RESUMEN
Hypersensitivity to sounds is one of the prevalent symptoms in individuals with Fragile X syndrome (FXS). It manifests behaviorally early during development and is often used as a landmark for treatment efficacy. However, the physiological mechanisms and circuit-level alterations underlying this aberrant behavior remain poorly understood. Using the mouse model of FXS (Fmr1 KO), we demonstrate that functional maturation of auditory brainstem synapses is impaired in FXS. Fmr1 KO mice showed a greatly enhanced excitatory synaptic input strength in neurons of the lateral superior olive (LSO), a prominent auditory brainstem nucleus, which integrates ipsilateral excitation and contralateral inhibition to compute interaural level differences. Conversely, the glycinergic, inhibitory input properties remained unaffected. The enhanced excitation was the result of an increased number of cochlear nucleus fibers converging onto one LSO neuron, without changing individual synapse properties. Concomitantly, immunolabeling of excitatory ending markers revealed an increase in the immunolabeled area, supporting abnormally elevated excitatory input numbers. Intrinsic firing properties were only slightly enhanced. In line with the disturbed development of LSO circuitry, auditory processing was also affected in adult Fmr1 KO mice as shown with single-unit recordings of LSO neurons. These processing deficits manifested as an increase in firing rate, a broadening of the frequency response area, and a shift in the interaural level difference function of LSO neurons. Our results suggest that this aberrant synaptic development of auditory brainstem circuits might be a major underlying cause of the auditory processing deficits in FXS.SIGNIFICANCE STATEMENT Fragile X Syndrome (FXS) is the most common inheritable form of intellectual impairment, including autism. A core symptom of FXS is extreme sensitivity to loud sounds. This is one reason why individuals with FXS tend to avoid social interactions, contributing to their isolation. Here, a mouse model of FXS was used to investigate the auditory brainstem where basic sound information is first processed. Loss of the Fragile X mental retardation protein leads to excessive excitatory compared with inhibitory inputs in neurons extracting information about sound levels. Functionally, this elevated excitation results in increased firing rates, and abnormal coding of frequency and binaural sound localization cues. Imbalanced early-stage sound level processing could partially explain the auditory processing deficits in FXS.
Asunto(s)
Vías Auditivas/fisiopatología , Percepción Auditiva , Tronco Encefálico/fisiopatología , Potenciales Evocados Auditivos del Tronco Encefálico , Síndrome del Cromosoma X Frágil/fisiopatología , Trastornos de la Audición/fisiopatología , Animales , Corteza Auditiva/fisiopatología , Potenciales Postsinápticos Excitadores , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Masculino , Ratones , Ratones NoqueadosRESUMEN
Naked mole-rats (Heterocephalus glaber) live in large eu-social, underground colonies in narrow burrows and are exposed to a large repertoire of communication signals but negligible binaural sound localization cues, such as interaural time and intensity differences. We therefore asked whether monaural and binaural auditory brainstem nuclei in the naked mole-rat are differentially adjusted to this acoustic environment. Using antibody stainings against excitatory and inhibitory presynaptic structures, namely the vesicular glutamate transporter VGluT1 and the glycine transporter GlyT2 we identified all major auditory brainstem nuclei except the superior paraolivary nucleus in these animals. Naked mole-rats possess a well structured medial superior olive, with a similar synaptic arrangement to interaural-time-difference encoding animals. The neighboring lateral superior olive, which analyzes interaural intensity differences, is large and elongated, whereas the medial nucleus of the trapezoid body, which provides the contralateral inhibitory input to these binaural nuclei, is reduced in size. In contrast, the cochlear nucleus, the nuclei of the lateral lemniscus and the inferior colliculus are not considerably different when compared to other rodent species. Most interestingly, binaural auditory brainstem nuclei lack the membrane-bound hyperpolarization-activated channel HCN1, a voltage-gated ion channel that greatly contributes to the fast integration times in binaural nuclei of the superior olivary complex in other species. This suggests substantially lengthened membrane time constants and thus prolonged temporal integration of inputs in binaural auditory brainstem neurons and might be linked to the severely degenerated sound localization abilities in these animals.
Asunto(s)
Vías Auditivas/anatomía & histología , Vías Auditivas/metabolismo , Tronco Encefálico/anatomía & histología , Tronco Encefálico/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Animales , Western Blotting , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Microscopía Confocal , Ratas Topo , Extractos de TejidosRESUMEN
The ventral nucleus of the lateral lemniscus (VNLL) provides a major inhibitory projection to the inferior colliculus (IC). Neurons in the VNLL respond with various firing patterns and different temporal precision to acoustic stimulation. The present study investigates the underlying intrinsic and synaptic properties of various cell types in different regions of the VNLL, using in vitro electrophysiological recordings from acute brain slices of mice and immunohistochemistry. We show that the biophysical membrane properties and excitatory input characteristics differed between dorsal and ventral VNLL neurons. Neurons in the ventral VNLL displayed an onset-type firing pattern and little hyperpolarization-activated current (Ih). Stimulation of lemniscal inputs evoked a large all-or-none excitatory response similar to Calyx of Held synapses in neurons in the lateral part of the ventral VNLL. Neurons that were located within the fiber tract of the lateral lemniscus, received several and weak excitatory input fibers. In the dorsal VNLL onset-type and sustained firing neurons were intermingled. These neurons showed large Ih and were strongly immunopositive for the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) subunit. Both neuron types received several excitatory inputs that were weaker and slower compared to ventrolateral VNLL neurons. Using a mouse model that expresses channelrhodopsin under the promotor of the vesicular GABA transporter (VGAT) suggests that dorsal and ventral neurons were inhibitory since they were all depolarized by light stimulation. The diverse membrane and input properties in dorsal and ventral VNLL neurons suggest differential roles of these neurons for sound processing.
Asunto(s)
Percepción Auditiva/fisiología , Tronco Encefálico/fisiología , Membrana Celular/fisiología , Fenómenos Electrofisiológicos/fisiología , Neuronas/fisiología , Animales , Tronco Encefálico/citología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Técnicas de Placa-Clamp , Transmisión Sináptica/fisiologíaRESUMEN
KCNQ5/Kv7.5, a low-threshold noninactivating voltage-gated potassium channel, is preferentially targeted to excitatory endings of auditory neurons in the adult rat brainstem. Endbulds of Held from auditory nerve axons on the bushy cells of the ventral cochlear nucleus (VCN) and calyces of Held around the principal neurons in the medial nucleus of the trapezoid body (MNTB) are rich in KCNQ5 immunoreactivity. We have previously shown that this synaptic distribution occurs at about the time of hearing onset. The current study tests whether this localization in excitatory endings depends on the peripheral activity carried by the auditory nerve. Auditory nerve activity was abolished by cochlear removal or intracochlear injection of tetrodotoxin (TTX). Presence of KCNQ5 was analyzed by immunocytochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. After cochlear removal, KCNQ5 immunoreactivity was virtually undetectable at its usual location in endbulbs and calyces of Held in the anteroventral CN and in the MNTB, respectively, although it was found in cell bodies in the VCN. The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies. Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection. These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem. This may contribute to adaptations in the excitability and neurotransmitter release properties of these presynaptic endings under altered input conditions.
Asunto(s)
Enfermedades Auditivas Centrales/etiología , Enfermedades Auditivas Centrales/patología , Tronco Encefálico/patología , Enfermedades Cocleares/complicaciones , Canales de Potasio KCNQ/metabolismo , Neuronas/metabolismo , Anestésicos Locales/farmacología , Animales , Calbindina 2/metabolismo , Enfermedades Cocleares/inducido químicamente , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Femenino , Fluoresceínas , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Degeneración Nerviosa/etiología , Neuronas/efectos de los fármacos , ARN Mensajero , Ratas , Ratas Wistar , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
Kv7.5/KCNQ5, a voltage-dependent potassium channel that generates a subthreshold K+ current (also called M-current), is localized in excitatory endings of auditory brainstem nuclei in the adult rat. Here, we focus on how specific targeting develops from birth to adulthood in the rat. We first analyzed by immunocytochemistry the distribution of KCNQ5 during postnatal development of neurons in the anteroventral cochlear nucleus (AVCN) and their targets in the medial nucleus of the trapezoid body (MNTB). From postnatal days (P) 0 to 12, KCNQ5 immunoreactivity was restricted to cell bodies, whereas from P13 onward a shift in labeling pattern was seen, with KCNQ5 immunoreactivity becoming confined to synaptic endings in both the AVCN and MNTB. The developmental synaptic targeting was also accompanied by a downregulation of KCNQ5 transcripts in the cochlear nucleus from P13 onward, as seen with quantitative reverse transcriptase polymerase chain reaction. We further tested whether auditory nerve activity at hearing onset (approximately P12) regulates synaptic targeting of the channel. Cochleae were removed at P10, before hearing onset. In the MNTB, 3 days after cochlear ablation, at P13, KCNQ5 immunoreactivity was seen in calyces of Held, as in normal age-matched controls. However, immunolabeling virtually disappeared from MNTB calyces 40 days after cochlear ablation but reappeared in the somata of neurons in AVCN. These findings suggest that synaptic targeting of KCNQ5 in brainstem auditory neurons occurs around the time of hearing onset, regardless of auditory nerve activity. However, long-term synaptic localization after hearing onset depends on peripheral input.
Asunto(s)
Tronco Encefálico/crecimiento & desarrollo , Tronco Encefálico/fisiología , Canales de Potasio KCNQ/metabolismo , Neuronas/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Vías Auditivas/crecimiento & desarrollo , Vías Auditivas/fisiología , Percepción Auditiva/fisiología , Western Blotting , Cóclea/crecimiento & desarrollo , Cóclea/lesiones , Cóclea/fisiología , Nervio Coclear/crecimiento & desarrollo , Nervio Coclear/fisiología , Núcleo Coclear/crecimiento & desarrollo , Núcleo Coclear/fisiología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Microscopía Confocal , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
KCNQ, also called Kv7, is a family of voltage-dependent potassium channels with important roles in excitability regulation. Of its five known subunits, KCNQ5/Kv7.5 is extensively expressed in the central nervous system and it contributes to the generation of M-currents. The distribution of KCNQ5 was analyzed in auditory nuclei of the rat brainstem by high-resolution immunocytochemistry. Double labeling with anti-KCNQ5 antibodies and anti-synaptophysin or anti-syntaxin, which mark synaptic endings, or anti-microtubule-associated protein 2 (MAP2) antibodies, which mark dendrites, were used to analyze the subcellular distribution of KCNQ5 in neurons in the cochlear nucleus, superior olivary complex, nuclei of the lateral lemniscus, and inferior colliculus. An abundance of KCNQ5 labeling in punctate structures throughout auditory brainstem nuclei along with colocalization with such synaptic markers suggests that a preferred localization of KCNQ5 is in synaptic endings in these auditory nuclei. Punctate KCNQ5 immunoreactivity virtually disappeared from the cochlear nucleus after cochlea removal, which strongly supports localization of this channel in excitatory endings of the auditory nerve. Actually, neither glycinergic endings, labeled with an anti-glycine transporter 2 (GlyT2) antibody, nor gamma-aminobutyric acid (GABA)ergic endings, labeled with an anti-glutamic acid decarboxylase (GAD65) antibody, contained KCNQ5 immunoreactivity, suggesting that KCNQ5 is mostly in excitatory endings throughout the auditory brainstem. Overlap of KCNQ5 and MAP2 labeling indicates that KCNQ5 is also targeted to dendritic compartments. These findings predict pre- and postsynaptic roles for KCNQ5 in excitability regulation in auditory brainstem nuclei, at the level of glutamatergic excitatory endings and in dendrites.