Deiters Cells Research Articles

Corti fluid is a medium for outer hair cell force transmission.

The mammalian cochlea amplifies sounds selectively to improve frequency resolution. However, vibrations around the outer hair cells (OHCs) are amplified non-selectively. The mechanism of the selective or non-selective amplification is unknown. This study demonstrates that active force transmission through the extracellular fluid in the organ of Corti (Corti fluid) can explain how the cochlea achieves selective sound amplification despite the non-frequency-selective action of OHCs. Computational model simulations and experiments with excised cochleae from young gerbils of both sexes were exploited. OHC motility resulted in characteristic off-axis motion of the joint between the OHC and Deiters cell (ODJ). Incorporating the Corti fluid dynamics was critical to account for the ODJ motion due to OHC motility. The incorporation of pressure transmission through the Corti fluid resulted in three distinct frequency tuning patterns depending on sites in the organ of Corti. In the basilar membrane, the responses were amplified near the best-responding frequency (BF). In the ODJ region, the responses were amplified non-selectively. In the reticular lamina, the responses were amplified near the BF but suppressed in lower frequencies. The suppressive effect of OHCs was further examined by observing the changes in tuning curves due to local inhibition of OHC motility. The frequency response of the reticular lamina resembled neural tuning, such as the hypersensitivity of tuning-curve tails after hair cell damage. Our results demonstrate how active OHCs exploit the elastic frame and viscous fluid in the organ of Corti to amplify and suppress cochlear vibrations for better frequency selectivity.Significance Statement Active outer hair cells have been considered to selectively amplify the basilar membrane vibrations near the sound's tonotopic location. However, recent observations from different labs showed that outer hair cells' action is non-selective-it spreads over the broad span of traveling waves. These observations challenge the existing theory pegged to basilar-membrane mechanics. The motion at the joint between the outer hair cell and the Deiters (ODJ) cell holds the key to account for the non-selective action of outer hair cells. We show that the characteristic motions at the ODJ are explained coherently when Corti fluid acts as the medium for outer hair cell force transmission. Our results demonstrate how non-selective outer hair cell action produces selective neural responses.

Single cell RNA sequencing provides novel cellular transcriptional profiles and underlying pathogenesis of presbycusis

Age-related hearing loss (ARHL) or presbycusis is associated with irreversible progressive damage in the inner ear, where the sound is transduced into electrical signal; but the detailed mechanism remains unclear. Here, we sought to determine the potential molecular mechanism involved in the pathogeneses of ARHL with bioinformatics methods. A single-cell transcriptome sequencing study was performed on the cochlear samples from young and aged mice. Detection of identified cell type marker allowed us to screen 18 transcriptional clusters, including myeloid cells, epithelial cells, B cells, endothelial cells, fibroblasts, T cells, inner pillar cells, neurons, inner phalangeal cells, and red blood cells. Cell-cell communications were analyzed between young and aged cochlear tissue samples by using the latest integration algorithms Cellchat. A total of 56 differentially expressed genes were screened between the two groups. Functional enrichment analysis showed these genes were mainly involved in immune, oxidative stress, apoptosis, and metabolic processes. The expression levels of crucial genes in cochlear tissues were further verified by immunohistochemistry. Overall, this study provides new theoretical support for the development of clinical therapeutic drugs.

Hearing restoration by gene replacement therapy for a multisite-expressed gene in a mouse model of human DFNB111 deafness

Gene therapy has made significant progress in the treatment of hereditary hearing loss. However, most research has focused on deafness-related genes that are primarily expressed in hair cells with less attention given to multisite-expressed deafness genes. MPZL2, the second leading cause of mild-to-moderate hereditary deafness, is widely expressed in different inner ear cells. We generated a mouse model with a deletion in the Mpzl2 gene, which displayed moderate and slowly progressive hearing loss, mimicking the phenotype of individuals with DFNB111. We developed a gene replacement therapy system mediated by AAV-ie for efficient transduction in various types of cochlear cells. AAV-ie-Mpzl2 administration significantly lowered the auditory brainstem response and distortion product otoacoustic emission thresholds of Mpzl2-/- mice for at least seven months. AAV-ie-Mpzl2 delivery restored the structural integrity in both outer hair cells and Deiters cells. This study suggests the potential of gene therapy for MPZL2-related deafness and provides a proof of concept for gene therapy targeting other deafness-related genes that are expressed in different cell populations in the cochlea.

Transgenic Tg(Kcnj10-ZsGreen) fluorescent reporter mice allow visualization of intermediate cells in the stria vascularis

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen’s, Deiters’ and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Abnormal Innervation, Demyelination, and Degeneration of Spiral Ganglion Neurons as Well as Disruption of Heminodes are Involved in the Onset of Deafness in Cx26 Null Mice.

GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness. For individuals suffering from severe to profound GJB2-related deafness, cochlear implants have emerged as the sole remedy for auditory improvement. Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation. Thus, we generated a conditional knockout mouse model (Cx26-CKO) in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter. The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter's cells, which represented the extreme form of human deafness caused by GJB2 gene mutations. In addition, multiple pathological changes in the peripheral auditory nervous system were found, including abnormal innervation, demyelination, and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice. These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice.

Regional differences in cochlear nonlinearity across the basal organ of Corti of gerbil: Regional differences in cochlear nonlinearity

Auditory sensation is based in nanoscale vibration of the sensory tissue of the cochlea, the organ of Corti complex (OCC). Motion within the OCC is now observable due to optical coherence tomography. In a previous study (Cooper et al., 2018), the region that includes the electro-motile outer hair cells (OHC) and Deiters cells (DC) was observed to move with larger amplitude than the basilar membrane (BM) and surrounding regions and was termed the "hotspot." In addition to this quantitative distinction, the hotspot moved qualitatively differently than the BM, in that its motion scaled nonlinearly with stimulus level at all frequencies, evincing sub-BF activity. Sub-BF activity enhances non-BF motion; thus the frequency tuning of the OHC/DC region was reduced relative to the BM. In this work we further explore the motion of the gerbil basal OCC and find that regions that lack significant sub-BF activity include the BM, the medial and lateral OCC, and the reticular lamina (RL) region. The observation that the RL region does not move actively sub-BF (already observed in Cho and Puria 2022), suggests that hair cell stereocilia are not exposed to sub-BF activity in the cochlear base. The observation that the lateral and RL regions move approximately linearly sub-BF indicates that linear forces dominate non-linear OHC-based forces on these components at sub-BF frequencies. A complex difference analysis was performed to reveal the internal motion of the OHC/DC region and showed that amplitude structure and phase shifts in the directly measured OHC/DC motion emerge due to the internal OHC/DC motion destructively interfering with BM motion.

Otoacoustic emissions reveal the micromechanical role of organ-of-Corti cytoarchitecture in cochlear amplification

The intricate, crystalline cytoarchitecture of the mammalian organ of Corti presumably plays an important role in cochlear amplification. As currently understood, the oblique, Y-shaped arrangement of the outer hair cells (OHCs) and phalangeal processes of the Deiters cells serves to create differential "push-pull" forces that drive the motion of the basilar membrane via the spatial feedforward and/or feedbackward of OHC forces. In concert with the cochlear traveling wave, the longitudinal separation between OHC sensing and forcing creates phase shifts that yield a form of negative damping, amplifying waves as they propagate. Unlike active forces that arise and act locally, push-pull forces are inherently directional-whereas forward-traveling waves are boosted, reverse-traveling waves are squelched. Despite their attractions, models based on push-pull amplification must contend with otoacoustic emissions (OAEs), whose existence implies that amplified energy escapes from the inner ear via mechanisms involving reverse traveling waves. We analyze hybrid local/push-pull models to determine the constraints that reflection-source OAEs place on the directionality of cochlear wave propagation. By implementing a special force-mixing control knob, we vary the mix of local and push-pull forces while leaving the forward-traveling wave unchanged. Consistency with stimulus-frequency OAEs requires that the active forces underlying cochlear wave amplification be primarily local in character, contradicting the prevailing view. By requiring that the oblique cytoarchitecture produce predominantly local forces, we reinterpret the functional role of the Y-shaped geometry, proposing that it serves not as a push-pull amplifier, but as a mechanical funnel that spatially integrates local OHC forces.

PARP inhibitor rescues hearing and hair cell impairment in Cx26‐null mice

AbstractGJB2 (encoding connexin26, Cx26) mutation is the most common genetic cause of hereditary deafness. Cochlear sensory hair cell (HC) death is the core pathologic phenomenon of GJB2‐related deafness. However, mechanism‐based therapy is still obscure. A targeted‐cell conditional Gjb2 knockout mouse model was established in which the Cx26 in Deiters cells and pillar cells were knocked out at birth. We explored the mechanism of HC death caused by deficiency of GJB2 in supporting cells (SCs), which exhibited moderate deafness, early HC death without SCs, and a decrease in distortion product otoacoustic emission. Here, a DNA damage response was observed in HCs during the onset of hearing loss. Apoptosis, necroptosis, and ferroptosis do not contribute to the death of HCs. We identified and demonstrated that parthanatos, a poly‐(ADP‐ribose) polymerase (PARP)‐dependent and caspase‐independent form of cell death, is a mechanism of HC degeneration. Furthermore, we observed that the use of PARP inhibitors effectively alleviated both deafness and HC loss. Our study reveals the specific mechanism of HC death caused by GJB2 deficiency. These findings demonstrate that targeting parthanatos is an HC protective strategy in the prevention of GJB2‐related hereditary deafness.

Ca2+ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3-Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Distribution of ciliary adaptor proteins tubby and TULP3 in the organ of Corti.

Tubby-like proteins are membrane-associated adaptors that mediate directional trafficking into primary cilia. In inner ear sensory epithelia, cilia-including the hair cell's kinocilium-play important roles as organizers of polarity, tissue architecture and cellular function. However, auditory dysfunction in tubby mutant mice was recently found to be related to a non-ciliary function of tubby, the organization of a protein complex in sensory hair bundles of auditory outer hair cells (OHCs). Targeting of signaling components into cilia in the cochlea might therefore rather rely on closely related tubby-like proteins (TULPs). In this study, we compared cellular and subcellular localization of tubby and TULP3 in the mouse inner ear sensory organs. Immunofluorescence microscopy confirmed the previously reported highly selective localization of tubby in the stereocilia tips of OHCs and revealed a previously unnoticed transient localization to kinocilia during early postnatal development. TULP3 was detected in the organ of Corti and vestibular sensory epithelium, where it displayed a complex spatiotemporal pattern. TULP3 localized to kinocilia of cochlear and vestibular hair cells in early postnatal development but disappeared subsequently before the onset of hearing. This pattern suggested a role in targeting ciliary components into kinocilia, possibly related to the developmental processes that shape the sensory epithelia. Concurrent with loss from kinocilia, pronounced TULP3 immunolabeling progressively appeared at microtubule bundles in non-sensory Pillar (PCs) and Deiters cells (DC). This subcellular localization may indicate a novel function of TULP proteins associated with the formation or regulation of microtubule-based cellular structures.

Cochlear amplification in the short-wave region by outer hair cells changing organ-of-Corti area to amplify the fluid traveling wave

Many details of the operation of the mammalian cochlea are known, but how they all work together to produce cochlear amplification is not understood. Outer-hair-cell (OHC) motility produces two kinds of amplification: non-propagating amplification (NPA) that is from local OHCs, and traveling-wave amplification (TWA) that increases basilar-membrane (BM) motion. Proposed here are a series of hypotheses that provide a new explanation, the “OoC-area-pump”, for TWA: (1) In the short-wave region OHC vibrations cause cyclic longitudinal motion of fluid in the organ of Corti (OoC) and peri‑Deiters-cell tissue, (2) the longitudinal motion changes the local OoC area, which (3) by reticular-lamina (RL) movement drives the fluid in scala media in a way that amplifies the fluid-pressure traveling wave. (4) At the NPA-TWA changeover frequency, an abrupt change in the OoC frequency-wavenumber relationship is due to positive feedback between TWA and the mode of cochlear motion that is dominant, aided by focusing of the pressure traveling wave. It is hypothesized that OoC radial expansion and radial force from the Deiters-cell phalangeal process act to advance RL and/or lateral-compartment phase. Finally, it is hypothesized that human and lab-animal frequency tuning have similar bandwidths in distance along the cochlea because their traveling-wave wavelengths are similar in the corresponding short-wave regions. Experiments are needed to test these hypotheses and to determine for TWA whether the OoC-area-pump hypothesis replaces or supplements the “OHCs-act-on-BM” hypothesis. Several tests are outlined that can be done with current methodology. A key step in the evolution of mammalian hearing was the development of the complex OoC anatomy, including Deiters cells and OoC fluid spaces that allow local wide-band NPA to produce TWA that enables small local increments of gain to accumulate in the traveling wave and sharpen tuning.

Deiters Cells Act as Mechanical Equalizers for Outer Hair Cells.

The outer hair cells in the mammalian cochlea are cellular actuators essential for sensitive hearing. The geometry and stiffness of the structural scaffold surrounding the outer hair cells will determine how the active cells shape mammalian hearing by modulating the organ of Corti (OoC) vibrations. Specifically, the tectorial membrane and the Deiters cell are mechanically in series with the hair bundle and soma, respectively, of the outer hair cell. Their mechanical properties and anatomic arrangement must determine the relative motion among different OoC structures. We measured the OoC mechanics in the cochleas acutely excised from young gerbils of both sexes at a resolution fine enough to distinguish the displacement of individual cells. A three-dimensional finite element model of fully deformable OoC was exploited to analyze the measured data in detail. As a means to verify the computer model, the basilar membrane deformations because of static and dynamic stimulations were measured and simulated. Two stiffness ratios have been identified that are critical to understand cochlear physics, which are the stiffness of the tectorial membrane with respect to the hair bundle and the stiffness of the Deiters cell with respect to the outer hair cell body. Our measurements suggest that the Deiters cells act like a mechanical equalizer so that the outer hair cells are constrained neither too rigidly nor too weakly.SIGNIFICANCE STATEMENT Mammals can detect faint sounds thanks to the action of mammalian-specific receptor cells called the outer hair cells. It is getting clearer that understanding the interactions between the outer hair cells and their surrounding structures such as the tectorial membrane and the Deiters cell is critical to resolve standing debates. Depending on theories, the stiffness of those two structures ranges from negligible to rigid. Because of their perceived importance, their properties have been measured in previous studies. However, nearly all existing data were obtained ex situ (after they were detached from the outer hair cells), which obscures their interaction with the outer hair cells. We quantified the mechanical properties of the tectorial membrane and the Deiters cell in situ.

Regenerated hair cells in the neonatal cochlea are innervated and the majority co-express markers of both inner and outer hair cells.

After a damaging insult, hair cells can spontaneously regenerate from cochlear supporting cells within the first week of life. While the regenerated cells express several markers of immature hair cells and have stereocilia bundles, their capacity to differentiate into inner or outer hair cells, and ability to form new synaptic connections has not been well-described. In addition, while multiple supporting cell subtypes have been implicated as the source of the regenerated hair cells, it is unclear if certain subtypes have a greater propensity to form one hair cell type over another. To investigate this, we used two CreER mouse models to fate-map either the supporting cells located near the inner hair cells (inner phalangeal and border cells) or outer hair cells (Deiters’, inner pillar, and outer pillar cells) along with immunostaining for markers that specify the two hair cell types. We found that supporting cells fate-mapped by both CreER lines responded early to hair cell damage by expressing Atoh1, and are capable of producing regenerated hair cells that express terminal differentiation markers of both inner and outer hair cells. The majority of regenerated hair cells were innervated by neuronal fibers and contained synapses. Unexpectedly, we also found that the majority of the laterally positioned regenerated hair cells aberrantly expressed both the outer hair cell gene, oncomodulin, and the inner hair cell gene, vesicular glutamate transporter 3 (VGlut3). While this work demonstrates that regenerated cells can express markers of both inner and outer hair cells after damage, VGlut3 expression appears to lack the tight control present during embryogenesis, which leads to its inappropriate expression in regenerated cells.

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 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 that 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 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 mechanoelectrical transduction 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 (which 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 modeling 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.

The Acute Effects of Furosemide on Na-K-Cl Cotransporter-1, Fetuin-A and Pigment Epithelium-Derived Factor in the Guinea Pig Cochlea.

BackgroundFurosemide is a loop diuretic used to treat edema; however, it also targets the Na-K-Cl cotransporter-1 (NKCC1) in the inner ear. In very high doses, furosemide abolishes the endocochlear potential (EP). The aim of the study was to gain a deeper understanding of the temporal course of the acute effects of furosemide in the inner ear, including the protein localization of Fetuin-A and PEDF in guinea pig cochleae.Material and MethodAdult guinea pigs were given an intravenous injection of furosemide in a dose of 100 mg per kg of body weight. The cochleae were studied using immunohistochemistry in controls and at four intervals: 3 min, 30 min, 60 min and 120 min. Also, cochleae of untreated guinea pigs were tested for Fetuin-A and PEDF mRNA using RNAscope® technology.ResultsAt 3 min, NKCC1 staining was abolished in the type II fibrocytes in the spiral ligament, followed by a recovery period of up to 120 min. In the stria vascularis, the lowest staining intensity of NKCC1 presented after 30 min. The spiral ganglion showed a stable staining intensity for the full 120 min. Fetuin-A protein and mRNA were detected in the spiral ganglion type I neurons, inner and outer hair cells, pillar cells, Deiters cells and the stria vascularis. Furosemide induced an increased staining intensity of Fetuin-A at 120 min. PEDF protein and mRNA were found in the spiral ganglia type I neurons, the stria vascularis, and in type I and type II fibrocytes of the spiral ligament. PEDF protein staining intensity was high in the pillar cells in the organ of Corti. Furosemide induced an increased staining intensity of PEDF in type I neurons and pillar cells after 120 min.ConclusionThe results indicate rapid furosemide-induced changes of NKCC1 in the type II fibrocytes. This could be part of the mechanism that causes reduction of the EP within minutes after high dose furosemide injection. Fetuin-A and PEDF are present in many cells of the cochlea and probably increase after furosemide exposure, possibly as an otoprotective response.

Microstructural interactions contribute to the hotspot in the living cochlea

The mechanism of the active cochlea relies on a complex interaction between microstructures in the organ of Corti. A significant longitudinal vibration "hotspot" was recently observed in the high-frequency region of the living gerbil cochlea between the Deiters cells and the outer hair cells. A similar phenomenon was also found in guinea pigs with a relatively smaller magnitude. The cause is unknown, but one hypothesis is that this phenomenon is due to the structural constraints between different microstructures. It is not easy to explain the mechanism of hotspots directly from experimental observations. It may also be difficult to image or test if the hotspot will occur in the low-frequency region in the cochlea. We built two three-dimensional finite element models corresponding to the high- and low-frequency regions in the guinea pig cochlea. Responses of the organ of Corti to passive acoustic and outer hair cell electrical excitation were calculated. The two excitations were then superimposed to predict the active response of the organ of Corti. The hotspot phenomenon in the experiment was reproduced and analyzed in-depth about influencing factors. Our results indicate that hotspots appear in the low-frequency region of the cochlea as well. We hypothesize that the hotspot is a locally originated phenomenon in the cochlea, and the traveling wave further enhances the response to low-frequency excitation. The movement of outer hair cells inclined in the longitudinal direction is the leading cause of the hotspot.

Observation in inner ear of tree shrew using scanning electron microscope and the Atoh1 distribution in cochlea.

This study aimed to observe the ultrastructure on the surface of the inner ear of a normal tree shrew using scanning electron microscope (SEM). The specimens of cochlea, macula utriculi, macula sacculi, and crista ampullaris of the normal adult tree shrew were collected and observed by SEM. We used immunofluorescence for cochlear protein Atoh1 staining. We observed that cochlea of the tree shrew is centered on the cochlear axis, circling about 3.5 times from bottom to top of the cochlea. The organ of Corti is located between medial and lateral grooves, including inner and outer hair cells as well as supporting cells. Maculae staticae include macula of saccule and macula of utricle, and the surface of macula is covered with a large number of otoliths. We found a gelatinous layer below the otoliths, followed by the layer of the honeycomb structure. The hair cell cilia of macula and crista ampullaris include one kinocilium and more stereocilia. There is no obvious cross structure but numerous hair cell cilia on semicircular canal crista ampullaris. Immunofluorescence staining showed that protein Atoh1 is mainly distributed in the nucleus of the cochlea's inner and outer hair cells. The observation of the inner ear structure under SEM elucidate the fine surface morphological structure of the entire cochlea, the vestibular maculae staticae, and crista ampullaris, providing new insight into the structure and function of the inner ear of tree shrew. HIGHLIGHTS: This article is the first to describe the inner ear ultrastructure of a small primate tree shrew by scanning electron microscopy (SEM). Under an SEM, the phalangeal processes of Deiter cells in tree shrews were observed to be connected to the tip of a neighboring hair cell, which was different from that of Deiters' cells in guinea pigs, and this crossed one hair cell, and connected to the tip of the third hair cell. It was observed that the crista ampullaris of tree shrews were horseshoe-shaped, and similar to that of humans and monkeys, this had no obvious "cross-shaped hump" structure. Tree shrew's ABR threshold value curve conforms to the mammalian U-shaped curve, wave III is the main wave of ARB, its sensory frequency may be higher 8 kHz, and the characteristics of the stereocilia of tree shrew we have observed may be related to the perception of higher frequency hearing.

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration.

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

ATP and ACh Evoked Calcium Transients in the Neonatal Mouse Cochlear and Vestibular Sensory Epithelia.

Hair cells in the mammalian inner ear sensory epithelia are surrounded by supporting cells which are essential for function of cochlear and vestibular systems. In mice, support cells exhibit spontaneous intracellular Ca2+ transients in both auditory and vestibular organs during the first postnatal week before the onset of hearing. We recorded long lasting (>200 ms) Ca2+ transients in cochlear and vestibular support cells in neonatal mice using the genetic calcium indicator GCaMP5. Both cochlear and vestibular support cells exhibited spontaneous intracellular Ca2+ transients (GCaMP5 ΔF/F), in some cases propagating as waves from the apical (endolymph facing) to the basolateral surface with a speed of ∼25 μm per second, consistent with inositol trisphosphate dependent calcium induced calcium release (CICR). Acetylcholine evoked Ca2+ transients were observed in both inner border cells in the cochlea and vestibular support cells, with a larger change in GCaMP5 fluorescence in the vestibular support cells. Adenosine triphosphate evoked robust Ca2+ transients predominantly in the cochlear support cells that included Hensen’s cells, Deiters’ cells, inner hair cells, inner phalangeal cells and inner border cells. A Ca2+ event initiated in one inner border cells propagated in some instances longitudinally to neighboring inner border cells with an intercellular speed of ∼2 μm per second, and decayed after propagating along ∼3 cells. Similar intercellular propagation was not observed in the radial direction from inner border cell to inner sulcus cells, and was not observed between adjacent vestibular support cells.