RESUMEN
Type I spiral ganglion neurons (SGNs) transmit sound information from cochlear hair cells to the CNS. Using transcriptome analysis of thousands of single neurons, we demonstrate that murine type I SGNs consist of subclasses that are defined by the expression of subsets of transcription factors, cell adhesion molecules, ion channels, and neurotransmitter receptors. Subtype specification is initiated prior to the onset of hearing during the time period when auditory circuits mature. Gene mutations linked to deafness that disrupt hair cell mechanotransduction or glutamatergic signaling perturb the firing behavior of SGNs prior to hearing onset and disrupt SGN subtype specification. We thus conclude that an intact hair cell mechanotransduction machinery is critical during the pre-hearing period to regulate the firing behavior of SGNs and their segregation into subtypes. Because deafness is frequently caused by defects in hair cells, our findings have significant ramifications for the etiology of hearing loss and its treatment.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Audición/fisiología , Mecanotransducción Celular , Neuronas/fisiología , Transducción de Señal , Ganglio Espiral de la Cóclea/fisiología , Animales , Análisis por Conglomerados , Marcadores Genéticos , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Mutación , Neuroglía/fisiología , Análisis de Secuencia de ARNRESUMEN
Intrinsically generated neural activity propagates through the developing auditory system to promote maturation and refinement of sound processing circuits prior to hearing onset. This early patterned activity is induced by non-sensory supporting cells in the organ of Corti, which are highly interconnected through gap junctions containing connexin 26 (Gjb2). Although loss of function mutations in Gjb2 impair cochlear development and are the most common cause of congenital deafness, it is not known if these variants disrupt spontaneous activity and the developmental trajectory of sound processing circuits in the brain. Here, we show in a new mouse model of Gjb2-mediated congenital deafness that cochlear supporting cells adjacent to inner hair cells (IHCs) unexpectedly retain intercellular coupling and the capacity to generate spontaneous activity, exhibiting only modest deficits prior to hearing onset. Supporting cells lacking Gjb2 elicited coordinated activation of IHCs, leading to coincident bursts of activity in central auditory neurons that will later process similar frequencies of sound. Despite alterations in the structure of the sensory epithelium, hair cells within the cochlea of Gjb2-deficient mice were intact and central auditory neurons could be activated within appropriate tonotopic domains by loud sounds at hearing onset, indicating that early maturation and refinement of auditory circuits was preserved. Only after cessation of spontaneous activity following hearing onset did progressive hair cell degeneration and enhanced auditory neuron excitability manifest. This preservation of cochlear spontaneous neural activity in the absence of connexin 26 may increase the effectiveness of early therapeutic interventions to restore hearing.
Asunto(s)
Cóclea , Sordera , Ratones , Animales , Conexina 26 , Cóclea/fisiología , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas Internas/fisiología , Sordera/genéticaRESUMEN
Spontaneous bursts of electrical activity in the developing auditory system arise within the cochlea before hearing onset and propagate through future sound-processing circuits of the brain to promote maturation of auditory neurons. Studies in isolated cochleae revealed that this intrinsically generated activity is initiated by ATP release from inner supporting cells (ISCs), resulting in activation of purinergic autoreceptors, K+ efflux, and subsequent depolarization of inner hair cells. However, it is unknown when this activity emerges or whether different mechanisms induce activity during distinct stages of development. Here we show that spontaneous electrical activity in mouse cochlea from both sexes emerges within ISCs during the late embryonic period, preceding the onset of spontaneous correlated activity in inner hair cells and spiral ganglion neurons, which begins at birth and follows a base to apex developmental gradient. At all developmental ages, pharmacological inhibition of P2Y1 purinergic receptors dramatically reduced spontaneous activity in these three cell types. Moreover, in vivo imaging within the inferior colliculus revealed that auditory neurons within future isofrequency zones exhibit coordinated neural activity at birth. The frequency of these discrete bursts increased progressively during the postnatal prehearing period yet remained dependent on P2RY1. Analysis of mice with disrupted cholinergic signaling in the cochlea indicate that this efferent input modulates, rather than initiates, spontaneous activity before hearing onset. Thus, the auditory system uses a consistent mechanism involving ATP release from ISCs and activation of P2RY1 autoreceptors to elicit coordinated excitation of neurons that will process similar frequencies of sound.SIGNIFICANCE STATEMENT In developing sensory systems, groups of neurons that will process information from similar sensory space exhibit highly correlated electrical activity that is critical for proper maturation and circuit refinement. Defining the period when this activity is present, the mechanisms responsible and the features of this activity are crucial for understanding how spontaneous activity influences circuit development. We show that, from birth to hearing onset, the auditory system relies on a consistent mechanism to elicit correlate firing of neurons that will process similar frequencies of sound. Targeted disruption of this activity will increase our understanding of how these early circuits mature and may provide insight into processes responsible for developmental disorders of the auditory system.
Asunto(s)
Vías Auditivas/crecimiento & desarrollo , Vías Auditivas/fisiología , Receptores Purinérgicos/fisiología , Adenosina Trifosfato/metabolismo , Animales , Señalización del Calcio/fisiología , Cóclea/crecimiento & desarrollo , Cóclea/fisiología , Femenino , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas Internas/fisiología , Colículos Inferiores/fisiología , Células Laberínticas de Soporte/fisiología , Masculino , Ratones , Sistema Nervioso Parasimpático/efectos de los fármacos , Sistema Nervioso Parasimpático/fisiología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/fisiología , Retina/fisiología , Ganglio Espiral de la Cóclea/fisiologíaRESUMEN
Sensory information is represented by small neuronal ensembles in sensory cortices. Neuronal activity shows high trial-by-trial variability in that repeated presentation of the same stimulus, e. g., multiple presentations of the same sound activate differing ensembles in the auditory cortex (AC). How the differing ensembles interact to selectively activate to process incoming sound inputs with reduced energy is unknown. Efficient processing of complex acoustic signals requires that these sparsely distributed neuronal ensembles actively interact in order to provide a constant percept. Here, we probe interactions within and across ensembles by combining in vivo 2-photon Ca2+ imaging and holographic optogenetic stimulation to study how increased activity of single cells level affects the cortical network. We stimulated a small number of neurons sharing the same frequency preference alongside the presentation of a target pure tone, further increasing their tone-evoked activity. We found that other non-stimulated co-tuned neurons decreased their tone-evoked activity while non co-tuned neurons were unaffected. This shows that co-tuned ensembles communicated and balanced their total activity across the network. The rebalanced activity due to external stimulation remained constant. These effects suggest that co-tuned ensembles in AC interact and rapidly rebalance their activity to maintain encoding homeostasis, and that the rebalanced network is persistent.
RESUMEN
Neurons that process sensory information exhibit bursts of electrical activity during development, providing early training to circuits that will later encode similar features of the external world. In the mammalian auditory system, this intrinsically generated activity emerges from the cochlea prior to hearing onset, but its role in maturation of auditory circuitry remains poorly understood. We show that selective suppression of cochlear supporting cell spontaneous activity disrupts patterned burst firing of central auditory neurons without affecting cell survival or acoustic thresholds. However, neurons in the inferior colliculus of these mice exhibit enhanced acoustic sensitivity and broader frequency tuning, resulting in wider isofrequency laminae. Despite this enhanced neural responsiveness, total tone-responsive regions in the auditory cortex are substantially smaller. Thus, disruption of pre-hearing cochlear activity causes profound changes in neural encoding of sound, with important implications for restoration of hearing in individuals who experience reduced activity during this critical developmental period.
Asunto(s)
Corteza Auditiva , Colículos Inferiores , Ratones , Animales , Colículos Inferiores/fisiología , Corteza Auditiva/fisiología , Cóclea , Audición , Neuronas/fisiología , MamíferosRESUMEN
Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.
Asunto(s)
Calcio , Empalme del ARN , Empalme Alternativo/genética , Secuencia de Bases , Exones/genética , Regulación de la Expresión Génica , Intrones/genéticaRESUMEN
Astrocytes play an essential role in the development of neural circuits by positioning transporters and receptors near synapses and secreting factors that promote synaptic maturation. However, the mechanisms that coordinate astrocyte and neural maturation remain poorly understood. Using in vivo imaging in unanesthetized neonatal mice, we show that bursts of neuronal activity passing through nascent sound processing networks reliably induce calcium transients in astrocytes. Astrocyte transients were dependent on intense neuronal activity and constrained to regions near active synapses, ensuring close spatial and temporal coordination of neuron and astrocyte activity. Astrocyte responses were restricted to the pre-hearing period and induced by synergistic activation of two metabotropic glutamate receptors, mGluR5 and mGluR3, which promoted IP3R2-dependent calcium release from intracellular stores. The widespread expression of these receptors by astrocytes during development and the prominence of neuronal burst firing in emerging neural networks may help coordinate the maturation of excitatory synapses.
Asunto(s)
Astrocitos/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Señalización del Calcio/fisiología , Ratones , Sinapsis/fisiologíaRESUMEN
Neurons in developing sensory pathways exhibit spontaneous bursts of electrical activity that are critical for survival, maturation and circuit refinement. In the auditory system, intrinsically generated activity arises within the cochlea, but the molecular mechanisms that initiate this activity remain poorly understood. We show that burst firing of mouse inner hair cells prior to hearing onset requires P2RY1 autoreceptors expressed by inner supporting cells. P2RY1 activation triggers K+ efflux and depolarization of hair cells, as well as osmotic shrinkage of supporting cells that dramatically increased the extracellular space and speed of K+ redistribution. Pharmacological inhibition or genetic disruption of P2RY1 suppressed neuronal burst firing by reducing K+ release, but unexpectedly enhanced their tonic firing, as water resorption by supporting cells reduced the extracellular space, leading to K+ accumulation. These studies indicate that purinergic signaling in supporting cells regulates hair cell excitability by controlling the volume of the extracellular space.
As the brain develops, billions of cells respond to genetic and environmental cues to form the trillions of connections that make up its neural networks. However, before these brain circuits can respond to real life stimuli, their connections are refined by bursts of electrical activity. For example, sensory cells in the ear produce bursts of spontaneous electrical activity that mimic those made by sounds. This activity allows the neural network in the hearing system to 'practice' responding to sounds. However, the origin of these electrical bursts is unusual as they do not start in the sensory cells themselves, but are initiated by the non-sensory cells around them. Past research has shown that as the ear develops these non-sensory cells, or supporting cells, release regular doses of a molecule called ATP. The supporting cells then detect their own ATP release using specialized receptor proteins on their surface. This self-stimulation causes the supporting cells to release potassium ions that interact with the sensory cells and trigger bursts of electrical activity. However, the identity of this ATP-detecting receptor was not known, and without this information it was unclear how the electrical activity starts and why it happens in rhythmic bursts. To fill this knowledge gap, Babola et al. measured electrical activity in ear cells isolated from mice, and examined nerve cell activity in live mice during this critical stage of development. This revealed that the bursts of activity in the ear depend on a receptor called P2RY1 which can be found on the supporting cells located next to sensory cells. When P2RY1 is activated it triggers the release of calcium ions inside the supporting cells. This opens channels in the cell membrane, allowing the potassium ions to flow out and electrically activate the sensory cells. But, when the potassium ions leave the supporting cells, water is drawn out with them, causing the cells to shrink and the space around the cells to get bigger. As a result, the released potassium ions disperse more quickly, moving away from the sensory cells and stopping the burst in electrical activity. Conversely, when P2RY1 is inhibited, this causes the supporting cells to swell, trapping potassium ions near the sensory cells and making them fire continuously. This indicates that bursts in electrical activity are controlled by the rhythmic swelling and shrinking of supporting cells. Although supporting cells cannot detect sound themselves, they seem to play a crucial role in developing the hearing system. A better understanding of these cells could therefore aid research into hearing problems without a known cause such as hypersensitivity to sound, tinnitus, and complex auditory processing disorders in children.
Asunto(s)
Espacio Extracelular/fisiología , Células Ciliadas Auditivas Internas/fisiología , Audición/fisiología , Células Laberínticas de Soporte/fisiología , Receptores Purinérgicos P2Y1/metabolismo , Potenciales de Acción , Animales , Calcio/metabolismo , Femenino , Masculino , Ratones , Neuronas/fisiología , Potasio/metabolismo , Ratas , Receptores Purinérgicos P2Y1/genética , Transducción de Señal , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/fisiologíaRESUMEN
Neurons in the developing auditory system exhibit spontaneous bursts of activity before hearing onset. How this intrinsically generated activity influences development remains uncertain, because few mechanistic studies have been performed in vivo. We show using macroscopic calcium imaging in unanesthetized mice that neurons responsible for processing similar frequencies of sound exhibit highly synchronized activity throughout the auditory system during this critical phase of development. Spontaneous activity normally requires synaptic excitation of spiral ganglion neurons (SGNs). Unexpectedly, tonotopic spontaneous activity was preserved in a mouse model of deafness in which glutamate release from hair cells is abolished. SGNs in these mice exhibited enhanced excitability, enabling direct neuronal excitation by supporting cell-induced potassium transients. These results indicate that homeostatic mechanisms maintain spontaneous activity in the pre-hearing period, with significant implications for both circuit development and therapeutic approaches aimed at treating congenital forms of deafness arising through mutations in key sensory transduction components.