Deiters Cells Research Articles

Cell-cell junctions: a target of acoustic overstimulation in the sensory epithelium of the cochlea

BackgroundExposure to intense noise causes the excessive movement of the organ of Corti, stretching the organ and compromising sensory cell functions. We recently revealed changes in the transcriptional expression of multiple adhesion-related genes during the acute phases of cochlear damage, suggesting that the disruption of cell-cell junctions is an early event in the process of cochlear pathogenesis. However, the functional state of cell junctions in the sensory epithelium is not clear. Here, we employed graded dextran-FITC, a macromolecule tracer that is impermeable to the organ of Corti under physiological conditions, to evaluate the barrier function of cell junctions in normal and noise-traumatized cochlear sensory epithelia.ResultsExposure to an impulse noise of 155 dB (peak sound pressure level) caused a site-specific disruption in the intercellular junctions within the sensory epithelium of the chinchilla cochlea. The most vulnerable sites were the junctions among the Hensen cells and between the Hensen and Deiters cells within the outer zone of the sensory epithelium. The junction clefts that formed in the reticular lamina were permeable to 40 and 500 but not 2,000 kDa dextran-FITC macromolecules. Moreover, this study showed that the interruption of junction integrity occurred in the reticular lamina and also in the basilar membrane, a site that had been considered to be resistant to acoustic injury. Finally, our study revealed a general spatial correlation between the site of sensory cell damage and the site of junction disruption. However, the two events lacked a strict one-to-one correlation, suggesting that the disruption of cell-cell junctions is a contributing, but not the sole, factor for initiating acute sensory cell death.ConclusionsImpulse noise causes the functional disruption of intercellular junctions in the sensory epithelium of the chinchilla cochlea. This disruption occurs at an early phase of cochlear damage. Understanding the role of this disruption in cochlear pathogenesis will require future study.

Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.

Deiters cells tread a narrow path—The Deiters cells-basilar membrane junction—

Deiters cells extend from the basilar membrane to the reticular lamina and, together with pillar cells and outer hair cells, structurally define the micro-architecture of the organ of Corti. Studying vibrotome sections of the mouse organ of Corti with confocal and scanning electron microscopy we found that the basal pole of every Deiters cell, independently of their position in the organ of Corti and along the cochlear spiral, attached to the basilar membrane within a 15.1 ± 0.3 μm-wide stripe running the length of the cochlear spiral adjacent to the row of outer pillar cells. All Deiters cells' basal poles had similar diameter and general morphology, and distributed on the stripe in a precise arrangement with a center-to-center distance of 7.1 ± 0.3 μm between neighbor cells of the same row and 5.9 ± 0.4 μm for neighbor cells in adjacent rows. Complete detachment of Deiters cells revealed an elliptical imprint on the top surface of the basilar membrane consisting of a smaller central structure with a very smooth surface surrounded by a rougher area, suggesting the presence of two different anchoring junctions. These previously unidentified morphological features of Deiters cells could be critical for the mechanical response of the organ of Corti.

Age-DependentIn VivoConversion of Mouse Cochlear Pillar and Deiters' Cells to Immature Hair Cells by Atoh1 Ectopic Expression

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

A Novel Atoh1 “Self-Terminating” Mouse Model Reveals the Necessity of Proper Atoh1 Level and Duration for Hair Cell Differentiation and Viability

Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

TAK1 Expression in the Cochlea: A Specific Marker for Adult Supporting Cells

Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker's organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters' cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.

Expression and Function of Sox21 During Mouse Cochlea Development

The development of the inner ear is an orchestrated process of morphogenesis with spatiotemporally controlled generations of individual cell types. Recent studies have revealed that the Sox gene family, a family of evolutionarily conserved HMG-type transcriptional factors, is differentially expressed in each cell type of the mammalian inner ear and plays critical roles in cell-fate determination during development. In this study, we examined the expression pattern of Sox21 in the developing and adult murine cochlea. Sox21 was expressed throughout the sensory epithelium in the early otocyst stage but became restricted to supporting cells during adulthood. Interestingly, the expression in adults was restricted to the inner phalangeal, inner border, and Deiters' cells: all of these cells are in direct contact with hair cells. Evaluations of the auditory brainstem-response revealed that Sox21(-/-) mice suffered mild hearing impairments, with an increase in hair cells that miss their appropriate planar cell polarity. Taken together with the previously reported critical roles of SoxB1 families in the morphogenesis of inner ear sensory and neuronal cells, our results suggest that Sox21, a counteracting partner of the SoxB1 family, controls fine-tuned cell fate decisions. Also, the characteristic expression pattern may be useful for labelling a particular subset of supporting cells.

Localization of fatty acid binding proteins (FABPs) in the cochlea of mice

Various fatty acids (FAs) are involved in many different functions in the organism as a source of energy, as essential ingredients of membranous lipids as well as intracellular signaling molecules. Intracellular fatty acid binding proteins (FABPs) comprise a family of soluble lipid binding proteins with low molecular masses and which can make long chain FAs soluble to allow intracellular translocation in the aqueous cytosol. To clarify the possible involvement of FAs and FABPs in hearing function, the present study investigated the localization of FABPs in the cochlea of adult mice using immunohistochemical procedures. Among various FABP species, H (heart-type)-FABP was localized in inner and outer pillar cells and outer phalangeal cells, while B (brain-type)-FABP was localized in border cells and cells of Hensen, and fibrocytes in the spiral limbus and spiral prominence. In the spiral ganglion, moderate to low H-FABP immunoreactivity was observed in almost all neurons, while B-FABP immunoreactivity was found in satellite cells. The discrete localization of the two FABPs in different non-receptor cells in the Organ of Corti suggests that the FABP species and/or their ligands, FAs, play important roles in the regulation of the hearing function.

Is halofantrine ototoxic? Experimental study on guinea pig cochlea model

Halofantrine is a newly developed antimalarial drug used for the treatment of Plasmodium falciparum malaria. The introduction of this drug has been delayed because of its possible side effects, and due to insufficient studies on adverse reactions in humans. There have been no studies investigating its effect on hearing. Thirty guinea pigs were divided into three groups: a control group, a halofantrine therapeutic dose group and a halofantrine double therapeutic dose group. One cochlea specimen from each animal was stained with haematoxylin and eosin and the other with toluidine blue. No changes were detected in the control group. The halofantrine therapeutic dose group showed loss and distortion of inner hair cells and inner phalangeal cells, and loss of spiral ganglia cells. In the halofantrine double therapeutic dose group, the inner and outer hair cells were distorted and there was loss of spiral ganglia cells. Halofantrine has mild to moderate pathological effects on cochlea histology, and can be considered an ototoxic drug.

Calcium transients evoked by ATP in the cells of the mouse organ of Corti

Event Abstract Back to Event Calcium transients evoked by ATP in the cells of the mouse organ of Corti Máté Aller1*, Tamás Horváth1, Gábor Polonyi2, Ádám Fekete1, György Halmos3, Attila Heinrich1, Beáta Sperlágh1, Balázs Lendvai4, E. S. Vizi1 and Tibor Zelles1 1 HAS, Institute of Experimental Medicine, Hungary 2 Semmelweis University, Department of Oto-rhino-laryngology, Hungary 3 University Medical Center , Department of Otolaryngology, Netherlands 4 Gedeon Richter Ltd., Hungary The process of converting the mechanical sound waves into electrical signals is developed by the co-work of functionally different subunits of the inner ear. The organ of Corti is the effective transducer, where the functions of the different cell types are modulated by ATP, which generates [Ca2+]i changes in these cells. We have developed a method of fluorescence calcium imaging in acute hemicochlea preparation, using dissected mature mouse cochlea that keeps the original tissue organization. After bulk loading the preparation with the calcium dye Fura-2/AM, we could evoke repeatable calcium transients in the inner hair cells, pillar cells, Deiters cells, and Hensen cells by perfusion of ATP. Here, we show that simultaneously measured basal [Ca2+]i differs in the specialized cells of the organ of Corti of 15-21 days old mice. We observed the lowest basal [Ca2+]i in Deiters cells (47 nM ± 3 nM, n=87), and the highest in Hensen cells (87 nM ± 5 nM, n=71). Application of 50M ATP caused calcium transients with the lowest peak in inner hair cells (103 nM ± 9 nM, n=35), and the highest in Hensen cells (245 nM ± 13 nM, n=71). The response was dose-dependent in the applied 1M to 100M ATP range. In order to get more information about the purinergic receptors involved, we also applied ATP during a perfusion of PPADS, and performed RT-PCR analysis of P2X and P2Y receptors in the organ of Corti. To specify whether these calcium transients are connected more to internal stores or they are depending on the external source, we applied CPA, the specific inhibitor of Ca2+-ATPase of the internal stores, and calcium free buffer together with the ATP. Remarkable differences could be observed between the different cell types. By using nifedipine, we also found that L-type calcium channels possibly plays a role in the ATP evoked calcium transients in the Hensen cells. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Sensory and motor systems Citation: Aller M, Horváth T, Polonyi G, Fekete Á, Halmos G, Heinrich A, Sperlágh B, Lendvai B, Vizi ES and Zelles T (2010). Calcium transients evoked by ATP in the cells of the mouse organ of Corti. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00060 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2010; Published Online: 21 Apr 2010. * Correspondence: Máté Aller, HAS, Institute of Experimental Medicine, Budapest, Hungary, allerm@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Máté Aller Tamás Horváth Gábor Polonyi Ádám Fekete György Halmos Attila Heinrich Beáta Sperlágh Balázs Lendvai E. S Vizi Tibor Zelles Google Máté Aller Tamás Horváth Gábor Polonyi Ádám Fekete György Halmos Attila Heinrich Beáta Sperlágh Balázs Lendvai E. S Vizi Tibor Zelles Google Scholar Máté Aller Tamás Horváth Gábor Polonyi Ádám Fekete György Halmos Attila Heinrich Beáta Sperlágh Balázs Lendvai E. S Vizi Tibor Zelles PubMed Máté Aller Tamás Horváth Gábor Polonyi Ádám Fekete György Halmos Attila Heinrich Beáta Sperlágh Balázs Lendvai E. S Vizi Tibor Zelles Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Organ of Corti Micromachine Enables Hair Bundles to Deform the Stiff Basilar Membrane

The organ of Corti (OC) is a highly organized structure in the mammalian cochlea that houses the sensory hair cells. It is believed that the OC functions to optimize force transmission from the outer hair cell (OHC) to the basilar membrane and the inner hair cell. Recent studies reveal that the OC cannot be considered as a rigid body and has a complex mode of deformation. We developed a 3-D finite element model of the OC to dissect its mechanics. Geometric and mechanical information was taken from the gerbil cochlea at 2 and 10 mm from the stapes, positions encoding high and low frequencies respectively, and in each case several hundred microns longitudinal extent was simulated. The model included all structurally significant components: OHCs, pillar cells, Deiters cells and reticular lamina. The model was validated by reproducing experimental results on point stiffness and longitudinal space constant measured at the basilar membrane and response to current steps through the OC. Deformation of the OC by two different active OHC forces (the OHC somatic force and the stereociliary-based force) was then simulated. A surprising result was that despite smaller magnitude, the stereociliary-based force (0.1and 0.7 nN at apex and base) was nearly as effective as the somatic force (10 nN) in displacing the basilar membrane. The results also suggested that for the active forces to work efficiently the radial stiffness of the tectorial membrane must be comparable to or greater than the hair bundle stiffness. Funded by NIH RO1 DC01362.

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

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

Cell-Specific Inducible Gene Recombination in Postnatal Inner Ear Supporting Cells and Glia

Recent studies indicate that supporting cells play important roles in inner ear development, function, and regeneration after injury, but the molecular mechanisms underlying these processes remain poorly understood. Inducible cell-specific gene recombination in supporting cells could be a powerful tool to study the roles of specific molecules in these cells. Here we tested the feasibility, effectiveness, and cell specificity of inducible Cre-mediated gene recombination in the postnatal inner ear using mice that express an inducible form of Cre (CreER(T)) under the transcriptional control of the proteolipid protein (PLP) promoter. We assessed the pattern of tamoxifen-induced gene recombination in the inner ear using the ROSA26-LacZ reporter line, in which the beta-galactosidase gene is expressed only after Cre-mediated excision of a loxP-flanked stop cassette. Recombination was detected in cochlear inner phalangeal cells, supporting cells surrounding hair cells in vestibular maculae and cristae. Recombination also occurred in Schwann cells. We also found that this CreER(T) line can be used to increase and decrease the levels of expression of a trophic factor, brain-derived neurotrophic factor, specifically in supporting cells. These results show that PLP/CreER(T) mice are a powerful tool to dissect gene function in inner ear supporting cells.

PTEN attenuates PIP 3/Akt signaling in the cochlea of the aging CBA/J mouse

We have previously reported the activation of cell death pathways in the sensory cells of the aging cochlea. Here we investigate age-associated changes in survival mechanisms focusing on phosphatidylinositol 3,4,5-trisphosphate (PIP 3)/Akt signaling. The animal model is the CBA/J mouse of 18 months of age prior to the onset of major functional loss (ABR thresholds, 26 ± 8 dB SPL) which is compared to young animals of 3 months of age (ABR thresholds, 19 ± 7 dB SPL). Immunostaining on cochlear cryosections revealed a wide-spread distribution of PIP 3 in the cochlea which was markedly attenuated in old animals in inner and outer hair cells, Deiters cells and pillar cells. Protein levels of the lipid phosphatase PTEN which regulates PIP 3 increased in those cells with aging while its mRNA did not, suggesting an age-related reduction of PTEN degradation. Furthermore, staining intensity of phosphorylated PTEN (ser380) and its nuclear localization increased. Consistent with a reduction of PIP 3, the phosphorylation of the downstream target Akt at threonine 308 significantly decreased in outer hair cells. The results suggest a decline of the survival capacity of aging outer hair cells due to a decrease in PIP 3/Akt signaling caused by an increase of PTEN.

Cochlear injuries induced by the combined exposure to noise and styrene

Workers exposed to industrial solvents are also frequently exposed to mechanical noise. In this study, a combination of a continuous noise (100 dB SPL) and an impact noise (110 dB SPL) was used to mimic the noise exposure in the workplace. A noise band of 10–20 kHz was used to induce a cochlear injury in the same cochlear region in the rat as styrene exposure. Styrene levels of 300 and 400 mg/kg were applied to induce outer hair cell (OHC) loss limited to the third row of the middle turn, but without significant cochlear functional loss. The combined exposures of the noise and styrene for 3 weeks caused greater threshold shifts than the noise alone, although the styrene alone did not induce significant threshold shift. Correspondingly, the combined exposures induced OHC losses that were greater than the summated OHC losses induced by the noise and styrene exposure alone. Apoptosis in Deiters cells was also examined after a short-term exposure (7 days) to a combined exposure of a high-level styrene (800 mg/kg) and the noise. The styrene-noise synergistic interaction was also observed in the Deiters cells. The synergistic interaction between the noise and styrene suggests that each of the exposures alone (noise or styrene) may cause stress, temporary alteration, or nonlethal injury in cochlear cells and the combined exposure strengthens the stress leading to cell death.

Unique expression of connexins in the human cochlea

Mutations in the genes GJB2 and GJB6, which encode the proteins Connexin 26 (Cx26) and Connexin 30 (Cx30), have been linked to nonsyndromic prelingual deafness in humans. These proteins may form so-called gap junctions (GJ) or transcellular pathways between cells. The pathogenesis of deafness due to GJ Connexin mutations remains unclear partly because examinations performed in the human ear are infrequent. Here we analysed the expression and distribution of Cx26 and Cx30 in five fresh normal human cochleae taken out at occasional surgery. Immunohistochemistry including confocal microscopy in decalcified specimen showed that these proteins are widely expressed in the human cochlea. In the lateral wall there was strong antibody co-labeling for Cx26 and Cx30 that support the existence of channels comprising heteromeric Cx26/Cx30 connexons. In the organ of Corti there were some co-labeling in the supporting cell area including mainly the Claudius cells and Deiter cells of these two Cxs, apart from isolated Cx26 and Cx30 labeling in the same area, suggestive of both homomeric/homotypic pattern and hybrid pattern (heteromeric or heterotypic). Cx30, Cx26 and Connexin 36 (Cx36) immunoreactivity was also associated with spiral ganglion type I neurons, the latter being a gap junction protein specific to neurons. Gap–junction-based electrical synapses are not known to occur in mammalian auditory system other than in bats where they may play a role for fast electrical nerve transmission useful for echolocation. Their potential role in the processing of human auditory nerve signaling as well as non-GJ roles of the connexins in human cochlea is discussed.

Notch signaling and Hes labeling in the normal and drug-damaged organ of Corti

During the development of the inner ear, the Notch cell signaling pathway is responsible for the specification of the pro-sensory domain and influences cell fate decisions. It is assumed that Notch signaling ends during maturity and cannot be reinitiated to alter the fate of new or existing cells in the organ of Corti. This is in contrast to non-mammalian species which reinitiate Delta1–Notch1 signaling in response to trauma in the auditory epithelium, resulting in hair cell regeneration through transdifferentiation and/or mitosis. We report immunohistochemical data and Western protein analysis showing that in the aminoglycoside-damaged guinea pig organ of Corti, there is an increase in proteins involved in Notch activation occurring within 24 h of a chemical hair cell lesion. The signaling response is characterized by the increased presence of Jagged1 ligand in pillar and Deiters cells, Notch1 signal in surviving supporting cell nuclei, and the absence of Jagged2 and Delta-like1. The pro-sensory bHLH protein Atoh1 was absent at all time points following an ototoxic lesion, while the repressor bHLH transcription factors Hes1 and Hes5 were detected in surviving supporting cell nuclei in the former inner and outer hair cell areas, respectively. Notch pathway proteins peaked at 2 weeks, decreased at 1 month, and nearly disappeared by 2 months. These results indicate that the mammalian auditory epithelium retains the ability to regulate Notch signaling and Notch-dependent Hes activity in response to cellular trauma and that the signaling is transient. Additionally, since Hes activity antagonizes the transcription of pro-sensory Atoh1, the presence of Hes after a lesion may prohibit the occurrence of transdifferentiation in the surviving supporting cells.

Identification and characterization of pannexin expression in the mammalian cochlea

The gap junction in vertebrates is encoded by the connexin gene family. Recently, a new gene family termed pannexin (Panx) has been identified in vertebrates and found to encode gap junctional proteins as well. To date, three pannexin isoforms (Panx1, 2, and 3) have been cloned from mouse and human genomes. In this study, expression of pannexins in the mouse and rat cochlea was investigated. Polymerase chain reaction and Western blot analysis showed that all three pannexin isoforms were expressed in the cochlea. Immunofluorescent staining showed that Panx1 expression was extensive. In the organ of Corti, Panx1 labeling was found in supporting cells, including pillar cells, Hensen cells, Claudius cells, and Boettcher cells. Both surface plaque-like punctate labeling and diffuse-cytoplasmic labeling were visible. However, the labeling was weak and rare in Deiters cells. No labeling was found in the hair cells. Intense labeling for Panx1 was also observed in the interdental cells in the spiral limbus, the inner and outer sulcus cells, and the type II fibrocytes in the spiral prominence and central region in the cochlear lateral wall. In addition, Panx1 labeling was detectable in Reissner's membrane and strial blood vessel cells. Panx2 labeling was restricted to the basal cells in the stria vascularis and was also detectable in the spiral ganglion neurons. However, no overlapping labeling for Panx1 and Panx2 was observed. Finally, Panx3 labeling was exclusively observed in the cochlear bone. Thus, Panx1, 2, and 3 are abundantly expressed in the mammalian cochlea and demonstrate distinct cellular distributions. Like connexins, they may play an important role in hearing.

Endocochlear potential depends on Cl− channels: mechanism underlying deafness in Bartter syndrome IV

Human Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a beta-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in mice now reveals that the positive potential, but not the high potassium concentration, of the scala media depends on the presence of these channels in the epithelium of the stria vascularis. The reduced driving force for K(+)-entry through mechanosensitive channels into sensory hair cells entails a profound congenital hearing loss and subtle vestibular symptoms. Although retaining all cell types and intact tight junctions, the thickness of the stria is reduced early on. Cochlear outer hair cells degenerate over several months. A collapse of endolymphatic space was seen when mice had additionally renal salt and fluid loss due to partial barttin deletion in the kidney. Bsnd(-/-) mice thus demonstrate a novel function of Cl(-) channels in generating the endocochlear potential and reveal the mechanism leading to deafness in human Bartter syndrome IV.

Mitochondrial dysfunction disrupts trafficking of Kir4.1 in spiral ganglion satellite cells

The inward-rectifier K(+) channel Kir4.1 is responsible for maintaining cochlear homeostasis and restoring neural excitability. The large-conductance calcium-activated K(+) channel (BK(Ca)) plays a key role in phase locking signals in the mammalian inner ear. To evaluate the influence of mitochondrial dysfunction on the expression and subcellular localization of these channels, 3-nitropropionic acid (3-NP) was administered to rat round window membranes for 30 min. Auditory brainstem response was measured both before and 2 hr after 3-NP administration. Immunofluorescent confocal microscopy was used to measure the expression and subcellular localization of Kir4.1 and BK(Ca). Alexa Fluor 568-labeled bovine serum albumin (BSA) was applied to round window membranes as a tracer to explore the cochlear distribution of drug delivery and was detected in the lateral wall, spiral ganglion, cochlear nerve, and organ of Corti. Hearing loss of 23 (+/-4.4 SE) and 58 (+/-6.7 SE) dB developed in rats treated with 0.3 and 0.5 mol/liter of 3-NP, respectively. BK(Ca) was visualized in the cellular membrane and cytoplasm in the upper and middle region of inner hair cells, and it was not affected by 3-NP. Kir4.1 was detected in intermediate cells of the stria, Deiter's cells, and spiral ganglion satellite cells. Kir4.1 failed to reach the perineural cytoplasm of the satellite cells after 3-NP treatment. The results of this study suggest that mitochondrial dysfunction disrupts trafficking of Kir4.1 in spiral ganglion satellite cells.