RESUMO
Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450â 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.
Assuntos
Homeostase , Organogênese , Tretinoína/metabolismo , Vestíbulo do Labirinto/embriologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Camundongos , Camundongos Knockout , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/metabolismo , Vestíbulo do Labirinto/citologiaRESUMO
The mammalian vestibular epithelia exhibit a remarkably stereotyped organization featuring cellular characteristics under planar cell polarity (PCP) control. PCP mechanisms are responsible for the organization of hair cell morphologic polarization vectors, and are thought to be responsible for the postsynaptic expression of the calcium-binding protein calretinin that defines the utricular striola and cristae central zone. However, recent analyses revealed that subtle differences in the topographic expression of oncomodulin, another calcium-binding protein, reflects heterogeneous factors driving the subtle variations in expression. Calbindin represents a third calcium-binding protein that has been previously described to be expressed in both hair cells and afferent calyces in proximity to the utricular striola and crista central zone. The objective of the present investigation was to determine calbindin's topographic pattern of expression to further elucidate the extent to which PCP mechanisms might exert control over the organization of vestibular neuroepithelia. The findings revealed that calbindin exhibited an expression pattern strikingly similar to oncomodulin. However, within calyces of the central zone calbindin was colocalized with calretinin. These results indicate that organizational features of vestibular epithelia are governed by a suite of factors that include PCP mechanisms as well others yet to be defined.
Assuntos
Calbindina 1/biossíntese , Calbindina 2/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Neuroepiteliais/metabolismo , Vestíbulo do Labirinto/metabolismo , Animais , Calbindina 1/metabolismo , Calbindina 2/metabolismo , Polaridade Celular/fisiologia , Células Ciliadas Auditivas/citologia , Camundongos Endogâmicos C57BL , Células Neuroepiteliais/citologia , Vestíbulo do Labirinto/citologiaRESUMO
Objective: To investigate the feasibility of inducing human amniotic fluid stem cells into functional neurons by supporting contact co-culture depended on feeder layer from mouse vestibular supporting cells. Methods: Human amniotic fluid stem cells were isolated to culture. The vestibular tissues were obtained from the newborn C57BL/6J mouse by enzymatic digestion and cell culture, the hollow spheres were selected to prepare a monolayer feeder layer. The nGFP-labeled amniotic fluid stem cells were planted on the surface of the feeder layer to form the supporting contact co-culture without adding any exogenous nerve growth factor and neuronal signal inducing factor, and detected the expression of Tuj1 and PSD95, and investigated whether there were ion channels in neurons by FM1-43. Human amniotic fluid stem cells and mouse vestibular supporting cells, which were differentiated separately, and Transwell coculture was used as the control group. Results: The feeder layer expressed the special marker P27(kip1)of the inner ear supporting cell. The nGFP-labeled amniotic fluid stem cells were inoculated on the feeder layer, and (52.0±3.0)% of the nGFP cells expressed Tuj1,which had typical neurons morphological characteristics[protrusion length (110.7±6.2) µm]; the feeder layer cells were differentiated separately, of which (1.1±0.6) % expressed Tuj1 in the control group; the amniotic fluid stem cells were differentiated independently without typical neuron morphological features [protrusion length (16±4.1) µm], of which (92.0±1.0) % expressed Tuj1. The amniotic fluid stem cells and the feeder layer were co-cultured in Transwell: although (92.0±1.0)% of amniotic fluid stem cells had the expression of Tuj1, which had no typical neurons morphological feature[protrusion length (17±4.5) µm], only (1.2±0.9) % of Tuj1 were observed in the feeder layer. Conclusion: By supporting contact co-culture, the feeder layer from the vestibular supporting cells can successfully differentiate human amniotic fluid stem cells into neurons.
Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Técnicas de Cocultura , Neurônios/citologia , Células-Tronco/citologia , Vestíbulo do Labirinto/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Células Alimentadoras , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Projetos PilotoRESUMO
Over the last few decades, several studies have been conducted to identify the mechanisms involved in spontaneous functional recovery following peripheral vestibular damage. Different reactive processes occur at both the central and peripheral levels over the first few hours after the loss of the peripheral vestibular input. The restoration of the electrophysiological homeostasis between opposite vestibular nuclei is one of the key mechanisms of central compensation. This is achieved through a mosaic of biochemical events within the vestibular nuclei that each occur with their own kinetics. At the same time, under specific conditions, strong synaptic plasticity may take place within the vestibular sensory organs. It is thought that this reactive plasticity can contribute to the repair of damaged contacts between hair cells and fibres of the vestibular nerve, thus gradually restoring peripheral sensory input. These different plastic phenomena seem to reproduce those observed during development. Research is now needed to identify the cellular and molecular mechanisms that support this spontaneous peripheral repair process, with the ambition 1 day to be able to control it and stimulate the restoration of gait and balance.
Assuntos
Adaptação Fisiológica/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Humanos , Recuperação de Função Fisiológica/fisiologia , Núcleos Vestibulares/citologia , Núcleos Vestibulares/fisiologia , Vestíbulo do Labirinto/citologiaRESUMO
Computational capability and connectivity are key elements for understanding how central vestibular neurons contribute to gaze-stabilizing eye movements during self-motion. In the well-characterized and segmentally distributed hindbrain oculomotor network of goldfish, we determined afferent and efferent connections along with discharge patterns of descending octaval nucleus (DO) neurons during different eye motions. Based on activity correlated with horizontal eye and head movements, DO neurons were categorized into two complementary groups that either increased discharge during both contraversive (type II) eye (e) and ipsiversive (type I) head (h) movements (eIIhI) or vice versa (eIhII). Matching time courses of slow-phase eye velocity and corresponding firing rates during prolonged visual and head rotation suggested direct causality in generating extraocular motor commands. The axons of the dominant eIIhI subgroup projected either ipsi- or contralaterally and terminated in the abducens nucleus, Area II, and Area I with additional recurrent collaterals of ipsilaterally projecting neurons within the parent nucleus. Distinct feedforward commissural pathways between bilateral DO neurons likely contribute to the generation of eye velocity signals in eIhII cells. The shared contribution of DO and Area II neurons to eye velocity storage likely represents an ancestral condition in goldfish that is clearly at variance with the task separation between mammalian medial vestibular and prepositus hypoglossi neurons. This difference in signal processing between fish and mammals might correlate with a larger repertoire of visuo-vestibular-driven eye movements in the latter species that potentially required a shift in sensitivity and connectivity within the hindbrain-cerebello-oculomotor network. NEW & NOTEWORTHY We describe the structure and function of neurons within the goldfish descending octaval nucleus. Our findings indicate that eye and head velocity signals are processed by vestibular and Area II velocity storage integrator circuitries whereas the velocity-to-position Area I neural integrator generates eye position solely. This ancestral condition differs from that of mammals, in which vestibular neurons generally lack eye position signals that are processed and stored within the nucleus prepositus hypoglossi.
Assuntos
Encéfalo/fisiologia , Movimentos Oculares , Neurônios/fisiologia , Vestíbulo do Labirinto/fisiologia , Potenciais de Ação , Animais , Encéfalo/citologia , Carpa Dourada , Tempo de Reação , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/inervaçãoRESUMO
It is commonly assumed that the brain's neural coding strategies are adapted to the statistics of natural stimuli. Specifically, to maximize information transmission, a sensory neuron's tuning function should effectively oppose the decaying stimulus spectral power, such that the neural response is temporally decorrelated (i.e. 'whitened'). However, theory predicts that the structure of neuronal variability also plays an essential role in determining how coding is optimized. Here, we provide experimental evidence supporting this view by recording from neurons in early vestibular pathways during naturalistic self-motion. We found that central vestibular neurons displayed temporally whitened responses that could not be explained by their tuning alone. Rather, computational modeling and analysis revealed that neuronal variability and tuning were matched to effectively complement natural stimulus statistics, thereby achieving temporal decorrelation and optimizing information transmission. Taken together, our findings reveal a novel strategy by which neural variability contributes to optimized processing of naturalistic stimuli.
Assuntos
Movimentos da Cabeça/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Primatas/fisiologia , Vestíbulo do Labirinto/fisiologia , Algoritmos , Animais , Macaca fascicularis/fisiologia , Macaca mulatta/fisiologia , Masculino , Modelos Neurológicos , Movimento (Física) , Vestíbulo do Labirinto/citologiaRESUMO
Vestibular function was established early in vertebrates and has remained, for the most part, unchanged. In contrast, each group of tetrapods underwent independent evolutionary processes to solve the problem of hearing on land, resulting in a remarkable mixture of conserved, divergent and convergent features that define extant auditory systems. The vestibuloacoustic nuclei of the hindbrain develop from a highly conserved ground plan and provide an ideal framework on which to address the participation of developmental processes to the evolution of neuronal circuits. We employed an electroporation strategy to unravel the contribution of two dorsoventral and four axial lineages to the development of the chick hindbrain vestibular and auditory nuclei. We compare the chick developmental map with recently established genetic fate-maps of the developing mouse hindbrain. Overall, we find considerable conservation of developmental origin for the vestibular nuclei. In contrast, a comparative analysis of the developmental origin of hindbrain auditory structures echoes the complex evolutionary history of the auditory system. In particular, we find that the developmental origin of the chick auditory interaural time difference circuit supports its emergence from an ancient vestibular network, unrelated to the analogous mammalian counterpart.
Assuntos
Tronco Encefálico/embriologia , Núcleo Coclear/embriologia , Núcleos Vestibulares/embriologia , Vestíbulo do Labirinto/embriologia , Animais , Vias Auditivas/citologia , Vias Auditivas/embriologia , Vias Auditivas/metabolismo , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Embrião de Galinha , Galinhas , Núcleo Coclear/citologia , Núcleo Coclear/metabolismo , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Rombencéfalo/citologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Especificidade da Espécie , Núcleos Vestibulares/citologia , Núcleos Vestibulares/metabolismo , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/metabolismoRESUMO
Parallel fibers in the molecular layer of the vertebrate cerebellum mediate slow spike conduction in the transverse plane. In contrast, electrophysiological recordings have indicated that rapid spike conduction exists between the lateral regions of the cerebellar cortex of the red-ear pond turtle (Trachemys scripta). The anatomical basis for this commissure is now examined in that species using neuronal tracing techniques. Fluorescently tagged dextrans and lipophilic carbocyanine dyes placed in one lateral edge of this nonfoliated cortex are transported across the midline of living brains in vitro and along the axonal membranes of fixed tissues, respectively. Surprisingly, the labeled commissural axons traversed the cortex within the Purkinje cell layer, and not in the white matter of the molecular layer or the white matter below the granule cell layer. Unlike thin parallel fibers that exhibit characteristic varicosities, this commissure is composed of smooth axons of large diameter that also extend beyond the cerebellar cortex via the cerebellar peduncles. Double labeling with myelin basic protein antibody demonstrated that these commissural axons are ensheathed with myelin. In contrast to this transverse pathway, an orthogonal myelinated tract was observed along the cerebellar midline. The connections of this transverse commissure with the lateral cerebellum, the vestibular nuclear complex, and the cochlear vestibular ganglia indicate that this commissure plays a role in bilateral vestibular connectivity.
Assuntos
Axônios/ultraestrutura , Cerebelo/citologia , Bainha de Mielina/ultraestrutura , Fibras Nervosas Mielinizadas/ultraestrutura , Células de Purkinje/ultraestrutura , Tartarugas/anatomia & histologia , Animais , Cerebelo/fisiologia , Cóclea/citologia , Cóclea/ultraestrutura , Imuno-Histoquímica , Proteína Básica da Mielina/química , Núcleos da Rafe/citologia , Núcleos da Rafe/ultraestrutura , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/ultraestrutura , Substância Branca/ultraestruturaRESUMO
The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.
Assuntos
Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Vestíbulo do Labirinto/metabolismo , Animais , Orelha Interna/citologia , Células Ciliadas Auditivas/citologia , Camundongos , Vestíbulo do Labirinto/citologiaRESUMO
There is substantial evidence that loss of vestibular function impairs spatial learning and memory related to hippocampal (HPC) function, as well as increasing evidence that striatal (Str) plasticity is also implicated. Since the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor is considered essential to spatial memory, previous studies have investigated whether the expression of HPC NMDA receptors changes following vestibular loss; however, the results have been contradictory. Here we used a novel flow cytometric method to quantify the number of neurons expressing NMDA receptors in the HPC and Str following bilateral vestibular loss (BVL) in rats. At 7 and 30 days post-op., there was a significant increase in the number of HPC neurons expressing NMDA receptors in the BVL animals, compared to sham controls (Pâ¯≤â¯0.004 and Pâ¯≤â¯0.0001, respectively). By contrast, in the Str, at 7 days there was a significant reduction in the number of neurons expressing NMDA receptors in the BVL group (Pâ¯≤â¯0.05); however, this difference had disappeared by 30 days post-op. These results suggest that BVL causes differential changes in the number of neurons expressing NMDA receptors in the HPC and Str, which may be related to its long-term impairment of spatial memory.
Assuntos
Corpo Estriado/metabolismo , Citometria de Fluxo/métodos , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/biossíntese , Vestíbulo do Labirinto/metabolismo , Animais , Corpo Estriado/citologia , Orelha Interna/citologia , Orelha Interna/metabolismo , Orelha Interna/cirurgia , Expressão Gênica , Hipocampo/citologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/cirurgiaRESUMO
Intratympanic gentamicin (ITG) has been used to treat refractory Ménière's disease. Disequilibrium after ITG was still a challenge for some patients, and the underlying mechanism is poorly understood. Our previous study demonstrated that gentamicin distributed in the bilateral vestibular efferent neurons (VEN) after ITG; however, does it lead to VEN damage and cause further disequilibrium in patients following ITG? In this study, we observed severe damaged gentamicin-positive neurons of VEN and severe fractured myelin layer plates around neural fibers when viewed under transmission electron microscopy at day 3 after ITG. At day 30, neurons of VEN presented with relatively normal structures. Compared with the control group, the total number of choline acetyltransferase (CHAT) immunolabeling neurons in bilateral VEN showed a significant decrease both at day 3 and day 30. However, there was no significant difference in the total number of CHAT immunolabeling neurons between day 3 and day 30. It indicates that gentamicin is not only retrogradely transported into bilateral VEN, but also results in the degeneration of VEN after ITG. These findings may be related to patients' disequilibrium symptom after ITG. Furthermore, we speculate that VEN may play a role in vestibular compensation.
Assuntos
Antibacterianos/efeitos adversos , Gentamicinas/efeitos adversos , Doença de Meniere/tratamento farmacológico , Neurônios Eferentes/efeitos dos fármacos , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Feminino , Imunofluorescência/métodos , Gentamicinas/administração & dosagem , Cobaias , Imuno-Histoquímica/métodos , Injeção Intratimpânica , Masculino , Doença de Meniere/patologia , Microscopia Eletrônica de Transmissão/métodos , Neurônios Eferentes/patologia , Vestíbulo do Labirinto/inervação , Vestíbulo do Labirinto/patologiaRESUMO
Specific pharmacological blockade of KCNQ (Kv7) channels with XE991 rapidly (within 20â¯min) and profoundly alters inner ear gravity receptor responses to head motion (Lee et al., 2017). We hypothesized that these effects were attributable to the suppression of K+ secretion following blockade of KCNQ1-KCNE1 channels in vestibular dark cells and marginal cells. To test this hypothesis, K+ secretion was independently inhibited by blocking the Na+-K+-2Cl- cotransporter (NKCC1, Slc12a2) rather than KCNQ1-KCNE1 channels. Acute blockade of NKCC1 with ethacrynic acid (40â¯mg/kg) eliminated auditory responses (ABRs) within approximately 70â¯min of injection, but had no effect on vestibular gravity receptor function (VsEPs) over a period of 2â¯h in the same animals. These findings show that, vestibular gravity receptors are highly resistant to acute disruption of endolymph secretion unlike the auditory system. Based on this we argue that acute suppression of K+ secretion alone does not likely account for the rapid profound effects of XE991 on gravity receptors. Instead the effects of XE991 likely require additional action at KCNQ channels located within the sensory epithelium itself.
Assuntos
Ácido Etacrínico/farmacologia , Gravitação , Movimentos da Cabeça , Canais de Potássio KCNQ/metabolismo , Potássio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Vestíbulo do Labirinto/efeitos dos fármacos , Animais , Antracenos/farmacologia , Endolinfa/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Canais de Potássio KCNQ/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Bloqueadores dos Canais de Potássio/farmacologia , Via Secretória , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fatores de Tempo , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/metabolismoRESUMO
At most synapses in the brain, short-term plasticity dynamically modulates synaptic strength. Rapid frequency-dependent changes in synaptic strength have key roles in sensory adaptation, gain control and many other neural computations. However, some auditory, vestibular and cerebellar synapses maintain constant strength over a wide range of firing frequencies, and as a result efficiently encode firing rates. Despite its apparent simplicity, frequency-invariant transmission is difficult to achieve because of inherent synaptic nonlinearities. Here we study frequency-invariant transmission at synapses from Purkinje cells to deep cerebellar nuclei and at vestibular synapses in mice. Prolonged activation of these synapses leads to initial depression, which is followed by steady-state responses that are frequency invariant for their physiological activity range. We find that synaptotagmin 7 (Syt7), a calcium sensor for short-term facilitation, is present at both synapses. It was unclear why a sensor for facilitation would be present at these and other depressing synapses. We find that at Purkinje cell and vestibular synapses, Syt7 supports facilitation that is normally masked by depression, which can be revealed in wild-type mice but is absent in Syt7 knockout mice. In wild-type mice, facilitation increases with firing frequency and counteracts depression to produce frequency-invariant transmission. In Syt7-knockout mice, Purkinje cell and vestibular synapses exhibit conventional use-dependent depression, weakening to a greater extent as the firing frequency is increased. Presynaptic rescue of Syt7 expression restores both facilitation and frequency-invariant transmission. Our results identify a function for Syt7 at synapses that exhibit overall depression, and demonstrate that facilitation has an unexpected and important function in producing frequency-invariant transmission.
Assuntos
Inibição Neural , Plasticidade Neuronal , Sinapses/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Animais , Percepção Auditiva , Cálcio/metabolismo , Cerebelo/citologia , Cerebelo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Terminações Pré-Sinápticas/metabolismo , Células de Purkinje/metabolismo , Sinaptotagminas/deficiência , Sinaptotagminas/genética , Vestíbulo do Labirinto/citologia , Vestíbulo do Labirinto/metabolismoRESUMO
One of the most obvious manifestations of polarity in epithelia is the subdivision of the cell surface by cell junctions into apical and basolateral domains. crumbs genes are among key regulators of this form of polarity. Loss of crumbs function disrupts the apical cell junction belt and crumbs overexpression expands the apical membrane size. Crumbs proteins contain a single transmembrane domain and localize to cell junction area at the apical surface of epithelia. In some tissues, they are also found in cilia. To test their role in ciliogenesis, we investigated mutant phenotypes of zebrafish crumbs genes. In zebrafish, mutations of three crumbs genes, oko meduzy/crb2a, crb3a, and crb2b, affect cilia length in a subset of tissues. In oko meduzy (ome), this is accompanied by accumulation of other Crumbs proteins in the ciliary compartment. Moreover, intraflagellar transport (IFT) particle components accumulate in the ciliary shaft of ome;crb3a double mutants. Consistent with the above, Crb3 knockdown in mammalian cells affects the dynamics of IFT particle movement. These findings reveal crumbs-dependent mechanisms that regulate the localization of ciliary proteins, including Crumbs proteins themselves, and show that crumbs genes modulate intraflagellar transport and cilia elongation.
Assuntos
Polaridade Celular , Células Epiteliais/citologia , Proteínas de Membrana/metabolismo , Vestíbulo do Labirinto/citologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Cílios , Células Epiteliais/metabolismo , Proteínas de Membrana/genética , Camundongos , Transporte Proteico , Proteínas Supressoras de Tumor/metabolismo , Vestíbulo do Labirinto/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The Vestibular Microphonic (VM) has only featured in a handful of publications, mostly involving non-mammalian and ex vivo models. The VM is the extracellular analogue of the vestibular hair cell receptor current, and offers a tool to monitor vestibular hair cell activity in vivo. OBJECTIVE: To characterise features of the VM measured in vivo in guinea pigs, using a relatively simple experimental setup. METHODS: The VM, evoked by bone-conducted vibration (BCV), was recorded from the basal surface of either the utricular or saccular macula after surgical removal of the cochlea, in 27 guinea pigs. RESULTS: The VM remained after vestibular nerve blockade, but was abolished following end-organ destruction or death. The VM reversed polarity as the recording electrode tracked across the utricular or saccular macula surface, or through the utricular macula. The VM could be evoked by BCV stimuli of frequencies between 100 Hz and 5 kHz, and was largest to vibrations between 600 Hz and 800 Hz. Experimental manipulations demonstrated a reduction in the VM amplitude with maculae displacement, or rupture of the utricular membrane. CONCLUSIONS: Results mirror those obtained in previous ex vivo studies, and further demonstrate that vestibular hair cells are sensitive to vibrations of several kilohertz. Changes in the VM with maculae displacement or rupture suggest utricular hydrops may alter vestibular hair cell sensitivity due to either mechanical or ionic changes.
Assuntos
Condução Óssea , Potenciais Evocados Auditivos , Células Ciliadas Auditivas/fisiologia , Vestíbulo do Labirinto/fisiologia , Estimulação Acústica , Animais , Feminino , Cobaias , Masculino , Mecanotransdução Celular , Fatores de Tempo , Vestíbulo do Labirinto/citologia , VibraçãoRESUMO
We sought to establish a more efficient technique for induction of inner ear hair cell-like cells (HC-like cells) from embryonic stem cells (ES cells) by using a combination of two previously reported methods; ST2 stromal cell-conditioned medium, known to be favorable for HC-like cell induction (HIST2 method), and ES cells with transfer of the Math1 gene (Math1-ES cells). Math1-ES cells carrying Tet-inducible Math1 were cultured for 14days with doxycycline in conditioned medium from cultures of ST2 stromal cells following formation of 4-day embryoid bodies (EBs). Although each of the previously introduced methods have been reported to induce approximately 20% HC-like cells and 10% HC-like cells in their respective populations in EB outgrowths at the end of the culture periods, the present combined method was able to generate approximately 30% HC-like cells expressing HC-related markers (myosin6, myosin7a, calretinin, α9AchR, Brn3c), which showed remarkable formation of stereocilia-like structures. Analysis of expressions of marker genes specific for cochlear (Lmod3, Emcn) and vestibular (Dnah5, Ptgds) cells indicated that our HIST2 method may lead to induction of cochlear- and vestibular-type cells. In addition, continuous Math1 induction by doxycycline without use of the HIST2 method preferentially induced cochlear markers with negligible effects on vestibular marker induction.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Meios de Cultivo Condicionados/farmacologia , Células Ciliadas Auditivas Internas/citologia , Células-Tronco Embrionárias Murinas/citologia , Transfecção , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Células Cultivadas , Cóclea/citologia , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mecanotransdução Celular , Camundongos , Miosinas/metabolismo , Estereocílios/metabolismo , Células Estromais/metabolismo , Vestíbulo do Labirinto/citologiaRESUMO
The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (â¼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis.
Assuntos
Separação Celular/métodos , Meios de Cultura/metabolismo , Células Endoteliais/fisiologia , Citometria de Fluxo , Macrófagos/fisiologia , Melanócitos/fisiologia , Pericitos/fisiologia , Vestíbulo do Labirinto/citologia , Fatores Etários , Animais , Biomarcadores/metabolismo , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/metabolismo , Macrófagos/metabolismo , Melanócitos/metabolismo , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Fenótipo , Cultura Primária de Células , Fatores de TempoRESUMO
Stimulation of vestibular efferent neurons excites calyx and dimorphic (CD) afferents. This excitation consists of fast and slow components that differ >100-fold in activation kinetics and response duration. In the turtle, efferent-mediated fast excitation arises in CD afferents when the predominant efferent neurotransmitter acetylcholine (ACh) activates calyceal nicotinic ACh receptors (nAChRs); however, it is unclear whether the accompanying efferent-mediated slow excitation is also attributed to cholinergic mechanisms. To identify synaptic processes underlying efferent-mediated slow excitation, we recorded from CD afferents innervating the turtle posterior crista during electrical stimulation of efferent neurons, in combination with pharmacological probes and mechanical stimulation. Efferent-mediated slow excitation was unaffected by nAChR compounds that block efferent-mediated fast excitation, but were mimicked by muscarine and antagonized by atropine, indicating that it requires ACh and muscarinic ACh receptor (mAChR) activation. Efferent-mediated slow excitation or muscarine application enhanced the sensitivity of CD afferents to mechanical stimulation, suggesting that mAChR activation increases afferent input impedance by closing calyceal potassium channels. These observations were consistent with suppression of a muscarinic-sensitive K+-current, or M-current. Immunohistochemistry for putative M-current candidates suggested that turtle CD afferents express KCNQ3, KCNQ4, and ERG1-3 potassium channel subunits. KCNQ channels were favored as application of the selective antagonist XE991 mimicked and occluded efferent-mediated slow excitation in CD afferents. These data highlight an efferent-mediated mechanism for enhancing afferent sensitivity. They further suggest that the clinical effectiveness of mAChR antagonists in treating balance disorders may also target synaptic mechanisms in the vestibular periphery, and that KCNQ channel modulators might offer similar therapeutic value.SIGNIFICANCE STATEMENT Targeting the efferent vestibular system (EVS) pharmacologically might prove useful in ameliorating some forms of vestibular dysfunction by modifying ongoing primary vestibular input. EVS activation engages several kinetically distinct synaptic processes that profoundly alter the discharge rate and sensitivity of first-order vestibular neurons. Efferent-mediated slow excitation of vestibular afferents is of considerable interest given its ability to elevate afferent activity over an extended time course. We demonstrate for the first time that efferent-mediated slow excitation of vestibular afferents is mediated by muscarinic acetylcholine receptor (mAChR) activation and the subsequent closure of KCNQ potassium channels. The clinical effectiveness of some anti-mAChR drugs in treating motion sickness suggest that we may, in fact, already be targeting the peripheral EVS.
Assuntos
Colinérgicos/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios Aferentes/fisiologia , Neurônios Eferentes/fisiologia , Receptores Muscarínicos/metabolismo , Transmissão Sináptica/fisiologia , Vestíbulo do Labirinto/citologia , Análise de Variância , Animais , Biofísica , Calbindina 2/metabolismo , Estimulação Elétrica , Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais Evocados/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Canais de Potássio KCNQ/metabolismo , Masculino , Vias Neurais/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Eferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Transmissão Sináptica/efeitos dos fármacos , TartarugasRESUMO
Disorders of hearing and balance are most commonly associated with damage to cochlear and vestibular hair cells or neurons. Although these cells are not capable of spontaneous regeneration, progenitor cells in the hearing and balance organs of the neonatal mammalian inner ear have the capacity to generate new hair cells after damage. To investigate whether these cells are restricted in their differentiation capacity, we assessed the phenotypes of differentiated progenitor cells isolated from three compartments of the mouse inner ear - the vestibular and cochlear sensory epithelia and the spiral ganglion - by measuring electrophysiological properties and gene expression. Lgr5+ progenitor cells from the sensory epithelia gave rise to hair cell-like cells, but not neurons or glial cells. Newly created hair cell-like cells had hair bundle proteins, synaptic proteins and membrane proteins characteristic of the compartment of origin. PLP1+ glial cells from the spiral ganglion were identified as neural progenitors, which gave rise to neurons, astrocytes and oligodendrocytes, but not hair cells. Thus, distinct progenitor populations from the neonatal inner ear differentiate to cell types associated with their organ of origin.
Assuntos
Diferenciação Celular/fisiologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Vestibulares/citologia , Células-Tronco Neurais/citologia , Gânglio Espiral da Cóclea/citologia , Vestíbulo do Labirinto/citologia , Animais , Células Cultivadas , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologiaRESUMO
The vestibular and geniculate ganglia of the ear in experimental animals carry both of the tyrosine hydroxylase (TH)-positive sympathetic neurons and the neuronal nitric oxide synthase (nNOS)-positive parasympathetic neurons. With an aid of immunohistochemistry, we examined these ganglia as well as the horizontal part of the facial nerve using specimens from 10 formalin-fixed elderly cadavers. The submandibular ganglion from the same cadavers was used for the positive control for both markers. Although there was a nonspecific reaction in nuclei for the present antibody of nNOS, these ganglia were unlikely to contain either nNOS- or TH-positive neurons. However, we did not deny a possibility that the absence was a result of degeneration with aging. In contrast, the facial nerve horizontal part consistently contained both of TH-positive- and nNOS-positive fibers. These fibers might regulate blood supply to the facial nerve and the dysregulation leads to edema to elevate pressure on the nerve within its osseous canal.