RESUMO
How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.
Assuntos
Espaço Extracelular/química , Ácido Hialurônico/farmacologia , Morfogênese , Especificidade de Órgãos , Pressão , Canais Semicirculares/citologia , Canais Semicirculares/embriologia , Actomiosina/metabolismo , Animais , Anisotropia , Comportamento Animal , Matriz Extracelular/metabolismo , Ácido Hialurônico/biossíntese , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Pressão Osmótica , Canais Semicirculares/diagnóstico por imagem , Comportamento Estereotipado , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Although many mathematical methods were used to analyze the neural activity under sinusoidal stimulation within linear response range in vestibular system, the reliabilities of these methods are still not reported, especially in nonlinear response range. Here we chose nonlinear least-squares algorithm (NLSA) with sinusoidal model to analyze the neural response of semicircular canal neurons (SCNs) during sinusoidal rotational stimulation (SRS) over a nonlinear response range. Our aim was to acquire a reliable mathematical method for data analysis under SRS in vestibular system. Our data indicated that the reliability of this method in an entire SCNs population was quite satisfactory. However, the reliability was strongly negatively depended on the neural discharge regularity. In addition, stimulation parameters were the vital impact factors influencing the reliability. The frequency had a significant negative effect but the amplitude had a conspicuous positive effect on the reliability. Thus, NLSA with sinusoidal model resulted a reliable mathematical tool for data analysis of neural response activity under SRS in vestibular system and more suitable for those under the stimulation with low frequency but high amplitude, suggesting that this method can be used in nonlinear response range. This method broke out of the restriction of neural activity analysis under nonlinear response range and provided a solid foundation for future study in nonlinear response range in vestibular system.
Assuntos
Algoritmos , Neurônios/fisiologia , Canais Semicirculares/citologia , Animais , Chinchila , Feminino , Análise dos Mínimos Quadrados , Dinâmica não Linear , Reprodutibilidade dos TestesRESUMO
Objetivos: Analizar la ontogenia del canal semicircular superior y del tegmen tympani y determinar si hay factores embriológicos comunes que expliquen la dehiscencia asociada de ambos. Métodos: Se han analizado 77 series embriológicas humanas de edades comprendidas entre las 6 semanas y recién nacidos. Las preparaciones estaban cortadas en serie y teñidas con la técnica de tricrómico de Martins. Resultados: La prolongación tegmentaria del tegmen tympani y el canal semicircular superior se originan de la misma estructura, la cápsula ótica, y poseen el mismo tipo de osificación endocondral; mientras que la prolongación escamosa del tegmen tympani se desarrolla desde la escama del temporal y su osificación es de tipo directa o intramembranosa. En la osificación de la prolongación tegmentaria colaboran los núcleos de osificación de los canales semicirculares superior, externo y accesorio del tegmen, los cuales por crecimiento se extienden hasta la prolongación tegmentaria, este hecho sumado a que ambas estructuras comparten una capa común de periostio externo podría explicar la coexistencia de falta de cobertura ósea en el tegmen y en el canal. Conclusión: El desarrollo del canal semicircular y tegmen tympani podrían explicar las causas de la asociación de ambas dehiscencias (AU)
Objectives: To analyze the ontogeny of the superior semicircular canal and tegmen tympani and determine if there are common embryological factors explaining both associated dehiscence. Methods: We analyzed 77 human embryological series aged between 6 weeks and newborn. Preparations were serially cut and stained with Masson's trichrome technique. Results: The tegmental prolongation of tegmen tympani and superior semicircular canal originate from the same structure, the otic capsule, and have the same type of endochondral ossification; while the extension of the squamous prolongation of tegmen tympani runs from the temporal squama and ossification is directly of intramembranous type. The nuclei of ossification of the superior and external semicircular canals and accessory of tegmen collaborate in the ossification of the tegmental extension and by growth extend to the tegmental prolongation. This fact plus the fact that both structures share a common layer of external periosteum could explain the coexistence of lack of bone coverage in tegmen and superior semicircular canal. Conclusion: The development of the semicircular canal and tegmen tympani could explain the causes of the association of both dehiscences (AU)
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Canais Semicirculares/anatomia & histologia , Canais Semicirculares/citologia , Embriologia/métodos , Embriologia/tendências , Osso Temporal/embriologia , Feto/embriologia , Membrana Timpânica/embriologia , Perfuração da Membrana Timpânica/embriologia , Pesquisas com Embriões , Ductos Semicirculares/anatomia & histologia , Osteogênese/fisiologiaRESUMO
In the present study we combined electrophysiology with optical heat pulse stimuli to examine thermodynamics of membrane electrical excitability in mammalian vestibular hair cells and afferent neurons. We recorded whole cell currents in mammalian type II vestibular hair cells using an excised preparation (mouse) and action potentials (APs) in afferent neurons in vivo (chinchilla) in response to optical heat pulses applied to the crista (ΔT ≈ 0.25°C per pulse). Afferent spike trains evoked by heat pulse stimuli were diverse and included asynchronous inhibition, asynchronous excitation, and/or phase-locked APs synchronized to each infrared heat pulse. Thermal responses of membrane currents responsible for APs in ganglion neurons were strictly excitatory, with Q10 ≈ 2. In contrast, hair cells responded with a mix of excitatory and inhibitory currents. Excitatory hair cell membrane currents included a thermoelectric capacitive current proportional to the rate of temperature rise (dT/dt) and an inward conduction current driven by ΔT An iberiotoxin-sensitive inhibitory conduction current was also evoked by ΔT, rising in <3 ms and decaying with a time constant of â¼24 ms. The inhibitory component dominated whole cell currents in 50% of hair cells at -68 mV and in 67% of hair cells at -60 mV. Responses were quantified and described on the basis of first principles of thermodynamics. Results identify key molecular targets underlying heat pulse excitability in vestibular sensory organs and provide quantitative methods for rational application of optical heat pulses to examine protein biophysics and manipulate cellular excitability.
Assuntos
Potenciais de Ação/efeitos da radiação , Células Ciliadas Vestibulares/efeitos da radiação , Temperatura Alta , Potenciais da Membrana/fisiologia , Células Receptoras Sensoriais/efeitos da radiação , Animais , Biofísica , Cálcio/metabolismo , Chinchila , Capacitância Elétrica , Feminino , Células Ciliadas Vestibulares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Modelos Neurológicos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais Semicirculares/citologia , Células Receptoras Sensoriais/fisiologiaRESUMO
The vestibular system operates in a push-pull fashion using signals from both labyrinths and an intricate bilateral organization. Unilateral vestibular lesions cause well-characterized motor deficits that are partially compensated over time and whose neural correlates have been traced in the mean response modulation of vestibular nuclei cells. Here we compare both response gains and neural detection thresholds of vestibular nuclei and semicircular canal afferent neurons in intact vs. unilateral-lesioned macaques using three-dimensional rotation and translation stimuli. We found increased stimulus-driven spike count variability and detection thresholds in semicircular canal afferents, although mean responses were unchanged, after contralateral labyrinth lesion. Analysis of trial-by-trial spike count correlations of a limited number of simultaneously recorded pairs of canal afferents suggests increased noise correlations after lesion. In addition, we also found persistent, chronic deficits in rotation detection thresholds of vestibular nuclei neurons, which were larger in the ipsilesional than the contralesional brain stem. These deficits, which persisted several months after lesion, were due to lower rotational response gains, whereas spike count variability was similar in intact and lesioned animals. In contrast to persistent deficits in rotation threshold, translation detection thresholds were not different from those in intact animals. These findings suggest that, after compensation, a single labyrinth is sufficient to recover motion sensitivity and normal thresholds for the otolith, but not the semicircular canal, system.
Assuntos
Potenciais Somatossensoriais Evocados , Percepção de Movimento , Canais Semicirculares/fisiologia , Núcleos Vestibulares/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Macaca mulatta , Masculino , Neurônios Aferentes/fisiologia , Rotação , Canais Semicirculares/citologia , Limiar Sensorial , Núcleos Vestibulares/citologia , Vestíbulo do Labirinto/citologiaRESUMO
AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.
Assuntos
Orelha/fisiologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXE/metabolismo , Canais Semicirculares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Ensaio de Desvio de Mobilidade Eletroforética , Embrião não Mamífero/citologia , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXE/genética , Canais Semicirculares/citologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
In hair cells dissected from the frog crista ampullaris, the combination of a calcium-dependent (IKCa) and a purely voltage-dependent component (IKV) gives rise to the delayed potassium current complex (IKD). These currents have been recently reported to display slow depolarization-induced inactivation and biphasic inactivation removal by hyperpolarization. The amplitude and inactivation kinetics of both IKCa and IKV are drastically modulated by a previously unrecognized mechanism of protein phosphorylation (sensitive to kinase inhibitors H89 and KT5823), which does not interfere with the transient potassium current (IA) or the calcium current (ICa). IKD amplitude was stable in cells patched with pipettes containing 8 mM ATP or under perforated-patch; under these conditions, a 10 min treatment with 10 µM H89 or 1-10 µM KT5823 reduced IKD amplitude by a mean of 67% at +40 mV. Similarly affected was the isolated IKV component (ICa blocked with Cd(2+)). Thus, a large potassium conductance can be activated by depolarization, but it is made available to the cell to a variable extent that depends on membrane potential and protein kinase activity. The total gKD ranged 4.6-44.0 nS in control cells, according to the level of steady-state inactivation, and was reduced to 1.4-2.7 nS after protein kinase inhibition. When sinusoidal membrane potential changes in the -70/-10 mV range were applied, to mimic receptor response to hair bundle deflection, IKD proved the main current dynamically activated and the only one regulated by PK: H89 decreased the total outward charge during each cycle by 60%. Phosphorylation appears to control both the amount of IKCa and IKV conductance activated by depolarization and the fraction thereof which can be rescued by removal of inactivation. The balance between the depolarizing transduction current and the repolarizing potassium current, and eventually the transmitter release at the cytoneural junction, are therefore modulated by a phosphorylation-mediated process.
Assuntos
Células Ciliadas Auditivas/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Proteínas Quinases/metabolismo , Rana esculenta/fisiologia , Canais Semicirculares/metabolismo , Animais , Cádmio/farmacologia , Cálcio/metabolismo , Carbazóis/farmacologia , Cátions Bivalentes , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Isoquinolinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Canais Semicirculares/citologia , Canais Semicirculares/efeitos dos fármacos , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
Several experiments suggest an important role for store-released Ca²âº in hair cell organs: drugs targeting IP3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRß in the ampulla only.
Assuntos
Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais Semicirculares/metabolismo , Animais , Cafeína , Cálcio/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rana esculenta , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canais Semicirculares/citologia , Distribuição TecidualRESUMO
In the last two decades, we have focused on establishing a reliable technique for focal stimulation of vestibular receptors to evaluate neural connectivity. Here, we summarize the vestibular-related neuronal circuits for the vestibulo-ocular reflex, vestibulocollic reflex, and vestibulospinal reflex arcs. The focal stimulating technique also uncovered some hidden neural mechanisms. In the otolith system, we identified two hidden neural mechanisms that enhance otolith receptor sensitivity. The first is commissural inhibition, which boosts sensitivity by incorporating inputs from bilateral otolith receptors, the existence of which was in contradiction to the classical understanding of the otolith system but was observed in the utricular system. The second mechanism, cross-striolar inhibition, intensifies the sensitivity of inputs from both sides of receptive cells across the striola in a single otolith sensor. This was an entirely novel finding and is typically observed in the saccular system. We discuss the possible functional meaning of commissural and cross-striolar inhibition. Finally, our focal stimulating technique was applied to elucidate the different constructions of axonal projections from each vestibular receptor to the spinal cord. We also discuss the possible function of the unique neural connectivity observed in each vestibular receptor system.
Assuntos
Vias Neurais/fisiologia , Membrana dos Otólitos/fisiologia , Reflexo Vestíbulo-Ocular/fisiologia , Canais Semicirculares/fisiologia , Animais , Humanos , Vias Neurais/citologia , Membrana dos Otólitos/citologia , Canais Semicirculares/citologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Nervo Vestibular/citologia , Nervo Vestibular/fisiologia , Núcleos Vestibulares/citologia , Núcleos Vestibulares/fisiologiaRESUMO
The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (â¼34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics. The I(KCa) component of the delayed I(KD) was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca(2+) independent fraction of I(KD), I(KV), and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both I(Ca) and I(KD) amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of I(KD) inactivation, although the changes were scaled to the reduced I(KD) amplitude. From these observations, it is expected that hair cells exposed to gentamicin 'in vivo' become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca(2+) inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis.
Assuntos
Antibacterianos/toxicidade , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Gentamicinas/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Canais Semicirculares/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas/metabolismo , Transporte de Íons , Potenciais da Membrana , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Rana esculenta , Canais Semicirculares/citologia , Canais Semicirculares/metabolismo , Fatores de TempoRESUMO
Sensory hair cells are the essential mechanotransducers of the inner ear, responsible not only for the transduction of sound and motion stimuli but also, remarkably, for nanomechanical amplification of sensory stimuli. Here we show that semicircular canal hair cells generate a mechanical nonlinearity in vivo that increases sensitivity to angular motion by amplification at low stimulus strengths. Sensitivity at high stimulus strengths is linear and shows no evidence of amplification. Results suggest that the mechanical work done by hair cells contributes approximately 97 zJ/cell of amplification per stimulus cycle, improving sensitivity to angular velocity stimuli below approximately 5 degrees /s (0.3-Hz sinusoidal motion). We further show that mechanical amplification can be inhibited by the brain via activation of efferent synaptic contacts on hair cells. The experimental model was the oyster toadfish, Opsanus tau. Physiological manifestation of mechanical amplification and efferent control in a teleost vestibular organ suggests the active motor process in sensory hair cells is ancestral. The biophysical basis of the motor(s) remains hypothetical, but a key discriminating question may involve how changes in somatic electrical impedance evoked by efferent synaptic action alter function of the motor(s).
Assuntos
Células Ciliadas da Ampola/fisiologia , Mecanotransdução Celular , Canais Semicirculares/citologia , Animais , Batracoidiformes/fisiologia , Movimento (Física)RESUMO
The rodent vestibular system is immature at birth. During the first postnatal week, vestibular type I and type II hair cells start to acquire their characteristic morphology and afferent innervation. We have studied postnatal changes in the membrane properties of type I hair cells acutely isolated from the semicircular canals (SCC) of gerbils and rats using whole cell patch clamp and report for the first time developmental changes in ionic conductances in these cells. At postnatal day (P) 5 immature hair cells expressed a delayed rectifier K(+) conductance (G(DR)) which activated at potentials above approximately -50 mV in both species. Hair cells also expressed a transient Na(+) conductance (G(Na)) with a mean half-inactivation of approximately -90 mV. At P6 in rat and P7 in gerbil, a low-voltage activated K(+) conductance (G(K,L)) was first observed and conferred a low-input resistance, typical of adult type I hair cells, on SCC type I hair cells. G(K,L) expression in hair cells increased markedly during the second postnatal week and was present in all rat type I hair cells by P14. In gerbil hair cells, G(K,L) appeared later and was present in all type I hair cells by P19. During the third postnatal week, G(Na) expression declined and was absent by the fourth postnatal week in rat and the sixth postnatal week in gerbils. Understanding the ionic changes associated with hair cell maturation could help elucidate development and regeneration mechanisms in the inner ear.
Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Canais de Potássio/metabolismo , Canais Semicirculares/crescimento & desenvolvimento , Canais Semicirculares/metabolismo , Canais de Sódio/metabolismo , 4-Aminopiridina/farmacologia , Envelhecimento/fisiologia , Animais , Interpretação Estatística de Dados , Eletrofisiologia , Gerbillinae , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Canais Semicirculares/citologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/crescimento & desenvolvimentoRESUMO
The sensations of sound and motion generated by the inner ear are controlled by the brain through extensive centripetal innervation originating within the brain stem. In the semicircular canals, brain stem efferent neurons make synaptic contacts with mechanosensory hair cells and with the dendrites of afferent neurons. Here, we examine the relative contributions of efferent action on hair cells and afferents. Experiments were performed in vivo in the oyster toadfish, Opsanus tau. The efferent system was activated via electrical pulses to the brain stem and sensory responses to motion stimuli were quantified by simultaneous voltage recording from afferents and intracellular current- and/or voltage-clamp recordings from hair cells. Results showed synaptic inputs to both afferents and hair cells leading to relatively long-latency intracellular signaling responses: excitatory in afferents and inhibitory in hair cells. Generally, the net effect of efferent action was an increase in afferent background discharge and a simultaneous decrease in gain to angular motion stimuli. Inhibition of hair cells was likely the result of a ligand-gated opening of a major basolateral conductance. The reversal potential of the efferent-evoked current was just below the hair cell resting potential, thus resulting in a small hyperpolarization. The onset latency averaged about 90 ms and latency to peak response was 150-400 ms. Hair cell inhibition often outlasted afferent excitation and, in some cases, latched hair cells in the "off" condition for >1 s following cessation of stimulus. These features endow the animal with a powerful means to adjust the sensitivity and dynamic range of motion sensation.
Assuntos
Vias Aferentes/fisiologia , Células Ciliadas Auditivas/fisiologia , Percepção de Movimento/fisiologia , Canais Semicirculares/citologia , Potenciais de Ação/fisiologia , Animais , Batracoidiformes , Fenômenos Biofísicos/fisiologia , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais da Membrana/fisiologia , Inibição Neural/fisiologia , Técnicas de Patch-Clamp/métodos , Estimulação Física/métodos , Tempo de Reação/fisiologia , Canais Semicirculares/fisiologiaRESUMO
Using multiunit recording of action potentials from the whole nerve with the aid of external perfusion, we investigated the effects of dopamine (DOP) agonists that are involved in modulatory actions on synaptic transmission in the isolated labyrinth preparations of frogs. The external application of DOP (0.1-1 mM), the D(1) agonist chloro-APB hydrobromide (CAPB, 50-100 microM) and the D2 agonist quinerolane (QUI, 50-100 microM) induced a dose-dependent and reversible decline in the resting discharge frequency. In this concentration range, the potency of applied CAPB considerably exceeded that of QUI. AMPA, NMDA and ACPD responses were inhibited by the D1 and D2 agonists, implicating both subtypes of DOP receptors in the modulation of both ionotropic and metabotropic glutamate receptors. The inhibitory action of the DOP agonists on L-glutamate responses persisted in a high Mg2+ solution in conditions of selective activation of the postsynaptic membrane. The results obtained suggest that DOP may interact with both D1 and D2 receptor subtypes, most likely located postsynaptically on the afferent nerve fibers. This dopaminergic control mechanism may result in the reduction of the activated firing rate, thus preventing over-excitation and excitotoxic injury of the afferent dendrites after the external application of L-glutamate and excessive receptor stimulation.
Assuntos
Vias Aferentes/metabolismo , Dopamina/metabolismo , Células Ciliadas Vestibulares/metabolismo , Canais Semicirculares/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Vias Aferentes/citologia , Animais , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Células Ciliadas Vestibulares/citologia , Rana temporaria , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Canais Semicirculares/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Fish otoliths consist of >90% calcium carbonate, the accretion of which depends on acellular endolymph. This study confirms the presence of plasma membrane calcium ATPase 1a isoform (Atp2b1a) in the auditory and vestibular system of a teleost fish. As shown by in situ hybridization, zebrafish atp2b1a is expressed mainly in larval otic placode and lateral-line neuromast as well as in the hair cells within the adult zebrafish inner ear chamber. Zebrafish atp2b1a knockdown by antisense morpholinos reduced the number of hair cells and produced malformation of semicircular canals and smaller otoliths. These defects coincide with unbalanced body orientation. The formation of smaller otoliths in atp2b1a morphants may stem from an impairment of calcium supply in the endolymph. However, otolith formation persists in most morphants, suggesting that other zebrafish Atp2b isoforms or paracellular pathways may also transport calcium into the endolymph. These results suggest that Atp2b1a plays an important role for normal development of the auditory and vestibular system as well as calcium transport in the inner ear of zebrafish.
Assuntos
ATPases Transportadoras de Cálcio/fisiologia , Membrana Celular/enzimologia , Membrana dos Otólitos/embriologia , Canais Semicirculares/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Sinalização do Cálcio/genética , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Clonagem Molecular , Membrana dos Otólitos/citologia , Fenótipo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Canais Semicirculares/citologia , Análise de Sequência de Proteína , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Within the vestibular system of virtually all vertebrate species, gravity and linear acceleration are detected via coupling of calcified masses to the cilia of mechanosensory hair cells. The mammalian ear contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while otolith matrix protein (OMP) is the most abundant matrix protein found in the otoliths of teleost fish. We have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 was detected in the ear between 15 hpf and 72 hpf, and was restricted primarily to the macula and the developing epithelial pillars of the semicircular canals. Expression of otoc1 was also detected in epiphysis, optic stalk, midbrain, diencephalon, flexural organ, and spinal cord. During embryogenesis, expression of otoc1 mRNA preceded the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produced a variety of aberrant otolith phenotypes. Our results suggest that Otoc1 may serve to nucleate calcium carbonate mineralization of aragonitic otoliths.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Membrana dos Otólitos/embriologia , Vestíbulo do Labirinto/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Carbonato de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Regulação para Baixo/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento/genética , Minerais/metabolismo , Dados de Sequência Molecular , Membrana dos Otólitos/citologia , Membrana dos Otólitos/metabolismo , Filogenia , Biossíntese de Proteínas/genética , RNA Antissenso/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais Semicirculares/citologia , Canais Semicirculares/embriologia , Canais Semicirculares/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/isolamento & purificaçãoRESUMO
We have reported that calretinin and calbindin staining of calyxes in the apical region of the cristae is reduced or absent in old gerbils (>or=35 months) that had normal numbers of hair cells [Kevetter GA, Leonard RB (2002) Decreased expression of calretinin and calbindin in the labyrinth of old gerbils. Brain Res 957:362-365]. Here we examine the ability of primary afferents in aged gerbils to carry a tracer injected into the vestibular nuclear complex to their terminals in the cristae. Calyxes throughout the cristae were well labeled in a young animal with such an injection. In the aged animals, many calyxes were only partially filled or not filled at all. In some cases labeled axons were also missing from the stroma underlying the missing calyxes. There is a strong correspondence between the region where the calyxes were not filled and the absence of calretinin immunostaining. To determine if afferents from the cristae are functionally abnormal, we recorded from their axons and attempted to activate them with natural stimulation. Among afferents that could be activated, we encountered many afferents that had spontaneous activity but could not be modulated with natural stimulation. When tested, the firing rate of these afferents could be modulated with galvanic stimulation, and/or they could be activated by pulsed electrical stimulation. We also encountered afferents that had no spontaneous activity. The presence of these axons was revealed by an injury discharge that could not be modulated with natural stimulation. When tested, these axons could be activated with pulsed electrical stimulation. In some instances we encountered two or more such afferents in a row, an event we have not seen in young animals. We suggest that the simplest explanation for these observations is that calyxes are being lost in old animals.
Assuntos
Envelhecimento/fisiologia , Gerbillinae/fisiologia , Canais Semicirculares/citologia , Canais Semicirculares/fisiologia , Nervo Vestibular/fisiologia , Animais , Calbindina 2 , Estimulação Elétrica , Potenciais Evocados/fisiologia , Potenciais Evocados/efeitos da radiação , Gerbillinae/anatomia & histologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Nervo Vestibular/citologiaRESUMO
Some aspects of Ca(2+) channel modulation in hair cells isolated from semicircular canals of the frog (Rana esculenta) have been investigated using the whole-cell technique and intra and extracellular solutions designed to modify the basic properties of the Ca(2+) macrocurrent. With 1 mM ATP in the pipette solution, about 60% of the recorded cells displayed a Ca(2+) current constituted by a mix of an L and a drug-resistant (R2) component; the remaining 40% exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca-dependent manner. If the pipette ATP was raised to 10 mM, cells exhibiting the R1 current fraction displayed an increase of both the R1 and L components by approximately 280 and approximately 70%, respectively, while cells initially lacking R1 showed a similar increase in the L component with R1 becoming apparent and raising up to a mean amplitude of approximately 44 pA. In both cell types the R2 current fraction was negligibly affect by ATP. The current run-up was unaffected by cyclic nucleotides, and was not triggered by 10 mM ATPgammaS, ADP, AMP or GTP. Long-lasting depolarisations (>5 s) produced a progressive, reversible decay in the inward current despite the presence of intracellular ATP. Ca(2+) channel blockade by Cd(2+) unmasked a slowly activating outward Cs(+) current flowing through a non-Ca(2+) channel type, which became progressively unblocked by prolonged depolarisation even though Cs(+) and TEA(+) were present on both sides of the channel. The outward current waveform could be erroneously ascribed to a Ca- and/or voltage dependence of the Ca(2+) macrocurrent.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , Células Ciliadas Vestibulares/fisiologia , Ativação do Canal Iônico/fisiologia , Rana esculenta/fisiologia , Canais Semicirculares/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Células Ciliadas Vestibulares/efeitos dos fármacos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Canais Semicirculares/citologiaRESUMO
The low Ca(2+) concentration ([Ca(2+)]) of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported (Yamauchi et al., Biochem Biophys Res Commun 331: 1353-1357, 2005) that the epithelial Ca(2+) channels TRPV5 and TRPV6 (transient receptor potential types 5 and 6) are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D(3), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca(2+)], and transepithelial potential of the utricle were measured in Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4(-/-) mice exhibit reduced pH and utricular endolymphatic potential and increased [Ca(2+)]. Monolayers of primary cultures of rat semicircular canal duct cells were grown on permeable supports, and cellular uptake of (45)Ca(2+) was measured individually from the apical and basolateral sides. Net uptake of (45)Ca(2+) was greater after incubation with 1,25-dihydroxyvitamin D(3). Net (45)Ca(2+) absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum, and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca(2+)] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca(2+) absorption.
Assuntos
Proteínas de Transporte de Ânions/deficiência , Cálcio/metabolismo , Antiportadores de Cloreto-Bicarbonato/deficiência , Ducto Endolinfático/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Absorção/efeitos dos fármacos , Ácidos/metabolismo , Animais , Calcitriol/farmacologia , Cálcio/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio , Células Cultivadas , Eletrofisiologia , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Concentração Osmolar , Ratos , Ratos Wistar , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/fisiopatologia , Canais Semicirculares/citologia , Canais Semicirculares/efeitos dos fármacos , Canais Semicirculares/metabolismo , Transportadores de SulfatoRESUMO
The effect of human and rabbit neutrophilic defensins NP-1 and amonoglycoside antibiotic gentamicin on the synaptic transmission in the afferent synapse of isolated vestibular apparatus of the frog has been comparatively studied. Both defensins proved active in the concentration range of 0.0001 to 1 nM and efficiently decreased the impulse frequency in the afferent nerve fibers in a concentration-dependent manner. No significant differences in the efficiency of rabbit and human defensin NP-1 have been revealed in these experiments. Gentamicin also had an inhibitory effect on the afferent discharge in the concentration range of 10-500 microM (0.5-25 mg/kg). The inhibitory effect of gentamicin on the impulse activity of the vestibular nerve was observed at therapeutic doses. The excitatory effect of the putative neurotransmitter L-glutamate was considerably inhibited by defensin NP-1. These findings suggest that the mechanism of defensin action involves a modification of the synaptic transmission the hair receptor and is mediated by L-glutamate.