Surface Of Outer Hair Cells Research Articles

AFM Images of Outer Hair Cells' Lateral Plasma Membrane: An Autocorrelation Function Analysis

Our atomic force microscopic study of the cytosolic surface of outer hair cells' lateral plasma revealed images of membrane particles with tip-geometry- corrected diameter of ∼10 nm [1], consistent with 10-nm particles reported by earlier EM studies. These particles were aligned preferentially in one direction and a much weaker alignment consistent with hexagonal packing. The immunoreactivity of these particles to prestin-antibody revealed that these particles involve prestin, a member of the SLC26 family of anion transporters associated with electromotility of outer hair cells. This observation together with reports that prestin forms tetramers consistent with the dimension of these 10-nm particles prompts a question: Are 10-nm particles tetramers of prestin? To address this question, we examined autocorrelation function of AFM images for the detailed structure of these particles. If the slice plain of the peak is adjusted to the dimension that matches the particles, the contours should reveal shapes of the particle. We found the contour at the corresponding height is approximately square, consistent with tetramer symmetry. However, the maximum width of the central peak corresponded to ∼8.2 nm, somewhat smaller than the size of the particles obtained by section analysis. This difference can be attributed to blurring effect of noise. In summary, our observation is consistent with a hypothesis that 10-nm particles are prestin tetramers. [1] Organization of membrane motor in outer hair cells: an atomic force microscopic study, G. Sinha, F. Sabri, E. Dimitriadis, K. Iwasa, Pfugers Archiv European J. of Physiology, 2009.

Distribution of Prestin on Outer Hair Cell Basolateral Surface

Prestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility and is expressed on the OHC surface. Previous studies revealed that OHC electromotility and its associated nonlinear capacitance were mainly located at the OHC lateral wall and absent at the apical cuticular plate and the basal nucleus region. Immunofluorescent staining for prestin also failed to demonstrate prestin expression at the OHC basal ends in whole–mount preparation of the organ of Corti. However, there lacks a definitive demonstration of the pattern of prestin distribution. The OHC lateral wall has a trilaminate organization and is composed of the plasma membrane, cortical lattice, and subsurface cisternae. In this study, the location of prestin proteins in dissociated OHCs was examined using immunofluorescent staining and confocal microscopy. We found that prestin was uniformly expressed on the basolateral surface, including the basal pole. No staining was seen on the cuticular plate and stereocilia. When co–stained with a membrane marker di–8–ANEPPS, prestin–labeling was found to be in the outer layer of the OHC lateral wall. After separating the plasma membrane from the underlying subsurface cisternae using a hypotonic extracellular solution, prestin–labeling was found to be in the plasma membrane, not the subsurface cisternae. The data show that prestin is expressed in the plasma membrane on the entire OHC basolateral surface.

The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities

Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916-19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bullet-shaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 x 77 x 115 A, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.

Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear

Connexin gap junctions play an important role in hearing function, but the mechanism by which this contribution occurs is unknown. Connexins in the cochlea are expressed only in supporting cells; no connexin expression occurs in auditory sensory hair cells. A gap junctional channel is formed by two hemichannels. Here, we show that connexin hemichannels in the cochlea can release ATP at levels that account for the submicromolar concentrations measured in the cochlear fluids in vivo. The release could be increased 3- to 5-fold by a reduction of extracellular Ca2+ or an increase in membrane stress, and blocked by gap junctional blockers. We also demonstrated that extracellular ATP at submicromolar levels apparently affected outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining cochlear sensitivity to sound stimulation in mammals. ATP reduced OHC electromotility and the slope factor of the voltage dependence and shifted the operating point to reduce the active amplifier gain. ATP also reduced the generation of distortion products. Immunofluorescent staining showed that purinergic receptors P2x2 and P2x7 were distributed on the OHC surface. Blockage of P2 receptors eliminated the effect of ATP on the OHC electromotility. The data revealed that there is a hemichannel-mediated, purinergic intercellular signaling pathway between supporting cells and hair cells in the cochlea to control hearing sensitivity. The data also demonstrated a potential source of ATP in the cochlea.

Claudin 14 knockout mice, a model for autosomal recessive deafness DFNB29, are deaf due to cochlear hair cell degeneration.

Tight junctions (TJs) create ion-selective paracellular permeability barriers between extracellular compartments. In the organ of Corti of the inner ear, TJs of the reticular lamina separate K(+)-rich endolymph and Na(+)-rich perilymph. In humans, mutations of the gene encoding claudin 14 TJ protein cause profound deafness but the underlying pathogenesis is unknown. To explore the role of claudin 14 in the inner ear and in other tissues we created a mouse model by a targeted deletion of Cldn14. In the targeted allele a lacZ cassette is expressed under the Cldn14 promoter. In Cldn14-lacZ heterozygous mice beta-galactosidase activity was detected in cochlear inner and outer hair cells and supporting cells, in the collecting ducts of the kidney, and around the lobules of the liver. Cldn14-null mice have a normal endocochlear potential but are deaf due to rapid degeneration of cochlear outer hair cells, followed by slower degeneration of the inner hair cells, during the first 3 weeks of life. Monolayers of MDCK cells expressing claudin 14 show a 6-fold increase in the transepithelial electrical resistance by decreasing paracellular permeability for cations. In wild type mice, claudin 14 was immunolocalized at hair cell and supporting cell TJs. Our data suggest that the TJ complex at the apex of the reticular lamina requires claudin 14 as a cation-restrictive barrier to maintain the proper ionic composition of the fluid surrounding the basolateral surface of outer hair cells.

Ultrastructural damage to the organ of corti during acute experimental Escherichia coli and pneumococcal meningitis in guinea pigs.

Experimental meningitis was induced in 16 pigmented guinea pigs by subarachnoid inoculation of mid log-phase 1 x 10(9) E. coli K-12 (n = 8) or 5 x 10(7) Streptococcus pneumoniae type 2 (n = 8). Animals were killed at various times between 3 and 12 h after inoculation and the ultrastructure of the organ of Corti (including the basilar membrane) was examined with high resolution scanning electron microscopy. Both E. coli and S. pneumoniae induced meningitis and invaded scala tympani. In both types of meningitis the apical surface of inner supporting cells developed craters. inner hair cell stereocilia were also disrupted. In pneumococcal meningitis both these lesions were more pronounced but in addition there were breaks in the junctions between inner hair cells and their adjacent supporting cells and there was ballooning and rupture of the apical surface of outer hair cells. Damage to the organ of Corti after bacterial invasion of the inner ear may be one of the mechanisms by which bacterial meningitis can cause deafness. The more severe cochlear lesions induced by S.pneumoniae may explain the higher incidence of deafness after pneumococcal meningitis.

Transitory endolymph leakage induced hearing loss and tinnitus: depolarization, biphasic shortening and loss of electromotility of outer hair cells.

There are types of deafness and tinnitus in which ruptures or massive changes in the ionic permeability of the membranes lining the endolymphatic space [e.g., of the reticular lamina (RL)] are believed to allow potassium-rich endolymph to deluge the low [K+] perilymphatic fluid (e.g., in the small spaces of Nuel). This would result in a K+ intoxication of sensory and neural structures. Acute attacks of Ménière's disease have been suggested to be an important example for this event. The present study investigated the effects of transiently elevated [K+] due to the addition of artificial endolymph to the basolateral cell surface of outer hair cells (OHC) in replicating endolymph-induced K+ intoxication of the perilymph in the small spaces of Nuel. The influence of K+ intoxication of the basolateral OHC cell surface on the transduction was then examined. Intoxication resulted in an inhibition of the physiological repolarizing K+ efflux from hair cells. This induced unwanted depolarizations of the hair cells, interfering with mechanoelectrical transduction. A pathological longitudinal OHC shortening was also found, with subsequent compression of the organ of Corti possibly influencing the micromechanics of the mechanically active OHC. Both micromechanical and electrophysiological alterations are proposed to contribute to endolymph leakage induced attacks of deafness and possibly also to tinnitus. Moreover, repeated or long-lasting K+ intoxications of OHC resulted in a chronic and complete loss of OHC motility. This is suggested to be a pathophysiological basis in some patients with chronic hearing loss resulting from Ménière's syndrome.

Further studies on the ultrastructure of the cochlea

Hearing research is important not only for clinical, professional and military medicine, but also for toxicology, gerontology and genetics. Ultrastructure of the cochlea attracts much attention of electron microscopists, (1―3) but the research lags far behind that of the other parts of the organnism. On the basis of careful microdissection, technical improvment and accurate observation, we have got some new findings which have not been reported in the literature.We collected four cochleas from human corpses. Temporal bones dissected 1 h after death and cochleas perfused with fixatives 4 h after death were good enough in terms of preservation of fine structures. SEM:The apical surface of OHCs (Outer hair cells) and DTs (Deiters cells) is narrower than that of IPs (Inner pillar cells). The mosaic configuration of the reticular membrane is not typical. The stereocilia of IHCs (Inner hair cells) are not uniform and some kinocilia could be seen on the OHCs in adults. The epithelial surface of RM (Reissner’s membrane) is not smooth and no mesh could be seen on the mesothelial surface of RM. TEM.

Ionic environment of cochlear hair cells.

The scala media of the adult cochlea in mammals comprises a morphologically closed compartment sealed with tight junctions of the intermediate to tight types. The unique ionic composition of endolymph is maintained by the stria vascularis through active reabsorption of sodium and active secretion of potassium against ionic gradients. The subtectorial space is only a partially closed compartment which communicates with the endolymph via holes in the tectorial membrane at its outer insertion to the organ of Corti. Hardesty's membrane divides the subtectorial space into two compartments: one facing the surfaces of inner hair cells and one facing the surfaces of outer hair cells. In the study of comparative anatomy, hair cells, e.g. in the lizard, basilar papilla are of two types: those covered with a tectorial membrane and those being free-standing lacking the tectorial membrane. The ionic environment of the hair cell surface seems to be the same, independent of whether covered with a tectorial membrane or not. The tectorial membrane itself is semipermeable to ions in the endolymphatic space. Only the surface structures of the hair cell with the sensory hairs facing the subtectorial space are exposed to the high concentration of potassium, whereas the remaining parts of the hair cell are surrounded by a fluid having a more normal extracellular type of ionic composition (cortilymph/perilymph). During embryonic development the ionic composition of endolymph develops in parallel with the morphologic maturation of the stria vascularis. A completely mature composition of endolymph is reached before any electrophysiological potentials in the cochlea can be elicited. The sensory hair surface of hair cells has reached a mature morphology prior to the maturation of endolymph. In several species the tectorial membrane is morphologically only partially mature when the increase of the potassium concentration of endolymph starts. Drugs primarily affecting the stria vascularis causing a transient change of the ionic composition of endolymph result in a transient dysfunction of inner ear potentials. If the ionic changes persist for longer time, morphological changes can occur in both the stria vascularis and the hair cells of the organ of Corti. Whether such changes are primarily caused by the ototoxic drug itself or by changes in the ionic composition of endolymph has to be explored further.

Postnatal maturation of cochlear sensory hairs in the mouse.

The surface morphology of inner hair cells in the cochlea of the mouse is fully developed at birth. At this time a regular pattern of elongated microvilli covers the surfaces of outer hair cells at all levels of the cochlea. The sensory hair on outer hair cells undergo a morphological maturation during the first days postnatally. A mature surface morphology is reached 5-7 days after birth. There are two gradients in hair cell maturation in the cochlea: (1) basal to apical, and (2) from inner hair cells to the 3rd row of outer hair cells. Polarization of sensory hairs on an outer hair cell, i.e. polar differentiation of the sensory hairs in the radial direction, occurs as a stepwise increase in length of those facing the stria vascularis.

The tectorial membrane of the rat.

AbstractHistochemical, x‐ray analytical and scanning and transmission electron microscopical procedures have been utilized to determine the chemical nature, physical appearance and attachments of the tectorial membrane in normal rats and to correlate these results with biochemical data on protein‐carbohydrate complexes. Additionally, pertinent histochemical and ultrastructural findings in chemically sympathectomized rats are considered. The results indicate that the tectorial membrane is a viscous, complex, colloid of glycoprotein(s) possessing some oriented molecules and an ionic composition different from either endolymph or perilymph. It is attached to the reticular laminar surface of the organ of Corti and to the tips of the outer hair cells; it is attached to and enclose the hairs of the inner hair cells. A fluid compartment may exist within the limbs of the “W” formed by the hairs on each outer hair cell surface. Present biochemical concepts of viscous glycoproteins suggest that they are polyelectrolytes interacting physically to form complex networks. They possess characteristics making them important in fluid and ion transport. Furthermore, the macromolecular configuration assumed by such polyelectrolytes is unstable and subject to change from stress or shifts in pH or ions. Thus, the attachments of the tectorial membrane to the hair cells may play an important role in the transduction process at the molecular level.

Activity of acetylcholinesterase on the endolymphatic surface of outer hair cells.

By means of the Karnovsky's modification of Koelle's thiocholine method for acetylcholinesterase (AChE), it has been demonstrated electronmicro-scopically that AChE is located on the endolymphatic surface of outer hair cells and not on the supporting cells in the organ of Corti of the guinea pig cochlea. The tissue from the middle part of the basal turn was used in all experiments. The identification of AChE was confirmed by use of appropriate concentration of selective inhibitors of cholines-terase. From the inhibitory effect of fixatives on AChE activity, it was obvious that the activity on the surface of outer hair cells was lower than the activity on the large nerve endings at the bottom of the hair cells.