditioning-related protection can be explained by fundamental and
persistent changes to the targets of the MOC neurons, the OHCs
themselves. Clearly, an increase in the contribution of OHCs to
amplification of motion in the cochlear duct would be expected to
enhance both DPOAEs and CAPs. In the original conditioning experiments,
Canlon and colleagues (1988) proposed such an
hypothesis, suggesting that the motile OHCs might be "trained" to
tolerate higher exposure levels. Such ideas grew from the
now-discounted notion that motility arose from actin-myosin
interactions and an analogy to the use-induced changes observed in
skeletal muscle. Nevertheless, fundamental changes to OHCs isolated
from conditioned cochleae have been identified. These changes include
increases in total membrane content in the basal poles of OHCs
(Canlon et al. 1993
) and changes in calbindin 28-k
immunoreactivity (Canlon and Fransson 1994
) and F-actin
labeling (Hu and Henderson 1997
). In physiological
experiments, such OHCs demonstrated altered responsiveness to ATP
(Chen et al. 1995
). Present understanding of the
functional consequences of such alterations on in vivo cochlear
responsiveness is limited and further investigation will be required to
determine the precise locus of the conditioning-related changes.
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ACKNOWLEDGMENTS |
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We thank M. C. Brown and J. J. Guinan for comments on the manuscript.
This research was supported by National Institute of Deafness and Other Communication Disorders Grants F32 DC-00180 and R01 DC-00188.
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FOOTNOTES |
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Present address and address for reprint requests: S. G. Kujawa, Dept. Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Bloedel Hearing Research Ctr, Box 357923, Bldg. CHDD, Seattle, WA 98195.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 September 1998; accepted in final form 25 March 1999.
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REFERENCES |
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