Ultrastructural damage in cochleas used for studies of basilar membrane mechanics

Cat cochleas used for interferometric studies of basilar membrane mechanics were examined with the electron microscope. The structures most severely damaged in the experimental cochleas are the outer hair cells and the radial afferent fibers to the inner hair cells. Since the basilar membrane and other supporting structures appear to be normal, mechanical changes observed in the experimental cochleas are most probably due to outer hair cell damage. Individual animals with varying degrees of damage showed large differences in the frequency of basilar membrane resonance at the same place in the cochlea. Shifts in tuning of this magnitude could occur as a consequence of hair cell damage only if the stiffness of the stereocilia and associated structures was greater initially than the stiffness of the basilar membrane and gradually decreased with damage. The present series of observations, therefore, suggest that the stiffness of the outer hair cell stereocilia determines basilar membrane tuning.     

Rotated stereociliary bundles and their relationship with the tectorial membrane in the guinea pig cochlea

Hair cells with rotated stereociliary bundles have been observed in the cochleae of control and kanamycin-treated guinea pigs. The affected outer hair cell bundles have a variable degree of rotation, with some being completely reversed. The inner hair cells are more rarely affected, and only small areas of an individual inner hair cell bundle are abnormal. In counts from ten cochleae, the number of outer hair cells with rotated bundles was most commonly between 10% and 20%, with almost 27% of all outer hair cells affected in the most extreme case. The rotated outer hair cell bundles often have distorted outlines but in other respects closely resemble normal bundles. In particular, they have the usual gradation in stereociliary height, intracellular cross-links and intercellular links to adjacent normally-orientated bundles. There are also corresponding imprints in the tectorial membrane which match the pattern of the stereocilia. In kanamycin-treated guinea pigs, imprints of both normal and rotated hair bundles are present, even when the corresponding bundle is absent, and there are frequently remnants of stereocilia inserted in the imprints. These observations suggest that, apart from their abnormal orientation, the rotated bundles are similar to normal bundles in both their organization and association with the tectorial membrane. The implications of this with respect to transduction and cochlear mechanics are discussed.     

Neural tuning in the granite spiny lizard

Scanning electron microscopy was used to examine the basilar papilla of the granite spiny lizard. The papilla contains three distinct hair cell populations: an apical and a basal population with free-standing cilia, and a central population with a tectorial membrane. In the free-standing populations, stereocilium length decreases towards the ends of the papilla. Ciliary tuft morphology differs in the free-standing and the tectorial membrane populations, except that several of the free-standing hair cells with the shortest stereocilia have a tuft morphology like the hair cells in the tectorial membrane population. On the basis of single-fiber physiology, auditory nerve fibers can be divided into a low characteristic frequency (CF) and a high CF population. Mappings of the tonotopic organization of the nerve demonstrated two groups of high CF fibers that correspond to the two free-standing hair cell populations. The low CF fibers are associated with the tectorial membrane hair cell population. Fiber CF correlated with hair cell cilium length, not position on basilar membrane, for hair cells with free-standing cilia. Tonotopic organization of high CF fibers could be predicted reasonably well from the histogram of fiber CFs.     

Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma

Damage to the tectorial membrane caused by acoustic trauma was examined with scanning and transmission electron microscopy immediately after exposure and at selected time points over a 10 day recovery period. At 0 h of recovery the structure of the tectorial membrane overlying the region of hair cell damage was severely disrupted and connections between the membrane and the basilar papilla were lost. By 24 h of recovery, regeneration of the tectorial membrane was evident in the secretion of new matrix materials by the supporting cells of the basilar papilla. By 10 days of recovery a new honeycomb-like matrix had replaced the segment of damaged tectorial membrane, re-established connections with hair cell stereocilia and become fused with adjacent regions of undamaged tectorial membrane. However, the regenerated segment included only the honeycomb-like structure of the lower layer of the normal tectorial membrane. The laterally-oriented fibers which form the upper layer of the membrane were not regenerated over the damaged region. These findings indicate that the tectorial membrane is regenerated in parallel with the hair cells during recovery from acoustic trauma but the full extent of this recovery and its effect on cochlear function are not yet clear.     

Tectorial membrane. II: Stiffness measurements in vivo

The tectorial membrane is assumed to play a crucial role in the stimulation of the cochlear hair cells and was thought for decades to serve as a stiff anchor for the tips of the hair-cell stereocilia, particularly those belonging to the OHCs. Yet, its stiffness has never been measured under conditions approximating its normal environment in live animals. We have developed a method for doing this. The tectorial membrane is approached through the lateral wall of scala media. The bony cochlear capsule is removed along scala media over somewhat less than 1/4 turn, and the underlying spiral ligament and stria vascularis are carefully reflected. With the help of a three axial hydraulic manipulator, a flexible micropipette filled with isotonic KCl is inserted into the tectorial membrane at one of two different angles and moved either transversally, away from the basilar membrane, or radially, toward or away from the modiolus. This causes the tectorial membrane to be deformed and the micropipette to bend. The micropipette stiffness is calibrated on an instrument of a new kind, so as to convert the bend into force. The calibration allows us to determine the point stiffness of the tectorial membrane from the amount of micropipette bend. The stiffness of the tectorial membrane per unit length has been calculated from the point stiffness with the help of the deformation pattern. Transversal and radial stiffness magnitudes have been determined in the second cochlear turn in Mongolian gerbils. Both are smaller by almost an order of magnitude than the corresponding aggregate stiffness of the OHC stereocilia. As a consequence, the tectorial membrane cannot act as a stiff anchor for the stereocilia but only as a mass load, except at relatively low sound frequencies where mass effects are negligible. This means that the classical model of shear motion between the tectorial membrane and the reticular lamina must be replaced.     

小鸡和豚鼠耳蜗毛细胞β-肌动蛋白分布比较

目的 :比较小鸡基底乳头和豚鼠耳蜗毛细胞 β-肌动蛋白 (β- actin)分布的特点。方法 :应用免疫组织化学方法观察小鸡和豚鼠耳蜗中β- actin免疫反应活性。结果 :鸡基底乳头高、矮毛细胞的静纤毛 ,盖膜根部附着处缘上皮细胞胞浆 ,豚鼠耳蜗三排外毛细胞胞浆 ,内、外支柱细胞胞浆和指状突β- actin免疫反应阳性。结论 :小鸡和豚鼠耳蜗毛细胞具有相同结构蛋白β- actin,但两种动物之间存在明显的分布差异。     

Wever and Lawrence revisited: effects of nulling basilar membrane movement on concomitant whole-nerve action potential

It has been assumed for decades that mechanically stimulating hair cells, both inner and outer (IHC, OHC), leads to CM and subsequent neural activity. A test of that assumption was attempted in this experiment. Tone-pips of 300 msec duration at 4 or 5 kc/s with fast rise times were simultaneously presented to the cochleae of 10 chinchillas, through the external meatus and a hole drilled into the scala tympani. A round-window electrode allowed the recording of CM and computer-averaged whole-nerve action potentials (CAP). Stimulus levels and relative phase could be adjusted to yield CAPs of similar amplitude and shape to either stimulus alone. When the two stimuli were combined, the vectorial CM could be changed by about 30 db between maximum and minimum levels when delta phi was changed by 180 degrees. However, the combined CAP was relatively insensitive to delta phi. If basilar membrane motion was minimized at CM minimum, the data mean that some other principle than basilar membrane motion must underlie or generate neural activity. These data are not consistent with the traditional view that basilar membrane motion underlies sensitivity and frequency discrimination, and are congruent with theories of sensitivity of hair cells or their stereocilia to direct acoustic or electric stimulation, with basilar membrane mechanical stimulation assigned some secondary role. The author offers an electromodel comprising one system of basilar membrane motion of supramolecular dimensions leading to mechanical stimulation of OHCs and their large CM, and a second parallel system excited by the same stapes displacements but of submolecular dimensions leading to a propagated acoustic wave through the cochlear partition and to acoustic----electric transduction by the tectorial membrane; the output of that membrane is picked up in the fluids of the subtectorial space by the electro-sensitive IHCs and analyzed by them in some unknown manner for frequency. These IHCs are then the sole direct precursors of neural activity. A seeming anomaly was found in that at delta phi = CM minimum, when the traditional model would predict reduced basilar membrane movement, a reduced CM, consequent reduction in neural activity, and an increase in the latency of the N1 component of the CAP, latency was in fact slightly but uniformly decreased. It was suggested that in this phase condition the larger CM may have been correlated with the suppressive action of the OHCs upon the IHCs.     

Recent developments in cochlear physiology

Recent findings in cochlear physiology have caused many of our long held ideas about how sound is analyzed by the ear to be reevaluated. This article describes changes which have occurred in three classical ideas of cochlear transduction: (1) There is a gradient of frequency representation along the cochlea with high frequencies being represented at the base and lower frequencies represented progressively toward the apex. It is now known that the specific frequency which is represented at a given location along the cochlea is not invariant but changes systematically during the normal development of hearing. (2) The place code and frequency tuning along the cochlea are due to the conventional traveling wave of von Békésy and basilar membrane mechanics. Experiments in nonmammalian vertebrates which lack a traveling wave have shown that other mechanisms, including the mechanical resonance of hair cell stereocilia, may contribute to tonotopic organization and frequency tuning. It is possible that hair cell stereocilia also contribute to frequency representation and tuning in the mammalian cochlea. (3) The vibration of the basilar membrane to sound is determined by its passive mechanical properties. It is now known that the response of the basilar membrane, and that of the cochlear partition as a whole, is influenced by physiological processes which utilize metabolic energy. The active processes are likely expressed through the motile activity of outer hair cells.     

Elemental composition of individual cells and tissues in the cochlea

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.     

Dimensions of the cochlear stereocilia in man and the guinea pig

The tuning properties of the basilar membrane and the presence of acoustic emissions from the cochlea suggest that an energy consuming, mechanically active cochlear amplifier exists. Some models of this amplifier demand a mechanical resonator within the cochlea. The lengths of the stereocilia of the inner and outer hair cells in man and the guinea pig have been measured from scanning electron micrographs using a stereometric technique. In both species there is a linear increase in the length of the longest inner hair cell stereocilia with distance along the cochlea. There are, however, marked differences between the dimensions of the outer hair cell stereocilia in the two species. In man, there is an increase in length which is is a hyperbolic function of distance along the cochlear duct. The picture is more complicated in the guinea pig. This could account for some of the differences in auditory physiology between the two groups. The mechanical resonance properties of the human OHC stereocilia have been assessed, and, with certain assumptions, these properties are such that resonance of the stereocilia of the OHCs could form part of the cochlea amplifier, at least in man.     

A micromechanical model of the cochlea with radial movement of the tectorial membrane.

The function of the tectorial membrane in the cochlear micromechanics is uncertain. In modeling approaches some models have assumed it to be a resonator that participates in the sharp tuning mechanisms of the cochlea with its mass coupled to the ciliary stiffness of outer hair cells, being driven by the shear force between the reticular lamina and itself. This paper presents a different type of micromechanical model which assumes that the tectorial membrane is driven by a lymphatic fluid flow that can be shown to have a substantial radial component. It also assumes that the reticular lamina is relatively stiff and thereby restrains the top end of outer hair cells that exert a force to the basilar membrane via Deiters cells. When combined with a three-dimensional block model, it can simulate the sharp tuning mechanisms of the cochlea well.     

Stereociliary bundle morphology in organotypic cultures of the mouse cochlea

Organotypic cultures of the neonatal mouse cochlea have a band of hair cells consisting of 3-5 rows of outer hair cells and a single row of inner hair cells. The outer hair cell stereociliary bundles show progressive differentiation from the apical to the basal ends of the band. Undifferentiated apical bundles have a disk-like array of short stereocilia resembling microvilli. Partially differentiated bundles are hemispherical with poorly organized rows of thickly clustered stereocilia, which gradually increase in height in the direction of the kinocilium. More differentiated bundles remain hemispherical with many microvilli-like stereocilia, but have highly organized rows of sterocilia along the side nearest to the kinocilium, and well-defined height increments between the rows. Highly-differentiated, basal bundles usually have a 'V' or 'W' shape, although some can be almost polygonal. The basal bundles have 4-5 regular rows of stereocilia with a well-defined gradient in height across the rows, and very few microvilli-like stereocilia. Cross-links are only consistently observed in more differentiated bundles, where the rows of stereocilia are regular and have significant height increments across them. The links show a wide variety of forms and orientations not previously observed in other preparations. Spoke-like arrays of links project from the upper regions of many stereocilia and other stereocilia appear to bear distinct tip-to-side links, although with a variety of orientations. A similar variety of cross-links is observed in early postnatal cochleae in vivo, but not in the cochleae of adult mice, indicating that this variety may be a transient feature of sterociliary bundle development. In vitro, inner hair cell stereociliary bundles are often covered by overlying material from the developing tectorial membrane. The variations in morphology of inner hair cell bundles and their cross-links are similar to those of the outer hair cell bundles.     

The inner ear as an electrosensory sense organ]

A model for cochlear perception is introduced based on the consideration that the organ of Corti is an electrosensory organ like that found in fish physiology during phylogenetic development. The inner hair cells work as electroreceptors, the outer hair cells as electrocytes. A layer of potassium ions on the lower surface of the tectorial membrane causes the excitation of the inner hair cells as soon as contact with a stereocilium occurs. A model for the basilar membrane motion, based on mathematical considerations and in accordance with the typical tuning curves of single auditory nerve fibres can serve as basis to explain the results of frequency and intensity analyses if we assume an electric excitation of the inner hair 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.     

A newly identified surface coat on cochlear hair cells

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.     

Motility of cochlear outer hair cells.

A recent exciting discovery in the physiology of hearing has been that fine tuning and regulatory properties of the mammalian auditory system reside in the micromechanics of the basilar membrane. While the inner hair cells are considered primary afferent transducers, the outer hair cells are postulated to modulate transduction based on their motile properties. These new insights have impacted on our view of auditory processing and are also leading to improved diagnostic procedures (such as those based on otoacoustic emissions) and a better understanding of sensorineural pathology. Outer hair cell motility may be separated into two categories: fast and slow. Fast motility is voltage-driven and frequency-following and provides positive feedback to the motion of the basilar membrane. Slow motility (shape changes measured in milliseconds to seconds) can be triggered through depolarization by potassium, osmotic effects, mechanical stimulation, efferent neurotransmitters, and the elevation of intracellular second messengers and calcium ions. These slow changes may be superimposed upon the fine-tuning provided by the fast motility. This paper discusses the mechanism underlying the shape changes induced by different stimuli, and assesses the role of fast and slow motility in the physiology and pathology of auditory transduction.     

豚鼠耳蜗盖膜下面的超微结构

目的 利用扫描电镜技术详细地观察豚鼠盖膜下的超微结构,为耳蜗的感音机制提供新的认识.方法 用S-4800型超高分辨率扫描电子显微镜观察了6只豚鼠12只耳蜗的盖膜.结果 (1)耳蜗各圈盖膜下面均可观察到外毛细胞静纤毛压迹,这种压迹多半与静纤毛最高排的形态一致,第一圈呈“W”型,由基底圈到顶圈,逐渐由“W”型渐变为“V”型及不规则的簇状.压迹为一排圆形的小凹,将盖膜下表面表层辐射状纤维断开,只有最高排的静纤毛才与盖膜接触形成压迹,压迹排列呈串珠一样.(2)盖膜下内毛细胞相应的位置有一条较深的波浪状沟槽,呈线性结构,沟槽比外毛细胞静纤毛的压迹浅而宽,由耳蜗的基底圈到顶圈这种压迹逐渐呈带状.(3)盖膜下的纤维由盖膜的外缘到盖膜与内毛细胞静纤毛压迹之间以蜗轴呈辐射状排列,但在外毛细胞静纤毛压迹的位置被这些压迹所阻断,而总的辐射方向和纤维走行并没有改变,纤维非常的精细,每根纤维之间又相互交错相连.内、外毛细胞压迹之间,柱细胞顶部的盖膜纤维很细,均匀呈细丝状.而内毛细胞与螺旋缘之间,即内沟上方的盖膜下方的纤维更加细长.结论 通过对盖膜超微结构的观察,盖膜下内外毛细胞静纤毛的压迹充分说明毛细胞静纤毛与盖膜的接触.盖膜下的纤维由盖膜的外缘到柱细胞和柱细胞到盖膜下内毛细胞静纤毛带状压迹之间也就是内、外毛细胞压迹之间,柱细胞顶部的盖膜的纤维与内毛细胞顶部盖膜带状结构到螺旋缘之间的纤维的粗细都不一样.这种纤维的分布特点,可能与盖膜在声音调谐方面所起的作用有一定的关系.     

Elektromechanische Transduktion

BACKGROUND: The somatic electromotility of the outer hair cells can be induced by an extracellular electrical field. This enables us to investigate the electromechanically induced motion of the organ of Corti. METHODS: The electrically induced motion of the guinea-pig organ of Corti was measured with a laser Doppler vibrometer in three cochlear turns at ten radial positions on the reticular lamina (RL) and six on each of the upper and lower surfaces of the tectorial membrane (TM). RESULTS AND CONCLUSIONS: We found a complex vibration pattern of the RL and TM, leading to a stimulus synchronous modulation of the depth of the subtectorial space in the region of the inner hair cells (IHCs). This modulation causes radial fluid motion inside the space up to at least 3 kHz. This motion is capable of deflecting the IHC stereocilia and provides an amplification mechanism additional to that associated with basilar-membrane motion.     

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otolithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles. The second layer formed by a columnar organization of filaments secures the otolithic membrane above the surface of the epithelium. The long columnar filaments are organized in parallel to the stereocilia bundles and are anchored to the apical surface of the supporting cells. The zonula adherens at the apical region of each supporting cell displays a thick polygonal bundle of actin filaments forming at the surface of the epithelium a transcellular honeycomb organization that provides mechanical ground support for the columnar filament layer. The dominant aspect of this columnar filament layer indicates that it may also have an important role in attenuating the force of inertia of the large otolithic mass during acceleration, screening stresses that would be directed to an effective bending of the stereocilia bundles.     

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.