Lawrence C. Erway

Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses

The common occurrence of hearing loss in both humans and mice, and the anatomical and functional similarities of their inner ears, attest to the potential of mice being used as models to study inherited hearing loss. A large-scale, auditory screening project is being undertaken at The Jackson Laboratory (TJL) to identify mice with inherited hearing disorders. To assess hearing sensitivity, at least five mice from each inbred strain had auditory brainstem response (ABR) thresholds determined. Thus far, we have screened 80 inbred strains of mice; 60 of them exhibited homogeneous ABR threshold values not significantly different from those of the control strain CBA/CaJ. This large database establishes a reliable reference for normal hearing mouse strains. The following 16 inbred strains exhibited significantly elevated ABR thresholds before the age of 3 months: 129/J, 129/ReJ, 129/SvJ, A/J, ALR/LtJ, ALS/LtJ, BUB/BnJ, C57BLKS/J, C57BR/cdJ, C57L/J, DBA/2J, I/LnJ, MA/MyJ, NOD/LtJ, NOR/LtJ, and SKH2/J. These hearing impaired strains may serve as models for some forms of human non-syndromic hearing loss and aid in the identification of the underlying genes.

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl− and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K+ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1−/− ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl− channel, were reduced in jejunum, cecum, and trachea of Nkcc1−/− mice, indicating that NKCC1 contributes to cAMP-induced Cl− secretion. In contrast, secretion of gastric acid in adult Nkcc1−/− stomachs and enterotoxin-stimulated fluid secretion in the intestine of sucklingNkcc1−/− mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K+ movements involved in generation of the K+-rich endolymph and the endocochlear potential.

Balance and Hearing Deficits in Mice with a Null Mutation in the Gene Encoding Plasma Membrane Ca2+-ATPase Isoform 2

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

A major gene affecting age-related hearing loss in C57BL/6J mice

A major gene responsible for age-related hearing loss (AHL) in C57BL/6J mice was mapped by analyses of a (C57BL/6J x CAST/Ei) x C57BL/6J backcross. AHL, as measured by elevated auditory-evoked brainstem response (ABR) thresholds, segregated among backcross mice as expected for a recessive, primarily single-gene trait. Both qualitative and quantitative linkage analyses gave the same genetic map position for the AHL gene (Ahl on chromosome 10, near D10Mit5. Marker assisted selection was then used to produce congenic lines of C57BL/6J that contain different CAST-derived segments of chromosome 10. ABR test results and cochlear histopathology of aged progenitors of these congenic lines are presented. Ahl is the first gene causing late-onset, non-syndromic hearing loss that has been reported in the mouse.

Genetics of age-related hearing loss in mice. III. Susceptibility of inbred and F1 hybrid strains to noise-induced hearing loss

Some humans and mice are genetically predisposed to age-related hearing loss (AHL), and others are variously susceptible to noise-induced hearing loss (NIHL). The inbred C57BL/6J (B6) mice exhibit AHL at an early age, whereas the inbred CBA/CaJ (CB) mice do not. The B6 mice are much more susceptible to NIHL than are the CB mice (Shone et al., 1991; Li, 1992a). The B6 mice possess an Ahl gene which maps to chromosome 10 (Erway et al., 1995). This study was designed, using these two inbred strains plus two F1 hybrid strains of mice, to begin to test the hypothesis that the Ahl genotypes may influence the susceptibility to NIHL. These strains of mice (with putative genotypes) are: inbred CB (+/+) and B6 (Ahl/Ahl); hybrid CBB6F1 (+/Ahl) and B6D2F1 (Ahl/Ahl; D2 represents inbred DBA/2J). Twenty-four mice of each of these four strains were exposed to noise (110 dB for 0, 1 or 2 h) and tested for auditory-evoked brainstem response (ABR) thresholds. The CB and CBB6F1 strains of mice did not differ significantly from each other, exhibiting mostly temporary threshold shifts. The B6 and B6D2F1 strains of mice did not differ significantly from each other, but did exhibit permanent threshold shifts. These results support the hypothesis that genetic predisposition to AHL may be revealed at a younger age by NIHL. This suggests that it may be possible to use the NIHL to distinguish segregating genotypes (+/Ahl vs. Ahl/Ahl) among backcross progeny and thereby to identify and map single genes for AHL.

Genetic basis for susceptibility to noise-induced hearing loss in mice

The C57BL/6J (B6) and DBA/2J (D2) inbred strains of mice exhibit an age-related hearing loss (AHL) due to a recessive gene (Ahl) that maps to Chromosome 10. The Ahl gene is also implicated in the susceptibility to noise-induced hearing loss (NIHL). The B6 mice (Ahl/Ahl) are more susceptible to NIHL than the CBA/CaJ (CB) mice (+(Ahl)). The B6xD2.F(1) hybrid mice (Ahl/Ahl) are more susceptible to NIHL than the CBxB6.F(1) mice (+/Ahl) [Erway et al., 1996. Hear. Res. 93, 181-187]. These genetic effects implicate the Ahl gene as contributing to NIHL susceptibility. The present study demonstrates segregation for the putative Ahl gene and mapping of such a gene to Chromosome 10, consistent with other independent mapping of Ahl for AHL in 10 strains of mice [Johnson et al., 2000. Genomics 70, 171-180]. The present study was based on a conventional cross between two inbred strains, CBxB6.F(1) backcrossed to B6 with segregation for the putative +/Ahl:Ahl/Ahl. These backcross progeny were exposed to 110 dB SPL noise for 8 h. All of the progeny were tested for auditory evoked brainstem responses and analyzed for any significant permanent threshold shift of NIHL. Cluster analyses were used to distinguish the two putative genotypes, the least affected with NIHL (+/Ahl) and most affected with PTS (Ahl/Ahl). Approximately 1/2 of the backcross progeny exhibited PTS, particularly at 16 kHz. These mice were genotyped for two D10Mit markers. Quantitative trait loci analyses (log of the odds=15) indicated association of the genetic factor within a few centiMorgan of the best evidence for Ahl [Johnson et al., 2000. Genomics 70, 171-180]. All of the available evidence supports a role for the Ahl gene in both AHL and NIHL among these strains of mice.

Genetics of age-related hearing loss in mice. II: Strain differences and effects of caloric restriction on cochlear pathology and evoked response thresholds

The effects of genotype and diet on age-related hearing loss were evaluated using auditory brainstem response (ABR) thresholds and post-mortem cochlear histopathology in 5 inbred mouse strains, CBA/H-T6J (CH), DBA/2J (D2), C57BL/6J (B6), BALB/cByJ (BY) and WB/ReJ (WB), and their 10 F1 hybrid strains. The mice had been maintained since weaning on either a high-energy (HE) control diet or low-energy (LE) calorically restricted diet. ABR thresholds were obtained when the mice were 23 months old; the mice were allowed to age until they died from natural causes prior to obtaining the histological material. The severity of post-mortem cochlear pathology in mice maintained with the HE diet supports our earlier genetic model which postulated that B6, BY, and WB strains each possessed a different recessive allele causing age-related hearing loss, D2 mice possessed all 3 genes, and CH mice possessed none. The histopathology indicates that the genes act at the cochlear level. Dietary restriction resulted in increased longevity in a number of strains, but age-related changes in cochlear pathology were not ameliorated in any of these; indeed, in some strains long-lived LE mice exhibited severe cochlear degeneration. In strains for which longevity was not extended by caloric restriction, only B6 mice exhibited an ameliorative effect of the LE diet on cochlear pathology. ABRs in 23-month-olds indicated a slowing of age-related hearing loss in LE mice of 3 F1 hybrid strains.

Genetics of age-related hearing loss in mice. IV. Cochlear pathology and hearing loss in 25 BXD recombinant inbred mouse strains

The effects of three putative genes which contribute to age-related hearing loss (AHL genes) were evaluated using auditory brainstem response (ABR) thresholds and post-mortem cochlear histopathology in 25 recombinant BXD inbred mouse strains, originally derived from C57BL/6J (B6) and DBA/2J (D2) progenitor strains. All BXD strains showed substantial elevation of ABR thresholds and loss of spiral ganglion cells (SGCs) during the first year of life. The findings are consistent with our genetic model in which D2 and B6 inbred strains both possess the Ahl (age-related hearing loss) gene, whereas D2 possesses two additional chromosomal loci with AHL genes (Ahl2 and Ahl3). The between-strain distribution in the severity of SGC loss and ABR threshold elevations suggests that the severity of hearing loss is determined in large part by the number of AH L genes an animal possesses and by additional genetic background effects. The present findings also demonstrate that, because BXD strains vary substantially in the rate and severity of progressive hearing loss (but are genetically closely related), they can provide powerful animal models for developmental studies of AHL.

Vestibular responses to linear acceleration are absent in otoconia-deficient C57BL/6JEi-het mice

UNLABELLED Vestibular evoked potentials (VsEPs) were measured in normal mice and in mice homozygous for the head tilt mutation (het/het, abbr. het). The het mice lack otoconia, the inertial mass critical for natural stimulation of inner ear gravity receptors. Our findings demonstrate that vestibular neural responses to pulsed linear acceleration are absent in het mice. THE RESULTS (1) confirm that adequate sensory stimuli fail to activate gravity receptors in the het model; and (2) serve as definitive evidence that far-field vestibular responses to pulsed linear acceleration depend critically on otolith end organs. The C57BL/6JEi-het mouse may be an excellent model of gravity receptor sensory deprivation.

Deficiency in plasma membrane calcium ATPase isoform 2 increases susceptibility to noise-induced hearing loss in mice.

Susceptibility to noise-induced hearing loss (NIHL) is poorly understood at the genetic level. Mice homozygous for a null mutation in the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene are deaf (Kozel et al., 1998). PMCA2 is expressed on outer hair cell stereocilia (Furuta et al., 1998). Fridberger et al. (1998) observed that the outer hair cell cytoplasmic Ca2+ concentration rises following acoustic overstimulation. We hypothesized that Pmca2+/- mice may be more susceptible to NIHL. Since the auditory brainstem response (ABR) thresholds of Pmca2+/- mice vary with the presence of a modifier locus (Noben-Trauth et al., 1997), Pmca2+/- mice were outcrossed to normal hearing CAST/Ei mice. The pre-exposure ABR thresholds of the resulting Pmca2+/+ and Pmca2+/- siblings were indistinguishable. Groups of these mice were exposed to varying intensities of broadband noise, and ABR threshold shifts were calculated. Fifteen days following an 8 h, 113 dB noise exposure, the Pmca2+/- mice displayed significant (P < or = 0.0007) permanent threshold shifts at 16 and 32 kHz that were 15 or 25 dB greater than those observed in Pmca2+/+ littermates. Pmca2 may be the first gene with a known mutated protein product that confers increased susceptibility to NIHL.