Motion microscopy for visualizing and quantifying small motions

Although the human visual system is remarkable at perceiving and interpreting motions, it has limited sensitivity, and we cannot see motions that are smaller than some threshold. Although difficult to visualize, tiny motions below this threshold are important and can reveal physical mechanisms, or be precursors to large motions in the case of mechanical failure. Here, we present a “motion microscope,” a computational tool that quantifies tiny motions in videos and then visualizes them by producing a new video in which the motions are made large enough to see. Three scientific visualizations are shown, spanning macroscopic to nanoscopic length scales. They are the resonant vibrations of a bridge demonstrating simultaneous spatial and temporal modal analysis, micrometer vibrations of a metamaterial demonstrating wave propagation through an elastic matrix with embedded resonating units, and nanometer motions of an extracellular tissue found in the inner ear demonstrating a mechanism of frequency separation in hearing. In these instances, the motion microscope uncovers hidden dynamics over a variety of length scales, leading to the discovery of previously unknown phenomena

Recovery kinetics of dual AAV-mediated human otoferlin expression

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5′ portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3′ portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5′ to 3′ vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression

Tectorial membrane porosity controls spread of excitation and tuning in the cochlea

Modifications of genes that encode proteins found exclusively in the tectorial membrane (TM) alter mechanical properties and produce a wide range of hearing deficits. However, the changes in TM physical properties responsible for these deficits remain unclear. In particular, the cochlear tuning of Tectb[superscript −/−] mice is significantly sharper than that of Tecta[superscript Y1870C/+] mice, even though the stiffnesses of Tecta[superscript Y1870C/+] and Tectb[superscript −/−] TMs are similarly reduced relative to wild-type TMs. Here we show that differences in TM wave properties that are governed by shear viscosity account for these differences in tuning. The shear viscosity of the TM results from the interaction of interstitial fluid with the porous structure of the TM’s macromolecular matrix. In basal regions of the cochlea, nanoscale pores of Tecta[superscript Y1870C/+] TMs are significantly larger than those of Tectb[superscript −/−] TMs. The larger pores in Tecta[superscript Y1870C/+] TMs gives rise to lower shear viscosity (by ∼70%), which in turn, reduces wave speed and increases wave decay constants relative to Tectb[superscript −/−] TM wave properties. These results demonstrate the importance of TM porosity in cochlear tuning and that TM porosity, not stiffness, underlies the striking differences in hearing between Tecta[superscript Y1870C/+] and Tectb[superscript −/−] mice.National Institutes of Health (U.S.) (Grant R01-DC00238)National Science Foundation (U.S.) (Grant 1122374

Age-related degradation of tectorial membrane dynamics with loss of CEACAM16

Studies of genetic disorders of sensorineural hearing loss have been instrumental in delineating mechanisms that underlie the remarkable sensitivity and selectivity that are hallmarks of mammalian hearing. For example, genetic modifications of TECTA and TECTB, which are principal proteins that comprise the tectorial membrane (TM), have been shown to alter auditory thresholds and frequency tuning in ways that can be understood in terms of changes in the mechanical properties of the TM. Here, we investigate effects of genetic modification targeting CEACAM16, a third important TM protein. Loss of CEACAM16 has been recently shown to lead to progressive reductions in sensitivity. Whereas age-related hearing losses have previously been linked to changes in sensory receptor cells, the role of the TM in progressive hearing loss is largely unknown. Here, we show that TM stiffness and viscosity are significantly reduced in adult mice that lack functional CEACAM16 relative to age-matched wild-type controls. By contrast, these same mechanical properties of TMs from juvenile mice that lack functional CEACAM16 are more similar to those of wild-type mice. Thus, changes in hearing phenotype align with changes in TM material properties and can be understood in terms of the same TM wave properties that were previously used to characterize modifications of TECTA and TECTB. These results demonstrate that CEACAM16 is essential for maintaining TM mechanical and wave properties, which in turn are necessary for sustaining the remarkable sensitivity and selectivity of mammalian hearing with increasing age