The functional age of hearing loss in a mouse model of presbycusis. I. Behavioral assessments

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Hearing Research 183 (2003) 44^56 www.elsevier.com/locate/heares

The functional age of hearing loss in a mouse model of presbycusis. I. Behavioral assessments Cynthia A. Prosen a

a;


, Dawn J. Dore a , Bradford J. May

b

Department of Psychology, 306 Gries Hall, Northern Michigan University, Marquette, MI 49855, USA b Department of Otolaryngology-HNS, Johns Hopkins University, Baltimore, MD 21205-2195, USA Received 20 December 2002; accepted 10 June 2003

Abstract Presbycusis is a common form of hearing loss that progresses from high to low frequencies with advancing age. C57BL/6J mice experience a rapid progression of presbycusis-like hearing deficits and thus provide a convenient animal model for evaluating behavioral, physiological and anatomical correlates of the disorder. Previous studies of C57BL/6J mice have relied on short-term observations of age-matched subject groups to reconstruct a time course for auditory pathologies. Such statistical approaches are weakened by the variability of hearing thresholds in young mice and the inconsistent timing of degenerative effects in older mice. The present study was designed to resolve these ambiguities by tracking the hearing abilities of individual C57BL/6J mice from age 16 weeks until the onset of hearing loss in specific listening conditions. Testing at frequencies of 8 and 16 kHz in quiet confirmed the high-to-low frequency progression that is characteristic of presbycusis. Often the hearing loss developed in two phases, one gradual and the other abrupt. Testing in noise revealed deficits that were first manifested as threshold instability and then an increased susceptibility to masking. These changes occurred before hearing loss in quiet. CBA/CaJ mice did not show significant loss during a similar period of observation. Our findings provide a well-ordered chronology for isolating the functional consequences of multiple cochlear pathologies that arise during the time course of presbycusis. This neurobehavioral assessment is termed the functional age of hearing loss. Neuroanatomical assessments of behaviorally characterized C57BL/6J mice are presented in the companion paper [Hear. Res. 183 (2003) 29^36]. = 2003 Elsevier B.V. All rights reserved. Key words: Presbycusis; Animal auditory psychophysics; Signal detection in noise

1. Introduction Persons aged 67 and older account for nearly half of all diagnoses of hearing loss in the United States (Peters and Moore, 1992). Among the elderly, presbycusis is the most common etiology for hearing impairment (Johnson et al., 1997). The disorder is characterized by a progressive loss of auditory sensitivity that advances from high to low frequencies (Parham, 1997; Phillips et al., 2000).

* Corresponding author. Tel.: +1 (906) 227 2941; Fax: +1 (906) 227 2954. E-mail address: [email protected] (C.A. Prosen). Abbreviations: ABR, auditory brainstem response; CR10 , critical ratio at 10-dB sensation level; SPL, sound pressure level

Auditory frequency selectivity also declines with presbycusis (Patterson et al., 1982) and may lead to an increased susceptibility to masking noise (Pichora-Fuller et al., 1995; Frisina and Frisina, 1997; Cohn, 1999). These de¢cits are especially debilitating because they disrupt normal auditory processing at sound levels well above threshold. Conventional hearing aids may restore cochlear sensitivity but do not improve the listener’s ability to distinguish meaningful sounds from background noise (Trychin, 1997). The diverse clinical manifestations of presbycusis imply multiple cochlear etiologies. Our studies are based on the premise that individual degenerative e¡ects follow an orderly time course and are revealed by discrete events in the progression of hearing loss. Consequently, speci¢c pathologies may be isolated by relating neuroanatomical measures to categorical perceptual de¢cits

0378-5955 / 03 / $ ^ see front matter = 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00211-9

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like the inability to detect signals in noise. Furthermore, hypothesized structure-function correlations may be enhanced by de¢ning audiological metrics that are most closely associated with unique pathological states. The objective of our present study was to begin to identify these predictive behavioral criteria for C57BL/ 6J mice. C57BL/6J mice exhibit presbycusis-like behavioral de¢cits that advance rapidly to profound deafness during the ¢rst year of life (Mikaelian, 1979; Henry and Chole, 1980). This mouse strain provides a convenient model for exploring anatomical and physiological correlates of presbycusis that may take decades to manifest in humans (Li and Borg, 1991; Spongr et al., 1997). A complete characterization of the functional stages of presbycusis in C57BL/6J mice remains problematic because previous behavioral studies have focused on the reconstruction of a general time course of hearing loss from short-term surveys of age-matched subjects. As an instrument for predicting dynamic hearing changes in individual mice, these behavioral descriptions are constrained by the variability of normal baseline thresholds in young mice and by di¡erences in the ensuing rates of hearing loss in older mice. Our study investigated function-based alternatives to the current age-based chronologies of presbycusis in C57BL/6J mice. It was assumed that mice reach de¢nitive stages of hearing loss at di¡erent ages but follow the same progression of impairment. Consequently, insights into the generalized mechanisms of presbycusis may be gained by classifying the functional status of individual mice in terms of current auditory performance under multiple listening conditions. This behavioral approach is designated the ‘functional age’ of hearing loss.

2. Materials and methods All of the following procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Northern Michigan University. 2.1. Subjects The progression of age-related hearing loss was assessed in 15 female C57BL/6J mice. Additional control experiments determined the normative hearing thresholds of nine female CBA/CaJ mice. Recent electrophysiological studies suggest that age-related hearing loss proceeds at a faster rate among female C57BL/6 mice (Henry, 2002). These e¡ects have not been reported for C57BL/6J or CBA/CaJ mice (Zheng et al., 1999). Our studies are restricted to female mice to avoid behavioral disruptions that may arise from mixing the scent of

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male and female animals in the vivarium or testing apparatus. All subjects were obtained from Jackson Labs at an approximate age of 5 weeks. No obvious e¡ects of acoustic over-exposure were noted in the initial baseline thresholds of CBA/CaJ mice or the more sensitive C57BL/6J mice. Presumably, any sound exposure during transportation was equal for both subject groups. The mice were maintained on a reverse day^night cycle and given free access to food throughout the course of experiments. A schedule of water deprivation was established to allow the reinforcement of tone-detection behaviors with water rewards. Fully trained mice obtained the majority of their daily water intake during behavioral sessions. An additional watering period was provided in the evening to maintain normal body weights. C57BL/6J mice were transported to Johns Hopkins University for histological examination immediately after the termination of psychophysical experiments. The results of these anatomical assessments are presented in the companion paper by Francis et al. (2003). 2.2. Behavioral apparatus and stimulus generation The design of our behavioral apparatus is illustrated in Fig. 1A and has been fully described in previous reports (Prosen et al., 2000; May et al., 2002). An array of photocells monitored the subject’s position in the testing cage. Pure-tone stimuli were delivered from a speaker above the head while the subject occupied the listening area of the cage. The subject indicated the detection of the tones by crossing to the response area of the cage and was rewarded by activation of a water dipper. Pure-tone signals and broadband masking noise were generated with digital-to-analog converters and gated with electronic switches (Tucker-Davis Technologies). Stimulus levels were controlled by progammable attenuators (Tucker-Davis Technologies) and an audio ampli¢er (Crown Audio). Stimulus waveforms were transduced by a high-frequency speaker (Realistic Super Tweeter). The testing cage was isolated from extraneous environmental sounds by a sound-attenuation chamber. The inner walls of the chamber were lined with anechoic foam (Sonex). During the presentation of auditory stimuli, the subject remained within the 8.5U10 cm listening area of the partitioned cage. Sound levels at test frequencies varied by no more than 5 dB within the listening area. The overhead location of the speaker minimized the e¡ects of head orientation on sound energy propagating to the tympanic membrane.

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2.3. Behavioral testing Behavioral contingencies of the tone-detection task are summarized in Fig. 1B. The mouse initiated each testing cycle by remaining in the listening area of the cage for 2^7 s. Successful completion of the waiting period was followed by the presentation of a 3-s trial interval. Repeating tone bursts comprised 75% of all trials in the daily session. The target stimuli were 250 ms in duration (10 ms rise/fall times) and repeated at a rate of 2 bursts/s. Subjects registered a correct detection (hit) by moving to the response area of the cage before the termination of the tone presentations, and were rewarded with 3 s of access to the 0.01 ml water dipper. Tone presentations were withheld on 25% of the trial intervals to monitor the probability of false positive responses. A correct response to these so-called catch trials (rejection) required that the subject remain in the listening area for the 3-s trial interval. Correct rejections produced an immediate tone trial to create the opportunity for water rewards. The tone presentations following catch trials were limited to the loudest stimulus level in the session and did not contribute to threshold calculations.

Response errors were followed by 5-s time-out intervals. These brief interruptions of the behavioral cycle decreased the probability of incorrect responses by delaying access to water rewards. Three types of error were possible. Subjects might leave the listening area during a waiting period (early) or catch trial (false alarm), or they might fail to enter the response area before the completion of a tone trial (miss). Six to eight weeks of training were needed to achieve stable performance under the ¢nal behavioral parameters of the tone-detection task. Data obtained during the training period were not included in threshold calculations. 2.4. Methods for assessing hearing thresholds Hearing sensitivity was determined by measuring the e¡ects of stimulus level on tone-detection rates. Each mouse was tested with a range of stimulus levels that re£ected its recent performance. Low stimulus levels were selected to elicit the near-chance detection scores that were needed for threshold calculations. High levels were easily detected and ensured an ample supply of water rewards. Stimulus levels were ¢xed within the trial interval and changed at random between trials according to the method of constant stimuli (Niemiec and Moody, 1995). The subject’s current hearing sensitivity was tracked in terms of average threshold across a minimum of ¢ve consecutive sessions. To be accepted as stable performance, individual sessions were required to have false alarm rates less than 35% and combined hit rates greater than 65%. Threshold variability was limited to less than 10 dB across sessions, with no pronounced upward or downward trends. These criteria for stability allowed reliable threshold determinations without excluding most changes in sensitivity during critical stages of hearing loss. Signal detection thresholds were derived from the summed hit and false alarm rates for all sessions meeting the stability criteria. The resulting response probabilities were transformed to dP statistics as described in Eq. 1: d 0 ¼ zðPhit Þ3zðPfalse alarm Þ

Fig. 1. The tone-detection task. (A) Mice were trained to move from the listening area to the response area during tone presentations. (B) Correct responses were rewarded with water. Incorrect responses were punished with time-out intervals. False positive responses were monitored by presenting catch trials without tone presentations.

ð1Þ

z(Phit ) is the z score for the percentage of hits at a tone level and z(Pfalse alarm ) is the z score for the percentage of false alarms. The detection threshold was de¢ned as the tone presentation level associated with dP = 1. This value was interpolated from psychometric functions relating global dP scores to stimulus level. Behavioral thresholds were obtained at test frequencies of 8 and 16 kHz. The most sensitive region of the mouse audiogram spans the 4-octave range of frequencies between 4 and 64 kHz (Ehret, 1975). The 8-kHz

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stimulus condition was selected to represent a relative low frequency in this species. The 16-kHz stimulus condition was the highest frequency that could be measured with our psychophysical procedures prior to age-related threshold shifts. In combination, the two frequencies were used to track the relative onset of high-to-low frequency hearing loss. Continuous broadband noise was added to the testing procedure after subjects achieved stable thresholds in quiet. The power spectrum of the background noise was determined by the frequency response characteristics of the output speaker, which deviated by less than P 5 dB at frequencies between 5 and 20 kHz. Noise levels were gradually increased over several sessions until the masked threshold was elevated 10 dB relative to performance in quiet. The e¡ects of background noise are described in terms of the signal-to-noise ratio at the 10-dB masked threshold. This critical ratio measure is designated CR10 , as de¢ned by Eq. 2: CR10 ¼

Detection threshold in noise ðdB SPLÞ Noise spectrum level ðdB per HzÞ

ð2Þ

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3.1. Baseline measures of hearing sensitivity Initial detection thresholds required 6^8 weeks of training to reach optimum performance. The delay of the ¢rst reliable assessment of auditory sensitivity raises the possibility that early onset hearing loss may have corrupted our best estimates of normal hearing in C57BL/6J mice. Independent baselines for assessing the magnitude of functional impairments in the C57BL/6J strain were obtained by testing CBA/CaJ mice under identical stimulus conditions. The threshold stability of three CBA/CaJ mice is described in Fig. 2. Although these results span a range of ages from approximately 4^9 months, the mice showed no signs of presbycusis-like hearing loss during this time period. That is, the higher-frequency threshold of mouse CBAau is not signi¢cantly elevated relative to the lower-frequency thresholds of mice CBAel and CBAga. None of the subjects displayed threshold elevations at later stages of training. Results from mouse CBAga illustrate how age-related hearing loss was distinguished from other forms of behavioral instability. The performance of this sub-

The noise spectrum level for the calculation is speci¢ed in dB per Hz and refers to energy in the frequency component matching the pure-tone signal. C57BL/6J mice exhibited highly variable masking e¡ects at the onset of hearing loss, making it impossible to achieve the requisite 10-dB threshold shift without some adjustments of background noise levels. Reported CR10 values are restricted to data from behavioral sessions where noise levels varied by less than 3 dB. In other regards, the sessions contributing to the assessment of hearing thresholds in noise were required to meet the same stability criteria as testing in quiet.

3. Results Comparisons of auditory thresholds from the same C57BL/6J mice in di¡erent listening conditions suggested that hearing loss occurred in discrete stages. The earliest impairments were observed when testing was conducted in background noise. Subsequent loss involved threshold elevations in quiet. In both instances, the impairments proceeded from high to low frequencies. The time course for the a¡ected listening condition often began with a gradual threshold elevation and ended with abrupt severe loss at test frequencies. The behavioral de¢cits that de¢ne this presbycusis-like sequence of hearing impairments were observed in all C57BL/6J mice and no control subjects from the CBA/ CaJ strain.

Fig. 2. Baseline tone-detection thresholds of representative CBA/CaJ mice. Mice CBAel and CBAga were tested with 8-kHz tones (b), mouse CBAau with 16-kHz tones (a). The threshold of mouse CBAga was replicated after ¢ve weeks of testing in background noise. Data indicate sessions meeting criteria for stability, as described in Section 2.

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ject ¢rst reached the criteria for stability at age 18 weeks. At this time, detection scores produced an 8-kHz threshold of 29 dB SPL. A downward trend of the daily thresholds suggested the subject was still learning to optimize performance in the task. Testing was continued until systematic threshold changes were no longer obvious at 22 weeks of age. The detection scores of these later sessions produced a threshold of 24.3 dB SPL. CBA/CaJ mice often showed additional learning effects in quiet after being tested with the more di⁄cult tone-in-noise task. Mouse CBAga experienced 5 weeks of testing in background noise before the collection of the second series of 8-kHz thresholds in Fig. 2. The average threshold for the post-noise tests was 19 dB SPL, which represents a 5-dB enhancement of the subject’s best previous threshold. C57BL/6J mice also produced stable detection thresholds prior to the onset of hearing loss. Representative data for 16-kHz tones are shown in Fig. 3. Mouse C57ra ¢rst reached the criteria for stability at 24 weeks of age. Although this subject’s threshold (39.6 dB SPL) was almost 15 dB higher than the least sensitive CBA/ CaJ threshold, daily performance did not indicate advancing hearing loss (upward trends) or learning e¡ects (downward trends). Unlike CBA/CaJ mice, the thresholds of C57BL/6J mice did not improve when testing in quiet was replicated after exposure to the tone-in-noise task. The initial thresholds of mouse C57ph suggest strong learning e¡ects until age 23 weeks. Subsequent performance remained stable for approximately 11 weeks. Thresholds collected during this period (29.1 dB SPL) were lower than average for C57BL/6J mice but higher

Fig. 3. Baseline tone-detection thresholds of representative C57BL/ 6J mice. These data were obtained before the onset of age-related hearing loss. Plotting conventions are described in Fig. 2.

Fig. 4. Distribution of baseline hearing thresholds in CBA/CaJ and C57BL/6J mice. The boxplots are divided by the median score of each sample. The upper and lower limits of the boxes indicate the interquartile range (i.e. middle 50% of the distributions). Error bars extend to the full range of thresholds. Numerical labels show the number of mice in each group.

than the 16-kHz thresholds of all CBA/CaJ mice. Once again, no learning e¡ects were evident during post-noise threshold replications. Fig. 4 summarizes the distribution of baseline thresholds for our entire sample of behaviorally characterized mice. On average, CBA/CaJ mice were more sensitive to tone frequencies of 16 kHz (20.9 dB) than 8 kHz (23.4 dB). This threshold di¡erence is predicted by the general shape of audibility curves in laboratory mice (see multiple sources provided by Fay, 1988), and by our previous measures of tone-detection thresholds in the control strain (May et al., 2002). The range of thresholds varied between subjects by less than 12 dB at both test frequencies. This uniformity establishes the reliability of our psychophysical methods. A presbycusis-like hearing loss is implied by the distribution of thresholds from C57BL/6J mice in Fig. 4. Relative to CBA/CaJ mice, these subjects displayed decreases in sensitivity at both 16 and 8 kHz, and increases in inter-subject variability. The average 16-kHz threshold of C57BL/6J mice was 13.4 dB higher than the thresholds of the control mice. The 8-kHz thresholds of the two strains showed a smaller 2.5-dB di¡erence, suggesting that the apical advancement of cochlear degeneration had not yet reached this location in the C57BL/6J mice. The 16-kHz thresholds of C57BL/6J mice extended from 21.5^45.4 dB SPL. This range represents a twofold increase in threshold variability relative to CBA/ CaJ mice. The most sensitive thresholds were similar in

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both subject groups, but the distribution of C57BL/6J mice also includes less sensitive thresholds that may re£ect pathology. Di¡erences between the two strains were smaller for the more apically encoded 8-kHz thresholds, where the most sensitive thresholds of C57BL/6J mice corresponded well to baseline values for CBA/CaJ mice. The frequency dependency of these e¡ects suggests that baseline threshold variability is observed between C57BL/6J mice because subjects show individual di¡erences in the onset or rate of impairment at early stages of hearing loss. An alternative explanation for the variability of baseline measures in the C57BL/6J strain is that the same progression of hearing loss was followed in each mouse and higher thresholds were simply recorded in older subjects. This possibility exists because the mice progressed to ¢nal behavioral parameters at di¡erent rates. At the completion of baseline measures, the age of C57BL/6J mice ranged from 19 to 32 weeks for 16kHz thresholds and 22 to 40 weeks for 8-kHz thresholds. Potential age e¡ects were addressed by exploring correlations between the subject’s age at threshold determination and the resulting threshold value. Scatterplots of this analysis are shown with comparison data from CBA/CaJ mice in Fig. 5. In general, 16-kHz thresholds were obtained from C57BL/6J mice at younger ages than 8-kHz thresholds. This sampling bias re£ects our intention to characterize behavioral performance at the higher test frequency as

Fig. 5. Age versus threshold correlations in CBA/CaJ and C57BL/6J mice. The 16-kHz thresholds of CBA/CaJ mice were intentionally biased toward older subjects to con¢rm the stability of hearing sensitivity in our control strain. The 16-kHz thresholds of C57BL/6J mice were collected in younger subjects to minimize the e¡ects of age-related hearing loss on baseline measures.

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early as possible in the sequence of hearing loss. Most of these subjects were subsequently tested at the lower frequency. In some instances, 8-kHz baselines were obtained at younger ages by alternating between test frequencies. The age-threshold analysis in Fig. 5 does not indicate a conspicuous trend toward greater hearing loss in older mice. Instead, a considerable range of baseline thresholds is observed among age-matched subjects. These results point out the inherent problems of agebased surveys of hearing loss in the C57BL/6J strain. 3.2. A high-to-low frequency progression of hearing loss The relative time course of hearing loss at high and low frequencies was demonstrated by simultaneously testing some mice with both 16- and 8-kHz tones. These results are shown in Fig. 6. In comparison to other subjects in the C57BL/6J strain (Fig. 4), mouse C57mi displayed excellent sensitivity up to 22 weeks of age. At that time, a rapid decline in high-frequency hearing was indicated by an abrupt elevation of the 16-kHz threshold. Hearing remained normal at 8-kHz for two additional months. Mouse C57fr exhibited a similar progressive hearing loss at 16 kHz, but not at 8 kHz. The initial threshold improvements of this subject at both 16 and 8 kHz suggest prolonged learning e¡ects during several months of training. Detection of the high-frequency tone brie£y stabilized around 28 weeks of age and then sharply elevated. By contrast, 8-kHz thresholds continued to improve until behavioral assessments were ended at age 34 weeks to allow the collection of histological data. The onset of low-frequency hearing loss was substantially delayed in some subjects. This e¡ect is illustrated by mouse C57ma, who failed to display signi¢cant threshold elevations until 48 weeks of age. Given these behavioral results, inter-subject variability is likely to confound any age-matched sampling of the anatomical or physiological correlates of presbycusis. A more comprehensive examination of the relative timing of high- and low-frequency hearing loss was completed by calculating the age at which each subject ¢rst reached a 10-dB threshold elevation at 16 or 8 kHz. Criterion threshold shifts were based on each subject’s own best thresholds to compensate for the heterogeneous baseline data from C57BL/6J mice (Fig. 4). Results of the age versus threshold shift analysis are presented in Fig. 7. Data are only shown for C57BL/6J mice because no CBA/CaJ mice exhibited the requisite 10-dB hearing loss at ages that ranged from 20^60 weeks (Fig. 5). By contrast, the majority of C57BL/6J mice experienced 10-dB threshold shifts at 16 kHz (10/ 11) and 8 kHz (9/12). The four C57BL/6J mice that did

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not show signi¢cant threshold elevations represent instances where normal hearing subjects were transferred to the histological study (Francis et al., 2003). Three general characteristics of hearing loss were revealed by the distribution of subject ages at the onset of 10-dB threshold shifts. First, high-frequency impairments preceded low-frequency loss. The median ages for 16- and 8-kHz threshold shifts were 27 and 33 weeks, respectively. Second, the frequency of ongoing threshold elevations varied within age-matched groups of mice. For example, approximately equal numbers of middle age mice (27^33 weeks) were entering the ¢rst stages of hearing loss at 16 or 8 kHz. Third, the onset of hearing loss varied between subjects. In particular, low-frequency threshold shifts occurred as early as 27 weeks and as late as 54 weeks. These results support the C57BL/6J strain as an adequate model of age-related hearing loss in humans, but also stress the ambiguities of age-based investigations of presbycusis in laboratory mice.

Fig. 7. Age at onset of hearing loss in C57BL/6J mice. Results are based on the occurrence of a 10-dB threshold shift relative to each subject’s baseline thresholds at the two test frequencies.

3.3. Early hearing loss in background noise

Fig. 6. E¡ects of frequency on age-related hearing loss in C57BL/6J mice. Subjects C57mi and C57fr showed a high-to-low frequency progression of threshold elevations when tested with 16- and 8-kHz tones. Subject C57ma was tested exclusively with 8-kHz tones and did not exhibit signi¢cant low-frequency hearing loss before age 45 weeks.

Noise masking e¡ects were investigated after the collection of stable baseline thresholds in quiet. This delay was necessary for the calculation of CR10 scores that re£ected hearing loss in background noise relative to individual performance under quiet conditions. Control experiments were performed on CBA/CaJ mice to evaluate the potential in£uence of early onset hearing loss on the thresholds of C57BL/6J mice. Typical results are shown in Fig. 8. Like these representative subjects, the remaining mice in the control group displayed no major changes in masking sensitivity at ages ranging from 24.9 to 63 weeks. The high-frequency hearing of mouse CBAlu remained stable in background noise at ages up to 55 weeks. Fig. 9 compares the statistical distribution of CR10 scores for C57BL/6J mice to the CBA/CaJ controls. The 16-kHz thresholds of C57BL/6J mice were determined at ages from 23^29 weeks, while 8-kHz thresholds were obtained at ages from 28^45 weeks. The close agreement of median 8-kHz thresholds between the subject groups con¢rms that baseline tone-in-noise experiments were completed at low frequencies before the onset of age-related changes in masking. The CR10 thresholds of both subject groups were higher at 16 kHz than at 8 kHz. The potentiation of masking e⁄ciency at high frequencies is presumed to re£ect the increasing bandwidth of auditory ¢lters. Masking e¡ects of this nature are well known in hu-

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mans and have been previously demonstrated in laboratory mice (Ehret, 1975, 1976). Age-related de¢cits in auditory frequency selectivity were expected to be revealed by further increases in CR10 thresholds. Although di¡erences between subject groups were not statistically signi¢cant at this early stage of impairment, the imminent loss of 16-kHz frequency selectivity was predicted by the elevation of the median CR10 threshold of C57BL/6J mice. This ¢nding is intriguing because it suggests that listening de¢cits in background noise may follow the same base-to-apex progression as hearing loss in quiet. A better indication of age-related changes in the susceptibility of C57BL/6J mice to masking noise was gained by conducting within-subject assessments of CR10 thresholds over prolonged testing periods. Representative results are summarized in Fig. 10. These results are limited to tone-in-noise thresholds that met our criteria for behavioral stability. Quiet thresholds were collected at random intervals during these tests to demonstrate the speci¢city of listening de¢cits in background noise. The behavioral thresholds reveal auditory de¢cits that were con¢ned to listening in background noise. Mouse C57to displayed episodic behavioral disruptions in noise that were not observed during simultaneous testing in quiet. After an initial learning e¡ect, mouse C57ja exhibited a sharp elevation in the 16-kHz CR10 at

Fig. 8. Baseline CR10 thresholds in representative CBA/CaJ mice.

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Fig. 9. Distribution of baseline CR10 thresholds in CBA/CaJ and C57BL/6J mice. Boxplot conventions are described in Fig. 4.

25 weeks of age. A progressive hearing loss was not observed in this subject under quiet conditions until 5 weeks later. The delay between hearing loss in noise and quiet tended to be longer and more variable at low frequencies. For example, mouse C57ma showed an initial elevation of the 8-kHz CR10 threshold at age 31 weeks and no signi¢cant change in quiet thresholds until age 49 weeks. C57BL/6J mice exhibited a loss of behavioral stability in background noise before showing elevated CR10 thresholds. Fig. 11 illustrates the relative timing of these distinct patterns of impairment by comparing the 8-kHz CR10 thresholds of mouse C57gr with cumulative stable sessions. This record was produced by assigning a binary score of 0 (not stable) or 1 (stable) to daily performances and then adding the scores as a function of session number. Criteria for behavioral stability are described in Section 2. The threshold and stability measures of mouse C57gr suggest three stages of progressive impairment. Sessions 1^7 were marked by behavioral instability and variable thresholds while the subject learned to perform the detection task in the presence of background noise. Sessions 7^18 were characterized by stable performance and gradually decreasing CR10 values. At age 30.9 weeks (session 18), an abrupt loss of behavioral stability was observed in the absence of CR10 threshold elevations. A return to stable performance is seen after session 32 because stimulus levels were increased to accommodate the subject’s advancing loss of hearing sensitivity in quiet and in noise at the test frequency. Fig. 12 presents statistical descriptions for the onset of instability in background noise. This analysis was

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limited to C57BL/6J mice that produced stable thresholds for at least ¢ve consecutive sessions. Only three mice met this condition when tests were conducted with 16-kHz tones. Age correlations for hearing de¢cits in quiet and noise are indicated by the scatterplot in Fig. 12A. This comparison is only available for mice that were ¢rst tested in noise early enough to capture the onset of behavioral instability and then evaluated in quiet long enough to demonstrate 10-dB threshold shifts. All data points fall to the left of the unity line in this plot, con¢rming the precedence of noise instability over

Fig. 10. E¡ects of age on CR10 thresholds in C57BL/6J mice. The speci¢city of listening de¢cits in background noise is indicated by the stability of concurrent performance under quiet conditions (Q).

Fig. 11. Time course of increased noise instability and elevation of CR10 thresholds. Results were obtained with 8-kHz tones from mouse C57gr. The cumulative record plots the total number of stable thresholds as a function of session number. Criteria for assessing behavioral stability are de¢ned in Section 2.

Fig. 12. Age distribution for the onset of noise instability in C57BL/6J mice. (A) Correlations of hearing loss in quiet and background noise. Data points to the left of the unity line indicate the onset of noise instability before threshold shifts in quiet. (B) Histograms showing a high-to-low frequency progression of performance de¢cits in background noise. Arrows note the median age of 16and 8-kHz threshold shifts under quiet testing conditions.

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hearing loss in quiet. This relationship suggests potential links between the two forms of impairment. Highfrequency de¢cits were observed before low-frequency de¢cits under both quiet and noise conditions. In addition, mice that showed early impairments in background noise were the ¢rst subjects to experience threshold elevations under quiet conditions. E¡ects of frequency on behavioral instability in background noise are summarized by the histograms in Fig. 12B. The breakdown of behavioral performance in background noise also appears to occur ¢rst at the higher frequency. Although our sample is small (n = 3), these events a¡ected all mice in the subject group within a narrow range of ages. Hearing de¢cits at the lower frequency developed at later and more variable ages. Regardless of frequency, the onset of behavioral instability in noise was distributed across an age range below the median age of 10-dB threshold shifts in quiet (arrows).

4. Discussion Genetic variation within the C57BL/6J strain is constrained by selective inbreeding. Phenotypic variation was minimized among the behavioral subjects of the present study by maintaining the mice in an environmentally controlled vivarium. Nevertheless, our subjects demonstrated signi¢cant di¡erences in their baseline measures of auditory sensitivity and the timing of progressive functional impairments. These ¢ndings call into question any experimental approach to the mechanisms of presbycusis that is predicated on the strict age dependency of cochlear degenerative e¡ects. In lieu of predictive age-based methods, C57BL/6J mice manifest orderly changes in behavioral performance that provide an alternative chronology for classifying the functional age of hearing loss in mouse models of presbycusis. 4.1. Baseline measures of auditory sensitivity Our descriptions of the timing and magnitude of hearing loss in C57BL/6J mice were based on withinsubject measures of behavioral performance. This experimental design requires an accurate estimate of auditory sensitivity prior to impairment. Such baseline measures are made di⁄cult by the early onset of highfrequency threshold elevations in the C57BL/6J strain and the training periods required by our behavioral paradigms. The reliability of our psychophysical approach in the absence of hearing loss was established by experiments with CBA/CaJ mice. After the completion of initial baseline thresholds, these control subjects did not show major changes in hearing sensitivity over months

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of subsequent testing except when thresholds in quiet improved after exposure to more di⁄cult noise listening conditions (Fig. 2). In addition, tone-detection thresholds in this strain showed little inter-subject variability and matched previously reported psychophysical results in laboratory mice (e.g. He¡ner and Masterton, 1980; Birch et al., 1968). The long-term threshold stability of CBA/CaJ mice has been independently con¢rmed with auditory brainstem response (ABR) measures (Zheng et al., 1999; Yoshida et al., 2000; May et al., 2002). C57BL/6J mice displayed a larger range of baseline thresholds than CBA/CaJ controls (Fig. 4). This intersubject variability was in£ated by a subset of subjects with relatively high thresholds and therefore may re£ect individual di¡erences in the onset of hearing loss. Changes in hearing sensitivity have been reported to occur in the C57BL/6J strain by age 2 months (Henry and Chole, 1980; Willott and Bross, 1996), 3 months (Mikaelian et al., 1974), or 8 months (Zheng et al., 1999). ABR measures in C57BL/6J mice indicate elevations of 16-kHz thresholds relative to CBA/Ca controls as early as 8 weeks of age (Li and Borg, 1991). Mice in our study showed gradual improvements in behavioral performance that delayed the collection of optimal baseline thresholds until 20 weeks of age (Fig. 5). Once baseline thresholds were determined, C57BL/ 6J mice maintained stable performance for weeks before clear indications of developing hearing de¢cits. In particular, although initial 8-kHz thresholds were elevated relative to CBA/CaJ controls (Fig. 4), C57BL/6J mice did not show systematically increasing threshold shifts at this frequency before 27 weeks of age. Some subjects maintained stable performance until almost one year of age. 4.2. Time course of age-related threshold shifts The suitability of C57BL/6J mice as an animal model of presbycusis was veri¢ed by the documentation of a pervasive high-to-low frequency hearing loss. Age-related de¢cits of at least 10 dB were observed in every C57BL/6J mouse, except when behavioral measures were stopped prior to the onset of marked sensitivity changes to provide functionally intact cochleae for histological evaluations (4/23 mice). Individual subjects displayed initial threshold shifts at 16 kHz and later de¢cits at 8 kHz when concurrently tested at both frequencies (Fig. 6). Presbycusis-like patterns of hearing loss also were demonstrated by comparing the distribution of subject ages at the time of 16- and 8-kHz threshold shifts for our entire sample of C57BL/6J mice (Fig. 7). Numerous reports have commented on the inter-subject variability of hearing loss in C57BL/6J mice (Li and Borg, 1991; Mizuta et al., 1993; Parham, 1997). In our

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study, this variability increased as hearing loss progressed to more apical cochlear frequencies. Threshold elevations began to appear at 16 kHz by age 21 weeks and were present in all subjects by age 33 weeks. Although this 3-month scattering of initial threshold shifts would be su⁄cient to confound most age-based anatomical studies of cochlear degeneration, even greater age di¡erences were observed at 8 kHz where the onset of hearing loss varied from 27 to 54 weeks. Low-frequency hearing loss appeared to follow a biphasic time course in some subjects. This phenomenon is illustrated by long-term changes in the 8-kHz thresholds of mouse C57ma (Fig. 6). During the 27 to 50 week period when most C57BL/6J mice lost sensitivity at 8 kHz, this subject exhibited several months of slowly increasing thresholds and then rapidly increasing deafness. Li and Borg (1991) have described similar patterns of ABR threshold shifts in C57BL/6J mice. The initial slow phase of low-frequency hearing loss is expected to lead to our criterion of 10-dB threshold shifts at older and more variable ages. By contrast, high-frequency threshold elevations occurred abruptly in younger mice and were more constrained in age (e.g. C57mi in Fig. 6). Recent ABR studies have demonstrated an age dependency for simultaneous high and low frequency hearing loss in C57BL/6J mice (Hequembourg and Liberman, 2001). High-frequency threshold shifts appear to result from degenerative e¡ects that spread from cochlear hair cells to the spiral ganglion. Damage patterns in the basal cochlea of C57BL/6J mice are consistent with these presbycusis-like structural changes. By contrast, ABR threshold elevations emerge at low frequencies before C57BL/6J mice show signi¢cant hair cell loss in the cochlear apex. Instead, the functional impairment is correlated with ¢brocyte degeneration in the lateral wall of the cochlea. Because the spiral ligament plays an important role in the cycling of potassium ions during the transduction process, disruption of cochlear homeostasis may represent an alternate mechanism for hearing loss in the C57BL/6J strain. The biphasic time course of threshold elevations in behaviorally characterized mice may re£ect gradually developing abnormalities of endolymphatic ionic compositions followed by abrupt neural degenerative e¡ects. 4.3. Time course of noise-related de¢cits The ability to resolve auditory frequency information declines with age (Patterson et al., 1982). Perceptual de¢cits of this nature are exacerbated by hearing loss (Florentine et al., 1980; Tyler and Tye-Murray, 1986) and therefore are presumed to re£ect a broadening of peripheral auditory ¢lters with diminished hair cell survival (Evans and Harrison, 1976; Moore, 1997; Peters

and Moore, 1992). Even when auditory signals are clearly audible, these impairments lead to disruptions of speech recognition (Smoorenburg, 1992) and listening in background noise (Leek and Summers, 1996). Our study evaluated age-related changes in the frequency selectivity of C57BL/6J mice in terms of CR10 thresholds. This experimental approach is based on the assumption that increases in auditory ¢lter bandwidth are manifested in behavioral performance as elevated masked thresholds (Fletcher, 1940). Critical ratio procedures are now relatively uncommon in human psychoacoustic studies because more informative methods like the notched-noise paradigms of Patterson (1976) simultaneously predict the bandwidth and shape of auditory ¢lters. Nevertheless, CR10 measures provide a useful context for characterizing the time course of rapidly advancing noise de¢cits in less e⁄cient animal paradigms because they are capable of estimating ¢lter bandwidth with a minimum number of stimulus manipulations. Age-related changes in the CR10 thresholds of C57BL/6J mice replicated previous clinical observations that absolute threshold shifts in quiet are accompanied by an increased susceptibility to noise masking e¡ects. Although the onset of listening de¢cits in noise followed the same high-to-low frequency progression as threshold elevations (Fig. 12B), the two forms of hearing loss could be distinguished by separate time courses and therefore may re£ect di¡erent underlying pathologies (Fig. 12A). Multiple sources of sensorineural hearing loss have been noted in previous physiological studies of sound-exposed cats where permanent shifts in auditory nerve thresholds are associated with a loss of cochlear inner hair cells and changes in frequency tuning with outer hair cell stereocilia damage (Liberman and Dodds, 1984). Our behavioral results are correlated with outer hair cell survival rates in the companion study by Francis et al. (2003). 4.4. The functional age of hearing loss A major ¢nding of this study is the observation that the perceptual landmarks of presbycusis are conserved in C57BL/6J mice despite inter-subject variation in the age at which the degenerative e¡ects occur. Behavioral characterization of this progressive hearing loss is signi¢cant for presbycusis research because it provides a well-de¢ned chronology for correlating functional impairments with the morphological status of the cochlea. Each threshold measure was characterized over several months in individual subjects. Consequently, the mice were tested on partial subsets of the stimulus conditions that de¢ne the functional age of hearing loss. Shared stimulus conditions allowed us to combine complementary results across subjects into a complete chronology.

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Fig. 13 presents a graphic representation of the psychophysical criteria that identify four generalized stages of hearing loss in C57BL/6J mice. Functional age 1 is de¢ned by listening de¢cits that are con¢ned to higher frequencies in noise. Speci¢cally, mice ¢rst developed a lack of stability and then displayed increased masked thresholds when tested with 16-kHz tones in background noise. These de¢cits were noted at chronological ages between 23 and 26.5 weeks. Functional age 2 is characterized by the elevation of high-frequency detection thresholds in quiet. Our subjects reached this stage of hearing loss for 16-kHz tones at chronological ages of 22^31 weeks. Although the distribution of subject ages was similar for functional ages 1 and 2, impairments in noise always preceded impairments in quiet when mice were simultaneously tested under both conditions. This pattern is illustrated by individual data from mouse C57ja in Fig. 13. The transitions to functional ages 3 and 4 are indicated by hearing de¢cits at low frequencies. C57BL/6J mice reached these criteria for 8-kHz tones at chronological ages of 27^51 weeks. As a result of considerable timing variations among subject groups, no clear age di¡erence was noted for the onset of hearing loss in quiet and noise. Again, this ambiguity was resolved by comparing the behavioral performance of individual mice under multiple stimulus conditions. Data from mouse C57ma are presented in Fig. 13 to demonstrate the orderly progression of presbycusis-like hearing loss from high to low frequencies, and from noisy to quiet listening environments. Long-term behavioral analyses are not conducive to laboratory studies where it is necessary to evaluate the hearing competency of individual mice at very young chronological ages. To a ¢rst approximation, pure-tone thresholds in quiet can be estimated by fast and reliable ABR techniques. Unfortunately, the de¢cits in auditory discrimination, noise masking, and speech comprehension that accompany presbycusis are not well predicted

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by simple threshold measures, and more importantly cannot be corrected by ampli¢cation strategies that consider only the magnitude of hearing loss (Miller et al., 1999). As more complex discrimination tests begin to provide additional perceptual links to the cochlear pathologies of mouse models of presbycusis (Prosen et al., 2000; May et al., 2002), a practical goal of this research will be to enhance the e⁄ciency of the behavioral approach by identifying the most informative paradigms. Our current studies of age-related hearing loss in C57BL/6J have focused on potential links between auditory behavior and cochlear pathology. These interpretations are somewhat tenuous because they ignore the added complexity of central processing de¢cits. The principal auditory nuclei of aging C57BL/6J mice di¡er not only in their degree of functional degeneration but also in their principal sound processing roles (Willott et al., 1991; Walton et al., 1995). As a result, the perceptual demands of the psychophysical task are likely to in£uence the magnitude of performance de¢cits. Conversely, speci¢c patterns of perceptual impairments under multiple listening conditions are capable of providing insights into the discrete anatomical sources of functional degeneration. This behavioral neuroanatomical approach is fundamental to clinical audiology and may prove equally important in directing future histological evaluations of the peripheral and central mechanisms of presbycusis in animal models.

Acknowledgements This research was sponsored by NIDCD grant R15 DC04405 (C.A.P.). Undergraduate students at Northern Michigan University conducted the behavioral experiments. Some ¢ndings were previously presented at the 24th Annual Midwinter Research Meeting of the Association for Research in Otolaryngology. References

Fig. 13. Age range for the onset of functional de¢cits under di¡erent testing conditions. Symbols show individual thresholds under multiple conditions for mice C57ma (a) and C57ja (b). This continuum of progressive impairment de¢nes the functional age of hearing loss in C57BL/6J mice.

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