Imaging electrically evoked micromechanical motion within the organ of corti of the excised gerbil cochlea

The outer hair cell (OHC) of the mammalian inner ear exhibits an unusual form of somatic motility that can follow membrane-potential changes at acoustic frequencies. The cellular forces that produce this motility are believed to amplify the motion of the cochlear partition, thereby playing a key role in increasing hearing sensitivity. To better understand the role of OHC somatic motility in cochlear micromechanics, we developed an excised cochlea preparation to visualize simultaneously the electrically-evoked motion of hundreds of cells within the organ of Corti (OC). The motion was captured using stroboscopic video microscopy and quantified using cross-correlation techniques. The OC motion at approximately 2-6 octaves below the characteristic frequency of the region was complex: OHC, Deiter's cell, and Hensen's cell motion were hundreds of times larger than the tectorial membrane, reticular lamina (RL), and pillar cell motion; the inner rows of OHCs moved antiphasic to the outer row; OHCs pivoted about the RL; and Hensen's cells followed the motion of the outer row of OHCs. Our results suggest that the effective stimulus to the inner hair cell hair bundles results not from a simple OC lever action, as assumed by classical models, but by a complex internal motion coupled to the RL.     

Evidence for a highly elastic shell-core organization of cochlear outer hair cells by local membrane indentation

Cochlear outer hair cells (OHCs) are thought to play an essential role in the high sensitivity and sharp frequency selectivity of the hearing organ by generating forces that amplify the vibrations of this organ at frequencies up to several tens of kHz. This tuning process depends on the mechanical properties of the cochlear partition, which OHC activity has been proposed to modulate on a cycle-by-cycle basis. OHCs have a specialized shell-core ultrastructure believed to be important for the mechanics of these cells and for their unique electromotility properties. Here we use atomic force microscopy to investigate the mechanical properties of isolated living OHCs and to show that indentation mechanics of their membrane is consistent with a shell-core organization. Indentations of OHCs are also found to be highly nonhysteretic at deformation rates of more than 40 microm/s, which suggests the OHC lateral wall is a highly elastic structure, with little viscous dissipation, as would appear to be required in view of the very rapid changes in shape and mechanics OHCs are believed to undergo in vivo.     

Model of cochlear microphonic explores the tuning and magnitude of hair cell transduction current

The mammalian cochlea relies on the active forcing of sensory outer hair cells (OHCs) to amplify traveling wave responses along the basilar membrane. These forces are the result of electromotility, wherein current through the OHCs leads to conformational changes in the cells that provide stresses on surrounding structures. OHC transducer current can be detected via the voltage in the scala tympani (the cochlear microphonic, CM), and the CM can be used as an indicator of healthy cochlear operation. The CM represents a summation of OHC currents (the inner hair cell contribution is known to be small) and to use CM to probe the properties of OHC transduction requires a model that simulates that summation. We developed a finite element model for that purpose. The pattern of current generators (the model input) was initially based on basilar membrane displacement, with the current size based on in vitro data. The model was able to reproduce the amplitude of experimental CM results reasonably well when the input tuning was enhanced slightly (peak increased by ∼6 dB), which can be regarded as additional hair bundle tuning, and with a current/input value of 200–260 pA/nm, which is ∼4 times greater than the largest in vitro measures.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Detection of Cochlear Amplification and Its Activation

The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source.     

Detection of Cochlear Amplification and Its Activation

The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source.     

Modulation of Outer Hair Cell Electromotility by Cochlear Supporting Cells and Gap Junctions

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.     

The endocochlear potential alters cochlear micromechanics

Acoustic stimulation gates mechanically sensitive ion channels in cochlear sensory hair cells. Even in the absence of sound, a fraction of these channels remains open, forming a conductance between hair cells and the adjacent fluid space, scala media. Restoring the lost endogenous polarization of scala media in an in vitro preparation of the whole cochlea depolarizes the hair cell soma. Using both digital laser interferometry and time-resolved confocal imaging, we show that this causes a structural refinement within the organ of Corti that is dependent on the somatic electromotility of the outer hair cells (OHCs). Specifically, the inner part of the reticular lamina up to the second row of OHCs is pulled toward the basilar membrane, whereas the outer part (third row of OHCs and the Hensen's cells) unexpectedly moves in the opposite direction. A similar differentiated response pattern is observed for sound-evoked vibrations: restoration of the endogenous polarization decreases vibrations of the inner part of the reticular lamina and results in up to a 10-fold increase of vibrations of the outer part. We conclude that the endogenous polarization of scala media affects the function of the hearing organ by altering its geometry, mechanical and electrical properties.     

Outer hair cell somatic electromotility in vivo and power transfer to the organ of Corti

The active amplification of sound-induced vibrations in the cochlea, known to be crucial for auditory sensitivity and frequency selectivity, is not well understood. The outer hair cell (OHC) somatic electromotility is a potential mechanism for such amplification. Its effectiveness in vivo is putatively limited by the electrical low-pass filtering of the cell's transmembrane potential. However, the transmembrane potential is an incomplete metric. We propose and estimate two metrics to evaluate the effectiveness of OHC electromotility in vivo. One metric is the OHC electromechanical ratio defined as the amplitude of the ratio of OHC displacement to the change in its transmembrane potential. The in vivo electromechanical ratio is derived from the recently measured in vivo displacements of the reticular lamina and the basilar membrane at the 19 kHz characteristic place in guinea pigs and using a model. The ratio, after accounting for the differences in OHC vibration in situ due to the impedances from the adjacent structures, is in agreement with the literature values of the in vitro electromechanical ratio measured by others. The second and more insightful metric is the OHC somatic power. Our analysis demonstrates that the organ of Corti is nearly optimized to receive maximum somatic power in vivo and that the estimated somatic power could account for the active amplification.     

Evidence for outer hair cell driven oscillatory fluid flow in the tunnel of corti

Outer hair cell (OHC) somatic motility plays a key role in mammalian cochlear frequency selectivity and hearing sensitivity, but the mechanism of cochlear amplification is not well understood and remains a matter of controversy. We have visualized and quantified the effects of electrically evoked OHC somatic motility within the gerbil organ of Corti using an excised cochlear preparation. We found that OHC motility induces oscillatory motion of the medial olivocochlear fibers where they cross the tunnel of Corti (ToC) in their course to innervate the OHCs. We show that this motion is present at physiologically relevant frequencies and remains at locations distal to the OHC excitation point. We interpret this fiber motion to be the result of oscillatory fluid flow in the ToC. We show, using a simple one-dimensional hydromechanical model of the ToC, that a fluid wave within the tunnel can travel without significant attenuation for distances larger than the wavelength of the cochlear traveling wave at its peak. This ToC fluid wave could interact with the cochlear traveling wave to amplify the motion of the basilar membrane. The ToC wave could also provide longitudinal coupling between adjacent sections of the basilar membrane, and such coupling may be critical for cochlear amplification.     

Tuning of the outer hair cell motor by membrane cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.     

Genome-wide analysis of chicken snoRNAs provides unique implications for the evolution of vertebrate snoRNAs

Background

Although outer hair cells (OHCs) play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23) and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility.

Results

Using the membrane-based yeast two-hybrid system to screen a newly built cDNA library made predominantly from OHCs, we identified two completely different groups of potential protein partners using prestin and cdh23 as bait. These include both membrane bound and cytoplasmic proteins with 12 being de novo gene products with unknown function(s). In addition, some of these genes are closely associated with deafness loci, implying a potentially important role in hearing. The most abundant prey for prestin (38%) is composed of a group of proteins involved in electron transport, which may play a role in OHC survival. The most abundant group of cdh23 prey (55%) contains calcium-binding domains. Since calcium performs an important role in hair cell mechanoelectrical transduction and amplification, understanding the interactions between cdh23 and calcium-binding proteins should increase our knowledge of hair cell function at the molecular level.

Conclusion

The results of this study shed light on some protein networks in cochlear hair cells. Not only was a group of de novo genes closely associated with known deafness loci identified, but the data also indicate that the hair cell tip link interacts directly with calcium binding proteins. The OHC motor protein, prestin, also appears to be associated with electron transport proteins. These unanticipated results open potentially fruitful lines of investigation into the molecular basis of cochlear amplification.     

Cochlear outer hair cell bending in an external electric field.

We have used a high-resolution motion analysis system to reinvestigate shape changes in isolated guinea pig cochlear outer hair cells (OHCs) evoked by low-frequency (2-3 Hz) external electric stimulation. This phenomenon of electromotility is presumed to result from voltage-dependent structural changes in the lateral plasma membrane of the OHC. In addition to well-known longitudinal movements, OHCs were found to display bending movements when the alternating external electric field gradients were oriented perpendicular to the cylindrical cell body. The peak-to-peak amplitude of the bending movement was found to be as large as 0.7 microm. The specific sulfhydryl reagents, p-chloromercuriphenylsulfonic acid and p-hydroxymercuriphenylsulfonic acid, that suppress electrically evoked longitudinal OHCs movements, also inhibit the bending movements, indicating that these two movements share the same underlying mechanism. The OHC bending is likely to result from an electrical charge separation that produces depolarization of the lateral plasma membrane on one side of the cell and hyperpolarization on the other side. In the cochlea, OHC bending could produce radial distortions in the sensory epithelium and influence the micromechanics of the organ of Corti.     

A quantitative comparison of mechanoelectrical transduction in vestibular and auditory hair cells of neonatal mice.

Vestibular hair cells (VHCs) and cochlear outer hair cells (OHCs) of neonatal mice were stimulated by a fluid jet directed at their stereociliary bundles. Relations between the force exerted by the jet, bundle displacement, and the resulting transducer current were studied. The mean maximum transducer conductance in VHCs (2.6 nS) was about half that of the OHCs (5.5 nS), with the largest recorded values being 4.1 nS and 9.2 nS, respectively. In some OHCs activity of a single, 112 pS transducer channel was observed, allowing an estimate of the maximum number of channels: up to 36 in VHCs and 82 in OHCs, corresponding to about one transducer channel per tip link. The VHC bundles required about 330 nm of tip displacement to activate 90% of the maximum transducer conductance, compared to 150 nm for the OHC bundles. This corresponded to 2 deg of rotation about their pivots for both, due to the greater length of the VHC bundles. The VHC bundles'' translational stiffness was one-seventh of that of the OHCs. Conversion to rotational stiffness almost abolished this difference. Rotation of the hair bundle rather than translation determines the gating of the transducer channels, independent of bundle height or origin of the cells.     

Effect of membrane mechanics on charge transfer by the membrane protein prestin

Prestin was found in the membrane of outer hair cells (OHCs) located in the cochlea of the mammalian inner ear. These cells convert changes in the membrane potential into dimensional changes and (if constrained) to an active electromechanical force. The OHCs provide the ear with the mechanism of amplification and frequency selectivity that is effective up to tens of kHz. Prestin is a crucial part of the motor complex driving OHCs. Other cells transfected with prestin acquire electromechanical properties similar to those in the native cell. While the mechanism of prestin has yet to be fully understood, the charge transfer is its critical component. Here we investigate the effect of the mechanics of the surrounding membrane on electric charge transfer by prestin. We simulate changes in the membrane mechanics via the corresponding changes in the free energy of the prestin system. The free energy gradient enters a Fokker-Planck equation that describes charge transfer in our model. We analyze the effects of changes in the membrane tension and membrane elastic moduli. In the case of OHC, we simulate changes in the longitudinal and/or circumferential stiffness of the cell’s orthotropic composite membrane. In the case of cells transfected with prestin, we vary the membrane areal modulus. As a result, we show the effects of the membrane mechanics on the probabilistic characteristics of prestin-associated charge transfer for both stationary and high-frequency conditions. We compare our computational results with the available experimental data and find good agreement with the experiment.     

Manipulation of the Endocochlear Potential Reveals Two Distinct Types of Cochlear Nonlinearity

The mammalian hearing organ, the cochlea, contains an active amplifier to boost the vibrational response to low level sounds. Hallmarks of this active process are sharp location-dependent frequency tuning and compressive nonlinearity over a wide stimulus range. The amplifier relies on outer hair cell (OHC)-generated forces driven in part by the endocochlear potential, the ∼+80 mV potential maintained in scala media, generated by the stria vascularis. We transiently eliminated the endocochlear potential in vivo by an intravenous injection of furosemide and measured the vibrations of different layers in the cochlea’s organ of Corti using optical coherence tomography. Distortion product otoacoustic emissions were also monitored. After furosemide injection, the vibrations of the basilar membrane lost the best frequency (BF) peak and showed broad tuning similar to a passive cochlea. The intra-organ of Corti vibrations measured in the region of the OHCs lost the BF peak and showed low-pass responses but retained nonlinearity. This strongly suggests that OHC electromotility was operating and being driven by nonlinear OHC current. Thus, although electromotility is presumably necessary to produce a healthy BF peak, the mere presence of electromotility is not sufficient. The BF peak recovered nearly fully within 2 h, along with the recovery of odd-order distortion product otoacoustic emissions. The recovery pattern suggests that physical shifts in operating condition are a critical step in the recovery process.     

Lateral wall protein content mediates alterations in cochlear outer hair cell mechanics before and after hearing onset

Specialized outer hair cells (OHCs) housed within the mammalian cochlea exhibit active, nonlinear, mechanical responses to auditory stimulation termed electromotility. The extraordinary frequency resolution capacity of the cochlea requires an exquisitely equilibrated mechanical system of sensory and supporting cells. OHC electromotile length change, stiffness, and force generation are responsible for a 100-fold increase in hearing sensitivity by augmenting vibrational input to non-motile sensory inner hair cells. Characterization of OHC mechanics is crucial for understanding and ultimately preventing permanent functional deficits due to overstimulation or as a consequence of various cochlear pathologies. The OHCs' major structural assembly is a highly-specialized lateral wall. The lateral wall consists of three structures; a plasma membrane highly-enriched with the motor-protein prestin, an actin-spectrin cortical lattice, and one or more layers of subsurface cisternae. Technical difficulties in independently manipulating each lateral wall constituent have constrained previous attempts to analyze the determinants of OHCs' mechanical properties. Temporal separations in the accumulation of each lateral wall constituent during postnatal development permit associations between lateral wall structure and OHC mechanics. We compared developing and adult gerbil OHC axial stiffness using calibrated glass fibers. Alterations in each lateral wall component and OHC stiffness were correlated as a function of age. Reduced F-actin labeling was correlated with reduced OHC stiffness before hearing onset. Prestin incorporation into the PM was correlated with increased OHC stiffness at hearing onset. Our data indicate lateral wall F-actin and prestin are the primary determinants of OHC mechanical properties before and after hearing onset, respectively.     

Prestin-prestin and prestin-GLUT5 interactions in HEK293T cells

The remarkable hearing sensitivity and frequency selectivity in mammals is attributed to cochlear amplifier in the outer hair cells (OHCs). Prestin, a membrane protein in the lateral wall of OHC plasma membrane, is required for OHC electromotility and cochlear amplifier. In addition, GLUT5, a fructose transporter, is reported to be abundant in the plasma membrane of the OHC lateral wall and has been originally proposed as the OHC motor protein. Here we provide evidence of interactions between prestin/prestin and prestin/GLUT5 in transiently transfected HEK293T cells. We used a combination of techniques: (1) membrane colocalization by confocal microscopy, (2) fluorescence resonance energy transfer (FRET) by fluorescence activated cell sorting (FACS), (3) FRET by acceptor photobleaching, (4) FRET by fluorescence lifetime imaging (FRET-FLIM), and (5) coimmunoprecipitation. Our results suggest that homomeric and heteromeric prestin interactions occur in native OHCs to facilitate its electromotile function and that GLUT5 interacts with prestin for its elusive function.     

Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell's turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell's orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.