E-cadherin in the mature and developing organ of Corti of the mouse

Summary The reticular lamina of the mammalian cochlea is formed by the tightly joined apical surfaces of hair cells and supporting cells. This lamina creates a barrier separating endolymph and perilymph, two fluids of different composition. The preservation of this fluid barrier is a requirement for cochlear function. This study aimed to determine whether the calcium-dependent, cell adhesion molecule, E-cadherin was appropriately placed both temporally and spatially to contribute to the formation and maintenance of the reticular lamina. Cochleas aged E15 to P31 were stained immunocytochemically for E-cadherin. In the P31 organ of Corti, E-cadherin is localized to the apical intercellular junctions of supporting cells, but is absent from supporting cell-hair cell borders. During development, E-cadherin is present only on the apices and lateral edges of those cells which will eventually lie adjacent to fluid spaces — pillar, outer hair and Deiters cells. The molecule disappears from the lateral cell membranes at about the time in development that fluid spaces form. These data suggest that E-cadherin plays a role in maintaining the reticular lamina by mediating inter-supporting cell adhesion and raise the possibility that fluid space opening in the organ of Corti is facilitated by the down-regulation or redistribution of E-cadherin.     

Postnatal development of the organ of Corti in dominant-negative Gjb2 transgenic mice

Hereditary hearing loss is one of the most prevalent inherited human birth defects, affecting one in 2000. A strikingly high proportion (50%) of congenital bilateral nonsyndromic sensorineural deafness cases have been linked to mutations in the GJB2 coding for the connexin26. It has been hypothesized that gap junctions in the cochlea, especially connexin26, provide an intercellular passage by which K(+) are transported to maintain high levels of the endocochlear potential essential for sensory hair cell excitation. We previously reported the generation of a mouse model carrying human connexin26 with R75W mutation (R75W+ mice). The present study attempted to evaluate postnatal development of the organ of Corti in the R75W+ mice. R75W+ mice have never shown auditory brainstem response waveforms throughout postnatal development, indicating the disturbance of auditory organ development. Histological observations at postnatal days (P) 5-14 were characterized by i) absence of tunnel of Corti, Nuel's space, or spaces surrounding the outer hair cells, ii) significantly small numbers of microtubules in inner pillar cells, iii) shortening of height of the organ of Corti, and iv) increase of the cross-sectional area of the cells of the organ of Corti. Thus, morphological observations confirmed that a dominant-negative Gjb2 mutation showed incomplete development of the cochlear supporting cells. On the other hand, the development of the sensory hair cells, at least from P5 to P12, was not affected. The present study suggests that Gjb2 is indispensable in the postnatal development of the organ of Corti and normal hearing.     

Cytologic structures unique to deiters cells of the cochlea

Deiters cells in the gerbil cochlea disclosed unusual ultrastructural features. A sharp transition zone separated the cell body underlying outer hair cells into an upper compartment with numerous organelles and a lower part devoid of structures other than the microtubule stalk. Deiters cells exhibited a unique structure, the rosette complex, which consisted of a core of densely fibrillar trabeculae, enclosed in a filamentous meshwork and surrounded by tubulocisternal endoplasmic reticulum. The dense trabeculae radiated in columns downward from an apical translucent area toward a lucent zone around the nucleus. They also spread to the medial plasmalemma enveloping nerves and upward into the base of the phalanx. Frequent, small Golgi complexes bordered the tubular reticulum. The distinctive mitochondria of Deiters cells frequently paralleled the plasmalemma, revealed an elongated, often arched profile, and contained sparse, longitudinally aligned cristae. The stalk, composed of characteristic microtubule bundles resembling those in pillar cells, ascended from basal to apical plasmalemma of the cell body and into the phalanx and reticular lamina as previously described. The stalk appeared also to ramify into smaller microtubule bundles in apical cytosol penetrating the rosette complex. Nuclei in Deiters cells differed from those in hair cells in their location high in the cell and in showing chromatin dispersion indicative of more active protein synthesis. © 1993 Wiley-Liss, Inc.     

Delayed formation of actin filaments in the outer pillar head plate of VASP-/- mice

Pillar cells with their rich network of tubulin and actin filaments have been reported to contribute to the rigidity of the organ of Corti. As the earliest expression of the actin filament enhancer vasodilator-stimulated phosphoprotein (VASP) in the outer pillar head plate has been found to be associated with the onset of hearing, we tested hearing development in VASP-/- compared to wild-type mice. Performing measurements of auditory brainstem responses on postnatal days (P) P14 and P21, we detected statistically significantly higher thresholds in VASP-/- compared to wild-type mice at P14, but no hearing differences at P21. Staining for prestin and synaptophysin at P12 in morphologically regularly developed cochleae of VASP-/- mice provided an immature prestin protein pattern but no evidence of developmental delay in hair cell innervations. Regularly intense staining of actin filaments in the outer pillar head plate was found only in wild-type but not in VASP-/- mice at P14. At P21, intensive actin filament staining was also observed in the outer pillar head plates of VASP-/- mice. The delayed hearing development in VASP-/- mice is supposed to be caused by a delayed formation of actin filaments in the outer pillar head plate indicating the importance of appropriate pillar cell stiffness in cochlear mechanics.     

Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti.

Previous studies have demonstrated the importance of FGF signaling at several stages in the development of the cochlea. At early stages of embryogenesis, Fgfr1, Fgfr2, and several FGFs are critical for both the induction of the otic vesicle and the initial development of the sensory epithelium. At late stages of cochlear development, Fgfr3 is necessary for the development of the tunnel of Corti. To determine the stage of development when Fgfr3 is required, we examined the expression of Fgfr3 and Fgf8 at various developmental stages. We also re-examined the Fgfr3 -/- mouse with additional markers for developing supporting cells. We confirmed the previous analysis of the Fgfr3 -/- mice, indicating that there are deficiencies in support cell differentiation. Specifically, we find that the inner pillar cell never develops, while the outer pillar cell is stalled in its differentiation. In addition, we found an extra row of outer hair cells, and accompanying Deiters' cells, in the apical two thirds of the organ of Corti in the Fgfr3 mutant. Thus, in addition to controlling the fate decision between pillar cells and Deiters' cells, we find that Fgfr3 also regulates the width of the sensory epithelium.     

Immunoelectron microscopy identifies several types of GABA-containing efferent synapses in the guinea-pig organ of Corti

Using an immunoperoxidase technique, we have localized by light and electron microscopy GABA-immunostained fibers within a component of the efferent innervation of the organ of Corti. At the light microscopic level, GABA-immunostained fibers were observed within the inner spiral bundle (below the inner hair cells) and the tunnel spiral bundle. The immunostaining was clearly more intense in the upper turns than in the basal turns. Mostly in the upper turns, GABA-immunostained fibers were seen crossing the tunnel of Corti to reach the outer hair cells where they formed large immunostained patches at the base of the cells. Unevenly distributed throughout these upper turns, immunostained fibers were seen climbing along the outer hair cells and traveling near the non-sensorineural Hensen's cells. The electron microscopic observations of GABA-immunostained fibers in the upper turns allowed us to identify within the inner spiral bundle vesiculated varicosities synapsing with radial dendrites connected to the inner hair cells. In the outer hair cell area, the GABA-immunostained fibers made several kinds of synaptic contacts. They included a minor population of the large axosomatic synapses with the basal pole of the outer hair cells and many axodendritic synapses with the spiral dendrites connected to these cells. Occasionally, the GABA-immunostained climbing fibers also synapsed with the outer hair cells at a supranuclear level. These result confirm previous light microscopic data dealing with the projection of the GABA-immunostained fibers along the cochlear partition. Moreover, they extend them in characterizing several kinds of GABA-immunostained synapses. These latter findings agree with previous neurochemical electrophysiological data which suggests an efferent neurotransmitter role for GABA. Nevertheless, such an existence of an efferent innervation predominantly projecting to the upper turns of the cochlea adds another criterion distinguishing the "apical" from the "basal" cochlea.     

Choline acetyltransferase (ChAT) immunoelectron microscopy distinguishes at least three types of efferent synapses in the organ of Corti

Summary Using anatomical criteria, the olivo-cochlear fibers ending in the organ of Corti (efferent fibers) have recently been separated into two systems: a lateral system innervating principally the inner hair cell (IHC) area and a medial system innervating mainly the outer hair cells (OHCs). Electrophysiological and biochemical experiments suggest that acetylcholine may be a neurotransmitter of these efferent fibers. However, efferent synapses that use acetylcholine as neurotransmitter have not yet been identified at the electron microscopic level. Using a pre-embedding immunoelectron microscopic technique with a monoclonal antibody against choline acetyltransferase (ChAT), we localized ChAT-immunostained fibers below both the IHCs and OHCs. In the inner spiral bundle, one type of ChAT-immunostained fibers was vesiculated and formed axo-dendritic synapses with the afferent auditory dendrites contacting the inner hair cells. A second type of ChAT-immunostained fibers seen in the inner spiral bundle was unvesiculated. Unstained vesiculated varicosities synapsing with the auditory dendrites were also seen in the inner spiral bundle. At the OHC level, ChAT immunostaining was found in nearly all the terminals synapsing with the OHCs. The finding of two types of ChAT-immunostained efferent synapses in the organ of Corti, i.e. axodendritic synapses in the inner spiral bundle and axosomatic synapses with the OHCs, supports the hypothesis that both the lateral and the medial olivocochlear systems use acetylcholine as a neurotransmitter. The finding of numerous unstained synapses in the inner spiral bundle, and some below OHCs, together with previous data about putative cochlear neurotransmitters, suggests the possibility of additional non-cholinergic olivo-cochlear systems. It might soon appear useful to reclassify efferents according to the nature of the different neurotrans-mitters/ co-transmitters found in the various efferent synapses of the organ of Corti.     

Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment.

Deletion of fibroblast growth factor receptor 3 (Fgfr3) leads to hearing impairment in mice due to defects in the development of the organ of Corti, the sensory epithelium of the Cochlea. To examine the role of FGFR3 in auditory development, cochleae from Fgfr3(-/-) mice were examined using anatomical and physiological methods. Deletion of Fgfr3 leads to the absence of inner pillar cells and an increase in other cell types, suggesting that FGFR3 regulates cell fate. Defects in outer hair cell differentiation were also observed and probably represent the primary basis for hearing loss. Furthermore, innervation defects were detected consistent with changes in the fiber guidance properties of pillar cells. To elucidate the mechanisms underlying the effects of FGFR3, we examined the expression of Bmp4, a known target. Bmp4 was increased in Fgfr3(-/-) cochleae, and exogenous application of bone morphogenetic protein 4 (BMP4) onto cochlear explants induced a significant increase in the outer hair cells, suggesting the Fgf and Bmp signaling act in concert to pattern the cochlea.     

Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: reponse to loss of cell partners in the Bronx waltzer mouse

This report is concerned with control of cell shaping, positioning, and cytoskeletal integration in a highly ordered cochlear neuroepithelium. It is largely based on investigations of events that occur during abnormal morphogenesis of the organ of Corti in the Bronx waltzer (bv/bv) mutant mouse. The organ's sensory hair cells and adjacent supporting cells ordinarily construct a spatially elaborate and supracellularly integrated cytoskeletal framework. Large microtubule bundles are connected to cytoskeletal components in neighbouring cells by actin-containing meshworks that link them to substantial arrays of adherens junctions. In bv/bv mice, degeneration and loss of most inner hair cells and outer pillar cells occurs during organ development. These cells flank each side of a row of inner pillar cells that respond by upregulating assembly of their actin-containing meshworks. This only occurs in surface regions where they no longer contact cell types involved in construction of the cytoskeletal framework. The meshworks are larger and exhibit a more extensive sub-surface deployment than is normally the case. Hence, assembly of intercellular cytoskeletal connecting components can proceed without contact with appropriate cell neighbours but termination of assembly is apparently subject to a negative feedback control triggered by successful completion of intercellular connection with the correct cell neighbours. In addition, inner pillar cells compensate for loss of cell neighbours by interdigitating and overlapping each other more extensively than is usually the case to increase opportunities for generating adherens junctions. Certain adherens junctions in the organs of +/+ and bv/bv mice exhibit features that distinguish them from all previously described cell junctions. The dense plaques on their cytoplasmic faces are composed of aligned ridges. We suggest that they are called ribbed adherens junctions. Perturbations of cell shaping and positioning indicate that loss of inner hair cells is the primary consequence of the bv mutation. Most of the other abnormalities can be understood in terms of a secondary sequence of morphogenetic aberrations (precipitated by loss of inner hair cells). These aberrations provide new information about the ways in which supporting cells help to control hair cell positioning.     

Abnormal auditory brainstem responses and cochlear pathology in rats induced by an exaggerated styrene exposure regimen.

Groups of 12 male 42-day-old rats were exposed to 0 or 800 ppm styrene vapors for 14 hr/day, 5 days/week for 3 weeks. Tone-pip auditory brainstem responses (ABRs) at 4, 8, 16, and 30 kHz were obtained after the last exposure. ABRs were minimally affected at 4 kHz and moderately to severely affected at 8, 16, and 30 kHz as indicated by waveforms which had a decreased amplitude and increased latency as compared to the controls. Missing outer hair cell(s) were evident in the basal and lower middle turns of the organ of Corti. Outer hair cell loss was least in the first row and greatest in the second and third rows. Occasional inner hair cells were also missing in regions of severe outer hair cell loss. The distribution of hair cell loss within the cochlea was consistent with the pattern of ABR alterations. These data document mid-frequency auditory dysfunction in styrene-exposed young adult rats with significant damage to the organ of Corti following an exaggerated styrene exposure regimen.     

Regional specialization of the hair cell plasmalemma in the organ of corti

Nonjunctional and nonsynaptic membranes of hair cells in the chinchilla organ of Corti were examined using the freeze-fracture technique. Cy-toplasmic leaflets of the apical membranes of hair cells have particles, 6–12 nm in diameter, but many more particles are found on apical membranes of outer hair cells than on inner hair cells. Cytoplasmic leaflets of the lateral membranes of outer hair cells are covered with large particles, but the corresponding regions of the inner hair cell membrane have fewer particles and these are small or medium-sized. Two types of particle aggregate also distinguish this region of the inner hair cell. The first consists of patches of particles closely spaced in rec tilinear arrays. The second consists of parallel strands of widely spaced large particles similar in size, but not in distribution, to the large particles on the lateral membranes of the outer hair cells. The basal membrane of outer hair cells is distinguished from that of inner hair cells by plaques consisting of cross-hatched in-cisures in the external membrane leaflet. While the significance of these anatomical features is not yet apparent, they give the hair cell plasmalemma a richness of regional specialization found in few other cells and suggest that there are important functional differences between inner and outer hair cells.     

Tectorial membrane-organ of Corti relationship during cochlear development

The development of stereociliary attachment to the tectorial membrane was investigated in the mouse cochlea using transmission and scanning electron microscopy. At the 18th gestational day, only the major tectorial membrane can be identified covering the greater epithelial ridge and the inner hair cells in all turns. At the 19th gestational day, the minor tectorial membrane was first seen in the basal turn, over the outer hair cells. During early stages of development, the stereocilia of hair cells were surrounded by a loose fibrillar material underneath the tectorial membrane. After the 10th postnatal day, the outer hair cells' stereocilia were attached to Kimura's (or Hardesty's) membrane, while inner hair cells' stereociliary bundles were attached to the undersurface of the tectorial membrane near the Hensen's stripe. Between the 10th and the 14th postnatal days, the space between the inner hair cells and the first row of outer hair cells widened by virtue of the growth of the heads of pillar cells, and the inner hair cells' stereocilia were displaced towards the Hensen's stripe. After the 14th postnatal day, the inner hair cells' stereociliary bundles detached from the tectorial membrane, while the outer hair cells' stereocilia remained attached to it. The tip-link system, which connects the tips of the stereocilia to the next tallest stereocilia, is present at birth in the outer hair cells. The marginal pillar, that anchored the tectorial membrane to the underlying organ of Corti during development, first appeared on the 6th postnatal day and disappeared on the 14th–15th postnatal day. The present data together with other reports support the idea that although some structures, such as hair cells' stereocilia and innervation, are already formed early during development, the cochlear microarchitecture is not fully developed morphologically and ready to function normally until the end of the second postnatal week in the mouse.     

Stereocilia and tectorial membrane development in the rat cochlea

Summary Maturation of the rat cochlea, from postnatal days 2 to 60, was studied using scanning electron microscopyt (SEM), with emphasis on stereocilia and tectorial membrane (TM). Two days after birth, the organ of Corti was very immature. An adult appearance of its surface was observed by day 16 in the basal turn, and by the end of the 3rd postnatal week in the apex. Stereocilia started their development first on inner hair cells. By contrast, the apical pole of outer hair cells ended its maturation before that of inner hair cells. Top-links were detected very early in inner hair cell stereociliary development (postnatal day 2). Marginal pillars temporarily attached the TM to the organ of Corti; they disappeared first in the apical region. This transient attachment seems to play a role in the coupling of outer hair cells to the TM, as prints of their longest stereocilia appeared at the undersurface of the TM by the same time. Moreover, these prints were more clear and regular at the base than at the apex of the cochlea. Results are discussed in relation to ultrastructural and functional data on rat cochlea maturation.     

Potentiation of cochlear hair cell loss by acoustic stimulus and gentamicin in the guinea pig

We explored the possibility of synergism between a pure-tone stimulus and gentamicin in causing cochlear injury by analyzing hair cell loss. Guinea pigs receiving daily injections of gentamicin (200 mg/kg body wt.) for 1 week were exposed to a 2 kHz tone (95 dB SPL, 2 hours daily). Surface preparations of the spiral organ were studied by phase contrast microscopy, and the extent of hair cell loss in the entire organ of Corti was recorded in cytocochleograms. Gentamicin by itself was slightly ototoxic, damaging the innermost row of outer hair cells, whereas exposure to sound alone caused no hair cell loss. Combined antibiotic and acoustic exposure produced extensive cochlear damage. A few animals showed massive hair cell degeneration and collapse of the organ of Corti, except in the apical turn. The site of damage was possibly determined by the frequency of the sound stimulus. Thus, an intermittent tonal stimulus such as that used in the present experiment can be harmless by itself, but causes injury to cochlear hair cells in guinea pigs when administered in combination with gentamicin.     

Regulation of electromotility in the cochlear outer hair cell

Mechanosensory outer hair cells play an essential role in the amplification of sound-induced vibrations within the mammalian cochlea due to their ability to contract or elongate following changes of the intracellular potential. This unique property of outer hair cells is known as electromotility. Selective efferent innervation of these cells within the organ of Corti suggests that regulation of outer hair cell electromotility may be the primary function of the efferent control in the cochlea. A number of studies demonstrate that outer hair cell electromotility is indeed modulated by the efferent neurotransmitter, acetylcholine. The effects of acetylcholine on outer hair cells include cell hyperpolarization and a decrease of the axial stiffness, both mediated by intracellular Ca²⁺. This article reviews these results and considers other potential mechanisms that may regulate electromotility, such as direct modification of the plasma membrane molecular motors, alteration of intracellular pressure, and modification of intracellular chloride concentration.     

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.     

Tunnel crossing fibers and their synaptic connections within the inner hair cell region in the organ of corti in the maturing mouse

 Combined ultrastructural and immunocytochemical studies reveal that in the adolescent 12- to 17-day-old mouse the afferent tunnel crossing fibers that innervate outer hair cells receive synaptic contacts from three distinct sources: the GABAergic fibers (GABA= gamma-aminobutyric acid) of the lateral olivocochlear bundle, the non-GABAergic efferent tunnel crossing fibers, and the inner hair cells themselves. The GABAergic fibers give off collaterals that synapse with the afferent tunnel fibers as they cross the inner hair cell region. These collaterals also form synapses with afferent radial dendrites that are synaptically engaged with the inner hair cells. Vesiculated varicosities of nonGABAergic efferent tunnel fibers also synapse upon the outer spiral afferents. Most of this synaptic activity occurs within the inner pillar bundle. Distinctive for this region are synaptic aggregations in which several neuronal elements and inner hair cells are sequentially interconnected. Finally, most unexpected were the afferent ribbon synapses that inner hair cells formed en passant on the shafts of the apparent afferent tunnel fibers. The findings indicate that: (1) the afferent tunnel (i.e., outer spiral) fibers may be postsynaptic to both the inner and the outer hair cells; (2) the non-GABAergic efferent and the afferent tunnel fibers form extensive synaptic connections before exiting the inner pillar bundle; (3) the GABAergic component of the lateral olivocochlear system modulates synaptically both radial and outer spiral afferents. Accepted: 7 May 1998     

Ultrastructure indicative of ion transport in tectal,Deiters, and tunnel cells: differences between gerbil and chinchilla basal and apical cochlea

Ultrastructural examination revealed an epithelium of about five tectal cells (TCs) roofing the outer tunnel (OT) in the mid to upper, but not the basal, region of gerbil and chinchilla cochlea. Structures in TCs that are apparently specialized for retrieval of K(+) released into tunnel fluid from outer hair cells (OHCs) include surface fimbriae in the gerbil and canalicular reticulum in the chinchilla. A tunnel roof of organelle-rich TCs appeared to be better equipped for ion resorption than a roof composed of organelle-poor Hensen cells (HCs). Fimbriae, filopodia, and the cell body of TCs descended to contact the third Deiters cell (DC3) in the gerbil, and the hypertrophied DC3 phalanx rose to contact TCs in the chinchilla, which suggests a solute exchange between TCs and DCs. Previously unrecognized structures that are speculated to provide ATP ligand for cochlear purinoreceptors occurred in the chinchilla DC and gerbil TC. The observation of a microtubule stalk in DCs indicated that they also function in cochlear mechanics. A newly delineated lateral tunnel cell (LTC) intervened between the DC3 and HC in both species. The apicomedial plasmalemma of all DCs fitted closely to the base of OHCs and enveloped afferent nerves. The morphologic specializations reported here provide further support for the proposed transcellular lateral flow route for K(+) currents generated by sound exposure and neural activity. The previously demonstrated expansion of Boettcher cells, outer sulcus cell roots, type Il and IV fibrocytes, and apical microvilli on HCs and Claudius cells (CCs) in the base of the cochlea is postulated here to mediate a basal parallel current that could supply the increased K(+) transport required for the basally elevated electric potential (EP).     

Late developmental changes of the innervation densities of the myelinated fibres and the outer hair cell efferent fibres in the rat cochlea

The baso-apical distributions of the myelinated nerve fibres (representative for the inner hair cell afferent fibres) and the outer hair cell efferent fibres were studied during postnatal development of the rat cochlea. The myelinated fibres were counted in the primary osseos spiral lamina from semi-thin sections. The outer hair cell efferent fibres were counted in the tunnel of Corti by means of ultra-thin sections. The developmental changes of the myelinated fibres were investigated between 8 and 60 days after birth (DAB); those of the outer hair cell efferent fibres between 20 and 30 DAB. Between 12 DAB (onset of hearing) and 20 DAB the baso-apical distribution of the myelinated fibres does not change. Striking maturational changes occur late after the onset of hearing, between 20 and 30 DAB. The innervation density of the myelinated fibres increases in the lower middle region of the cochlea. In this region a maximum of innervation density appears. The efferent fibres to the outer hair cells show at 20 DAB a maximum of innervation density in the middle of the cochlea but between 20 and 30 DAB, the fibre density decreases in this region. During the same period the maximum of innervation density shifts towardsthe base. The change in the innervation densities of the myelinated fibres and the outer hair cell efferent fibres occurs late in development, after the onset of hearing, and after the organ of Corti shows an adult-like appearance.