RESUMO
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.
Assuntos
Mutação com Ganho de Função , Células Ciliadas Auditivas/metabolismo , Plasticidade Neuronal , Receptores Nicotínicos/genética , Animais , Sinalização do Cálcio , Retroalimentação Fisiológica , Feminino , Células Ciliadas Auditivas/fisiologia , Potenciais Pós-Sinápticos Inibidores , Ativação do Canal Iônico , Masculino , Camundongos , Neurônios Eferentes/metabolismo , Neurônios Eferentes/fisiologia , Receptores Nicotínicos/metabolismoRESUMO
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.
Assuntos
Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Audição/fisiologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Colinérgicos/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Audição/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismoRESUMO
Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the alpha9alpha10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from alpha10 nAChR gene (Chrna10) "knockout" mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the alpha10 nAChR subunit. The present results show that the alpha10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.
Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Receptores Nicotínicos/deficiência , Animais , Apamina/farmacologia , Cóclea/embriologia , Capacitância Elétrica , Audição/fisiologia , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/antagonistas & inibidoresRESUMO
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.
Assuntos
Acetilcolina/metabolismo , Colinérgicos/metabolismo , Células Ciliadas Auditivas/fisiologia , Neurônios Eferentes/fisiologia , Mutação Puntual , Receptores Nicotínicos/genética , Animais , Vias Auditivas/fisiologia , Limiar Auditivo/fisiologia , Cóclea/metabolismo , Modelos Animais de Doenças , Retroalimentação Fisiológica/fisiologia , Perda Auditiva Neurossensorial/prevenção & controle , Camundongos , Camundongos Mutantes , Canais de Potássio/fisiologia , Receptores Nicotínicos/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologiaRESUMO
In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Quinidina/farmacologia , Quinina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Antimaláricos/toxicidade , Cloroquina/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Quinidina/toxicidade , Quinina/toxicidade , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/efeitos dos fármacos , Xenopus laevisRESUMO
The neuregulin receptor tyrosine kinase Erb-b4, initially linked to early cardiac development, is shown here to play a critical role in adult cardiac function. In wild-type mice, Erb-b4 protein localized to Z lines and to intercalated disks, suggesting a role in subcellular and intercellular communications of cardiomyocytes. Conditional inactivation of erb-b4 in ventricular muscle cells led to a severe dilated cardiomyopathy, characterized by thinned ventricular walls with eccentric hypertrophy, reduced contractility, and delayed conduction. This cardiac dysfunction may account for premature death in adult erb-b4-knockout mice. This study establishes a critical role for Erb-b4 in the maintenance of normal postnatal cardiac structure and function.