Does the geometrical arrangement of the outer hair cell stereocilia perform a fluid-mechanical function?

CONCLUSIONS: Our experiments qualitatively show that the geometrical structure of the inner ear may have the features of a micro-pump. To further substantiate this hypothesis, additional experiments, particularly on in vivo preparations, are needed. OBJECTIVE: To introduce some new ideas about the functional purpose of the geometric arrangement of the outer hair cell stereocilia. Analogies to some recently developed valveless micro-pumps are pointed out. To illustrate these points, comparative experiments were performed using a simplified macro model. METHODS: Specific structures of the organ of Corti were simulated in a partially open, partially closed acrylic tank. This rough approximation allows the visualization of fluid flows that are generated as a result of the relative motions between the tectorial membrane and the reticular lamina. RESULTS: It was shown that the arrangement of the cochlear elements not only forces fluid to flow in a one-way direction, but also generates a fluid stream that flows through the "outlets" between each two V or W formations of stereocilia. These fluid streams are directed towards the inner hair cells.

Morphology of auditory hair cells in guinea pig cochlea after transgene expression.

It is very important to determine if recombinant adenoviral vector (Ad) can damage the auditory hair cells in guinea pig cochlea after transgene expression. In this study, the scanning electron microscope was used to determine if there was loss of the auditory hair cells after Ad.LacZ (Ad5 containing Escherichia coli galactosidase) was inoculated into the cochlea through the round window membrane. Seven days later all inner and outer hair cells were found to express the LacZ gene. Except for the sparse loss of outer hair cells in the basal turn and the second turn, there was insignificant loss in the other turns. All inner hair cells were present. The damage to auditory hair cells resulting from intracochlear inoculation of Ad is limited, and this vector can be used as one of the ideal delivery tools in gene therapy of the cochlea.

CGRP- and cholinergic-containing fibers project to guinea pig outer hair cells.

MU33, an antibody to calcitonin gene-related peptide (CGRP), was used to investigate the magnitude of CGRP-containing nerve fiber endings, compared to acetylcholinesterase (AChE)-containing nerve fiber endings on outer hair cells (OHCs). Results showed that CGRP-containing nerve fiber immunoreactivity mimicked the AChE fiber OHC staining pattern across the cochlea suggesting that CGRP can function as both a medial efferent (contacting primarily OHCs) and as a lateral efferent (contacting primarily inner hair cell afferents) neurotransmitter.

[The effects of quinine on active motile responses and fine structure of isolated outer hair cells from the Guinea pig cochlea]. / Der Einfluss von Chinin auf aktive Motilität und Feinstruktur isolierter äusserer Haarzellen der Meerschweinchenkochlea.

BACKGROUND: Large doses of quinine (as well as of salicylate) are known to produce reversible hearing loss and tinnitus. Cochlear outer hair cells seem to be the common site for the ototoxic effect of both drugs. METHODS: Isolated outer hair cells from the guinea pig cochlea were exposed to ototoxic doses of quinine hydrochloride (0.05-1.5 mmol/l). The cells were examined using tight-seal whole-cell recording techniques and transmission electron microscopy. RESULTS: Quinine exposure led to a hyperpolarization followed by a depolarization of the hair cells' membrane potential. It also caused a diminution of evoked rapid motile responses that was more apparent in response to hyperpolarizing than to depolarizing pulses. These effects were largely dose dependent and reversible. Ototoxic doses of quinine were not found to induce changes in turgor, shape or fine structure of outer hair cells such as those reported with ototoxic doses of salicylates in vitro. CONCLUSIONS: The present in vitro findings show that quinine (as well as salicylate) directly and reversibly affects cochlear outer hair cells. They also indicate that the underlying mechanisms of quinine ototoxicity are considerably different to that of salicylate although both substances clinically lead to identical symptoms.

Serotonergic innervation of the organ of Corti.

The olivocochlear efferent system of the mammalian cochlea, which is divided into two lateral and medial bundles, contains numerous neuroactive substances (acetylcholine, GABA, dopamine, enkephalins, dynorphins and CGRP). These have been located at the brainstem in neurons belonging to the lateral superior olive (lateral efferent system) or in neurons of the periolivary region around the medial superior olive and the trapezoid body (medial efferent system). All of these substances were found in well-characterized projections corresponding to lateral and medial nerve fibres and terminals which connect to the type I afferent dendrites and the outer hair cells, respectively. All could be involved in the modulation of the auditory process, as is suggested by the cochlear turnover increases observed in some of them (i.e. enkephalins or dopamine) induced by sound stimulation. Recently, the presence and distribution of serotonin-containing fibres has been included in the long list of cochlear neuroactive substances. However, its highly particular peripheral pattern of distribution together with the lack of response to sound stimulation could suggest that serotonergic fibres constitute a previously unknown cochlear innervation.

Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics.

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

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.

Contribution of membrane cholesterol to outer hair cell lateral wall stiffness.

The outer hair cell can be divided into three domains: the apex, the base, and the lateral wall. With the use of filipin, a polyene fluorescent antibiotic that binds to cholesterol, we found under fluorescence microscopy that the lateral wall membranes were less intensely stained than the apical and basal membranes. This difference in filipin fluorescence between the lateral walls and the ends diminished when cells were incubated in water-soluble cholesterol before staining, suggesting that exogenous cholesterol enters the lateral wall. Under confocal microscopy, we studied the incorporation pattern of a fluorescent cholesterol analogue, NBD-cholesterol. NBD-cholesterol did not stain the apical membranes whereas it intensely labeled the lateral wall. The micropipette aspiration technique was used to assess the effect of cholesterol on lateral wall stiffness. The lateral wall stiffness parameter of cells treated with water-soluble cholesterol (n = 23) was significantly higher than that of controls (n = 27): 0.76+/-0.24 (mean +/- SD) versus 0.46+/-0.10 (Student's t-test, p < 0.001). In conclusion, cholesterol has different distributions among outer hair cell membranes, and when water-soluble cholesterol is incorporated into the cells, the outer hair cell lateral wall stiffness parameter increases.

[High resolution scanning electron microscopy of isolated outer hair cells]. / Hochauflösende Rasterelektronenmikroskopie von isolierten ausseren Haarzellen.

Isolated hair cell preparations have gained wide acceptance as a model for studying physiological and molecular properties of the sensory cells involved in the hearing process. Ultrastructural details, such as stereocilia links, lateral membrane substructure or synaptic links are of crucial importance for normal sensory transduction. For this reason, we developed a high-resolution scanning electron microscopy (SEM) procedure to study the surface of isolated hair cells. Cells were mechanically and/or enzymatically separated, isolated and immobilized on cover slips by alcian blue and fixed by 2% glutardialdehyde or 1% OsO4. After dehydration, preparations were critical point-dried and sputter-coated with gold-palladium (2-4 nm). Up to 5 nm resolution was achieved. Optimal fixation kept the cells in their typical cylindrical forms. Preservation of the stereocilia and the apical plates of the outer hair cells depended strongly on the fixation process. Tip- and side-links were observed only sporadically because of the aggressive preparation procedure. The lateral plasma membranes of the cell bodies showed regular granular structures of 5-7 nm diameter at maximal magnification. The granular structure of the cell membrane seemed to correspond to putative transmembrane proteins believed to generate membrane-based motility. The remnants of the nerve endings and/or supporting cells usually covered the cell base. The preservation of the cells was better when enzymatic isolation was omitted. The technique used allowed for high resolution ultrastructural examination of isolated hair cells and, when combined with immunological labeling, may permit the identification of proteins at a molecular level.

Synaptophysin and GAP-43 proteins in efferent fibers of the inner ear during postnatal development.

A rearrangement of afferent and efferent fibers occurs in the postnatal development of the inner ear. Growth and synaptogenesis was explored during this critical period by immunohistochemically monitoring the expression of GAP-43 and synaptophysin. Both proteins were colocalized in efferent fibers beyond postnatal day 3 (pn3). Two distinct synaptophysin- and GAP-43-positive fibers innervated different parts of inner hair cells in the first and second postnatal weeks, respectively. GAP-43-positive efferents projecting to outer hair cells upregulated synaptophysin with base to apex gradient between postnatal day 5 and postnatal day 14. In efferents projecting to outer hair cells GAP-43 was downregulated about 6 days beyond synaptogenesis. In efferents projecting to inner hair cells, however, GAP-43 remained upregulated even beyond pn18, indicating continuous synapse replacement of this fiber type. Both proteins thus improved as excellent markers for growth and synaptogenesis of distinct postnatal efferent fibers.

Hyaluronic acid as a molecular filter and friction-reducing lubricant in the human inner ear.

Immunofluorescence for hyaluronic acid occurred intracellularly in morphologically highly specialized areas in the adult human inner ear, for instance in the cuticular plates of all types of hair cells, at the apposition between outer hair cells and Deiter's cell bodies and in the near-surface area of Hensen's cells. The cytoskeletal organization in these regions is characterized by tightly packed filamentous proteins. Under physiological stimulus these regions undergo micromechanical change, either actively moving (force generation) or passively vibrating with changes in elasticity. Hyaluronic acid might therefore act as a friction-reducing molecular lubricant. In the lateral wall of the cochlea an accumulation of hyaluronic acid occurred in the loose connective tissue of the spiral ligament, in particular close to the stria vascularis. Due to its complex molecular network, hyaluronic acid offers considerable resistance to bulk flow of water and may exclude molecules. The basal cell region of the stria vascularis is thus given additional support to minimize (seal?) the stria vascularis towards all other areas except the endolymphatic space. Here, hyaluronic acid could act as a molecular filter.

Calbindin-D28K localization in the primate inner ear.

The distribution of one of the calcium-binding proteins, calbindin-D28K (CB-D28K), was studied in the adult human and squirrel monkey inner ear by means of immunocytochemical methods. Inner and outer hair cells in the organ of Corti and vestibular hair cells showed CB-D28K immunoreactivity, though some vestibular hair cells were devoid of immunoreactivity. In the spiral and vestibular ganglion, immunoreactive cells were found in both the squirrel monkey and human. The present results indicate that CB-D28K is localized within afferent neuronal components in these sensory organs and may regulate Ca++ levels for optimal neurotransmission in the primate auditory and vestibular systems. This study also provides evidence of two nonneuronal localizations of CB-D28K in the squirrel monkey. Subpopulations of fibrocytes in the spiral ligament and vestibular end organs were enriched with CB-D28K, suggesting that these cells are possibly equipped with the function to regulate Ca++ concentration in the perilymphatic fluid. In the maculae, many CB-D28K-immunoreactive particles were found in the otoconial membrane, indicating that CB-D28K may participate in the formation of otoconia.

Microtubule-associated proteins in adult human sensory organs.

The distribution of microtubule-associated proteins MAP-1 and MAP-2 was analysed with immunomorphological techniques in the serially sectioned adult human membranous labyrinth. In the organ of Corti, monoclonal antibodies to MAP-1 did not stain. Positivity for MAP-2 occurred in the entire outer hair cell cytoplasm, in the inner hair cells (?), in the nerve fibres and in the cytoplasm of epithelial cells of the spiral prominence. In addition, staining for MAP-2 was identified in many (but not all) cells or Reissner's membrane. Immunofluorescence for MAP-1 occurred in the supporting cells of the cristae and maculae interpreted to be localized in the apical region adjacent to the sensory cells. Thus, the distribution of MAP-1 and MAP-2 in the adult human membranous labyrinth was the same as described for several animal species with regard to the cochlea. In contrast to such a pattern, both MAP-1 and MAP-2 were identified in the human vestibular organs, thus identifying a subpopulation of centrally located nerve calyces and possibly also the apical portion of vestibular hair cells.

Acute hyperpolarization and elongation of cochlear outer hair cells on superfusion with cis-platinum.

The acute effects of cis-platinum on isolated cochlear outer hair cells (OHC) were investigated with whole-cell patch-clamps and measurements of cell length changes. Our findings demonstrated that cis-platinum reversibly induced a hyperpolarization and cellular elongation. These results suggest that the effects produced are the result of an interaction between cis-platinum and transduction channels in OHC. These acute effects are distinctly different from the chronic, irreversible ones that are followed by death of the OHC. The exact mechanism of these chronic effects remains unknown as yet.