RESUMO
The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.
Assuntos
Vias Eferentes/fisiologia , Sistema da Linha Lateral/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sinapses/fisiologia , Animais , Bungarotoxinas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Regulação da Expressão Gênica , Células Ciliadas Auditivas/fisiologia , Antagonistas Nicotínicos/farmacologia , Oócitos , Estimulação Física , Receptores Nicotínicos/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Estricnina/farmacologia , Xenopus , Peixe-ZebraRESUMO
The organ of Corti, the auditory mammalian sensory epithelium, contains two types of mechanotransducer cells, inner hair cells (IHCs) and outer hair cells (OHCs). IHCs are involved in conveying acoustic stimuli to the CNS, while OHCs are implicated in the fine tuning and amplification of sounds. OHCs are innervated by medial olivocochlear (MOC) cholinergic efferent fibers. The functional characteristics of the MOC-OHC synapse during maturation were assessed by electrophysiological and pharmacological methods in mouse organs of Corti at postnatal day 11 (P11)-P13, hearing onset in altricial rodents, and at P20-P22 when the OHCs are morphologically and functionally mature. Synaptic currents were recorded in whole-cell voltage-clamped OHCs while electrically stimulating the MOC fibers. A progressive increase in the number of functional MOC-OHC synapses, as well as in their strength and efficacy, was observed between P11-13 and P20-22. At hearing onset, the MOC-OHC synapse presented facilitation during MOC fibers high-frequency stimulation that disappeared at mature stages. In addition, important changes were found in the VGCC that are coupled to transmitter release. Ca2+ flowing in through L-type VGCCs contribute to trigger ACh release together with P/Q- and R-type VGCCs at P11-P13, but not at P20-P22. Interestingly, N-type VGCCs were found to be involved in this process at P20-P22, but not at hearing onset. Moreover, the degree of compartmentalization of calcium channels with respect to BK channels and presynaptic release components significantly increased from P11-P13 to P20-P22. These results suggest that the MOC-OHC synapse is immature at the onset of hearing.SIGNIFICANCE STATEMENT The functional expression of both VGCCs and BK channels, as well as their localization with respect to the presynaptic components involved in transmitter release, are key elements in determining synaptic efficacy. In this work, we show dynamic changes in the expression of VGCCs and Ca2+-dependent BK K+ channels coupled to ACh release at the MOC-OHC synapse and their shift in compartmentalization during postnatal maturation. These processes most likely set the short-term plasticity pattern and reliability of the MOC-OHC synapse on high-frequency activity.
Assuntos
Células Ciliadas Auditivas Externas/fisiologia , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Órgão Espiral/crescimento & desenvolvimento , Sinapses/fisiologia , Animais , Canais de Cálcio/metabolismo , Feminino , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Eferentes/fisiologia , Órgão Espiral/fisiologiaRESUMO
In the mature mammalian cochlea, inner hair cells (IHCs) are mainly innervated by afferent fibers that convey sound information to the CNS. During postnatal development, however, medial olivocochlear (MOC) efferent fibers transiently innervate the IHCs. The MOC-IHC synapse, functional from postnatal day 0 (P0) to hearing onset (P12), undergoes dramatic changes in the sensitivity to acetylcholine (ACh) and in the expression of key postsynaptic proteins. To evaluate whether there are associated changes in the properties of ACh release during this period, we used a cochlear preparation from mice of either sex at P4, P6-P7, and P9-P11 and monitored transmitter release from MOC terminals in voltage-clamped IHCs in the whole-cell configuration. The quantum content increased 5.6× from P4 to P9-P11 due to increases in the size and replenishment rate of the readily releasable pool of synaptic vesicles without changes in their probability of release or quantum size. This strengthening in transmission was accompanied by changes in short-term plasticity properties, which switched from facilitation at P4 to depression at P9-P11. We have previously shown that at P9-P11, ACh release is supported by P/Q- and N-type voltage-gated calcium channels (VGCCs) and negatively regulated by BK potassium channels activated by Ca2+ influx through L-type VGCCs. We now show that at P4 and P6-P7, release is mediated by P/Q-, R- and L-type VGCCs. Interestingly, L-type VGCCs have a dual role: they both support release and fuel BK channels, suggesting that at immature stages presynaptic proteins involved in release are less compartmentalized.SIGNIFICANCE STATEMENT During postnatal development before the onset of hearing, cochlear inner hair cells (IHCs) present spontaneous Ca2+ action potentials that release glutamate at the first auditory synapse in the absence of sound stimulation. The IHC Ca2+ action potential frequency pattern, which is crucial for the correct establishment and function of the auditory system, is regulated by the efferent medial olivocochlear (MOC) system that transiently innervates IHCs during this period. We show here that developmental changes in synaptic strength and synaptic plasticity properties at the MOC-IHC synapse upon MOC fiber activation at different frequencies might be crucial for tightly shaping the pattern of afferent activity during this critical period.
Assuntos
Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas Internas/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Acetilcolina/metabolismo , Animais , Cóclea/metabolismo , Feminino , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos Endogâmicos BALB C , Plasticidade NeuronalRESUMO
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
We studied the functional activation of different polymorphic variants of the human dopamine D(4) receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D(4) receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D(4) receptor variants coupled to oocyte G(i/o) proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D(4) receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC(50)=1 nM) than on D4.4 (EC(50)=5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D(4) receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D(4) receptor variants (EC(50 NA)=50 nM, EC(50 5-HT)=1.5 microM). All monoamine effects were blocked by the specific dopaminergic D(4) antagonist (S)-(-)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D(4) receptors and related monoamine receptors revealed that dopamine D(4) receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D(4) receptors can be pharmacologically stimulated by any the three major central monoamines.
Assuntos
Dopamina/farmacologia , Norepinefrina/farmacologia , Receptores de Dopamina D4/efeitos dos fármacos , Serotonina/farmacologia , Alelos , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Eletrofisiologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Polimorfismo Genético , Receptores de Dopamina D4/metabolismo , Xenopus laevisRESUMO
The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4-EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4-EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD.