RESUMO
KCNQ4 is a voltage-gated K+ channel was reported to distribute over the basolateral surface of type 1 vestibular hair cell and/or inner surface of calyx and heminode of the vestibular nerve connected to the type 1 vestibular hair cells of the inner ear. However, the precise localization of KCNQ4 is still controversial and little is known about the vestibular phenotypes caused by KCNQ4 dysfunction or the specific role of KCNQ4 in the vestibular organs. To investigate the role of KCNQ4 in the vestibular organ, 6-g hypergravity stimulation for 24 h, which represents excessive mechanical stimulation of the sensory epithelium, was applied to p.W277S Kcnq4 transgenic mice. KCNQ4 was detected on the inner surface of calyx of the vestibular afferent in transmission electron microscope images with immunogold labelling. Vestibular function decrease was more severe in the Kcnq4p.W277S/p.W277S mice than in the Kcnq4+/+ and Kcnq4+/p.W277S mice after the stimulation. The vestibular function loss was resulted from the loss of type 1 vestibular hair cells, which was possibly caused by increased depolarization duration. Retigabine, a KCNQ activator, prevented hypergravity-induced vestibular dysfunction and hair cell loss. Patients with KCNQ4 mutations also showed abnormal clinical vestibular function tests. These findings suggest that KCNQ4 plays an essential role in calyx and afferent of type 1 vestibular hair cell preserving vestibular function against excessive mechanical stimulation.
Assuntos
Células Ciliadas Vestibulares , Canais de Potássio KCNQ , Camundongos Transgênicos , Animais , Canais de Potássio KCNQ/metabolismo , Canais de Potássio KCNQ/genética , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/patologia , Camundongos , Fenilenodiaminas/farmacologia , Carbamatos/farmacologia , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/fisiopatologiaRESUMO
Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.
Assuntos
Traumatismos por Explosões , Células Ciliadas Vestibulares , Gânglio Espiral da Cóclea , Animais , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/patologia , Ratos , Traumatismos por Explosões/prevenção & controle , Células Ciliadas Vestibulares/efeitos dos fármacos , Células Ciliadas Vestibulares/metabolismo , Masculino , Oxirredução , Ratos Sprague-Dawley , Cóclea/efeitos dos fármacos , Cóclea/patologia , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Estresse Oxidativo/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/prevenção & controle , Perda Auditiva Provocada por Ruído/patologiaRESUMO
Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.
Assuntos
Regeneração , Análise de Célula Única , Transcriptoma , Humanos , Animais , Regeneração/genética , Camundongos , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/citologia , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Orelha Interna/metabolismo , Orelha Interna/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Feminino , Perfilação da Expressão GênicaRESUMO
In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.
Assuntos
Células Ciliadas Auditivas Internas , Células Ciliadas Vestibulares , Animais , Camundongos , Células Ciliadas Auditivas Internas/metabolismo , Ácido Glutâmico/metabolismo , Audição/fisiologia , Células Ciliadas Vestibulares/metabolismo , Sinapses/metabolismo , Cóclea/metabolismo , Cálcio/metabolismoRESUMO
The mechanoelectrical transduction (MET) protein complex in the inner-ear hair cells is essential for hearing and balance perception. Calcium and integrin-binding protein 2 (CIB2) has been reported to be a component of MET complex, and loss of CIB2 completely abolishes MET currents in auditory hair cells, causing profound congenital hearing loss. However, loss of CIB2 does not affect MET currents in vestibular hair cells (VHCs) as well as general balance function. Here, we show that CIB2 and CIB3 act redundantly to regulate MET in VHCs, as MET currents are completely abolished in the VHCs of Cib2/Cib3 double knock-out mice of either sex. Furthermore, we show that Cib2 and Cib3 transcripts have complementary expression patterns in the vestibular maculae, and that they play different roles in stereocilia maintenance in VHCs. Cib2 transcripts are highly expressed in the striolar region, and knock-out of Cib2 affects stereocilia maintenance in striolar VHCs. In contrast, Cib3 transcripts are highly expressed in the extrastriolar region, and knock-out of Cib3 mainly affects stereocilia maintenance in extrastriolar VHCs. Simultaneous knock-out of Cib2 and Cib3 affects stereocilia maintenance in all VHCs and leads to severe balance deficits. Taken together, our present work reveals that CIB2 and CIB3 are important for stereocilia maintenance as well as MET in mouse VHCs.SIGNIFICANCE STATEMENT Calcium and integrin-binding protein 2 (CIB2) is an important component of mechanoelectrical transduction (MET) complex, and loss of CIB2 completely abolishes MET in auditory hair cells. However, MET is unaffected in Cib2 knock-out vestibular hair cells (VHCs). In the present work, we show that CIB3 could compensate for the loss of CIB2 in VHCs, and Cib2/Cib3 double knock-out completely abolishes MET in VHCs. Interestingly, CIB2 and CIB3 could also regulate VHC stereocilia maintenance in a nonredundant way. Cib2 and Cib3 transcripts are highly expressed in the striolar and extrastriolar regions, respectively. Stereocilia maintenance and balance function are differently affected in Cib2 or Cib3 knock-out mice. In conclusion, our data suggest that CIB2 and CIB3 are important for stereocilia maintenance and MET in mouse VHCs.
Assuntos
Células Ciliadas Vestibulares , Animais , Camundongos , Cálcio/metabolismo , Células Ciliadas Vestibulares/metabolismo , Integrinas , Camundongos Knockout , Estereocílios/metabolismoRESUMO
Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.
Assuntos
Células Ciliadas Vestibulares , Receptores Nicotínicos , Vestíbulo do Labirinto , Humanos , Camundongos , Animais , Idoso , Camundongos Endogâmicos C57BL , Vestíbulo do Labirinto/metabolismo , Células Ciliadas Vestibulares/metabolismo , Colinérgicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
Sox2 is a transcription factor that is necessary in the mammalian inner ear for development of sensory hair cells and supporting cells. Sox2 is expressed in supporting cells of adult mammals, but its function in this context is poorly understood. Given its role in the developing inner ear, we hypothesized that Sox2 is required in vestibular supporting cells for regeneration of type II hair cells after damage. Using adult mice, we deleted Sox2 from Sox9-CreER-expressing supporting cells prior to diphtheria toxin-mediated hair cell destruction and used fate-mapping to assess regeneration. In utricles of control mice with normal Sox2 expression, supporting cells regenerated nearly 200 hair cells by 3 weeks post-damage, which doubled by 12 weeks. In contrast, mice with Sox2 deletion from supporting cells had approximately 20 fate-mapped hair cells at 3 weeks post-damage, and this number did not change significantly by 12 weeks, indicating regeneration was dramatically curtailed. We made similar observations for saccules and ampullae. We found no evidence that supporting cells lacking Sox2 had altered cellular density, morphology, or ultrastructure. However, some Sox2-negative supporting cell nuclei appeared to migrate apically but did not turn on hair cell markers, and type I hair cell survival was higher. Sox2 heterozygotes also had reduced regeneration in utricles, but more hair cells were replaced than mice with Sox2 deletion. Our study determined that Sox2 is required in supporting cells for normal levels of vestibular hair cell regeneration but found no other major requirements for Sox2 in adult supporting cells.
Assuntos
Células Ciliadas Vestibulares , Fatores de Transcrição SOXB1 , Animais , Camundongos , Regulação da Expressão Gênica , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/fisiologia , Mamíferos , Regeneração , Sáculo e Utrículo , Fatores de Transcrição SOXB1/metabolismoRESUMO
Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanoelectrical transducer (MET) channel in auditory hair cells. Using single-channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. All mutations reduced the Ca2+ permeability of the MET channel, triggering hair cell apoptosis and deafness. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance, whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. Tmc1 p.M412K and Tmc1 p.T416K reduced only the Ca2+ permeability. The consequences of these mutations endorse TMC1 as the pore of the MET channel. The accessory subunits, LHFPL5 and TMIE, are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in outer hair cells of Lhfpl5 and Tmie knockout mice to determine the properties of the channels, which could still be gated by hair bundle displacement. Single-channel conductance was unaffected in Lhfpl5-/- but was reduced in Tmie-/-, implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5-/- were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel. Based on counts of numbers of stereocilia per bundle, we estimate that each PCDH15 and LHFPL5 monomer may contact two channels irrespective of location.
Assuntos
Células Ciliadas Vestibulares , Mecanotransdução Celular , Animais , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Mecanotransdução Celular/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Estereocílios/metabolismoRESUMO
A harmonized interplay between the central nervous system and the five peripheral end organs is how the vestibular system helps organisms feel a sense of balance and motion in three-dimensional space. The receptor cells of this system, much like their cochlear equivalents, are the specialized hair cells. However, research over the years has shown that the vestibular end organs and hair cells evolved very differently from their cochlear counterparts. The structurally unique calyceal synapse, which appeared much later in the evolutionary time scale, and continues to intrigue researchers, is now known to support several forms of synaptic neurotransmission. The conventional quantal transmission is believed to employ the ribbon structures, which carry several tethered vesicles filled with neurotransmitters. However, the field of vestibular hair cell synaptic molecular anatomy is still at a nascent stage and needs further work. In this review, we will touch upon the basic structure and function of the peripheral vestibular system, with the focus on the various modes of neurotransmission at the type I vestibular hair cells. We will also shed light on the current knowledge about the molecular anatomy of the vestibular hair cell synapses and vestibular synaptopathy.
Assuntos
Células Ciliadas Vestibulares , Cóclea , Células Ciliadas Vestibulares/metabolismo , Neurotransmissores , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin's structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin's contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin's chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.
Assuntos
Microscopia Crioeletrônica , Células Ciliadas Auditivas Externas/metabolismo , Transportadores de Sulfato/química , Animais , Proteínas de Transporte de Ânions , Sítios de Ligação , Células CHO , Cricetulus , Gerbillinae , Células Ciliadas Vestibulares/metabolismo , Transporte de Íons , Proteínas de Membrana/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Domínios ProteicosRESUMO
Loss of hair cells from vestibular epithelium results in balance dysfunction. The current therapeutic regimen for vestibular diseases is limited. Upon injury or Atoh1 overexpression, hair cell replacement occurs rapidly in the mammalian utricle, suggesting a promising approach to induce vestibular hair cell regeneration. In this study, we applied simultaneous gentamicin-mediated hair cell ablation and Atoh1 overexpression to induce neonatal utricular hair cell formation in vitro. We confirmed that type I hair cells were the primary targets of gentamicin. Furthermore, injury and Atoh1 overexpression promoted hair cell regeneration in a timely and efficient manner through robust viral transfection. Hair cells regenerated with type II characteristics in the striola and type I/II characteristics in non-sensory regions. Rare EdU+/myosin7a+ cells in sensory regions and robust EdU+/myosin7a+ signals in ectopic regions indicate that transdifferentiation of supporting cells in situ, and mitosis and differentiation of non-sensory epithelial cells in ectopic regions, are sources of regenerative hair cells. Distinct regeneration patterns in in situ and ectopic regions suggested robust plasticity of vestibular non-sensory epithelium, generating more developed hair cell subtypes and thus providing a promising stem cell-like source of hair cells. These findings suggest that simultaneously causing injury and overexpressing Atoh1 promotes hair cell regeneration efficacy and maturity, thus expanding the understanding of ectopic plasticity in neonatal vestibular organs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Gentamicinas/farmacologia , Células Ciliadas Vestibulares/efeitos dos fármacos , Sáculo e Utrículo/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/patologiaRESUMO
In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/ultraestrutura , Mecanotransdução Celular , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ratos , Sinapses/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/ultraestruturaRESUMO
Current clinical interest lies in the relationship between hearing loss and cognitive impairment. Previous work demonstrated that noise exposure, a common cause of sensorineural hearing loss (SNHL), leads to cognitive impairments in mice. However, in noise-induced models, it is difficult to distinguish the effects of noise trauma from subsequent SNHL on central processes. Here, we use cochlear hair cell ablation to isolate the effects of SNHL. Cochlear hair cells were conditionally and selectively ablated in mature, transgenic mice where the human diphtheria toxin (DT) receptor was expressed behind the hair-cell specific Pou4f3 promoter. Due to higher Pou4f3 expression in cochlear hair cells than vestibular hair cells, administration of a low dose of DT caused profound SNHL without vestibular dysfunction and had no effect on wild-type (WT) littermates. Spatial learning/memory was assayed using an automated radial 8-arm maze (RAM), where mice were trained to find food rewards over a 14-day period. The number of working memory errors (WME) and reference memory errors (RME) per training day were recorded. All animals were injected with DT during P30-60 and underwent the RAM assay during P90-120. SNHL animals committed more WME and RME than WT animals, demonstrating that isolated SNHL affected cognitive function. Duration of SNHL (60 versus 90 days post DT injection) had no effect on RAM performance. However, younger age of acquired SNHL (DT on P30 versus P60) was associated with fewer WME. This describes the previously undocumented effect of isolated SNHL on cognitive processes that do not directly rely on auditory sensory input.
Assuntos
Células Ciliadas Auditivas/fisiologia , Memória/fisiologia , Aprendizagem Espacial/fisiologia , Animais , Cognição/fisiologia , Surdez/metabolismo , Surdez/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/fisiologia , Audição/fisiologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/fisiopatologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ruído , Fator de Transcrição Brn-3C/metabolismoRESUMO
Cochlear hair cells in human beings cannot regenerate after loss; however, those in fish and other lower species can. Recently, the role of inflammation in hair cell regeneration has been attracting the attention of scientists. In the present study, we investigated how suppression of inflammatory factors affects hair cell regeneration and the functional recovery of regenerated hair cells in zebrafish. We killed hair cells in the lateral line of zebrafish larvae with CuSO4 to induce an inflammatory response and coapplied BRS-28, an anti-inflammatory agent to suppress the inflammation. The recovery of the hair cell number and rheotaxis was slower when CuSO4 and BRS-28 were coapplied than when CuSO4 was applied alone. The recovery of hair cell count lagged behind that of the calcium imaging signal during the regeneration. The calcium imaging signal in the neuromasts in the inflammation-inhibited group was weaker than that in the noninflammation-inhibited group at the early stage of regeneration, although it returned to normal at the late stage. Our study demonstrates that suppressing inflammation by BRS-28 delays hair cell regeneration and functional recovery when hair cells are damaged. We suspect that BRS-28 inhibits pro-inflammatory factors and thereby reduces the migration of macrophages to delay the regeneration of hair cells.
Assuntos
Células Ciliadas Vestibulares/citologia , Inflamação/genética , Regeneração/genética , Peixe-Zebra/genética , Animais , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sulfato de Cobre/farmacologia , Células Ciliadas Vestibulares/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Larva/genética , Larva/crescimento & desenvolvimento , Sistema da Linha Lateral/crescimento & desenvolvimento , Sistema da Linha Lateral/patologia , Macrófagos , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. Although analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.
Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Células Ciliadas Vestibulares/metabolismo , Sáculo e Utrículo/embriologia , Animais , Fator 8 de Crescimento de Fibroblasto/genética , Células Ciliadas Vestibulares/citologia , Camundongos , Camundongos Knockout , Sáculo e Utrículo/citologiaRESUMO
Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron-dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non-inactivating CaV 1.3 (L-type) Ca2+ channels in response to cell depolarization causes a local increase in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. The Ca2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca2+ sensor synaptotagmin-4 (Syt-4). Therefore, we studied the exocytotic Ca2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt-4 knockout mice using patch-clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high-order dependence on Ca2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt-4. However, we did find that Syt-4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low-frequency vestibular information, consistent with that observed at low-frequency mammalian auditory receptors.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Transmissão Sináptica , Sinaptotagminas/genética , Animais , Exocitose , Camundongos , Camundongos Knockout , Modelos Animais , Técnicas de Patch-Clamp/métodos , Sinaptotagminas/metabolismoRESUMO
Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell's soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.
Assuntos
Actinas/análise , Células Ciliadas Auditivas/citologia , Células Ciliadas Vestibulares/citologia , Estereocílios/ultraestrutura , Actinas/metabolismo , Animais , Células Ciliadas Auditivas/química , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Células Ciliadas Vestibulares/química , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/ultraestrutura , Audição , Humanos , Mecanotransdução Celular , Equilíbrio Postural , Estereocílios/química , Estereocílios/metabolismoRESUMO
Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link is expected to anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm, and the apparent filament diameter decreased from 6.4 to 4.8 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Células Ciliadas Vestibulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/genética , Animais , Glicoproteínas de Membrana/genética , Camundongos , Proteínas dos Microfilamentos/genéticaRESUMO
It has been over 60 years since peripheral efferent vestibular terminals were first identified in mammals, and yet the function of the efferent vestibular system remains obscure. One reason for the lack of progress may be due to our deficient understanding of the peripheral efferent synapse. Although vestibular efferent terminals were identified as cholinergic less than a decade after their anatomical characterization, the cellular mechanisms that underlie the properties of these synapses have had to be inferred. In this review we examine how recent mammalian studies have begun to reveal both nicotinic and muscarinic effects at these terminals and therefore provide a context for fast and slow responses observed in classic electrophysiological studies of the mammalian efferent vestibular system, nearly 40 years ago. Although incomplete, these new results together with those of recent behavioral studies are helping to unravel the mysterious and perplexing action of the efferent vestibular system. Armed with this information, we may finally appreciate the behavioral framework in which the efferent vestibular system operates.
Assuntos
Acetilcolina/metabolismo , Células Ciliadas Vestibulares/fisiologia , Neurônios Eferentes/fisiologia , Receptores Colinérgicos/metabolismo , Transmissão Sináptica/fisiologia , Nervo Vestibular/fisiologia , Animais , Células Ciliadas Vestibulares/metabolismo , Neurônios Eferentes/metabolismo , Nervo Vestibular/metabolismoRESUMO
The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.