Structural relationships of the unfixed tectorial membrane

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.     

Acoustic overstimulation alters the morphology of the tectorial membrane

After a permanent threshold shift was induced by exposing guinea pigs to a 1 kHz pure tone at 105 dB(A) for 72 h, light microscopic observations of freshly dissected and stained tectorial membranes showed an increased waviness and clumping of the fibers of the middle zone. Hensen's stripe was not seen as a continuous dense structure running through the middle zone but was at times discontinuous and curved. As measured from cross-sections of the cochlea, the thickness of the tectorial membrane was decreased after acoustic overstimulation. The stereocilia of the inner and outer hair cells lie directly under the middle zone. Visual detection levels of threshold of tectorial membrane movement was determined by stimulating the marginal zone of the tectorial membrane of isolated cochlear coils by an oscillating water jet. After acoustic overstimulation the tectorial membrane became more compliant. The tectorial membrane abnormalities were restricted to the regions of the cochlea that demonstrated a 40–50 dB hearing loss.     

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.     

Mechanical transduction in outer hair cells

The outer hair cells are responsible for the exquisite sensitivity, frequency selectivity and dynamic range of the cochlea. These cells are part of a mechanical feedback system involving the basilar membrane and tectorial membrane. Transverse displacement of the basilar membrane results in relative motion between the tectorial membrane and the reticular lamina, causing deflection of the stereocilia and modulation of the open probability of their transduction channels. The resulting current causes a change of membrane potential, which in turn produces mechanical force, that is fed back into the motion of the basilar membrane. Experiments were conducted to address mechanical transduction mechanisms in both the stereocilia and the basolateral cell membrane, as well as modes of coupling of the outer hair cell force to the organ of Corti.     

Hereditary deafness in the cat: An electron-microscopic study of the tectorial membrane

Summary The tectorial membrane is affected at an early stage of the cochlear degeneration in the hereditarily deaf white cat. The membrane first descends towards the organ of Corti with obliteration of the intervening sub-tectorial space in the basal coil during the second post-natal week. Both the microvilli of supporting and Hensen's cells, and the hair cell stereocilia make deep indentations on the under-surface of the membrane. Cells are found insinuated between the tectorial membrane and Corti's organ, and numerous cellular processes occur within the former. A phagocytic function would appear probable for these cells, which seem to originate from the internal sulcus region. The membrane is retracted into the latter around the 2-month stage. At all ages, small spherical structures, which may represent altered interdental cell secretions, are found within the membrane, these becoming calcified in older animals.This investigation has been supported by the Norwegian Research Conncil for Science and the Humanities through grant C.37.59-1     

Ionic coupling among cells in the organ of Corti.

Gap junctions have been demonstrated morphologically among the supporting cells of the mammalian organ of Corti but, in contradistinction to reptiles, evidence for their existence between the supporting cells and hair cells is equivocal. The literature is ambiguous with respect to electrical coupling and dye coupling among the supporting cells, and no coupling of either kind has been demonstrated for the hair cells. We found strong coupling of both kinds among the supporting cells in the cochleas of live Mongolian gerbils and a less stable coupling between the supporting cells and the outer hair cells. The electrical coupling was established by recording alternating receptor potentials in the hair cells and following their decrement in the population of Hensen's cells; the dye coupling, by injecting Lucifer yellow electrophoretically into the hair cells or the supporting cells and investigating its spread to the neighboring cells. The electrical recordings were made by means of microelectrodes filled with either 1.5 or 3 M KCl or 1 M LiCl with 6% Lucifer yellow, the latter used for dye injection. The electrode resistances ranged from about 20 to 60 M omega in the first instance, and from about 50 to 110 M omega, in the second. The electrodes were inserted into the organ of Corti through scala media according to the method of Dallos, Santos-Sacchi and Flock (1982) modified by us. The alternating potential in Hensen's cells was usually larger than in the outer tunnel of Corti and remained practically constant up to the outer margin of the Hensen's-cell population. Its phase was the same as in the outer hair cells. When the dye was injected into a Hensen's cell, it always spread to neighboring Hensen's cells and often to Deiter's cells. Dye injected into outer hair cells (identified according to anatomical and physiological criteria) also spread to Deiter's and Hensen's cells and, usually, to other outer hair cells. Stained cells were identified in surface preparations and, on two occasions, in serial sections from plastic embedded cochleas.     

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.     

Ultrastructure of the normal human organ of corti. new anatomical findings in surgical specimens

CONCLUSION: A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology. OBJECTIVE: To investigate the ultrastructure of human adult cochleae using SEM. MATERIAL AND METHODS: Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM. RESULTS: The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

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.     

Tectorial membrane. I: Static mechanical properties in vivo

Although the tectorial membrane in the mammalian cochlea plays a crucial role in hair-cell stimulation, its mechanical properties have never been investigated under conditions approximating those under which it normally functions. For this reason, we performed such investigations in live Mongolian gerbils. Access to the tectorial membrane was gained through the lateral wall in the second cochlear turn. As far as possible, sodium ions were kept away from the tectorial membrane by avoiding injury to Reissner's membrane, relieving the perilymphatic pressure, and rinsing the scala media with an isotonic KCl solution. The tectorial membrane was manipulated with a flexible micropipette in three, approximately orthogonal, directions. Under these conditions the membrane was found to be highly compliant and resilient, and to have a relatively high tensile strength. Its viscosity was low. Some of these attributes were altered by sodium ions, dyes, or death.     

Glycerol effect on the guinea pig tectorial membrane

Summary The tectorial membrane (Tm) of guinea pigs has been found to have an altered organization of its matrix fibers in response to intravenously administered glycerol. Following treatment, the Tm middle zone shows an increase in waviness and clumping of fibers in nonhydropic and several hydropic ears in contrast to nontreated control ears. Residue of the internal sulcus cells occasionally fills the subtectorial space. In the present study, additional investigations were performed with scanning electron microscopy in order to study the relationship between the Tm and the organ of Corti, as well as the relationship between Hensen's stripe and the inner hair cell. Present findings provide evidence for a connection between the inner hair cell stereocilia and Hensen's stripe which may be the molecular basis for the modulation of hearing during the glycerol test in a patient with Meniere's disease. Correspondence to: A. M. Meyer zum Gottesberge     

Incorporation of palmitic acid in the organ of Corti as revealed by autoradiography

The sites of incorporation of [3H]palmitic acid perfused through the scala tympani of the guinea pig cochlea were localized autoradiographically. The most active incorporation occurred in the lipid globules of Hensen's cells, followed by the hair cells, myelin of the cochlear nerve and other cells. It is speculated that the lipid globules of Hensen's cells act as a reservoir of the vitamin A esterified by fatty acids.     

The effects of quinine on the cochlear mechanics in the isolated temporal bone preparation

Quinine is known to induce a reversible hearing loss and to evoke motile responses of isolated outer hair cells. To study the effect of quinine, mechanical tuning curves of the Hensen's cells were measured in the isolated cochlea preparation in response to acoustical stimuli applied to the ear before and after application of the drug. It was shown that 0.5-4 mM quinine increased the vibration amplitude at the peak of the mechanical resonance curves and increased the sharpness of tuning. The time course of the event depended on whether the scala media was opened or not. The results show that quinine alters the micromechanical tuning of the organ of Corti.     

Ultrastructure of the normal human organ of Corti. New anatomical findings in surgical specimens

Conclusion A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology.

Objective To investigate the ultrastructure of human adult cochleae using SEM.

Material and methods Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM.

Results The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

Localization of the marginal zone of the tectorial membrane in situ, unfixed, and with in vivo-like ionic milieu.

In order to determine the normal positional relationship between the tectorial membrane and the organ of Corti, a preparation method was developed which made it possible to study the unfixed tectorial membrane in its normal position in relation to the cochlea and with in vivo-like ionic conditions. With this method, post-mortem changes visible with the light microscope were detectable after 60 to 90 min instead of the normal 30 min. When endolymph of artifical endolymph are present in the scala media, the marginal zone lies in close contact with the surface of the organ of Corti. If the endolymph is replaced by artificial perilymph, first the marginal zone of the tectorial membrane, and later the whole membrane shrinks. At this stage, latex particles suspended in the perilymph are free to enter the subtectorial space.     

Displacement of the reticular lamina with and without the tectorial membrane in the guinea pig cochlea

OBJECTIVE: In an attempt to establish the mechanical relationships between the reticular lamina and tectorial membrane, we studied the morphological changes of the reticular lamina on a micrometer scale when an in vitro preparation of guinea pig cochlea with and without tectorial membrane was exposed to a potassium-rich medium. MATERIAL AND METHODS: Using video-enhanced differential interference contrast microscopy, the radial displacement of the inner hair cells (IHCs) and outer hair cells (OHCs) in the reticular lamina was measured in real time after exposure to the potassium-rich medium for 3 min. RESULTS: The amplitude of the displacement of the OHCs in preparations with an intact tectorial membrane was half of that observed in those in which the tectorial membrane had been removed. A similar displacement response was also observed for the IHCs, although it was smaller than that for the OHCs. There was no significant difference in the amplitude of the displacement among the three rows of OHCs. CONCLUSIONS: These results suggest that the structure linking the OHCs to the pillar cells is very elastic and that the movement of the OHCs in situ is weakly mechanically coupled to the IHCs. The tectorial membrane provides increased compliance in the motion of the IHCs and OHCs.     

Imprints of the inner sensory cell hairs on the human tectorial membrane

Summary Imprints indicating possible direct inner sensory cell hair contact with the tectorial membrane were observed in the cochlea of a 77-year-old woman under a scanning electron microscope (SEM). The imprints were seen in the lower and upper basal cochlear turns but not in the apical and middle turns. The small dot of imprints numbered from a few up to 12 and were arranged in various forms rather than straight lines. Contact between the tectorial membrane and inner and outer sensory cell hairs of the human cochlea was discussed from the SEM findings found in this case.     

Modifications of cochlear microphonic frequency responses following transient changes of hydrostatic pressure in the perilymph

Cochlear microphonic potential was recorded with differential electrodes implanted in the various turns of the guinea-pig cochlea. Isointensity frequency responses were plotted in normal conditions and after excessive displacements of the cochlear partition. These displacements were provoked by changes of hydrostatic pressure in the perilymph of scala tympani or scala vestibuli. Typical modifications of the frequency response were observed. The most noticeable was a division in two parts of the response zone which suggested the existence of two resonance peaks. Scanning electron microscopy revealed that changes of hydrostatic pressure provoked alterations of the stereocilia in the outer rows of external hair cells, probably in relation with a decoupling of the tectorial membrane from the organ of Corti. These results are discussed in terms of possible alterations of cochlear micromechanics.     

High resolution scanning electron microscopy of the human organ of Corti. A study using freshly fixed surgical specimens

Scanning electron microscopy on immediately fixed human cochleae obtained during surgery for life-threatening petro-clival meningioma showed excellently preserved morphology. We compared the morphological findings with those from transmission electron microscopic sections of well preserved human and animal tissue. The characteristics of neural innervation, the pathways of the nerves through the organ of Corti and the intimate relation of nerves to supporting cells along their route could be studied in detail. The lateral membranes of Hensen and Claudius cells were folded creating a surface enlargement. Marginal pillars extended the distal end of the tectorial membrane and correspond to the marginal net or "randfasernetz" described earlier. Stereocilia imprints at the undersurface of the tectorial membrane go as far as to the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cell, attached to the marginal pillars, raises questions about differences in the excitation of the last row of outer hair cells. The complex nature of many supporting cells, stria vascularis and Reissner's membrane, intracellular complexities as well as surface features are described. Supernumerary inner hair cells were observed and the different arrangement of outer spiral fibres in contrast to findings in animals and variations of nerve fibres within the organ of Corti between apex and base are discussed.