[Ultrastructural study of ampulla of mouse inner ear].

Objective: To observe the ultrastructure of the ampulla, and analyze its physiological and pathological significance. Methods: In this study, 20 Kunming mice were used, and scanning electron microscopy was used to observe the ultrastructure of the ampulla of inner ear. Results: Otoconia was found among the cilia bundles of different haircell(intercilla otoconia of ampulla). The cupula was attached to the lateral wall of the ampulla, and easily to be separated; after separated, a kind of slender crystal(surface otoconia of ampulla) could be seen between the cupula and lateral wall of the ampulla, both sides of ampullary crest were covered with slender crystals too. On the canal side of the ampulla wall, there was more particulate matter attached to the wall near the bottom of ampullary crest, partially embedded in the wall, and less on the utricle side of the ampulla wall. Conclusions: The observation of the ultrastructure of the ampulla is helpful for better understanding the physiological functions of the semicircular canals and the ampulla, and better understanding the pathogenesis and solution of some vertigo diseases.

Ultrastructural Characterization of Stem Cell-Derived Replacement Vestibular Hair Cells Within Ototoxin-Damaged Rat Utricle Explants.

The auditory apparatus of the inner ear does not show turnover of sensory hair cells (HCs) in adult mammals; in contrast, there are many observations supporting low-level turnover of vestibular HCs within the balance organs of mammalian inner ears. This low-level renewal of vestibular HCs exists during normal conditions and it is further enhanced after trauma-induced loss of these HCs. The main process for renewal of HCs within mammalian vestibular epithelia is a conversion/transdifferentiation of existing supporting cells (SCs) into replacement HCs.In earlier studies using long-term organ cultures of postnatal rat macula utriculi, HC loss induced by gentamicin resulted in an initial substantial decline in HC density followed by a significant increase in the proportion of HCs to SCs indicating the production of replacement HCs. In the present study, using the same model of ototoxic damage to study renewal of vestibular HCs, we focus on the ultrastructural characteristics of SCs undergoing transdifferentiation into new HCs. Our objective was to search for morphological signs of SC plasticity during this process. In the utricular epithelia, we observed immature HCs, which appear to be SCs transdifferentiating into HCs. These bridge SCs have unique morphological features characterized by formation of foot processes, basal accumulation of mitochondria, and an increased amount of connections with nearby SCs. No gap junctions were observed on these transitional cells. The tight junction seals were morphologically intact in both control and gentamicin-exposed explants. Anat Rec, 303:506-515, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Regenerating hair cells in vestibular sensory epithelia from humans.

Human vestibular sensory epithelia in explant culture were incubated in gentamicin to ablate hair cells. Subsequent transduction of supporting cells with ATOH1 using an Ad-2 viral vector resulted in generation of highly significant numbers of cells expressing the hair cell marker protein myosin VIIa. Cells expressing myosin VIIa were also generated after blocking the Notch signalling pathway with TAPI-1 but less efficiently. Transcriptomic analysis following ATOH1 transduction confirmed up-regulation of 335 putative hair cell marker genes, including several downstream targets of ATOH1. Morphological analysis revealed numerous cells bearing dense clusters of microvilli at the apical surfaces which showed some hair cell-like characteristics confirming a degree of conversion of supporting cells. However, no cells bore organised hair bundles and several expected hair cell markers genes were not expressed suggesting incomplete differentiation. Nevertheless, the results show a potential to induce conversion of supporting cells in the vestibular sensory tissues of humans.

Severe streptomycin ototoxicity in the mouse utricle leads to a flat epithelium but the peripheral neural degeneration is delayed.

The damaged vestibular sensory epithelium of mammals has a limited capacity for spontaneous hair cell regeneration, which largely depends on the transdifferentiation of surviving supporting cells. Little is known about the response of vestibular supporting cells to a severe insult. In the present study, we evaluated the impact of a severe ototoxic insult on the histology of utricular supporting cells and the changes in innervation that ensued. We infused a high dose of streptomycin into the mouse posterior semicircular canal to induce a severe lesion in the utricle. Both scanning electron microscopy and light microscopy of plastic sections showed replacement of the normal cytoarchitecture of the epithelial layer with a flat layer of cells in most of the samples. Immunofluorescence staining showed numerous cells in the severely damaged epithelial layer that were negative for hair cell and supporting cell markers. Nerve fibers under the flat epithelium had high density at the 1 month time point but very low density by 3 months. Similarly, the number of vestibular ganglion neurons was unchanged at 1 month after the lesion, but was significantly lower at 3 months. We therefore determined that the mouse utricular epithelium turns into a flat epithelium after a severe lesion, but the degeneration of neural components is slow, suggesting that treatments to restore balance by hair cell regeneration, stem cell therapy or vestibular prosthesis implantation will likely benefit from the short term preservation of the neural substrate.

[The ultrastructure of utricular maculae of mouse].

Objective: Using scanning electron microscope to observe the ultrastructure of utricular maculae of mouse. Methods: Ten young (6 to 8 weeks) and ten old (>12 months) mice were executed, and their utricles were harvested and the specimens were processed, using scanning electron microscope to observe the structures of the utricles from the surface of otoconia layer to the roots of hair cell cilia. Results: Under the scanning electron microscope, several ultrastructures were observed, including otoconia layer, unstructured gelatinous extracellular matrix layer, honeycomb-like gelatinous extracellular matrix layer, inter-cilia otoconia and hair cell cilia associated with these structures. When compared with young mouse, the otoconia surface of aged mouse was smoother, the gelatinous extracellular matrix between the adjacent otoconias was thinner. Conclusions: Using SEM, ultrastructures can be clearly observed from surface otoconia layer to the roots of hair cell cilia. By the analysis of the ultrastructure of utricular maculae, it is helpful for investigation of the pathological mechanisms of vestibular diseases, such as otolith diseases.

Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid.

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ∼15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter.

Internacionalização, ciência e saúde: a medicina regenerativa global e os mercados paralelos / Internationalization, science and health: global regenerative medicine and the parallel markets

A medicina regenerativa implica em uma mudança de paradigma, a regeneração do organismo ao nível celular ou tecidual – um assunto contemporâneo controverso e de difícil estandardização. O artigo apresenta um resumo das tendências científicas, econômicas, sociais e de regulamentação global nessa área, analisadas em relação a dilemas teóricos relevantes em antropologia médica e sociologia da ciência e da saúde. Em especial, aqueles que tratam da construção de um ‘aparato coletivo de sentido’ para as novas entidades biológicas e ontológicas, a formação da cidadania biológica e a governança pela incerteza. Apresentam-se, também, evidências empíricas sobre um fenômeno chave para a governança e a regulamentação, qual seja a instalação de uma nova demanda transnacional em pesquisa e saúde através de mercados paralelos de óvulos e de terapias celulares em experimentação. Utilizam-se dados qualitativos coletados para uma pesquisa mais abrangente, resenhas jornalísticas e entrevistas com lideranças internacionais. Conclui-se com uma reflexão sobre a importância da governança internacional em ensaios clínicos e dos caminhos a serem explorados, visando uma harmonização da diversidade de práticas normativas.

Regenerative medicine involves a paradigm change due to organism regeneration at cellular and tissue level – a controversial contemporary issue and difficult to regulate. This article presents a summary of the main scientific, economic, social and regulatory global trends, analyzed according to relevant theoretical dilemmas in medical anthropology and in the sociology of science and health. This is especially true of the construction of a ‘collective frame of reference’ on the new biological and ontological entities, the shaping of biological citizenship, and governance through uncertainty. Empirical evidence is also presented on a key aspect in regulation and governance, namely the emergence of a new transnational demand in health research through the establishment of parallel markets for ova and experimental cellular therapies. Qualitative data collected for a broader research paper is analyzed, as well as journal reviews and information gathered during interviews with international leaders. The paper concludes with a discussion on the importance on international governance of clinical trials and on further exploration, towards a multilevel harmonization of a diversity of normative practices.

Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants.

We have studied aminoglycoside-induced vestibular hair-cell renewal using long-term culture of utricular macula explants from 4-day-old rats. Explanted utricles were exposed to 1 mM of gentamicin for 48 h, during 2nd and 3rd days in vitro (DIV), and then recovering in unsupplemented medium. Utricles were harvested at specified time points from the 2nd through the 28th DIV. The cellular events that occurred within hair cell epithelia during the culture period were documented from serial sectioned specimens. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) with the assistance of morphometric software. Ultrastructural observations were made from selected specimens with transmission electron microscopy (TEM). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs was 11% of the number of HCs observed in non-gentamicin-exposed control explants. At 28 DIV the HC density was 61% of the number of HCs observed in the control group explant specimens. Simultaneously with this increase in HCs there was a corresponding decline in the number of SCs within the epithelium. The proportion of HCs in relation to SCs increased significantly in the gentamicin-exposed explant group during the 5th to the 28th DIV period of culture. There were no significant differences in the volume estimations of the gentamicin-exposed and the control group explants during the observed period of culture. Morphological observations showed that gentamicin exposure induced extensive loss of HCs within the epithelial layer, which retained their intact apical and basal linings. At 7 to 14 DIV (i.e. 3-11 days after gentamicin exposure) a pseudostratified epithelium with multiple layers of disorganized cells was observed. At 21 DIV new HCs were observed that also possessed features resembling SCs. After 28 DIV a new luminal layer of HCs with several layers of SCs located more basally characterized the gentamicin-exposed epithelium. No mitoses were observed within the epithelial layer of any explants. Our conclusion is that direct transdifferentiation of SCs into HCs was the only process contributing to the renewal of HCs after gentamicin exposure in these explants of vestibular inner ear epithelia obtained from the labyrinths of 4-day-old rats.

Detection of human utricular otoconia degeneration in vital specimen and implications for benign paroxysmal positional vertigo.

Otoconia are assumed to be involved in inner ear disorders such as benign paroxysmal positional vertigo (BPPV). Up to now, the distinct structure and morphology of intact and degenerate human utricular otoconia has been only poorly investigated on vital specimen. In this study, human otoconia were obtained from the utricle in five patients undergoing translabyrinthine vestibular schwannoma surgery. Specimens were examined by environmental scanning electron microscopy. Intact and degenerate otoconia as well as fracture particles of otoconia and bone were analyzed by energy dispersive X-ray microanalysis (EDX) and powder X-ray diffraction (XRD). Intact otoconia reveal a uniform size showing characteristic symmetry properties. Degenerative changes can be observed at several stages with gradual minor and major changes in their morphology including fragment formation. EDX analyses reveal the characteristic chemical composition also for otoconia remnants. XRD shows that intact and degenerate otoconia as well as remnants consist of the calcite modification. In conclusion, electron microscopy serves as a standard method for morphological investigations of otoconia. Human utricular otoconia show a uniform outer morphology corresponding to a calcite-based nanocomposite. Morphological changes provide further evidence for degeneration of utricular otoconia in humans, which might be a preconditioning factor causing BPPV. In case of uncertain origin, particles can be clearly assigned to otoconial origin using EDX and XRD analyses.

Anomalous Brownian motion discloses viscoelasticity in the ear's mechanoelectrical-transduction apparatus.

The ear detects sounds so faint that they produce only atomic-scale displacements in the mechanoelectrical transducer, yet thermal noise causes fluctuations larger by an order of magnitude. Explaining how hearing can operate when the magnitude of the noise greatly exceeds that of the signal requires an understanding both of the transducer's micromechanics and of the associated noise. Using microrheology, we characterize the statistics of this noise; exploiting the fluctuation-dissipation theorem, we determine the associated micromechanics. The statistics reveal unusual Brownian motion in which the mean square displacement increases as a fractional power of time, indicating that the mechanisms governing energy dissipation are related to those of energy storage. This anomalous scaling contradicts the canonical model of mechanoelectrical transduction, but the results can be explained if the micromechanics incorporates viscoelasticity, a salient characteristic of biopolymers. We amend the canonical model and demonstrate several consequences of viscoelasticity for sensory coding.

Saccular-specific hair cell addition correlates with reproductive state-dependent changes in the auditory saccular sensitivity of a vocal fish.

The plainfin midshipman fish, Porichthys notatus, is a seasonal breeding teleost fish for which vocal-acoustic communication is essential for its reproductive success. Female midshipman use the saccule as the primary end organ for hearing to detect and locate "singing" males that produce multiharmonic advertisement calls during the summer breeding season. Previous work has shown that female auditory sensitivity changes seasonally with reproductive state; summer reproductive females become better suited than winter nonreproductive females to detect and encode the dominant higher harmonic components in the male's advertisement call, which are potentially critical for mate selection and localization. Here, we test the hypothesis that these seasonal changes in female auditory sensitivity are concurrent with seasonal increases in saccular hair cell receptors. We show that there is increased hair cell density in reproductive females and that this increase is not dependent on body size since similar changes in hair cell density were not found in the other inner ear end organs. We also observed an increase in the number of small, potentially immature saccular hair bundles in reproductive females. The seasonal increase in saccular hair cell density and smaller hair bundles in reproductive females was paralleled by a dramatic increase in the magnitude of the evoked saccular potentials and a corresponding decrease in the auditory thresholds recorded from the saccule. This demonstration of correlated seasonal plasticity of hair cell addition and auditory sensitivity may in part facilitate the adaptive auditory plasticity of this species to enhance mate detection and localization during breeding.

Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants.

A method for long term culture of utricular macula explants is demonstrated to be stable and reproducible over a period of 28 days in vitro (DIV). This culture system for four-day-old rat utricular maculae is potentially suitable for studies of hair cell loss, repair and regeneration processes as they occur in post-natal mammalian inner ear sensory epithelia. The cellular events that occur within utricular macula hair cell epithelia during 28 days of culture are documented from serial sections. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) and the assistance of morphometric computer software. Ultrastructural observations were made with transmission electron microscopy (TEM) for describing the changes in the fine detailed morphological characteristics that occurred in the explants related to time in vitro. After 2 DIV the density of HCs was 77%, at 21 DIV it was 69%, and at 28 DIV it was 52% of HCs present at explantation. Between 2 DIV and 28 DIV there was a 1.7% decrease of the vestibular macula HC density per DIV. The corresponding decrease of SC density within the utricular explants was less than 1% per DIV. The overall morphology of the epithelia, i.e. relationship of HCs to SCs, was well preserved during the first two weeks in culture. After this time a slight deterioration of the epithelia was observed and although type I and type II HCs were identified by TEM observations, these two HC types could no longer be distinguished from one another by LM observations. In preparations cultured for 21 DIV, SC nuclei were located more apical and further away from the basal membrane compared to their position in macula explants fixed immediately after dissection. The loss of cells that occurred was probably due to expulsion from the apical (i.e. luminal) surface of the sensory epithelia, but no lesions of the apical lining or ruptures of the basal membrane were observed. There were no significant changes in the volume of the vestibular HC comprising macular epithelium during the observation period of 28 DIV.

The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs.

Mammals experience permanent impairments from hair cell (HC) losses, but birds and other non-mammals quickly recover hearing and balance senses after supporting cells (SCs) give rise to replacement HCs. Avian HC epithelia express little or no E-cadherin, and differences in the thickness of F-actin belts at SC junctions strongly correlate with different species' capacities for HC replacement, so we investigated junctional cadherins in human and murine ears. We found strong E-cadherin expression at SC-SC junctions that increases more than sixfold postnatally in mice. When we cultured utricles from young mice with γ-secretase inhibitors (GSIs), striolar SCs completely internalized their E-cadherin, without affecting N-cadherin. Hes and Hey expression also decreased and the SCs began to express Atoh1. After 48 h, those SCs expressed myosins VI and VIIA, and by 72 h, they developed hair bundles. However, some scattered striolar SCs retained E-cadherin and the SC phenotype. In extrastriolar regions, the vast majority of SCs also retained E-cadherin and failed to convert into HCs even after long GSI treatments. Microscopic measurements revealed that the junctions between extrastriolar SCs were more developed than those between striolar SCs. In GSI-treated utricles as old as P12, differentiated striolar SCs converted into HCs, but such responses declined with age and ceased by P16. Thus, temporal and spatial differences in postnatal SC-to-HC phenotype conversion capacity are linked to the structural attributes of E-cadherin containing SC junctions in mammals, which differ substantially from their counterparts in non-mammalian vertebrates that readily recover from hearing and balance deficits through hair cell regeneration.

Histological effects of intratympanic gentamicin on the vestibular organ of guinea pigs.

BACKGROUND: Transtympanic administration of gentamicin may be suitable to achieve unilateral vestibular ablation, in order to control unilateral Ménière's disease. In low doses, gentamicin appears to affect selectively the vestibular system, with relative sparing of the cochlea. An experimental study on guinea pigs was conducted to determine what single dose of gentamicin would produce a unilateral vestibular organ lesion when applied to the middle ear. STUDY DESIGN: Experimental and prospective. METHODS: Four groups of guinea pigs received different gentamicin doses (1, 5, 10 and 25 mg) administered to the middle ear. The animals' vestibular organs were then assessed by scanning electron microscopy, in order to quantify the level of vestibular damage. RESULTS: Study of the utricular macula and the ampullar crista of the lateral semicircular canal revealed vestibular neuroepithelial lesions in all infused ears. CONCLUSIONS: The severity of the vestibular neuroepithelial lesions was dose-dependent. Lower gentamicin doses were observed to damage vestibular structures more than cochlear structures.

The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice.

The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle's longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.

Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis.

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a (dr)) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a "cochlear-gravistatic" endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.

Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.

Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration.

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.

Closure of supporting cell scar formations requires dynamic actin mechanisms.

In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6h), and allowed to recover (18h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules.

Control of hair cell excitability by vestibular primary sensory neurons.

In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting of synapses was prevented. Under this condition, the voltage-gated sodium current and the underlying action potentials persisted in a large proportion of nonafferented hair cells. We then studied whether impairment of nerve terminals in the utricle of adult rats may also affect hair cell excitability. We induced selective and transient damages of afferent terminals using glutamate excitotoxicity in vivo. The efficiency of the excitotoxic injury was attested by selective swellings of the terminals and underlying altered vestibular behavior. Under this condition, the sodium-based excitability transiently recovered in hair cells. These results indicate that the modulation of hair cell excitability depends on the state of the afferent terminals. In adult utricle hair cells, this property may be essential to set the conditions required for restoration of the sensory network after damage. This is achieved via re-expression of a biological process that occurs during synaptogenesis.