RESUMEN
Impairments of vision and hearing are highly prevalent conditions limiting the quality of life and presenting a major socioeconomic burden. For a long time, retinal and cochlear disorders have remained intractable for causal therapies, with sensory rehabilitation limited to glasses, hearing aids, and electrical cochlear or retinal implants. Recently, the application of gene therapy and optogenetics to eye and ear has generated hope for a fundamental improvement of vision and hearing restoration. To date, one gene therapy for the restoration of vision has been approved, and ongoing clinical trials will broaden its application including gene replacement, genome editing, and regenerative approaches. Moreover, optogenetics, i.e., controlling the activity of cells by light, offers a more general alternative strategy. Over little more than a decade, optogenetic approaches have been developed and applied to better understand the function of biological systems, while protein engineers have identified and designed new opsin variants with desired physiological features. Considering potential clinical applications of optogenetics, the spotlight is on the sensory systems, particularly the eye and ear. Multiple efforts have been undertaken to restore lost or hampered function in the eye and ear. Optogenetic stimulation promises to overcome fundamental shortcomings of electrical stimulation, namely, poor spatial resolution and cellular specificity, and accordingly to deliver more detailed sensory information. This review aims to provide a comprehensive reference on current gene therapeutic and optogenetic research relevant to the restoration of hearing and vision. We will introduce gene-therapeutic approaches and discuss the biotechnological and optoelectronic aspects of optogenetic hearing and vision restoration.
Asunto(s)
Pérdida Auditiva/terapia , Trastornos de la Visión/terapia , Humanos , Optogenética , Prótesis VisualesRESUMEN
Our sense of hearing enables the processing of stimuli that differ in sound pressure by more than six orders of magnitude. How to process a wide range of stimulus intensities with temporal precision is an enigmatic phenomenon of the auditory system. Downstream of dynamic range compression by active cochlear micromechanics, the inner hair cells (IHCs) cover the full intensity range of sound input. Yet, the firing rate in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it. As a population, spiral ganglion neurons with their respective individual coding fractions cover the entire audible range. How such "dynamic range fractionation" arises is a topic of current research and the focus of this review. Here, we discuss mechanisms for generating the diverse functional properties of SGNs and formulate testable hypotheses. We postulate that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound information and thus contributes to a population code for sound intensity.
Asunto(s)
Cóclea , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Internas/fisiología , Sonido , Sinapsis/fisiología , Ganglio Espiral de la CócleaRESUMEN
In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.
Asunto(s)
Células Ciliadas Auditivas Internas , Proteómica , Ratones , Animales , Células Ciliadas Auditivas Internas/metabolismo , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Auditory synaptopathy/neuropathy (AS/AN) is a distinct type of sensorineural hearing loss in which the cochlear sensitivity to sound (i.e. active cochlear amplification by outer hair cells) is preserved whereas sound encoding by inner hair cells and/or auditory nerve fibers is disrupted owing to genetic or environmental factors. Autosomal-dominant auditory neuropathy type 2 (AUNA2) was linked either to chromosomal bands 12q24 or 13q34 in a large German family in 2017. By whole-genome sequencing, we now detected a 5500 bp deletion in ATP11A on chromosome 13q34 segregating with the phenotype in this family. ATP11A encodes a P-type ATPase that translocates phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane. The deletion affects both isoforms of ATP11A and activates a cryptic splice site leading to the formation of an alternative last exon. ATP11A carrying the altered C-terminus loses its flippase activity for phosphatidylserine. Atp11a is expressed in fibers and synaptic contacts of the auditory nerve and in the cochlear nucleus in mice, and conditional Atp11a knockout mice show a progressive reduction of the spiral ganglion neuron compound action potential, recapitulating the human phenotype of AN. By combining whole-genome sequencing, immunohistochemistry, in vitro functional assays and generation of a mouse model, we could thus identify a partial deletion of ATP11A as the genetic cause of AUNA2.
Asunto(s)
Pérdida Auditiva Central , Pérdida Auditiva Sensorineural , Humanos , Ratones , Animales , Pérdida Auditiva Central/genética , Pérdida Auditiva Sensorineural/genética , Mutación , Células Ciliadas Auditivas Internas , Cromosomas , Transportadoras de Casetes de Unión a ATP/genéticaRESUMEN
Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.
Asunto(s)
Células Ciliadas Auditivas Internas , Neuropéptidos , Ratas , Animales , Audición/fisiología , Sinapsis/fisiología , Cóclea , Ganglio Espiral de la Cóclea/metabolismo , Proteínas del Citoesqueleto/metabolismoRESUMEN
BACKGROUND: Proper connectivity between type I spiral ganglion neurons (SGNs) and inner hair cells (IHCs) in the cochlea is necessary for conveying sound information to the brain in mammals. Previous studies have shown that type I SGNs are heterogeneous in form, function and synaptic location on IHCs, but factors controlling their patterns of connectivity are not well understood. RESULTS: During development, cochlear supporting cells and SGNs express Semaphorin-3A (SEMA3A), a known axon guidance factor. Mice homozygous for a point mutation that attenuates normal SEMA3A repulsive activity (Sema3aK108N ) show cochleae with grossly normal patterns of IHC innervation. However, genetic sparse labeling and three-dimensional reconstructions of individual SGNs show that cochleae from Sema3aK108N mice lacked the normal synaptic distribution of type I SGNs. Additionally, Sema3aK108N cochleae show a disrupted distribution of GLUA2 postsynaptic patches around the IHCs. The addition of SEMA3A-Fc to postnatal cochleae led to increases in SGN branching, similar to the effects of inhibiting glutamate receptors. Ca2+ imaging studies show that SEMA3A-Fc decreases SGN activity. CONCLUSIONS: Contrary to the canonical view of SEMA3A as a guidance ligand, our results suggest SEMA3A may regulate SGN excitability in the cochlea, which may influence the morphology and synaptic arrangement of type I SGNs.
Asunto(s)
Células Ciliadas Auditivas , Semaforina-3A , Animales , Ratones , Cóclea/metabolismo , Neuronas/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.
Asunto(s)
Aciltransferasas/fisiología , Calcio/metabolismo , Exocitosis/fisiología , Células Ciliadas Auditivas/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Vesículas Sinápticas/fisiología , Animales , Cóclea/citología , Cóclea/fisiología , Endocitosis , Femenino , Células Ciliadas Auditivas/citología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transmisión SinápticaRESUMEN
We obtain the total impulse in the scattering of nonspinning binaries in general relativity at fourth post-Minkowskian order, i.e., O(G^{4}), including linear, nonlinear, and hereditary radiation-reaction effects. We derive the total radiated spacetime momentum as well as the associated energy flux. The latter can be used to compute gravitational-wave observables for generic (un)bound orbits. We employ the ("in-in") Schwinger-Keldysh worldline effective field theory framework in combination with modern "multiloop" integration techniques from collider physics. The complete results are in agreement with various partial calculations in the post-Minkowskian and post-Newtonian expansion.
RESUMEN
The mammalian cochlea is a snail-shaped structure deeply that is embedded in the temporal bone and harbors the auditory sensory epithelium - the organ of Corti. Since the discovery of this remarkable hearing organ in the middle of the 19th century, generations of anatomists and physiologists have been attracted to study the structural and functional details of this intricate and delicate structure and thereby contributed to establishing our current understanding of peripheral sound encoding. Since these early days, the continued development of novel imaging technologies - both on light and electron microscopic level - has driven the auditory research field and now enables the visualization of cochlear structures across multiple scales with unprecedented clarity and exquisite detail. To honor these achievements, this review aims to provide a concise overview of current multi-scale imaging methodologies to investigate cochlear anatomy and cellular function in the peripheral auditory pathway. For this purpose, we will outline the technological concepts underlying these techniques - ranging from label-free to label-containing approaches - highlight their respective strengths and limitations and provide specific examples of their use in modern auditory research. We will focus on traditional as well as less conventional imaging techniques that present essential tools for unraveling the protein composition, nanoscale assembly, and physiology of the first auditory synapse and associated structures. In addition, we will introduce novel non-invasive large-scale methodologies that allow for high-resolution in situ imaging of the structurally-unperturbed cochlea and point out potential future applications. In combination, these techniques allow for a comprehensive multi-scale analysis of cochlear structure and function.
Asunto(s)
Cóclea , Audición , Animales , Audición/fisiología , Mamíferos , SinapsisRESUMEN
Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2µ (AP-2µ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2µ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2µ-deficient IHCs, indicating a further role of AP-2µ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/fisiología , Células Ciliadas Auditivas/fisiología , Vesículas Sinápticas/metabolismo , Potenciales de Acción , Animales , Potenciales Evocados Auditivos del Tronco Encefálico , Audición , Ratones Endogámicos C57BL , Ratones Transgénicos , Sinapsis/fisiología , Transmisión SinápticaRESUMEN
Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.
Asunto(s)
Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de la Membrana/genética , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Tomografía con Microscopio Electrónico , Exocitosis/fisiología , Femenino , Células Ciliadas Auditivas Internas/citología , Audición/genética , Audición/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Técnicas de Placa-ClampRESUMEN
Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was â¼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.
Asunto(s)
Membrana Celular/metabolismo , Clatrina/fisiología , Dinamina I/fisiología , Exocitosis/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Hidrazonas/farmacología , Naftoles/farmacología , Animales , Membrana Celular/efectos de los fármacos , Dinamina I/antagonistas & inhibidores , Dinamina I/genética , Exocitosis/efectos de los fármacos , Femenino , Células Ciliadas Auditivas Internas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Missense/fisiología , Órgano Espiral/citología , Órgano Espiral/metabolismoRESUMEN
Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.
Asunto(s)
Cóclea/citología , Cóclea/crecimiento & desarrollo , Células Ciliadas Auditivas/fisiología , Mecanorreceptores/fisiología , Proteínas de la Membrana/genética , Síndromes de Usher/genética , Estimulación Acústica , Factores de Edad , Oxidorreductasas de Alcohol/metabolismo , Animales , Animales Recién Nacidos , Asparagina/genética , Bario/farmacología , Fenómenos Biofísicos/genética , Cadherinas/genética , Línea Celular Transformada , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas/ultraestructura , Humanos , Lisina/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo/métodos , Mutación/genética , Fibras Nerviosas/patología , Fibras Nerviosas/ultraestructura , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Estimulación Física/métodos , Psicoacústica , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Receptores AMPA/metabolismo , Sinapsis/patología , Sinapsis/ultraestructura , Transfección , Síndromes de Usher/patología , Síndromes de Usher/fisiopatologíaRESUMEN
Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.
Asunto(s)
Exocitosis/fisiología , Proteínas de la Membrana/fisiología , Sinaptotagmina I/fisiología , Estimulación Acústica/métodos , Animales , Animales Recién Nacidos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Exocitosis/genética , Hipocampo/citología , Hipocampo/fisiología , Fusión de Membrana/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Inhibición Neural/genética , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Sinapsis/genética , Sinapsis/fisiología , Sinaptotagmina I/deficiencia , Sinaptotagmina I/genéticaRESUMEN
Hearing impairment is one of the most common disorders with a global burden and increasing prevalence in an ever-aging population. Previous research has largely focused on peripheral sensory perception, while the brain circuits of auditory processing and integration remain poorly understood. Mutations in the rdx gene, encoding the F-actin binding protein radixin (Rdx), can induce hearing loss in human patients and homozygous depletion of Rdx causes deafness in mice. However, the precise physiological function of Rdx in hearing and auditory information processing is still ill-defined. Here, we investigated consequences of rdx monoallelic loss in the mouse. Unlike the homozygous (-/-) rdx knockout, which is characterized by the degeneration of actin-based stereocilia and subsequent hearing loss, our analysis of heterozygous (+/-) mutants has revealed a different phenotype. Specifically, monoallelic loss of rdx potentiated the startle reflex in response to acoustic stimulation of increasing intensities, suggesting a gain of function relative to wildtype littermates. The monoallelic loss of the rdx gene also facilitated pre-pulse inhibition of the acoustic startle reflex induced by weak auditory pre-pulse stimuli, indicating a modification to the circuit underlying sensorimotor gating of auditory input. However, the auditory brainstem response (ABR)-based hearing thresholds revealed a mild impairment in peripheral sound perception in rdx (+/-) mice, suggesting minor aberration of stereocilia structural integrity. Taken together, our data suggest a critical role of Rdx in the top-down processing and/or integration of auditory signals, and therefore a novel perspective to uncover further Rdx-mediated mechanisms in central auditory information processing.
RESUMEN
Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology research indicate that neurotransmission depends on such optimization strategies. However, the molecular topography of the presynaptic active zone (AZ), where transmitter is released upon synaptic vesicle (SV) fusion, remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. This was facilitated by a novel sample immobilization technique that we name heat-assisted rapid dehydration (HARD), wherein a thin layer of rod synaptic terminals (spherules) was transferred onto glass coverslips from fresh retinal slices. Rod ribbon AZs were readily immunolabeled and imaged in 3D with a precision of a few nanometers. Our 3D-MINFLUX results indicate that the SV release site in rods is a molecular complex of bassoon-RIM2-ubMunc13-2-Cav1.4, which repeats longitudinally on both sides of the ribbon.
RESUMEN
Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.
Asunto(s)
Tomografía con Microscopio Electrónico , Optogenética , Ratones , Animales , Sinapsis/fisiología , Vesículas Sinápticas/ultraestructura , Células Ciliadas Auditivas Internas/fisiología , Exocitosis/fisiologíaRESUMEN
Recent studies reveal great diversity in the structure, function, and efferent innervation of afferent synaptic connections between the cochlear inner hair cells (IHCs) and spiral ganglion neurons (SGNs), which likely enables audition to process a wide range of sound pressures. By performing an extensive electron microscopic (EM) reconstruction of the neural circuitry in the mature mouse organ of Corti, we demonstrate that afferent SGN dendrites differ in abundance and composition of efferent innervation in a manner dependent on their afferent synaptic connectivity with IHCs. SGNs that sample glutamate release from several presynaptic ribbons receive more efferent innervation from lateral olivocochlear projections than those driven by a single ribbon. Next to the prevailing unbranched SGN dendrites, we found branched SGN dendrites that can contact several ribbons of 1-2 IHCs. Unexpectedly, medial olivocochlear neurons provide efferent innervation of SGN dendrites, preferring those forming single-ribbon, pillar-side synapses. We propose a fine-tuning of afferent and efferent SGN innervation.
Asunto(s)
Cóclea/citología , Dendritas/ultraestructura , Células Ciliadas Auditivas Internas/citología , Vías Nerviosas/citología , Neuronas/citología , Ganglio Espiral de la Cóclea/citología , Sinapsis/ultraestructura , Animales , Femenino , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Electrónica , Órgano Espiral/citologíaRESUMEN
Hearing relies on Ca(2+) influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca(2+) channel subunit Ca(V)beta(2) in hearing. Of the Ca(V)beta(1-4) mRNAs, IHCs predominantly contained Ca(V)beta(2). Hearing was severely impaired in mice lacking Ca(V)beta(2) in extracardiac tissues (Ca(V)beta(2)(-/-)). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in Ca(V)beta(2)(-/-) mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. Ca(V)beta(2)(-/-) IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing Ca(V)1.3 channels. Confocal Ca(2+) imaging revealed presynaptic Ca(2+) microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer Ca(V)1.3 channels. The coupling of the remaining Ca(2+) influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the Ca(V)beta(2)(-/-) mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in Ca(V)beta(2)(-/-) mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic Ca(V)1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca(2+) activated potassium channels in IHCs were impaired in the absence of Ca(V)beta(2). Moreover, we found the developmental efferent innervation to persist in Ca(V)beta(2)-deficient IHCs. In summary, Ca(V)beta(2) has an essential role in regulating the abundance and properties of Ca(V)1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.