Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants

Abstract

Our sense of gravitation and linear acceleration is mediated by stimulation of vestibular hair cells through displacement of otoconia in the utricle and saccule (the gravity receptor organ). We recently showed that otoconin-90 (Oc90) deletion led to formation of giant otoconia. In the present study, we determined the extent to which the giant otoconia affected balance and gravity receptor sensory input and compared the findings with other otoconia mutants. We employed a wide spectrum of balance behavioral tests, including reaching and air-righting reflexes, gait, swimming, beam-crossing, rotorod latencies, and a direct measure of gravity receptor input, vestibular evoked potentials (VsEPs). All tests on homozygous adult mutants consistently ranked the order of imbalance as (from worst to best) Nox3(het)<otopetrin 1(tlt)<Oc90 null<Oc90 wild type and C57Bl/6 mice using systematic statistical comparisons of the frequency of occurrence or the severity of abnormal functions. This order coincides with the degree of otoconia deficiencies and is consistent with VsEP measures. Notably, all mice (except Nox3(het)) showed remarkable learned adaptation to peripheral vestibular deficits by staying on the rotating rod significantly longer in each successive trial, and the rate and extent of such learned improvements ranked the same order as their initial balance ability. Despite the vestibular morbidity, Oc90 null mice had normal hearing, as measured by auditory brainstem responses (ABRs) and distortion products of otoacoustic emissions (DPOAEs). The study demonstrates that the remnant otoconia mass in Oc90 nulls does stimulate the gravity receptor organs, which was likely responsible for the improved balance performance relative to strains with absent otoconia. Furthermore, the combination of direct electrophysiological measures and a series of behavioral tests can be used to interpret the imbalance severity arising from altered inputs from the gravity receptor end organ.

Fig. 1

Imbalance behaviors of Oc90 null mice as compared with other otoconia mutants. Statistical comparisons are presented in Table 1 and Fig. 2. (A) Delayed development of the air-righting response of Oc90 null mice (P11 shown). At this age, wt pups right their body positions instantly, and it is not possible to photograph the landing process. (C) Oc90 null pups (P16) hold their heads and bodies in a tilted position. Compared with wt mice (D), Oc90 null mice (F) walk with ataxic gaits that have irregular shorter strides, wider stances and a non-linear curved pattern compared with wt littermate controls. Homozygous Otop1^tlt (J) and Nox3^het (L) mice have worse ataxic gaits than Oc90 null mice. (G) Oc90 null pups sometimes walk in a circular pattern, i.e. when they are put in a new environment and become disoriented. Otop1^tlt (K) and Nox3^het (M) mice show worse circular patterns under the same conditions.

Fig. 2

(A–C) Gait analysis of otoconia mutant mice. Compared with wt mice (n=18, P16), Oc90 null mice (n=16, P16) have wider stances of the hind limbs (hind limb base or HB), shorter strides of all limbs, a smaller LRA and a larger OL. Compared with age-matched C57 mice (n=18, P20), the gaits of homozygous Otop1^tlt mice (n=6, P20) show significant differences in all measurements including wider stances and shorter strides of all limbs, a smaller LRA and a larger OL. In comparison, the gaits of homozygous Nox3^het mice (n=6, P20) have similar strides and LRA compared with those of C57 mice, but show wider stances of all limbs and a larger OL. FB and HB, forelimb and hind limb base. LFS and RFS, left and right forelimb strides. LHS and RHS, left and right hind limb strides. * P<0.05; ** P<0.01; *** P<0.001. (D, E) Poor balance and coordination of Oc90 null mice on the accelerating rod as compared with other otoconia mutants (2–6 months old). The latencies on the rotorod are compared in Table 3. (D) Oc90 wt (n=7) and null (n=9) mice. (E) C57 (n=7), homozygous Otop1^tlt (n=8) and Nox3^het (n=8) mice. Oc90 null mice stayed on for a significantly shorter time than wt mice, Otop1^tlt mice even shorter and Nox3^het the shortest. All mice stayed longer with the progressing of each trial, with Oc90 wt and C57 improved the most (P<0.001). The rate and extent of learned adaptation for each strain of mice ranked the same order as their initial balance ability, with Nox3^het mice showing only limited improvement (E).

Fig. 3

Representative VsEP intensity series for Oc90 wt (left) and null (right) mice (4 months old). Response peaks P1, P2 and P3 are labeled where present. Stimulus intensity is described in dB re: 1.0 g/ms. As stimulus amplitude decreases, response peak latencies increase and peak amplitudes decrease until no response is seen at levels below threshold. Clearly evident here are the reduced response peak amplitudes (P1, P2 and P3) for the null mouse and elevated threshold. Thresholds for these animals were scored at −12.5 dB for the wt and at −1.5 dB for the KO. Total time represented for each waveform is 10 ms.

Fig. 4

SEM confirmation of giant otoconia in Oc90 null mice (P20) (B) or absent otoconia in homozygous Otop1^tlt (C) and Nox3^het mice (D) (P20) used in behavioral and VsEPs tests.

Fig. 5

Normal density, organization and appearance of stereocilia in the adult vestibule (the utricle is shown) of homozygous otoconia mutants as demonstrated by phalloidin-stained whole-mount vestibules (3 months old). The Oc90 null vestibule has been previously shown (Zhao et al., 2007). Insets show normal hair bundle size and organization in the homozygous mutant mice as demonstrated by SEM. Scale bars= 20 μm (A—D); 1 μm (insets). A, anterior; L, lateral.

Fig. 6

Normal hearing of Oc90 null mice (n=10 for wt, 8 for null, 4 months old). (A) Normal ABR thresholds of Oc90 null mice (only a selected set of frequencies is presented). (B) Normal DPOAE amplitudes of Oc90 null mice.