Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

Interconnections between the stereovilli of the fish inner ear

Summary We investigated at the electron-microscopic level the hair bundles of the macula organs in the inner ear of two species of teleost fish (Rutilus rutilus, Scardinius erythrophthalmus). All hairs (stereovilli) are interconnected by extracellular material that, similar to the cell coat, is well contrasted by application of tannic acid/osmium.The kinocilium is connected to the longest stereovilli via filaments. Filaments also connect the stereovilli at their bases. More distally all stereovilli are linked by thin filaments or very short solid connectors.The interconnections of the stereovilli in fish show greater variations, and are different in structural detail and distribution, compared to the interconnections described for amphibians and mammals. The interconnections are discussed in the context of their structural and functional significance and variations in terms of organ- and species specificity. Certain forms of interconnections are tentatively interpreted to be characteristic for developing hair bundles.     

Structures transmitting stimulatory force to the sensory hairs of vestibular ampullae of fishes and frog

Summary Serial sections of the vestibular ampullae of two species of fish and one species of frog were investigated by electron microscopy. The kinocilium is the only connection between the sensory cells and the auxiliary structure (cupula). The cupula possesses canals that traverse its entire height. Each canal contains a single kinocilium in its proximal part; distally, it is filled with material that stains with colloidal silver. The matrix of the cupula consists of filaments running perpendicular to the canals. These filaments do not stain with colloidal silver. The kinocilium is connected to the wall of the canal via structures that differ in the studied species of fish and frog. The filamentous links between the kinocilium and the longest stereovilli of the sensory hair bundle are similar in all the investigated species. The stereovilli are interconnected by basal and shaft links, and by horizontal and oblique tip connectors, similar to those described by other authors for macula organs and the organ of Corti, although differences in structural details, especially of the horizontal tip and the shaft connectors, are present. Some of these are species specific and some are related to the position of the sensory cell in the epithelium and/or specific to the organ (ampulla or macula organ). Some attachment sites of the links are associated with osmiophilic submembranous material. These differences in the structure, distribution and attachment sites of the links are possibly of functional importance.     

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli.
  New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.     

A Qualitative and Quantitative Study of a Sensory Epithelium in the Inner Ear of a Fish (Colisa labiosa; Anabantidae)

The macula lagenae of the anabantide fish Colisa labiosa was studied with light and transmission electron microscopy. (1) The sensory area is naturally divided in a central area (A) surrounded by a peripheral part (B). (2) Generally the central hair cells are separated by supporting cells, while the peripheral hair cells are found in groups. The cells of a group are not separated by supporting cells. (3) Tubuli-like structures, hexagonal in cross section, are found in all cells. In peripheral hair cells the longitudinally oriented tubuli-like structures are aggregated in thick bundles. (4) Variation in shape, electron density, stereocilia arrangement and size of mitochondria was found in different hair cells. (5) The central hair cells contain large accumulations of presynaptic bodies (10–44). Contrarily, the peripheral hair cells contain only a few pre-synaptic bodies (1–3). (6) The central hair cells are innervated by thick afferent (6–15 μm) and fine presumed efferent (less than 1 μm nerve fibres, while the peripheral hair cells are innervated by thin (1–6 μm) afferent nerve fibres only.     

Evidence for multiple,developmentally regulated isoforms of Ptprq on hair cells of the inner ear

Ptprq is a receptor‐like inositol lipid phosphatase associated with the shaft connectors of hair bundles. Three lines of evidence suggest Ptprq is a chondroitin sulfate proteoglycan: (1) chondroitinase ABC treatment causes a loss of the ruthenium‐red reactive, electron‐dense particles associated with shaft connectors, (2) chondroitinase ABC causes an increase in the electrophoretic mobility of Ptprq, and (3) hair bundles in the developing inner ear of wild‐type mice, but not those of Ptprq^?/? mice, react with monoclonal antibody (mAb) 473‐HD, an IgM that recognizes the dermatan‐sulfate‐dependent epitope DSD1. Two lines of evidence indicate that there may be multiple isoforms of Ptprq expressed in hair bundles. First, although Ptprq is expressed throughout the lifetime of most hair cells, hair bundles in the mouse and chick inner ear only express the DSD1 epitope transiently during development. Second, mAb H10, a novel mAb that recognizes an epitope common to several avian inner‐ear proteins including Ptprq, only stains mature hair bundles in the extrastriolar regions of the vestibular maculae. MAb H10 does not stain mature hair bundles in the striolar regions of the maculae or in the basilar papilla, nor does it stain immature hair bundles in any organ. Three distinct, developmentally regulated isoforms of Ptprq may therefore be expressed on hair bundles of the chick inner ear. Hair bundles in the mature chick ear that do not express the H10 epitope have longer shaft connectors than those that do, indicating the presence or absence of the H10 epitope on Ptprq may modulate the spacing of stereocilia. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 129‐141, 2011     

Cytoskeleton-membrane interactions in the cnidocil complex of hydrozoan nematocytes

Summary Each cnidocil complex of the hydrozoans Tubularia larynx and Hydra vulgaris consists of 9 or 7–10 large stereovilli (=stereocilia), respectively, and a modified cilium, the cnidocil. The cnidocils comprise the regular 9 microtubule doublets, up to 30 additional microtubules, as well as a central filament body. Adjacent stereovilli are linked together by intermembrane connectors forming the stereovillar cone. The distal tips of the stereovilli surround the cnidocil in a closed tubular arrangement measuring up to 0.7 m in length. Within this contact region the cnidocil is linked to the stereovillar tube by another set of intermembrane connectors, which seem to hold the cnidocil in a central position within the stereovillar cone. Stereovillar membrane and actin core are linked by 16-nm long cross bridges, which display a periodicity of 16 nm and emerge from the actin core. Within the cnidocils periodically arranged membrane-cytoskeleton bridges are uniformly restricted to the contact region. Here, 24-nm long cross bridges, which are spaced by a regular distance of 20 nm, interconnect the A-tubules of the microtubule doublets and the membrane. The cnidociliary membrane is differentiated into distinct domains as revealed by freeze-fracturing. Within the contact region of the nematocytes of Tubularia larynx, intramembrane particles are arranged in 9 rows of 700 nm length and 50 nm width, separated by particlefree areas. Intramembrane particles are irregularly distributed distal to the contact region. Considering recent physiological results we presume that the latter represent chemoreceptor units, while mechanical stimuli are transmitted via the intermembrane connectors and the microtubule-membrane bridges to mechanosensitive channels within the domain of the cnidociliary membrane in the contact region.     

Evidence for an auditory fovea in the New Zealand kiwi (Apteryx mantelli)

Kiwi are rare and strictly protected birds of iconic status in New Zealand. Yet, perhaps due to their unusual, nocturnal lifestyle, surprisingly little is known about their behaviour or physiology. In the present study, we exploited known correlations between morphology and physiology in the avian inner ear and brainstem to predict the frequency range of best hearing in the North Island brown kiwi. The mechanosensitive hair bundles of the sensory hair cells in the basilar papilla showed the typical change from tall bundles with few stereovilli to short bundles with many stereovilli along the apical-to-basal tonotopic axis. In contrast to most birds, however, the change was considerably less in the basal half of the epithelium. Dendritic lengths in the brainstem nucleus laminaris also showed the typical change along the tonotopic axis. However, as in the basilar papilla, the change was much less pronounced in the presumed high-frequency regions. Together, these morphological data suggest a fovea-like overrepresentation of a narrow high-frequency band in kiwi. Based on known correlations of hair-cell microanatomy and physiological responses in other birds, a specific prediction for the frequency representation along the basilar papilla of the kiwi was derived. The predicted overrepresentation of approximately 4-6 kHz matches potentially salient frequency bands of kiwi vocalisations and may thus be an adaptation to a nocturnal lifestyle in which auditory communication plays a dominant role.     

Chiton integument: Ultrastructure of the sensory hairs of Mopalia muscosa (Mollusca: Polyplacophora)

Summary The dorsal integument of the girdle of the chiton Mopalia muscosa is covered by a chitinous cuticle about 0.1 mm in thickness. Within the cuticle are fusiform spicules composed of a central mass of pigment granules surrounded by a layer of calcium carbonate crystals. Tapered, curved chitinous hairs with a groove on the mesial surface pass through the cuticle and protrude above the surface. The spicules are produced by specialized groups of epidermal cells called spiniferous papillae and the hairs are produced by trichogenous papillae. Processes of pigment cells containing green granules are scattered among the cells of each type of papilla and among the common epidermal cells.The wall or cortex of each hair is composed of two layers. The cortex surrounds a central medulla that contains matrix material of low density and from 1 to 20 axial bundles of dendrites. The number of bundles within the medulla varies with the size of the hair. Each bundle contains from 1 to 25 dendrites ensheathed by processes of supporting cells. The dendrites and supporting sheath arise from epidermal cells of the central part of the papilla. At the base of each trichogenous papilla are several nerves that pass into the dermis. Two questions remain unresolved. The function of the hairs is unknown, and we have not determined whether the sensory cells are primary sensory neurons or secondary sensory cells.     

Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

Scanning electron microscopic observations on the inner ear of the skate,Raja ocellata

The inner ear of the skate, Raja ocellata, was examined by scanning electron microscopy. The otolithic membranes have a gelatinous component and an endogenous class of otoconia. Cupulae are reticulate in form. The morphology and polarization of sensory cell hair bundles are described for the various regions of the labyrinth, and are compared with published observations on other species. In the otolithic maculae, the more centrally located receptor cells generally have longer sterecolia than the peripheral cells. The hair bundles of the lacinia are similar to those of the central portion of the sacculus and differed from those of the rest of the utricular macula. Hair bundles in the peripheral regions of all maculae and cristae are similar. The polarization pattern of the utriculus is similar to that of teleosts, while that of the lagena is less clearly dichotomized. The receptor cells of most of the sacculus are oriented in a bivertical direction, with cells in the anterior portion, and a few in the posterior region, being aligned longitudinally. The significance of morphology and polarization with respect to the functions of the otolithic organs is discussed. The relationship of cell processes of the ampullary receptors to the cupula is briefly considered.     

The cellular organization and nervous supply of the basilar papilla in the lizard,Calotes versicolor

Summary The basilar papilla of the lizard Calotes versicolor contains about 225 sensory cells. These are of two types: the short-haired type A cells in the ventral (apical) part of the organ, and the type B cells with long hair bundles, in the dorsal (basal) part of the organ. The type A cells are unidirectionally oriented and are covered by a tectorial membrane while the type B cells lack a covering structure and their hair bundles are oriented bidirectionally. Apart from those differences, the type A and type B cells are similar. They are columnar, and display the features common to most sensory cells in inner ear epithelia. The sensory cells are separated by supporting cells, which have long slender processes that keep the sensory cells apart. Close to the surface of the basilar papilla a terminal bar of specialized junctions interlocks adjacent cells. Below this, adjacent supporting cells are linked by an occluding junction.The cochlear nerve enters from the medial (neural) aspect. The fibres of the nerve lose their myelin sheaths as they enter the basilar papilla. Each sensory cell is associated with several nerve endings. All the nerves identified were afferent. Marked variations were seen between nerve endings in the basilar papilla, but no morphological equivalents of any functional differences were observed.This work is supported by grant no. B76-12X-00720-11A from the Swedish Medical Research Council, and by funds from the Karolinska Institute, Stockholm, Sweden.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Early peripheral sensory neurons in the development of trochozoan animals

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.     

Kinocilia mediate mechanosensitivity in developing zebrafish hair cells

Mechanosensitive cilia are vital to signaling and development across many species. In sensory hair cells, sound and movement are transduced by apical hair bundles. Each bundle is comprised of a single primary cilium (kinocilium) flanked by multiple rows of actin-filled projections (stereocilia). Extracellular tip links that interconnect stereocilia are thought to gate mechanosensitive channels. In contrast to stereocilia, kinocilia are not critical for hair-cell mechanotransduction. However, by sequentially imaging the structure of hair bundles and mechanosensitivity of individual lateral-line hair cells in?vivo, we uncovered a central role for kinocilia in mechanosensation during development. Our data demonstrate that nascent hair cells require kinocilia and kinocilial links for mechanosensitivity. Although nascent hair bundles have correct planar polarity, the polarity of their responses to mechanical stimuli is initially reversed. Later in development, a switch to correctly polarized mechanosensitivity coincides with the formation of tip links and the onset of tip-link-dependent mechanotransduction.     

Surface morphology of basilar papilla of the tufted duck Aythya fuligula,and domestic chicken Gallus gallus domesticus

Quantitative details of the surface morphology of the hearing organ, the Papilla basilaris, as seen in the scanning electron microscope are described for the tufted duck Aythya fuligula and for comparison for the domestic chicken Gallus gallus domesticus, for which some published information is already available. As in the other avian species investigated to date, each papilla shows a unique constellation of features. The papilla of the tufted duck is 3.5 mm long in the unfixed state and contains 8,200 sensory hair cells. It shows systematic changes in its surface features along the length and across the width of the sensory epithelium. In general, its features and those of the chicken Papilla basilaris can be described as relatively primitive in comparison with other species. The tufted duck papilla does, however, show one feature that has so far been found to be well developed only in advanced papillae; the number of stereovilli per hair cell bundle is generally much higher on hair cells of the neural than those on the abneural side. This difference is only weakly developed in the chicken. It is clear that features considered to be evolutionarily advanced were acquired independently of one another during evolution and that each bird species can show a mosaic of primitive and advanced features. © 1996 Wiley-Liss, Inc.     

Cellular distribution of parvalbumin immunoreactivity in the peripheral vestibular system of three rodents

The cellular distribution of parvalbumin immunoreactivity in the vestibular peripheral system of mouse, rat, and guinea pig was investigated by light and electron microscopy. Parvalbumin was found in all neurons of the vestibular ganglia of these species but in the sensory epithelia immunoreactivity was restricted to type I hair cells localized exclusively in the central areas. The very intense staining pattern was similar in the cristae ampullares and utricles of all three species but a faint immunoreaction was also detectable in sensory cells of peripheral areas of rat cristae. The parvalbumin-immunoreactive type I sensory cells are connected by nerve fibres of the calyx unit type which are known selectively to contain calretinin.     

Hair cells without supporting cells: further studies in the ear of the zebrafish mind bomb mutant

Each sensory hair cell in the ear is normally surrounded by supporting cells, which separate it from the next hair cell. In the mind bomb mutant, as a result of a failure of lateral inhibition, cells that would normally become supporting cells differentiate as hair cells instead, creating sensory patches that consist of hair cells only. This provides a unique opportunity to pinpoint the functions for which supporting cells are required in normal hair cell development. We find that hair cells in the mutant develop an essentially normal cytoskeleton, with a correctly structured hair bundle and well-defined planar polarity, and form apical junctional complexes with one another in standard epithelial fashion. They fail, however, to form a basal lamina or to adhere properly to the adjacent non-sensory epithelial cells, which overgrow them. The hair cells are eventually expelled from the ear epithelium into the underlying mesenchyme, losing their hair bundles in the process. It is not clear whether they undergo apoptosis: many cells staining strongly with the TUNEL procedure are seen but do not appear apoptotic by other criteria. Supporting cells, therefore, are needed to hold hair cells in the otic epithelium and, perhaps, to keep them alive, but are not needed for the construction of normal hair bundles or to give the hair bundles a predictable polarity. Moreover, supporting cells are not absolutely required as a source of materials for otoliths, which, though small and deformed, still develop in their absence.     

Structure and Innervation of the Inner Ear Sensory Organs in an Otophysine Fish,the Upside–down Catfish (Synodontis nigriventrisDavid)

All the sensory epithelia of the inner ear in the upside–down catfish (Synodontis nigriventrisDavid) were examined by light microscopy. The morphology of the membranous labyrinth and the orientation of the hair cells is similar to what has been found in other otophysine fishes. The sensory cells are of variable size both inter– and intraepithelially; particularly the macula sacculi is equipped with heterogeneous receptors. Regional differences in the hair cell density are presented for all the otolith organs plus the papilla neglecta. Nerve stainings reveal regional differentiation. The central areas are innervated by stout and stubbly nerve endings intermingled with a few thin nerve fibres while the peripheral parts are reached exclusively by thin axons. In the anterior region of the macula sacculi are found unique cup–shaped axon terminations which surround the basal parts of a single or a few sensory cells. The number and diameter range of the myelinated nerve fibres as well as the hair cell/axon ratio are presented. Electron microscopy demonstrates the presence of unmyelinated axons in all inner ear nerve ramuli.     

Histamine is a major mechanosensory neurotransmitter candidate in Drosophila melanogaster

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.