RESUMEN
Hearing is one of our most important means of communication. Disabling hearing loss (DHL) is a long-standing, unmet problem in medicine, and in many elderly people, it leads to social isolation, depression, and even dementia. Traditionally, major efforts to cure DHL have focused on hair cells (HCs). However, the auditory nerve is also important because it transmits electrical signals generated by HCs to the brainstem. Its function is critical for the success of cochlear implants as well as for future therapies for HC regeneration. Over the past two decades, cell transplantation has emerged as a promising therapeutic option for restoring lost auditory nerve function, and two independent studies on animal models show that cell transplantation can lead to functional recovery. In this article, we consider the approaches most likely to achieve success in the clinic. We conclude that the structure and biochemical integrity of the auditory nerve is critical and that it is important to preserve the remaining neural scaffold, and in particular the glial scar, for the functional integration of donor cells. To exploit the natural, autologous cell scaffold and to minimize the deleterious effects of surgery, donor cells can be placed relatively easily on the surface of the nerve endoscopically. In this context, the selection of donor cells is a critical issue. Nevertheless, there is now a very realistic possibility for clinical application of cell transplantation for several different types of hearing loss.
Asunto(s)
Trasplante de Células/métodos , Nervio Coclear/trasplante , Animales , HumanosRESUMEN
KEY POINTS: Age-related hearing loss is a progressive hearing loss involving environmental and genetic factors, leading to a decrease in hearing sensitivity, threshold and speech discrimination. We compared age-related changes in inner hair cells (IHCs) between four mouse strains with different levels of progressive hearing loss. The surface area of apical coil IHCs (9-12 kHz cochlear region) decreases by about 30-40% with age. The number of BK channels progressively decreases with age in the IHCs from most mouse strains, but the basolateral membrane current profile remains unchanged. The mechanoelectrical transducer current is smaller in mice harbouring the hypomorphic Cdh23 allele Cdh23ahl (C57BL/6J; C57BL/6NTac), but not in Cdh23-repaired mice (C57BL/6NTacCdh23+ ), indicating that it could contribute to the different progression of hearing loss among mouse strains. The degree of efferent rewiring onto aged IHCs, most likely coming from the lateral olivocochlea fibres, was correlated with hearing loss in the different mouse strains. ABSTRACT: Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea, transducing acoustic information into electrical signals that are relayed to the afferent neurons. Functional changes in IHCs are a potential cause of age-related hearing loss. Here, we have investigated the functional characteristics of IHCs from early-onset hearing loss mice harbouring the allele Cdh23ahl (C57BL/6J and C57BL/6NTac), from late-onset hearing loss mice (C3H/HeJ), and from mice corrected for the Cdh23ahl mutation (C57BL/6NTacCdh23+ ) with an intermediate hearing phenotype. There was no significant loss of IHCs in the 9-12 kHz cochlear region up to at least 15 months of age, but their surface area decreased progressively by 30-40% starting from â¼6 months of age. Although the size of the BK current decreased with age, IHCs retained a normal KCNQ4 current and resting membrane potential. These basolateral membrane changes were most severe for C57BL/6J and C57BL/6NTac, less so for C57BL/6NTacCdh23+ and minimal or absent in C3H/HeJ mice. We also found that lateral olivocochlear (LOC) efferent fibres re-form functional axon-somatic connections with aged IHCs, but this was seen only sporadically in C3H/HeJ mice. The efferent post-synaptic SK2 channels appear prior to the establishment of the efferent contacts, suggesting that IHCs may play a direct role in re-establishing the LOC-IHC synapses. Finally, we showed that the size of the mechanoelectrical transducer (MET) current from IHCs decreased significantly with age in mice harbouring the Cdh23ahl allele but not in C57BL/6NTacCdh23+ mice, indicating that the MET apparatus directly contributes to the progression of age-related hearing loss.
Asunto(s)
Células Ciliadas Auditivas Internas , Canales de Potasio de Gran Conductancia Activados por el Calcio , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
KEY POINTS: Age-related hearing loss (ARHL) is a very heterogeneous disease, resulting from cellular senescence, genetic predisposition and environmental factors (e.g. noise exposure). Currently, we know very little about age-related changes occurring in the auditory sensory cells, including those associated with the outer hair cells (OHCs). Using different mouse strains, we show that OHCs undergo several morphological and biophysical changes in the ageing cochlea. Ageing OHCs also exhibited the progressive loss of afferent and efferent synapses. We also provide evidence that the size of the mechanoelectrical transducer current is reduced in ageing OHCs, highlighting its possible contribution in cochlear ageing. ABSTRACT: Outer hair cells (OHCs) are electromotile sensory receptors that provide sound amplification within the mammalian cochlea. Although OHCs appear susceptible to ageing, the progression of the pathophysiological changes in these cells is still poorly understood. By using mouse strains with a different progression of hearing loss (C57BL/6J, C57BL/6NTac, C57BL/6NTacCdh23+ , C3H/HeJ), we have identified morphological, physiological and molecular changes in ageing OHCs (9-12 kHz cochlear region). We show that by 6 months of age, OHCs from all strains underwent a reduction in surface area, which was not a sign of degeneration. Although the ageing OHCs retained a normal basolateral membrane protein profile, they showed a reduction in the size of the K+ current and non-linear capacitance, a readout of prestin-dependent electromotility. Despite these changes, OHCs have a normal Vm and retain the ability to amplify sound, as distortion product otoacoustic emission thresholds were not affected in aged, good-hearing mice (C3H/HeJ, C57BL/6NTacCdh23+ ). The loss of afferent synapses was present in all strains at 15 months. The number of efferent synapses per OHCs, defined as postsynaptic SK2 puncta, was reduced in aged OHCs of all strains apart from C3H mice. Several of the identified changes occurred in aged OHCs from all mouse strains, thus representing a general trait in the pathophysiological progression of age-related hearing loss, possibly aimed at preserving functionality. We have also shown that the mechanoelectrical transduction (MET) current from OHCs of mice harbouring the Cdh23ahl allele is reduced with age, highlighting the possibility that changes in the MET apparatus could play a role in cochlear ageing.
Asunto(s)
Células Ciliadas Auditivas Externas , Emisiones Otoacústicas Espontáneas , Animales , Cadherinas , Cóclea , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
KEY POINTS: Age-related hearing loss (ARHL) is associated with the loss of inner hair cell (IHC) ribbon synapses, lower hearing sensitivity and decreased ability to understand speech, especially in a noisy environment. Little is known about the age-related physiological and morphological changes that occur at ribbon synapses. We show that the differing degrees of ARHL in four selected mouse stains is correlated with the loss of ribbon synapses, being most severe for the strains C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ -Repaired and lowest for C3H/HeJ. Despite the loss of ribbon synapses with age, the volume of the remaining ribbons increased and the size and kinetics of Ca2+ -dependent exocytosis in IHCs was unaffected, indicating the presence of a previously unknown degree of functional compensation at ribbon synapses. Although the age-related morphological changes at IHC ribbon synapses contribute to the different progression of ARHL, without the observed functional compensation hearing loss could be greater. ABSTRACT: Mammalian cochlear inner hair cells (IHCs) are specialized sensory receptors able to provide dynamic coding of sound signals. This ability is largely conferred by their ribbon synapses, which tether a large number of vesicles at the IHC's presynaptic active zones, allowing high rates of sustained synaptic transmission onto the afferent fibres. How the physiological and morphological properties of ribbon synapses change with age remains largely unknown. Here, we have investigated the biophysical and morphological properties of IHC ribbon synapses in the ageing cochlea (9-12 kHz region) of four mouse strains commonly used in hearing research: early-onset progressive hearing loss (C57BL/6J and C57BL/6NTac) and 'good hearing' strains (C57BL/6NTacCdh23+ and C3H/HeJ). We found that with age, both modiolar and pillar sides of the IHC exhibited a loss of ribbons, but there was an increased volume of those that remained. These morphological changes, which only occurred after 6 months of age, were correlated with the level of hearing loss in the different mouse strains, being most severe for C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ and absent for C3H/HeJ strains. Despite the age-related reduction in ribbon number in three of the four strains, the size and kinetics of Ca2+ -dependent exocytosis, as well as the replenishment of synaptic vesicles, in IHCs was not affected. The degree of vesicle release at the fewer, but larger, individual remaining ribbon synapses colocalized with the post-synaptic afferent terminals is likely to increase, indicating the presence of a previously unknown degree of functional compensation in the ageing mouse cochlea.
Asunto(s)
Cóclea , Células Ciliadas Auditivas Internas , Envejecimiento , Animales , Cadherinas , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , SinapsisRESUMEN
KEY POINTS: The physiological maturation of auditory hair cells and their innervation requires precise temporal and spatial control of cell differentiation. The transcription factor gata3 is essential for the earliest stages of auditory system development and for survival and synaptogenesis in auditory sensory afferent neurons. We show that during postnatal development in the mouse inner ear gata3 is required for the biophysical maturation, growth and innervation of inner hair cells; in contrast, it is required only for the survival of outer hair cells. Loss of gata3 in inner hair cells causes progressive hearing loss and accounts for at least some of the deafness associated with the human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. The results show that gata3 is critical for later stages of mammalian auditory system development where it plays distinct, complementary roles in the coordinated maturation of sensory hair cells and their innervation. ABSTRACT: The zinc finger transcription factor gata3 regulates inner ear development from the formation of the embryonic otic placode. Throughout development, gata3 is expressed dynamically in all the major cochlear cell types. Its role in afferent formation is well established but its possible involvement in hair cell maturation remains unknown. Here, we find that in heterozygous gata3 null mice (gata3+/- ) outer hair cells (OHCs) differentiate normally but their numbers are significantly lower. In contrast, inner hair cells (IHCs) survive normally but they fail to acquire adult basolateral membrane currents, retain pre-hearing current and efferent innervation profiles and have fewer ribbon synapses. Targeted deletion of gata3 driven by otoferlin-cre recombinase (gata3fl/fl otof-cre+/- ) in IHCs does not affect OHCs or the number of IHC afferent synapses but it leads to a failure in IHC maturation comparable to that observed in gata3+/- mice. Auditory brainstem responses in gata3fl/fl otof-cre+/- mice reveal progressive hearing loss that becomes profound by 6-7 months, whilst distortion product otoacoustic emissions are no different to control animals up to this age. Our results, alongside existing data, indicate that gata3 has specific, complementary functions in different cell types during inner ear development and that its continued expression in the sensory epithelium orchestrates critical aspects of physiological development and neural connectivity. Furthermore, our work indicates that hearing loss in human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome arises from functional deficits in IHCs as well as loss of function from OHCs and both afferent and efferent neurons.
Asunto(s)
Cóclea/metabolismo , Cóclea/fisiología , Factor de Transcripción GATA3/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/fisiología , Animales , Diferenciación Celular/fisiología , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/fisiología , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/fisiología , Audición/fisiología , Pérdida Auditiva/metabolismo , Pérdida Auditiva/fisiopatología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Ratones Transgénicos , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Sinapsis/metabolismoRESUMEN
Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca2+ signals from sensory and non-sensory cells.
Asunto(s)
Vías Aferentes , Canales de Calcio Tipo L/fisiología , Calcio/metabolismo , Cóclea/fisiología , Conexina 30/fisiología , Células Ciliadas Auditivas Externas/fisiología , Células Receptoras Sensoriales/fisiología , Potenciales de Acción , Animales , Señalización del Calcio , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores Purinérgicos P2X3/fisiología , Sinapsis/fisiologíaRESUMEN
Cell transplantation is an ambitious, but arguably realistic, therapy for repair of the nervous system. Cell delivery is a major challenge for clinical translation, especially given the apparently inhibitory astrogliotic environment in degenerated tissue. However, astrogliotic tissue also contains endogenous structural and biochemical cues that can be harnessed for functional repair. Minimizing damage to these cues during cell delivery could enhance cell integration. This theory is supported by studies with an auditory astrocyte scar model, in which cells delivered onto the surface of the damaged nerve were more successfully integrated in the host than those injected into the tissue. We consider the application of this less invasive approach for nerve injury and its potential application to some neurodegenerative disorders.
Asunto(s)
Trasplante de Células/métodos , Regeneración Nerviosa , Enfermedades Neurodegenerativas/terapia , Traumatismos del Sistema Nervioso/terapia , Animales , Humanos , Regeneración Nerviosa/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Traumatismos del Sistema Nervioso/fisiopatologíaRESUMEN
The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33). VOT-N33 cells expressing a Pax2-response element reporter were GFP-negative when untreated, but became GFP positive after forced differentiation or treatment with a potent LSD inhibitor. Pharmacological inhibition of LSD1 activity resulted in the enrichment of mono- and di-methylation of H3K4, upregulation of sensory neuronal genes and an increase in the number of sensory neurons in mouse inner ear organoids. Together, these results identify the LSD1/NuRD complex as a previously unrecognized modulator for Pax2-mediated neuronal differentiation in the inner ear.
Asunto(s)
Diferenciación Celular/fisiología , Oído Interno/citología , Histona Demetilasas/fisiología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Células-Madre Neurales/citología , Factor de Transcripción PAX2/metabolismo , Animales , Línea Celular , Oído Interno/metabolismo , Proteínas Fluorescentes Verdes/genética , Histona Demetilasas/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Unión ProteicaRESUMEN
In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.
Asunto(s)
Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Nociceptores/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Clonales , Ganglios Espinales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales de Sodio/metabolismo , Transfección , Veratridina/farmacologíaRESUMEN
Cell transplantation therapy has long been investigated as a therapeutic intervention for neurodegenerative disorders, including spinal cord injury, Parkinson's disease, and amyotrophic lateral sclerosis. Indeed, patients have high hopes for a cell-based therapy. However, there are numerous practical challenges for clinical translation. One major problem is that only very low numbers of donor cells survive and achieve functional integration into the host. Glial scar tissue in chronic neurodegenerative disorders strongly inhibits regeneration, and this inhibition must be overcome to accomplish successful cell transplantation. Intraneural cell transplantation is considered to be the best way to deliver cells to the host. We questioned this view with experiments in vivo on a rat glial scar model of the auditory system. Our results show that intraneural transplantation to the auditory nerve, preceded by chondroitinase ABC (ChABC)-treatment, is ineffective. There is no functional recovery, and almost all transplanted cells die within a few weeks. However, when donor cells are placed on the surface of a ChABC-treated gliotic auditory nerve, they autonomously migrate into it and recapitulate glia- and neuron-guided cell migration modes to repair the auditory pathway and recover auditory function. Surface transplantation may thus pave the way for improved functional integration of donor cells into host tissue, providing a less invasive approach to rescue clinically important neural tracts.
Asunto(s)
Proliferación Celular , Nervio Coclear/patología , Regeneración Nerviosa , Neuroglía/patología , Animales , Condroitina ABC Liasa/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Masculino , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapia , Ratas , Ratas Sprague-DawleyRESUMEN
Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca(2+) inflow through Ca(V)1.3 (L-type) Ca(2+) channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca(2+) currents in IHCs positioned at the middle turn (frequency â¼ 2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na(+) based extracellular solution), we found that the macroscopic Ca(2+) current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (Po: 0.024 in response to 500-ms depolarizing steps at â¼-18 mV). The value of Po was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca(2+) channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the Po and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca(2+) channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune Ca(V)1.3 Ca(2+) channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Cóclea/citología , Células Ciliadas Auditivas Internas/metabolismo , Animales , Exocitosis , Gerbillinae , Células Ciliadas Auditivas Internas/citología , Inmunohistoquímica , Iones/química , Potenciales de la Membrana , Técnicas de Placa-Clamp , Sodio/metabolismo , Sinapsis/metabolismoRESUMEN
Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a 'critical period' of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Neuronas Motoras/fisiología , Receptores Nicotínicos/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción , Animales , Cóclea/fisiología , Ratones , Ratones Noqueados , Receptores Nicotínicos/genética , Estereocilios , Sinaptotagmina II/genética , Sinaptotagmina II/fisiologíaRESUMEN
Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.
Asunto(s)
Células Ciliadas Auditivas/patología , Pérdida Auditiva/genética , Proteínas de Microfilamentos/deficiencia , Análisis de Varianza , Animales , Audiometría de Respuesta Evocada , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Microscopía Electrónica , Técnicas de Placa-ClampRESUMEN
The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.
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Potenciales de Acción/fisiología , Calcio/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Sinapsis/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Antibacterianos/farmacología , Doxiciclina/farmacología , Células Ciliadas Auditivas Internas/citología , Ratones , Ratones Transgénicos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Sinapsis/genéticaRESUMEN
Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca²âº entry through CaV1.3 Ca²âº channels. Despite the crucial role of CaV1.3 Ca²âº channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca²âº channel activity in adult gerbil IHCs. We found that Ca²âº channels are partially active at the IHC resting membrane potential (-60 mV). At -20 mV, the large majority (>70%) of Ca²âº channel first openings occurred with an estimated delay of about 50 µs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca²âº channels in IHCs showed a low mean open probability (0.21 at -20 mV), but this increased significantly (up to 0.91) when Ca²âº channel activity switched to a bursting modality. We propose that IHC Ca²âº channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca²âº channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse.
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Canales de Calcio Tipo L/fisiología , Células Ciliadas Auditivas Internas/fisiología , Sinapsis/fisiología , Animales , Gerbillinae , Técnicas In Vitro , Cinética , Sodio/fisiologíaRESUMEN
HYPOTHESIS: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. BACKGROUND: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. METHODS: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry. RESULTS: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. CONCLUSION: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.
Asunto(s)
Reprogramación Celular/fisiología , Células Ciliadas Auditivas/fisiología , Células Madre Mesenquimatosas/fisiología , Actinas/metabolismo , Células Madre Adultas/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Biomarcadores , Diferenciación Celular , Línea Celular , Separación Celular , Técnicas de Cocultivo , Dependovirus/genética , Vectores Genéticos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Ganglio Espiral de la Cóclea/citologíaRESUMEN
The mechanical properties of the mammalian organ of Corti determine its sensitivity to sound frequency and intensity, and the structure of supporting cells changes progressively with frequency along the cochlea. From the apex (low frequency) to the base (high frequency) of the guinea pig cochlea inner pillar cells decrease in length incrementally from 75-55 µm whilst the number of axial microtubules increases from 1,300-2,100. The respective values for outer pillar cells are 120-65 µm and 1,500-3,000. This correlates with a progressive decrease in the length of the outer hair cells from >100 µm to 20 µm. Deiters'cell bodies vary from 60-50 µm long with relatively little change in microtubule number. Their phalangeal processes reflect the lengths of outer hair cells but their microtubule numbers do not change systematically. Correlations between cell length, microtubule number and cochlear location are poor below 1 kHz. Cell stiffness was estimated from direct mechanical measurements made previously from isolated inner and outer pillar cells. We estimate that between 200 Hz and 20 kHz axial stiffness, bending stiffness and buckling limits increase, respectively,~3, 6 and 4 fold for outer pillar cells, ~2, 3 and 2.5 fold for inner pillar cells and ~7, 20 and 24 fold for the phalangeal processes of Deiters'cells. There was little change in the Deiters'cell bodies for any parameter. Compensating for effective cell length the pillar cells are likely to be considerably stiffer than Deiters'cells with buckling limits 10-40 times greater. These data show a clear relationship between cell mechanics and frequency. However, measurements from single cells alone are insufficient and they must be combined with more accurate details of how the multicellular architecture influences the mechanical properties of the whole organ.
Asunto(s)
Cobayas/anatomía & histología , Células Laberínticas de Soporte/citología , Animales , Fenómenos Biomecánicos , Tamaño de la Célula , Células Laberínticas de Soporte/fisiología , Células Laberínticas de Soporte/ultraestructura , Microscopía Electrónica de Transmisión , Microtúbulos/fisiología , Microtúbulos/ultraestructuraRESUMEN
Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+) action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca(V)1.3) Ca(2+) current (I(Ca)), IHCs also transiently express a large Na(+) current (I(Na)). We aimed to investigate the specific contribution of I(Na) to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I(Na) differ between low- and high-frequency IHCs. We show that I(Na) is highly temperature-dependent and activates at around -60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (-55 mV/-60 mV). However, in vivo the availability of I(Na) could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as -70 mV. When IHCs were held at -60 mV and I(Na) elicited using a simulated action potential as a voltage command, we found that I(Na) contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I(Na) is likely to be carried by the TTX-sensitive channel subunits Na(V)1.1 and Na(V)1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I(Na) in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.
Asunto(s)
Cóclea/fisiología , Células Ciliadas Auditivas Internas/fisiología , Activación del Canal Iónico , Canales de Sodio/fisiología , Potenciales de Acción , Animales , Inmunohistoquímica , RatasRESUMEN
Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.
Asunto(s)
Calcio/metabolismo , Cóclea/citología , Células Ciliadas Auditivas Internas/fisiología , Mecanotransducción Celular/fisiología , Potenciales de la Membrana/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Biofisica , Calcio/farmacología , Cóclea/crecimiento & desarrollo , Sulfato de Dihidroestreptomicina/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Endolinfa/metabolismo , Femenino , Glicinérgicos/farmacología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Mecanotransducción Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Técnicas de Placa-Clamp , Estimulación Física , Estricnina/farmacologíaRESUMEN
Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.