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Electrical coupling differs in the in vitro and in vivo organ of Corti.

Electrical communication between the supporting cells of the guinea pig organ of Corti was studied. For in vitro experiments, the inner ear was rapidly removed and placed in a heated perfusion chamber. Medium 199 was used. The bony cochlea and the lateral wall (spiral ligament and stria vascularis) were removed to expose the top two coils of the organ of Corti. In vivo experiments were performed upon anesthetized animals whose cochleas were exposed surgically. A tiny fenestra was made in the bony cochlea which permitted the passage of electrodes through the lateral wall and into the organ of Corti of the third turn. Coupling was assessed by impaling neighboring cells with 3 M KCl electrodes, and noting the spread of intracellularly injected current. Coupling ratios in the in vitro preparation were consistently greater than those obtained in vivo (0.58 +/- 0.17 vs. 0.104 +/- 0.064). Differences exist between the in vitro and in vivo preparations which might account for these results. In vivo the supporting cells are bathed in two different media, endolymph apically, and perilymph basally. Consequently, on their apical side the supporting cells are exposed to fluid high in K+, low in Ca2+ and at a potential of 80 mV, the endolymphatic potential. In vitro the cells are bathed on all sides in fluid similar to perilymph. Intermixing the fluids in an in vivo preparation, by tearing away the stria vascularis and Reissner's membrane, increases the magnitude of the coupling ratio (0.455 +/- 0.209). Thus the unique microenvironment of the inner ear maintains lower coupling ratios, and smaller space constants for the supporting cells.

Animals↗

Presynaptic fibres of spiral neurons and reciprocal synapses in the organ of Corti in culture.

Isolated segments of the newborn mouse organ of Corti were explanted together with the spiral ganglion components. Within the innervation provided by the spiral neurons, we observed presynaptic vesiculated nerve endings that form reciprocal ribbon-afferent/efferent synapses with inner hair cells. These intracochlear presynaptic fibres are characteristically located between adjoining inner hair cells, on the modiolar side, low and close to the supporting cells. The presynaptic fibres display different modes of synaptic connectivity, forming repetitive reciprocal synapses on single inner hair cells or on adjoining hair cells, or connecting adjoining inner hair cells through simultaneous efferent synapses. Many presynaptic fibres exhibit a distinctive ultrastructure: defined clusters of synaptic vesicles, dense core vesicles, coated vesicles, and mitochondria. These organelles occur focally at the synaptic sites; beyond the efferent synaptic specializations, the endings appear quite nondescript and afferent-like. We believe that the reciprocal synapses, although observed in cultures of the organ of Corti, represent real intracochlear synaptic arrangements providing a feedback mechanism between the primary sensory receptors and a special class of spiral ganglion cells that have yet to be recognized in the organ in situ.

Animals↗

Cellular growth and rearrangement during the development of the mammalian organ of Corti.

The sensory epithelium of the mammalian cochlea, the organ of Corti, is comprised of ordered rows of cells, including inner and outer hair cells. Recent results suggest that physical changes in the overall size and shape of the cochlear duct, including possible convergence and extension, could play a role in the development of this pattern. To examine this hypothesis, changes in cell size and distribution were determined for different regions of the cochlea duct during embryonic development. In addition, changes in the spatial distribution of sensory precursor cells were determined at different developmental time points based on expression of p27kip1. Unique changes in luminal surface area, cell density, and number of cell contacts were observed for each region of the duct. Moreover, the spatial distribution of p27kip1-positive cells changed from short and broad early in development, to long and narrow. These results are consistent with the hypothesis that convergence and extension plays a role in cellular patterning within the organ of Corti.

Animals↗

Ontogenesis of rat cochlea. A quantitative study of the organ of Corti.

A systematic quantitative set of data concerning the organ of Corti in developing Sprague-Dawley rats at intervals from 18 days of gestation to 10 days after birth (DAB) is provided in this study. Using phalloidin staining, the total number of inner and outer hair cells, the whole length of cochlea, as well as the diameter of inner and outer hair cells and the intercellular space between inner hair cells were determined in order to analyze the quantitative change of inner and outer hair cells during development and to explore some roles of the factors regulating the growth of cochlea. The results show that: (1) The length of cochlea approached its adult size by 7DAB. (2) The growth of the extreme part of the apex was responsible for the delayed elongation of the cochlea. (3) Growth in the cochlear length mainly results from an increase of cell diameter tempered by a decrease of intercellular space. (4) The adult size of inner and outer hair cells was obtained by 7-14DAB. (5) The final number of inner and outer hair cells was reached at 3DAB and remained constant through adulthood. No significant hair cell overproduction and cell death were observed during ontogenesis of the cochlea. The negligible importance of overproduction and missing hair cells during hair cell differentiation suggest that there is a precise regulation phenomenon for producing the right spatial organization of the organ of Corti.

Actins↗

The dynamic range of inner hair cell and organ of Corti responses.

Inner hair cell (IHC) and organ of Corti (OC) responses are measured from the apical three turns of the guinea pig cochlea, allowing access to regions with best, or most sensitive, frequencies at approximately 250, 1000, and 4000 Hz. In addition to measuring both ac and dc receptor potentials, the average value of the half-wave rectified response (AVEHR) is computed to better reflect the signal that induces transmitter release. This measure facilitates comparisons with single-unit responses in the auditory nerve. Although IHC ac responses exhibit compressive growth, response magnitudes at high levels depend on stimulus frequency. For example, IHCs with moderate and high best frequencies (BF) exhibit more linear responses below the BF of the cell, where higher sound-pressure levels are required to approach saturation. Because a similar frequency dependence is observed in extracellular OC responses, this phenomenon may originate in cochlear mechanics. At the most apical recording location, however, the pattern documented at the base of the cochlea is not seen in IHCs with low BFs around 250 Hz. In fact, more linear behavior is measured above the BF of the cell. These frequency-dependent features require modification of cochlear models that do not provide for longitudinal variations and generally depend on a single stage of saturation located at the synapse. Finally, behavior of dc and AVEHR responses suggests that a single IHC is capable of coding intensity over a large dynamic range [Patuzzi and Sellick, J. Acoust. Soc. Am. 74, 1734-1741 (1983); Smith et al., in Hearing--Physiological Bases and Psychophysics (Springer, Berlin, 1983); Smith, in Auditory Function (Wiley, New York, 1988)] and that information compiled over wide areas along the cochlear partition is not essential for loudness perception, consistent with psychophysical results [Viemeister, Hearing Res. 34, 267-274 (1988)].

Animals↗

Distribution of synaptic ribbons in the developing organ of Corti.

Studies of synaptogenesis in the developing organ of Corti in the intact mouse and in culture indicate that the inner and outer hair cells contain three populations of synaptic ribbons, i.e. ribbons adjacent to nerve fibres, free intracellular ribbons and misplaced ribbons apposed to non-neuronal elements. Ribbons adjacent to nerve fibres can be further classified into: ribbons synaptically engaged, ribbons participating in formation of presynaptic complexes only and ribbons that are not engaged to the hair cell membrane. In the developing innervated cultures the ribbon distributions are similar to those in the normal animal. Inner and outer hair cells differ in distribution of the ribbons. In the inner hair cells the ribbons adjacent to the nerve fibres are dominant (over 90%) and most of them (88%) are synaptically engaged. In the outer hair cells the presynaptic ribbons dominate the population (up to 60%) during the first postnatal week when the cells acquire afferent synaptic connections. This stage is followed by a marked reduction in the number of all ribbons. In the intact animal the rapid decrease results in a relative increase of misplaced and free ribbons. These changes are presumably due to the loss of some of the afferents. In the denervated hair cells the distribution of ribbons indicated the presence of conspicuous scatter. In the areas of incomplete denervation, however, the ribbons are apposed to the preserved fibres. Despite denervation, most of the ribbons develop the entire presynaptic complex in apposition to non-neuronal structures. The different populations of synaptic ribbons appear to reflect different stages in synapse formation. Possibly, the synaptic body originates in the interior of the hair cell and subsequently migrates to the cell membrane. In any case, a nerve fibre appears critical in influencing the location of the synaptic ribbon. At the apposition of the ribbon to the hair cell membrane, presynaptic densities are formed and the ribbon appears to become anchored. Typically, the nerve fibre membrane apposed to the presynaptic complex responds with the formation of postsynaptic densities.

Aging↗

Tissue culture of the organ of Corti and the isolated hair cells from the newborn guinea pig.

The organ of Corti and isolated hair cells from newborn guinea pig were cultured with the Rose chamber method. The hair cells could be maintained for more than 20 days after explantation when the organ of Corti was cultured as a whole, though the isolated hair cells swelled and degenerated within about 13 hours after explantation. By comparison between the tissue culture of the hair cells with the organ of Corti and isolated hair cell culture, the following conclusions were reached: (1) There are some functional correlations concerning the nutritional requirements and cell metabolism between the hair cells and the supporting cells in addition to the structural correlations. (2) There are no distinct fundamental differences between both inner and outer hair cells about their affectability as a single cell level.

Animals↗

Distribution of cyclic nucleotides in the organ of Corti.

Recent developments in radioimmunoassay technology have made possible measurements of cyclic nucleotides in individual specimens of the organ of Corti and its subdivisions. Steep longitudinal and transverse gradients of glycogen are known to exist in the organ of Corti of the guinea pig, with preferential accumulation in the outer hair cells of the apical turns. However, no significant longitudinal gradient of cyclic AMP was detectable in the organ of Corti, and the concentration of the compound was found to be nearly equal in the inner and outer hair cell layers. This is indirect evidence against the concept that cyclic AMP plays a role as "second messenger" in the control of glycogen metabolism of the organ of Corti. By contrast, the concentration of cyclic GMP was found to be consistently higher in the inner layer than in the outer layer of the organ of Corti, and to increase significantly in basal direction. This trend is remarkably similar to the distribution patterns of acetylcholinesterase, which may be considered as indirect evidence in favor of a possible role of cyclic GMP in the mediation of cholinergic effects.

Acetylcholinesterase↗

Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure.

Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells and/or non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 (GAL-1) antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication.

Amikacin↗

Pathomorphological peculiarities of damage to hair cells of the organ of corti in experimental sensorineural hearing loss.

Pathomorphology of the organ of Corti was studied on models of acute and chronic sensorineural damage to the acoustic analyzer. Peculiarities of hair cell degeneration, necrosis, and apoptosis in the organ were studied by light and scanning electron microscopy. The type of pathomorphological substrate in abnormalities of the organ of Corti depends on the intensity of the destructive exposure, but not on the nature of otopathological factors.

Animals↗

Immunocytochemical localization of 205 kDa microtubule-associated protein (205 kDa MAP) in the guinea pig organ of Corti.

We have studied the immunocytochemical localization of microtubule-associated proteins (MAPs) in the guinea pig organ of Corti. Using immunological methods with antibodies against MAP1A, MAP1B, MAP2, tau and 205 kDa MAP, we have identified 205 kDa MAP as a major MAP of the sensory epithelium in the organ of Corti. Immunoperoxidase microscopic study has shown that both cochlear hair cells and supporting cells reacted with anti-205 kDa MAP antibody. Immunoelectron microscopy revealed that 205 kDa MAP was associated with most microtubules in the sensory epithelial cells. It was also associated with the microtubules of bundle structures within supporting cells, suggesting that this MAP might form a part of cross-bridges between microtubules and between microtubules and actin filaments in the bundle structure. In contrast, MAP1A, MAP1B and tau, which are known to be expressed in neuronal tissue, were localized only in nerve fibers in the organ of Corti, not in the sensory epithelium. MAP2, which is known to be localized in dendrites and soma of nerve cells, was not distributed in nerve fibers in the organ of Corti. These results suggest possible roles of the 205 kDa MAP in the formation and maintenance of the highly polarized morphology of the epithelial cells of the organ of Corti, through stabilization and modulation of microtubule networks of these cells.

Animals↗

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants.

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for 1 month in a minimal essential medium. In contrast, under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 h. Hair cell counts show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than at the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) prevents most OHC losses. According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants. Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig organ of Corti cultures can also be prevented by treatment with several of the growth factors tested, i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1), and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders.

Animals↗

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

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. axo-dendritic synapses in the inner spiral bundle and axo-somatic synapses with the OHCs, supports the hypothesis that both the lateral and the medial olivo-cochlear 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 neurotransmitters/co-transmitters found in the various efferent synapses of the organ of Corti.

Animals↗

Ultrastructural localization of cadherin in the adult guinea-pig organ of Corti.

The apices of the majority of cells of the organ of Corti are connected together by junctional complexes to form the reticular lamina, a barrier that prevents the mixing of endolymph and perilymph. These complexes include tight junctions, adherens junctions and desmosomes. Further information is required about the identity and distribution of the molecules involved in these connections if the function and organization of the reticular lamina are to be well understood. One major category of molecules occurring in adherens junctions and desmosomes, and involved in the maintenance of tissue integrity, is the cadherins. However, although cadherin has been identified in junctions between supporting cells in the adult mammalian organ of Corti at the light microscopic level, its ultrastructural distribution has not so far been described. A post-embedding immunogold labelling technique has therefore been used in conjunction with a monoclonal antibody to cadherin to investigate its ultrastructural distribution in the adult guinea-pig reticular lamina. Immunolabelling is observed in hair cell-supporting cell junctions and in supporting cell-supporting cell junctions. In addition, there is more labelling associated with inner hair cell-supporting cell junctions than with outer hair cell-supporting cell junctions. This may indicate that the junctions associated with the two types of hair cell have different functional properties.

Animals↗

Intercellular fluid pathways in the organ of Corti of cat and man.

The intercellular junctions in the organ of Corti of cat and man were examined with the electron microscope. In contrast to the zone of tight junctions, or zonulae occludentes, which were present at the surface of cells lining the scala media, there was no tight junctional specialization among the cells of the tympanic lamina, perilymphatic lining cells of the scala vestibuli, basal processes of the supporting cells of the organ of Corti or cells of the spiral limbus and spiral ligament. These findings suggest the possibility of fluid continuity between the scala vestibuli and scala tympani all along the cochlear duct. Morphological evidence for an intercellular diffusion barrier was present only at the endolymphatic surfaces of the cochlear duct and between the processes of the basal cells of the stria vascularis in cat and man.

Animals↗

In vivo micromechanical measurements of the organ of Corti in the basal cochlear turn.

Cochlear mechanical measurements of organ of Corti motion are generally accomplished in the apical or basal turn as in vivo or in vitro studies. In the apex it is possible to observe and measure tectorial membrane vibration as well as vibrations of structures such as the reticular lamina or the basilar membrane (BM). However, compared to the basal turn, cochlear amplification and nonlinearity are not strong in the apex. Basal turn studies have typically been limited to point location measurements of the BM but improved technology for laser interferometry is now making possible the spatial mapping of BM motion. The 'complexity' of BM motion in the radial direction (particularly the phase variation) is important to new models of cochlear wave amplification. In future work it may be possible to learn about vibration of structures within the organ of Corti.

Animals↗

Ultrastructure of the organ of Corti in experimental hydrops.

The early ultrastructural changes of the organ of Corti in the guinea pig after obliteration of the endolymphatic sac and duct were examined in a time-sequence study. Initial loss of outer hair cells was followed by inner hair cell degeneration, both starting at the apical part of the cochlea. Morphological changes of the sensory cells were found to start at the endolymphatic surface and were characterized by a variety of sterociliary pathologies, distortion of the cuticular plate and a less prominent contrast-staining of glycocalyx. Further degeneration was marked by pathological changes of intracellular organelles and the afferent and efferent nerve endings. Details of the ultrastructural changes in the organ of Corti are discussed with regard to the pathophysiology of experimental hydrops.

Animals↗

Stimulus-related potassium changes in the organ of Corti of guinea-pig.

1. Potassium concentration was measured with double-barrelled K+-selective microelectrodes within the organ of Corti in the first turn of the guinea-pig cochlea. 2. Penetration of the electrode from scala tympani through the basilar membrane was accompanied by an increase in K+ resting level from 3.0 mmol/l in perilymph to 3.4 mmol/l in cortilymph (n = 8). K+ resting level was not significantly different in various extracellular regions of the organ of Corti. On penetration of the cuticular plate, the K+ level reached 140 mmol/l simultaneously with the occurrence of a +80 mV endocochlear potential. Impalement of hair cells and supporting cells was accompanied by an increase in K+ level, but intracellular K+ level was not systematically measured. 3. Stimulation with pure tones over the frequency range 500 Hz to 25 kHz produced changes in the K+ level in the organ of Corti. The magnitude of these changes was dependent on stimulus frequency and intensity. At high sound intensities the K+ level in the tunnel of Corti could increase by typically 1 mmol/l, while a maximum increase of 3 mmol/l with respect to the resting level was observed immediately adjacent to inner hair cells. 4. During brief exposures to moderate intensity, pure tone acoustic stimulation (10 s, less than 80 dB SPL (sound pressure level] of frequency 4 kHz or greater the K+ level in the extracellular fluid of the organ of Corti rose monotonically to a steady peak level. On cessation of the stimulus the K+ level fell monotonically with a time constant of about 2 s to a level close to the pre-stimulus level. In some cases this level was slightly above the pre-stimulus level. 5. For brief exposures to moderate intensity sound (10 s, less than 80 dB SPL) the extracellular potential in the organ of Corti became more positive. The amplitude of this sound-evoked change adapted during stimulation to a level approximately one-fifth of its initial value. Upon cessation of the stimulus the potential fell transiently below its pre-stimulus level, before recovering to that level. The time constant of these changes was between 2 and 3 s. 6. Iso-response tuning curves for the sound-evoked elevation in K+ level in the organ of Corti in animals in good condition were similar to iso-rate tuning curves for primary afferent fibres reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)

Acoustic Stimulation↗