RESUMEN
Survival of animals is dependent on the correct selection of an appropriate behavioral response to competing external stimuli. Theoretical models have been proposed and underlying mechanisms are emerging to explain how one circuit is selected among competing neural circuits. The evolutionarily conserved forebrain to midbrain habenulo-interpeduncular nucleus (Hb-IPN) pathway consists of cholinergic and non-cholinergic neurons, which mediate different aversive behaviors. Simultaneous calcium imaging of neuronal cell bodies and of the population dynamics of their axon terminals reveals that signals in the cell bodies are not reflective of terminal activity. We find that axon terminals of cholinergic and non-cholinergic habenular neurons exhibit stereotypic patterns of spontaneous activity that are negatively correlated and localize to discrete subregions of the target IPN. Patch-clamp recordings show that calcium bursts in cholinergic terminals at the ventral IPN trigger excitatory currents in IPN neurons, which precede inhibition of non-cholinergic terminals at the adjacent dorsal IPN. Inhibition is mediated through presynaptic GABAB receptors activated in non-cholinergic habenular neurons upon GABA release from the target IPN. Together, the results reveal a hardwired mode of competition at the terminals of two excitatory neuronal populations, providing a physiological framework to explore the relationship between different aversive responses.
Asunto(s)
Habénula , Terminales Presinápticos , Animales , Calcio/metabolismo , Colinérgicos/metabolismo , Habénula/fisiología , Terminales Presinápticos/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
In the developing central nervous system, electrical signaling is thought to rely exclusively on differentiating neurons as they acquire the ability to generate and propagate action potentials. Accordingly, neuroepithelial progenitors (NEPs), which give rise to all neurons and glial cells during development, have been reported to remain electrically passive. Here, we investigated the physiological properties of NEPs at the onset of spontaneous neural activity (SNA) initiating motor behavior in mouse embryonic spinal cord. Using patch-clamp recordings, we discovered that spinal NEPs exhibit spontaneous membrane depolarizations during episodes of SNA. These rhythmic depolarizations exhibited a ventral-to-dorsal gradient with the highest amplitude located in the floor plate, the ventral-most part of the neuroepithelium. Paired recordings revealed that NEPs are coupled via gap junctions and form an electrical syncytium. Although other NEPs were electrically passive, we discovered that floor-plate NEPs generated large Na+/Ca2+ action potentials. Unlike in neurons, floor-plate action potentials relied primarily on the activation of voltage-gated T-type calcium channels (TTCCs). In situ hybridization showed that all 3 known subtypes of TTCCs are predominantly expressed in the floor plate. During SNA, we found that acetylcholine released by motoneurons rhythmically triggers floor-plate action potentials by acting through nicotinic acetylcholine receptors. Finally, by expressing the genetically encoded calcium indicator GCaMP6f in the floor plate, we demonstrated that neuroepithelial action potentials are associated with calcium waves and propagate along the entire length of the spinal cord. Our work reveals a novel physiological mechanism to generate and propagate electrical signals across a neural structure independently from neurons.
Asunto(s)
Neuronas Motoras , Médula Espinal , Potenciales de Acción/fisiología , Animales , Canales de Calcio , Uniones Comunicantes , Ratones , Neuronas Motoras/fisiología , Médula Espinal/fisiologíaRESUMEN
Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical systems theory, we analyzed, for the first time, the mechanisms underlying the electrophysiological properties of V1R during early embryonic development of the mouse spinal cord locomotor networks (E11.5-E16.5). We found that these interneurons are subdivided into several functional clusters from E11.5 and then display an unexpected transitory involution process during which they lose their ability to sustain tonic firing. We demonstrated that the essential factor controlling the diversity of the discharge pattern of embryonic V1R is the ratio of a persistent sodium conductance to a delayed rectifier potassium conductance. Taken together, our results reveal how a simple mechanism, based on the synergy of two voltage-dependent conductances that are ubiquitous in neurons, can produce functional diversity in embryonic V1R and control their early developmental trajectory.
Asunto(s)
Potenciales de Acción , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Potasio/metabolismo , Células de Renshaw/metabolismo , Canales de Sodio/metabolismo , Sodio/metabolismo , Médula Espinal/metabolismo , Animales , Femenino , Glutamato Descarboxilasa/genética , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones Transgénicos , Modelos Neurológicos , Morfogénesis , Fenotipo , Médula Espinal/embriología , Teoría de Sistemas , Factores de TiempoRESUMEN
While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase (chata), vesicular acetylcholine transporters (vachta, vachtb), high-affinity choline transporter (hacta) and acetylcholinesterase (ache), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which continued during development. On the contrary, nAChR subunits remained highly expressed throughout the spinal cord. We found that protein and enzymatic activities of presynaptic cholinergic genes were also reduced in the rostral spinal cord. Our work demonstrating that cholinergic genes are initially expressed in the embryonic spinal cord, which is dynamically downregulated during development suggests that cholinergic signaling may play a pivotal role during the formation of intra-spinal locomotor circuit.
Asunto(s)
Sistema Nervioso Central/embriología , Regulación del Desarrollo de la Expresión Génica , Médula Espinal/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Sistema Nervioso Central/metabolismo , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Embrión no Mamífero , Larva/metabolismo , Neuronas Motoras/metabolismo , Neuronas/fisiología , Neurotransmisores/metabolismo , Médula Espinal/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Astrocytes are a major type of glial cell in the mammalian brain, essentially regulating neuronal development and function. Quantitative imaging represents an important approach to study astrocytic signaling in neural circuits. Focusing on astrocytic Ca2+ activity, a key pathway implicated in astrocye-neuron interaction, we here report a strategy combining fast light sheet fluorescence microscopy (LSFM) and correlative screening-based time series analysis, to map activity domains in astrocytes in living mammalian nerve tissue. Light sheet of micron-scale thickness enables wide-field optical sectioning to image astrocytes in acute mouse brain slices. Using both chemical and genetically encoded Ca2+ indicators, we demonstrate the complementary advantages of LSFM in mapping Ca2+ domains in astrocyte populations as compared to epifluorescence and two-photon microscopy. Our approach then revealed distinct kinetics of Ca2+ signals between cortical and hypothalamic astrocytes in resting conditions and following the activation of adrenergic G protein coupled receptor (GPCR). This observation highlights the activity heterogeneity across regionally distinct astrocyte populations, and indicates the potential of our method for investigating dynamic signals in astrocytes.
Asunto(s)
Astrocitos/fisiología , Encéfalo/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Animales , Ratones , Microscopía Fluorescente , Neuronas/fisiologíaRESUMEN
Microglia are known to regulate several aspects of the development of the central nervous system. When microglia colonize the spinal cord, from E11.5 in the mouse embryo, they interact with growing central axons of dorsal root ganglion sensory neurons (SNs), which suggests that they may have some functions in SN development. To address this issue, we analyzed the effects of embryonic macrophage ablation on the early development of SNs using mouse embryo lacking embryonic macrophages (PU.1 knock-out mice) and immune cell ablation. We discovered that, in addition to microglia, embryonic macrophages contact tropomyosin receptor kinase (Trk) C+ SN, TrkB+ SN, and TrkA+ SN peripheral neurites from E11.5. Deprivation of immune cells resulted in an initial reduction of TrkC+ SN and TrkB+ SN populations at E11.5 that was unlikely to be related to an alteration in their developmental cell death (DCD), followed by a transitory increase in their number at E12.5. It also resulted in a reduction of TrkA+ SN number during the developmental period analyzed (E11.5-E15.5), although we did not observe any change in their DCD. Proliferation of cells negative for brain fatty acid-binding protein (BFABP- ), which likely correspond to neuronal progenitors, was increased at E11.5, while their proliferation was decreased at E12.5, which could partly explain the alterations of SN subtype production observed from E11.5. In addition, we observed alterations in the proliferation of glial cell progenitors (BFABP+ cells) in the absence of embryonic macrophages. Our data indicate that embryonic macrophages and microglia ablation alter the development of SNs.
Asunto(s)
Ganglios Espinales/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Macrófagos/metabolismo , Microglía/metabolismo , Células Receptoras Sensoriales/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Muerte Celular , Citocinas/metabolismo , Embrión de Mamíferos , Femenino , Galectina 3/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 µm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.
Asunto(s)
Neuronas GABAérgicas/fisiología , Red Nerviosa/fisiología , Células de Renshaw/fisiología , Canales de Sodio/fisiología , Sodio/fisiología , Médula Espinal/embriología , Potenciales de Acción , Animales , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/citología , Comunicación Paracrina , Técnicas de Placa-Clamp , Riluzol/farmacología , Médula Espinal/citología , Sinapsis/fisiologíaRESUMEN
Virtually all oligodendrocyte precursors cells (OPCs) receive glutamatergic and/or GABAergic synapses that are lost upon their differentiation into oligodendrocytes in the postnatal and adult brain. Although OPCs are generated at mid-embryonic stages, several weeks before the onset of myelination, it remains unknown when and where OPCs receive their first synapses and become susceptible to the influence of neuronal activity. In the embryonic spinal cord, neuro-epithelial precursors in the pMN domain cease generating cholinergic motor neurons (MNs) to produce OPCs when the first synapses are formed in the ventral-lateral marginal zone. We discovered that when the first synapses form onto MNs, axoglial synapses also form onto the processes of neuro-epithelial precursors located in the marginal zone as they differentiate into OPCs. After leaving the neuro-epithelium, these pioneer OPCs preferentially accumulate in the marginal zone where they are contacted by functional glutamatergic and GABAergic synapses. Spontaneous activity of these axoglial synapses was significantly potentiated by cholinergic signaling acting through presynaptic nicotinic acetylcholine receptors. Moreover, we discovered that chronic nicotine treatment significantly increases early OPC proliferation and density in the marginal zone. Our results demonstrate that OPCs are contacted by functional synapses as soon as they emerge from their precursor domain and that embryonic spinal cord colonization by OPCs can be regulated by cholinergic signaling acting onto these axoglial synapses.
Asunto(s)
Axones/metabolismo , Células Precursoras de Oligodendrocitos/citología , Oligodendroglía/metabolismo , Sinapsis/patología , Animales , Diferenciación Celular/fisiología , Ratones , Neuronas Motoras/metabolismo , Neurogénesis/fisiología , Médula Espinal/metabolismo , Células Madre/fisiología , Sinapsis/fisiologíaRESUMEN
A remarkable feature of early neuronal networks is their endogenous ability to generate spontaneous rhythmic electrical activity independently of any external stimuli. In the mouse embryonic SC, this activity starts at an embryonic age of â¼ 12 d and is characterized by bursts of action potentials recurring every 2-3 min. Although these bursts have been extensively studied using extracellular recordings and are known to play an important role in motoneuron (MN) maturation, the mechanisms driving MN activity at the onset of synaptogenesis are still poorly understood. Because only cholinergic antagonists are known to abolish early spontaneous activity, it has long been assumed that spinal cord (SC) activity relies on a core network of MNs synchronized via direct cholinergic collaterals. Using a combination of whole-cell patch-clamp recordings and extracellular recordings in E12.5 isolated mouse SC preparations, we found that spontaneous MN activity is driven by recurrent giant depolarizing potentials. Our analysis reveals that these giant depolarizing potentials are mediated by the activation of GABA, glutamate, and glycine receptors. We did not detect direct nAChR activation evoked by ACh application on MNs, indicating that cholinergic inputs between MNs are not functional at this age. However, we obtained evidence that the cholinergic dependency of early SC activity reflects a presynaptic facilitation of GABA and glutamate synaptic release via nicotinic AChRs. Our study demonstrates that, even in its earliest form, the activity of spinal MNs relies on a refined poly-synaptic network and involves a tight presynaptic cholinergic regulation of both GABAergic and glutamatergic inputs.
Asunto(s)
Acetilcolina/metabolismo , Potenciales de Acción/fisiología , Uniones Comunicantes/fisiología , Ácido Glutámico/metabolismo , Glicina/metabolismo , Neuronas Motoras/fisiología , Red Nerviosa/fisiología , Médula Espinal/citología , Ácido gamma-Aminobutírico/metabolismo , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Colinérgicos/farmacología , Embrión de Mamíferos , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Ácido Glutámico/farmacología , Glicina/farmacología , Proteínas de Homeodominio/genética , Técnicas In Vitro , Ratones , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Red Nerviosa/efectos de los fármacos , Embarazo , Tetrodotoxina/farmacología , Factores de Transcripción/genética , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and ß4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.
Asunto(s)
Lateralidad Funcional/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Habénula/fisiología , Modelos Animales , Tegmento Mesencefálico/fisiología , Acetilcolina/metabolismo , Animales , Secuencia de Bases , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Cartilla de ADN/genética , Estimulación Eléctrica , Habénula/metabolismo , Hibridación in Situ , Larva/fisiología , Datos de Secuencia Molecular , Vías Nerviosas/fisiología , Optogenética , Técnicas de Placa-Clamp , Receptores Nicotínicos/metabolismo , Análisis de Secuencia de ARN , Tegmento Mesencefálico/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Pez CebraRESUMEN
We found that, during the formation of the mouse barrel cortex, NG2 cells received glutamatergic synapses from thalamocortical fibers and preferentially accumulated along septa separating the barrels. Sensory deprivation reduced thalamocortical inputs on NG2 cells and increased their proliferation, leading to a more uniform distribution in the deprived barrels. Thus, early sensory experience regulates thalamocortical innervation on NG2 cells, as well as their proliferation and distribution during development.
Asunto(s)
Células-Madre Neurales/fisiología , Corteza Somatosensorial/fisiología , Animales , Recuento de Células , Proliferación Celular , Proteínas de Unión al ADN , Oscuridad , Potenciales Postsinápticos Excitadores/fisiología , Glutamatos/fisiología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Fibras Nerviosas/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Oligodendroglía/fisiología , Técnicas de Placa-Clamp , Tálamo/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/fisiología , Vibrisas/inervación , Vibrisas/fisiologíaRESUMEN
Microglia are known to invade the mammalian spinal cord (SC) at an early embryonic stage. While the mechanisms underlying this early colonization of the nervous system are still unknown, we recently found that it is associated, at least partially, with the ability of microglia to proliferate at the onset of motoneuron developmental cell death and of synaptogenesis in mouse embryo (E13.5). In vitro studies have shown that the proliferation and activation of adult microglia can be influenced by the purinergic ionotropic receptor P2X7 via a coupling with Pannexin-1. By performing patch-clamp recordings in situ using a whole-mouse embryonic SC preparation, we show here that embryonic microglia already express functional P2X7R. P2X7R activation evoked a biphasic current in embryonic microglia, which is supposed to reflect large plasma membrane pore opening. However, although embryonic microglia express pannexin-1, this biphasic current was still recorded in microglia of pannexin-1 knock-out embryos, indicating that it rather reflected P2X7R intrinsic pore dilatation. More important, we found that proliferation of embryonic SC microglia, but not their activation state, depends almost entirely on P2X7R by comparing wild-type and P2X7R-/- embryos. Absence of P2X7R led also to a decrease in microglia density. Pannexin-1-/- embryos did not exhibit any difference in microglial proliferation, showing that the control of embryonic microglial proliferation by P2X7R does not depend on pannexin-1 expression. These results reveal a developmental role of P2X7R by controlling embryonic SC microglia proliferation at a critical developmental state in the SC of mouse embryos.
Asunto(s)
Diferenciación Celular/fisiología , Conexinas/metabolismo , Microglía/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Médula Espinal/citología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Antígenos CD/metabolismo , Biofisica , Receptor 1 de Quimiocinas CX3C , Caspasa 3/metabolismo , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Conexinas/deficiencia , Estimulación Eléctrica , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Antígeno Ki-67/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Técnicas de Placa-Clamp , Agonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , ARN Mensajero/metabolismo , Receptores de Quimiocina/genética , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Colorantes de Rosanilina , Médula Espinal/crecimiento & desarrolloRESUMEN
The cellular and molecular mechanisms that govern the response of the perinatal brain to injury remain largely unexplored. We investigated the role of white matter astrocytes in a rodent model of diffuse white matter injury produced by exposing neonatal mice to chronic hypoxia-a paradigm that mimics brain injury in premature infants. We demonstrate the absence of reactive gliosis in the immature white matter following chronic hypoxia, as determined by astrocyte proliferation index and glial fibrillary acidic protein levels. Instead, Nestin expression in astrocytes is transiently increased, and the glial-specific glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) are reduced. Finally, we demonstrate that Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling-which is important in both astrocyte development and response to injury-is reduced in the white matter following hypoxia, as well as in primary astrocytes exposed to hypoxia in vitro. Hypoxia and JAK/STAT inhibition reduce glutamate transporter expression in astrocytes, but unlike hypoxia JAK/STAT inhibition downregulates GLAST expression without affecting GLT-1, as demonstrated in vitro by treatment with JAK inhibitor I and in vivo by treatment with the JAK/STAT inhibitor AG490 [(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide]. Our findings (1) demonstrate specific changes in astrocyte function after perinatal hypoxia, which might contribute to the particular pathogenesis of perinatal white matter injury, (2) provide evidence that at least part of these changes result from a disturbance of the JAK/STAT pathway by hypoxia, and (3) identify JAK/STAT signaling as a potential therapeutic target to restore normal GLAST expression and uptake of glutamate after perinatal brain injury.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Astrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Hipoxia/patología , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Ácido Aspártico/metabolismo , Bromodesoxiuridina/metabolismo , Recuento de Células/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/genética , Gliosis/etiología , Gliosis/metabolismo , Gliosis/patología , Proteínas Fluorescentes Verdes/genética , Proteínas de Filamentos Intermediarios/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tritio/metabolismo , Tirfostinos/farmacologíaRESUMEN
During embryogenesis, the pallial-subpallial boundary (PSB) divides the two main progenitor domains in the telencephalon: the pallium, the major source of excitatory neurons, and the subpallium, the major source of inhibitory neurons. The PSB is formed at the molecular interface between the pallial (high Pax6+) and subpallial (high Gsx2+) ventricular zone (VZ) compartments. Initially, the PSB contains cells that express both Pax6 and Gsx2, but during later stages of development this boundary is largely refined into two separate compartments. In this study we examined the developmental mechanisms underlying PSB boundary formation and the postnatal consequences of conditional loss of Pax6 function at the PSB on neuronal fate in the amygdala and olfactory bulb, two targets of PSB-derived migratory populations. Our cell fate and time-lapse imaging analyses reveal that the sorting of Pax6+ and Gsx2+ progenitors during embryogenesis is the result of a combination of changes in gene expression and cell movements. Interestingly, we find that in addition to giving rise to inhibitory neurons in the amygdala and olfactory bulb, Gsx2+ progenitors generate a subpopulation of amygdala excitatory neurons. Consistent with this finding, targeted conditional ablation of Pax6 in Gsx2+ progenitors results in discrete local embryonic patterning defects that are linked to changes in the generation of subsets of postnatal excitatory and inhibitory neurons in the amygdala and inhibitory neurons in the olfactory bulb. Thus, in PSB progenitors, Pax6 plays an important role in the generation of multiple subtypes of neurons that contribute to the amygdala and olfactory bulb.
Asunto(s)
Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Sistema Límbico/citología , Sistema Límbico/crecimiento & desarrollo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Animales Recién Nacidos , Proteínas Bacterianas/genética , Embrión de Mamíferos , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas Luminiscentes/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas , Neuronas/clasificación , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Técnicas de Placa-Clamp , Proteínas Represoras/genética , Telencéfalo , Imagen de Lapso de Tiempo/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
It has been 10 years since the seminal work of Dwight Bergles and collaborators demonstrated that NG2 (nerve/glial antigen 2)-expressing oligodendrocyte progenitor cells (NG2 cells) receive functional glutamatergic synapses from neurons (Bergles et al., 2000), contradicting the old dogma that only neurons possess the complex and specialized molecular machinery necessary to receive synapses. While this surprising discovery may have been initially shunned as a novelty item of undefined functional significance, the study of neuron-to-NG2 cell neurotransmission has since become a very active and exciting field of research. Many laboratories have now confirmed and extended the initial discovery, showing for example that NG2 cells can also receive inhibitory GABAergic synapses (Lin and Bergles, 2004) or that neuron-to-NG2 cell synaptic transmission is a rather ubiquitous phenomenon that has been observed in all brain areas explored so far, including white matter tracts (Kukley et al., 2007; Ziskin et al., 2007; Etxeberria et al., 2010). Thus, while still being in its infancy, this field of research has already brought many surprising and interesting discoveries, and has become part of a continuously growing effort in neuroscience to re-evaluate the long underestimated role of glial cells in brain function (Barres, 2008). However, this area of research is now reaching an important milestone and its long-term significance will be defined by its ability to uncover the still elusive function of NG2 cells and their synapses in the brain, rather than by its sensational but transient successes at upsetting the old order established by neuronal physiology. To participate in the effort to facilitate such a transition, here we propose a critical review of the latest findings in the field of NG2 cell physiology--discussing how they inform us on the possible function(s) of NG2 cells in the brain--and we present some personal views on new directions the field could benefit from in order to achieve lasting significance.
Asunto(s)
Antígenos/metabolismo , Oligodendroglía/fisiología , Proteoglicanos/metabolismo , Células Madre/fisiología , Animales , Calcio/metabolismo , Diferenciación Celular , Humanos , Vaina de Mielina/fisiología , Regeneración Nerviosa/fisiología , Neuronas/fisiología , Sinapsis/metabolismoRESUMEN
BACKGROUND: The mammalian amygdala is composed of two primary functional subdivisions, classified according to whether the major output projection of each nucleus is excitatory or inhibitory. The posterior dorsal and ventral subdivisions of the medial amygdala, which primarily contain inhibitory output neurons, modulate specific aspects of innate socio-sexual and aggressive behaviors. However, the development of the neuronal diversity of this complex and important structure remains to be fully elucidated. RESULTS: Using a combination of genetic fate-mapping and loss-of-function analyses, we examined the contribution and function of Sonic hedgehog (Shh)-expressing and Shh-responsive (Nkx2-1+ and Gli1+) neurons in the medial amygdala. Specifically, we found that Shh- and Nkx2-1-lineage cells contribute differentially to the dorsal and ventral subdivisions of the postnatal medial amygdala. These Shh- and Nkx2-1-lineage neurons express overlapping and non-overlapping inhibitory neuronal markers, such as Calbindin, FoxP2, nNOS and Somatostatin, revealing diverse fate contributions in discrete medial amygdala nuclear subdivisions. Electrophysiological analysis of the Shh-derived neurons additionally reveals an important functional diversity within this lineage in the medial amygdala. Moreover, inducible Gli1CreER(T2) temporal fate mapping shows that early-generated progenitors that respond to Shh signaling also contribute to medial amygdala neuronal diversity. Lastly, analysis of Nkx2-1 mutant mice demonstrates a genetic requirement for Nkx2-1 in inhibitory neuronal specification in the medial amygdala distinct from the requirement for Nkx2-1 in cerebral cortical development. CONCLUSIONS: Taken together, these data reveal a differential contribution of Shh-expressing and Shh-responding cells to medial amygdala neuronal diversity as well as the function of Nkx2-1 in the development of this important limbic system structure.
Asunto(s)
Amígdala del Cerebelo/embriología , Amígdala del Cerebelo/metabolismo , Proteínas Hedgehog/genética , Neurogénesis/genética , Neuronas/metabolismo , Células Madre/metabolismo , Amígdala del Cerebelo/citología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural/genética , Neuronas/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Madre/citología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
We found that demyelinated axons formed functional glutamatergic synapses onto adult-born NG2(+) oligodendrocyte progenitor cells (OPCs) migrating from the subventricular zone after focal demyelination of adult mice corpus callosum. This glutamatergic input was substantially reduced compared with endogenous callosal OPCs 1 week after lesion and was lost on differentiation into oligodendrocytes. Therefore, axon-oligodendrocyte progenitor synapse formation is a transient and regulated step that occurs during remyelination of callosal axons.
Asunto(s)
Células Madre Adultas/fisiología , Cuerpo Calloso/fisiopatología , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Nicho de Células Madre/fisiología , Sinapsis/fisiología , Animales , Axones/fisiología , Diferenciación Celular , Movimiento Celular , Cuerpo Calloso/lesiones , Potenciales Postsinápticos Excitadores , Ácido Glutámico/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Neuronas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Progenitor cells expressing the proteoglycan NG2 represent approximately 5% of the total cells in the adult brain, and are found both in grey and white matter regions where they give rise to oligodendrocytes. The finding that these cells receive synaptic contacts from excitatory and inhibitory neurons has not only raised major interest in the possible roles of these synapses, but also stimulated further research on the developmental and cellular functions of NG2-expressing (NG2(+)) progenitors themselves in the context of neural circuit physiology. Here we review recent findings on the functional properties of the synapses on NG2(+) cells in grey and white matter regions of the brain. In this review article we make an attempt to integrate current knowledge on the cellular and developmental properties of NG2(+) progenitors with the functional attributes of their synapses, in order to understand the physiological relevance of neuron-NG2(+) progenitor signal transmission. We propose that, although NG2(+) progenitors receive synaptic contact in all brain regions where they are found, their synapses might have different developmental and functional roles, probably reflecting the distinct functions of NG2(+) progenitors in the brain.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/fisiología , Antígenos/metabolismo , Modelos Neurológicos , Neuronas/citología , Neuronas/fisiología , Proteoglicanos/metabolismo , Sinapsis/fisiología , Adulto , Animales , Diferenciación Celular , Proliferación Celular , Humanos , Neurotransmisores/metabolismo , Sinapsis/ultraestructuraRESUMEN
Several studies have provided evidence that NG2-expressing (NG2(+)) progenitor cells are anatomically associated to neurons in gray matter areas. By analyzing the spatial distribution of NG2(+) cells in the hilus of the mouse dentate gyrus, we demonstrate that NG2(+) cells are indeed closely associated to interneurons. To define whether this anatomical proximity reflected a specific physiological interaction, we performed patch-clamp recordings on hilar NG2(+) cells and interneurons between 3 and 21 postnatal days. We first observed that hilar NG2(+) cells exhibit spontaneous glutamatergic EPSCs (sEPSCs) whose frequency and amplitude increase during the first 3 postnatal weeks. At the same time, the rise time and decay time of sEPSCs significantly decreased, suggesting that glutamatergic synapses in NG2(+) cells undergo a maturation process that is reminiscent of what has been reported in neurons during the same time period. We also observed that hilar interneurons and associated NG2(+) cells are similarly integrated into the local network, receiving excitatory inputs from both granule cells and CA3 pyramidal neurons. By performing pair recordings, we found that bursts of activity induced by GABAergic antagonists were strongly synchronized between both cell types and that the amplitude of these bursts was positively correlated. Finally, by applying carbachol to increase EPSC activity, we observed that closely apposed cells were more likely to exhibit synchronized EPSCs than cells separated by >200 microm. The finding that NG2(+) cells are sensing patterns of activity arising in closely associated neurons suggests that NG2(+) cell function is finely regulated by the local network.