Characterization of vestibular dysfunction in the mouse model for Usher syndrome 1F

Abstract

The deaf-circling Ames waltzer (av) mouse harbors a mutation in the protocadherin 15 (Pcdh15) gene and is a model for inner ear defects associated with Usher syndrome type 1F. Earlier studies showed altered cochlear hair cell morphology in young av mice. In contrast, no structural abnormality consistent with significant vestibular dysfunction in young av mice was observed. Light and scanning electron microscopic studies showed that vestibular hair cells from presumptive null alleles Pcdh15(av-Tg) and Pcdh15(av-3J) are morphologically similar to vestibular sensory cells from control littermates, suggesting that the observed phenotype in these alleles might be a result of a central, rather than peripheral, defect. In the present study, a combination of physiologic and anatomic methods was used to more thoroughly investigate the source of vestibular dysfunction in Ames waltzer mice. Analysis of vestibular evoked potentials and angular vestibulo-ocular reflexes revealed a lack of physiologic response to linear and angular acceleratory stimuli in Pcdh15 mutant mice. Optokinetic reflex function was diminished but still present in the mutant animals, suggesting that the defect is primarily peripheral in nature. These findings indicate that the mutation in Pcdh15 results in either a functional abnormality in the vestibular receptor organs or that the defects are limited to the vestibular nerve. AM1-43 dye uptake has been shown to correlate with normal transduction function in hair cells. Dye uptake was found to be dramatically reduced in Pcdh15 mutants compared to control littermates, suggesting that the mutation affects hair cell function, although structural abnormalities consistent with significant vestibular dysfunction are not apparent by light and scanning electron microscopy in the vestibular neuroepithelia of young animals.

Fig. 1

Cross sections of the vestibule from a control animal (A) and a Pcdhl5^av-Tg mutant (B) at 1 year of age. In both micrographs, the arrow at lower right indicates the area of the saccular macula, and the arrow at upper left indicates the utricular macula. In the mutant, the saccular neuroepithelium has completely degenerated, whereas in the control, the saccular macula is normal. The utricular macula is of normal configuration in both the mutant and the control. Toluidine blue stain. The scale bar at lower right indicates 50 μm in both micrographs.

Fig. 2

Degeneration of saccular otoconia in the Pcdh15^av-Tg mutant as seen by scanning electron microscopy. A. Crystalline layer of the saccular otoconial membrane from a 30-day-old mutant showing fragmented and demineralized otoconial crystals. B. Normal otoconia from the saccule of a young-adult control mouse. The scale bars at lower right in both micrographs indicate 5 μm.

Fig. 3

Cross sections of the saccular macula from a control animal (A) and a Pcdhl5^av-Tg mutant (B) at 10 days after birth. In both cases, the saccular neuroepithelium is composed of normal-appearing sensory and supporting cells. Toluidine blue stain. The scale bar at lower right indicates 10 μm in both micrographs.

Fig. 4

Scanning electron micrographs showing stereocilia on vestibular sensory cells from control (A, C, E) and Pcdhl5^av-Tg mutant (B, D, F) inner ears at 10 days after birth. No dramatic differences in configuration of stereocilia are seen between control and mutant animals at this age in either the saccule (top row), utricle (middle row), or superior crista (bottom row). The scale bar at lower right in A and B indicates 5 μm; scale bars in the other micrographs all equal 1 μm.

Fig. 5

Representative VsEP waveforms for Pcdh15^av-J, Pcdh15^av-2J, and Pcdh15^av-Tg heterozygotes (+/− left panel) and homozygotes (−/− right panel). Stimulus intensity used to obtain these traces was +6 dB re: 1.0 g/ms. Total time for each waveform is 10 ms. The first three response peaks are marked in the left panel.

Fig. 6

Intensity functions for one heterozygote (left) and homozygote (right) of the Pcdhl5^av-2J mutation. The homozygote in this case showed circling behavior, normal swimming behavior, and VsEPs were present. VsEP threshold for this homozygote, however, was elevated in comparison to controls. Stimulus intensity is in dB re: 1.0 g/ms and is shown for each trace pair. The top two trace pairs were obtained at +6 dB re: 1.0 g/ms with (M) and without (NM) an intense wide band forward masker (100 dB SPL, 50–50,000 Hz).

Fig. 7

Plots of angular vestibulo-ocular reflex gain and phase versus stimulus frequency. Control animals and homozygous Pcdh15^av-3J alleles are plotted as circles and triangles, respectively. Rotation in light and darkness denoted by open and filled symbols, respectively. The phase plot for Pcdh15^av-3J in darkness is omitted because the phase calculation in the absence of a detectable response is spurious. Error bars span 1 standard deviation. The curves for Pcdh15^av-3J mutants (3J/3J) reflect the absence of an angular vestibulo-ocular reflex and a total reliance on visual inputs to drive compensatory eye movements in the light.

Fig. 8

Plot of horizontal optokinetic reflex gain versus stimulus velocity. Control animals and Pcdh15^av-3J mutants (3J/3J) are plotted as open and filled triangles, respectively. Error bars span 1 standard deviation. The speed-tuning curve of the Pcdh15^av-3J group is significantly attenuated, but the mutants still generate a robust optokinetic response.

Fig. 9

AM1-43 stained utricular maculae from P10 Pcdh15^av2J mutant and control mice. A relatively high fluorescence can be seen across the macular surface in the control mouse (A), compared to the mutant mouse (B). The box in A outlines an area in which the bodies of sensory cells are in focus. This area is shown at higher magnification in C. B. The overall fluorescence is significantly lower in the utricular maculae from mutant mice with a reduction in the number of stain-filled hair cells, compared to controls. The asterisk (*) marks autofluorescence of otoconia that did not come free of the surface at dissection. The box in B outlines the area that is shown at higher magnification (D). This area is in a similar focal plane as B, but lacks whole cell staining of recognizable hair cell bodies. Bar = 100 μm.