Calcium signaling in interdental cells during the critical developmental period of the mouse cochlea.

The tectorial membrane (TM), a complex acellular structure that covers part of the organ of Corti and excites outer hair cells, is required for normal hearing. It consists of collagen fibrils and various glycoproteins, which are synthesized in embryonic and postnatal development by different cochlear cell types including the interdental cells (IDCs). At its modiolar side, the TM is fixed to the apical surfaces of IDCs, which form the covering epithelium of the spiral limbus. We performed confocal membrane imaging and Ca2+ imaging in IDCs of the developing mouse cochlea from birth to postnatal day 18 (P18). Using the fluorescent membrane markers FM 4-64 and CellMask™ Deep Red on explanted whole-mount cochlear epithelium, we identified the morphology of IDCs at different z-levels of the spiral limbus. Ca2+ imaging of Fluo-8 AM-loaded cochlear epithelia revealed spontaneous intracellular Ca2+ transients in IDCs at P0/1, P4/5, and P18. Their relative frequency was lowest on P0/1, increased by a factor of 12.5 on P4/5 and decreased to twice the initial value on P18. At all three ages, stimulation of IDCs with the trinucleotides ATP and UTP at 1 and 10 µM elicited Ca2+ transients of varying amplitude and shape. Before the onset of hearing, IDCs responded with robust Ca2+ oscillations. At P18, after the onset of hearing, ATP stimulation either caused Ca2+ oscillations or an initial Ca2+ peak followed by a plateau while the UTP response was unchanged from that at pre-hearing stage. Parameters of spontaneous and nucleotide-evoked Ca2+ transients such as amplitude, decay time and duration were markedly reduced during cochlear development, whereas the kinetics of the Ca2+ rise did not show relevant changes. Whether low-frequency spontaneous Ca2+ transients are necessary for the formation and maintenance of the tectorial membrane e.g. by regulating gene transcription needs to be elucidated in further studies.

Otogelin, otogelin-like, and stereocilin form links connecting outer hair cell stereocilia to each other and the tectorial membrane.

The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.

Molecular organization and fine structure of the human tectorial membrane: is it replenished?

Auditory sensitivity and frequency resolution depend on the physical properties of the basilar membrane in combination with outer hair cell-based amplification in the cochlea. The physiological role of the tectorial membrane (TM) in hair cell transduction has been controversial for decades. New insights into the TM structure and function have been gained from studies of targeted gene disruption. Several missense mutations in genes regulating the human TM structure have been described with phenotypic expressions. Here, we portray the remarkable gradient structure and molecular organization of the human TM. Ultrastructural analysis and confocal immunohistochemistry were performed in freshly fixed human cochleae obtained during surgery. Based on these findings and recent literature, we discuss the role of human TMs in hair cell activation. Moreover, the outcome proposes that the α-tectorin-positive amorphous layer of the human TM is replenished and partly undergoes regeneration during life.

Microstructures in the organ of Corti help outer hair cells form traveling waves along the cochlear coil.

According to the generally accepted theory of mammalian cochlear mechanics, the fluid in the cochlear scalae interacts with the elastic cochlear partition to generate transversely oscillating displacement waves that propagate along the cochlear coil. Using a computational model of cochlear segments, a different type of propagating wave is reported, an elastic propagating wave that is independent of the fluid-structure interaction. The characteristics of the propagating wave observed in the model, such as the wavelength, speed, and phase lag, are similar to those observed in the living cochlea. Three conditions are required for the existence of the elastic propagating wave in the cochlear partition without fluid-interaction: 1), the stiffness gradient of the cochlear partition; 2), the elastic longitudinal coupling; and 3), the Y-shaped structure in the organ of Corti formed by the outer hair cell, the Deiters cell, and the Deiters cell phalangeal process. The elastic propagating waves in the cochlear partition disappeared without the push-pull action provided by the outer hair cell and Deiters cell phalangeal process. The results suggest that the mechanical feedback of outer hair cells, facilitated by the organ of Corti microstructure, can control the tuning and amplification by modulating the cochlear traveling wave.

Vibration responses of the organ of Corti and the tectorial membrane to electrical stimulation.

Coupling of somatic electromechanical force from the outer hair cells (OHCs) into the organ of Corti is investigated by measuring transverse vibration patterns of the organ of Cori and tectorial membrane (TM) in response to intracochlear electrical stimulation. Measurement places at the organ of Corti extend from the inner sulcus cells to Hensen's cells and at the lower (and upper) surface of the TM from the inner sulcus to the OHC region. These locations are in the neighborhood of where electromechanical force is coupled into (1) the mechanoelectrical transducers of the stereocilia and (2) fluids of the organ of Corti. Experiments are conducted in the first, second, and third cochlear turns of an in vitro preparation of the adult guinea pig cochlea. Vibration measurements are made at functionally relevant stimulus frequencies (0.48-68 kHz) and response amplitudes (<15 nm). The experiments provide phase relations between the different structures, which, dependent on frequency range and longitudinal cochlear position, include in-phase transverse motions of the TM, counterphasic transverse motions between the inner hair cell and OHCs, as well as traveling-wave motion of Hensen's cells in the radial direction. Mechanics of sound processing in the cochlea are discussed based on these phase relationships.

Tectorial membrane material properties in Tecta(Y)(1870C/+) heterozygous mice.

The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the α-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In Tecta(Y)(1870C/+) mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed a decrease in fixed charge concentration from -6.4±1.4 mmol/L in wild-types to -2.1±0.7 mmol/L in Tecta(Y)(1870C/+) TMs. TMs from Tecta(Y)(1870C/+) mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by 10±1.6 dB in Tecta(Y)(1870C/+) mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a corresponding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed.

The tectorial membrane: anatomical, biomechanical, and histological analysis.

There is minimal information in the literature regarding the tectorial membrane. Further, information in the literature regarding the anatomy and function of this structure is often contradictory. We performed the current study to elucidate further this structure's detailed anatomy, function, and histology. Thirteen adult cadavers underwent dissection of their tectorial membranes and detailed observations and measurements were made of them. Ranges of motion of the craniocervical junction were performed before and after transection of this structure. Histological analysis was performed on all membranes. The tectorial membrane was found to attach much more superiorly than previously described and was found to be firmly adherent to the cranial base and body of the axis but not to the posterior aspect of the odontoid process. The mean thickness of this membrane was found to be 1 mm. Flexion of the head made the tectorial membrane fully taut at 15 degrees and extension made it fully taut at 20 degrees; however, there was a buckling effect (redundant tectorial membrane) noted at the level of the odontoid process in extension. With the alar and transverse ligaments cut and with flexion of the head, the middle portion of this membrane was stretched over the odontoid process, thus acting as a "hammock" that inhibited the odontoid process from moving posteriorly. The tectorial membrane did not limit cervical flexion per se but rather helped to insure that the odontoid process did not impinge into the cervical canal. Lateral flexion was not found to be limited by this structure. Histologically, parallel collagen fibers with spindle-shaped fibrocytes were observed within this membrane and near its attachment to the posterior axis, the collagen fibers were noted to be more homogenous with larger non-spindled fibrocytes. At the cranial attachment of the tectorial membrane, multiple calcified areas were noted that interdigitated with the underlying bone. Also near this cephalic bony attachment, there was an increase in the number of elastic fibers, which were found running parallel with the surrounding Type III collagen fibers. The tectorial membrane was found to attach much more superiorly than previously described. We would propose that the tectorial membrane provides for a second line of defense, preventing the odontoid process from compressing the spinal cord and by doing so, secondarily limits movement of the craniocervical juncture. This hypothesis is strengthened by the finding of many elastic fibers in the tectorial membrane. To our knowledge, our study is the first to perform a detailed histological analysis of the tectorial membrane. We hope that these data are useful to the clinician who investigates this ligament of the craniocervical region.

Abnormal cochlea linked to deafness in transgenic mice expressing human cytokeratin K8.

The cytokeratin intermediate filaments have a relevant role in the proliferation and differentiation processes of epithelial cells. To provide information about the role of K8 cytokeratin during the auditory receptor differentiation, two groups of adult mice were used: TGK8-4 transgenic and control animals. The TGK8-4 transgenic mice contained 12 kb of K8 human cytokeratin (HK8) locus (Casanova et al., 1995, 1999). The functional activity of the auditory receptor was analyzed by auditory thresholds. Morphological studies demonstrate that the auditory receptors of the TGK8-4 transgenic mice are highly immature. Immunocytochemical studies were made by using two monoclonal antibodies: CAM 5-2 (recognizing K8 human cytokeratin) and Troma-1 (recognizing both mouse and human K8 cytokeratin). These demonstrated significant differences between the auditory receptors of the transgenic mice and the control mice. These functional and morphological differences clearly suggest that K8 cytokeratin has a relevant role during the differentiation and tridimensional organization of the sensory and the supporting cells of the auditory receptor.

Active nonlinear mechanics of the organ of Corti including the stereocilia-tectorial membrane complex.

There is a large amount of knowledge about the different components of the organ of Corti (OC), but little is known about how these components act together in vivo. To clarify the complex mechanical behavior of the OC, anatomic results are carefully analyzed and used to develop a finite element model of a short section of OC, which includes 8 outer hair cells (OHC) and their supporting structures. The modal analysis shows the frequency-dependent phase reversal of the radial stereocilia displacement. The transient computation confirms the amplification of OC displacements when the ability of the OHC to contract and elongate is considered. The inclusion of a nonlinear function describing the mechanoelectrical transduction in OHC amplifies and distorts the displacement of the OC when it is stimulated by a sinusoidal input pressure function. These results are in agreement with other psychoacoustic, electrophysiologic and otoacoustic measurements.

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane.

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a "major" and "minor" tectorial membrane (Lim, Hear Res 1986;22:117-146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified. In the present study, a novel monoclonal antibody (TC2) that recognizes a native epitope on glycosaminoglycans enriched in chondroitin-4-sulfate revealed a transient and restricted expression in the developing gerbil TM. The localization patterns suggest that Deiters' and pillar cells secrete a TC2-positive matrix prior to birth that later becomes incorporated into the marginal band and superior layer (cover net) of the TM. The developmental timecourse and patterns of TC2 reactivity suggest that this molecule may play a critical role in the fusion of the minor TM with the major TM.

Postnatal maturation of the organ of Corti in gerbils: morphology and physiological responses.

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period.

Development of the tectal cells in the mouse cochlea.

Tectal cells appear at birth in the outer part of the developing organ of Corti. At first they are attached to the basilar membrane, but later they ascend through the auditory epithelium. During the 1st postnatal week (coinciding with the development of the minor tectorial membrane), the newly formed tectal cells show several cytological characteristics suggesting increased metabolic and secretory activities, which include: (1) a large Golgi complex, (2) abundant amorphous material inside the cisterns of rough endoplasmic reticulum, and (3) dense granules inside the mitochondrial matrix. All these features gradually disappear, and by the 14th postnatal day the tectal cells show a dark cytoplasm and few and short microvilli. In addition, tectal cells were stained selectively by some lectins. These findings suggest that tectal cells may participate in the secretion of some components of the minor tectorial membrane, different from those produced by Deiters' cells, Hensen's cells and pillar cells.

Incorporation of D3H glucosamine to the adult and developing cochlear tectorial membrane of normal and hypothyroid rats.

The uptake of D-3H-glucosamine by the developing cochlea of normal and hypothyroid rats was examined using light microscopic radioautography. During postnatal development, normal and hypothyroid rat cochleas exhibited a layer of radiolabelling in the tectorial membrane (TM). This layer first appeared in the TM region which covers the spiral limbus and the Kölliker's organ (KO), then progressively reached the apical part of the TM covering the organ of Corti. Radiolabelling was significantly greater in hypothyroid than in normal cochleas. These findings suggests that the enormous size reached by the TM in the congenital hypothyroidism could be related to an increase of epithelial secretion, at least for carbohydrates. It also suggests that TM, in normal and hypothyroid cochleas, could be formed during development by the addition of successive layers. Older layers could be displaced upwards by the new ones. Cochleas of normal young adult rats, treated with D-3H-glucosamine, showed a very scarce and diffuse radiolabelling. Cochleas of hypothyroid young adult rats exhibited a thickened and distorted TM, which incorporated a significant amount of carbohydrates. These results suggest that TM secretion is highly reduced in young adult normal animals, while in young adult hypothyroid ones it is still active. During cochlear maturation, thyroxine seems to be necessary, not only for the synthesis of normal glycoproteins (as suggested by previous reports), but also for the control of glycoprotein secretion.

Tissue culture of the organ of Corti.

In 1975, Sobkowicz et al. (1) described long-term organotypic cultures of the organ of Corti of the newborn mouse. This paper provides detailed methods for dissection and maintenance of the isolated organ of Corti with its corresponding segment of spiral ganglion in culture. Descriptions and illustrations of cellular characteristics of the developing organ are carefully documented. The work is based on 19 years of experience and over one thousand cultures. Review of the literature and the application of the technique to research on the inner ear are provided.

Pilocarpine elicits the interdental cell secretory activity of the inner ear.

Subcutaneous injection of pilocarpine in guinea pigs resulted in the following ultrastructural changes: 1) the apical cavities of the interdental cells were filled with a substance indistinguishable from the overlying amorphous layer of the TM; 2) a great number of spherical structures appeared over the limbal portion of the tectorial membrane. In TEM photomicrographs these structures displayed the same appearance as the amorphous layer of the TM and were usually continuous to it; 3) the number of holes that decorate the upper surface of the limbal portion of the TM was dramatically increased and it was found that they connect the endolymphatic space to the apical cavities of the interdental cells; 4) there was an increase in the number of the small extracellular vesicles found in the clear spaces of the tectorial membrane. These facts suggest that pilocarpine stimulates the secretion of the interdental cells, confirming the existence of the secretory processes previously described (Prieto et al., 1990). These findings can be related to the turnover of the TM in the adult animal and, perhaps, to the secretion of some organic compound to the endolymph. We postulate that the actions of pilocarpine on the interdental cells are most probably mediated by the activation of muscarinic acetylcholine receptors in these cells.

Localization of anionic sulfate groups in the tectorial membrane.

Colloidal iron hydroxide (CIH) staining demonstrates the existence of anionic sulfate groups of glycoconjugates associated with several constituents of the tectorial membrane (TM). In the adult animal, labelling in the main body of the TM appears as long, electron-dense patches surrounding type A fibrils which show alternating stained and unstained zones. On the other hand, labelling of the fibrils of the matrix of the TM appears as single, CIH particles with no special arrangement. Some of the structurally distinct regions of the TM are also labelled (limbal zone, Hensen's stripe and inner portions of the cover net), while others are not (marginal band and outer portions of the cover net). Staining of type A fibrils in the major TM is already present in newborn animals; while, both the outermost region of the TM closest to the cells of the organ of Kölliker and the minor TM are not labelled. The implications of these distributions of sulfated glycoconjugates for the electrochemical properties of the TM are discussed.

Development of the tectorial membrane.

During development, the organization of the stereociliary bundles undergoes drastic changes from the microvilli-like nascent stereocilia to the 'W' formation of the step-like arrangement of the adult form. During this period the developing tectorial membrane (TM) establishes prescribed attachments with various substructures of the developing sensory ciliary bundles and supporting cells. The TM detaches from the supporting cells and inner hair cell stereociliary bundles as Kölliker's organ matures. The inter-connecting linkage system develops postnatally, and the 'tip-linkages' are already found in one-week-old mice, suggesting that the critical organization of the micromechanics of the stereocilia matures rapidly during the postnatal period. The TM develops in stages, and its development parallels that of the organ of Corti. The major TM is initially secreted by the greater epithelial ridge cells, and the minor TM is produced by the lesser epithelial ridge cells. The substructures of the TM are formed by the participation of a number of different supporting cells. During the active stage of production of the substructures by the supporting cells, these cells are intensely Alcian blue-PAS stained, indicating that the glycoconjugates are locally produced by these cells.

Guinea pig tectorial membrane profile in an in vitro cochlear preparation.

The guinea pig cochlea was examined under high-magnification light microscopy in an in vitro preparation. After extraction of the otic capsule, the bulla was opened widely and a small hole made into the fourth turn of the scala vestibuli. The organ of Corti was visualized under artificial endolymph at 600 X magnification. Added 1-micron titanium dioxide particles settled on the upper surface of the transparent tectorial membrane. Particle positions showed that much of this upper surface lay in a flat sheet that extended centrifugally almost to the Hensen's cells, giving the impression it was attached there. The sheet extended at least to the level of the inner hair cells, where a tectorial membrane thickness of about 40 micron was reached. Titanium dioxide particles were seen regularly in immediate proximity to the hair cell cilia, indicating that scala media is continuous with the subtectorial space. Upon mechanical manipulation, Hensen's cells proved to be extremely cohesive and elastic. It is suggested that hair cell stereocilia provide major mechanical connections for the tectorial membrane.

The developmental appearance of a major basilar papilla-specific protein in the chick.

The sensory epithelium of the chick cochlea, the basilar papilla, contains a major protein of approximately 23,000 daltons. This protein was as abundant as actin in the papilla, yet could not be found in significant quantities in any other cochlear tissue. The protein appeared at a time in development when other studies have shown that the chick embryo develops peripheral auditory competence. These observations suggest a role for this protein in cochlear function.