Long-Term Sound Conditioning Enhances Cochlear Sensitivity

Sharon G. Kujawa and M. Charles Liberman

Department of Otology and Laryngology, Harvard Medical School; and Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Schematic illustrating the timing of the various experimental treatments across the 4 groups of animals. Details related to each of the stimulation protocols can be found in METHODS.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2. Average (means ± SE) compound action potential (CAP) amplitudes as a function of increasing stimulus level (5-85 dB SPL in 5-dB steps) obtained for animals from each group (Control = 18; Condition Only = 26; Condition-Trauma = 12; Trauma-Only = 12). Responses are recorded as peak-to-peak amplitudes in microvolts and are shown in separate panels for stimulus frequencies between 2.78 and 12.14 kHz. Average values less than our standard criterion (10 µV p-p) have been removed from the graphs to allow clearer display of low-level responses.



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Fig. 3. Average (means ± SE) 2f1-f2 distortion product otoacoustic emission (DPOAE) amplitudes as a function of increasing stimulus level (L1 = L2; noise floor to ~75 dB SPL) for all animals from all groups (Control = 22; Condition Only = 26; Condition-Trauma = 24; Trauma Only = 20). Responses are recorded in dB SPL and are shown, along with their corresponding noise floors, in separate panels for f2 frequencies 2.78-12.14 kHz. Responses were acquired for stimuli incremented in 2-dB steps; they are displayed in 4-dB steps for viewing clarity. Average values less than the noise floor have been removed from the graphs to allow clearer display of low-level responses.



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Fig. 4. Average (means ± SE) CAP and DPOAE isoresponse contours for Control animals (n = 22). Data are plotted relative to the stimulus levels required to achieve the designated criterion levels of response. Contours representing near-threshold responses (CAP = 5 µV; DPOAE = -10 dB SPL isoresponse criteria) are compared with contours for high criterion levels of response (CAP = 50 µV; DPOAE +10 dB SPL) for CAP (A) and DPOAE (B) metrics. DPOAE data are expressed in terms of f2 stimulus frequency.

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 (triangle ) and Control (open circle ) 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 (triangle ), 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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Fig. 5. Average (means ± SE) CAP and DPOAE shifts from Control group (n = 22) values (normalized to 0) for animals in Condition-Only (n = 26), Condition-Trauma (n = 24), and Trauma-Only (n = 20) groups. CAP isoresponse shifts (5 µV; 50-µV amplitude criteria) are shown in A and B, respectively; DPOAE isoresponse shifts (-10; +10 dB SPL amplitude criteria) are shown in C and D. In constructing the averaged DPOAE functions, responses failing to meet the stated criterion amplitude at the highest stimulus level tested were assigned a value 1 dB greater than the maximum stimulus level employed; this was never necessary for the CAPs.

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 (black-triangle) than for Trauma-Only animals (triangle ). 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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Fig. 6. Average (means ± SE) CAP and DPOAE protection (Trauma-Only - Condition-Trauma) as a function of frequency and isoresponse criteria. Data obtained by CAP measures are illustrated A; those obtained by DPOAE measures are contained in B.

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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Fig. 7. DPOAEs at 2f1-f2 were recorded in response to primary-only (ipsilateral) stimulation (during the 1st 1,000 ms) and during primary + contralateral wideband noise stimulation (during the 2nd 1,000 ms). Ipsilateral (ipsi) and contralateral (contra) OC reflex strength was measured as shown in animals from Condition-Only (n = 7) and Control (n = 5) groups. Depending on the primary level combination, ipsi and contra OC effects could be negative (A) or positive (B) in sign. In this particular example, f2 = 10 kHz; L1 = 75, L2 = 70 (A) or 65 (B) (see text for further details).

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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Fig. 8. Average (means ± SE) ipsi and contra OC effect magnitudes in subgroups of Control (n = 8) and Condition-Only (n = 12) animals. Measures were obtained for f2 = 2-12 kHz and L1 = 60-80; L2 = 55-75 dB SPL incremented together in 5-dB steps. Effects could be positive or negative in sign as shown in Fig. 7; thus means shown here were calculated from the absolute values of the effects. Contralateral wideband noise was delivered at an overall level of 70 dB SPL during second 2 of a 2-s primary stimulation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society