miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.     

A model for amplification of hair-bundle motion by cyclical binding of Ca2+ to mechanoelectrical-transduction channels

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca²⁺ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system’s behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca²⁺ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken’s cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.     

Enkephalin-like immunoreactivity of olivocochlear nerve fibers in cochlea of guinea pig and cat

The distribution of enkephalin-like immunoreactivity in the cochlea of the guinea pig and cat was studied. Indirect immunofluorescence immunohistochemistry using antisera generated against a methionine enkephalin-bovine thyroglobulin conjugate was applied to surface preparations of the organ of Corti and cryostat sections of the whole of the cochlea. In the cochlear osseous spiral lamina, immunofluorescence was localized to unmyelinated fibers of the intraganglionic spiral bundle. In the organ of Corti, immunofluorescence was localized to a small number of fibers at inner hair cells, the inner spiral bundle, and tunnel spiral bundle, to tunnel crossing fibers at the level of the tunnel floor, to an occasional spiral outer fiber, and to the synaptic region of outer hair cells in the three rows of the basal turn of the cochlea. Less immunofluorescence was found in this region as one progressed towards the apex, with none seen at the apex. At the most apical region the inner spiral bundle became patchy and the tunnel spiral bundle developed arcades. There was no immunofluorescence found in spiral ganglion cells, in auditory nerve fibers, or in the hair cells of the organ of Corti. The findings were the same in cat as in guinea pig, the latter being studied in more detail. It was concluded that efferent, olivocochlear neurons of the cochlea, synapsing predominantly with primary auditory nerve fibers from the inner sensory cells or with the sensory cells, contain enkephalin-like immunoreactivity. Also, the findings indicate that endings of olivocochlear neurons that synapse predominantly with outer hair cells contain enkephalin-like immunoreactivity. It has previously been shown that olivocochlear neurons are likely to be cholinergic.     

Active control of waves in a cochlear model with subpartitions.

Multiscale asymptotic methods developed previously to study macromechanical wave propagation in cochlear models are generalized here to include active control of a cochlear partition having three subpartitions, the basilar membrane, the reticular lamina, and the tectorial membrane. Activation of outer hair cells by stereocilia displacement and/or by lateral wall stretching result in a frequency-dependent force acting between the reticular lamina and basilar membrane. Wavelength-dependent fluid loads are estimated by using the unsteady Stokes' equations, except in the narrow gap between the tectorial membrane and reticular lamina, where lubrication theory is appropriate. The local wavenumber and subpartition amplitude ratios are determined from the zeroth order equations of motion. A solvability relation for the first order equations of motion determines the subpartition amplitudes. The main findings are as follows: The reticular lamina and tectorial membrane move in unison with essentially no squeezing of the gap; an active force level consistent with measurements on isolated outer hair cells can provide a 35-dB amplification and sharpening of subpartition waveforms by delaying dissipation and allowing a greater structural resonance to occur before the wave is cut off; however, previously postulated activity mechanisms for single partition models cannot achieve sharp enough tuning in subpartitioned models.     

Developmental segregation in the afferent projections to mammalian auditory hair cells.

The mammalian ear contains two types of auditory receptors, inner and outer hair cells, that lie in close proximity to each other within the sensory epithelium of the cochlea. In adult mammals, these two classes of auditory hair cells are innervated by separate populations of afferent neurons that differ strikingly in their cellular morphology and their pattern of arborization within the cochlea. At present, it is unclear when or how these distinctive patterns of cochlear innervation emerge and become segregated during development. In the present study, an in vitro horseradish peroxidase labeling method was used to examine the formation of individual auditory neuron arbors at the same location within the apex of the developing gerbil cochlea. At birth, most cochlear neurons displayed peripheral arbors that embraced both inner and outer hair cell receptors. During the next 6 days, however, the arbors of individual cochlear afferents become confined to either the inner or outer hair cell zone, and thus there is a complete segregation of afferent innervation. This neural segregation occurs principally through the withdrawal of inappropriate connections to the outer hair cell system and is completed well before hearing commences.     

Evidence of tectorial membrane radial motion in a propagating mode of a complex cochlear model

Knowledge of vibratory patterns in the cochlea is crucial to understanding the stimulation of mechanosensory cells. Experiments to determine the motion of the cochlear partition and surrounding fluid are extremely challenging. As a result, the motion data are incomplete and often contradictory. The bending mechanism of hair bundles, thought to be related to the shear motion and endolymphatic flow between the tectorial membrane (TM) and reticular lamina (RL), is controversial. We, therefore, extend the frequency range of our previous hybrid analytical-finite-element approach to model the basal as well as apical regions of the guinea pig cochlea. We solve the fluid-solid interaction eigenvalue problem for the axial wavenumber, fluid pressure, and vibratory relative motions of the cochlear partition as a function of frequency. A simple monophasic vibratory mode of the basilar membrane is found at both ends of the cochlea. However, this simple movement is associated with a complex frequency-dependent relative deformation between the TM and the RL. We provide evidence of a radial component of TM motion that is out of phase with the RL and that facilitates the bending of outer hair cell stereocilia at appropriate frequencies at both the cochlear base and apex.     

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.     

Nanomechanics of the subtectorial space caused by electromechanics of cochlear outer hair cells

The stereocilia of the cochlear inner hair cells (IHCs) transduce vibrations into the sensory receptor current. Until now, mechanisms for deflecting these stereocilia have not been identified experimentally. Here, we identify a mechanism by using the electromechanical properties of the soma of the outer hair cell to produce an intracochlear, mechanical force stimulus. It is known that the soma of this cell generates mechanical force in response to a change of its transmembrane potential. In the present experiments, the force was induced by intracochlear electrical stimulation at frequencies that covered the entire functionally relevant range of 50 kHz. Vibration responses were measured in the transverse direction with a laser Doppler vibrometer. For frequencies up to approximately 3 kHz in the first three turns of the guinea-pig cochlea, the apical surface of the IHC and the opposing surface of the tectorial membrane were found to vibrate with similar amplitudes but opposite phases. At high frequencies, there was little relative motion between these surfaces in the transverse direction. The counterphasic motion up to approximately 3 kHz results in a pulsatile motion of the fluid surrounding the stereocilia of the IHCs. Based on physical principles of fluid flow between narrowly spaced elastic plates, we show that radial fluid motion is amplified relative to transverse membrane motion and that the radial motion is capable of bending the stereocilia. In conclusion, for frequencies up to at least 3 kHz, there appears to be direct fluid coupling between outer hair cells and IHCs.     

Stereocilia mediate transduction in vertebrate hair cells (auditory system/cilium/vestibular system).

The vertebrate hair cell is a sensory receptor that responds to mechanical stimulation of its hair bundle, which usually consists of numerous large microvilli (stereocilia) and a single true cilium (the kinocilium). We have examined the roles of these two components of the hair bundle by recording intracellularly from bullfrog saccular hair cells. Detachment of the kinocilium from the hair bundle and deflection of this cilium produces no receptor potentials. Mechanical stimulation of stereocilia, however, elicits responses of normal amplitude and sensitivity. Scanning electron microscopy confirms the assessments of ciliary position made during physiological recording. Stereocilia mediate the transduction process of the vertebrate hair cell, while the kinocilium may serve primarily as a linkage conveying mechanical displacements to the stereocilia.     

Sensitivity, polarity, and conductance change in the response of vertebrate hair cells to controlled mechanical stimuli.

Hair cells, the primary receptors of the auditory, vestibular, and lateral-line sensory systems, produce electrical signals in response to mechanical stimulation of their apical hair bundles. We employed an in vitro preparation and intracellular recording to investigate the transduction mechanism of hair cells in the sacculus from the inner ear of the bullfrog (Rana catesbeiana). When stimulated directly by mechanical deflection of their hair bundles, these cells gave graded responses up to 15 mV in amplitude; the peak sensitivity was about 20 mV/micron deflection. The depolarizing component of the receptor potential corresponding to stimuli directed towards the kinocilium. Depolarizing responses were associated with a membrane resistance decrease, and hyperpolarizing responses with a resistance increase. Action potentials, possibly calcium spikes, were occasionally evoked in hair cells by mechanical or electrical stimulation.     

Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells

In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.     

Resonant tectorial membrane motion in the inner ear: its crucial role in frequency tuning.

The tectorial membrane has long been postulated as playing a role in the exquisite sensitivity of the cochlea. In particular, it has been proposed that the tectorial membrane provides a second resonant system, in addition to that of the basilar membrane, which contributes to the amplification of the motion of the cochlear partition. Until now, technical difficulties had prevented vibration measurements of the tectorial membrane and, therefore, precluded direct evidence of a mechanical resonance. In the study reported here, the vibration of the tectorial membrane was measured in two orthogonal directions by using a novel method of combining laser interferometry with a photodiode technique. It is shown experimentally that the motion of the tectorial membrane is resonant at a frequency of 0.5 octave (oct) below the resonant frequency of the basilar membrane and polarized parallel to the reticular lamina. It is concluded that the resonant motion of the tectorial membrane is due to a parallel resonance between the mass of the tectorial membrane and the compliance of the stereocilia of the outer hair cells. Moreover, in combination with the contractile force of outer hair cells, it is proposed that inertial motion of the tectorial membrane provides the necessary conditions to allow positive feedback of mechanical energy into the cochlear partition, thereby amplifying and tuning the cochlear response.     

Synapses involving auditory nerve fibers in primate cochlea.

The anatomical mechanisms for processing auditory signals are extremely complex and incompletely understood, despite major advances already made with the use of electron microscopy. A major enigma, for example, is the presence in the mammalian cochlea of a double hair cell receptor system. A renewed attempt to discover evidence of synaptic coupling between the two systems in the primate cochlea, postulated from physiological studies, has failed. However, in the outer spiral bundle the narrow and rigid clefts seen between pairs of presumptive afferent fibers suggest the possibility of dendro-dendritic interaction confined to the outer hair cell system. The clustering of afferent processes within folds of supporting cells subjacent to outer hair cells is in contrast to the lack of such close associations in the inner hair cell region. The difference reinforces the suggestion of functional interaction of some sort between the outer hair cell afferent nerve processes.     

Vibration pattern of the organ of Corti up to 50 kHz: evidence for resonant electromechanical force

Electromechanical force derived from the soma of the outer hair cell has long been postulated as the basis of the exquisite sensitivity of the cochlea. The problem with this postulate is that the electrical source and mechanical load for the electromechanical outer hair cell might be severely attenuated and phase-shifted by the electrical impedance of the cell and the mechanical impedance of the organ of Corti, respectively. Until now, it has not been possible to experimentally derive the high-frequency electrically induced force at the reticular lamina when the cells are embedded within the organ of Corti. In the study reported here, we succeeded in determining the frequency spectrum of the force up to 50 kHz. This was achieved by measuring both the electrically induced velocity and the mechanical impedance at different radial positions on the reticular lamina without tectorial membrane and with clamped basilar membrane. Velocity was measured with a laser interferometer and impedance, with a magnetically driven atomic force cantilever. The electromechanical force, normalized to the electric current density, exhibited a broad amplitude maximum at 7-20 kHz with a quality factor, Q(3dB), of 0.6 - 0.8. The displacement response was independent of frequency up to 10-20 kHz. The force response compensates for the viscoelastic impedance of the organ of Corti, extending the amplitude response of the organ to high frequencies. It is proposed that the electrical phase response of the cell is compensated with Zwislocki's original mechanism of a parallel resonance in the tectorial membrane-stereocilia complex.     

Negative hair-bundle stiffness betrays a mechanism for mechanical amplification by the hair cell

Hearing and balance rely on the ability of hair cells in the inner ear to sense miniscule mechanical stimuli. In each cell, sound or acceleration deflects the mechanosensitive hair bundle, a tuft of rigid stereocilia protruding from the cell's apical surface. By altering the tension in gating springs linked to mechanically sensitive transduction channels, this deflection changes the channels' open probability and elicits an electrical response. To detect weak stimuli despite energy losses caused by viscous dissipation, a hair cell can use active hair-bundle movement to amplify its mechanical inputs. This amplificatory process also yields spontaneous bundle oscillations. Using a displacement-clamp system to measure the mechanical properties of individual hair bundles from the bullfrog's ear, we found that an oscillatory bundle displays negative slope stiffness at the heart of its region of mechanosensitivity. Offsetting the hair bundle's position activates an adaptation process that shifts the region of negative stiffness along the displacement axis. Modeling indicates that the interplay between negative bundle stiffness and the motor responsible for mechanical adaptation produces bundle oscillation similar to that observed. Just as the negative resistance of electrically excitable cells and of tunnel diodes can be embedded in a biasing circuit to amplify electrical signals, negative stiffness can be harnessed to amplify mechanical stimuli in the ear.     

Receptor synapses without synaptic ribbons in the cochlea of the cat.

This report describes recepto-neural junctions thought to be chemical synapses without synaptic ribbons or vesicle aggregates. Such synapses occur in the cat between the outer hair cells of the cochlea and auditory nerve fibers. These synapses are formed by flat or indented junctions having symmetric membrane complexes associated with smooth endoplasmic reticulum and coated vesicles in the outer hair cell cytoplasm. Inner hair cells have receptoneural junctions with asymmetric membrane complexes; synaptic ribbons and vesicles gather at one type of junction, while the other type associates with endoplasmic reticulum. Gap junctions are not seen at cochlear synapses but do link supporting cells. The results suggest that inner and outer hair cell synapses have different properties and raise questions about the role of synaptic vesicles and endoplasmic reticulum in the storage and release of transmitters.     

The transmembrane inner ear (Tmie) protein is essential for normal hearing and balance in the zebrafish

Little is known about the proteins that mediate mechanoelectrical transduction, the process by which acoustic and accelerational stimuli are transformed by hair cells of the inner ear into electrical signals. In our search for molecules involved in mechanotransduction, we discovered a line of deaf and uncoordinated zebrafish with defective hair-cell function. The hair cells of mutant larvae fail to incorporate fluorophores that normally traverse the transduction channels and their ears lack microphonic potentials in response to vibratory stimuli. Hair cells in the posterior lateral lines of mutants contain numerous lysosomes and have short, disordered hair bundles. Their stereocilia lack two components of the transduction apparatus, tip links and insertional plaques. Positional cloning revealed an early frameshift mutation in tmie, the zebrafish ortholog of the mammalian gene transmembrane inner ear. The mutant line therefore affords us an opportunity to investigate the role of the corresponding protein in mechanoelectrical transduction.     

Calcium-binding sites on sensory processes in vertebrate hair cells.

Vertebrate lateral line and vestibular systems center their function on highly mechanosensitive hair cells. Each hair cell is equipped with one kinocilium (which resembles a motile cilium) and 50-100 actin-containing stereocilia (which resemble microvilli) at the site of stimulus reception. This report describes electron-microscopic localization of calcium-binding sites on the sensory processes of vertebrate hair cells. Using the Oschman-Wall technique for calcium localization [Oschman, J. L. & Wall, B. J. (1972) J. Cell Biol. 55, 58-73] together with electron-probe x-ray microanalysis of thin sections, we observed: (i) calcium- and iron-containing deposits in the region of the ciliary necklace in goldfish lateral line hair cells, (ii) calcium deposits upon the surface of stereocilia of hair cells of the bullfrog inner ear, and (iii) calcium deposits upon stereocilia of hair cells of the guinea pig vestibular system.