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1.
J Neurosci ; 39(36): 7037-7048, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31217330

RESUMO

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this maturation process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells. In this work, we used an α9 cholinergic nicotinic receptor knock-in mouse model (of either sex) with enhanced medial efferent activity (Chrna9L9'T, L9'T) to further understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III of auditory brainstem responses (which represents synchronized activity of synapses within the superior olivary complex) was smaller in L9'T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a mediolateral (ML) axis. The topographic organization of MNTB physiological properties observed in wildtype (WT) was abolished in L9'T mice. Additionally, electrophysiological recordings in slices indicated MNTB synaptic alterations. In vivo multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9'T The present results indicate that the transient cochlear efferent innervation to inner hair cells during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the properties of synaptic transmission at a central auditory nucleus.SIGNIFICANCE STATEMENT Cochlear inner hair cells of altricial mammals display spontaneous electrical activity before hearing onset. The pattern and firing rate of these cells are crucial for the correct maturation of the central auditory pathway. A descending efferent innervation from the CNS contacts the hair cells during this developmental window. The present work shows that genetic enhancement of efferent function disrupts the orderly topographic distribution of biophysical and synaptic properties in the auditory brainstem and causes severe synaptic dysfunction. This work adds to the notion that the transient efferent innervation to the cochlea is necessary for the correct establishment of the central auditory circuitry.

2.
J Neurosci ; 39(18): 3360-3375, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30755493

RESUMO

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.

3.
J Neurosci ; 38(16): 3939-3954, 2018 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-29572431

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.

4.
Open Biol ; 3(11): 130163, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24350389

RESUMO

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a 'critical period' of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Neurônios Motores/fisiologia , Receptores Nicotínicos/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação , Animais , Cóclea/fisiologia , Camundongos , Camundongos Knockout , Receptores Nicotínicos/genética , Estereocílios , Sinaptotagmina II/genética , Sinaptotagmina II/fisiologia
5.
J Neurosci ; 33(39): 15477-87, 2013 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24068816

RESUMO

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca(2+)-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca(2+)-activated small-conductance type 2 (SK2)K(+) channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA(B(1a,2)) receptors [GABA(B(1a,2))Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA(B)Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA(B1)-GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA(B1a) isoform selectively inhibits release at efferent cholinergic synapses.


Assuntos
Células Ciliadas Auditivas/fisiologia , Potenciais Pós-Sinápticos Inibidores , Receptores de GABA-B/metabolismo , Sinapses/fisiologia , Acetilcolina/metabolismo , Animais , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Estimulação Elétrica , Células Ciliadas Auditivas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Eferentes/fisiologia , Receptores de GABA-B/genética , Sinapses/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Ácido gama-Aminobutírico/metabolismo
6.
J Assoc Res Otolaryngol ; 10(3): 397-406, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19452222

RESUMO

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/metabolismo
7.
J Assoc Res Otolaryngol ; 10(2): 221-32, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19252947

RESUMO

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 & inibidores
8.
J Neurochem ; 103(6): 2651-64, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17961150

RESUMO

Acetylcholine is a key neurotransmitter of the inner ear efferent system. In this study, we identify two novel nAChR subunits in the inner ear: α1 and γ, encoded by Chrna1 and Chrng, respectively. In situ hybridization shows that the messages of these two subunits are present in vestibular and cochlear hair cells during early development. Chrna1 and Chrng expression begin at embryonic stage E13.5 in the vestibular system and E17.5 in the organ of Corti. Chrna1 message continues through P7, whereas Chrng is undetectable at post-natal stage P6. The α1 and γ subunits are known as muscle-type nAChR subunits and are surprisingly expressed in hair cells which are sensory-neural cells. We also show that ATOH1/MATH1, a transcription factor essential for hair cell development, directly activates CHRNA1 transcription. Electrophoretic mobility-shift assays and supershift assays showed that ATOH1/E47 heterodimers selectively bind on two E boxes located in the proximal promoter of CHRNA1. Thus, Chrna1 could be the first transcriptional target of ATOH1 in the inner ear. Co-expression in Xenopus oocytes of the α1 subunit does not change the electrophysiological properties of the α9α10 receptor. We suggest that hair cells transiently express α1γ-containing nAChRs in addition to α9α10, and that these may have a role during development of the inner ear innervation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Orelha Interna/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Receptores Nicotínicos/biossíntese , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Orelha Interna/embriologia , Ensaio de Desvio de Mobilidade Eletroforética , Fenômenos Eletrofisiológicos , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/genética , Gravidez , RNA/biossíntese , RNA/genética , Receptores Nicotínicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Xenopus laevis
9.
Eur J Pharmacol ; 562(3): 165-73, 2007 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-17350612

RESUMO

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 Drogas , 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 laevis
10.
Eur J Neurosci ; 24(9): 2429-38, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17100831

RESUMO

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.


Assuntos
Encéfalo/metabolismo , Cromossomos Artificiais Bacterianos , Neurônios/metabolismo , Receptores de Dopamina D4/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Técnicas de Patch-Clamp
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