RESUMEN
"Growing old" is the most common cause of hearing loss. Age-related hearing loss (ARHL) (presbycusis) first affects the ability to understand speech in background noise, even when auditory thresholds in quiet are normal. It has been suggested that cochlear denervation ("synaptopathy") is an early contributor to age-related auditory decline. In the present work, we characterized age-related cochlear synaptic degeneration and hair cell loss in mice with enhanced α9α10 cholinergic nicotinic receptors gating kinetics ("gain of function" nAChRs). These mediate inhibitory olivocochlear feedback through the activation of associated calcium-gated potassium channels. Cochlear function was assessed via distortion product otoacoustic emissions and auditory brainstem responses. Cochlear structure was characterized in immunolabeled organ of Corti whole mounts using confocal microscopy to quantify hair cells, auditory neurons, presynaptic ribbons, and postsynaptic glutamate receptors. Aged wild-type mice had elevated acoustic thresholds and synaptic loss. Afferent synapses were lost from inner hair cells throughout the aged cochlea, together with some loss of outer hair cells. In contrast, cochlear structure and function were preserved in aged mice with gain-of-function nAChRs that provide enhanced olivocochlear inhibition, suggesting that efferent feedback is important for long-term maintenance of inner ear function. Our work provides evidence that olivocochlear-mediated resistance to presbycusis-ARHL occurs via the α9α10 nAChR complexes on outer hair cells. Thus, enhancement of the medial olivocochlear system could be a viable strategy to prevent age-related hearing loss.
Asunto(s)
Envejecimiento/fisiología , Cóclea , Células Ciliadas Auditivas Externas , Presbiacusia , Complejo Olivar Superior , Animales , Cóclea/fisiología , Cóclea/fisiopatología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Retroalimentación Fisiológica/fisiología , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/fisiología , Ratones , Emisiones Otoacústicas Espontáneas/fisiología , Presbiacusia/fisiopatología , Presbiacusia/prevención & control , Complejo Olivar Superior/citología , Complejo Olivar Superior/fisiologíaRESUMEN
Gain control of the auditory system operates at multiple levels. Cholinergic medial olivocochlear (MOC) fibers originate in the brainstem and make synaptic contacts at the base of the outer hair cells (OHCs), the final targets of several feedback loops from the periphery and higher-processing centers. Efferent activation inhibits OHC active amplification within the mammalian cochlea, through the activation of a calcium-permeable α9α10 ionotropic cholinergic nicotinic receptor (nAChR), functionally coupled to calcium activated SK2 potassium channels. Correct operation of this feedback requires careful matching of acoustic input with the strength of cochlear inhibition (Galambos, 1956; Wiederhold and Kiang, 1970; Gifford and Guinan, 1987), which is driven by the rate of MOC activity and short-term facilitation at the MOC-OHC synapse (Ballestero et al., 2011; Katz and Elgoyhen, 2014). The present work shows (in mice of either sex) that a mutation in the α9α10 nAChR with increased duration of channel gating (Taranda et al., 2009) greatly elongates hair cell-evoked IPSCs and Ca2+ signals. Interestingly, MOC-OHC synapses of L9'T mice presented reduced quantum content and increased presynaptic facilitation. These phenotypic changes lead to enhanced and sustained synaptic responses and OHC hyperpolarization upon high-frequency stimulation of MOC terminals. At the cochlear physiology level these changes were matched by a longer time course of efferent MOC suppression. This indicates that the properties of the MOC-OHC synapse directly determine the efficacy of the MOC feedback to the cochlea being a main player in the "gain control" of the auditory periphery.SIGNIFICANCE STATEMENT Plasticity can involve reciprocal signaling across chemical synapses. An opportunity to study this phenomenon occurs in the mammalian cochlea whose sensitivity is regulated by efferent olivocochlear neurons. These release acetylcholine to inhibit sensory hair cells. A point mutation in the hair cell's acetylcholine receptor that leads to increased gating of the receptor greatly elongates IPSCs. Interestingly, efferent terminals from mutant mice present a reduced resting release probability. However, upon high-frequency stimulation transmitter release facilitates strongly to produce stronger and far longer-lasting inhibition of cochlear function. Thus, central neuronal feedback on cochlear hair cells provides an opportunity to define plasticity mechanisms in cholinergic synapses other than the highly studied neuromuscular junction.
Asunto(s)
Mutación con Ganancia de Función , Células Ciliadas Auditivas/metabolismo , Plasticidad Neuronal , Receptores Nicotínicos/genética , Animales , Señalización del Calcio , Retroalimentación Fisiológica , Femenino , Células Ciliadas Auditivas/fisiología , Potenciales Postsinápticos Inhibidores , Activación del Canal Iónico , Masculino , Ratones , Neuronas Eferentes/metabolismo , Neuronas Eferentes/fisiología , Receptores Nicotínicos/metabolismoRESUMEN
During a critical developmental period, cochlear inner hair cells (IHCs) exhibit sensory-independent activity, featuring action potentials in which Ca2+ ions play a fundamental role in driving both spiking and glutamate release onto synapses with afferent auditory neurons. This spontaneous activity is controlled by a cholinergic input to the IHC, activating a specialized nicotinic receptor with high Ca2+ permeability, and coupled to the activation of hyperpolarizing SK channels. The mechanisms underlying distinct excitatory and inhibitory Ca2+ roles within a small, compact IHC are unknown. Making use of Ca2+ imaging, afferent auditory bouton recordings, and electron microscopy, the present work shows that unusually high intracellular Ca2+ buffering and "subsynaptic" cisterns provide efficient compartmentalization and tight control of cholinergic Ca2+ signals. Thus, synaptic efferent Ca2+ spillover and cross-talk are prevented, and the cholinergic input preserves its inhibitory signature to ensure normal development of the auditory system.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Cóclea/fisiología , Células Ciliadas Auditivas Internas/citología , Sinapsis/fisiología , Acetilcolina/farmacología , Potenciales de Acción , Animales , Vías Auditivas/fisiología , Estimulación Eléctrica , Femenino , Ácido Glutámico/metabolismo , Audición , Masculino , Ratones , Neuronas/fisiología , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/fisiología , Transducción de SeñalRESUMEN
Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.
Asunto(s)
Calcio/metabolismo , Receptores Nicotínicos/genética , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Señalización del Calcio , Membrana Celular/metabolismo , Células Cultivadas , Pollos , Evolución Molecular , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Agonistas Nicotínicos/farmacología , Permeabilidad , Ratas , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Xenopus laevisRESUMEN
In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHC function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked IPSCs (eIPSCs) are mediated solely by α9α10 nAChRs functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1 Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies >10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier.
Asunto(s)
Cóclea/citología , Nervio Coclear/fisiología , Células Ciliadas Auditivas/fisiología , Inhibición Neural/fisiología , Sinapsis/fisiología , Estimulación Acústica , Animales , Animales Recién Nacidos , Biofisica , Quelantes , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica , Femenino , Glicinérgicos/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Técnicas In Vitro , Indoles/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Inhibición Neural/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Péptidos/farmacología , Antagonistas de la Serotonina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Estricnina/farmacología , Sinapsis/efectos de los fármacos , Temperatura , Tetrodotoxina/farmacología , Factores de Tiempo , TropisetrónRESUMEN
Mechanosensory hair cells of the organ of Corti transmit information regarding sound to the central nervous system by way of peripheral afferent neurons. In return, the central nervous system provides feedback and modulates the afferent stream of information through efferent neurons. The medial olivocochlear efferent system makes direct synaptic contacts with outer hair cells and inhibits amplification brought about by the active mechanical process inherent to these cells. This feedback system offers the potential to improve the detection of signals in background noise, to selectively attend to particular signals, and to protect the periphery from damage caused by overly loud sounds. Acetylcholine released at the synapse between efferent terminals and outer hair cells activates a peculiar nicotinic cholinergic receptor subtype, the alpha9alpha10 receptor. At present no pharmacotherapeutic approaches have been designed that target this cholinergic receptor to treat pathologies of the auditory system. The potential use of alpha9alpha10 selective drugs in conditions such as noise-induced hearing loss, tinnitus and auditory processing disorders is discussed.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Receptores Nicotínicos/fisiología , Acetilcolina/metabolismo , Animales , Trastornos de la Percepción Auditiva/tratamiento farmacológico , Trastornos de la Percepción Auditiva/metabolismo , Cóclea/anatomía & histología , Cóclea/fisiología , Dislexia/tratamiento farmacológico , Dislexia/metabolismo , Pérdida Auditiva/tratamiento farmacológico , Pérdida Auditiva/etiología , Humanos , Ruido/efectos adversos , Núcleo Olivar/fisiología , Subunidades de Proteína/fisiología , Transmisión Sináptica , Acúfeno/tratamiento farmacológico , Acúfeno/metabolismoRESUMEN
Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.
Asunto(s)
Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/metabolismo , Audición/fisiología , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Animales , Colinérgicos/farmacología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Audición/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Factor de Transcripción Brn-3C/genética , Factor de Transcripción Brn-3C/metabolismoRESUMEN
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.
Asunto(s)
Acetilcolina/metabolismo , Colinérgicos/metabolismo , Células Ciliadas Auditivas/fisiología , Neuronas Eferentes/fisiología , Mutación Puntual , Receptores Nicotínicos/genética , Animales , Vías Auditivas/fisiología , Umbral Auditivo/fisiología , Cóclea/metabolismo , Modelos Animales de Enfermedad , Retroalimentación Fisiológica/fisiología , Pérdida Auditiva Sensorineural/prevención & control , Ratones , Ratones Mutantes , Canales de Potasio/fisiología , Receptores Nicotínicos/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiologíaRESUMEN
The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.
Asunto(s)
Células Ciliadas Auditivas/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Receptores Colinérgicos/efectos de los fármacos , Rianodina/farmacología , Acetilcolina/farmacología , Animales , Pollos , Relación Dosis-Respuesta a Droga , Células Ciliadas Auditivas/fisiología , Ratones , Canales de Potasio Calcio-Activados/efectos de los fármacos , Canales de Potasio Calcio-Activados/fisiología , Subunidades de Proteína/análisis , Ratas , Receptores Colinérgicos/fisiología , Receptores Nicotínicos/análisis , Xenopus laevisRESUMEN
Before the onset of hearing, a transient efferent innervation is found on inner hair cells (IHCs). This synapse is inhibitory and mediated by a nicotinic cholinergic receptor (nAChR) probably formed by the alpha9 and alpha10 subunits. We analysed the pharmacological and biophysical characteristics of the native nAChR using whole-cell recordings from IHCs in acutely excised apical turns of the rat organ of Corti. Nicotine did not activate but rather blocked the acetylcholine (ACh)-evoked currents with an IC50 of 1 +/- 0.1 microM. Antagonists of non-cholinergic receptors such as strychnine, bicuculline and ICS-205930 blocked ACh-evoked responses with an IC50 of 8.6 +/- 0.8 nM, 59 +/- 4 nM and 0.30 +/- 0.02 microM, respectively. The IHC nAChR was both permeable to (P(Ca)/P(Na) = 8 +/- 0.9) and modulated by external Ca2+. ACh-evoked currents were potentiated by Ca2+ up to 500 microM but were reduced by higher concentrations of this cation. Ba2+ mimicked the effects of Ca2+ whereas Mg2+ only blocked these currents. In addition, elevation of extracellular Ca2+ reduced the amplitude of spontaneous synaptic currents without affecting their time course. The receptor had an EC50 for ACh of 60.7 +/- 2.8 microM in 0.5 mM Ca2+. In the absence of Ca2+, the EC50 for ACh increased, suggesting that potentiation by Ca2+ involves changes in the apparent affinity for the agonist. These pharmacological and biophysical characteristics of the IHC nAChR closely resemble those of the recombinant alpha9alpha10 nAChR, reinforcing the hypothesis that the functional nAChR at the olivocochlear efferent-IHC synapse is composed of both the alpha9 and alpha10 subunits.
Asunto(s)
Potenciales de Acción/fisiología , Vías Auditivas/fisiología , Células Ciliadas Auditivas Internas/fisiología , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Receptores Nicotínicos/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Adaptación Fisiológica/fisiología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Células Ciliadas Auditivas Internas/embriología , Homeostasis/fisiología , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both alpha9 and alpha10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that alpha9 mRNA is expressed in adult IHCs but that alpha10 mRNA disappears after the onset of hearing. In the present study, antibodies to alpha10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of alpha10, but not alpha9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.