Local cochlear mechanical responses revealed through outer hair cell receptor potential measurements.

Sensory hair cells, including the sensorimotor outer hair cells, which enable the sensitive, sharply tuned responses of the mammalian cochlea, are excited by radial shear between the organ of Corti and the overlying tectorial membrane. It is not currently possible to measure directly in vivo mechanical responses in the narrow cleft between the tectorial membrane and organ of Corti over a wide range of stimulus frequencies and intensities. The mechanical responses can, however, be derived by measuring hair cell receptor potentials. We demonstrate that the seemingly complex frequency- and intensity-dependent behavior of outer hair cell receptor potentials could be qualitatively explained by a two degrees of freedom system with local cochlear partition and tectorial membrane resonances strongly coupled by the outer hair cell stereocilia. A local minimum in the receptor potential below the characteristic frequency should always be observed at a frequency where the tectorial membrane mechanical impedance is minimal, i.e., at the presumed tectorial membrane resonance frequency. The tectorial membrane resonance frequency might, however, shift with stimulus intensity in accordance with a shift in the maximum of the tectorial membrane radial mechanical responses to lower frequencies, as observed in experiments.

A Gap-Junction Mutation Reveals That Outer Hair Cell Extracellular Receptor Potentials Drive High-Frequency Cochlear Amplification.

Cochlear amplification enables the enormous dynamic range of hearing through amplifying cochlear responses to low- to moderate-level sounds and compressing them to loud sounds. Amplification is attributed to voltage-dependent electromotility of mechanosensory outer hair cells (OHCs) driven by changing voltages developed across their cell membranes. At low frequencies, these voltage changes are dominated by intracellular receptor potentials (RPs). However, OHC membranes have electrical low-pass filter properties that attenuate high-frequency RPs, which should potentially attenuate amplification of high-frequency cochlear responses and impede high-frequency hearing. We made in vivo intracellular and extracellular electrophysiological measurements from the organ of Corti of male and female mice of the CBA/J strain, with excellent high-frequency hearing, and from the CD-1 mouse strain, which has sensitive hearing below 12 kHz but loses high-frequency hearing within a few weeks postpartum. The CD-1 mouse strain was transfected with an A88V mutation of the connexin 30 gap-junction protein. By blocking the action of the GJ protein to reduce input resistance, the mutation increased the OHC extracellular RP (ERP) magnitude and rescued high-frequency hearing. However, by increasing the organ of Corti resistance, the mutation rescued high-frequency hearing through preserving the OHC extracellular RP (ERP) magnitude. We measured the voltage developed across the basolateral membranes of OHCs, which controls their electromotility, for low- to high-frequency sounds in male and female mice of the CD-1 strain that expressed the A88V mutation. We demonstrate that ERPs, not RPs, drive OHC motility and cochlear amplification at high frequencies because at high frequencies, ERPs are not frequency attenuated, exceed RPs in magnitude, and are appropriately timed to provide cochlear amplification.SIGNIFICANCE STATEMENT Cochlear amplification, which enables the enormous dynamic range of hearing, is attributed to voltage-dependent electromotility of the mechanosensory outer hair cells (OHCs) driven by sound-induced voltage changes across their membranes. OHC intracellular receptor potentials are electrically low-pass filtered, which should hinder high-frequency hearing. We measured the intracellular and extracellular voltages that control OHC electromotility in vivo in a mouse strain with impaired high-frequency hearing. A gap-junction mutation of the strain rescued high-frequency hearing, increased organ of Corti resistance, and preserved large OHC extracellular receptor potentials but reduced OHC intracellular receptor potentials and impaired low-frequency hearing. We concluded intracellular potentials drive OHC motility at low frequencies and extracellular receptor potentials drive OHC motility and cochlear amplification at high frequencies.

Drug distribution along the cochlea is strongly enhanced by low-frequency round window micro vibrations.

The cochlea's inaccessibility and complex nature provide significant challenges to delivering drugs and other agents uniformly, safely and efficiently, along the entire cochlear spiral. Large drug concentration gradients are formed along the cochlea when drugs are administered to the middle ear. This undermines the major goal of attaining therapeutic drug concentration windows along the whole cochlea. Here, utilizing a well-known physiological effect of salicylate, we demonstrate a proof of concept in which drug distribution along the entire cochlea is enhanced by applying round window membrane low-frequency micro vibrations with a probe that only partially covers the round window. We provide evidence of enhanced drug influx into the cochlea and cochlear apical drug distribution without breaching cochlear boundaries. It is further suggested that ossicular functionality is not required for the effective drug distribution we report. The novel method presented here of local drug delivery to the cochlea could be implemented when ossicular functionality is absent or impeded and can be incorporated in clinically approved auditory protheses for patients who suffer with conductive, sensorineural or mixed hearing loss.

Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound.

The detection of different frequencies in sound is accomplished with remarkable precision by the basilar membrane (BM), an elastic, ribbon-like structure with graded stiffness along the cochlear spiral. Sound stimulates a wave of displacement along the BM with maximal magnitude at precise, frequency-specific locations to excite neural signals that carry frequency information to the brain. Perceptual frequency discrimination requires fine resolution of this frequency map, but little is known of the intrinsic molecular features that demarcate the place of response on the BM. To investigate the role of BM microarchitecture in frequency discrimination, we deleted extracellular matrix protein emilin 2, which disturbed the filamentous organization in the BM. Emilin2 -/- mice displayed broadened mechanical and neural frequency tuning with multiple response peaks that are shifted to lower frequencies than normal. Thus, emilin 2 confers a stiffness gradient on the BM that is critical for accurate frequency resolution.

Local Drug Delivery to the Entire Cochlea without Breaching Its Boundaries.

The mammalian cochlea is one of the least accessible organs for drug delivery. Systemic administration of many drugs is severely limited by the blood-labyrinth barrier. Local intratympanic administration into the middle ear would be a preferable option in this case, and the only option for many newly emerging classes of drugs, but it leads to the formation of drug concentration gradients along the extensive, narrow cochlea. The gradients are orders of magnitude and well outside the therapeutic windows. Here we present an efficient, quick, and simple method of cochlear pumping, through large-amplitude, low-frequency reciprocal oscillations of the stapes and round window, which can consistently and uniformly deliver drugs along the entire length of the intact cochlea within minutes without disrupting the cochlear boundaries. The method should facilitate novel ways of approaching the treatment of inner ear disorders because it overcomes the challenge of delivering therapeutics along the entire cochlear length.

Drug Diffusion Along an Intact Mammalian Cochlea.

Intratympanic drug administration depends on the ability of drugs to pass through the round window membrane (RW) at the base of the cochlea and diffuse from this location to the apex. While the RW permeability for many different drugs can be promoted, passive diffusion along the narrowing spiral of the cochlea is limited. Earlier measurements of the distribution of marker ions, corticosteroids, and antibiotics demonstrated that the concentration of substances applied to the RW was two to three orders of magnitude higher in the base compared to the apex. The measurements, however, involved perforating the cochlear bony wall and, in some cases, sampling perilymph. These manipulations can change the flow rate of perilymph and lead to intake of perilymph through the cochlear aqueduct, thereby disguising concentration gradients of the delivered substances. In this study, the suppressive effect of salicylate on cochlear amplification via block of the outer hair cell (OHC) somatic motility was utilized to assess salicylate diffusion along an intact guinea pig cochlea in vivo. Salicylate solution was applied to the RW and threshold elevation of auditory nerve responses was measured at different times and frequencies after application. Resultant concentrations of salicylate along the cochlea were calculated by fitting the experimental data using a mathematical model of the diffusion and clearing of salicylate in a tube of variable diameter combined with a model describing salicylate action on cochlear amplification. Concentrations reach a steady-state at different times for different cochlear locations and it takes longer to reach the steady-state at more apical locations. Even at the steady-state, the predicted concentration at the apex is negligible. Model predictions for the geometry of the longer human cochlea show even higher differences in the steady-state concentrations of the drugs between cochlear base and apex. Our findings confirm conclusions that achieving therapeutic drug concentrations throughout the entire cochlear duct is hardly possible when the drugs are applied to the RW and are distributed via passive diffusion. Assisted methods of drug delivery are needed to reach a more uniform distribution of drugs along the cochlea.

Transgenic Tmc2 expression preserves inner ear hair cells and vestibular function in mice lacking Tmc1.

Recent work has demonstrated that transmembrane channel-like 1 protein (TMC1) is an essential component of the sensory transduction complex in hair cells of the inner ear. A closely related homolog, TMC2, is expressed transiently in the neonatal mouse cochlea and can enable sensory transduction in Tmc1-null mice during the first postnatal week. Both TMC1 and TMC2 are expressed at adult stages in mouse vestibular hair cells. The extent to which TMC1 and TMC2 can substitute for each other is unknown. Several biophysical differences between TMC1 and TMC2 suggest these proteins perform similar but not identical functions. To investigate these differences, and whether TMC2 can substitute for TMC1 in mature hair cells, we generated a knock-in mouse model allowing Cre-inducible expression of Tmc2. We assayed for changes in hair cell sensory transduction and auditory and vestibular function in Tmc2 knockin mice (Tm[Tmc2]) in the presence or absence of endogenous Tmc1, Tmc2 or both. Our results show that expression of Tm[TMC2] restores sensory transduction in vestibular hair cells and transiently in cochlear hair cells in the absence of TMC1. The cellular rescue leads to recovery of balance but not auditory function. We conclude that TMC1 provides some additional necessary function, not provided by TMC2.

Self-assembled nanoformulation of methylprednisolone succinate with carboxylated block copolymer for local glucocorticoid therapy.

A new self-assembled formulation of methylprednisolone succinate (MPS) based on a carboxylated trifunctional block copolymer of ethylene oxide and propylene oxide (TBC-COOH) was developed. TBC-COOH and MPS associated spontaneously at increased concentrations in aqueous solutions to form almost monodisperse mixed micelles (TBC-COOH/MPS) with a hydrodynamic diameter of 19.6 nm, zeta potential of -27.8 mV and optimal weight ratio ∼1:6.3. Conditions for the effective formation of TBC-COOH/MPS were elucidated by comparing copolymers and glucocorticoids with different structure. The micellar structure of TBC-COOH/MPS persisted upon dilution, temperature fluctuations and interaction with blood serum components. TBC-COOH increased antiradical activity of MPS and promoted its intrinsic cytotoxicity in vitro attributed to enhanced cellular availability of the mixed micelles. Intracellular transportation and hydrolysis of MPS were analyzed using optimized liquid chromatography tandem mass spectrometry with multiple reaction monitoring which showed increased level of both MPS and methylprednisolone in neuronal cells treated with the formulated glucocorticoid. Our results identify TBC-COOH/MPS as an advanced in situ prepared nanoformulation and encourage its further investigation for a potential local glucocorticoid therapy.

Rippling pattern of distortion product otoacoustic emissions evoked by high-frequency primaries in guinea pigs.

The origin of ripples in distortion product otoacoustic emission (DPOAE) amplitude which appear at specific DPOAE frequencies during f1 tone sweeps using fixed high frequency f2 (>20 kHz) in guinea pigs is investigated. The peaks of the ripples, or local DPOAE amplitude maxima, are separated by approximately half octave intervals and are accompanied by phase oscillations. The local maxima appear at the same frequencies in DPOAEs of different order and velocity responses of the stapes and do not shift with increasing levels of the primaries. A suppressor tone had little effect on the frequencies of the maxima, but partially suppressed DPOAE amplitude when it was placed close to the f2 frequencies. These findings agree with earlier observations that the maxima occur at the same DPOAE frequencies, which are independent of the f2 and the primary ratio, and thus are likely to be associated with DPOAE propagation mechanisms. Furthermore, the separation of the local maxima by approximately half an octave may suggest that the maxima are due to interference of the travelling waves along the basilar membrane at the frequency of the DPOAE. It is suggested that the rippling pattern appears because of interaction between DPOAE reverse travelling waves with standing waves formed in the cochlea.

Amplification mode differs along the length of the mouse cochlea as revealed by connexin 26 deletion from specific gap junctions.

The sharp frequency tuning and exquisite sensitivity of the mammalian cochlea is due to active forces delivered by outer hair cells (OHCs) to the cochlear partition. Force transmission is mediated and modulated by specialized cells, including Deiters' cells (DCs) and pillar cells (PCs), coupled by gap-junctions composed of connexin 26 (Cx26) and Cx30. We created a mouse with conditional Cx26 knock-out (Cx26 cKO) in DCs and PCs that did not influence sensory transduction, receptor-current-driving-voltage, low-mid-frequency distortion-product-otoacoustic-emissions (DPOAEs), and passive basilar membrane (BM) responses. However, the Cx26 cKO desensitizes mid-high-frequency DPOAEs and active BM responses and sensitizes low-mid-frequency neural excitation. This functional segregation may indicate that the flexible, apical turn cochlear partition facilitates transfer of OHC displacements (isotonic forces) for cochlear amplification and neural excitation. DC and PC Cx26 expression is essential for cochlear amplification in the stiff basal turn, possibly through maintaining cochlear partition mechanical impedance, thereby ensuring effective transfer of OHC isometric forces.

A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics.

Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells.

Modified protein expression in the tectorial membrane of the cochlea reveals roles for the striated sheet matrix.

The tectorial membrane (TM) of the mammalian cochlea is a complex extracellular matrix which, in response to acoustic stimulation, displaces the hair bundles of outer hair cells (OHCs), thereby initiating sensory transduction and amplification. Here, using TM segments from the basal, high-frequency region of the cochleae of genetically modified mice (including models of human hereditary deafness) with missing or modified TM proteins, we demonstrate that frequency-dependent stiffening is associated with the striated sheet matrix (SSM). Frequency-dependent stiffening largely disappeared in all three TM mutations studied where the SSM was absent either entirely or at least from the stiffest part of the TM overlying the OHCs. In all three TM mutations, dissipation of energy is decreased at low (<8 kHz) and increased at high (>8 kHz) stimulus frequencies. The SSM is composed of polypeptides carrying fixed charges, and electrostatic interaction between them may account for frequency-dependent stiffness changes in the material properties of the TM. Through comparison with previous in vivo measurements, it is proposed that implementation of frequency-dependent stiffening of the TM in the OHC attachment region facilitates interaction among tones, backward transmission of energy, and amplification in the cochlea.

A novel mechanism of cochlear excitation during simultaneous stimulation and pressure relief through the round window.

The round window (RW) membrane provides pressure relief when the cochlea is excited by sound. Here, we report measurements of cochlear function from guinea pigs when the cochlea was stimulated at acoustic frequencies by movements of a miniature magnet which partially occluded the RW. Maximum cochlear sensitivity, corresponding to subnanometre magnet displacements at neural thresholds, was observed for frequencies around 20 kHz, which is similar to that for acoustic stimulation. Neural response latencies to acoustic and RW stimulation were similar and taken to indicate that both means of stimulation resulted in the generation of conventional travelling waves along the cochlear partition. It was concluded that the relatively high impedance of the ossicles, as seen from the cochlea, enabled the region of the RW not occluded by the magnet, to act as a pressure shunt during RW stimulation. We propose that travelling waves, similar to those owing to acoustic far-field pressure changes, are driven by a jet-like, near-field component of a complex pressure field, which is generated by the magnetically vibrated RW. Outcomes of research described here are theoretical and practical design principles for the development of new types of hearing aids, which use near-field, RW excitation of the cochlea.

Tonotopic morphometry of the lamina reticularis of the guinea pig cochlea with associated microstructures and related mechanical implications.

Morphometry of the lamina reticularis of the guinea pig cochlea was performed using scanning electron microscopy. Seventy-four geometrical parameters of the lamina reticularis, the bundles of stereocilia, and individual stereocilia, in all rows of hair cells and within the individual hair cells, were measured at ten equally spaced locations along the longitudinal direction of the cochlea. Variations of the parameters versus the longitudinal coordinate were statistically analyzed and fitted with polynomials (constant, linear, or quadratic). Our data show that a unique set of geometrical parameters of inner and outer hair cells is typical for every frequency-dependent position at the lamina reticularis. Morphology of the outer hair cell structures varies more than respective parameters of the inner hair cells. Mechanical modeling using the obtained geometrical parameters provides a novel glance at the mechanical characteristics with respect to the cochlear tonotopy.

A mouse model for human deafness DFNB22 reveals that hearing impairment is due to a loss of inner hair cell stimulation.

The gene causative for the human nonsyndromic recessive form of deafness DFNB22 encodes otoancorin, a 120-kDa inner ear-specific protein that is expressed on the surface of the spiral limbus in the cochlea. Gene targeting in ES cells was used to create an EGFP knock-in, otoancorin KO (Otoa(EGFP/EGFP)) mouse. In the Otoa(EGFP/EGFP) mouse, the tectorial membrane (TM), a ribbon-like strip of ECM that is normally anchored by one edge to the spiral limbus and lies over the organ of Corti, retains its general form, and remains in close proximity to the organ of Corti, but is detached from the limbal surface. Measurements of cochlear microphonic potentials, distortion product otoacoustic emissions, and basilar membrane motion indicate that the TM remains functionally attached to the electromotile, sensorimotor outer hair cells of the organ of Corti, and that the amplification and frequency tuning of the basilar membrane responses to sounds are almost normal. The compound action potential masker tuning curves, a measure of the tuning of the sensory inner hair cells, are also sharply tuned, but the thresholds of the compound action potentials, a measure of inner hair cell sensitivity, are significantly elevated. These results indicate that the hearing loss in patients with Otoa mutations is caused by a defect in inner hair cell stimulation, and reveal the limbal attachment of the TM plays a critical role in this process.

The vestibular system mediates sensation of low-frequency sounds in mice.

The mammalian inner ear contains sense organs responsible for detecting sound, gravity and linear acceleration, and angular acceleration. Of these organs, the cochlea is involved in hearing, while the sacculus and utriculus serve to detect linear acceleration. Recent evidence from birds and mammals, including humans, has shown that the sacculus, a hearing organ in many lower vertebrates, has retained some of its ancestral acoustic sensitivity. Here we provide not only more evidence for the retained acoustic sensitivity of the sacculus, but we also found that acoustic stimulation of the sacculus has behavioral significance in mammals. We show that the amplitude of an elicited auditory startle response is greater when the startle stimuli are presented simultaneously with a low-frequency masker, including masker tones that are outside the sensitivity range of the cochlea. Masker-enhanced auditory startle responses were also observed in otoconia-absent Nox3 mice, which lack otoconia but have no obvious cochlea pathology. However, masker enhancement was not observed in otoconia-absent Nox3 mice if the low-frequency masker tones were outside the sensitivity range of the cochlea. This last observation confirms that otoconial organs, most likely the sacculus, contribute to behavioral responses to low-frequency sounds in mice.

The dynein-tubulin motor powers active oscillations and amplification in the hearing organ of the mosquito.

The design principles and specific proteins of the dynein-tubulin motor, which powers the flagella and cilia of eukaryotes, have been conserved throughout the evolution of life from algae to humans. Cilia and flagella can support both motile and sensory functions independently, or sometimes in parallel to each other. In this paper we show that this dual sensory-motile role of eukaryotic cilia is preserved in the most sensitive of all invertebrate hearing organs, the Johnston's organ of the mosquito. The Johnston's organ displays spontaneous oscillations, which have been identified as being a characteristic of amplification in the ears of mosquitoes and Drosophila. In the auditory organs of Drosophila and vertebrates, the molecular basis of amplification has been attributed to the gating and adaptation of the mechanoelectrical transducer channels themselves. On the basis of their temperature-dependence and sensitivity to colchicine, we attribute the molecular basis of spontaneous oscillations by the Johnston's organ of the mosquito Culex quinquefasciatus, to the dynein-tubulin motor of the ciliated sensillae. If, as has been claimed for insect and vertebrate hearing organs, spontaneous oscillations epitomize amplification, then in the mosquito ear, this process is independent of mechanotransduction.