3.1. Inbred strains of mice with normal ABR thresholds
As a reference for normal hearing, we repeatedly tested CBA/CaJ mice at four ages from 9 to 39 weeks (, ). The mean thresholds for CBA/CaJ mice were 36, 24, 15, and 39 dB SPL for the click, 8 kHz, 16 kHz, and 32 kHz stimuli, respectively. We then defined as hearing impaired those inbred strains that exhibited average ABR thresholds at least 15 dB SPL above these mean values, for any of the four acoustic stimuli at any age of testing. An increase of 15 dB is at least three standard deviations above the CBA/CaJ mean values for all four test stimuli () and is, therefore, a conservative criterion for significance. Thus, we considered any strain with average ABR threshold values above 55 (for click stimulus), 40 (for 8 kHz), 35 (for 16 kHz), or 60 (for 32 kHz) dB SPL to be hearing impaired. Strains with average ABR values below these values for all four test stimuli were considered normal hearing.
Fig. 1 Determination of ABR thresholds. Normal ABR patterns for a 32 week old CBA/CaJ mouse (A) induced by click stimuli of 65, 55, 45, 35 and 30 dB SPL. The ABR threshold for this mouse was estimated to be 35 dB SPL. (There are usually 4 or 5 response peaks, (more ...)
ABR thresholds (dB SPL), means (shown in boldface) and associated standard deviations (S.D.) of standard strain CBA/CaJ
Inbred strains with normal hearing are arranged alphabetically by their strain designations in . The mice tested were composed of two major age groups, one about 4–12 weeks of age, the other about 25–33 weeks of age. If the old retired breeders (25–33 weeks of age) did not exhibit elevated ABR thresholds, we assumed that they had normal hearing at younger ages. Mice from some strains were tested at more than one age. Arithmetic means and standard deviations were computed for each strain/age group.
Inbred mouse strains with normal ABR thresholds (dB SPL). A total of 430 mice from 60 strains or substrains were tested
In , we report results for 430 normal hearing mice from 60 different inbred strains, thus establishing a reliable reference for normal ABR thresholds. Despite the extensive genetic variability among inbred strains, as well as the variability in ages (7–39 weeks) of the mice tested, the grand means and standard deviations (in dB SPL) of the normal hearing mice were remarkably uniform for each tested stimulus (, bottom line): click (38 ± 2.7) and tone bursts of 8 kHz (29 ± 3.4), 16 kHz (18 ± 4.2), and 32 kHz (44 ± 3.2). These grand means were very similar to those obtained from the control CBA/CaJ strain () and may serve as references for comparing other strains of mice judged to exhibit a significant elevation of ABR thresholds.
To be certain that the grand means did not obscure some age-related differences, the normal hearing mice were divided into two groups according to their age when tested: 196 were tested at 7–14 weeks of age, and 223 were tested at 16–39 weeks of age (). No significant differences in ABR thresholds were observed between these two different age groups (P > 0.05). For the mice tested at 7–14 weeks, the mean ABR thresholds were 38 (click), 28 (8 kHz), 17 (16 kHz) and 43 (32 kHz) dB SPL; for the mice tested at 16–39 weeks, the average values were 39, 30, 18, and 44, respectively. Likewise, no significant gender differences in thresholds were observed between female and male mice (P > 0.05). For female mice, the mean ABR thresholds were 38 (click), 29 (8 kHz), 18 (16 kHz), and 44 (32 kHz) dB SPL; for male mice, the average values were 40, 30, 19, and 44, respectively.
3.2. Inbred strains of mice with elevated ABR thresholds
All strains with mean ABR threshold values above 55 (click), 40 (8 kHz), 35 (16 kHz), or 60 (32 kHz) dB SPL were considered hearing impaired and are presented in . For reference, the grand means for ABR thresholds of normal hearing mouse strains are listed at the top of . Nineteen strains or substrains of mice exhibited elevated ABR thresholds before 13 weeks of age, for either one, two or all of the four stimuli (). Sixteen strains with normal hearing when tested before 13 weeks of age exhibited hearing loss when tested at older ages. These 16 strains with late-onset hearing loss are denoted by asterisks in and results for older, hearing-impaired mice from these strains are presented in .
Inbred strains with elevated ABR thresholds (dB SPL). A total of 326 mice from 19 strains with early onset hearing loss and 82 mice from 16 strains with late onset hearing loss were tested
Severity and time of onset of hearing impairment varied among strains (). To classify strains according to the severity of their hearing loss, we define here three levels of impairment as measured by ABR thresholds: 20–40 dB SPL above normal means = mild impairment, 41–60 dB above normal means = intermediate impairment, and greater than 60 dB above normal means = severe impairment. With these criteria, strains 129/J, 129/ReJ, 129/SvJ, C57BR/cdJ, C57/J, DBA/2HaSmn, I/LnJ, and MA/MyJ have mild hearing impairment; strains A/J, DBA/2J, and SKH2/J intermediate impairment; and strains BUB/BnJ, NOR/LtJ, and NOD/LtJ severe impairment before 13 weeks of age.
Fig. 2 ABR audiometric profiles for inbred strains with significant hearing impairment. For each strain and age of mice tested, ABR threshold values (in dB SPL) are plotted against the auditory stimulus frequency (in kHz). The normal hearing CBA/CaJ control (more ...)
ABR thresholds increased with age for all of the hearing impaired inbred strains (; ). For example, the A/J strain () exhibits a mild impairment at 8–10 weeks, an intermediate impairment at 13–30 weeks, and a severe impairment at 40 weeks of age. Although the NOD/LtJ () strain already exhibits intermediate hearing impairment at 3 weeks, it further progresses to severe hearing loss by 9 weeks of age.
For some strains, ABR thresholds varied significantly depending on the stimulus frequency. Different strains exhibited different frequency sensitivities. For example, the A/J strain () is most vulnerable to hearing loss at 16 kHz, whereas the C57BR/cdJ () and C57L/J () strains are least vulnerable to loss at 16 kHz.
Among 326 hearing impaired mice (), as was the case with normal hearing mice, there were no statistically significant gender differences in ABR thresholds (P > 0.05). For female mice, the average ABR thresholds were 71 (click), 65 (8 kHz), 61 (16 kHz), and 82 (32 kHz) dB SPL; for male mice, the average values were 74, 68, 64, and 86, respectively.
3.3. Allelism tests
Different inbred strains identified with elevated ABR thresholds may share some of the same underlying genes for hearing loss because of common ancestry. For recessive genes, allelic complementation can be tested by examining F1 hybrids for hearing loss. If hearing loss is due to the same gene loci in both strains tested, then the F1 hybrids inheriting these non-complementing recessive alleles will exhibit hearing loss. If the hearing loss genes are different between the strains, they will complement each other and the F1 hybrids will hear normally.
All F1 hybrids between the good hearing strain CAST/Ei and strains identified with hearing impairment (including 129/ReJ, A/J, ALR/J, BUB/BnJ, C57BR/cdJ, C57L/J, DBA/2J, and NOD/LtJ) exhibited normal hearing, even at advanced ages, indicating that the genes contributing to hearing loss are recessive (data not shown). To see if the inbred strains 129/ReJ, A/J, and NOD.NON-H2nb1 share any hearing loss genes with DBA/2J, we generated F1 hybrids and measured their ABR thresholds. At 3 months of age, both the 129/ReJ and A/J strains appear to complement the DBA/2J strain (the F1 hybrids have normal ABR thresholds; ), suggesting that these two strains do not share all of their hearing loss genes with DBA/ 2J. At 29 weeks of age, however, (DBA/2JUA/J) F1 hybrids do exhibit significant hearing loss (), suggesting that these two strains share at least one gene contributing to hearing loss. The elevated ABR thresholds of (NOD.NON-H2nb1×DBA/2J) and (NOD.NON-H2nb1×ALR/LtJ) F1 hybrids, even by 13 weeks of age (), indicate that these three strains probably share all of the same hearing loss genes.
Allelism tests for recessive hearing loss genes