Effects of PMCA2 mutation on DPOAE amplitudes and latencies in deafwaddler mice1

Hearing Research 151 (2001) 205^220 www.elsevier.com/locate/heares

E¡ects of PMCA2 mutation on DPOAE amplitudes and latencies in deafwaddler mice1 Dawn Konrad-Martin b

a;

*, Susan J. Norton

b;c

, Kelley E. Mascher c , Bruce L Tempel

b;d

a Department of Speech and Hearing Sciences, University of Washington, Seattle, WA 98195, USA Department of Otolaryngology and Head and Neck Surgery, University of Washington School of Medicine, Seattle, WA 98195, USA c Audiology Research, Children's Hospital and Regional Medical Center, Seattle, WA 98105, USA d The Virginia Merrill Bloedel Hearing Research Center, University of Washington School of Medicine, Seattle, WA 98195, USA

Received 24 February 2000; accepted 29 September 2000

Abstract The deafwaddler (dfw) mouse mutant is caused by a spontaneous mutation in the gene that encodes a plasma membrane Ca2‡ ATPase (type 2), PMCA2 (Street et al., 1998. Nat. Genet. 19, 390^394), which is expressed in cochlear and vestibular hair cells. Distortion product otoacoustic emission (DPOAE) amplitudes and latencies were examined in control mice, deafwaddler mutants, and controls treated with the drug furosemide. Furosemide causes a transient reduction of DPOAEs (Mills et al., 1993. J. Acoust. Soc. Am. 94, 2108^2122). We wanted to determine whether DPOAEs obtained in furosemide-treated mice were similar or different from results obtained in +/dfw mice. DPOAE amplitude and phase were measured as a function of f2 /f1 ratio. These data were converted into waveforms using inverse fast Fourier transform, and their average latency was used to estimate DPOAE group delay. Homozygous deafwaddlers did not produce DPOAEs. Heterozygous deafwaddlers (+/dfw) had increased DPOAE thresholds and reduced amplitudes at high frequencies, compared to controls. To the extent that DPOAEs depend on functional outer hair cells (OHCs), abnormal DPOAEs in +/dfw mice suggest that PMCA2 is important for OHC function at high frequencies. Similar to the effects of furosemide, the mutation reduced DPOAEs for low-level stimuli ; in contrast to furosemide, the mutation altered DPOAEs elicited by high levels. ß 2001 Elsevier Science B.V. All rights reserved. Key words: Mutant mouse; Distortion product otoacoustic emission; Furosemide

1. Introduction Genetic mutations a¡ecting auditory function in mice have been useful for understanding the molecular biology underlying human deafness (Petit, 1996; Steel and Brown, 1996). One example is the deafwaddler mouse mutant, which has auditory and vestibular impairment. Deafwaddler (dfw) is caused by a spontaneous mutation in the Atp2b2 gene which encodes the plasma mem-

* Corresponding author. BTNRH 555 North 30th Street, Omaha, NE 68131, USA. Tel.: +1 (402) 498-6705; Fax: +1 (402) 498-6351; E-mail: [email protected] 1 Portions of this paper were presented at the Mid-winter Meeting of the Association for Research in Otolaryngology, February, 1997, 1998.

brane Ca2‡ ATPase (type 2) protein, PMCA2 (Street et al., 1998). PMCA2 is localized to outer hair cell (OHC) and inner hair cell (IHC) stereocilia and cuticular plates in mouse. It is also detected in IHC basolateral membrane and in spiral ganglion cells (Street et al., 1998). Auditory brainstem responses (ABR) are absent in homozygote deafwaddler mice (dfw/dfw), indicating that they are profoundly deaf (Norton et al., 1996). In heterozygous deafwaddlers (+/dfw), distortion product otoacoustic emissions (DPOAEs), cochlear microphonic potentials, compound action potentials, and ABRs have increased thresholds and reduced amplitudes at high frequencies (Norton et al., 1996). Anatomical damage in +/dfw mice is not readily apparent at the transmission electron microscopy level (Pujol et al., 1997), and there is no visible balance defect. It was hypothesized that hearing loss in +/dfw mice may be

0378-5955 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 2 2 8 - 8

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primarily an OHC disorder. This led to the view that examination of DPOAEs in +/dfw mice might shed light on the role of PMCA2 in cochlear function. DPOAEs are acoustic signals recorded in the ear canal in response to a pair of tones, f1 and f2 , where f1 6 f2 . At low to moderate levels of stimulation, DPOAE generation depends on the integrity of the OHC system (Brownell, 1990; Kemp, 1978; Norton et al., 1991; Probst, 1990; Whitehead et al., 1992a). The primary source of DPOAEs appears to be intermodulation near the f2 site, where the basilar membrane traveling waves produced by f1 and f2 overlap maximally (Brown and Kemp, 1984; Siegel et al.; 1982). For the 2f1 ^f2 DPOAE, functions generated by plotting amplitude as a function of f2 /f1 (or the DP frequency) are parabolic. In the DP literature, these functions are often called `¢lter functions'. The largest DP amplitude is obtained when the ratio of the stimulating frequencies leads to a DP located one-half octave below f2 (Brown and Gaskill, 1990; Harris et al., 1989). Amplitude decreases as the two stimulating frequencies diverge or converge relative to this point. The relationship between OHC function and ¢lter functions is not known. Therefore, it was not known a priori what f2 /f1 would be the most sensitive indicator of OHC dysfunction in dfw mice. Some investigators attribute the ¢lter function shape to a second cochlear ¢lter augmenting the DPOAE response at a location one-half octave below f2 (Brown and Gaskill, 1990), and have proposed the tectorial membrane as the second ¢lter because of its connections to the basilar membrane via the OHC stereocilia. This led to hypotheses that the bandwidth of the DPOAE ¢lter function is related to cochlear frequency selectivity (Allen and Fahey, 1993; Brown et al., 1993a; Brown et al., 1993b), or basilar membrane suppression (Kanis and de Boer, 1997). Some investigators have suggested that the ¢lter function shape is caused by interactions among multiple emission sources (Brown et al., 1992; Stover et al., 1996 ; Kemp and Knight, 1999). These sources may include (1) nonlinearities near the f2 place and (2) a stimulus frequency OAE corresponding to the DP frequency (Furst and Lapid, 1988; Gaskill and Brown, 1996 ; Kemp, 1979; Kemp and Brown, 1983; Martin et al., 1987 ; Wilson, 1980; Zwicker and Harris, 1990 ; Stover et al., 1996). Stover et al. (1996) converted ¢lter functions obtained using a ¢xed-f2 /swept-f1 paradigm to the time domain using inverse fast Fourier transform (IFFT). This analysis separates response components by their phase characteristics, providing the opportunity to test hypotheses about multiple sources of ear canal DPOAEs. If more than one location contributes to DPOAE level and phase, ¢lter functions as well as DPOAE latency might be altered by experimental ma-

nipulations that di¡erentially a¡ect OHCs at those locations (Stover et al., 1996). The latency of the OAE is thought to re£ect the round trip travel time to the emission generator site, including the time it takes for the emission to be generated. Onset latency determined (using a pulsed stimulus) from the DPOAE waveform in the time domain varies systematically as a function of frequency and level (Whitehead et al., 1996). Similar variations occur for phase-slope group delays, which can be obtained using a swept-tone paradigm (either the f2 frequency is ¢xed and f1 is varied, or f1 is ¢xed and f2 is varied) (e.g. Kimberley et al., 1993 ; O'Mahoney and Kemp, 1995 ; Whitehead et al., 1996). In addition, group delay can be determined by the center of energy of the IFFT without potential errors due to phase unwrapping, a necessary step for calculating phase-slope group delay (Stover et al., 1996). To explain level-dependent OAE latencies, it was proposed that latency depends on traveling wave velocity (Neely et al., 1988), and/or auditory ¢lter `build-up time' (Kimberley et al., 1993). If group delay was in£uenced by either of these mechanisms, both stimulus level and OHC dysfunction at the OAE generation site might a¡ect group delay. The experiments reported here were aimed at providing information about the e¡ects of dfw on OHC system function in adult mice as inferred from DPOAE amplitude and group delay measurements obtained using a ¢xed-f2 /swept-f1 paradigm. The ¢rst experiment compares responses obtained in control mice and dfw mutants. In the second experiment, DPOAEs are examined in furosemide-treated control mice. Furosemide causes a transient reduction of the endocochlear potential (e.g. Evans and Klinke, 1982), which alters the resting voltage across OHCs. This in turn causes a systematic reduction of DPOAE amplitudes within a given furosemide-treated animal (Mills et al., 1993). Filter functions and group delays obtained in furosemidetreated mice were used to help clarify the interpretation of data obtained in dfw mice. The overall goal was to determine the extent to which DPOAEs are altered in dfw mice as a function of stimulus frequency and level. 2. Materials and methods 2.1. Animals Data from 49 mice are presented. Mice were approximately 2 months old and were raised in a colony of C3HeB/FeJ-dfw mice maintained at the University of Washington since 1989. Twenty mice were used as controls (13 were C3HeB/FeJ mice and seven were unaffected littermates (+/+)). Twelve heterozygous (+/dfw) and eight homozygous deafwaddlers (dfw/dfw) were

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studied. Five C3HeB/FeJ and four +/+ mice were treated with furosemide. Data from 5^8 animals per genotype were obtained per condition. Breeding pairs were genotyped by PCR analysis of tail clip DNA (Street et al., 1998). A few mice were characterized phenotypically (based on DP-grams) before molecular genotyping was available. The experimental protocol was approved by the Animal Care and Use Committee at Children's Hospital and Regional Medical Center and the University of Washington. 2.2. Equipment and procedures Anesthetic was delivered by intramuscular injection of ketamine (Ketaset: 15 mg/kg) and xylazine (Rompun : 5mg/kg), and supplemented with one-third the initial dosage every 30^45 min. The left pinna and most of the ear canal were removed. Animal core body temperature was held at 36^37.5³C using a custom heating pad with a micro-processor-based body temperature controller. DPOAEs were measured using a custom-designed system described elsewhere (Mills et al., 1993; Mills and Rubel, 1994). It is based on a host computer, and includes a DSP board, a 4-channel A/D converter, a 4channel 20-bit D/A converter, and an array processor (all by Tucker Davis Technologies). The two stimulating frequencies were generated by separate D/A channels and sent to separate digital attenuators and separate channels of a power ampli¢er (Crown D75). Customized piezoelectric tweeters (Radio Shack, 401383) delivered the signal to a brass cone seated against the animal's left ear. The coupler also contained ports for the calibration microphone (Larsen-Davis) and measurement microphone (Etymotic Research 10B). The system was calibrated at the beginning and at several times throughout an experiment. The high-frequency limit of the measurement system was approximately 50 kHz. The physical dimensions of the calibration setup make it impossible to be sure of absolute sound pressure levels (SPLs) at the highest frequencies due to standing waves. However, calibrations were repeatable within an animal and similar across animals, even at high frequencies. 2.3. Stimulus and recording parameters DPOAEs were elicited using three stimulus paradigms, each employing two tones called `primaries' (f2 and f1 ). The acoustic waveform in the ear canal was sampled at 139 kHz for 4 s if stimulus levels were 6 50 dB SPL or f2 s were s 24 kHz. Otherwise, the measurement window was 2 s. The magnitude and phase of the 2f1 3f2 DPOAE were captured using Fourier transform techniques and saved for further analyses.

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To generate DP-grams, DPOAE amplitude was measured as f2 was varied in 1 kHz steps over frequencies spanning most of the mouse hearing range (1^50 kHz) for a ¢xed f2 /f1 (1.22). L1 was 65 dB SPL and L2 was 55 dB SPL. In another paradigm, DPOAE amplitude for ¢xed f2 s was measured as a function of L1 where f2 /f1 = 1.22 (DPOAE input^output (I/O) functions). Finally, DPOAE amplitudes were measured as a function of f2 /f1 ratio (1.01^1.4). For these `¢lter function' measurements, f2 was held constant and f1 was varied. The f1 step sizes were 50 Hz for f2 = 6^12 kHz, and 100 Hz for the higher f2 s. Although the point of maximal overlap between primary frequencies changes with f1 , all conditions, to some extent, share a common generator site because of the steep slope of the displacement pattern on the apical side of f2 . Filter function data were used to calculate DPOAE latency estimates (described below). For ¢lter functions and I/O functions, f2 = 6, 9, 12, 15, 18, 24, 30, 36, and 48 kHz. Responses were elicited for L1 ranging from 30 to 90 dB SPL ; L2 was always 10 dB below L1. 2.4. Analysis : DPOAE latency DPOAE amplitude and phase data from an entire f1 -sweep were placed in their correct frequency positions within a large bu¡er (1024). The resulting `spectrum' of real and imaginary numbers served as the input to an IFFT. Thus, the output of the complex IFFT is the response magnitude with respect to derived time. The pseudo-time representations shown throughout this paper are the absolute IFFT magnitude and will be referred to as the `IFFT waveform'. The time delay corresponding to the center of energy was calculated (delay = 4(timeUpressure2 )/4pressure2 ). Although not shown, the more common phase-slope method for determining group delay was used in pilot studies in control and dfw mice, with similar results. 2.5. Furosemide treatment In nine control mice, emission measurements were obtained prior to, and up to 2 h following subcutaneous administration of furosemide (150^200 mg/kg). Furosemide intoxication results in a transient decrease of the endocochlear potential by disabling a Na‡ /Cl3 /K‡ cotransporter in the stria vascularis (Greger and Wangemann, 1987). Because e¡ects of this treatment vary over time, an abbreviated number of conditions were tested for each animal. Filter functions were constructed at one f2 , either 12, 24, or 30 kHz. Data from at least two furosemide-treated animals were obtained per f2 . Stimulus levels corresponded to 20 dB below and 10 dB above the in£ection point in the individual's DPOAE I/O function, which was around 70 dB SPL.

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The f1 step sizes used to generate ¢lter functions in furosemide-treated animals were 100 Hz steps for 12 kHz, 200 Hz steps for 24 kHz, and 300 Hz steps for 30 kHz. 3. Results 3.1. Untreated controls and dfw mutants 3.1.1. DP-grams DP-grams were obtained for a moderate stimulus intensity as one measure of OHC function at the start, and several times throughout each experiment. As expected based on a pilot study (Norton et al., 1996), +/+, +/dfw, and dfw/dfw mice produced di¡erent DP-grams. Fig. 1 compares mean DP-grams ( þ 1 S.D.) obtained in untreated control mice, +/dfw mice, and dfw/dfw mice. For controls (top panel), DPOAE amplitude increased from f2 = 6 kHz to f2 = 15 kHz, and remained constant thereafter. Emission amplitudes were similar in +/dfw (middle panel) and control mice for f2 s up to 15 kHz, above which mean DPOAE amplitudes in +/dfw mice were reduced. Responses in +/dfw mice were usually absent above about 35 kHz. In dfw/dfw mice (bottom panel), responses were absent at all f2 s at this stimulus intensity. 3.1.2. DP I/O functions DPOAE I/O functions in laboratory animals exhibit a compressive nonlinearity below about 70 dB SPL, which gives way to a steep, linear slope at higher levels of stimulation. Only the high-level segment is observed following experimental manipulations known to disrupt OHC function (Norton et al., 1991; Kim, 1980; Anderson and Kemp, 1979; Mills et al., 1993; Mills and Rubel, 1994; Long and Tubis, 1988). Median DPOAE amplitudes are shown in Fig. 2 as a function of L1. Amplitudes in +/dfw mice were reduced compared to controls at f2 s v 9 kHz. Both the low-level and high-level portions of I/O functions were reduced in +/dfw mice, compared to controls. At 48 kHz, responses were reduced or absent in +/dfw mice. Responses in dfw/dfw mice were generally in the noise £oor. Multiple regression analyses were performed to examine frequency-speci¢c e¡ects in +/dfw mice. DPOAE responses were subdivided into three groups based on f2 ; data from dfw/dfw were excluded from this analysis. For the lowest f2 group (6, 9 and 12 kHz), e¡ects of genotype (P = 0.001) and level (P 6 0.001) were signi¢cant. For the second group (15, 18, and 24 kHz), there was an e¡ect of level (P 6 0.001) and a level by genotype interaction (P 6 0.001), perhaps re£ecting larger di¡erences at higher levels at 24 kHz. At the highest

Fig. 1. Comparison of mean DP-grams across genotype. DP-grams were obtained by plotting DPOAE amplitude by f2 , where f1 and f2 were varied together. The f2 /f1 = 1.22. Respective panels represent data for control mice (+/+ and C3HeB/FeJ), heterozygous deafwaddlers (+/dfw), and homozygous deafwaddlers (dfw/dfw). Squares indicate mean DPOAE amplitude. Error bars give standard deviations. Gray circles represent the mean noise.

f2 s (30, 36, and 48 kHz), genotype (P = 0.005) and level (P 6 0.001) were also related to DP amplitude. There was a signi¢cant level by genotype interaction (P 6 0.001), as responses from both genotypes tended to converge at the noise £oor when low stimulus levels were used.

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Fig. 2. Mean I/O functions for a range of f2 s (noted in each panel). The parameter within a panel is genotype, including controls (solid lines), +/dfw mice (long dashes), and dfw/dfw mice (asterisks). The ¢fth and 95th percentile ranges of the data are given for controls (shaded areas) and +/dfw mice (areas enclosed by short dashes). L2 was always 10 dB below L1.

3.1.3. DP ¢lter functions The f2 /f1 commonly used to elicit DPOAEs (around 1.22) produces high-level responses in normal ears (Probst, 1990). However, the ideal ratio for detecting OHC dysfunction is not known and may depend on

the con¢guration of the hearing loss (Stover et al., 1999). Related to this issue is the possibility that there is a relationship between ¢lter function shape and basilar membrane tuning (Brown et al., 1993a,b), and/or interactions among multiple emission sources (Brown

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Fig. 3. Changes in amplitude as a function of primary frequency ratio (f2 /f1 ) and DPOAE frequency (`¢lter functions'). To obtain these functions, f2 was ¢xed and f1 was varied. Examples are from a control for a moderate (top panel) and high (bottom panel) stimulus level where the f2 was 24 kHz. At a moderate level, ¢lter functions displayed the characteristic `peaked' shape. Phase delay is given as DPOAE phase relative to the stimulus phase (DPOAEÖ3(2f1 Ö3f2 Ö)) and is shown prior to unwrapping. Comparing the top and bottom panels, it can be seen that phase slope depends on stimulus intensity. Phase tended to change linearly with DPOAE frequency for moderate stimulus levels (top panel); responses to high-level tones (bottom panel) were not as well behaved.

et al., 1993a ; Kemp and Knight, 1999; Stover et al., 1996 ; Faulstich and Kossl, 2000). OHC function, as evident in DP-grams and I/O functions, decreases as frequency increases (Figs. 1 and 2) in +/dfw mice.

This situation provides the opportunity to examine whether a relationship exists between OHC function and ¢lter function shape. Further, if second source activity is present in mice, relative contributions to the DPOAE by distortion at f2 and the stimulus frequency OAE arising one-half octave below it (at the 2f1 3f2 place) may di¡er in +/dfw and control mice. Therefore, DPOAE amplitude was measured as a function of f2 /f1 . Fig. 3 shows DPOAE amplitude (solid line) and noise (long dashes) from a typical control animal as a function of the 2f1 3f2 DP frequency (lower X-axis), and f2 / f1 (upper X-axis). Data are for a moderate (top) and high (bottom) stimulus intensity. In both control and +/ dfw mice, ¢lter functions had a parabolic shape. Consistent with the literature, the amplitude minimum where f2 /f1 approaches unity was most pronounced below 70 dB SPL (Gaskill and Brown, 1990). Above about 70^80 dB SPL, ¢lter functions were relatively £at. The £attening out of ¢lter functions at high levels might re£ect spread of excitation on the basilar membrane and/or changes in the relative contributions by emission generators (Stover et al., 1996). Like guinea pig (Brown and Gaskill, 1990) and gerbil (Faulstich and Kossl, 2000), ¢lter functions in mice do not show much ¢ne structure, suggesting that any contribution to the DP from the 2f1 3f2 place is limited (Heitmann et al., 1997). Fig. 4 depicts median ¢lter functions for mice grouped by genotype. DPOAE amplitudes were more variable among +/dfw mice than controls. Median amplitudes were similar in +/dfw mice and control mice when f2 99 kHz. Above 9 kHz, amplitudes were reduced for +/dfw mice, even for high stimulus levels. For 18 kHz, large f2 /f1 ratios tended to reveal greater DPOAE amplitude di¡erences relative to the control group. As a result, the ¢lter function peak sometimes appeared to be shifted toward lower f2 /f1 ratios (higher DP frequencies) in +/dfw mice. At 30 kHz, the notch in ¢lter functions where f2 /f1 approaches 1 may be larger in +/dfw mice than controls for similar input levels. Multiple regression analysis, excluding data from dfw/dfw mice, was used to examine e¡ects of genotype, stimulus level and their interactions. To best ¢t the DP amplitude data, a squared term was included for f2 /f1 . Individual regressions were performed on three subgroups based on f2 . For the lowest frequencies, DP amplitude was a¡ected by L1 (P = 0.001). At 15^24 and 30^48 kHz, there were stimulus level e¡ects (P = 0.001 for both groups) and a genotype by level interaction (P = 0.01 and P = 0.025, respectively). Stimulus levels (L1) necessary for 80% of the animals to produce a response 6 dB above the noise £oor are given in Fig. 5. The parameter in this ¢gure is genotype. These criteria were met for three or more points taken from the peak region of the ¢lter function. At f2 s above

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Fig. 4. Comparison of ¢lter functions by genotype. The three columns are for f2 = 9, 18, and 30 kHz, respectively. Rows represent the presentation level (L1). L2 was always 10 dB below L1. Median ¢lter functions are shown for controls (solid lines) and +/dfw mice (long dashes). The ¢fth and 95th percentile ranges of the data are also given (controls = shaded areas; +/dfw mice = areas enclosed by short dashes).

30 kHz, the stimulus levels required to meet criteria for the +/dfw group were 30^40 dB higher on average compared to the normal control group.

Fig. 5. Level required for DPOAE response as a function of f2 and genotype. Symbols indicate the L1 required for a response by 80% of animals within a group. For a response to be considered present, at least three DPOAE measurements near the peak of the ¢lter function (f2 /f1 = 1.15^1.25) had to be 6 dB above the noise. Circles and triangles represent data obtained in controls and +/dfw mice, respectively.

3.1.4. IFFT waveforms DPOAE group delay was examined in control mice and +/dfw mice in order to investigate possible e¡ects of the deafwaddler mutation on latencies. It has been hypothesized that, similar to an electronic ¢lter, the more sharply tuned an auditory ¢lter, the longer the delay imposed by the ¢ltering mechanism (Ruggero, 1992). OHC function is thought to increase the tuning of the basilar membrane response, whereas OHC damage results in relatively broad tuning. Since the dfw mutation might a¡ect OHC function, DPOAE group delay might be shorter in +/dfw mice than controls at high frequencies where DP amplitudes are reduced. Fig. 6 displays IFFT waveforms (energy as a function of derived time). Data were obtained from a typical control (left) and +/dfw mouse (right) for an f2 of 9 kHz, at a range of levels (60^90 dB SPL). Figs. 7 and 8 follow the same format, but are for f2 s of 18 and 30 kHz, respectively.

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Fig. 6. Representative IFFT waveforms from a control (left) and +/dfw mouse (right) for a relatively low frequency (f2 = 9 kHz). An individual IFFT waveform is an IFFT of the ¢lter function data. Absolute IFFT magnitude is given in energy by derived time. Rows depict increasing stimulus intensity from 60 to 90 dB SPL. The Y-axis is an auto-ranged scale, given on each plot.

IFFT waveforms obtained in mice had fewer peaks than has been reported for humans (Stover et al., 1996), their number depending on the f2 frequency. The shape of the IFFT waveforms was similar in control and +/ dfw mice. Below about 18 kHz, delays associated with individual peaks appeared to be constant with changes in L1, whereas the relative dominance of the peaks shifted systematically with level. The ¢rst peak was most prominent at high levels. At f2 s above about 18 kHz, it was di¤cult to resolve more than one peak, perhaps due to the frequency resolution of the measurements. For high f2 s, the peak became more broad and the center of energy of the IFFT waveform tended to shift with decreasing stimulus level. Thus, e¡ects of level are generally consistent with data obtained in humans (Stover et al., 1996) ; however, the present results

suggest fewer sources and/or re£ections contribute to the DPOAE in mice. DPOAE group delay was determined by the energyweighted average of the IFFT waveform (i.e. each point in time was weighted by the IFFT magnitude at that point). Fig. 9 shows median group delays for controls (left column) and +/dfw mice (right column). The three rows contain responses to low, moderate and high frequencies, respectively. Group delay changed systematically with stimulus level and frequency, and in +/dfw mice was abnormally long at frequencies having abnormal amplitudes. At low frequencies (6, 9, and 12 kHz), there was an e¡ect of stimulus level (P = 0.001). At moderate f2 s (15, 18, and 24 kHz), there were e¡ects of level (P = 0.001) and genotype (P = 0.02). At the highest f2 s (30, 36, and 48 kHz), delays depended on

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Fig. 7. The same format as Fig. 6, but for f2 = 18 kHz.

level (P = 0.001) and there was a level by genotype interaction (P = 0.001). 3.2. Furosemide-treated control mice In nine control mice, DPOAE ¢lter functions and group delays were examined following furosemide treatment for f2 = 12, 24, and 30 kHz. Furosemide intoxication reduces DPOAEs by reducing the power supply to the OHCs (e.g. Evans and Klinke, 1982; Greger and Wangemann, 1987). Because e¡ects of furosemide are transient, serial DPOAE measurements in a given animal presumably re£ect the endocochlear potential changing along a continuum. Thus, mechanisms underlying furosemide intoxication and the dfw mutation affect OHC activity as viewed by DPOAEs; however,

there is no evidence to suggest that the mechanisms are linked. We wanted to determine whether changes in DPOAEs obtained at various times after the administration of furosemide were similar or di¡erent from results obtained in +/dfw mice. Fig. 10 shows ¢lter functions from two mice for f2 = 24 kHz. The parameter is time relative to the furosemide injection. Responses were obtained for moderate (top row) and high (bottom row) stimulus levels. The broadening of ¢lter functions in untreated control and +/dfw mice as a function of increasing stimulus level was not mimicked by furosemide treatment. After administration of the drug, ¢lter functions elicited by moderate levels shifted down in amplitude, usually dropping into the noise £oor. For two subjects (including animal 9657, right column), post-injection DP am-

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Fig. 8. The same format as Figs. 6 and 7, but for f2 = 30 kHz.

plitudes began to recover following an initial decline. In all but one case (animal 9657), furosemide changed the overall amplitude, but not the shape of ¢lter functions in response to moderate-level primaries. Little or no change was observed in DPOAE amplitudes elicited by high stimulus levels, which were interleaved with moderate levels, and therefore could be compared at a similar time after administration of furosemide. Thus, there were di¡erences between results in furosemidetreated controls and observations in +/dfw mice. Group delays were calculated from IFFT waveforms measured in furosemide-treated mice. Data were obtained from at least two mice at each f2 . For moderate-level stimuli, furosemide intoxication decreased DPOAE delays in three mice, increased delays in ¢ve mice, and had no e¡ect on delays in one mouse. Furo-

semide-induced latency changes in individual mice were small ( = 0.1 ms for ¢ve out of nine mice). The average post-injection change in response delay was an increase of 0.02 and 0.03 ms for moderate- and high-level primaries, respectively. Fig. 11 shows IFFT waveforms (left column) and ¢lter functions (right column) for one mouse prior to (top row), and at three time points after, the administration of furosemide. In this example, f2 was 12 kHz and L1 was 55 dB SPL. Following the drug injection, peaks in IFFT waveforms decreased in amplitude; relative amplitudes of the two major peaks remained constant, as did their respective delays. However, there was a slight increase (0.11 ms) in the average latency (asterisks) with time, which appeared to be related to changes in the relative magnitudes of the multiple peaks.

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Fig. 9. Comparison of the group delay corresponding to the center of energy in IFFT waveforms (energy-weighted average of waveform time) for control (left) and +/dfw (right) mice. Median values are shown as a function of L1 level. The three rows show group delays obtained for low-, moderate- and high-frequency stimuli. The parameter within a panel is f2 .

4. Discussion The deafwaddler phenotype is caused by a point mutation in the gene encoding the plasma membrane Ca2‡ ATPase protein, PMCA2. This point mutation causes a highly conserved glycine residue to be changed to a serine, presumably resulting in a signi¢cant reduction in the Ca2‡ pumping activity in dfw mutants (Street et al., 1998). The speci¢c role of PMCA2 in hearing

remains undetermined. In bull frog saccular hair cells, PMCA2 appears to produce a transduction-dependent outward Ca2‡ current, which when blocked by PMCA inhibitors, increases Ca2‡ concentrations in the stereocilia bundle (Yamoah et al., 1998). Results from previous studies show that both IHC and OHC function is a¡ected in dfw/dfw mice, which do not respond to sound (Norton et al., 1996). Results from the present study show that PMCA2 is important for the genera-

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Fig. 10. Filter functions obtained in two furosemide-treated control mice (columns 1 and 2, respectively). The parameter is time relative to the administration of furosemide. Responses shown here were obtained using a moderate (top panels) and a high (bottom panels) stimulus intensity level.

tion of DPOAEs. In the OHCs, PMCA2 is localized to stereocilia and cuticular plates, suggesting that e¡ects on DPOAE might be due to changes in OHC transduction.

compared to controls at frequencies which had grossly abnormal amplitudes (15^48 kHz). (6) There was not a systematic e¡ect of furosemide treatment on response delay.

4.1. Summary of results

4.2. Level and frequency e¡ects

(1) Homozygous deafwaddlers did not have DPOAEs. (2) Filter functions were parabolic in untreated control animals, +/dfw mice, and furosemidetreated controls. However, for each of these groups, ¢lter functions became less stereotyped at high stimulus levels. (3) While ¢lter function shapes were largely independent of genotype, DPOAE amplitudes were not: amplitudes were reduced across the entire f1 -sweep in +/dfw mice for f2 frequencies of 9^48 kHz, at all stimulus levels. Amplitude di¡erences between control and +/dfw mice increased with increasing frequency. (4) In contrast, DPOAE amplitudes in furosemide-treated controls were reduced for moderate (55^60 dB SPL), but not for high (80^90 dB SPL) stimulus intensities. (5) DPOAE response delays in +/dfw mice were long

The fact that DPOAEs were abnormal in +/dfw mice, regardless of stimulus intensity, suggests that both highlevel and low-level emissions depend on PMCA2. Emissions elicited by high-level stimuli are generally robust, surviving anoxia (Kim, 1980), treatment with furosemide (Anderson and Kemp, 1979; Mills et al., 1993) and salicylates (Anderson and Kemp, 1979; Long and Tubis, 1988), and even death (when measured immediately post-mortem) (Norton et al., 1991). However, high-level emissions require the presence of OHCs, as they are absent in mutant mice with no OHCs (Schrott et al., 1991). Additionally, even high-level DPOAEs were abolished by sulfhydryl reagents applied to the round window which also blocked OHC electromotility (Frolenkov et al., 1998). In the present study, dfw was

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Fig. 11. Representative IFFT waveforms (left) and corresponding ¢lter functions (right) from a furosemide-treated control mouse. Asterisks indicate the average IFFT waveform delay. The top row corresponds to the una¡ected control condition. Subsequent rows depict increasing time relative to administration of furosemide. The f2 = 12 kHz and L1 = 55 dB SPL. The length of the time window containing the IFFT waveform is inversely related to the frequency spacing of the input spectrum (in this case, the 2f1 3f2 step size), which for furosemide-treated mice was up to three times larger than for controls. As a result, average delays will be relatively short in furosemide-treated mice compared to the untreated mice.

more detrimental to DPOAEs than furosemide, which did not a¡ect high-level emissions. DPOAEs in dfw/dfw mice and in +/dfw mice for high f2 s were consistent with severe OHC abnormalities, which in +/dfw mice would not have been predicted based on preliminary anatomical results (Pujol et al., 1997). DPOAEs in +/dfw mice were abnormal at high frequencies, but appeared to be normal at low frequencies. It is not known whether the distribution of PMCA2 is graded along the frequency axis of the cochlea (this has not yet been examined at high resolution) or if the function of PMCA2 is most critical for high frequencies. In guinea pig and gerbil, high-frequency emissions

were particularly vulnerable to manipulations of the endocochlear potential (Brown and Pye, 1975 ; Mills et al., 1993; Mills and Rubel, 1994). Additionally, the rate of transduction-dependent K‡ (and presumably also Ca2‡ ) turnover may be relatively high at the base of the cochlea (Rattay et al., 1998), which would be consistent with a greater e¡ect of dfw at high f2 s. 4.3. Filter functions Results from the present study suggest that hearing loss, per se, does not lead to wider DPOAE ¢lter functions. This view is supported by other studies. Filter

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functions in furosemide-treated adult gerbils and untreated pups were indistinguishable from those of untreated adult controls (Mills and Rubel, 1997). The characteristic shape of ¢lter functions was present in rabbits following noise exposure (Ohlms et al., 1991). There were no changes in bandwidths of ¢lter functions recorded in humans during temporary noise-induced threshold shift (Engdahl and Kemp, 1996) or following administration of high dosages of aspirin (Brown et al., 1993b). These data suggest that a link between mechanism(s) underlying cochlear frequency selectivity and DPOAE ¢lter function shape does not exist, or that an additional mechanism in£uences ¢lter function shape that is not directly linked to cochlear sensitivity or tuning. Interactions between DPOAE sources arising near f2 and DP sites may in£uence the ¢lter function shape. This possibility was examined by Stover et al. (1999), who obtained ¢lter function data from listeners with normal hearing and with frequency-speci¢c hearing loss. The amplitude dip at closely spaced primary frequencies was absent in people with steeply rising audiograms in which f1 and f2 resulted in 2f1 3f2 frequencies located within the impaired region of hearing. This ¢nding suggests that functioning OHCs at the 2f1 3f2 place may be necessary to produce the characteristic parabolic function. For a given stimulus intensity, mild to moderate hearing loss at the f2 frequency caused a decrease in DPOAE amplitudes across f2 /f1 ratios; however, the functions remained parabolic. Similar to the mechanism by which a spontaneous OAE (SOAE) is generated, the second source activity may arise as a re£ection when the apically traveling DP encounters discontinuities near its characteristic place on the basilar membrane (Shera and Zweig, 1993). In humans, rapid variations in DPOAE level and phase (and therefore group delay) with frequency have been taken as evidence for interaction between the two DPOAE sources described above (e.g. Talmadge et al., 1988). Rodents exhibit less DPOAE ¢ne structure than humans and SOAEs are not usually found in rodents (Mills and Rubel, 1997; Faulstich and Kossl, 2000). However, some evidence suggests that more than one source contributes to DPOAEs measured in the ear canals of rodents. Filter functions for f2 s between 1.8 and 16 kHz in gerbil had a large peak, and a small second peak at low f2 /f1 s (Faulstich and Kossl, 2000). Abrupt variations in the phase accompanied the f2 /f1 delineating these two ¢lter function peaks. Our IFFT waveforms are consistent with the twosource hypothesis. Figs. 6 and 11, for f2 = 9 and 12 kHz, show IFFT waveforms having at least two peaks. The inability to distinguish more than one peak at f2 s above 15 kHz might have been due to the frequency resolution of the measurement. However, multi-

ple peaks in the IFFT (beyond two) were uncommon in mice, suggesting that contributions by a secondary source are reduced in mice compared to humans. If interaction between two sources produces the peaked ¢lter function shape in mice, furosemide intoxication and the dfw mutation might not alter the ¢lter function shape, only its overall amplitude, as long as generators at f2 and 2f1 3f2 are a¡ected equally (and are still producing energy at the DP frequency). Where the dfw mutation exerts a steeply graded e¡ect along the cochlea (i.e. in the basal region), contributions from the f2 source might decrease relative to the 2f1 3f2 place source, which is slightly lower in frequency. Filter functions in +/dfw mice were generally similar in shape to those obtained in controls. These results suggest that interactions between multiple components are either affected very little by the con¢guration of the hearing loss in +/dfw mice, or that DPOAEs in mice are dominated by the distortion component of the emission and the DP site component has little in£uence on the ¢lter function shape. 4.4. DPOAE latencies Associated with the loss of OHC-assisted basilar membrane vibration is a downward shift in characteristic frequency (CF), a reduction in frequency tuning and CF-speci¢c nonlinearities (Cody and Johnstone, 1981). Therefore, in the present experiment, DPOAE latencies in +/dfw mice and furosemide-treated control mice were predicted to decrease. The observation that latencies in furosemide-treated controls were similar to pre-injection values was not completely unexpected due to the short basilar membrane in mouse as compared to other mammals, and since only small changes in DPOAE group delay were obtained in furosemidetreated gerbils (Mills and Rubel, 1997). In contrast, the ¢nding that DPOAE latencies obtained in +/dfw mice at high f2 s were longer than those of controls was puzzling. One might hypothesize that the changes caused by the dfw mutation alter the interaction between multiple components of the emission. For example, any decrease in delay caused by OHC dysfunction at f2 in +/dfw mice may have been o¡set by the relatively robust response at the 2f1 3f2 place, due to the sloping con¢guration of hearing loss. Thus, emission generators having longer latencies may have dominated group delays in +/dfw mutants, whereas the f2 source may have dominated responses in control mice. The fact that very little ¢ne structure is present in mouse DPOAE ¢lter functions tempers this argument, suggesting that any contribution by the 2f1 3f2 source is small, at least compared to observations made in humans (Heitmann et al., 1997). Another possible explanation might be that the mutation results in a tonotopic shift-

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ing of high frequencies towards the apex. However, latencies at 36 kHz in +/dfw mice were longer near DPOAE threshold than those obtained at any level in controls for frequencies above about 12 kHz, weakening this argument. We cannot rule out the possibility that di¡erences in group delays between the two groups might be related to lower signal-to-noise ratios in +/dfw mice. Group delay was determined by the weighted average of IFFT waveform, such that each time point was weighted by its corresponding magnitude. Therefore, noise will contribute more to group delays for DPOAEs having low compared to high signal-to-noise ratios. 5. Conclusions The deafwaddler mutation disrupts cochlear OHC function, leading to a decrease or elimination of DPOAEs. DPOAEs in +/dfw mice suggest that PMCA2 is critical for high-frequency hearing. At affected frequencies, DPOAE amplitudes were reduced regardless of the stimulus intensity in +/dfw mice, whereas furosemide intoxication in control mice did not alter DPOAEs to high-level stimuli. This presumably re£ects the di¡erent mechanisms underlying abnormal OHC system function in dfw mutants and furosemide-treated control mice. Acknowledgements We thank Michael Gorga, Stephen Neely, and three anonymous reviewers for their comments on earlier versions of this manuscript. Thanks to Brandon Warren and Mark Martin for computer programming assistance. Research was supported by HR 5806 from Children's Hospital and Regional Medical Center, Seattle, WA, USA, and T32 DC00033 awarded to the University of Washington by the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. Support for the dfw mouse colony and genotyping was given by R01 DC02739. Manuscript preparation and data analyses were supported in part by a postdoctoral training Grant (T32 DC00013) awarded to Boys Town National Research Hospital. References Allen, J.B., Fahey, P.F., 1993. A second cochlear-frequency map that correlates distortion product and neural tuning measurements. J. Acoust. Soc. Am. 94, 809^816. Anderson, S.D., Kemp, D.T., 1979. The evoked cochlear mechanical

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