- Email: [email protected]
Characteristic findings of auditory brainstem response and otoacoustic emission in the Bronx waltzer mouse
Brain & Development 28 (2006) 617–624 www.elsevier.com/locate/braindev
Characteristic findings of auditory brainstem response and otoacoustic emission in the Bronx waltzer mouse Masumi Inagaki
a,*
, Kaori Kon a, Seiko Suzuki a, Naoko Kobayashi a, Makiko Kaga a, Eiji Nanba b
a
b
Department of Developmental Disorders, National Institute of Mental Health, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawa Higashi, Kodaira 187-8553, Japan Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-machi, Yonago 683-8503, Japan Received 16 January 2006; received in revised form 1 April 2006; accepted 10 April 2006
Abstract Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were evaluated serially from 1 to 22 months in Bronx waltzer homozygotes (bv/bv), heterozygotes (+/bv) and control (+/+) mice, which were differentiated by means of PCR of marker DNA (D5Mit209). The wave IV threshold of the click-evoked ABR was higher than the DPOAE threshold with the DP growth method in each bv/bv, although the two thresholds were almost the same in the +/+ group. The DP value at 2f1 f2 in the bv/bv showed an apparent decrease at 2 to 3 months of age with 80 dB SPL stimulation using f2 frequency 7996 Hz and frequency ratio f2/f1 = 1.22, compared to control or heterozygote mice. It was characteristic that the 2f2 f1 DP signal-to-noise ratio (SNR) value was more preserved from 80 to 60 dB SPL than the 2f1 f2 DP value at f2 frequency 7996 Hz in most bv/bv, however, control mice showed almost the same levels of 2f1 f2 and 2f2 f1 SNR value at both f2 frequencies of 6006 and 7996 Hz. The preservation of a substantial 2f2 f1 DP suggested that it would be generated basal to the primary-tone place on the basilar membrane and there might be a reflection of the unique function of the remaining outer hair cells in the Bronx waltzer mice. These findings suggest that combination of ABR with DPOAE could offer useful information about differentiating the mechanism of hair cell dysfunction of the hereditary hearing impairment in the clinical fields. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Auditory brainstem response (ABR); Bronx waltzer mouse; Hair cell function; Otoacoustic emission (OAE)
1. Introduction The homozygous Bronx waltzer (bv/bv) mouse is known as a hearing-impairment model with selective inner hair and pillar cell damage in the cochlea
Abbreviations used: ABR, auditory brainstem response; OAE, otoacoustic emission; DPOAE, 2f1 f2 or 2f2 f1 distortion product otoacoustic emission; SNR, signal-to-noise ratio; PCR, polymerase chain reaction. * Corresponding author. E-mail address: [email protected] (M. Inagaki). 0387-7604/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2006.04.006
occurring just before birth [1–3]. Since about 70–80% of the inner hair cells (IHCs) degenerate and eventually die, moderate to severe hearing impairment follows [4]. In contrast, the outer hair cells (OHCs) in the cochlea are completely present but disarranged with folding of the lateral wall at the site of IHC degeneration [5,6]. Elevation of the threshold of both the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) has been reported separately in detail [7–9], which suggests the presence of hair cell dysfunction. A recent study has also revealed that an abnormal neuronal response pattern in the inferior colliculus of
618
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
the Bronx waltzer mouse, i.e., elevation of the threshold of the characteristic frequency and broad frequency tuning, is produced on an addition of OHC damage to IHC degeneration [6]. These findings suggest that the bv mutation affects both types of hair cells at morphological and functional levels to some extent at different stages. However, serial changes in each hair cell function in the Bronx waltzer mouse have not been well addressed. In this study, we compared click- and high frequency tone burst-evoked ABRs with DPOAEs by means of DP growth method in the mutant mice from 1 to 22 months of age. And we discussed whether the characteristic findings of the hair cell function in this kind of mutant mouse could provide or not new information about pathophysiology of hereditary hearing impairment in human adult or newborn hearing screening test.
2. Materials and methods 2.1. Animals Homozygous Bronx waltzer mice of both sexes have been bred at the National Institute of Mental Health, National Center of Neurology and Psychiatry (NCNP) in Japan since 1998. The breeding bv/bv pairs were obtained through the courtesy of Dr. H.M. Sobkowicz, Department of Neurology, University of Wisconsin, Madison, WI, USA, who had received the original mutant mice that had arisen at Albert Einstein College [10]. The mice were housed individually with a 12 h/ 12 h light dark cycle with food and water ad libitum in an EBAC-S breeding apparatus (CL-5351; CLEA-Japan, Co. Ltd., Japan), with which a constant temperature and humidity can be maintained. Tympanic membranes were examined prior to the experiment. All physiological procedures were performed under anesthesia using intraperitoneal injection of sodium pentobarbi-
tal (50 mg/kg body weight) supplemented as required during experiments. All experimental and euthanasia procedures conformed fully to the guidelines for animal care stipulated by the animal committee of the National Institute of Neuroscience, NCNP. Although bv/bv mutant mice were originally maintained in a CBA/J background [10], the details of the genetic background have been obscure in the colony [11,12]. So, one of the authors (EN) identified the genotype of each animal using a recently reported method [13]. Briefly, genomic DNA was prepared from the tail of each living animal or deep frozen samples by a standard technique. Mice were genotyped by PCR using the closest marker (D5Mit209) to the bv mutation. PCR was carried out under the following conditions: 35 cycles of 1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C, with final extension for 5 min at 72 °C. The sequences of the forward and reverse primers were TCTGAGCAA GGTCGTCCAC and CCCTGTCTCAAGATAAAA, respectively. PCR products of the homozygote gave two 359 bp bands, designated as bv/bv, and heterozygote gave two different bands (359 bp and 325 bp), typed as +/bv, on 3% agarose gel electrophoresis (Fig. 1). In the present study, physiological data from bv/bv, +/bv and +/+ animals (ddY and ICR strains), which were purchased from CLEA-Japan, were analyzed [14,15]. Moreover, a commercially available mouse strain, DBA/2J, which has been reported to show early onset progressive sensorineural hearing loss [16,17], was also used as positive control. 2.2. Auditory brainstem responses (ABRs) ABR examination was performed after anesthesia as above described in a quiet room. Needle electrodes were inserted subcutaneously in the retro auricular (active), vertex (reference) and nasial (ground) areas. Two kinds of sound stimuli were used; an alternate click sound of
Fig. 1. Identification of the genotype of the Bronx waltzer mice. There was a difference in the size of the polymerase chain reaction (PCR) product of marker DNA (D5Mit209) between homozygous (bv/bv) and heterozygous bv (bv/+). Homozygotes gave two 359 bp bands, and heterozygotes two different bands (359 bp and 325 bp) on 3% agarose gel electrophoresis. D5Mit209 is localized at 0.39 cm from the bv locus. M4: /X174/HaeIII.
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
0.1 ms duration for all mice, and tone bursts with a rise and fall time of 1 ms and a plateau of 1 ms of 8, 16, 24, or 32 kHz frequency for some mice. The click sound was presented monaurally through an ear tube masked with white noise for the contra-lateral ear canal. The click sound had substantial energy in the range of 2–8 kHz. A maximum sound pressure level (SPL) of 135 dB was employed for all stimuli. The tone bursts, which were delivered with a sound stimulator (DPS-725; Diamedical System Co. Ltd., Japan), were delivered at the rate of 10/s and started at the intensity of 100 dB SPL through a speaker (PT-RIII; Pioneer, Japan) situated 20 cm in front of the animal. Evoked potentials were band pass-filtered (100– 3000 Hz) by the preamplifier and 500 traces were averaged using an averager (MEB 9104; Nihon Kohden, Co. Ltd., Japan). Upward deflection was defined as positive polarity. Mice were kept warm during the experiment. The auditory threshold was taken as the minimum signal amplitude necessary to evoke clear potentials of wave II or IV. If no ABR waves could be obtained with click of an intensity of 135 dB SPL, a nominal threshold of 140 dB was assigned. Totals of 55 bv/bv and 55 +/+ mice in the different age group were examined. 2.3. Otoacoustic emission (OAE) OAE measurements were conducted with a DP Echoport of ILO 92 system (Otodynamics, Co., Ltd., UK) just after the ABR examination. The DP growth method was applied to each animal using a standard probe. The DPOAE stimuli consisted of two primary frequencies, f1 and f2. Two equilevel (L1 = L2) primary signals at 7996 or 6006 Hz (f2) and 6555 or 4919 Hz (f1) with a fixed f2/f1 ratio of 1.22 were presented. At least 32 consecutive responses were averaged. Threshold measures in the form of input/output functions were obtained by decreasing the primary tone from 80 to 45 dB SPL in 5-dB steps. Detection of DPOAE thresholds at 2f1 f2 was defined as the intensity of the primaries that elicited a DPOAE whose amplitude was 2 dB greater than the noise level. Also, in elder mice, DPOAE levels of more than +2 SD above the noise floor were considered valid. The 2f2 f1 as well as 2f1 f2 DPOAE amplitude minus noise level (S/N value) at each stimulus sound level in four mouse-groups (bv/bv, +/bv, +/+ and DBA/2J) mouse were also calculated at the age of 2–5 months. 2.4. Statistical analysis Statistical evaluation was performed among mouse groups using analysis of variance (ANOVA) and Fisher’s Protected Least Significant Difference (PLSD) for post hoc comparison with a commercially available soft-
619
ware package (StatView ver. 4.5; Abacus Co. Ltd., USA). Statistical significance was accepted when the p value was less than 0.05.
3. Results 3.1. ABR Control mice showed an ABR wave with 4 or 5 positive peaks in response to the click sound. The basic waveform comprised high amplitude waves I and II, and lower amplitude wave IV. The ABR waveform in response to the tone bursts was similar to that to the click sound stimuli. The mean (±SD) thresholds of wave IV for young control mice were 43 (±5), 22 (±19), 20 (±6), 22 (±15) and 38 (±16) dB SPL for the click sound, 8, 16, 24 and 32 kHz tone burst stimuli, respectively (Fig. 2). Heterozygous mice had almost the same level of the IV threshold as control. However, homozygous Bronx waltzer mice showed significant (p < 0.001) elevation of the click sound threshold value at a young age (2 to 3 months of age). DBA/2J mice (n = 10) at the age of 2–5 months also showed apparent elevation of IV threshold (mean ± SD; 98 ± 18 dB SPL) for the click sound. In bv/bv, threshold of tone bursts was also elevated equally by around 60 dB from the control value (p < 0.001). There was little change in the average threshold of click-evoked ABR for 2 years of age. The latencies of waves I, II and IV are shown in Table 1. Wave latencies were not significantly prolonged in the young Bronx waltzer mice compared with the controls. Elder homozygotes showed similar findings as to the latency of each wave without significant prolongation.
Fig. 2. ABR threshold of Bronx waltzer and control mice. The ABR thresholds (circles) of the mutants (n = 6) in response to the click sound and higher frequency tone bursts, i.e., 8000, 16,000, 24,000 and 32,000 Hz, were in the range of 80–110 dB SPL at 2 to 3 months of age. The mean thresholds (triangles) in the control mice (n = 5) with the tone bursts (8000, 16,000 and 24,000 Hz) were equally at around 20 dB SPL. Bars represent 1 SD.
620
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
Table 1 Wave latencies of click-evoked ABR in control and mutant mice a
Control (ddY) bv/bva bv/bvb
n
Peak latency of wave I
Peak latency of wave II
Peak latency of wave IV
I–IV Interpeak latency
11 17 6
1.11 (0.09) 1.15 (0.13) 1.06 (0.21)
1.93 (0.17) 2.03 (0.14) 1.94 (0.13)
3.61 (0.25) 3.81 (0.35) 3.63 (0.28)
2.50 (0.18) 2.60 (0.60) 2.58 (0.23)
Data (ms) are shown as means (SD) at the intensity of 135 dB SPL. a Age range: 1–3 months of age. b Age range: 12–13 months of age.
3.2. DPOAE Data were collected in the form of DPOAE input/ output (I/O) functions. Although the noise amplitudes at the f2 frequency of 7996 Hz were less than 5 dB SPL on average, those at 6006 Hz were relatively high. At 2 to 3 months of age, the DP value at 2f1 f2 in response to 80 dB SPL stimuli (f2 frequency = 7996 Hz) in the homozygous mutants (n = 17) showed an apparent decrease (from 5.1 to 12.0 dB) compared to the control (n = 15) value (18.8–30.3 dB), which were at the same levels until about 2 years old (Fig. 3). The DP levels at 2f1 f2 did not exceed the noise floor in more than half the bv/bv group after 6 months old. Distribution of signal minus noise value (S/N value) of 2f1 f2 DPOAE at the stimulus intensity of 75 dB SPL was similar in the ddY group ( 10 to 35 dB SPL) as in the heterozygous bv group (5–38 dB SPL). On the other hand, the pattern of 2f2 f1 DP value (S/N value) at f2 frequencies of 7996 Hz was different among mouse strain examined. In the homozygous group, 2f2 f1 S/N value was preserved with a decrease of 2f1 f2 value especially in the 65–80 dB SPL stimuli, (Fig. 4a). Control mice such as ddY and heterozygous mice showed almost the same DP value of 2f2 f1 and 2f1 f2 (Figs. 4b and c), although both had a reversal
pattern showing 2f2 f1 value P2f1 f2 value (2f2 f1 dominant pattern) only at the stimulus intensity of 80 dB SPL. There was no statistically significant difference in the 2f2 f1 value between the homozygous mutants (22.3 ± 13.7 dB SPL at the stimulus intensity of 75 dB SPL) and control (ddY and heterozygotes) ears (18.0 ± 10.9 dB SPL and 23.6 ± 18.8, respectively) at the f2 frequency of 7996 Hz. DBA mice of which ABR threshold was elevated from 80–110 dB SPL also had reduced level of both 2f1 f2 and 2f2 f1 DP value at every stimulus intensity of 80–45 dB SPL. At f2 frequencies of 6006 Hz, both 2f2 f1 and 2f1 f2 value in the homozygous bv group reduced more than control group (Fig. 5). There was neither 2f2 f1 nor 2f1 f2 DP in DBA mice at f2 frequencies of 6006 Hz at stimulus intensity of 80 dB SPL. Scatter plots of the ABR and DPOAE threshold values in the same cases at the age of 2 to 3 months showed that the wave IV threshold of click-evoked ABRs was higher than the DP threshold in each bv/bv (mean thresholds; ABR = 110 dB SPL, DPOAE = 75 dB SPL), although the two thresholds were almost the same value (mean threshold; ABR = 60 dB SPL, DPOAE = 55 dB SPL) in the control group (Fig. 6).
4. Discussion
Fig. 3. Changes in the 2f1 f2 DP level in control and mutant ears. There was a significant decrease in the DP level at the f2 frequency of 7996 Hz in the Bronx waltzer mice (circles) as compared to in the control animals (squares) from 2 to 22 months of age. All data were recorded through the left ear with the intensity of 80 dB SPL.
Mice can hear over a range of frequencies between 500 and 120,000 Hz, however, normal mice are known to be most sensitive to the sound with frequencies of 12–24 kHz [18]. The present results as to the threshold pattern of ABR in response to tone bursts in control mice are consistent with these findings [19]. The Bronx waltzer homozygotes showed little frequency-dependent sensitivity, i.e., about 60 dB elevation at any frequency, which is also consistent with a previous report [7] demonstrating threshold elevation of the ABR without frequency dependence and a median threshold value of around 90 dB SPL. The ABR seems to exhibit little progress in bv/bv. The discrepancy between the ABR and OAE thresholds and selective atrophy of auditory system may support the idea that morphological and functional degeneration in the IHCs is the primary damage in this type of mutation [20].
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
621
Fig. 4. Signal-to-noise ratio of DP at a frequency of 7996 Hz. Mean and standard deviation values of signal-to-noise ratio (SNR) of 2f1 f2 (triangles) and 2f2 f1 (circles) DPs at an f2 frequency of 7996 Hz with stimulus intensity from 80 to 45 dB SPL were plotted in the figures. Homozygous Bronx waltzer mice (a) showed a relatively preserved SNR of 2f2 f1 at an f2 frequency of 7996 Hz as compared to the values of heterozygous bv (b) and control groups (c). DBA/2J had decreased levels of both 2f1 f2 and 2f2 f1 SNR (d).
Fig. 5. Signal-to-noise ratio of DP at a frequency of 6555 Hz. Mean and SD values of signal-to-noise ratio (SNR) of 2f1 f2 (triangles) and 2f2 f1 (circles) at an f2 frequency of 6555 Hz with stimulus intensity from 80 to 45 dB SPL were plotted. Although homozygous bv showed 2f2 f1 P 2f1 f2 pattern at stimulus intensity of 75 and 80 dB SPL (a), there were no remarkable changes between 2f1 f2 (triangles) and 2f2 f1 (circles) DPs among three mouse groups (+/bv, (b); +/+, (c); DBA/2J, (d)).
622
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
Fig. 6. Scatter plots of the ABR and OAE threshold values in the same cases in different mouse groups. The wave IV threshold of click-evoked ABR was higher than the 2f1 f2 DP threshold in each bv/bv, although the two thresholds were almost the same in the +/+ group. Circles, bv/bv mice (n = 30); triangles; control (ddY) mice (n = 30).
On the other hand, the decreased 2f1 f2 DP level at an f2 frequency of 9 to 10 kHz or reduced amplitude of the cochlear microphonics in bv/bv is thought to be due to outer hair cell (OHC) dysfunction [9,21]. In this study, DPOAEs were collected primarily at an f2 frequency of 7996 and 6006 Hz. These test ranges just begin to describe hearing capability of the mouse. In mice with age-related hearing loss these frequencies are not affected until severe progression of OHC loss has occurred. Consequently, with the present protocol it would be impossible to demonstrate serial changes exactly that typically begin at much higher frequencies. However, a recent report on abnormal ‘frequency tuning curves’ in the inferior colliculus indicates alteration of the OHC function in bv/bv in wide range of frequency from 4 to 70 kHz [6]. It was characteristic that the 2f2 f1 DP value was higher than the 2f1 f2 DP value at the f2 frequency of 7996 Hz in most bv/bv for 1 year. The fact that the 2f2 f1 DP level in bv/bv was almost the same as that in control suggests the preserved function of the region of 2f2 f1 DP generation. Moreover, the signal-to-noise ratios (SNRs) at 2f2 f1 were more than those at 2f1 f2 at lower stimulus intensity in bv/bv. And DBA/2J mouse exhibited a diminished level of both 2f1 f2 and 2f2 f1 SNR at any intensity. Anesthetized rodents such as guinea pigs and mice are known to show a 2f1 f2 > 2f2 f1 DP amplitude pattern with geometric frequencies at 5600 and 8000 Hz in general [22]. The amplitudes for 2f2 f1 were known to be lower than for 2f1 f2 also in human subjects with normal hearing [23]. The SNRs are sometimes larger at 2f2 f1 compared to 2f1 f2, however, for f2 frequency higher than 1 kHz the SNR is reported to be greater at
2f1 f2 [24]. So, it is speculated that some OHCs are active but that less organized OHCs may lead to different action in frequency tuning in the present Bronx waltzer mice. A reduction in the level of 2f1 f2 with an increase in 2f2 f1 DPOAE has been observed in rabbits following brief exposure to excessive sound at frequencies higher than f2 [22]. These phenomena are similar to the present findings for the Bronx waltzer mouse and might be related to physiological vulnerability of the cochlear regions basal to f2. Since it is hypothesized that 2f2 f1 DP is generated basal to the primary-tone (f2) place on the basilar membrane [25], OHCs in such areas might excessively vibrate the basilar membrane with specific frequency tones in young Bronx waltzer mice. Or there could be a functional elimination of the secondary emission source basal to f2, which is in addition to the 2f1 f2 and cancels the 2f2 f1 in the mutant cochlea. These results suggest that hair cell function, especially in OHCs, could fluctuate in the adult period. Further analyses of the function of the OHCs in the organ of Corti, which is not affected directly by the bv mutation, with mechanical nonlinearity in the external canal are necessary to elucidate the mechanism of suppression or enhancement of various types of DPOAE. In newborn hearing screening program, transient evoked OAE (TEOAE) and/or automated ABR (AABR) have been adopted in several countries [26– 28]. It is easier to connect the subject to the device and measurements were quicker for OAE than for AABR. However, AABR is highly reliable, and has an advantage that both ears can be tested simultaneously. It is well known that TEOAE measurement is easy to administer and leads to the real time assessment. Some investigators have pointed out that use of a 2-step screening is better than one step screening using TEOAE in terms of the efficacy and determination of human permanent hearing loss, though it is still controversial [29–31]. On the other hand, DPOAE is superior to TEOAE in determination of frequency specific function in the inner ear. And DPOAE has also been prevailing examination for hearing evaluation in children and useful for detecting the cochlear function of pediatric patients with neurological disorders [32–34]. Detailed examination of both OAE and ABR is necessary to diagnose a noticeable clinical entity such as auditory neuropathy or auditory nerve disease [35,36]. Present findings in the Bronx waltzer mice suggest the frequency specific dysfunction of the both hair cells of the organ of Corti in the hereditary hearing disorders. Therefore, combination of the two measurements, i.e., ABR and DPOAE, might be suitable for evaluating the heritable hearing disorder in early infancy as well as in the newborn period. Moreover, a large family of Czech descent with slowly progressive hearing loss has been reported to have a similar genetic background to the Bronx waltzer [2,37].
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
So, age related changes of both 2f1 f2 and 2f2 f1 DP values would be a diagnostic clue for the specific type of the nonsyndromic hereditary hearing impairment with bv mutation or DFNA25 locus [37,38] in adulthood.
Acknowledgements This study was presented in part at the IERASG meeting held in the Canary Islands, Spain, in June 2003. The authors would like to express their deep thanks to Ms. R. Honma, Ms. Y. Tamura, Ms. R. Ohta and Ms. N. Endo for their technical assistance, and to Prof. K. Kaga (Department of Otolaryngology, University of the Tokyo) for critical reading of the manuscript. And authors wish to thank Prof. H.M. Sobkowicz, University of Wisconsin, for her warm encouragement. This study was supported in part by the Health and Labour Sciences Research Grants (H12-Sensory-006; H16-Kokoro-001) for Research on Psychiatric and Neurological Diseases and Mental Health from the Ministry of Health, Labour and Welfare in Japan.
References [1] Whitlon DS, Gabel C, Zhang X. Cochlear inner hair cells exist transiently in the fetal Bronx Waltzer (bv/bv) mouse. J Comp Neurol 1996;364:515–22. [2] Bussoli TJ, Kelly A, Steel KP. Localization of the bronx waltzer (bv) deafness gene to mouse chromosome 5. Mamm Genome 1997;8:714–7. [3] Tucker JB, Mackie JB, Bussoli TJ, Steel KP. Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: response to loss of cell partners in the Bronx waltzer mouse. J Neurocytol 1999;28:1017–34. [4] Deol MS. The inner ear in bronx waltzer mice. Acta Otolaryngol 1981;92:331–6. [5] Kong WJ, Scholtz AW, Hussl B, Kammen-Jolly K, SchrottFischer A. Localization of efferent neurotransmitters in the inner ear of the homozygous Bronx waltzer mutant mouse. Hear Res 2002;167:136–55. [6] Sterbing SJ, Schrott-Fischer A. Neuronal responses in the inferior colliculus of mutant mice (Bronx waltzer) with hereditary inner hair cell loss. Hear Res 2003;177:91–9. [7] Schrott A, Stephan K, Spoendlin H. Hearing with selective inner hair cell loss. Hear Res 1989;40:213–9. [8] Schrott A, Puel JL, Rebillard G. Cochlear origin of 2f1 f2 distortion products assessed by using 2 types of mutant mice. Hear Res 1991;52:245–53. [9] Horner KC, Lenoir M, Bock GR. Distortion product otoacoustic emissions in hearing-impaired mutant mice. J Acoust Soc Am 1985;78:1603–11. [10] Deol MS, Gluecksohn-Waelsch S. The role of inner hair cells in hearing. Nature 1979;278:250–2. [11] Sobkowicz HM, Inagaki M, August BK, Slapnick SM. Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse. J Neurocytol 1999;28:17–38. [12] Sobkowicz HM, August BK, Slapnick SM. Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture. Int J Dev Neurosci 2002;20:537–54.
623
[13] Cheong MA, Steel KP. Early development and degeneration of vestibular hair cells in bronx waltzer mutant mice. Hear Res 2002;164:179–89. [14] Dabdoub A, Donohue MJ, Brennan A, Wolf V, Montcouquiol M, Sassoon DA, et al. Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea. Development 2003;130:2375–84. [15] Miura H, Qiao H, Kitagami T, Ohta T. Fluvoxamine, a selective serotonin reuptake inhibitor, suppresses tetrahydrobiopterin in the mouse hippocampus. Neuropharmacology 2004;46:340–8. [16] Willott JF, Erway LC, Archer JR, Harrison DE. Genetics of agerelated hearing loss in mice. II. Strain differences and effects of caloric restriction on cochlear pathology and evoked response thresholds. Hear Res 1995;88:143–55. [17] Turner JG, Willott JF. Exposure to an augmented acoustic environment alters auditory function in hearing-impaired DBA/ 2J mice. Hear Res 1998;118:101–13. [18] Ehret G. Psychoacoustics. In: Willott JF, editor. Auditory psychobiology of the mouse. Springfield, IL: Charles C Thomas; 1983. p. 13–53. [19] Zheng QY, Johnson KR, Erway LC. Assessment of hearing in 80 inbred strains of mice by ABR threshold analysis. Hear Res 1999;130:94–107. [20] Keithley EM, Feldman ML. The spiral ganglion and hair cells of Bronx waltzer mice. Hear Res 1983;12:381–91. [21] Bock GR, Yates GK. Cochlear electrophysiology in the bronx waltzer mutant mouse. J Physiol (Proceedings of the Physiological Society) 1982;332:P20–1. [22] Martin GK, Stagner BB, Jassir D, Telischi FF, Lonsbury-Martin BL. Suppression and enhancement of distortion-product otoacoustic emissions by interference tones above f2. I. Basic findings in rabbits. Hear Res 1999;136:105–23. [23] Wable J, Collet L, Chery-Croze S. Phase delay measurements of distortion product otoacoustic emissions at 2f1 f2 and 2f2 f1 in human ears. J Acoust Soc Am 1996;100:2228–35. [24] Gorga MP, Nelson K, Davis T, Dorn PA, Neely ST. Distortion product otoacoustic emission test performance when both 2f1 f2 and 2f2 f1 are used to predict auditory status. J Acoust Soc Am 2000;107:2128–35. [25] Lonsbury-Martin BL, Martin GK, Probst R, Coats AC. Acoustic distortion products in rabbit ear canal. I. Basic features and physiological vulnerability. Hear Res 1987;28:173–89. [26] Hall 3rd JW, Smith SD, Popelka GR. Newborn hearing screening with combined otoacoustic emissions and auditory brainstem responses. J Am Acad Audiol 2004;15:414–25. [27] Meier S, Narabayashi O, Probst R, Schmuziger N. Comparison of currently available devices designed for newborn hearing screening using automated auditory brainstem and/or otoacoustic emission measurements. Int J Pediatr Otorhinolaryngol 2004;68:927–34. [28] Korres S, Nikolopoulos TP, Komkotou V, et al. Newborn hearing screening: effectiveness, importance of high-risk factors, and characteristics of infants in the neonatal intensive care unit and well-baby nursery. Otol Neurotol 2005;26:1186–90. [29] Lin HC, Shu MT, Lee KS, et al. Comparison of hearing screening programs between one step with transient evoked otoacoustic emissions (TEOAE) and two steps with TEOAE and automated auditory brainstem response. Laryngoscope 2005;115:1957–62. [30] Johnson JL, White KR, Widen JE, et al. A multisite study to examine the efficacy of the otoacoustic emission/automated auditory brainstem response newborn hearing screening protocol: introduction and overview of the study. Am J Audiol 2005;14:S178–85. [31] Gravel JS, White KR, Johnson JL, et al. A multisite study to examine the efficacy of the otoacoustic emission/automated auditory brainstem response newborn hearing screening protocol:
624
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
recommendations for policy, practice, and research. Am J Audiol 2005;14:S217–28. [32] Ferber-Viart C, Duclaux R, Dubreuil C, Sevin F, Collet L, Berthier JC. Otoacoustic emissions and brainstem auditory evoked potentials in children with neurological afflictions. Brain Dev 1994;16:213–8. [33] Ochi A, Yasuhara A, Kobayashi Y. Comparison of distortion product otoacoustic emissions with auditory brain-stem response for clinical use in neonatal intensive care unit. Electroencephalogr Clin Neurophysiol 1998;108:577–83. [34] Kon K, Inagaki M, Kaga M, Sasaki M, Hanaoka S. Otoacoustic emission in patients with neurological disorders who have auditory brainstem response abnormality. Brain Dev 2000;22:327–35.
[35] Starr A, Picton TW, Sininger Y, Hood LJ, Berlin CI. Auditory neuropathy. Brain 1996;119(Pt. 3):741–53. [36] Kaga K, Nakamura M, Shinogami M, Tsuzuku T, Yamada K, Shindo M. Auditory nerve disease of both ears revealed by auditory brainstem responses, electrocochleography and otoacoustic emissions. Scand Audiol 1996;25:233–8. [37] Greene CC, McMillan PM, Barker SE, et al. DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24. Am J Hum Genet 2001;68:254–60. [38] Thirlwall AS, Brown DJ, McMillan PM, Barker SE, Lesperance MM. Phenotypic characterization of hereditary hearing impairment linked to DFNA25. Arch Otolaryngol Head Neck Surg 2003;129:830–5.
Characteristic findings of auditory brainstem response and otoacoustic emission in the Bronx waltzer mouse Masumi Inagaki
a,*
, Kaori Kon a, Seiko Suzuki a, Naoko Kobayashi a, Makiko Kaga a, Eiji Nanba b
a
b
Department of Developmental Disorders, National Institute of Mental Health, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawa Higashi, Kodaira 187-8553, Japan Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-machi, Yonago 683-8503, Japan Received 16 January 2006; received in revised form 1 April 2006; accepted 10 April 2006
Abstract Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were evaluated serially from 1 to 22 months in Bronx waltzer homozygotes (bv/bv), heterozygotes (+/bv) and control (+/+) mice, which were differentiated by means of PCR of marker DNA (D5Mit209). The wave IV threshold of the click-evoked ABR was higher than the DPOAE threshold with the DP growth method in each bv/bv, although the two thresholds were almost the same in the +/+ group. The DP value at 2f1 f2 in the bv/bv showed an apparent decrease at 2 to 3 months of age with 80 dB SPL stimulation using f2 frequency 7996 Hz and frequency ratio f2/f1 = 1.22, compared to control or heterozygote mice. It was characteristic that the 2f2 f1 DP signal-to-noise ratio (SNR) value was more preserved from 80 to 60 dB SPL than the 2f1 f2 DP value at f2 frequency 7996 Hz in most bv/bv, however, control mice showed almost the same levels of 2f1 f2 and 2f2 f1 SNR value at both f2 frequencies of 6006 and 7996 Hz. The preservation of a substantial 2f2 f1 DP suggested that it would be generated basal to the primary-tone place on the basilar membrane and there might be a reflection of the unique function of the remaining outer hair cells in the Bronx waltzer mice. These findings suggest that combination of ABR with DPOAE could offer useful information about differentiating the mechanism of hair cell dysfunction of the hereditary hearing impairment in the clinical fields. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Auditory brainstem response (ABR); Bronx waltzer mouse; Hair cell function; Otoacoustic emission (OAE)
1. Introduction The homozygous Bronx waltzer (bv/bv) mouse is known as a hearing-impairment model with selective inner hair and pillar cell damage in the cochlea
Abbreviations used: ABR, auditory brainstem response; OAE, otoacoustic emission; DPOAE, 2f1 f2 or 2f2 f1 distortion product otoacoustic emission; SNR, signal-to-noise ratio; PCR, polymerase chain reaction. * Corresponding author. E-mail address: [email protected] (M. Inagaki). 0387-7604/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2006.04.006
occurring just before birth [1–3]. Since about 70–80% of the inner hair cells (IHCs) degenerate and eventually die, moderate to severe hearing impairment follows [4]. In contrast, the outer hair cells (OHCs) in the cochlea are completely present but disarranged with folding of the lateral wall at the site of IHC degeneration [5,6]. Elevation of the threshold of both the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) has been reported separately in detail [7–9], which suggests the presence of hair cell dysfunction. A recent study has also revealed that an abnormal neuronal response pattern in the inferior colliculus of
618
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
the Bronx waltzer mouse, i.e., elevation of the threshold of the characteristic frequency and broad frequency tuning, is produced on an addition of OHC damage to IHC degeneration [6]. These findings suggest that the bv mutation affects both types of hair cells at morphological and functional levels to some extent at different stages. However, serial changes in each hair cell function in the Bronx waltzer mouse have not been well addressed. In this study, we compared click- and high frequency tone burst-evoked ABRs with DPOAEs by means of DP growth method in the mutant mice from 1 to 22 months of age. And we discussed whether the characteristic findings of the hair cell function in this kind of mutant mouse could provide or not new information about pathophysiology of hereditary hearing impairment in human adult or newborn hearing screening test.
2. Materials and methods 2.1. Animals Homozygous Bronx waltzer mice of both sexes have been bred at the National Institute of Mental Health, National Center of Neurology and Psychiatry (NCNP) in Japan since 1998. The breeding bv/bv pairs were obtained through the courtesy of Dr. H.M. Sobkowicz, Department of Neurology, University of Wisconsin, Madison, WI, USA, who had received the original mutant mice that had arisen at Albert Einstein College [10]. The mice were housed individually with a 12 h/ 12 h light dark cycle with food and water ad libitum in an EBAC-S breeding apparatus (CL-5351; CLEA-Japan, Co. Ltd., Japan), with which a constant temperature and humidity can be maintained. Tympanic membranes were examined prior to the experiment. All physiological procedures were performed under anesthesia using intraperitoneal injection of sodium pentobarbi-
tal (50 mg/kg body weight) supplemented as required during experiments. All experimental and euthanasia procedures conformed fully to the guidelines for animal care stipulated by the animal committee of the National Institute of Neuroscience, NCNP. Although bv/bv mutant mice were originally maintained in a CBA/J background [10], the details of the genetic background have been obscure in the colony [11,12]. So, one of the authors (EN) identified the genotype of each animal using a recently reported method [13]. Briefly, genomic DNA was prepared from the tail of each living animal or deep frozen samples by a standard technique. Mice were genotyped by PCR using the closest marker (D5Mit209) to the bv mutation. PCR was carried out under the following conditions: 35 cycles of 1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C, with final extension for 5 min at 72 °C. The sequences of the forward and reverse primers were TCTGAGCAA GGTCGTCCAC and CCCTGTCTCAAGATAAAA, respectively. PCR products of the homozygote gave two 359 bp bands, designated as bv/bv, and heterozygote gave two different bands (359 bp and 325 bp), typed as +/bv, on 3% agarose gel electrophoresis (Fig. 1). In the present study, physiological data from bv/bv, +/bv and +/+ animals (ddY and ICR strains), which were purchased from CLEA-Japan, were analyzed [14,15]. Moreover, a commercially available mouse strain, DBA/2J, which has been reported to show early onset progressive sensorineural hearing loss [16,17], was also used as positive control. 2.2. Auditory brainstem responses (ABRs) ABR examination was performed after anesthesia as above described in a quiet room. Needle electrodes were inserted subcutaneously in the retro auricular (active), vertex (reference) and nasial (ground) areas. Two kinds of sound stimuli were used; an alternate click sound of
Fig. 1. Identification of the genotype of the Bronx waltzer mice. There was a difference in the size of the polymerase chain reaction (PCR) product of marker DNA (D5Mit209) between homozygous (bv/bv) and heterozygous bv (bv/+). Homozygotes gave two 359 bp bands, and heterozygotes two different bands (359 bp and 325 bp) on 3% agarose gel electrophoresis. D5Mit209 is localized at 0.39 cm from the bv locus. M4: /X174/HaeIII.
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
0.1 ms duration for all mice, and tone bursts with a rise and fall time of 1 ms and a plateau of 1 ms of 8, 16, 24, or 32 kHz frequency for some mice. The click sound was presented monaurally through an ear tube masked with white noise for the contra-lateral ear canal. The click sound had substantial energy in the range of 2–8 kHz. A maximum sound pressure level (SPL) of 135 dB was employed for all stimuli. The tone bursts, which were delivered with a sound stimulator (DPS-725; Diamedical System Co. Ltd., Japan), were delivered at the rate of 10/s and started at the intensity of 100 dB SPL through a speaker (PT-RIII; Pioneer, Japan) situated 20 cm in front of the animal. Evoked potentials were band pass-filtered (100– 3000 Hz) by the preamplifier and 500 traces were averaged using an averager (MEB 9104; Nihon Kohden, Co. Ltd., Japan). Upward deflection was defined as positive polarity. Mice were kept warm during the experiment. The auditory threshold was taken as the minimum signal amplitude necessary to evoke clear potentials of wave II or IV. If no ABR waves could be obtained with click of an intensity of 135 dB SPL, a nominal threshold of 140 dB was assigned. Totals of 55 bv/bv and 55 +/+ mice in the different age group were examined. 2.3. Otoacoustic emission (OAE) OAE measurements were conducted with a DP Echoport of ILO 92 system (Otodynamics, Co., Ltd., UK) just after the ABR examination. The DP growth method was applied to each animal using a standard probe. The DPOAE stimuli consisted of two primary frequencies, f1 and f2. Two equilevel (L1 = L2) primary signals at 7996 or 6006 Hz (f2) and 6555 or 4919 Hz (f1) with a fixed f2/f1 ratio of 1.22 were presented. At least 32 consecutive responses were averaged. Threshold measures in the form of input/output functions were obtained by decreasing the primary tone from 80 to 45 dB SPL in 5-dB steps. Detection of DPOAE thresholds at 2f1 f2 was defined as the intensity of the primaries that elicited a DPOAE whose amplitude was 2 dB greater than the noise level. Also, in elder mice, DPOAE levels of more than +2 SD above the noise floor were considered valid. The 2f2 f1 as well as 2f1 f2 DPOAE amplitude minus noise level (S/N value) at each stimulus sound level in four mouse-groups (bv/bv, +/bv, +/+ and DBA/2J) mouse were also calculated at the age of 2–5 months. 2.4. Statistical analysis Statistical evaluation was performed among mouse groups using analysis of variance (ANOVA) and Fisher’s Protected Least Significant Difference (PLSD) for post hoc comparison with a commercially available soft-
619
ware package (StatView ver. 4.5; Abacus Co. Ltd., USA). Statistical significance was accepted when the p value was less than 0.05.
3. Results 3.1. ABR Control mice showed an ABR wave with 4 or 5 positive peaks in response to the click sound. The basic waveform comprised high amplitude waves I and II, and lower amplitude wave IV. The ABR waveform in response to the tone bursts was similar to that to the click sound stimuli. The mean (±SD) thresholds of wave IV for young control mice were 43 (±5), 22 (±19), 20 (±6), 22 (±15) and 38 (±16) dB SPL for the click sound, 8, 16, 24 and 32 kHz tone burst stimuli, respectively (Fig. 2). Heterozygous mice had almost the same level of the IV threshold as control. However, homozygous Bronx waltzer mice showed significant (p < 0.001) elevation of the click sound threshold value at a young age (2 to 3 months of age). DBA/2J mice (n = 10) at the age of 2–5 months also showed apparent elevation of IV threshold (mean ± SD; 98 ± 18 dB SPL) for the click sound. In bv/bv, threshold of tone bursts was also elevated equally by around 60 dB from the control value (p < 0.001). There was little change in the average threshold of click-evoked ABR for 2 years of age. The latencies of waves I, II and IV are shown in Table 1. Wave latencies were not significantly prolonged in the young Bronx waltzer mice compared with the controls. Elder homozygotes showed similar findings as to the latency of each wave without significant prolongation.
Fig. 2. ABR threshold of Bronx waltzer and control mice. The ABR thresholds (circles) of the mutants (n = 6) in response to the click sound and higher frequency tone bursts, i.e., 8000, 16,000, 24,000 and 32,000 Hz, were in the range of 80–110 dB SPL at 2 to 3 months of age. The mean thresholds (triangles) in the control mice (n = 5) with the tone bursts (8000, 16,000 and 24,000 Hz) were equally at around 20 dB SPL. Bars represent 1 SD.
620
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
Table 1 Wave latencies of click-evoked ABR in control and mutant mice a
Control (ddY) bv/bva bv/bvb
n
Peak latency of wave I
Peak latency of wave II
Peak latency of wave IV
I–IV Interpeak latency
11 17 6
1.11 (0.09) 1.15 (0.13) 1.06 (0.21)
1.93 (0.17) 2.03 (0.14) 1.94 (0.13)
3.61 (0.25) 3.81 (0.35) 3.63 (0.28)
2.50 (0.18) 2.60 (0.60) 2.58 (0.23)
Data (ms) are shown as means (SD) at the intensity of 135 dB SPL. a Age range: 1–3 months of age. b Age range: 12–13 months of age.
3.2. DPOAE Data were collected in the form of DPOAE input/ output (I/O) functions. Although the noise amplitudes at the f2 frequency of 7996 Hz were less than 5 dB SPL on average, those at 6006 Hz were relatively high. At 2 to 3 months of age, the DP value at 2f1 f2 in response to 80 dB SPL stimuli (f2 frequency = 7996 Hz) in the homozygous mutants (n = 17) showed an apparent decrease (from 5.1 to 12.0 dB) compared to the control (n = 15) value (18.8–30.3 dB), which were at the same levels until about 2 years old (Fig. 3). The DP levels at 2f1 f2 did not exceed the noise floor in more than half the bv/bv group after 6 months old. Distribution of signal minus noise value (S/N value) of 2f1 f2 DPOAE at the stimulus intensity of 75 dB SPL was similar in the ddY group ( 10 to 35 dB SPL) as in the heterozygous bv group (5–38 dB SPL). On the other hand, the pattern of 2f2 f1 DP value (S/N value) at f2 frequencies of 7996 Hz was different among mouse strain examined. In the homozygous group, 2f2 f1 S/N value was preserved with a decrease of 2f1 f2 value especially in the 65–80 dB SPL stimuli, (Fig. 4a). Control mice such as ddY and heterozygous mice showed almost the same DP value of 2f2 f1 and 2f1 f2 (Figs. 4b and c), although both had a reversal
pattern showing 2f2 f1 value P2f1 f2 value (2f2 f1 dominant pattern) only at the stimulus intensity of 80 dB SPL. There was no statistically significant difference in the 2f2 f1 value between the homozygous mutants (22.3 ± 13.7 dB SPL at the stimulus intensity of 75 dB SPL) and control (ddY and heterozygotes) ears (18.0 ± 10.9 dB SPL and 23.6 ± 18.8, respectively) at the f2 frequency of 7996 Hz. DBA mice of which ABR threshold was elevated from 80–110 dB SPL also had reduced level of both 2f1 f2 and 2f2 f1 DP value at every stimulus intensity of 80–45 dB SPL. At f2 frequencies of 6006 Hz, both 2f2 f1 and 2f1 f2 value in the homozygous bv group reduced more than control group (Fig. 5). There was neither 2f2 f1 nor 2f1 f2 DP in DBA mice at f2 frequencies of 6006 Hz at stimulus intensity of 80 dB SPL. Scatter plots of the ABR and DPOAE threshold values in the same cases at the age of 2 to 3 months showed that the wave IV threshold of click-evoked ABRs was higher than the DP threshold in each bv/bv (mean thresholds; ABR = 110 dB SPL, DPOAE = 75 dB SPL), although the two thresholds were almost the same value (mean threshold; ABR = 60 dB SPL, DPOAE = 55 dB SPL) in the control group (Fig. 6).
4. Discussion
Fig. 3. Changes in the 2f1 f2 DP level in control and mutant ears. There was a significant decrease in the DP level at the f2 frequency of 7996 Hz in the Bronx waltzer mice (circles) as compared to in the control animals (squares) from 2 to 22 months of age. All data were recorded through the left ear with the intensity of 80 dB SPL.
Mice can hear over a range of frequencies between 500 and 120,000 Hz, however, normal mice are known to be most sensitive to the sound with frequencies of 12–24 kHz [18]. The present results as to the threshold pattern of ABR in response to tone bursts in control mice are consistent with these findings [19]. The Bronx waltzer homozygotes showed little frequency-dependent sensitivity, i.e., about 60 dB elevation at any frequency, which is also consistent with a previous report [7] demonstrating threshold elevation of the ABR without frequency dependence and a median threshold value of around 90 dB SPL. The ABR seems to exhibit little progress in bv/bv. The discrepancy between the ABR and OAE thresholds and selective atrophy of auditory system may support the idea that morphological and functional degeneration in the IHCs is the primary damage in this type of mutation [20].
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
621
Fig. 4. Signal-to-noise ratio of DP at a frequency of 7996 Hz. Mean and standard deviation values of signal-to-noise ratio (SNR) of 2f1 f2 (triangles) and 2f2 f1 (circles) DPs at an f2 frequency of 7996 Hz with stimulus intensity from 80 to 45 dB SPL were plotted in the figures. Homozygous Bronx waltzer mice (a) showed a relatively preserved SNR of 2f2 f1 at an f2 frequency of 7996 Hz as compared to the values of heterozygous bv (b) and control groups (c). DBA/2J had decreased levels of both 2f1 f2 and 2f2 f1 SNR (d).
Fig. 5. Signal-to-noise ratio of DP at a frequency of 6555 Hz. Mean and SD values of signal-to-noise ratio (SNR) of 2f1 f2 (triangles) and 2f2 f1 (circles) at an f2 frequency of 6555 Hz with stimulus intensity from 80 to 45 dB SPL were plotted. Although homozygous bv showed 2f2 f1 P 2f1 f2 pattern at stimulus intensity of 75 and 80 dB SPL (a), there were no remarkable changes between 2f1 f2 (triangles) and 2f2 f1 (circles) DPs among three mouse groups (+/bv, (b); +/+, (c); DBA/2J, (d)).
622
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
Fig. 6. Scatter plots of the ABR and OAE threshold values in the same cases in different mouse groups. The wave IV threshold of click-evoked ABR was higher than the 2f1 f2 DP threshold in each bv/bv, although the two thresholds were almost the same in the +/+ group. Circles, bv/bv mice (n = 30); triangles; control (ddY) mice (n = 30).
On the other hand, the decreased 2f1 f2 DP level at an f2 frequency of 9 to 10 kHz or reduced amplitude of the cochlear microphonics in bv/bv is thought to be due to outer hair cell (OHC) dysfunction [9,21]. In this study, DPOAEs were collected primarily at an f2 frequency of 7996 and 6006 Hz. These test ranges just begin to describe hearing capability of the mouse. In mice with age-related hearing loss these frequencies are not affected until severe progression of OHC loss has occurred. Consequently, with the present protocol it would be impossible to demonstrate serial changes exactly that typically begin at much higher frequencies. However, a recent report on abnormal ‘frequency tuning curves’ in the inferior colliculus indicates alteration of the OHC function in bv/bv in wide range of frequency from 4 to 70 kHz [6]. It was characteristic that the 2f2 f1 DP value was higher than the 2f1 f2 DP value at the f2 frequency of 7996 Hz in most bv/bv for 1 year. The fact that the 2f2 f1 DP level in bv/bv was almost the same as that in control suggests the preserved function of the region of 2f2 f1 DP generation. Moreover, the signal-to-noise ratios (SNRs) at 2f2 f1 were more than those at 2f1 f2 at lower stimulus intensity in bv/bv. And DBA/2J mouse exhibited a diminished level of both 2f1 f2 and 2f2 f1 SNR at any intensity. Anesthetized rodents such as guinea pigs and mice are known to show a 2f1 f2 > 2f2 f1 DP amplitude pattern with geometric frequencies at 5600 and 8000 Hz in general [22]. The amplitudes for 2f2 f1 were known to be lower than for 2f1 f2 also in human subjects with normal hearing [23]. The SNRs are sometimes larger at 2f2 f1 compared to 2f1 f2, however, for f2 frequency higher than 1 kHz the SNR is reported to be greater at
2f1 f2 [24]. So, it is speculated that some OHCs are active but that less organized OHCs may lead to different action in frequency tuning in the present Bronx waltzer mice. A reduction in the level of 2f1 f2 with an increase in 2f2 f1 DPOAE has been observed in rabbits following brief exposure to excessive sound at frequencies higher than f2 [22]. These phenomena are similar to the present findings for the Bronx waltzer mouse and might be related to physiological vulnerability of the cochlear regions basal to f2. Since it is hypothesized that 2f2 f1 DP is generated basal to the primary-tone (f2) place on the basilar membrane [25], OHCs in such areas might excessively vibrate the basilar membrane with specific frequency tones in young Bronx waltzer mice. Or there could be a functional elimination of the secondary emission source basal to f2, which is in addition to the 2f1 f2 and cancels the 2f2 f1 in the mutant cochlea. These results suggest that hair cell function, especially in OHCs, could fluctuate in the adult period. Further analyses of the function of the OHCs in the organ of Corti, which is not affected directly by the bv mutation, with mechanical nonlinearity in the external canal are necessary to elucidate the mechanism of suppression or enhancement of various types of DPOAE. In newborn hearing screening program, transient evoked OAE (TEOAE) and/or automated ABR (AABR) have been adopted in several countries [26– 28]. It is easier to connect the subject to the device and measurements were quicker for OAE than for AABR. However, AABR is highly reliable, and has an advantage that both ears can be tested simultaneously. It is well known that TEOAE measurement is easy to administer and leads to the real time assessment. Some investigators have pointed out that use of a 2-step screening is better than one step screening using TEOAE in terms of the efficacy and determination of human permanent hearing loss, though it is still controversial [29–31]. On the other hand, DPOAE is superior to TEOAE in determination of frequency specific function in the inner ear. And DPOAE has also been prevailing examination for hearing evaluation in children and useful for detecting the cochlear function of pediatric patients with neurological disorders [32–34]. Detailed examination of both OAE and ABR is necessary to diagnose a noticeable clinical entity such as auditory neuropathy or auditory nerve disease [35,36]. Present findings in the Bronx waltzer mice suggest the frequency specific dysfunction of the both hair cells of the organ of Corti in the hereditary hearing disorders. Therefore, combination of the two measurements, i.e., ABR and DPOAE, might be suitable for evaluating the heritable hearing disorder in early infancy as well as in the newborn period. Moreover, a large family of Czech descent with slowly progressive hearing loss has been reported to have a similar genetic background to the Bronx waltzer [2,37].
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
So, age related changes of both 2f1 f2 and 2f2 f1 DP values would be a diagnostic clue for the specific type of the nonsyndromic hereditary hearing impairment with bv mutation or DFNA25 locus [37,38] in adulthood.
Acknowledgements This study was presented in part at the IERASG meeting held in the Canary Islands, Spain, in June 2003. The authors would like to express their deep thanks to Ms. R. Honma, Ms. Y. Tamura, Ms. R. Ohta and Ms. N. Endo for their technical assistance, and to Prof. K. Kaga (Department of Otolaryngology, University of the Tokyo) for critical reading of the manuscript. And authors wish to thank Prof. H.M. Sobkowicz, University of Wisconsin, for her warm encouragement. This study was supported in part by the Health and Labour Sciences Research Grants (H12-Sensory-006; H16-Kokoro-001) for Research on Psychiatric and Neurological Diseases and Mental Health from the Ministry of Health, Labour and Welfare in Japan.
References [1] Whitlon DS, Gabel C, Zhang X. Cochlear inner hair cells exist transiently in the fetal Bronx Waltzer (bv/bv) mouse. J Comp Neurol 1996;364:515–22. [2] Bussoli TJ, Kelly A, Steel KP. Localization of the bronx waltzer (bv) deafness gene to mouse chromosome 5. Mamm Genome 1997;8:714–7. [3] Tucker JB, Mackie JB, Bussoli TJ, Steel KP. Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: response to loss of cell partners in the Bronx waltzer mouse. J Neurocytol 1999;28:1017–34. [4] Deol MS. The inner ear in bronx waltzer mice. Acta Otolaryngol 1981;92:331–6. [5] Kong WJ, Scholtz AW, Hussl B, Kammen-Jolly K, SchrottFischer A. Localization of efferent neurotransmitters in the inner ear of the homozygous Bronx waltzer mutant mouse. Hear Res 2002;167:136–55. [6] Sterbing SJ, Schrott-Fischer A. Neuronal responses in the inferior colliculus of mutant mice (Bronx waltzer) with hereditary inner hair cell loss. Hear Res 2003;177:91–9. [7] Schrott A, Stephan K, Spoendlin H. Hearing with selective inner hair cell loss. Hear Res 1989;40:213–9. [8] Schrott A, Puel JL, Rebillard G. Cochlear origin of 2f1 f2 distortion products assessed by using 2 types of mutant mice. Hear Res 1991;52:245–53. [9] Horner KC, Lenoir M, Bock GR. Distortion product otoacoustic emissions in hearing-impaired mutant mice. J Acoust Soc Am 1985;78:1603–11. [10] Deol MS, Gluecksohn-Waelsch S. The role of inner hair cells in hearing. Nature 1979;278:250–2. [11] Sobkowicz HM, Inagaki M, August BK, Slapnick SM. Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse. J Neurocytol 1999;28:17–38. [12] Sobkowicz HM, August BK, Slapnick SM. Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture. Int J Dev Neurosci 2002;20:537–54.
623
[13] Cheong MA, Steel KP. Early development and degeneration of vestibular hair cells in bronx waltzer mutant mice. Hear Res 2002;164:179–89. [14] Dabdoub A, Donohue MJ, Brennan A, Wolf V, Montcouquiol M, Sassoon DA, et al. Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea. Development 2003;130:2375–84. [15] Miura H, Qiao H, Kitagami T, Ohta T. Fluvoxamine, a selective serotonin reuptake inhibitor, suppresses tetrahydrobiopterin in the mouse hippocampus. Neuropharmacology 2004;46:340–8. [16] Willott JF, Erway LC, Archer JR, Harrison DE. Genetics of agerelated hearing loss in mice. II. Strain differences and effects of caloric restriction on cochlear pathology and evoked response thresholds. Hear Res 1995;88:143–55. [17] Turner JG, Willott JF. Exposure to an augmented acoustic environment alters auditory function in hearing-impaired DBA/ 2J mice. Hear Res 1998;118:101–13. [18] Ehret G. Psychoacoustics. In: Willott JF, editor. Auditory psychobiology of the mouse. Springfield, IL: Charles C Thomas; 1983. p. 13–53. [19] Zheng QY, Johnson KR, Erway LC. Assessment of hearing in 80 inbred strains of mice by ABR threshold analysis. Hear Res 1999;130:94–107. [20] Keithley EM, Feldman ML. The spiral ganglion and hair cells of Bronx waltzer mice. Hear Res 1983;12:381–91. [21] Bock GR, Yates GK. Cochlear electrophysiology in the bronx waltzer mutant mouse. J Physiol (Proceedings of the Physiological Society) 1982;332:P20–1. [22] Martin GK, Stagner BB, Jassir D, Telischi FF, Lonsbury-Martin BL. Suppression and enhancement of distortion-product otoacoustic emissions by interference tones above f2. I. Basic findings in rabbits. Hear Res 1999;136:105–23. [23] Wable J, Collet L, Chery-Croze S. Phase delay measurements of distortion product otoacoustic emissions at 2f1 f2 and 2f2 f1 in human ears. J Acoust Soc Am 1996;100:2228–35. [24] Gorga MP, Nelson K, Davis T, Dorn PA, Neely ST. Distortion product otoacoustic emission test performance when both 2f1 f2 and 2f2 f1 are used to predict auditory status. J Acoust Soc Am 2000;107:2128–35. [25] Lonsbury-Martin BL, Martin GK, Probst R, Coats AC. Acoustic distortion products in rabbit ear canal. I. Basic features and physiological vulnerability. Hear Res 1987;28:173–89. [26] Hall 3rd JW, Smith SD, Popelka GR. Newborn hearing screening with combined otoacoustic emissions and auditory brainstem responses. J Am Acad Audiol 2004;15:414–25. [27] Meier S, Narabayashi O, Probst R, Schmuziger N. Comparison of currently available devices designed for newborn hearing screening using automated auditory brainstem and/or otoacoustic emission measurements. Int J Pediatr Otorhinolaryngol 2004;68:927–34. [28] Korres S, Nikolopoulos TP, Komkotou V, et al. Newborn hearing screening: effectiveness, importance of high-risk factors, and characteristics of infants in the neonatal intensive care unit and well-baby nursery. Otol Neurotol 2005;26:1186–90. [29] Lin HC, Shu MT, Lee KS, et al. Comparison of hearing screening programs between one step with transient evoked otoacoustic emissions (TEOAE) and two steps with TEOAE and automated auditory brainstem response. Laryngoscope 2005;115:1957–62. [30] Johnson JL, White KR, Widen JE, et al. A multisite study to examine the efficacy of the otoacoustic emission/automated auditory brainstem response newborn hearing screening protocol: introduction and overview of the study. Am J Audiol 2005;14:S178–85. [31] Gravel JS, White KR, Johnson JL, et al. A multisite study to examine the efficacy of the otoacoustic emission/automated auditory brainstem response newborn hearing screening protocol:
624
M. Inagaki et al. / Brain & Development 28 (2006) 617–624
recommendations for policy, practice, and research. Am J Audiol 2005;14:S217–28. [32] Ferber-Viart C, Duclaux R, Dubreuil C, Sevin F, Collet L, Berthier JC. Otoacoustic emissions and brainstem auditory evoked potentials in children with neurological afflictions. Brain Dev 1994;16:213–8. [33] Ochi A, Yasuhara A, Kobayashi Y. Comparison of distortion product otoacoustic emissions with auditory brain-stem response for clinical use in neonatal intensive care unit. Electroencephalogr Clin Neurophysiol 1998;108:577–83. [34] Kon K, Inagaki M, Kaga M, Sasaki M, Hanaoka S. Otoacoustic emission in patients with neurological disorders who have auditory brainstem response abnormality. Brain Dev 2000;22:327–35.
[35] Starr A, Picton TW, Sininger Y, Hood LJ, Berlin CI. Auditory neuropathy. Brain 1996;119(Pt. 3):741–53. [36] Kaga K, Nakamura M, Shinogami M, Tsuzuku T, Yamada K, Shindo M. Auditory nerve disease of both ears revealed by auditory brainstem responses, electrocochleography and otoacoustic emissions. Scand Audiol 1996;25:233–8. [37] Greene CC, McMillan PM, Barker SE, et al. DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24. Am J Hum Genet 2001;68:254–60. [38] Thirlwall AS, Brown DJ, McMillan PM, Barker SE, Lesperance MM. Phenotypic characterization of hereditary hearing impairment linked to DFNA25. Arch Otolaryngol Head Neck Surg 2003;129:830–5.