Optogenetics Reveals Roles for Supporting Cells in Force Transmission to and From Outer Hair Cells in the Mouse Cochlea.

Outer hair cells (OHCs) of the organ of Corti (OoC), acting as bidirectional cellular mechanoelectrical transducers, generate, receive, and exchange forces with other major elements of the cochlear partition, including the sensory inner hair cells (IHCs). Force exchange is mediated via a supporting cell scaffold, including Deiters' (DC) and outer pillar cells (OPC), to enable the sensitivity and exquisite frequency selectivity of the mammalian cochlea and to transmit its responses to the auditory nerve. To selectively activate DCs and OPCs in male and female mice, we conditionally expressed in them a hyperpolarizing halorhodopsin (HOP), a light-gated inward chloride ion pump, and measured extracellular receptor potentials (ERPs) and their DC component (ERPDCs) from the cortilymph, which fills the OoC fluid spaces, and compared the responses with similar potentials from HOP-/- littermates. The compound action potentials (CAP) of the auditory nerve were measured as an indication of IHC activity and transmission of cochlear responses to the CNS. HOP light-activated hyperpolarization of DCs and OPCs suppressed cochlear amplification through changing the timing of its feedback, altered basilar membrane (BM) responses to tones at all measured levels and frequencies, and reduced IHC excitation. HOP activation findings reported here complement recent studies that revealed channelrhodopsin activation depolarized DCs and OPCs and effectively bypassed, rather than blocked, the control of OHC mechanical and electrical responses to sound and their contribution to timed and directed electromechanical feedback to the mammalian cochlea. Moreover, our findings identify DCs and OPCs as potential targets for the treatment of noise-induced hearing loss.

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.

Auditory sensory range of male mosquitoes for the detection of female flight sound.

Male mosquitoes detect and localize conspecific females by their flight-tones using the Johnston's organ (JO), which detects antennal deflections under the influence of local particle motion. Acoustic behaviours of mosquitoes and their JO physiology have been investigated extensively within the frequency domain, yet the auditory sensory range and the behaviour of males at the initiation of phonotactic flights are not well known. In this study, we predict a maximum spatial sensory envelope for flying Culex quinquefasciatus by integrating the physiological tuning response of the male JO with female aeroacoustic signatures derived from numerical simulations. Our sensory envelope predictions were tested with a behavioural assay of free-flying males responding to a female-like artificial pure tone. The minimum detectable particle velocity observed during flight tests was in good agreement with our theoretical prediction formed by the peak JO sensitivity measured in previous studies. The iso-surface describing the minimal detectable particle velocity represents the quantitative auditory sensory range of males and is directional with respect to the female body orientation. Our results illuminate the intricacy of the mating behaviour and point to the importance of observing the body orientation of flying mosquitoes to understand fully the sensory ecology of conspecific communication.

In vivo optogenetics reveals control of cochlear electromechanical responses by supporting cells.

Cochlear sensitivity, essential for communication and exploiting the acoustic environment, results from sensory-motor outer hair cells (OHCs) operating in a structural scaffold of supporting cells and extracellular cortilymph (CL) within the organ of Corti (OoC). Cochlear sensitivity control is hypothesized to involve interaction between the OHCs and OoC supporting cells (e.g., Deiters' cells (DCs) and outer pillar cells (OPCs)), but this has never been established in vivo Here, we conditionally expressed channelrhodopsins (ChR2) specifically in male and female mouse DCs and OPCs. illumination of the OoC activated the nonselective ChR2 cation conductance and depolarized DCs when measured in vivo and in isolated OoC. Measurements of sound-induced cochlear mechanical and electrical responses revealed OoC illumination suppressed the normal functions of OoC supporting cells transiently and reversibly. OoC illumination blocked normally occurring continuous minor adjustments of tone-evoked basilar membrane (BM) displacements over their entire dynamic range and OHC voltage responses to tones at levels and frequencies subject to cochlear amplification. OoC illumination altered the OHC MET conductance operating point, which reversed the asymmetry of OHC voltage responses to high level tones. OoC illumination accelerated recovery from temporary loud sound-induced acoustic desensitization. We concluded that DCs and OPCs are involved in both the control of cochlear responses that are essential for normal hearing, and the recovery from temporary acoustic desensitization. This is the first direct in vivo evidence for the interdependency of the structural, mechanical, and electrochemical arrangements of OHCs and OoC supporting cells that together provide fine control of cochlear responses.Significance statement:A striking feature of the mammalian cochlear sensory epithelium, the organ of Corti, is the cellular architecture and supporting cell arrangement that provides a structural scaffold for the sensory-motor outer hair cells. The role of the supporting cell scaffold, however, has never been elucidated in vivo, although in vitro and modelling studies indicate the scaffold is involved in exchange of forces between the outer hair cells and the organ of Corti. We used in vivo techniques, including optogenetics, that do not disrupt arrangements between the outer hair cells and supporting cells, but selectively, transiently, and reversibly interfere with supporting cell normal function. We revealed the supporting cells provide continuous adjustment of cochlear sensitivity, which is instrumental in normal hearing.

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.

Aerodynamic imaging by mosquitoes inspires a surface detector for autonomous flying vehicles.

Some flying animals use active sensing to perceive and avoid obstacles. Nocturnal mosquitoes exhibit a behavioral response to divert away from surfaces when vision is unavailable, indicating a short-range, mechanosensory collision-avoidance mechanism. We suggest that this behavior is mediated by perceiving modulations of their self-induced airflow patterns as they enter a ground or wall effect. We used computational fluid dynamics simulations of low-altitude and near-wall flights based on in vivo high-speed kinematic measurements to quantify changes in the self-generated pressure and velocity cues at the sensitive mechanosensory antennae. We validated the principle that encoding aerodynamic information can enable collision avoidance by developing a quadcopter with a sensory system inspired by the mosquito. Such low-power sensing systems have major potential for future use in safer rotorcraft control systems.

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.

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.

Pre-copula acoustic behaviour of males in the malarial mosquitoes Anopheles coluzzii and Anopheles gambiae s.s. does not contribute to reproductive isolation.

We reveal that males of two members of the Anopheles gambiae s.l. species complex, Anopheles coluzzii and Anopheles gambiae s.s. (hereafter A. gambiae), which are both malaria vectors, perform a stereotypical acoustic behaviour in response to pure tones at frequencies that encompass the frequency range of the female's flight-tones. This behaviour resembles that described for Culex quinquefasciatus and consists of phonotactic flight initiated by a steep increase in wing-beat frequency (WBF) followed by rapid frequency modulation (RFM) of WBF when in close proximity to the sound source. RFM was elicited without acoustic feedback or the presence of a live female, but it appears to be a stereotypic behaviour in the immediate lead up to copula formation. RFM is an independent and different behavioural process from harmonic convergence interactions used by male-female pairs for mate recognition at earlier stages of mating. Acoustic threshold for RFM was used to plot behavioural audiograms from free-flying A coluzzii and A gambiae males. These audiograms were almost identical (minima ∼400 Hz) and encompassed the WBF ranges of A coluzzii (378-601 Hz) and A gambiae (373-590 Hz) females, indicating that males of the two species share similar frequency tuning and range. Furthermore, no differences were found between the two species in their WBFs, RFM behaviour or harmonic convergence ratios. These results indicate that assortative mating between A coluzzii and A gambiae is unlikely to be based on male-specific acoustic behaviours during RFM. The significance of these findings in relation to possible mechanisms for assortative mating is discussed.

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.

A role for acoustic distortion in novel rapid frequency modulation behaviour in free-flying male mosquitoes.

We describe a new stereotypical acoustic behaviour by male mosquitoes in response to the fundamental frequency of female flight tones during mating sequences. This male-specific free-flight behaviour consists of phonotactic flight beginning with a steep increase in wing-beat frequency (WBF) followed by rapid frequency modulation (RFM) of WBF in the lead up to copula formation. Male RFM behaviour involves remarkably fast changes in WBF and can be elicited without acoustic feedback or physical presence of the female. RFM features are highly consistent, even in response to artificial tones that do not carry the multi-harmonic components of natural female flight tones. Comparison between audiograms of the robust RFM behaviour and the electrical responses of the auditory Johnston's organ (JO) reveals that the male JO is tuned not to the female WBF per se but, remarkably, to the difference between the male and female WBFs. This difference is generated in the JO responses as a result of intermodulation distortion products (DPs) caused by non-linear interaction between male-female flight tones in the vibrations of the antenna. We propose that male mosquitoes rely on their own flight tones in making use of DPs to acoustically detect, locate and orientate towards flying females. We argue that the previously documented flight-tone harmonic convergence of flying male and female mosquitoes could be a consequence of WBF adjustments so that DPs generated through flight-tone interaction fall within the optimal frequency ranges for JO detection.

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.

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.