RESUMO
Inner hair cells (IHCs) of the cochlea use ribbon synapses to transmit auditory information faithfully to spiral ganglion neurons (SGNs). In the present study, we used genetic disruption of the presynaptic scaffold protein bassoon in mice to manipulate the morphology and function of the IHC synapse. Although partial-deletion mutants lacking functional bassoon (Bsn(ΔEx4/5)) had a near-complete loss of ribbons from the synapses (up to 88% ribbonless synapses), gene-trap mutants (Bsn(gt)) showed weak residual expression of bassoon and 56% ribbonless synapses, whereas the remaining 44% had a loosely anchored ribbon. Patch-clamp recordings and synaptic CaV1.3 immunolabeling indicated a larger number of Ca(2+) channels for Bsn(gt) IHCs compared with Bsn(ΔEx4/5) IHCs and for Bsn(gt) ribbon-occupied versus Bsn(gt) ribbonless synapses. An intermediate phenotype of Bsn(gt) IHCs was also found by membrane capacitance measurements for sustained exocytosis, but not for the size of the readily releasable vesicle pool. The frequency and amplitude of EPSCs were reduced in Bsn(ΔEx4/5) mouse SGNs, whereas their postsynaptic AMPA receptor clusters were largely unaltered. Sound coding in SGN, assessed by recordings of single auditory nerve fibers and their population responses in vivo, was similarly affected in Bsn(gt) and Bsn(ΔEx4/5) mice. Both genotypes showed impaired sound onset coding and reduced evoked and spontaneous spike rates. In summary, reduced bassoon expression or complete lack of full-length bassoon impaired sound encoding to a similar extent, which is consistent with the comparable reduction of the readily releasable vesicle pool. This suggests that the remaining loosely anchored ribbons in Bsn(gt) IHCs were functionally inadequate or that ribbon independent mechanisms dominated the coding deficit.
Assuntos
Cóclea/citologia , Células Ciliadas Auditivas Internas/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Sinapses/fisiologia , Estimulação Acústica , Acústica , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Oxirredutases do Álcool , Animais , Limiar Auditivo/fisiologia , Biofísica , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ácido Egtázico/farmacologia , Estimulação Elétrica , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Emissões Otoacústicas Espontâneas/genética , Técnicas de Patch-Clamp , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismoRESUMO
Sound coding at hair cell ribbon synapses is tightly regulated by Ca(2+). Here, we used patch-clamp, fast confocal Ca(2+) imaging and modeling to characterize synaptic Ca(2+) signaling in cochlear inner hair cells (IHCs) of hearing mice. Submicrometer fluorescence hotspots built up and collapsed at the base of IHCs within a few milliseconds of stimulus onset and cessation. They most likely represented Ca(2+) microdomains arising from synaptic Ca(2+) influx through Ca(V)1.3 channels. Synaptic Ca(2+) microdomains varied substantially in amplitude and voltage dependence even within single IHCs. Testing putative mechanisms for the heterogeneity of Ca(2+) signaling, we found the amplitude variability unchanged when blocking mitochondrial Ca(2+) uptake or Ca(2+)-induced Ca(2+) release, buffering cytosolic Ca(2+) by millimolar concentrations of EGTA, or elevating the Ca(2+) channel open probability by the dihydropyridine agonist BayK8644. However, we observed substantial variability also for the fluorescence of immunolabeled Ca(V)1.3 Ca(2+) channel clusters. Moreover, the Ca(2+) microdomain amplitude correlated positively with the size of the corresponding synaptic ribbon. Ribbon size, previously suggested to scale with the number of synaptic Ca(2+) channels, was approximated by using fluorescent peptide labeling. We propose that IHCs adjust the number and the gating of Ca(V)1.3 channels at their active zones to diversify their transmitter release rates.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio , Células Ciliadas Auditivas Internas/fisiologia , Transmissão Sináptica , Animais , Citosol , Audição/fisiologia , Camundongos , MitocôndriasRESUMO
Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.
Assuntos
Vias Auditivas/fisiologia , Percepção Auditiva/fisiologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/metabolismo , Potenciais de Ação , Animais , Cálcio/metabolismo , Cóclea/citologia , Endocitose , Exocitose , Audição/fisiologia , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Complexins (CPXs I-IV) presumably act as regulators of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, but their function in the intact mammalian nervous system is not well established. Here, we explored the role of CPXs in the mouse auditory system. Hearing was impaired in CPX I knock-out mice but normal in knock-out mice for CPXs II, III, IV, and III/IV as measured by auditory brainstem responses. Complexins were not detectable in cochlear hair cells but CPX I was expressed in spiral ganglion neurons (SGNs) that give rise to the auditory nerve. Ca(2+)-dependent exocytosis of inner hair cells and sound encoding by SGNs were unaffected in CPX I knock-out mice. In the absence of CPX I, the resting release probability in the endbulb of Held synapses of the auditory nerve fibers with bushy cells in the cochlear nucleus was reduced. As predicted by computational modeling, bushy cells had decreased spike rates at sound onset as well as longer and more variable first spike latencies explaining the abnormal auditory brainstem responses. In addition, we found synaptic transmission to outlast the stimulus at many endbulb of Held synapses in vitro and in vivo, suggesting impaired synchronization of release to stimulus offset. Although sound encoding in the cochlea proceeds in the absence of complexins, CPX I is required for faithful processing of sound onset and offset in the cochlear nucleus.
Assuntos
Vias Auditivas/fisiologia , Núcleo Coclear/metabolismo , Audição/fisiologia , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Nervo Coclear/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapses/metabolismoRESUMO
Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). Postsynaptic recordings from this synapse in prehearing animals had delivered strong indications for synchronized release of several vesicles. The underlying mechanism, however, remains unclear. Here, we used presynaptic membrane capacitance measurements to test whether IHCs release vesicles in a statistically independent or dependent (coordinated) manner. Exocytic changes of membrane capacitance (deltaC(m)) were repeatedly stimulated in IHCs of prehearing and hearing mice by short depolarizations to preferentially recruit the readily releasable pool of synaptic vesicles. A compound Poisson model was devised to describe hair cell exocytosis and to test the analysis. From the trial-to-trial fluctuations of the deltaC(m) we were able to estimate the apparent size of the elementary fusion event (C(app)) at the hair cell synapse to be 96-223 aF in immature and 55-149 aF in mature IHCs. We also approximated the single vesicle capacitance in IHCs by measurements of synaptic vesicle diameters in electron micrographs. The results (immature, 48 aF; mature, 45 aF) were lower than the respective C(app) estimates. This indicates that coordinated exocytosis of synaptic vesicles occurs at both immature and mature hair cell synapses. Approximately 35% of the release events in mature IHCs and approximately 50% in immature IHCs were predicted to involve coordinated fusion, when assuming a geometric distribution of elementary sizes. In summary, our presynaptic measurements indicate coordinated exocytosis but argue for a lesser degree of coordination than suggested by postsynaptic recordings.
Assuntos
Exocitose/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
Hearing relies on faithful sound coding at hair cell ribbon synapses, which use Ca2+-triggered glutamate release to signal with submillisecond precision. Here, we investigated stimulus-secretion coupling at mammalian inner hair cell (IHC) synapses to explore the mechanisms underlying this high temporal fidelity. Using nonstationary fluctuation analysis on Ca2+ tail currents, we estimate that IHCs contain approximately 1700 Ca2+ channels, mainly of CaV1.3 type. We show by immunohistochemistry that the CaV1.3 Ca2+ channels are localized preferentially at the ribbon-type active zones of IHCs. We argue that each active zone holds approximately 80 Ca2+ channels, of which probably <10 open simultaneously during physiological stimulation. We then manipulated the Ca2+ current by primarily changing single-channel current or open-channel number. Effects on exocytosis of the readily releasable vesicle pool (RRP) were monitored by membrane capacitance recordings. Consistent with the high intrinsic Ca2+ cooperativity of exocytosis, RRP exocytosis changed nonlinearly with the Ca2+ current when varying the single-channel current. In contrast, the apparent Ca2+ cooperativity of RRP exocytosis was close to unity when primarily manipulating the number of open channels. Our findings suggest a Ca2+ channel-release site coupling in which few nearby CaV1.3 channels impose high nanodomain [Ca2+] on release sites in IHCs during physiological stimulation. We postulate that the IHC ribbon synapse uses this Ca2+ nanodomain control of exocytosis to signal with high temporal precision already at low sound intensities.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Exocitose/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Canais de Cálcio Tipo L/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Camundongos , Nanoestruturas , Estrutura Terciária de Proteína/fisiologia , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose , Células Ciliadas Auditivas Internas/metabolismo , Modelos Neurológicos , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Animais , Exocitose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.
Assuntos
Canais de Cálcio/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Canais de Cálcio/genética , Potenciais Pós-Sinápticos Excitadores/genética , Exocitose/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Sinapses/genética , Vesículas Sinápticas/genéticaRESUMO
Cochlear inner hair cells (IHCs) transmit acoustic information to spiral ganglion neurons through ribbon synapses. Here we have used morphological and physiological techniques to ask whether synaptic mechanisms differ along the tonotopic axis and within IHCs in the mouse cochlea. We show that the number of ribbon synapses per IHC peaks where the cochlea is most sensitive to sound. Exocytosis, measured as membrane capacitance changes, scaled with synapse number when comparing apical and midcochlear IHCs. Synapses were distributed in the subnuclear portion of IHCs. High-resolution imaging of IHC synapses provided insights into presynaptic Ca(2+) channel clusters and Ca(2+) signals, synaptic ribbons and postsynaptic glutamate receptor clusters and revealed subtle differences in their average properties along the tonotopic axis. However, we observed substantial variability for presynaptic Ca(2+) signals, even within individual IHCs, providing a candidate presynaptic mechanism for the divergent dynamics of spiral ganglion neuron spiking.
Assuntos
Cóclea/citologia , Cóclea/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Estimulação Acústica/métodos , Oxirredutases do Álcool , Animais , Animais Recém-Nascidos , Calbindinas , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Proteínas Correpressoras , Proteínas de Ligação a DNA/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Gerbillinae , Células Ciliadas Auditivas Internas/ultraestrutura , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Técnicas de Patch-Clamp , Fosfoproteínas/metabolismo , Psicoacústica , Receptores de AMPA/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Sinapses/ultraestrutura , Fatores de TempoRESUMO
Our auditory system is capable of perceiving the azimuthal location of a low frequency sound source with a precision of a few degrees. This requires the auditory system to detect time differences in sound arrival between the two ears down to tens of microseconds. The detection of these interaural time differences relies on network computation by auditory brainstem neurons sharpening the temporal precision of the afferent signals. Nevertheless, the system requires the hair cell synapse to encode sound with the highest possible temporal acuity. In mammals, each auditory nerve fibre receives input from only one inner hair cell (IHC) synapse. Hence, this single synapse determines the temporal precision of the fibre. As if this was not enough of a challenge, the auditory system is also capable of maintaining such high temporal fidelity with acoustic signals that vary greatly in their intensity. Recent research has started to uncover the cellular basis of sound coding. Functional and structural descriptions of synaptic vesicle pools and estimates for the number of Ca(2+) channels at the ribbon synapse have been obtained, as have insights into how the receptor potential couples to the release of synaptic vesicles. Here, we review current concepts about the mechanisms that control the timing of transmitter release in inner hair cells of the cochlea.
Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Audição/fisiologia , Sinapses/fisiologia , Vias Aferentes/fisiologia , Animais , Vias Auditivas/fisiologia , Canais de Cálcio/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Mecanorreceptores/fisiologia , Camundongos , SomRESUMO
Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.
Assuntos
Cegueira/patologia , Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Doenças Neurodegenerativas/patologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Sinapses/ultraestrutura , Animais , Cegueira/genética , Cegueira/metabolismo , Proteínas de Transporte/genética , Proteínas de Choque Térmico HSP40/deficiência , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/fisiologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retina/patologiaRESUMO
KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.