Inner-ear morphology of the New Zealand Kiwi (Apteryx mantelli) suggests high-frequency specialization

The sensory systems of the New Zealand kiwi appear to be uniquely adapted to occupy a nocturnal ground-dwelling niche. In addition to well-developed tactile and olfactory systems, the auditory system shows specializations of the ear, which are maintained along the central nervous system. Here, we provide a detailed description of the auditory nerve, hair cells, and stereovillar bundle orientation of the hair cells in the North Island brown kiwi. The auditory nerve of the kiwi contained about 8,000 fibers. Using the number of hair cells and innervating nerve fibers to calculate a ratio of average innervation density showed that the afferent innervation ratio in kiwi was denser than in most other birds examined. The average diameters of cochlear afferent axons in kiwi showed the typical gradient across the tonotopic axis. The kiwi basilar papilla showed a clear differentiation of tall and short hair cells. The proportion of short hair cells was higher than in the emu and likely reflects a bias towards higher frequencies represented on the kiwi basilar papilla. The orientation of the stereovillar bundles in the kiwi basilar papilla showed a pattern similar to that in most other birds but was most similar to that of the emu. Overall, many features of the auditory nerve, hair cells, and stereovilli bundle orientation in the kiwi are typical of most birds examined. Some features of the kiwi auditory system do, however, support a high-frequency specialization, specifically the innervation density and generally small size of hair-cell somata, whereas others showed the presumed ancestral condition similar to that found in the emu.

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.

GATA Factors Regulate Inner Ear Development and Midbrain Neurogenesis

The zinc-finger transcription factors GATA2 and GATA3 in vertebrates belong to the six-member family that are essential regulators in the development of various organs. The aim of this study was to gain new information of the roles of GATA2 and GATA3 in inner ear morphogenesis and of the function of GATA2 in neuronal fate specification in the midbrain using genetically modified mouse and chicken embryos as models. A century ago the stepwise process of inner ear epithelial morphogenesis was described, but the molecular players regulating the cellular differentiation of the otic epithelium are still not fully resolved. This study provided novel data on GATA factor roles in several developmental processes during otic development. The expression analysis in chicken suggested that GATA2 and GATA3 possess redundant roles during otic cup and vesicle formation, but complementary cell-type specific functions during vestibular and cochlear morphogenesis. The comparative analysis between mouse and chicken Gata2 and Gata3 expression revealed many conserved aspects, especially during later stages of inner ear development, while the expression was more divergent at early stages. Namely, expression of both Gata genes was initiated earlier in chicken than mouse otic epithelium relative to the morphogenetic stages. Likewise, important differences concerning Gata3 expression in the otic cup epithelium were detected between mouse and chicken, suggesting that distinct molecular mechanisms regulate otic vesicle closure in different vertebrate species. Temporally distinct Gata2 and Gata3 expression was also found during otic ganglion formation in mouse and chicken. Targeted inactivation of Gata3 in mouse embryos caused aberrant morphology of the otic vesicle that in severe cases was disrupted into two parts, a dorsal and a ventral vesicle. Detailed analyses of Gata3 mutant embryos unveiled a crucial role for GATA3 in the initial inner ear morphogenetic event, the invagination of the otic placode. A large-scale comparative expression analysis suggested that GATA3 could control cell adhesion and motility in otic epithelium, which could be important for early morphogenesis. GATA3 was also identified as the first factor to directly regulate Fgf10 expression in the otic epithelium and could thus influence the development of the semicircular ducts. Despite the serious problems in the early inner ear development, the otic sensory fate establishment and some vestibular hair cell differentiation was observable in pharmacologically rescued Gata3-/- embryos. Cochlear sensory differentiation was, however, completely blocked so that no auditory hair cells were detected. In contrast to the early morphogenetic phenotype in Gata3-/- mutants, conditional inactivation of Gata2 in mouse embryos resulted in a relatively late growth defect of the three semicircular ducts. GATA2 was required for the proliferation of the vestibular nonsensory epithelium to support growing of the three ducts. Concurrently, with the role in epithelial semicircular ducts, GATA2 was also required for the mesenchymal cell clearance from the vestibular perilymphatic region between the membranous labyrinth and bony capsule. The gamma-aminobutyric acid-secreting (GABAergic) neurons in the midbrain are clinically relevant since they contribute to fear, anxiety, and addiction regulation. The molecular mechanisms regulating the GABAergic neuronal development, however, are largely unknown. Using tissue-specific mutagenesis in mice, GATA2 was characterized as a critical determinant of the GABAergic neuronal fate in the midbrain. In Gata2-deficient mouse midbrain, GABAergic neurons were not produced, instead the Gata2-mutant cells acquired a glutamatergic neuronal phenotype. Gain-of-function experiments in chicken also revealed that GATA2 was sufficient to induce GABAergic differentiation in the midbrain.

Wave digital filter model of the entire auditory periphery

A model of the auditory periphery assembled from analog network submodels of all the relevant anatomical structures is described. There is bidirectional coupling between networks representing the outer ear, middle ear and cochlea. A simple voltage source representation of the outer hair cells provides level-dependent basilar membrane curves. The networks are translated into efficient computational modules by means of wave digital filtering. A feedback unit regulates the average firing rate at the output of an inner hair cell module via a simplified modelling of the dynamics of the descending paths to the peripheral ear. This leads to a digital model of the entire auditory periphery with applications to both speech and hearing research.

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.

NO3- transport across the plasma membrane of Arabidopsis thaliana root hairs:Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO₃^-challenge and to quantify transport activity. The NO₃^--associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4-6 days postgermination. In 6-day-old seedlings, additions of 5-100 μm NO₃^-to the bathing medium resulted in membrane depolarizations of 8-43 mV, and membrane voltage (V_m) recovered on washing NO₃^-from the bath. Voltage clamp measurements carried out immediately before and following NO₃^-additions showed that the NO₃^--evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (-300 to +50 mV). Both membrane depolarizations and NO₃^--evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for K_m of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm^-2. The NO₃^-current showed a pronounced voltage sensitivity within the normal physiological range between -250 and -100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4-8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pH_o and [NO₃^-]_o. At a constant pH_o of 6.1, depolarization from -250 to -150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO₃^-. By contrast, the same depolarization effected an approximate 20% fall in the K_m for transport as a function in [H⁺]_o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO₃^-binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H⁺) per NO₃^-anion transported across the membrane. The results concur with previous studies showing a high-affinity NO₃^-transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO₃^-transport at the plant plasma membrane. © 1995 Springer-Verlag New York Inc.

Cable correction of membrane currents recorded from root hairs of Arabidopsis thaliana L.

Membrane currents were recorded under voltage clamp from root hairs of Arabidopsis thaliana L. using the two-electrode method. Concurrent measurements of membrane voltage distal to the point of current injection were also carried out to assess the extent of current dissipation along the root hair axis. Estimates of the characteristic cable length, λ, showed this parameter to be a function both of membrane voltage and of substrate concentration for transport. The mean value for λ at 0 mV was 103 ± 20 μm (n=17), but ranged by as much as 6-fold in any one cell for membrane voltages from -300 to +40 mV and was affected by 0.25 to 3-fold at any one voltage on raising [K⁺]₀ from 0.1 to 10 mol m^-3. Current dissipation along the length of the cells lead to serious distortions of the current-voltage [I-V) characteristic, including consistent underestimates of membrane current as well as a general linearization of the I-V curve and a masking of conductance changes in the presence of transported substrates. In some experiments, microelectrodes were also placed in neighbouring epidermal cells to record the extent of intercellular coupling. Even with current-passing microelectrodes placed at the base of root hairs, coupling was ≤5% (voltage deflection of the epidermal cell ≤5% that recorded at the site of current injection), indicating an appreciable resistance to current passage between cells. These results demonstrate the feasibility of using root hairs as a 'single-cell model' in electrophysiological analyses of transport across the higher-plant plasma membrane; they also confirmed the need to correct for the cable properties of these cells on a cell-by-cell basis. © 1994 Oxford University Press.

The effect of Nystatin on the inner ear : an experimental guinea pig study

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde.

Immunohistochemistry of synergistic effects of kanamycin and furosemide in the murine model

Adult CBA/CaJ mice were injected with large single dose of both kanamycin and furosemide in order to study hair cell lesion and macrophage recruitment within the cochlea.

Distortion product otoacoustic emissions in the bullfrog

Recovery of distortion product otoacoustic emissions in the bullfrog after noise exposure does not correlate with hair cell damage noted on the amphibian papilla.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Isolation of sphere-forming stem cells from the mouse inner ear

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

Distribution of phytolithes in CRP sediment cores from the Ross Sea, Antarctica

Phytoliths (siliceous plant microfossils) have been recovered from Cenozoic sediments (c. 34 to 17 Ma) in the CRP-2/2A and CRP-3 drillholes cored off Cape Roberts, Victoria Land Basin, Antarctica. The phytolith assemblages are sparse, but well-preserved and dominated by spherical forms similar to those of modern trees or shrubs. Rare phytoliths comparable to modern grass forms are also present. However, due to the paucity of phytolith data, any interpretations made are necessarily tentative. The assemblages of CRP-2/2A and the upper c. 250 m of CRP-3 are interpreted as representing a predominantly woody vegetation, including Nothofagus and Libocedrus with local areas of grass in the more exposed locations. A cool climate is interpreted to have prevailed throughout both cores. However, beneath c. 250 metres below sea floor in CRP-3, the dominant woody vegetation is supplemented by pockets of Palmae, ?Proteaceae and 'warm' climate grasses. This association represents vegetation growth in sheltered, moist sites - possibly north-facing mid-slopes or the coastal fringe. It may also represent remnant vegetation that grew in moist, temperate conditions during the Middle to Late Eocene, previously interpreted from the Southern McMurdo Sound erratics and lower part of the CIROS-1 drillhole. The phytolith analysis compares well to the terrestrial palynomorph record from both cores and provides additional independent taxonomic and climatic interpretations.

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.