Norma B. Slepecky

Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells

The distribution of actin, alpha-actinin, fimbrin, tropomyosin and tubulin in the apical region of inner and outer hair cells was studied by immunofluorescent localization of antibodies to these proteins. The macromolecular distribution of actin and alpha-actinin was studied using post-embedding immunoelectron microscopic techniques. Actin is present in the stereocilia and cuticular plate of both inner and outer hair cells. Antibodies to actin were localized with fluorescence and colloidal gold. Colloidal gold particles were distributed uniformly over the stereocilia, stereocilia rootlets and cuticular plate. Fimbrin is present in the stereocilia and the cuticular plate. Immunofluorescent label was more intense over the cuticular plate of outer hair cells than over the cuticular plate of inner hair cells. Alpha-actinin is present in the cuticular plate only. At the ultrastructural level, antibodies to alpha-actinin were labeled throughout the cuticular plate, with larger accumulations of colloidal gold over the electron dense bodies in the cuticular plate, as well as over the electron dense region at the junctional complex. There was no label over the electron dense portion of the stereocilia rootlets. Tropomyosin is observed in the area of the stereocilia rootlets by immunofluorescent techniques, but like fimbrin, the antigenic sites of tropomyosin did not withstand processing for ultrastructural localization. Tubulin is not present in the apical region of inner or outer hair cells, although its presence could be documented in the hair cell body and in the supporting cells.

Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients

Whole mounts and tissue sections of the organ of Corti from two representative mammalian species, the Mongolian gerbil (Meriones unguiculatus) and the guinea pig (Cavea porcellus) were probed with antibodies to cytoskeletal and calcium-binding proteins (actin, tubulin, including post-translational modifications, spectrin, fimbrin, calmodulin, parvalbumin, calbindin, S-100 and calretinin). All of the proteins tested were expressed in both species. New findings include the following. Actin is present in large accumulations in cell bodies of the Deiters cells under the outer hair cells (OHC), as well as in the filament networks previously described. These accumulations are more prominent in the apical turns. Tubulin is present in sensory cells in the tyrosinated (more dynamic) form, while tubulin in the supporting cells is post-translationally modified, indicating greater stability. Fimbrin, present in the stereocilia of both IHCs and OHCs, is similar to the isoform of fimbrin found in the epithelial cells of the intestine (fimbrin-I), which implies that actin bundling by fimbrin is reduced in the presence of increased calcium. Parvalbumin appears to be an IHC-specific calcium-binding protein in the gerbil as well as in the guinea pig; labeling displays a longitudinal gradient, with hair cells at the apex staining intensely and hair cells at the base staining weakly. Calbindin displays a similar longitudinal gradient, with staining intense in the IHCs and OHCs at the apex and weak to absent in the base. In the middle turns of the guinea pig cochlea, OHCs in the first row near the pillar cells lose immunoreactivity to calbindin before those in the second and third rows. Calmodulin is found throughout the whole cochlea in the IHCs and OHCs in the stereocilia, cuticular plate, and cell body. Calretinin is present in IHCs and Deiters cells in both species, as well as the tectal cell (modified Hensen cell) in the gerbil. S-100 is a supporting cell-specific calcium-binding protein which has not been localized in the sensory cells of these two species. The supporting cells containing S-100 include the inner border, inner phalangeal, pillar, Deiters, tectal (in gerbil) and Hensen cells, where labeling displays a longitudinal gradient decreasing in intensity towards the apex (opposite to what has been seen with labeling for other proteins in the cochlea).

Overview of mechanical damage to the inner ear: noise as a tool to probe cochlear function.

The majority of experiments causing mechanical damage to the cochlea involve the use of sound pressure waves to cause overstimulation. This presentation is an overview of the research during the past years on the structural damage produced by noise. The effect of noise on the cochlea depends on the type of noise exposure-impulse or continuous. Experiments have been conducted to determine the effect of increasing intensity, the effect of increasing duration, and the effect of equal energy presented over varying periods of time. The initial mechanism of damage, the progression of damage over time, and the ability of hair cells to recover are discussed. Noise has been used as a tool to probe cochlear function by selectively damaging regions along the length of the sensory epithelium and by selectively damaging one of the two types of hair cells. Results obtained from these types of experiments have given us information on cochlear mechanics, as well as of stereocilia micromechanics and transduction. Information on susceptibility of hair cells to noise confirms previous results, suggesting the presence of structural and metabolic gradients both longitudinally and radially within the sensory epithelium. Moreover, noise lesions have been used to map the afferent innervation pattern to the cochlear nucleus, and noise studies show correlation of hair cell damage with efferent innervation pattern.

Actin-binding and microtubule-associated proteins in the organ of Corti

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.

The cell coat of inner ear sensory and supporting cells as demonstrated by ruthenium red

The ultrastructure of the cell coat of sensory and supporting cells of chinchilla and lizard inner ears was studied using ruthenium red. On the apical surface of both cell types, in both animals, the glycoproteins in the cell coat stain positively with this cationic dye. The apical surface of the sensory hair cells displays no regional variations in cell coat thickness. The uniform staining along the length of the stereocilia is not influenced by the normal presence or absence of a tectorial membrane. Although no micro-domains in the glycoproteins that stain positively with ruthenium red were observed that might correlate with the ultrastructural localization of sites of initiation of the transduction event, the cell coat material on the apical cell surface might play an important role in sequestering ions (particularly calcium) which are required for the transduction process.

Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti

Calcium is thought to play a major signaling role in outer hair cells to control metabolism, cytoskeletal integrity, cell shape and cell excitability. For this to happen, in resting cells the concentration of free calcium ions must be maintained at low levels so that focal increases can trigger specific events. In this paper, the localization of calcium, calcium-binding and calcium-dependent regulatory proteins in sensory cells from the guinea pig inner ear was demonstrated using immunocytochemical and histochemical techniques. We found the calcium buffer and/or calcium sensor proteins calmodulin, calbindin and calsequestrin predominantly in sensory cells and that when present, these proteins can be enriched in the outer hair cells. Calmodulin is found in the stereocilia, in the cuticular plate and in the cytoplasm and calbindin is found only in the cuticular plate and cytoplasm of both the inner and outer hair cells. The staining for these proteins in the outer hair cells is homogeneous, with no apparent compartmentalization along the lateral wall. Calsequestrin, thought to store and release calcium from membrane bound intracellular storage sites is found only in the cytoplasm of outer hair cells. There, it has a more punctuate staining pattern than does calmodulin or calbindin suggesting that it may be present in calciosomes rather than soluble in the cytoplasm. We did not detect caldesmon and S-100. Using the potassium pyroantimonate technique, we found precipitates containing calcium ions distributed throughout the cytoplasm of outer hair cells, with no evidence that the subsurface cisterns along the lateral wall act as calcium storage sites. Thus, calcium in resting cells is found in the cytoplasm along with calbindin and calmodulin and appears to have a punctate distribution consistent with a co-localization with calsequestrin. The implications of this distribution with respect to the slow shortening and elongation seen in outer hair cells are discussed.

Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

SummaryThe distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S1. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S1. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.

Localization of Type II, IX and V Collagen in the Inner Ear

Types II and IX collagen are traditionally considered cartilage collagens; however, within the inner ear, types II and IX collagen have a more diverse distribution. In the adult gerbil, type II collagen is the major fibrillar component. In the otic capsule it is present surrounding the osteocytes embedded and branching in the periosteal layer, in the cartilaginous rests of the enchondral layer, and in the endosteal layer bordering the membranous labyrinth. In the regions of the sensory cells, type II collagen is found in the osseous spiral lamina, the connective tissue of the spiral limbus, the subepithelial tissue of the maculae in the vestibule and the cristae in the ampullae, and in the spiral ligament. It is present in the non-cartilaginous and acellular structures of the tectorial membrane over the cochlear hair cells and the vestibular membrane lining the semicircular canals. Type IX collagen, when present, in all cases co-localizes with type II collagen but is found in more limited regions. It is found only in the cartilaginous rests of the enchondral bone, the tectorial membrane and the vestibular membrane. Type V-like collagen, a connective tissue collagen, is found to have a complementary localization to types II and IX collagen within the interstitial bone of the otic capsule, the osseous spiral lamina and the tectorial membrane, but it is absent from the vestibular membrane. This report is the first documenting the co-localization of types II and IX collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron-microscopic localization of type II, IX, and V collagen in the organ of Corti of the gerbil

SummaryThe presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.

Correlation of Audiometric Data with Changes in Cochlear Hair Cell Stereocilia Resulting from Impulse Noise Trauma

In a previous experiment, after chinchillas had been exposed to impulse noise trauma, plastic-embedded surface preparations of the organ of Corti were examined with the light microscope. A consistent relationship between cochlear hair cell loss and hearing loss was not found (Hamernik et al., 1980). In the present study, four cochleas from that experiment were sectioned and examined with the transmission electron microscope to determine if their were consistent patterns of damage to the sensory cells at the ultrastructural level that would more closely correlate with the audiometric data. Alterations of the outer hair cell stereocilia were found when threshold was elevated 15 to 30 dB. The membranes of the stereocilia appeared loose and wrinkled and the stereocilia were no longer erect. In some cases, predominantly in the first row of outer hair cells, stereocilia were missing and in other cases, stereocilia were fused. Within these giant stereocilia, the rootlets of the individual stereocilia had disintegrated. Other alterations in sensory cell ultrastructure, though present, had no consistent pattern and could not be related to changes in hearing thresholds. Only the changes in the outer hair cell stereocilia appeared to correlate with hearing loss and the degree of damage was reflected in the amount of threshold elevation.