Postnatal development of the organ of Corti in cats: a light microscopic morphometric study

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.     

Vital staining of the hearing organ: visualization of cellular structure with confocal microscopy

Cells inside the intact organ of Corti were labelled with fluorescent probes reflecting various aspects of structure and function. The dyes were introduced into the perilymphatic space by perfusion of the scala tympani of the temporal bone from the guinea-pig maintained in isolation. The dyes were able to diffuse through the basilar membrane and into the organ of Corti where they were spontaneously absorbed by the sensory and supporting cells. Confocal microscopic observation was made through an opening in the apex of the cochlea. A number of different dyes were used; a carbocyanine dye which stains mitochondria; two styryl dyes which are absorbed by the cell membranes and calcein, a cytoplasmic marker that fluoresces in vital cells. Extracellular space was stained by a cell-impermeant Dextran fluorescein. The most striking finding was that the membrane dyes preferentially stained the sensory cells and neural elements whereas the staining of the supporting cells was faint. The cytoplasmic dye in general stained sensory and supporting cells to the same extent. By tilting the organ, a view could be obtained from the side like a radial section through the organ. Outer and inner hair cells with their sensory hairs, nerve fibres and nerve endings, especially under the inner hair cells, could be seen in profile. Introduction of a high molecular weight Dextran into the endolymphatic space outlined the tectorial membrane which was seen in negative contrast. The simultaneous perfusion with a membrane dye stained the hair cells and their sensory hairs. Merging of the two images gave the possibility to examine, in the living tissue, the cilia to tectorial membrane relationship. Of general interest is the finding that the membrane dyes preferentially stained the sensory and neural elements of the nervous system, represented here by the hair cells and nerve fibres of the inner ear.     

Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?

In the adult mammalian cochlea, post-injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper-sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post-treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre-existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear-shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper-sensitivity to antibiotic.     

The level dependence of response phase: observations from cochlear hair cells

Hair cell responses are recorded from third turn of the guinea pig cochlea in order to define the relationship between hair cell depolarization and position of the basilar membrane. Because the latter is determined locally, using the cochlear microphonic recorded in the organ of Corti (OC) fluid space, no corrections are required to compensate traveling wave and/or synaptic delays. At low levels, inner hair cells (IHC) depolarize near basilar membrane velocity to scala vestibuli reflecting the free standing nature of their stereocilia. At high levels, the time of depolarization changes rapidly from velocity to scala vestibuli to the scala tympani phase of the basilar membrane response. This change in response phase, recorded in the fundamental component of the IHC response, is associated with a decrease in response magnitude. The absence of this behavior in OC and outer hair cell responses implies that basilar membrane mechanics may not be responsible for these response patterns. Because these features are reminiscent of the magnitude notches and the large phase shifts observed in single unit responses at high stimulus levels, they provide the IHC correlates of these phenomena.     

Quantitative measures reflect degeneration, but not regeneration, in the deafness mouse organ of Corti

The deafness mouse (dn/dn) is a well known model of hereditary deafness uncomplicated by behavioral and motor disturbances. The organ of Corti in this mouse develops a normal complement of sensory and supporting cell structures, yet animals homozygous for this gene never demonstrate any hearing capacity. They are profoundly deaf from birth. Soon after development, the organ of Corti rapidly degenerates, most sensory cells having vanished by 50 days of age. Published observations have suggested that apical regions of the organ of Corti may regenerate some supporting cell structures by 90 days of age. We have quantified changes in organ of Corti structure from 15 to 130 days of age using several different measures. Measures of peak height and total cross-sectional area. as well as a subjective rating scale, all demonstrate consistent degenerative changes during this time period. No evidence for regeneration of supporting or sensory cell structures is noted, although a surprising degree of variability is present in all regions of the organ of Corti which may account for previous claims.     

Time course of nerve-fiber regeneration in the noise-damaged mammalian cochlea

The time course of events which are essential for nerve-fiber regeneration in the mammalian cochlea was determined using a group of chinchillas that had been exposed for 3.5 hr to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. The animals recovered from 40 min (0 days) to 100 days at which times their inner ears were fixed and the organs of Corti prepared for phase-contrast and bright-field microscopy as plastic-embedded flat preparations. Selected areas identified in the flat preparations were semi-thick and thin sectioned at radial or tangential angles for examination by bright-field and transmission electron microscopy. The following time-ordered events appeared critical for nerve-fiber regeneration: (1) The area of the basilar membrane in which regeneration had a possibility of occurring showed signs of severe injury. Outer hair cells degenerated first followed by outer pillars, inner pillars, inner hair cells and other supporting cells; (2) Myelinated nerve fibers in the osseous spiral lamina became fragmented, starting at the distal ends of the fibers. This degeneration gradually extended back to Rosenthal's canal; (3) Fibrous processes, originating from Schwann-like cells in the osseous spiral lamina, extended laterally on the basilar membrane; (4) Schwann cells lined up medial to the habenulae perforata in the areas of severest damage, apparently ready to migrate through the habenulae onto the basilar membrane; (5) Schwann-cell nuclei appeared on the basilar membrane beneath the developing layer of squamous epithelium which was in the process of replacing the degenerated portion of the organ of Corti; (6) Regenerated nerve fibers with thin myelin sheaths or a simple investment of Schwann cell cytoplasm appeared in areas of total loss of the organ of Corti; and (7) The myelin sheaths on the regenerated nerve fibers gradually became thicker.     

Labyrinthine anomalies in trisomy 13 mosaicism

The temporal bone histopathology of a 2-month-old male with a bilateral cleft palate, cleft lip and polydactylia due to trisomy 13 mosaicism was studied. Anomalies in the ear involved a partial absence of the superior semicircular canal, an abnormally wide lateral semicircular canal, underdeveloped cochlear coils, an abnormally broad modiolus, an undeveloped organ of Corti and an underdeveloped basilar membrane in the basal turn of the cochlea, an unusually wide cochlear aqueduct, and deformed stapedial crura.     

The development of the reticular lamina in the hamster: an examination of transitory features and their functional roles

This study examines the development of the reticular lamina in the Syrian golden hamster postriatally from birth to adulthood at 2 day intervals using the scanning electron microscope. During this period, numerous transitory features emerged whose roles were concerned primarily with the development of the tectorial membrane (TM). The principal findings were as follows. (1) The surface of the developing organ of Corti produced all the fibrous material composing the minor tectorial membrane (mTM) including radial and longitudinal fiber bundles which formed the skeleton of the TM, and spongy, amorphous material which formed its intervening ground substance. (2) Throughout most of the cochlear spiral, radial fiber bundles were seen extending from the microvilli of supporting cells and projecting toward the major tectorial membrane (MTM). In most of the basal turn, but not in the apical turn, these radial bundles were interwoven with longitudinal fiber bundles which emerged from the surface of Hensen's cells. These findings indicate that the architecture of the TM is more complex in the basal turn than in the apex. (3) Increases in the dimensions of the reticular lamina resulted from the emergence of pillar cell headplates and growth in the diameter of hair cells and supporting cells. The emergence of pillar cell headplates was the principal factor contributing to increases in the radial dimension of the reticular lamina. This emergence was most dramatic between 10 and 12 days after birth (DAB) after the mTM completed its growth. Since the mTM appears to be bound medially to the MTM and laterally to the marginal pillars by 10 DAB, it seems likely that the growth of the reticular lamina after 10 DAB causes some stretching of the mTM both radially and longitudinally. (4) Completion of outer hair cell stereocilia growth at 8 DAB was followed by loss of supporting cell attachments of the TM (trabeculae) by 10 DAB, and coincided with the formation of marginal pillars from the third row of supporting cells. It is suggested that the formation of marginal pillars may be required for coupling of the TM to the tips of outer hair cell stereocilia and for induction of radial tension of the mTM. (5) Removal of the marginal pillar attachments occurred following completion of hair cell growth. (6) All structures on the reticular lamina appeared to have adult-like characteristics by 20 DAB.     

Terminal dendritic sprouting and reactive synaptogenesis in the postnatal organ of Corti in culture

Synaptogenesis in the organ of Corti between the primary receptors, the inner hair cells, and the peripheral processes of their afferent spiral ganglion neurons in the mouse lasts for 5 days postnatally (Sobkowicz et al. [1986] J. Neurocytol. 15:693-714). The transplantation of the organ into culture at the fifth postnatal day induces a reactive sprouting of dendritic terminals and an extensive formation of new ribbon synapses within 24 hours. This reactive synaptogenesis differs strikingly from the primary synaptogenesis and has been seen thus far only in the inner hair cells. The synaptically engaged neuronal endings sprout a multitude of filopodia that intussuscept the inner hair cells. The filopodial tips contain a heavy electron-dense matter that appears to attract the synaptic ribbons, which form new synaptic contacts with the growing processes. The intensity of the filopodial growth and synaptogenesis subsides in about 3 days; the filopodia undergo resorption, leaving behind fibrous cytoplasmic plaques mostly stored in the supranuclear part of the hair cells. However, occasional filopodial growth and formation of new synaptic connections continued. The data demonstrate that any disruption or disturbance of the initial synaptic contacts between the inner hair cells and their afferent neurons caused by transplantation results in prompt synaptic reacquisition. Furthermore, we suggest that the transitory phase of terminal sprouting and multiribbon synapse formation manifests a trophic dependence that develops postnatally between the synaptic cells.     

Postnatal depression and social supports in Vietnamese, Arabic and Anglo-Celtic mothers

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation.     

Cochlear dysfunction in the jerker mouse.

Surface preparations showed that the "jerker" mutant mouse has a normal total number of cochlear hair cells when young but that these progressively degenerate with increasing age. However, no gross 8th nerve action potentials or cochlear microphonics could be detected at the round window in 12–20 day old mutants, although many hair cells still appeared to be intact at these ages. Light microscopy of surface preparations is apparently a poor indicator of the functional state of hair cells, at least in genetically determined inner-ear defects. The endocochlear potential (EP) was significantly higher in the mutants than in controls during the maturation of the cochlea. During anoxia induced in adults, EP fell to a significantly less negative value in mutants than in controls. This abnormality in the anoxia potential probably reflects an organ of Corti abnormality. (30 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sodium current expression during postnatal development of rat outer hair cells

Outer hair cells of the cultured organ of Corti from newborn rats (0-11 days after birth) were studied in the whole-cell patch-clamp configuration. A voltage-activated sodium current was detected in 97% (n = 109) of the cells at 0-9 days after birth. The properties of this current were: (1) its activation and inactivation kinetics were fast and voltage-dependent, (2) the voltage at half-maximum activation was -45.0 mV, (3) its steady-state inactivation was temperature-sensitive (the half-inactivating voltage was -92.6 mV at 23 degrees C and -84.8 mV at 37 degrees C), (4) the reversal potential (80 mV) was close to the sodium equilibrium potential and currents could be abolished by the removal of extracellular sodium, and (5) tetrodotoxin blocked the current with a Kd of 474 nmol/l. Current amplitudes were up to 1.7 nA at room temperature. Mean current amplitudes showed a developmental time course with a maximum at postnatal days 3 and 7 for outer hair cells from the basal and apical part of the cochlea, respectively. In current-clamp mode cells had membrane potentials of -59.7 +/- 11.7 mV (n = 9). When cells were hyperpolarized by constant current injection, depolarizing currents were able to trigger action potentials. At 18 days after birth, sodium currents were greatly reduced and barely detectable. The results show that, unlike adult outer hair cells, immature outer hair cells regularly express voltage-gated sodium channels. However, due to mismatching of the sodium current inactivation range and membrane potential in vitro, a physiological function appears questionable.     

G protein Gi2 alpha in the cochlea: cloning and selective occurrence in receptor cells

A cDNA library was made from the mouse cochlea and screened with a G protein-cDNA like molecule obtained from cochlear tissue by polymerase chain reaction. The nucleotide sequence of a clone, named cochlear Gi2 alpha, had 99.2% identity to mouse macrophage Gi2 alpha. Using an antibody which is selective for Gi2 alpha, expression of the cochlear Gi2 alpha was localized in outer and inner hair cells of the organ of Corti. Possible functional roles of this G protein in hair cells are discussed.     

Upper airway motor outputs during vomiting versus swallowing in the decerebrate cat

Hair cells in the basilar papilla of birds have the capacity to regenerate after injury. Methods commonly used to induce cochlear damage are systemic application of ototoxic substances such as aminoglycoside antibiotics or loud sound. Both methods have disadvantages. The systemic application of antibiotics results in damage restricted to the basal 50% of the papilla and has severe side effects on the kidneys. Loud sound damages only small parts of the papilla and is restricted to the short hair cells. The present study was undertaken to determine the effect of local aminoglycoside application on the physiology and morphology of the avian basilar papilla. Collagen sponges loaded with gentamicin were placed at the round window of the cochlea in adult pigeons. The time course of hearing thresholds was determined from auditory brain stem responses elicited with pure tone bursts within a frequency range of 0.35-5.565 kHz. The condition of the basilar papilla was determined from scanning electron micrographs. Five days after application of the collagen sponges loaded with gentamicin severe hearing loss, except for the lowest frequency tested, was observed. Only at the apical 20% of the basilar papilla hair cells were left intact, all other hair cells were missing or damaged. At all frequencies there was little functional recovery until day 13 after implantation. At frequencies above 1 kHz functional recovery occurred at a rate of up to 4 dB/day until day 21, beyond that day recovery continued at a rate below 1 dB/day until day 48 at the 5.6 kHz. Below 1 kHz recovery occurred up to day 22, the recovery rate was below 2 dB/day. A residual hearing loss of about 15-25 dB remained at all frequencies, except for the lowest frequency tested. At day 20 new hair cells were seen on the basilar papilla. At day 48 the hair cells appeared to have recovered fully, except for the orientation of the hair cell bundles. The advantage of the local application of the aminoglycoside drug over systemic application is that it damages almost all hair cells in the basilar papilla and it has no toxic side effects. The damage is more extensive than with systemic application.     

Pressure-induced basilar membrane position shifts and the stimulus-evoked potentials in the low-frequency region of the guinea pig cochlea

We have used the guinea pig isolated temporal bone preparation to investigate changes in the non-linear properties of the tone-evoked cochlear potentials during reversible step displacements of the basilar membrane towards either the scala tympani or the scala vestibuli. The position shifts were produced by changing the hydrostatic pressure in the scala tympani. The pressures involved were calculated from measurements of the fluid flow through the system, and the cochlear DC impedance calculated (1.5 x 10(11) kg m-4 s-1, n = 10). Confocal microscopic visualization of the organ of Corti showed that pressure increases in the scala tympani caused alterations of the position of the reticular lamina and stereocilia bundles. For low pressures, there was a sigmoidal relation between the DC pressure applied to the scala tympani (and thus the position shift of the organ of Corti) and the amplitude of the summating potential. The cochlear microphonic potential also showed a pronounced dependence on the applied pressure: pressure changes altered the amplitude of the fundamental as well as its harmonics. In addition, the sound pressure level at which the responses began to saturate was increased, implying a transition towards a linear behaviour. An increase of the phase lag of the cochlear microphonic potential was seen when the basilar membrane was shifted towards the scala vestibuli. We have also measured the intracochlear DC pressure using piezoresistive pressure transducers. The results are discussed in terms of changes in the non-linear properties of cochlear transduction. In addition, the implications of these results for the pathophysiology and diagnosis of Meniérè's disease are discussed.     

Compression, gain, and nonlinear distortion in an active cochlear model with subpartitions

The propagation of inhomogeneous, weakly nonlinear waves is considered in a cochlear model having two degrees of freedom that represent the transverse motions of the tectorial and basilar membranes within the organ of Corti. It is assumed that nonlinearity arises from the saturation of outer hair cell active force generation. I use multiple scale asymptotics and treat nonlinearity as a correction to a linear hydroelastic wave. The resulting theory is used to explain experimentally observed features of the response of the cochlear partition to a pure tone, including: the amplification of the response in a healthy cochlea vs a dead one; the less than linear growth rate of the response to increasing sound pressure level; and the amount of distortion to be expected at high and low frequencies at basal and apical locations, respectively. I also show that the outer hair cell nonlinearity generates retrograde waves.     

The postnatal development of F-actin in tension fibroblasts of the spiral ligament of the gerbil cochlea

The tension fibroblasts of the spiral ligament of the mammalian cochlea are thought to create radial tension on the basilar membrane. Their postnatal development was investigated in the gerbil (Meriones unguiculatus) with confocal fluorescence microscopy using phallotoxin as a specific marker for F-actin. In the adult cochlea, tension fibroblasts were restricted to the basal cochlear turn and were arranged in 2-4 rows in the marginal region of the spiral ligament. They contained intensely stained parallel bundles of F-actin. In upper cochlear turns, the marginal region of the spiral ligament was occupied by sparsely distributed, unobtrusively labeled fibrocytes, the bone lining cells. The spiral ligament of young postnatal stages (newborn--6 days after birth (DAB)) lacked F-actin labeling patterns that are characteristic for tension fibroblasts in the adult. Rather, the whole inner surface of the otic capsule throughout all cochlear turns was outlined by cell layers with distinct but diffuse cytoplasmic F-actin label. These cells may represent perichondrial fibrocytes. Around 9 DAB, the perichondrium revealed changes in morphology and F-actin patterns that indicate a further differentiation into tension fibroblasts (basal turn) or bone lining cells (more apical turns). At 12 DAB, around onset of hearing, adult-like bone lining cells were found in the marginal regions of the spiral ligament of upper cochlear turns. In the basal turn, tension fibroblasts were present, but their F-actin cytoskeleton was not fully developed. During the following days, F-actin label increased in tension fibroblasts and reached adult-like configuration at 17 DAB, coinciding with mature hearing characteristics. The role of tension fibroblasts in development of hearing characteristics is discussed.     

Frequency tuning of basilar membrane and auditory nerve fibers in the same cochleae

Responses to tones of a basilar membrane site and of auditory nerve fibers innervating neighboring inner hair cells were recorded in the same cochleae in chinchillas. At near-threshold stimulus levels, the frequency tuning of auditory nerve fibers closely paralleled that of basilar membrane displacement modified by high-pass filtering, indicating that only relatively minor signal transformations intervene between mechanical vibration and auditory nerve excitation. This finding establishes that cochlear frequency selectivity in chinchillas (and probably in mammals in general) is fully expressed in the vibrations of the basilar membrane and renders unnecessary additional ("second") filters, such as those present in the hair cells of the cochleae of reptiles.     

Cochlear outer hair cell bending in an external electric field

We have used a high-resolution motion analysis system to reinvestigate shape changes in isolated guinea pig cochlear outer hair cells (OHCs) evoked by low-frequency (2-3 Hz) external electric stimulation. This phenomenon of electromotility is presumed to result from voltage-dependent structural changes in the lateral plasma membrane of the OHC. In addition to well-known longitudinal movements, OHCs were found to display bending movements when the alternating external electric field gradients were oriented perpendicular to the cylindrical cell body. The peak-to-peak amplitude of the bending movement was found to be as large as 0.7 microm. The specific sulfhydryl reagents, p-chloromercuriphenylsulfonic acid and p-hydroxymercuriphenylsulfonic acid, that suppress electrically evoked longitudinal OHCs movements, also inhibit the bending movements, indicating that these two movements share the same underlying mechanism. The OHC bending is likely to result from an electrical charge separation that produces depolarization of the lateral plasma membrane on one side of the cell and hyperpolarization on the other side. In the cochlea, OHC bending could produce radial distortions in the sensory epithelium and influence the micromechanics of the organ of Corti.     

Measurements and a model of the outer hair cell hydraulic conductivity

The dimensions of the apical surfaces of hair cells were measured in guinea pigs, aged from 3 weeks before term to 25 weeks after birth. In the basal two-thirds of the cochlea, the apical surfaces of the outer hair cells and their supporting cells changed with age, shrinking in a direction radial across the cochlear duct. There was an associated widening of the angle of the 'V' of the rows of stereocilia. Further apically, between 12 and 16 mm from the base of the cochlea, the outer hair cells and their supporting cells underwent the opposite change, becoming wider in a radial direction with age. The changes were seen before birth and continued for more than 3 weeks after birth. The results suggest that the guinea pig cochlea continues certain developmental processes for a considerable time after birth.