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.

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.

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.

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.

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.

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.

Outer hair cell somatic, not hair bundle, motility is the basis of the cochlear amplifier.

Sensitivity, dynamic range and frequency tuning of the cochlea are attributed to amplification involving outer hair cell stereocilia and/or somatic motility. We measured acoustically and electrically elicited basilar membrane displacements from the cochleae of wild-type and Tecta(DeltaENT/DeltaENT) mice, in which stereocilia are unable to contribute to amplification near threshold. Electrically elicited responses from Tecta(DeltaENT/DeltaENT) mice were markedly similar to acoustically and electrically elicited responses from wild-type mice. We conclude that somatic, and not stereocilia, motility is the basis of cochlear amplification.

Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane.

Frequency tuning in the cochlea is determined by the passive mechanical properties of the basilar membrane and active feedback from the outer hair cells, sensory-effector cells that detect and amplify sound-induced basilar membrane motions. The sensory hair bundles of the outer hair cells are imbedded in the tectorial membrane, a sheet of extracellular matrix that overlies the cochlea's sensory epithelium. The tectorial membrane contains radially organized collagen fibrils that are imbedded in an unusual striated-sheet matrix formed by two glycoproteins, alpha-tectorin (Tecta) and beta-tectorin (Tectb). In Tectb(-/-) mice the structure of the striated-sheet matrix is disrupted. Although these mice have a low-frequency hearing loss, basilar-membrane and neural tuning are both significantly enhanced in the high-frequency regions of the cochlea, with little loss in sensitivity. These findings can be attributed to a reduction in the acting mass of the tectorial membrane and reveal a new function for this structure in controlling interactions along the cochlea.

A deafness mutation isolates a second role for the tectorial membrane in hearing.

Alpha-tectorin (encoded by Tecta) is a component of the tectorial membrane, an extracellular matrix of the cochlea. In humans, the Y1870C missense mutation in TECTA causes a 50- to 80-dB hearing loss. In transgenic mice with the Y1870C mutation in Tecta, the tectorial membrane's matrix structure is disrupted, and its adhesion zone is reduced in thickness. These abnormalities do not seriously influence the tectorial membrane's known role in ensuring that cochlear feedback is optimal, because the sensitivity and frequency tuning of the mechanical responses of the cochlea are little changed. However, neural thresholds are elevated, neural tuning is broadened, and a sharp decrease in sensitivity is seen at the tip of the neural tuning curve. Thus, using Tecta(Y1870C/+) mice, we have genetically isolated a second major role for the tectorial membrane in hearing: it enables the motion of the basilar membrane to optimally drive the inner hair cells at their best frequency.

Role of the tectorial membrane revealed by otoacoustic emissions recorded from wild-type and transgenic Tecta(deltaENT/deltaENT) mice.

Distortion product otoacoustic emissions (DPOAE) were recorded from wild-type mice and mutant Tecta(deltaENT/deltaENT) mice with detached tectorial membranes (TM) under combined ketamine/xylaxine anesthesia. In Tecta(deltaENT/deltaENT) mice, DPOAEs could be detected above the noise floor only when the levels of the primary tones exceeded 65 dB SPL. DPOAE amplitude decreased with increasing frequency of the primaries in Tecta(deltaENT/deltaENT) mice. This was attributed to hair cell excitation via viscous coupling to the surrounding fluid and not by interaction with the TM as in the wild-type mice. Local minima and corresponding phase transitions in the DPOAE growth functions occurred at higher DPOAE levels in wild-type than in Tecta(deltaENT/deltaENT) mice. In less-sensitive Tecta(deltaENT/deltaENT) mice, the position of the local minima varied nonsystematically with frequency or no minima were observed. A bell-like dependence of the DPOAE amplitude on the ratio of the primaries was recorded in both wild-type and Tecta(deltaENT/deltaENT) mice. However, the pattern of this dependence was different in the wild-type and Tecta(deltaENT/deltaENT) mice, an indication that the bell-like shape of the DPOAE was produced by a combination of different mechanisms. A nonlinear low-frequency resonance, revealed by nonmonotonicity of the phase behavior, was seen in the wild-type but not in Tecta(deltaENT/deltaENT) mice.

One source for distortion product otoacoustic emissions generated by low- and high-level primaries.

Distortion product otoacoustic emissions (DPOAE) elicited by tones below 60-70 dB sound pressure level (SPL) are significantly more sensitive to cochlear insults. The vulnerable, low-level DPOAE have been associated with the postulated active cochlear process, whereas the relatively robust high-level DPOAE component has been attributed to the passive, nonlinear macromechanical properties of the cochlea. However, it is proposed that the differences in the vulnerability of DPOAEs to high and low SPLs is a natural consequence of the way the cochlea responds to high and low SPLs. An active process boosts the basilar membrane (BM) vibrations, which are attenuated when the active process is impaired. However, at high SPLs the contribution of the active process to BM vibration is small compared with the dominating passive mechanical properties of the BM. Consequently, reduction of active cochlear amplification will have greatest effect on BM vibrations and DPOAEs at low SPLs. To distinguish between the "two sources" and the "single source" hypotheses we analyzed the level dependence of the notch and corresponding phase discontinuity in plots of DPOAE magnitude and phase as functions of the level of the primaries. In experiments where furosemide was used to reduce cochlear amplification, an upward shift of the notch supports the conclusion that both the low- and high-level DPOAEs are generated by a single source, namely a nonlinear amplifier with saturating I/O characteristic.