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PubMed2004

Selective acquisition of individual cell types in the vestibular periphery for molecular biology studies.

OBJECTIVES: To develop a method for characterizing the transcriptome of individual cell types in the inner ear sensory epithelia. STUDY DESIGN: We employed the technique of laser capture microdissection to obtain enriched populations of hair cells and supporting cells. The respective mRNAs were extracted, reverse transcribed, and amplified using PCR. RESULTS: We were able to isolate RNAs with good integrity from enriched cell populations obtained with laser capture microscopy and amplify specific mRNA targets. CONCLUSIONS: We can now investigate the molecular differences between the different cell types in the inner ear sensory epithelia as identified by morphological criteria. SIGNIFICANCE: Analysis of gene expression profiles in the inner ear cell types has been hampered by the small size of this tissue and by the compact histoarchitecture of the sensory epithelia; however, the present technique offers new possibilities for the analysis of transcriptomes in the vestibular periphery using available high-throughput gene expression analysis methods.

Animals↗
PubMed2002

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells.

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.

Aging↗
PubMed1978

The ultrastructure of the basilar papilla of the chick.

The basilar papilla of the chicken was studied by transmission and scanning electron microscopy. It is composed of two distinct types of hair cells, a tall hair cell (THC) and a short hair cell (SHC). Gradual transitions exist between these two forms. The THC occurs mainly in the anterior part of the neuroepithelium and is situated on the superior fibro-cartilagenous plate, while the SHC dominates in the posterior part and is found on the free basilar membrane. The THC is a tall columnar cell with an elongated hexagonal apical surface. One kinocilium and 150 to 220 stereocilia protrude from the center of its surface. The THC makes synaptic contact with several large afferent nerve terminals and with small bouton-shaped efferent terminals. The SHC is short and thick, having a much wider surface area than the THC. From the inferior part of the surface of the SHC 90 to 120 stereocilia protrude, whereas the kinocilium is lacking. The nucleus is located near the base of the cell. Small afferent nerve terminals and extremely large efferent nerve endings synapse with the SHC.

Animals↗
PubMed1983

Sustentacular cells of the organ of Corti--the tectal cells of the outer tunnel.

The cells which form the roof of the outer tunnel of the organ of Corti were studied by light microscopy and scanning and transmission electron microscopy. In the mustache bat, Pteronotus p. parnellii, the cells are characterized by: (1) a unique position in the roof and along the lateral wall of the outer tunnel; (2) no contact with the basilar membrane; (3) isolation of adjacent cell bodies; (4) an extensive endolymphatic surface with a sparse population of short microvilli; (5) a loose association with the adjacent mat of polypous surface projections on the outer tunnel surface of the first row of Hensen's cells; and (6) a darkly staining cytoplasm. These cells occur in certain other mammals (cats and mice) and have been classified previously as Hensen's or Deiters' cells, but since they lack the distinct morphological characteristics of either of these types of cells, it is suggested that they be recognized as a distinct cell type, the tectal cells of the outer tunnel.

Animals↗
PubMed1984

Fine structure of cochlear innervation in the cat.

Examination of adult and juvenile cat cochleas by electron microscopy and semi-serial sections permitted identification of the cytological features characteristic of the afferent and efferent nerve fiber populations identified in Golgi impregnations of the cochlea. This study demonstrated the distribution of synaptic contacts made by these fiber populations. As in the Golgi findings, radial and outer spiral afferent fibers were identified in well separated zones of the inner spiral bundle. The trunks of the outer spiral fibers, containing many microtubules and few neurofilaments, at first coursed spirally below the inner hair cells on the proximal face of the inner pillar, turned abruptly between adjacent pillar cells and entered the tunnel without branching. Radial afferents, containing many neurofilaments and a few microtubules, coursed through the inner spiral bundle, maintaining a radial or oblique orientation and proceeded directly toward the inner hair cells. Efferent fibers in the region of the inner spiral bundle were distinguishable by size, by orientation, and, to a lesser extent, by cytology. Small (1 micron) efferent fibers, containing few neurofilaments, an occasional microtubule, and mitochondria, occurred in the inner and tunnel spiral bundles and formed large varicosities, which contacted radial afferents. A separate population of much thicker efferents, containing many neurofilaments, mitochondria and dense-cored vesicles, but no microtubules, did not enter the inner spiral bundle but coursed directly to the level of the tunnel spiral bundle on the proximal face of the inner pillar cells. These fibers crossed the tunnel at the level of the tunnel spiral bundle and, upon reaching the outer hair cells, formed large synaptic contacts on outer hair cells and on outer spiral fibers as well. Some of these efferent fibers also synapse on afferent fibers while crossing the tunnel. The findings agree with previous observations with the Golgi method showing that entirely separate populations of spiral ganglion cells innervate the inner and outer hair cells. Likewise, there are efferent fibers innervating only inner or outer hair cells, but the probability of efferent fibers to both inner and outer hair cells cannot be ruled out.

Animals↗
PubMed1987

Auditory stereocilia in the alligator lizard.

The normal anatomy of stereociliary tufts in the basilar papilla of the alligator lizard is described and demonstrated with scanning electron micrographs. Stereociliary tufts in the tectorial region differ from those in the free-standing region in several ways. Tectorial stereociliary tufts are short (less than 10 micron in height), unidirectional in orientation, and covered with a tectorial membrane. Free-standing stereociliary tufts are very tall (up to 38 micron in height), bidirectional in orientation and not covered by a tectorial membrane or any other tectorial substance. The heights of the stereociliary tufts along the length and across the width of the basilar papilla were measured in serial light and transmission electron micrographs. Free-standing stereociliary tufts decrease progressively in height along the length of the basilar membrane, being tallest at the apical end and shortest at the basal end. Tectorial stereociliary tufts do not increase progressively along the length of the basilar membrane but do increase progressively across the width of the basilar membrane, being shortest on the neural side. Free-standing stereociliary tufts are structurally simple being a prominent specialization of the lizard cochlea. Tectorial stereociliary tufts are structurally more complicated conforming more closely to the general anatomical pattern of vertebrate auditory hair cells.

Animals↗
PubMed2002

In vivo and in vitro localization of brain-derived neurotrophic factor, fibroblast growth factor-2 and their receptors in the bullfrog vestibular end organs.

The inner ear sensory epithelia of vertebrates are composed mainly of supporting cells and hair cells (HCs). Brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) are trophins that are believed to play an essential role in the development and innervation of inner ear epithelia. Both trophins also may play a crucial role in the maintenance and regeneration of hair cells in the adult vertebrate ear. In the bullfrog vestibular system, hair cells are produced throughout life, and the epithelia regenerates following ototoxicity. The expression of BDNF and FGF-2 in the vestibular organs of the adult bullfrog was investigated at a cellular level both in histological sections and in vitro in dissociated cell cultures. In histological sections of the crista ampullaris, in situ hybridization and immunocytochemical techniques demonstrated that HCs express both BDNF and its receptor trkB, while the supporting cells express the receptor trkB alone. Following dissociation and in vitro cell culture no changes in the pattern of BDNF and trkB receptor were observed. Immunocytochemical studies demonstrated that in vivo hair cells express FGF-2 and the receptors FGFR-1 and FGFR-2 while supporting cells do not express either molecule. Following dissociation, HCs continue to express FGF-2 and its two receptors, while supporting cells upregulate the expression of FGF-2 and its receptor FGFR-2. These data confirm the potential role of BDNF and FGF-2 trophic regulation of the sensory epithelia of the adult inner ear. The findings suggest that BDNF has a role in the maintenance of the vestibular epithelia while FGF-2 may regulate the proliferation of supporting cells.

Animals↗
PubMed1996

[Bromodeoxyuridine immunohistochemical observation of sensory epithelium repair after ototoxic destruction in the crista ampullaris of guinea pigs].

In this in vivo study we investigated the proliferation of crista ampullaris in guinea pigs after Gentamicin intoxication by BrdU immunohistochemical observation and semi-thin sections. The experimental animals were given a single intraperitonial injection of BrdU at a dose of 150 mg/kg body weight before subsequently killed at 1, 3, 7, 14 and 22 day post-treatment (PT). Supporting cell proliferation was evident by BrdU immunoreactivity in the chromatin material of dividing cells at 7 and 14 day PT. The results demonstrated that the crista ampullaris of guinea pigs had repaired by supporting cell mitosis.

Animals↗
PubMed1990

Ultrastructure of the supporting cells of the paratympanic organ in the chicken: a preliminary study.

Electron microscopic studies reveal that the supporting cells of the paratympanic organ in the chicken have a fine structure characterised by mitochondria, RER and a well developed Golgi apparatus; furthermore, several vesicles with a clear content can be observed in the apical cytoplasm. The ultrastructure of supporting cells indicates high metabolic activity. The possible metabolic and mechanical function of the supporting cells are discussed.

Animals↗
PubMed2006

Embryonic inner ear cells use migratory mechanisms to establish cell patterns in vitro.

The hair cells of the sensory epithelium in the inner ear are among the most precisely organized cells in vertebrates. The mechanisms that lead to this orderly arrangement are only beginning to be understood. It has been suggested that hair cells use migratory mechanisms to help achieve their final position in the organ of Corti. The small size and complex organization of the intact inner ear have made it difficult to monitor changes in hair cell location over time in vivo. In the present study, an established in vitro assay of dissociated, embryonic inner ear cells was used to monitor how hair cells reorganize over time. The hair cell specific marker myosin-VI demonstrated that hair cell precursors from both cochlear and vestibular regions reorganized into specific patterns between 3-24 hr in vitro. In contrast to the unlabeled cells, the myosin-VI-positive cells extended processes while establishing the hair cell patterning within an aggregate. These studies support the hypothesis that hair cell precursors actively migrate to help achieve final patterning within the inner ear sensory epithelium.

Animals↗
PubMed1994

Cytoskeletal organization of the vestibular sensory epithelia: saponin perfusion method for observing intracellular structures by scanning electron microscopy.

The cytoskeletal organization of the guinea pig vestibular sensory epithelial cells were investigated by the use of saponin perfusion method using scanning electron microscopy. The skeletal framework of a cell is composed of thin (actin or intermediate filaments) and thick filaments (microtubules). The membrane bound organelles such as nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, etc. were also well demonstrated. This made it possible to investigate the three-dimensional structures of cytoskeletons as well as their complex interactions with various membranes bound organelles. It is therefore suspected that this technique may provide us further information about distribution, topographic relationships, and the functional role of cytoskeletons.

Actin Cytoskeleton↗
PubMed1997

Morphological evidence for supporting cell to hair cell conversion in the mammalian utricular macula.

The possible origin of the immature hair cells that appear in the utricular maculae of guinea pigs following gentamicin-induced hair cell death was investigated. Guinea pigs were continuously infused with bromodeoxyuridine, to label proliferating cells and their progeny, for 2 weeks after inducing damage to the inner ear on one side with gentamicin. The opposite ear in each animal served as control. Serial sections were cut through the entire utricular maculae of both ears of each animal and the number of labelled cells in the epithelium and underlying connective tissue was counted. Label was present in cells in the sensory epithelium in the utricles from the drug exposed ears but not in the controls. The nuclei of cells in the underlying connective tissue were also labelled in both ears. Some of the labelled nuclei in the epithelium were at the level normally occupied by hair cells, but most were at the level of supporting cell nuclei. However, the total number of labelled nuclei in the sensory epithelium was small; the maximum was 12 in one animal. The number of labelled nuclei in the connective tissue of the treated ears was significantly greater than the number in the untreated ear. This confirms that cell proliferation is stimulated in the mature mammalian utricular macula after hair cell loss, but the extent to which it occurs appears to be insufficient to explain the recovery in hair cell numbers which is observed. Detailed thin section studies of the utricular maculae of gentamicin-treated animals over a prolonged post-treatment period were also performed. In utricles which had suffered damage, there were cells which, like supporting cells but unlike hair cells, were resting on basement membrane, but which possessed at their apical ends organized bundles of microvilli similar to immature hair cell stereocilia. Other cells with more obvious stereocilia remained in contact with the basement membrane via and a small feet process. In still other cells, where a stereociliary bundle was obvious and almost mature in appearance, there was a foot process extending towards the basement membrane but not quite in contact, suggesting it had just detached. All these cells were contacted by nerve endings and specialization of the membranes were apparent at the site of cell-neurone contact. The morphological characteristics of these cells are consistent with phenotypic conversion of supporting cells into hair cells and this may account for some of the hair cell production in the mature mammalian vestibular sensory epithelia after hair cell death.

Animals↗
PubMedDate not supplied

Morphological and molecular changes in the inner hair cell region of the rat cochlea after amikacin treatment.

This study investigates the morphological and molecular changes that occur in the inner hair cell area of the rat cochlea following aminoglycoside treatment. Rats were injected daily with 500 mg/kg of amikacin between postnatal day 9 (PND9) and PND16. Cochleae were examined at PND16 to PND120 using both scanning and transmission electron microscopy and molecular fluorescent labeling. The inner hair cells showed obvious signs of apoptosis in response to amikacin treatment and most of them were missing by one week after the end of the aminoglycoside exposure period. Concomitantly, the epithelium became scarred as the surrounding supporting cells expanded and filled the space vacated by the missing IHCs. The mid-basolateral region of these modified supporting cells was surrounded by many afferent and efferent terminals. However, these cells expressed neither calbindin nor SNAP25, proteins that are both expressed by IHCs in the normal, untreated organ of Corti in the rat. In addition, these supporting cells remained attached to the basal lamina by a thin cytoplasmic process. The supporting cells surrounding the inner hair cells therefore appear unable to convert directly into inner hair cells following aminoglycoside induced hair-cell loss but may be able to provide trophic support for the remaining afferent and efferent neurites.

Actins↗
PubMed2006

Regulation of cell fate in the sensory epithelia of the inner ear.

The sensory epithelia of the inner ear contain mechanosensory hair cells and non-sensory supporting cells. Both classes of cell are heterogeneous, with phenotypes varying both between and within epithelia. The specification of individual cells as distinct types of hair cell or supporting cell is regulated through intra- and extracellular signalling pathways that have been poorly understood. However, new methodologies have resulted in significant steps forward in our understanding of the molecular pathways that direct cells towards these cell fates.

Animals↗
PubMed1982

Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells.

Several structural and contractile proteins have been searched for with immunohistochemical methods using antibodies directed against these proteins. Three types of preparations from the guinea pig have been used: isolated stereocilia from the utricle, organ of Corti fragments obtained by cellular dissociation and 0.2-1 micrometer sections obtained by cryoultramicrotomy. The main finding is that different sets of proteins compose the cytoskeleton in supporting cells and the mechanoreceptor structures of the sensory cells. Thus, actin was found in association with fimbrin in the mechanoreceptive region of hair cells, whereas supporting cells, although rich in actin, did not reveal fimbrin. Instead tubulin was seen together with actin in supporting cells which also exhibited prekeratin. Fimbrin appears to function as a protein capable of making bundles and networks from actin filaments. Its exclusive presence in the mechanosensitive region of the sensory cells is possibly related to the function of these cells as mechanoreceptors.

Actins↗
PubMed1990

Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a post-embryonic fish.

The sources of new hair cells, support cells, and Schwann cells were identified in the statoacoustic end organs of normal post-embryonic fish (Astronotus ocellatus). S-phase cells, defined as cells that take up 3H-thymidine in preparation for mitosis, and their progeny were visualized using autoradiography. Two types of S-phase cells were found: 'embryonic-like neuroepithelial' (NE) cells and 'basally located S-phase' (BLS) cell. The NE cells had elongated nuclei and processes extending basally and apically. Thirty minutes after the thymidine injection labeled NE cell nuclei were found between hair cell nuclei and support cell nuclei. After two and four hours survival some labeled NE nuclei were closer to the lumen, where they divided. One labeled support cell was seen after four hours survival and one labeled hair cell after nine hours survival. Significant numbers of labeled support and hair cells were not seen until 24 h survival. These results led us to identify NE cells as the immediate source of new hair cells and support cells. The BLS cells had small nuclei with little surrounding cytoplasm. Shortly after the thymidine injection BLS cells were found between the hair cell nuclei and the basement membrane, and they underwent mitosis in this position. The BLS cells resemble intra-epithelial Schwann cells, except that they are not curved around axonal profiles. We suggest that the BLS cells are Schwann cell precursors.

Animals↗