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.

Nanoscale Poroelasticity of the Tectorial Membrane Determines Hair Bundle Deflections.

Stereociliary imprints in the tectorial membrane (TM) have been taken as evidence that outer hair cells are sensitive to shearing displacements of the TM, which plays a key role in shaping cochlear sensitivity and frequency selectivity via resonance and traveling wave mechanisms. However, the TM is highly hydrated (97% water by weight), suggesting that the TM may be flexible even at the level of single hair cells. Here we show that nanoscale oscillatory displacements of microscale spherical probes in contact with the TM are resisted by frequency-dependent forces that are in phase with TM displacement at low and high frequencies, but are in phase with TM velocity at transition frequencies. The phase lead can be as much as a quarter of a cycle, thereby contributing to frequency selectivity and stability of cochlear amplification.

Anisotropic Material Properties of Wild-Type and Tectb-/- Tectorial Membranes.

The tectorial membrane (TM) is an extracellular matrix that is directly coupled with the mechanoelectrical receptors responsible for sensory transduction and amplification. As such, the TM is often hypothesized to play a key role in the remarkable sensory abilities of the mammalian cochlea. Genetic studies targeting TM proteins have shown that changes in TM structure dramatically affect cochlear function in mice. Precise information about the mechanical properties of the TMs of wild-type and mutant mice at audio frequencies is required to elucidate the role of the TM and to understand how these genetic mutations affect cochlear mechanics. In this study, images of isolated TM segments are used to determine both the radial and longitudinal motions of the TM in response to a harmonic radial excitation. The resulting longitudinally propagating radial displacement and highly spatially dependent longitudinal displacement are modeled using finite-element models that take into account the anisotropy and finite dimensions of TMs. An automated, least-square fitting algorithm is used to find the anisotropic material properties of wild-type and Tectb-/- mice at audio frequencies. Within the auditory frequency range, it is found that the TM is a highly viscoelastic and anisotropic structure with significantly higher stiffness in the direction of the collagen fibers. Although no decrease in the stiffness in the fiber direction is observed, the stiffness of the TM in shear and in the transverse direction is found to be significantly reduced in Tectb-/- mice. As a result, TMs of the mutant mice tend to be significantly more anisotropic within the frequency range examined in this study. The effects of the Tectb-/- mutation on the TM's anisotropic material properties may be responsible for the changes in cochlear tuning and sensitivity that have been previously reported for these mice.

The Tectorial Membrane: Mechanical Properties and Functions.

The tectorial membrane (TM) is widely believed to play a critical role in determining the remarkable sensitivity and frequency selectivity that are hallmarks of mammalian hearing. Recently developed mouse models of human hearing disorders have provided new insights into the molecular, nanomechanical mechanisms that underlie resonance and traveling wave properties of the TM. Herein we review recent experimental and theoretical results detailing TM morphology, local poroelastic and electromechanical interactions, and global spread of excitation via TM traveling waves, with direct implications for cochlear mechanisms.

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.

Geometric Requirements for Tectorial Membrane Traveling Waves in the Presence of Cochlear Loads.

Recent studies suggest that wave motions of the tectorial membrane (TM) play a critical role in determining the frequency selectivity of hearing. However, frequency tuning is also thought to be limited by viscous loss in subtectorial fluid. Here, we analyze effects of this loss and other cochlear loads on TM traveling waves. Using a viscoelastic model, we demonstrate that hair bundle stiffness has little effect on TM traveling waves calculated with physiological parameters, that the limbal attachment can cause small (<20%) increases in TM wavelength, and that viscous loss in the subtectorial fluid can cause small (<20%) decreases in TM wave decay constants. However, effects of viscous loss in the subtectorial fluid are significantly increased if TM thickness is decreased. In contrast, increasing TM thickness above its physiological range has little effect on the wave, suggesting that the TM is just thick enough to maximize the spatial extent of the TM traveling wave.

Tectorial Membrane Traveling Waves Underlie Sharp Auditory Tuning in Humans.

Our ability to understand speech requires neural tuning with high frequency resolution, but the peripheral mechanisms underlying sharp tuning in humans remain unclear. Sharp tuning in genetically modified mice has been attributed to decreases in spread of excitation of tectorial membrane traveling waves. Here we show that the spread of excitation of tectorial membrane waves is similar in humans and mice, although the mechanical excitation spans fewer frequencies in humans-suggesting a possible mechanism for sharper tuning.

Longitudinal spread of mechanical excitation through tectorial membrane traveling waves.

The mammalian inner ear separates sounds by their frequency content, and this separation underlies important properties of human hearing, including our ability to understand speech in noisy environments. Studies of genetic disorders of hearing have demonstrated a link between frequency selectivity and wave properties of the tectorial membrane (TM). To understand these wave properties better, we developed chemical manipulations that systematically and reversibly alter TM stiffness and viscosity. Using microfabricated shear probes, we show that (i) reducing pH reduces TM stiffness with little change in TM viscosity and (ii) adding PEG increases TM viscosity with little change in TM stiffness. By applying these manipulations in measurements of TM waves, we show that TM wave speed is determined primarily by stiffness at low frequencies and by viscosity at high frequencies. Both TM viscosity and stiffness affect the longitudinal spread of mechanical excitation through the TM over a broad range of frequencies. Increasing TM viscosity or decreasing stiffness reduces longitudinal spread of mechanical excitation, thereby coupling a smaller range of best frequencies and sharpening tuning. In contrast, increasing viscous loss or decreasing stiffness would tend to broaden tuning in resonance-based TM models. Thus, TM wave and resonance mechanisms are fundamentally different in the way they control frequency selectivity.

Porosity controls spread of excitation in tectorial membrane traveling waves.

Cochlear frequency selectivity plays a key role in our ability to understand speech, and is widely believed to be associated with cochlear amplification. However, genetic studies targeting the tectorial membrane (TM) have demonstrated both sharper and broader tuning with no obvious changes in hair bundle or somatic motility mechanisms. For example, cochlear tuning of Tectb(-/-) mice is significantly sharper than that of Tecta(Y1870C/+) mice, even though TM stiffnesses are similarly reduced relative to wild-type TMs. Here we show that differences in TM viscosity can account for these differences in tuning. In the basal cochlear turn, nanoscale pores of Tecta(Y1870C/+) TMs are significantly larger than those of Tectb(-/-) TMs. The larger pore size reduces shear viscosity (by ∼70%), thereby reducing traveling wave speed and increasing spread of excitation. These results demonstrate the previously unrecognized importance of TM porosity in cochlear and neural tuning.

Electrokinetic properties of the mammalian tectorial membrane.

The tectorial membrane (TM) clearly plays a mechanical role in stimulating cochlear sensory receptors, but the presence of fixed charge in TM constituents suggests that electromechanical properties also may be important. Here, we measure the fixed charge density of the TM and show that this density of fixed charge is sufficient to affect mechanical properties and to generate electrokinetic motions. In particular, alternating currents applied to the middle and marginal zones of isolated TM segments evoke motions at audio frequencies (1-1,000 Hz). Electrically evoked motions are nanometer scaled (∼5-900 nm), decrease with increasing stimulus frequency, and scale linearly over a broad range of electric field amplitudes (0.05-20 kV/m). These findings show that the mammalian TM is highly charged and suggest the importance of a unique TM electrokinetic mechanism.

Tectorial membrane material properties in Tecta(Y)(1870C/+) heterozygous mice.

The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the α-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In Tecta(Y)(1870C/+) mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed a decrease in fixed charge concentration from -6.4±1.4 mmol/L in wild-types to -2.1±0.7 mmol/L in Tecta(Y)(1870C/+) TMs. TMs from Tecta(Y)(1870C/+) mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by 10±1.6 dB in Tecta(Y)(1870C/+) mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a corresponding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed.

Tectorial membrane travelling waves underlie abnormal hearing in Tectb mutant mice.

Remarkable sensitivity and exquisite frequency selectivity are hallmarks of mammalian hearing, but their underlying mechanisms remain unclear. Cochlear insults and hearing disorders that decrease sensitivity also tend to broaden tuning, suggesting that these properties are linked. However, a recently developed mouse model of genetically altered hearing (Tectb(-/-)) shows decreased sensitivity and sharper frequency selectivity. In this paper, we show that the Tectb mutation reduces the spatial extent and propagation velocity of tectorial membrane (TM) travelling waves and that these changes in wave propagation are likely to account for all of the hearing abnormalities associated with the mutation. By reducing the spatial extent of TM waves, the Tectb mutation decreases the spread of excitation and thereby increases frequency selectivity. Furthermore, the change in TM wave velocity reduces the number of hair cells that effectively couple energy to the basilar membrane, which reduces sensitivity. These results highlight the importance of TM waves in hearing.

Col11a2 deletion reveals the molecular basis for tectorial membrane mechanical anisotropy.

The tectorial membrane (TM) has a significantly larger stiffness in the radial direction than other directions, a prominent mechanical anisotropy that is believed to be critical for the proper functioning of the cochlea. To determine the molecular basis of this anisotropy, we measured material properties of TMs from mice with a targeted deletion of Col11a2, which encodes for collagen XI. In light micrographs, the density of TM radial collagen fibers was lower in Col11a2 -/- mice than wild-types. Tone-evoked distortion product otoacoustic emission and auditory brainstem response measurements in Col11a2 -/- mice were reduced by 30-50 dB independent of frequency as compared with wild-types, showing that the sensitivity loss is cochlear in origin. Stress-strain measurements made using osmotic pressure revealed no significant dependence of TM bulk compressibility on the presence of collagen XI. Charge measurements made by placing the TM as an electrical conduit between two baths revealed no change in the density of charge affixed to the TM matrix in Col11a2 -/- mice. Measurements of mechanical shear impedance revealed a 5.5 +/- 0.8 dB decrease in radial shear impedance and a 3.3 +/- 0.3 dB decrease in longitudinal shear impedance resulting from the Col11a2 deletion. The ratio of radial to longitudinal shear impedance fell from 1.8 +/- 0.7 for TMs from wild-type mice to 1.0 +/- 0.1 for those from Col11a2 -/- mice. These results show that the organization of collagen into radial fibrils is responsible for the mechanical anisotropy of the TM. This anisotropy can be attributed to increased mechanical coupling provided by the collagen fibrils. Mechanisms by which changes in TM material properties may contribute to the threshold elevation in Col11a2 -/- mice are discussed.

Plastic masters-rigid templates for soft lithography.

We demonstrate a simple process for the fabrication of rigid plastic master molds for soft lithography directly from (poly)dimethysiloxane devices. Plastics masters (PMs) provide a cost-effective alternative to silicon-based masters and can be easily replicated without the need for cleanroom facilities. We have successfully demonstrated the use of plastics micromolding to generate both single and dual-layer plastic structures, and have characterized the fidelity of the molding process. Using the PM fabrication technique, world-to-chip connections can be integrated directly into the master enabling devices with robust, well-aligned fluidic ports directly after molding. PMs provide an easy technique for the fabrication of microfluidic devices and a simple route for the scaling-up of fabrication of robust masters for soft lithography.

Frequency-dependent shear impedance of the tectorial membrane.

Microscale mechanical probes were designed and bulk-fabricated for applying shearing forces to biological tissues. These probes were used to measure shear impedance of the tectorial membrane (TM) in two dimensions. Forces were applied in the radial and longitudinal directions at frequencies ranging from 0.01-9 kHz and amplitudes from 0.02-4 microN. The force applied was determined by measuring the deflection of the probes' cantilever arms. TM impedance in the radial direction had a magnitude of 63 +/- 28 mN x s/m at 10 Hz and fell with frequency by 16 +/- 0.4 dB/decade, with a constant phase of -72 +/- 6 degrees . In the longitudinal direction, impedance was 36 +/- 9 mN x s/m at 10 Hz and fell by 19 +/- 0.4 dB/decade, with a constant phase of -78 +/- 4 degrees . Impedance was nearly constant as a function of force except at the highest forces, for which it fell slightly. These results show that the viscoelastic properties of the TM extend over a significant range of audio frequencies, consistent with a poroelastic interpretation of TM mechanics. The shear modulus G' determined from these measurements was 17-50 kPa, which is larger than in species with a lower auditory frequency range. This value suggests that hair bundles cannot globally shear the TM, but most likely cause bulk TM motion.

Otoacoustic emissions in humans, birds, lizards, and frogs: evidence for multiple generation mechanisms.

Many non-mammalian ears lack physiological features considered integral to the generation of otoacoustic emissions in mammals, including basilar-membrane traveling waves and hair-cell somatic motility. To help elucidate the mechanisms of emission generation, this study systematically measured and compared evoked emissions in all four classes of tetrapod vertebrates using identical stimulus paradigms. Overall emission levels are largest in the lizard and frog species studied and smallest in the chicken. Emission levels in humans, the only examined species with somatic hair cell motility, were intermediate. Both geckos and frogs exhibit substantially higher levels of high-order intermodulation distortion. Stimulus frequency emission phase-gradient delays are longest in humans but are at least 1 ms in all species. Comparisons between stimulus-frequency emission and distortion-product emission phase gradients for low stimulus levels indicate that representatives from all classes except frog show evidence for two distinct generation mechanisms analogous to the reflection- and distortion-source (i.e., place- and wave-fixed) mechanisms evident in mammals. Despite morphological differences, the results suggest the role of a scaling-symmetric traveling wave in chicken emission generation, similar to that in mammals, and perhaps some analog in the gecko.

Longitudinally propagating traveling waves of the mammalian tectorial membrane.

Sound-evoked vibrations transmitted into the mammalian cochlea produce traveling waves that provide the mechanical tuning necessary for spectral decomposition of sound. These traveling waves of motion that have been observed to propagate longitudinally along the basilar membrane (BM) ultimately stimulate the mechano-sensory receptors. The tectorial membrane (TM) plays a key role in this process, but its mechanical function remains unclear. Here we show that the TM supports traveling waves that are an intrinsic feature of its visco-elastic structure. Radial forces applied at audio frequencies (2-20 kHz) to isolated TM segments generate longitudinally propagating waves on the TM with velocities similar to those of the BM traveling wave near its best frequency place. We compute the dynamic shear storage modulus and shear viscosity of the TM from the propagation velocity of the waves and show that segments of the TM from the basal turn are stiffer than apical segments are. Analysis of loading effects of hair bundle stiffness, the limbal attachment of the TM, and viscous damping in the subtectorial space suggests that TM traveling waves can occur in vivo. Our results show the presence of a traveling wave mechanism through the TM that can functionally couple a significant longitudinal extent of the cochlea and may interact with the BM wave to greatly enhance cochlear sensitivity and tuning.

Doppler optical coherence microscopy for studies of cochlear mechanics.

The possibility of measuring subnanometer motions with micron scale spatial resolution in the intact mammalian cochlea using Doppler optical coherence microscopy (DOCM) is demonstrated. A novel DOCM system is described that uses two acousto-optic modulators to generate a stable 500-kHz heterodyne frequency. Images and motion measurements are obtained using phase-resolved analysis of the interference signal. The DOCM system permits imaging with micron-scale resolution and 85-dB sensitivity and motion measurements with 100-kHz bandwidth, directional discrimination, and 30-pm/Hz(0.5) noise floor. Images and motion measurements are presented that demonstrate the ability to resolve motions of structures of interest in a mammalian cochlea in vitro including the basilar membrane, reticular lamina, tectorial membrane, and outer hair cells.

Poroelastic bulk properties of the tectorial membrane measured with osmotic stress.

The equilibrium stress-strain relation and the pore radius of the isolated tectorial membrane (TM) of the mouse were determined. Polyethylene glycol (PEG), with molecular mass (MM) in the range 20-511 kDa, added to the TM bathing solution was used to exert an osmotic pressure. Strain on the TM induced by isosmotic PEG solutions of different molecular masses was approximately the same for MM > or = 200 kDa. However, for MM < or = 100 kDa, the TM strain was appreciably smaller. We infer that for the smaller molecular mass, PEG entered the TM and exerted a smaller effective osmotic pressure. The pore radius of the TM was estimated as 22 nm. The equilibrium stress-strain relation of the TM was measured using PEG with a molecular mass of 511 kDa. This relation was nonlinear and was fit with a power function. In the radial cochlear direction, the transverse stiffness of the TM was 20% stiffer in the inner than in the outer region. TM segments from the basal region had a larger transverse stiffness on average compared to sections from the apical-middle region. These measurements provide a quantitative basis for a poroelastic model of the TM.