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The sense of balance relies on vestibular hair cells, which detect head motions. Mammals have two types of vestibular hair cell, I and II, with unique morphological, molecular, and physiological properties. Furthermore, each hair cell type synapses on a unique form of afferent nerve terminal. Little is known about the mechanisms in mature animals that maintain the specific features of each hair cell type or its post-synaptic innervation. We found that deletion of the transcription factor Sox2 from type II hair cells in adult mice of both sexes caused many cells in utricles to acquire features unique to type I hair cells and to lose type II-specific features. This cellular transdifferentiation, which included changes in nuclear size, chromatin condensation, soma and stereocilium morphology, and marker expression, resulted in a significantly higher proportion of type I-like hair cells in all epithelial zones. Furthermore, Sox2 deletion from type II hair cells triggered non-cell autonomous changes in vestibular afferent neurons; they retracted bouton terminals (normally present on only type II cells) from transdifferentiating hair cells and replaced them with a calyx terminal (normally present on only type I cells). These changes were accompanied by significant expansion of the utricle's central zone, called the striola. Our study presents the first example of a transcription factor required to maintain the type-specific hair cell phenotype in adult inner ears. Furthermore, we demonstrate that a single genetic change in type II hair cells is sufficient to alter the morphology of their post-synaptic partners, the vestibular afferent neurons.SIGNIFICANCE STATEMENT:The sense of balance relies on two types of sensory cells in the inner ear - type I and type II hair cells. These two cell types have unique properties. Furthermore, their post-synaptic partners, the vestibular afferent neurons, have differently shaped terminals on type I versus type II hair cells. We show that the transcription factor Sox2 is required to maintain the cell-specific features of type II hair cells and their post-synaptic terminals in adult mice. This is the first evidence of a molecule that maintains the phenotypes of hair cells and, non-cell autonomously, their post-synaptic partners in mature animals.
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Aging, disease, and trauma can lead to loss of vestibular hair cells and permanent vestibular dysfunction. Previous work showed that, following acute destruction of â¼95% of vestibular hair cells in adult mice, â¼20% regenerate naturally (without exogenous factors) through supporting cell transdifferentiation. There is, however, no evidence for the recovery of vestibular function. To gain insight into the lack of functional recovery, we assessed functional differentiation in regenerated hair cells for up to 15 months, focusing on key stages in stimulus transduction and transmission: hair bundles, voltage-gated conductances, and synaptic contacts. Regenerated hair cells had many features of mature type II vestibular hair cells, including polarized mechanosensitive hair bundles with zone-appropriate stereocilia heights, large voltage-gated potassium currents, basolateral processes, and afferent and efferent synapses. Regeneration failed, however, to recapture the full range of properties of normal populations, and many regenerated hair cells had some properties of immature hair cells, including small transduction currents, voltage-gated sodium currents, and small or absent HCN (hyperpolarization-activated cyclic nucleotide-gated) currents. Furthermore, although mouse vestibular epithelia normally have slightly more type I hair cells than type II hair cells, regenerated hair cells acquired neither the low-voltage-activated potassium channels nor the afferent synaptic calyces that distinguish mature type I hair cells from type II hair cells and confer distinctive physiology. Thus, natural regeneration of vestibular hair cells in adult mice is limited in total cell number, cell type diversity, and extent of cellular differentiation, suggesting that manipulations are needed to promote full regeneration with the potential for recovery of vestibular function.SIGNIFICANCE STATEMENT Death of inner ear hair cells in adult mammals causes permanent loss of hearing and balance. In adult mice, the sudden death of most vestibular hair cells stimulates the production of new hair cells but does not restore balance. We investigated whether the lack of systems-level function reflects functional deficiencies in the regenerated hair cells. The regenerated population acquired mechanosensitivity, voltage-gated channels, and afferent synapses, but did not reproduce the full range of hair cell types. Notably, no regenerated cells acquired the distinctive properties of type I hair cells, a major functional class in amniote vestibular organs. To recover vestibular system function in adults, we may need to solve how to regenerate the normal variety of mature hair cells.
Assuntos
Diferenciação Celular/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Regeneração/fisiologia , Sinapses/fisiologia , Animais , Camundongos , Camundongos Knockout , Transmissão Sináptica/fisiologiaRESUMO
DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons and patch-clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25. KEY POINTS: The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs). The VGLUT3-p.A211V variant is associated with human deafness DFNA25. Mutant mice carrying the VGLUT3-p.A211V variant show progressive hearing loss. IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres. These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages.
Assuntos
Estereocílios , Sinapses , Animais , Cóclea , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , CamundongosRESUMO
Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.
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Células Ciliadas Auditivas Internas/metabolismo , Sistema da Linha Lateral/inervação , Sistema da Linha Lateral/metabolismo , Mecanotransdução Celular , Sinapses/metabolismo , Animais , Células Ciliadas Auditivas Internas/ultraestrutura , Sistema da Linha Lateral/ultraestrutura , Membranas/metabolismo , Mutação/genética , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Peixe-ZebraRESUMO
Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglycoside antibiotics.
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Aminoglicosídeos/efeitos adversos , Proteína 1 de Troca de Ânion do Eritrócito/genética , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Mutação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Aminoglicosídeos/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Mapeamento Cromossômico , Resistência a Medicamentos/genética , Genótipo , Células Ciliadas Auditivas/ultraestrutura , Íons/metabolismo , Dados de Sequência Molecular , Neomicina/farmacologia , Fenótipo , Transporte Proteico , Alinhamento de Sequência , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term 'meisosomes,' in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.
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Proteínas de Caenorhabditis elegans , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epiderme/metabolismo , Células Epidérmicas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Matriz Extracelular/metabolismoRESUMO
Sox2 is a transcription factor that is necessary in the mammalian inner ear for development of sensory hair cells and supporting cells. Sox2 is expressed in supporting cells of adult mammals, but its function in this context is poorly understood. Given its role in the developing inner ear, we hypothesized that Sox2 is required in vestibular supporting cells for regeneration of type II hair cells after damage. Using adult mice, we deleted Sox2 from Sox9-CreER-expressing supporting cells prior to diphtheria toxin-mediated hair cell destruction and used fate-mapping to assess regeneration. In utricles of control mice with normal Sox2 expression, supporting cells regenerated nearly 200 hair cells by 3 weeks post-damage, which doubled by 12 weeks. In contrast, mice with Sox2 deletion from supporting cells had approximately 20 fate-mapped hair cells at 3 weeks post-damage, and this number did not change significantly by 12 weeks, indicating regeneration was dramatically curtailed. We made similar observations for saccules and ampullae. We found no evidence that supporting cells lacking Sox2 had altered cellular density, morphology, or ultrastructure. However, some Sox2-negative supporting cell nuclei appeared to migrate apically but did not turn on hair cell markers, and type I hair cell survival was higher. Sox2 heterozygotes also had reduced regeneration in utricles, but more hair cells were replaced than mice with Sox2 deletion. Our study determined that Sox2 is required in supporting cells for normal levels of vestibular hair cell regeneration but found no other major requirements for Sox2 in adult supporting cells.
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Células Ciliadas Vestibulares , Fatores de Transcrição SOXB1 , Animais , Camundongos , Regulação da Expressão Gênica , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/fisiologia , Mamíferos , Regeneração , Sáculo e Utrículo , Fatores de Transcrição SOXB1/metabolismoRESUMO
Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.
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Vias Auditivas/fisiologia , Percepção Auditiva/fisiologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/metabolismo , Potenciais de Ação , Animais , Cálcio/metabolismo , Cóclea/citologia , Endocitose , Exocitose , Audição/fisiologia , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Recent studies demonstrated that reversible continuous noise exposure may induce a temporary threshold shift (TTS) with a permanent degeneration of auditory nerve fibers, although hair cells remain intact. To probe the impact of TTS-inducing impulse noise exposure on hearing, CBA/J Mice were exposed to noise impulses with peak pressures of 145 dB SPL. We found that 30 min after exposure, the noise caused a mean elevation of ABR thresholds of ~30 dB and a reduction in DPOAE amplitude. Four weeks later, ABR thresholds and DPOAE amplitude were back to normal in the higher frequency region (8-32 kHz). At lower frequencies, a small degree of PTS remained. Morphological evaluations revealed a disturbance of the stereociliary bundle of outer hair cells, mainly located in the apical regions. On the other hand, the reduced suprathreshold ABR amplitudes remained until 4 weeks later. A loss of synapse numbers was observed 24 h after exposure, with full recovery two weeks later. Transmission electron microscopy revealed morphological changes at the ribbon synapses by two weeks post exposure. In addition, increased levels of oxidative stress were observed immediately after exposure, and maintained for a further 2 weeks. These results clarify the pathology underlying impulse noise-induced sensory dysfunction, and suggest possible links between impulse-noise injury, cochlear cell morphology, metabolic changes, and hidden hearing loss.
RESUMO
The vestibular organs of reptiles, birds, and mammals possess Type I and Type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a Type I or Type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in Type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse utricles between postnatal days 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to Type I hair cells) increased in all regions of the utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in Type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER T2 :Rosa26 tdTomato mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were Type II. Our findings show that most neonatally-derived hair cells become Type II, and many Type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.
Assuntos
Células Ciliadas Auditivas/citologia , Sáculo e Utrículo/citologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C57BL , Terminações Nervosas/ultraestrutura , FenótipoRESUMO
In the cochlea, the mammalian auditory organ, fibrocytes of the mesenchymal nonsensory regions play important roles in cochlear physiology, including the maintenance of ionic and hydric components in the endolymph. Occurrence of human deafness in fibrocyte alterations underlines their critical roles in auditory function. We recently described a novel gene, Otos, which encodes otospiralin, a small protein of unknown function that is produced by the fibrocytes of the cochlea and vestibule. We now have generated mice with deletion of Otos and found that they show moderate deafness, with no frequency predominance. Histopathology revealed a degeneration of type II and IV fibrocytes, while hair cells and stria vascularis appeared normal. Together, these findings suggest that impairment of fibrocytes caused by the loss in otospiralin leads to abnormal cochlear physiology and auditory function. This moderate dysfunction may predispose to age-related hearing loss.
Assuntos
Cóclea/citologia , Cóclea/fisiologia , Surdez/fisiopatologia , Proteínas/metabolismo , Animais , Limiar Auditivo , Cóclea/patologia , Cóclea/fisiopatologia , Surdez/genética , Surdez/patologia , Eletrofisiologia , Marcação de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas/genéticaRESUMO
Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of neomycin exposure. Animals exposed to a low, 25-microM concentration of neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of neomycin (50-200 microM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage.
Assuntos
Células Ciliadas Auditivas/efeitos dos fármacos , Sistema da Linha Lateral/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neomicina/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/fisiopatologia , Sistema da Linha Lateral/patologia , Sistema da Linha Lateral/fisiopatologia , Microscopia Eletrônica , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Inibidores da Síntese de Proteínas/toxicidade , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Fatores de Tempo , Peixe-ZebraRESUMO
This report summarizes recent neuropharmacological data at the IHC afferent/efferent synaptic complex: the type of Glu receptors and transporter involved and the modulation of this fast synaptic transmission by the lateral efferents. Neuropharmacological data were obtained by coupling the recording of cochlear potentials and single unit of the auditory nerve with intra-cochlear applications of drugs (multi-barrel pipette). We also describe the IHC afferent/efferent functioning in pathological conditions. After acoustic trauma or ischemia, acute disruption of IHC-auditory dendrite synapses are seen. However, a re-growth of the nerve fibres and a re-afferentation of the IHC were completely done 5 days after injury. During this synaptic repair, multiple presynaptic bodies were commonly found, either linked to the membrane or "floating" in ectopic positions. In the meantime, the lateral efferents directly contact the IHCs. The demonstration that NMDA receptors blockade delayed the re-growth of neurites suggests a neurotrophic role of NMDA receptors in pathological conditions.
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Nervo Coclear/metabolismo , Otopatias/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Plasticidade Neuronal , Regeneração , Sinapses/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Nervo Coclear/efeitos dos fármacos , Otopatias/induzido quimicamente , Otopatias/patologia , Otopatias/fisiopatologia , Potenciais Evocados Auditivos/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Humanos , Receptores de AMPA/metabolismo , Sinapses/efeitos dos fármacos , Fatores de TempoRESUMO
Sensorineural hearing loss is commonly caused by damage to cochlear sensory hair cells. Coinciding with hair cell degeneration, the peripheral fibres of type I spiral ganglion neurons (SGNs) that normally form synaptic connections with the inner hair cell gradually degenerate. We examined the time course of these degenerative changes in type I SGNs and their satellite Schwann cells at the ultrastructural level in guinea pigs at 2, 6, and 12 weeks following aminoglycoside-induced hearing loss. Degeneration of the peripheral fibres occurred prior to the degeneration of the type I SGN soma and was characterised by shrinkage of the fibre followed by retraction of the axoplasm, often leaving a normal myelin lumen devoid of axoplasmic content. A statistically significant reduction in the cross-sectional area of peripheral fibres was evident as early as 2 weeks following deafening (p < 0.001, ANOVA). This was followed by a decrease in type I SGN density within Rosenthal's canal that was statistically significant 6 weeks following deafening (p < 0.001, ANOVA). At any time point examined, few type I SGN soma were observed undergoing degeneration, implying that once initiated, soma degeneration was rapid. While there was a significant reduction in soma area as well as changes to the morphology of the soma, the ultrastructure of surviving type I SGN soma appeared relatively normal over the 12-week period following deafening. Satellite Schwann cells exhibited greater survival traits than their type I SGN; however, on loss of neural contact, they reverted to a non-myelinating phenotype, exhibiting an astrocyte-like morphology with the formation of processes that appeared to be searching for new neural targets. In 6- and 12-week deafened cochlea, we observed cellular interaction between Schwann cell processes and residual SGNs that distorted the morphology of the SGN soma. Understanding the response of SGNs, Schwann cells, and the complex relationship between them following aminoglycoside deafening is important if we are to develop effective therapeutic techniques designed to rescue SGNs.
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Surdez/patologia , Órgão Espiral/ultraestrutura , Células de Schwann/ultraestrutura , Gânglio Espiral da Cóclea/ultraestrutura , Aminoglicosídeos , Animais , Surdez/induzido quimicamente , CobaiasRESUMO
Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. This study examines intracellular pathways involved in hair cell death induced by CDDP exposure in vivo to develop effective therapeutic strategies to protect the auditory receptor from CDDP-initiated hearing loss. Guinea pigs were treated with systemic administration of CDDP. Cochlear hair cells from CDDP-treated animals exhibited classic apoptotic alterations in their morphology. Several important signaling events that regulate the death of CDDP-injured cochlear hair cells were identified. CDDP treatment induced the activation and redistribution of cytosolic Bax and the release of cytochrome c from injured mitochondria. Activation of caspase-9 and caspase-3, but not caspase-8, was detected after treatment with CDDP, and the cleavage of fodrin by activated caspase-3 was observed within damaged hair cells. Intracochlear perfusions with caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD-fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (JNK) signaling pathway is activated in response to CDDP toxicity, intracochlear perfusion of d-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but instead potentiated the ototoxic effects of CDDP. The results of the present study show that blocking a critical step in apoptosis may be a useful strategy to prevent harmful side effects of CDDP ototoxicity in patients having to undergo chemotherapy.
Assuntos
Inibidores de Caspase , Cisplatino/toxicidade , Inibidores de Cisteína Proteinase/farmacologia , Perda Auditiva/prevenção & controle , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citocromos c/metabolismo , Feminino , Cobaias , Células Ciliadas Auditivas/efeitos dos fármacos , Perda Auditiva/induzido quimicamente , Perda Auditiva/enzimologia , Isoenzimas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2RESUMO
Salicylate, the active component of aspirin, is known to induce tinnitus. However, the site and the mechanism of generation of tinnitus induced by salicylate remains unclear. Here, we developed a behavioral procedure to measure tinnitus in rats. The behavioral model was based on an active avoidance paradigm in which rats had to display a motor task (i.e., to jump on a climbing pole when hearing a sound). Giving salicylate led to a decrease in the percentage of correct responses (score) and a drastic increase in the number of false positive responses (i.e., animals execute the motor task during a silent period). Presentation of the sound at a constant perceptive level prevents decrease of the score, leading to the proposal that score is related to hearing performance. In contrast, the increase of false positive responses remained unchanged. In fact, animals behaved as if they hear a sound, indicating that they are experiencing tinnitus. Mefenamate in place of salicylate also increased the number of false positive responses, suggesting that salicylate-induced tinnitus is related to an inhibition of cyclooxygenase. One physiological basis of salicylate ototoxicity is likely to originate from altered arachidonic acid metabolism. Because arachidonic acid potentiates NMDA receptor currents, we tested the involvement of cochlear NMDA receptors in the occurrence of tinnitus. Application of NMDA antagonists into the perilymphatic fluids of the cochlea blocked the increase in pole-jumping behavior induced by salicylate, suggesting that salicylate induces tinnitus through activation of cochlear NMDA receptors.
Assuntos
Cóclea/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Salicilatos , Zumbido/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Cóclea/fisiopatologia , Nervo Coclear/efeitos dos fármacos , Nervo Coclear/fisiopatologia , Condicionamento Clássico , Inibidores de Ciclo-Oxigenase , Modelos Animais de Doenças , Eletrodos Implantados , Eletrochoque , Reações Falso-Positivas , Feminino , Ácido Mefenâmico , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Ratos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/metabolismo , Zumbido/fisiopatologiaRESUMO
Mesenchymal nonsensory regions of the inner ear are important structures surrounding the neurosensory epithelium that are believed to participate in the ionic homeostasis of the cochlea and vestibule. We report here the discovery of otospiralin, an inner ear-specific protein that is produced by fibrocytes from these regions, including the spiral ligament and spiral limbus in the cochlea and the maculae and semicircular canals in the vestibule. Otospiralin is a novel 6.4 kDa protein of unknown function that shares a protein motif with the gag p30 core shell nucleocapsid protein of type C retroviruses. To evaluate its functional importance, we downregulated otospiralin by cochlear perfusion of antisense oligonucleotides in guinea pigs. This led to a rapid threshold elevation of the compound action potentials and irreversible deafness. Cochlear examination by transmission electron microscopy revealed hair cell loss and degeneration of the organ of Corti. This demonstrates that otospiralin is essential for the survival of the neurosensory epithelium.
Assuntos
Surdez/metabolismo , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/genética , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Motivos de Aminoácidos/genética , Animais , Limiar Auditivo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Cóclea/patologia , Nervo Coclear/fisiologia , Surdez/induzido quimicamente , Surdez/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Orelha Interna/efeitos dos fármacos , Orelha Interna/patologia , Fibroblastos/metabolismo , Cobaias , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Especificidade de Órgãos , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
Riluzole has been reported to protect against the deleterious effect of cerebral ischemia by blocking glutamatergic neurotransmission. Here, we investigated whether acoustic trauma-induced cochlear excitotoxicity could be attenuated by riluzole. Cumulative intracochlear perfusion of riluzole completely abolished single-nerve fiber activity in the guinea pig cochlea and the compound action potential of the auditory nerve. Guinea pigs treated with riluzole (100 microM) showed significantly less hearing threshold shift than untreated guinea pigs, and presented no sign of dendritic damage in the cochlea observable by electron microscopy. When coapplied with glutamate, riluzole did not prevent glutamate-induced swelling of auditory nerve dendrites, suggesting that the protective effect of riluzole was mediated principally by inhibition of glutamate release from sensory inner hair cells.
Assuntos
Estimulação Acústica/efeitos adversos , Perda Auditiva Provocada por Ruído/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Riluzol/uso terapêutico , Estimulação Acústica/métodos , Doença Aguda , Animais , Cóclea/efeitos dos fármacos , Cóclea/patologia , Cobaias , Perda Auditiva Provocada por Ruído/patologia , Fármacos Neuroprotetores/farmacologia , Ruído/efeitos adversos , Riluzol/farmacologiaRESUMO
We investigated the expression patterns of several cell adhesion molecules (CAMs) during rat cochlea ontogeny, from embryo day 16 to adulthood, with the use of immunohistochemistry: neural cadherin (N-cad) and polysialic acid neural CAM (PSA-NCAM) as two different neural CAM paradigms; epithelial cadherin (E-cad), which was restricted to the epitheloid phenotype; and the cytoplasmic domain-free truncated-cadherin (T-cad). We made the following observations. (1) T-cad was present in all types of fibrocyte and in subdomains within the pillar cells. (2) E- and N-cad were expressed with mutually exclusive patterns and did not overlap with T-cad. All cochlear epithelial cells, including the sensory outer hair cells (OHCs), were E-cad-positive, except for the negative inner hair cells (IHCs) and the nonsensory Kölliker's organ domain close to the IHCs. N-cad expression appeared first in the developing IHCs and then in the neighboring Kölliker's organ in an increasingly mediolateral gradient in opposition to the E-cad gradient. The OHCs, which are never N-cad positive, intensively expressed E-cad, as did the Hensen cells at the beginning of their differentiation. (3) The cadherin-linked molecule beta-catenin, absent in fibrocytes, was detected in all epithelial cell membranes and was prominent in the E-cad-rich modiolar extremity of Kölliker's organ. (4) Gradual PSA-NCAM expression was observed in the lateral portion of Kölliker's organ, and the intense PSA-NCAM expression was seen surrounding the IHCs. As development proceeded, PSA-NCAM immunoreactivity progressively became restricted to the basal poles of the IHCs, where it remained in the adult rat cochlea, suggesting a synaptic plasticity. Synaptic plasticity in rat cochlea and hypotheses about T-cad functions and neosensory features of the Kölliker's organ are discussed.
Assuntos
Caderinas/biossíntese , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Proteínas do Citoesqueleto/biossíntese , Molécula L1 de Adesão de Célula Nervosa/biossíntese , Ácidos Siálicos/biossíntese , Transativadores/biossíntese , Animais , Caderinas/análise , Cóclea/química , Proteínas do Citoesqueleto/análise , Regulação da Expressão Gênica/fisiologia , Camundongos , Molécula L1 de Adesão de Célula Nervosa/análise , Órgão Espiral/química , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/metabolismo , Ratos , Ratos Wistar , Ácidos Siálicos/análise , Transativadores/análise , beta CateninaRESUMO
The outer hair cells (OHCs) from the mammalian organ of Corti are assumed to enhance the sensitivity and the selectivity of the cochlea via an electromotile response to sound stimulation. These OHC mechanical changes feed energy back into the cochlea before completion of the transduction process by inner hair cells. OHC electromotility is thought to depend on specific transmembrane motor proteins. Electron microscopy has been used previously to image the OHC lateral plasma membrane, where voltage sensors and motors are located. A very specific and regular organization of membrane particles has been described, together with an equally specific submembraneous meshwork of cytoskeleton anchored to the plasma membrane. To confirm and extend these observations, we have used, for the first time on the OHC lateral wall, atomic force microscopy (AFM). As a result of an improved tapping mode technique as well as the unique ultrastructural organization of the OHC plasma membrane, we have obtained high-resolution three-dimensional (3D) images of a markedly enhanced quality, allowing high-resolution 3D imaging. Tapping-mode AFM confirmed the presence of regularly aligned particles (presumably transmembrane proteins) on both faces of the OHC plasma membrane. It also revealed the presence of markedly different membrane domains, smooth and undulating. The differences between these zones probably are due to local differences in cytoskeleton-membrane interactions. Moreover, 3D reconstructions allowed us to distinguish between globular and pore-like particles, a distinction that may be of great functional significance.