Structure of outer hair cell stereocilia side and attachment links in the chinchilla cochlea.

The structure and symmetry of chinchilla outer hair cell (OHC) stereocilia side and attachment links were investigated by transmission electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. The side links run laterally between and across the rows of the stereocilia and connect the stereocilia together within the bundle. Attachment links form a crown-like array around the tips of only the tallest OHC stereocilia and attach these stereocilia to the Type B fibrils of the tectorial membrane. Computer averaging of the side links from tannic acid-treated tissues showed a central dense region of the link between adjacent stereocilia and a smaller dense portion at the plasma membrane end of the link. Computer averaging of Cuprolinic blue-treated tissues showed low electron density of the central region of the link, and the plasma membrane ends of the link were electron dense. After tannic acid treatment, the attachment links showed a diffused radial distribution around the tips of the tallest OHC stereocilia. After Cuprolinic blue treatment, the attachment links appeared as electron-dense, membrane-bound granular structures arranged with radial symmetry. The central regions of the side links are reactive to tannic acid. These regions appear to contain neutral and basic residues of proteins and participate in side-by-side association of the side links in regular aggregates. Cuprolinic blue-reactive regions of the side and attachment links appear to contain acidic sulfated residues of glycoproteins or proteoglycans, which may be involved in the attachment of these links to the stereocilium membrane.     

Structure of outer hair cell stereocilia links in the chinchilla

The structure of side, tip, and “attachment” links of chinchilla outer hair cell (OHC) stereocilia was studied by transmission and scanning electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. Tannic acid, which interacts with many different types of proteins and glycoproteins irrespective of their electrical charge, showed strong reactivity for the central area of the side links and weak reactivity for the marginal area of these links adjacent to the stereocilia membrane. Tannic acid treatment revealed the tip links as thin strands, about 5 nm thick. Attachment links were poorly visualized after tannic acid treatment and appeared as sparse filamentous strands at tips of the tallest OHC stereocilia. Cuprolinic blue, at a high critical electrolyte concentration, reacted with strongly negative, primarily sulfated, carbohydrate residues of glycoconjugate macromolecules. In contrast to the tannic acid treatment, the central portions of the OHC stereocilia side links were unstained after Cuprolinic blue treatment; however, membrane-associated ends of these links were darkly stained. The tip links showed a similar appearance as after tannic acid treatment; however, Cuprolinic blue revealed an electron-dense substructure at both ends of its insertion into the stereocilia. Cuprolinic blue reactive structures were also observed as attachment links only at the tips of the OHC stereocilia of the tallest row in each bundle. These structures formed a crown-like array around the tip of each stereocilium. Their primary function appears to be attachment of type B fibrils of the tectorial membrane to the tallest OHC stereocilia. Cuprolinic blue reactive structures of the side, tip, and attachment links appear to contain acidic, sulfated residues of proteoglycans or glycoproteins. These structures may function as connective elements between the stereocilia links and the hair cell cytoskeleton.     

Divalent counterions tether membrane-bound carbohydrates to promote the cohesion of auditory hair bundles

The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg²⁺ was a more potent mediator of attachment than was Ca²⁺. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing.     

Characteristics of echolocating bats’ auditory stereocilia length,compared with other mammals

The stereocilia of the Organ of Corti in 4 different echolocating bats, Myotis adversus, Murina leucogaster, Nyctalus plancyi (Nyctalus velutinus), and Rhinolophus ferrumequinum were observed by using scanning electron microscopy (SEM). Stereocilia lengths were estimated for comparison with those of non-echolocating mammals. The specialized lengths of outer hair cells (OHC) stereocilia in echolocating bats were shorter than those of non-echolocating mammals. The specialized lengths of inner hair cells (IHC) stereocilia were longer than those of outer hair cells stereocilia in the Organ of Corti of echolocating bats. These characteristics of the auditory stereocilia length of echolocating bats represent the fine architecture of the electromotility process, helping to adapt to high frequency sound and echolocation.     

Stereocilium injury mediates hair bundle stiffness loss and recovery following intense water-jet stimulation

Inner ear hair cells exhibit many pathologies following exposure to intense sound, and the hair bundle is a major site of damage. This paper measures in vitro hair bundle motion on chick cochlear hair cells after intense in vitro and in vivo stimulation to explore the nature of hair bundle injury. Hair bundle stiffness, as well as relative and asymmetric motion of individual stereocilia, is controlled largely by the extracellular tip links, and a change in hair bundle motion was used to assess tip-link destruction following overstimulation. Intense in vitro stimulation caused a loss in stiffness that fully recovered within 10 min post-exposure. Relative and asymmetric stereocilia motion, however, were unchanged following the exposure, implying that tip links remained intact while the core or rootlet of the stereocilia were damaged and subsequently repaired. Intense and prolonged in vivo sound exposures produced stereocilia movements, measured in vitro, that were indicative of damage to stereocilia and tip links. Finally, the relative susceptibility of hair bundles to overstimulation was addressed by comparing stiffness loss with morphological features in the hair bundles. The loss of stiffness significantly increased as the amount of curvature in the hair bundle contour increased.     

Characteristics of echolocating bats’ auditory stereocilia length, compared with other mammals

The stereocilia of the Organ of Corti in 4 different echolocating bats, Myotis adversus, Murina leucogaster, Nyctalus plancyi (Nyctalus velutinus), and Rhinolophus ferrumequinum were observed by using scanning electron microscopy (SEM). Stereocilia lengths were estimated for comparison with those of non-echolocating mammals. The specialized lengths of outer hair cells (OHC) stereocilia in echolocating bats were shorter than those of non-echolocating mammals. The specialized lengths of inner hair cells (IHC) stereocilia were longer than those of outer hair cells stereocilia in the Organ of Corti of echolocating bats. These characteristics of the auditory stereocilia length of echolocating bats represent the fine architecture of the electromotility process, helping to adapt to high frequency sound and echolocation. These authors contributed equally to this work Supported by the National Natural Science Foundation of China (Grant No. 30430120) and Foundation of President of the Chinese Academy of Sciences     

Mechanical properties and consequences of stereocilia and extracellular links in vestibular hair bundles

Although knowledge of the fine structure of vestibular hair bundles is increasing, the mechanical properties and functional significance of those structures remain unclear. In 2004, Bashtanov and colleagues reported the contribution of different extracellular links to bundle stiffness. We simulated Bashtanov's experimental protocol using a three-dimensional finite element bundle model with geometry measured from a typical striolar hair cell. Unlike any previous models, we separately consider two types of horizontal links: shaft links and upper lateral links. Our most important results are as follows. First, we identified the material properties required to match Bashtanov's experiment: stereocilia Young's modulus of 0.74 GPa, tip link assembly (gating spring) stiffness of 5,300 pN/microm, and the combined stiffness of shaft links binding two adjacent stereocilia of 750 approximately 2,250 pN/microm. Second, we conclude that upper lateral links are likely to have nonlinear mechanical properties: they have minimal stiffness during small bundle deformations but stiffen as the bundle deflects further. Third, we estimated the stiffness of the gating spring based on our realistic three-dimensional bundle model rather than a conventional model relying on the parallel arrangement assumption. Our predicted stiffness of the gating spring was greater than the previous estimation.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Dynamic changes in hair cell stereocilia and cochlear transduction after noise exposure

The structures of cochlear transduction include stereocilia at the apical surface of hair cells and their connection to the tectorial membrane. The transduction site is one of the loci for noise-induced cochlear damage. Although stereocilia are susceptible to noise, it has been found that in the inner ears of avians, this fragile structure is largely self-repairing and is associated with recovery of hearing sensitivity after noise exposure, as observed in the difference between the temporal threshold shift (TTS) and the permanent threshold shift (PTS). In the mammalian cochleae, however, threshold shifts measured in the auditory brainstem responses (ABR) did not parallel the chronological changes in the stereocilia on hair cells. It is unclear how the morphological recovery of the stereocilia on the mammalian hair cells is correlated with the changes in cochlear transduction that can be assessed by measuring receptor potential. In the present study, guinea pigs were exposed to a broadband noise of 110 dB SPL for 2 h. Auditory sensitivity was evaluated using ABR and cochlear transduction was assessed using cochlear microphonics (CM). Stereocilia morphology was quantified at different time points after the noise and compared with the control. The noise produced a TTS of 55.69 ± 14.13 dB in frequency-averaged ABR thresholds. The threshold shift was reduced to 9.58 ± 11.75 dB SPL 1 month later with virtually no loss of hair cells. Damage to the stereocilia immediately after noise exposure was found to be associated with depression of CM amplitude. Virtually no abnormal stereocilia were observed 1 month after the noise in association with a fully recovered CM.     

Gelsolin Plays a Role in the Actin Polymerization Complex of Hair Cell Stereocilia

A complex of proteins scaffolded by the PDZ protein, whirlin, reside at the stereocilia tip and are critical for stereocilia development and elongation. We have shown that in outer hair cells (OHCs) whirlin is part of a larger complex involving the MAGUK protein, p55, and protein 4.1R. Whirlin interacts with p55 which is expressed exclusively in outer hair cells (OHC) in both the long stereocilia that make up the stereocilia bundle proper as well as surrounding shorter microvilli that will eventually regress. In erythrocytes, p55 forms a tripartite complex with protein 4.1R and glycophorin C promoting the assembly of actin filaments and the interaction of whirlin with p55 indicates that it plays a similar role in OHC stereocilia. However, the components directly involved in actin filament regulation in stereocilia are unknown. We have investigated additional components of the whirlin interactome by identifying interacting partners to p55. We show that the actin capping and severing protein, gelsolin, is a part of the whirlin complex. Gelsolin is detected in OHC where it localizes to the tips of the shorter rows but not to the longest row of stereocilia and the pattern of localisation at the apical hair cell surface is strikingly similar to p55. Like p55, gelsolin is ablated in the whirler and shaker2 mutants. Moreover, in a gelsolin mutant, stereocilia in the apex of the cochlea become long and straggly indicating defects in the regulation of stereocilia elongation. The identification of gelsolin provides for the first time a link between the whirlin scaffolding protein complex involved in stereocilia elongation and a known actin regulatory molecule.     

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.     

Power Dissipation in the Subtectorial Space of the Mammalian Cochlea Is Modulated by Inner Hair Cell Stereocilia

The stereocilia bundle is the mechano-transduction apparatus of the inner ear. In the mammalian cochlea, the stereocilia bundles are situated in the subtectorial space (STS)—a micrometer-thick space between two flat surfaces vibrating relative to each other. Because microstructures vibrating in fluid are subject to high-viscous friction, previous studies considered the STS as the primary place of energy dissipation in the cochlea. Although there have been extensive studies on how metabolic energy is used to compensate the dissipation, much less attention has been paid to the mechanism of energy dissipation. Using a computational model, we investigated the power dissipation in the STS. The model simulates fluid flow around the inner hair cell (IHC) stereocilia bundle. The power dissipation in the STS because of the presence IHC stereocilia increased as the stimulating frequency decreased. Along the axis of the stimulating frequency, there were two asymptotic values of power dissipation. At high frequencies, the power dissipation was determined by the shear friction between the two flat surfaces of the STS. At low frequencies, the power dissipation was dominated by the viscous friction around the IHC stereocilia bundle—the IHC stereocilia increased the STS power dissipation by 50- to 100-fold. There exists a characteristic frequency for STS power dissipation, CF_STS, defined as the frequency where power dissipation drops to one-half of the low frequency value. The IHC stereocilia stiffness and the gap size between the IHC stereocilia and the tectorial membrane determine the characteristic frequency. In addition to the generally assumed shear flow, nonshear STS flow patterns were simulated. Different flow patterns have little effect on the CF_STS. When the mechano-transduction of the IHC was tuned near the vibrating frequency, the active motility of the IHC stereocilia bundle reduced the power dissipation in the STS.     

Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism''s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca²⁺-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.     

Characteristics of echolocating bats’auditory stereocilia length, compared with other mammals

The stereocilia of the Organ of Corti in 4 different echolocating bats, Myotis adversus, Murina leuco-gaster, Nyctalus plancyi (Nyctalus velutinus), and Rhinolophus ferrumequinum were observed by using scanning electron microscopy (SEM). Stereocilia lengths were estimated for comparison with those of non-echolocating mammals. The specialized lengths of outer hair cells (OHC) stereocilia in echolocating bats were shorter than those of non-echolocating mammals. The specialized lengths of inner hair cells (IHC) stereocilia were longer than those of outer hair cells stereocilia in the Organ of Corti of echolocating bats. These characteristics of the auditory stereocilia length of echolocating bats represent the fine architecture of the electromotility process, helping to adapt to high frequency sound and echolocation.     

Voltage responses to tones of outer hair cells in the basal turn of the guinea-pig cochlea: significance for electromotility and desensitization.

Voltage responses were recorded from outer hair cells (OHCS) in the basal coil of the guinea-pig cochlea in response to tones at frequencies above the characteristic frequency (CF) presented together with a 100 Hz tone at 80 dB or 85 dB sound pressure level (SPL). The amplitude and polarity of voltage responses to a 100 Hz, 85 dB SPL tone were altered when presented together with tones at frequencies above CF according to the frequency and level of the high-frequency tone, OHC phasic (ac) (greater than 500 microV) but not tonic (dc) voltage responses were elicited by the high-frequency tone. Thus the responses of OHCS to low-frequency tones can be altered when presented together with a high-frequency tone without an apparent dc change in membrane potential. Recordings were made from an OHC during cochlear desensitization through exposure to an intense tone. The maximum voltage response to high-level low-frequency tones remained unchanged, although the OHC response to high-frequency tones became less sensitive to low-level stimuli and more linear as a function of level. It is suggested that desensitization is associated with a change in the mechanical properties of the cochlea, possibly associated with the OHCS themselves, and not with inactivation of the transducer channels. The amplitude of the OHC ac voltage response was measured at neural threshold, and the consequences of these measurements on hair cell electromotility are considered.     

Estimation of optimal insertion angle in a mammalian outer hair cell stereocilium

Optimal insertion angle of mammalian stereocilia is estimated from the finite element analysis of the tip motion of outer hair cells (OHCs) stereocilia. The OHC stereocilia motion in the acousticolateral system appears to result in the mechanoelectrical transduction channels. Deflection of the hair bundle towards the tallest row of stereocilia causes increased probability of opening of ion channels. In this work, we focus on one of the physical features of the OHC stereocilium, the initial insertion angle of the tallest row into the tectorial membrane (TM), and its effects on the stereocilia's deflection motion. A three-dimensional model was built for the tallest stereocilium and the TM at the region where the best frequency was 500Hz. The mechanical interactions between the embedded stereocilia and the TM have been implemented into the finite element simulation. We found that, the optimum insertion angle of the tallest stereocilium into the TM was 69.8°, where the stereocilium is maximally deflected. This quantity is consistent with the histological observation obtained from the literature.     

Simulation of the response of the inner hair cell stereocilia bundle to an acoustical stimulus

Mammalian hearing relies on a cochlear hydrodynamic sensor embodied in the inner hair cell stereocilia bundle. It is presumed that acoustical stimuli induce a fluid shear-driven motion between the tectorial membrane and the reticular lamina to deflect the bundle. It is hypothesized that ion channels are opened by molecular gates that sense tension in tip-links, which connect adjacent stepped rows of stereocilia. Yet almost nothing is known about how the fluid and bundle interact. Here we show using our microfluidics model how each row of stereocilia and their associated tip links and gates move in response to an acoustical input that induces an orbital motion of the reticular lamina. The model confirms the crucial role of the positioning of the tectorial membrane in hearing, and explains how this membrane amplifies and synchronizes the timing of peak tension in the tip links. Both stereocilia rotation and length change are needed for synchronization of peak tip link tension. Stereocilia length change occurs in response to accelerations perpendicular to the oscillatory fluid shear flow. Simulations indicate that nanovortices form between rows to facilitate diffusion of ions into channels, showing how nature has devised a way to solve the diffusive mixing problem that persists in engineered microfluidic devices.     

Putative immunolocalization of the mechanoelectrical transduction channels in mammalian cochlear hair cells.

Hair cells bear an apical bundle of stereocilia arranged in serried rows. Deflection of the bundle controls the opening and closing of mechanoelectrical transduction channels, thereby altering the conductance across the apical plasma membrane. Two locations for these channels have been proposed in the bundle, either near the bases of the stereocilia or towards their tips. One hypothesis that is consistent with the latter possibility suggests that fine extracellular filaments, which run between the tips of the shorter stereocilia and the sides of the taller stereocilia behind, operate the channels. Determining the precise position of the channels is essential to test this hypothesis. We have therefore attempted to localize them immunocytochemically. Because hair-cell transduction is amiloride sensitive, the channels may have an amiloride-binding site associated with them. We have therefore used a polyclonal antibody raised against another amiloride-sensitive ion channel to hunt for them. This antibody recognizes a 62-64 kDa band in immunoblots of cochlear tissue, and produces discrete labelling in the hair bundle. This is most concentrated just below the tips of the shorter stereocilia, coinciding with a region of specialization in the closely apposed membranes of the short and tall stereocilia but not with either end of the tip link.     

Conservation of endocochlear potential in mice with profound hearing loss induced by co-administration of kanamycin and furosemide

Research in mammalian hair cell regeneration is hampered by a lack of in vivo model of adult mouse inner ear injury. In the present study we investigated the effects of a combination of a single dose of aminoglycoside followed by a loop diuretic in adult mice. The auditory brainstem response threshold shift, extent and defining characteristics of the cochlear lesion were assessed and verified at different time points post-treatment. Our data indicated that this drug combination caused the rapid and extensive death of outer hair cells (OHCs). OHC death presented throughout the cochlea that commenced in the basal turn by 24 h and progressed apically. In contrast, inner hair cell (IHC) loss was delayed and mild. Terminal deoxynucleotidyl transferase dUTP nick end labelling-positive nuclei demonstrated that the majority of OHCs died via an apoptotic pathway. Auditory threshold shifts of up to 90 dB SPL indicated a profound hearing loss. In addition, the endocochlear potential (EP) in the drug-treated animals displayed a significant decline at 12 h post-treatment followed by recovery by 48 h post-treatment. Despite this recovery, there was a significant and progressive decrease in strial vascularis thickness, which was predominantly due to atrophy of marginal cells. The present study reproduced an adult mouse model of aminoglycoside-induced hearing loss. The mechanism underlying the recovered EP in the model with extensive hair cell death is discussed.     

Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.