RESUMEN
Long-term memories are believed to be encoded by unique transcriptional signatures in the brain. The expression of immediate early genes (IEG) promotes structural and molecular changes required for memory consolidation. Recent evidence has shown that the brain is equipped with mechanisms that not only promote, but actively constrict memory formation. However, it remains unknown whether IEG expression may play a role in memory suppression. Here we uncovered a novel function of the IEG neuronal PAS domain protein 4 (Npas4), as an inducible memory suppressor gene of highly salient aversive experiences. Using a contextual fear conditioning paradigm, we found that low stimulus salience leads to monophasic Npas4 expression, while highly salient learning induces a biphasic expression of Npas4 in the hippocampus. The later phase requires N-methyl-D-aspartate (NMDA) receptor activity and is independent of dopaminergic neurotransmission. Our in vivo pharmacological and genetic manipulation experiments suggested that the later phase of Npas4 expression restricts the consolidation of a fear memory and promote behavioral flexibility, by facilitating fear extinction and the contextual specificity of fear responses. Moreover, immunofluorescence and electrophysiological analysis revealed a concomitant increase in synaptic input from cholecystokinin (CCK)-expressing interneurons. Our results demonstrate how salient experiences evoke unique temporal patterns of IEG expression that fine-tune memory consolidation. Moreover, our study provides evidence for inducible gene expression associated with memory suppression as a possible mechanism to balance the consolidation of highly salient memories, and thereby to evade the formation of maladaptive behavior.
RESUMEN
The LINC00473 (Lnc473) gene has previously been shown to be associated with cancer and psychiatric disorders. Its expression is elevated in several types of tumors and decreased in the brains of patients diagnosed with schizophrenia or major depression. In neurons, Lnc473 transcription is strongly responsive to synaptic activity, suggesting a role in adaptive, plasticity-related mechanisms. However, the function of Lnc473 is largely unknown. Here, using a recombinant adeno-associated viral vector, we introduced a primate-specific human Lnc473 RNA into mouse primary neurons. We show that this resulted in a transcriptomic shift comprising downregulation of epilepsy-associated genes and a rise in cAMP response element-binding protein (CREB) activity, which was driven by augmented CREB-regulated transcription coactivator 1 nuclear localization. Moreover, we demonstrate that ectopic Lnc473 expression increased neuronal excitability as well as network excitability. These findings suggest that primates may possess a lineage-specific activity-dependent modulator of CREB-regulated neuronal excitability.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Neuronas , Primates , Animales , Humanos , Ratones , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Epilepsia/genética , Neuronas/metabolismo , Primates/genéticaRESUMEN
Differences in the learning associated transcriptional profiles between mouse strains with distinct learning abilities could provide insight into the molecular basis of learning and memory. The inbred mouse strain DBA/2 shows deficits in hippocampus-dependent memory, yet the transcriptional responses to learning and the underlying mechanisms of the impairments are unknown. Comparing DBA/2J mice with the reference standard C57BL/6N mouse strain we verify an enhanced susceptibility to kainic acid induced seizures, confirm impairments in hippocampus-dependent spatial memory tasks and uncover additional behavioral abnormalities including deficits in hippocampus-independent learning. Surprisingly, we found no broad dysfunction of the DBA/2J strain in immediate early gene (IEG) activation but instead report brain region-specific and gene-specific alterations. The learning-associated IEGs Arc, c-Fos, and Nr4a1 showed no DBA/2J deficits in basal or synaptic activity induced gene expression in hippocampal or cortical primary neuronal cultures or in the CA1, CA3, or retrosplenial cortex following spatial object recognition (SOR) training in vivo. However, the parietal cortex showed reduced and the dentate gyrus showed enhanced SOR-evoked induction of most IEGs. All DBA/2J hippocampal regions exhibited elevated basal expression of inhibin ß A (Inhba) and a learning-associated superinduction of the transcription factor neuronal Per-Arnt-Sim domain protein 4 (Npas4) known to regulate the synaptic excitation-inhibition balance. In line with this, CA1 pyramidal neurons of DBA/2J mice showed fewer inhibitory and more excitatory miniature postsynaptic currents but no alteration in most other electrophysiological properties or gross dendritic morphology. The dysregulation of Npas4 and Inhba expression and synaptic connectivity may underlie the cognitive deficits and increased susceptibility to seizures of DBA/2J mice.
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Cognición , Hipocampo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Subunidades beta de Inhibinas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
KEY POINTS: We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be â¼77 nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. ABSTRACT: Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83 nM resulting in a block of more than 82% of ES-NMDARs during a 5 min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.
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Maleato de Dizocilpina/farmacología , Ácido Glutámico/análisis , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Región CA1 Hipocampal/fisiología , Técnicas In Vitro , Técnicas de Placa-Clamp , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinapsis/fisiologíaRESUMEN
Coordinated collective electrochemical signals in multicellular assemblies, such as ion fluxes, membrane potentials, electrical gradients, and steady electric fields, play an important role in cell and tissue spatial organization during many physiological processes like wound healing, inflammatory responses, and hormone release. This mass of electric actions cumulates in an en masse activity within cell collectives which cannot be deduced from considerations at the individual cell level. However, continuously sampling en masse collective electrochemical actions of the global electrochemical activity of large-scale electrically coupled cellular assemblies with intracellular resolution over long time periods has been impeded by a lack of appropriate recording techniques. Here we present a bioelectrical interface consisting of low impedance vertical gold nanoelectrode interfaces able to penetrate the cellular membrane in the course of cellular adhesion, thereby allowing en masse recordings of intracellular electrochemical potentials that transverse electrically coupled NRK fibroblast, C2C12 myotube assemblies, and SH-SY5Y neuronal networks of more than 200,000 cells. We found that the intracellular electrical access of the nanoelectrodes correlates with substrate adhesion dynamics and that penetration, stabilization, and sealing of the electrode-cell interface involves recruitment of surrounding focal adhesion complexes and the anchoring of actin bundles, which form a caulking at the electrode base. Intracellular recordings were stable for several days, and monitoring of both basal activity as well as pharmacologically altered electric signals with high signal-to-noise ratios and excellent electrode coupling was performed.
RESUMEN
Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies.SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.
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Señalización del Calcio/fisiología , Núcleo Celular/fisiología , Dendritas/ultraestructura , Extinción Psicológica/fisiología , Consolidación de la Memoria/fisiología , Plasticidad Neuronal/fisiología , Factor D de Crecimiento Endotelial Vascular/metabolismo , Animales , Calcio , Dendritas/fisiología , Masculino , Recuerdo Mental/fisiología , Ratones , Ratones Endogámicos C57BL , Retención en Psicología/fisiología , Transducción de Señal/fisiologíaRESUMEN
The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by calcium/calmodulin signaling in the nucleus. Recent studies have drawn attention to a new family of transcriptional regulators, the so-called calmodulin-binding transcription activator (CAMTA) proteins. CAMTAs are expressed at particularly high levels in the mouse and human brain, and we reasoned that, as calmodulin-binding transcription factors, CAMTAs may regulate the formation of long-term memory by coupling synaptic activity and calcium/calmodulin signaling to memory-related transcriptional responses. This hypothesis is supported by genetic studies that reported a correlation between Camta gene polymorphisms or mutations and cognitive capability in humans. Here, we show that acute knockdown of CAMTA1, but not CAMTA2, in the hippocampus of adult mice results in impaired performance in two memory tests, contextual fear conditioning and object-place recognition test. Short-term memory and neuronal morphology were not affected by CAMTA knockdown. Gene expression profiling in the hippocampus of control and CAMTA knockdown mice revealed a number of putative CAMTA1 target genes related to synaptic transmission and neuronal excitability. Patch clamp recordings in organotypic hippocampal slice cultures provided further evidence for CAMTA1-dependent changes in electrophysiological properties. In summary, our study provides experimental evidence that confirms previous human genetic studies and establishes CAMTA1 as a regulator of long-term memory formation.
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Proteínas de Unión al Calcio/fisiología , Hipocampo/fisiología , Memoria a Largo Plazo/fisiología , Transactivadores/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Células Cultivadas , Condicionamiento Clásico , Dendritas/fisiología , Miedo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BL , Células Piramidales/citología , Reconocimiento en Psicología , Transmisión Sináptica , Transactivadores/genéticaRESUMEN
Both synaptic N-methyl-d-aspartate (NMDA) receptors and voltage-operated calcium channels (VOCCs) have been shown to be critical for nuclear calcium signals associated with transcriptional responses to bursts of synaptic input. However the direct contribution to nuclear calcium signals from calcium influx through NMDA receptors and VOCCs has been obscured by their concurrent roles in action potential generation and synaptic transmission. Here we compare calcium responses to synaptically induced bursts of action potentials with identical bursts devoid of any synaptic contribution generated using the pre-recorded burst as the voltage clamp command input to replay the burst in the presence of blockers of action potentials or ionotropic glutamate receptors. Synapse independent replays of bursts produced nuclear calcium responses with amplitudes around 70% of their original synaptically generated signals and were abolished by the L-type VOCC blocker, verapamil. These results identify a major direct source of nuclear calcium from local L-type VOCCs whose activation is boosted by NMDA receptor dependent depolarization. The residual component of synaptically induced nuclear calcium signals which was both VOCC independent and NMDA receptor dependent showed delayed kinetics consistent with a more distal source such as synaptic NMDA receptors or internal stores. The dual requirement of NMDA receptors and L-type VOCCs for synaptic activity-induced nuclear calcium dependent transcriptional responses most likely reflects a direct somatic calcium influx from VOCCs whose activation is amplified by synaptic NMDA receptor-mediated depolarization and whose calcium signal is boosted by a delayed input from distal calcium sources mostly likely entry through NMDA receptors and release from internal stores. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
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Potenciales de Acción/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Hipocampo/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Hipocampo/citología , Ratones , Neuronas/citología , Técnicas de Placa-Clamp , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Signal-regulated changes in cell size affect cell division and survival and therefore are central to tissue morphogenesis and homeostasis. In this respect, GABA receptors (GABA(A)Rs) are of particular interest because allowing anions flow across the cell membrane modulates the osmolyte flux and the cell volume. Therefore, we have here investigated the hypothesis that GABA may regulate neural stem cell proliferation by inducing cell size changes. We found that, besides neuroblasts, also neural precursors in the neonatal murine subependymal zone sense GABA via GABA(A) Rs. However, unlike in neuroblasts, where it induced depolarization-mediated [Ca(2+)](i) increase, GABA(A) Rs activation in precursors caused hyperpolarization. This resulted in osmotic swelling and increased surface expression of epidermal growth factor receptors (EGFRs). Furthermore, activation of GABA(A) Rs signaling in vitro in the presence of EGF modified the expression of the cell cycle regulators, phosphatase and tensin homolog and cyclin D1, increasing the pool of cycling precursors without modifying cell cycle length. A similar effect was observed on treatment with diazepam. We also demonstrate that GABA and diazepam responsive precursors represent prominin(+) stem cells. Finally, we show that as in in vitro also in in vivo a short administration of diazepam promotes EGFR expression in prominin(+) stem cells causing activation and cell cycle entry. Thus, our data indicate that endogenous GABA is a part of a regulatory mechanism of size and cell cycle entry of neonatal stem cells. Our results also have potential implications for the therapeutic practices that involve exposure to GABA(A) Rs modulators during neurodevelopment.
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Antígenos CD/metabolismo , Ciclo Celular/efectos de los fármacos , Glicoproteínas/metabolismo , Células-Madre Neurales/metabolismo , Péptidos/metabolismo , Receptores de GABA-A/metabolismo , Transducción de Señal , Antígeno AC133 , Animales , Animales Recién Nacidos , Proliferación Celular , Células Cultivadas , Diazepam/farmacología , Receptores ErbB/biosíntesis , Receptores ErbB/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Presión Osmótica/fisiología , Precursores de Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Calcium is the major intracellular messenger linking synaptic activity in neurons to gene expression to control diverse functions including adaptive responses to synaptic activity as well as survival and death (Bading et al. 1993; Hardingham et al. 1997; Chawla and Bading 2001; West et al. 2001; Zhang et al. 2007; Flavell and Greenberg 2008; Mellstrom et al. 2008; Redmond 2008; Wayman et al. 2008; Bootman et al. 2009; Zhang et al. 2009; Hardingham and Bading 2010). Calcium entry at the synapse acts locally to activate signaling cascades which regulate posttranslational modifications essential for synaptic plasticity, such as the insertion of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic membrane (Soderling 2000; Malinow and Malenka 2002; Ehrlich and Malinow 2004). Synaptic activity can also evoke calcium signals in the nucleus which regulate gene pools largely through the phosphorylation of cAMP response element-binding protein (CREB) and its coactivator, CREB-binding protein (CBP) (Bading et al. 1993; Hardingham et al. 1997; Hardingham et al. 1999; Hu et al. 1999; Hardingham et al. 2001b; Impey et al. 2002; Zhang et al. 2009). Distinct mechanisms have been proposed to mediate synaptically generated calcium signals in subcompartments of pyramidal neurons; N-methyl-D -aspartate receptors (NMDARs) and ryanodine receptors have been implicated in the spine, inositol 3,4,5 triphosphate (IP3) receptors in the dendrites, and L-type voltage-gated calcium channels (VGCCs) at the soma and nucleus, although both NMDARs and IP3 receptors can also contribute to somatic and nuclear calcium signals under certain stimulation conditions (Nakamura et al. 1999; Bardo et al. 2006; Raymond and Redman 2006; Watanabe et al. 2006; Hong and Ross 2007; Hagenston et al. 2008; Bengtson et al. 2010). We review here the calcium signaling pathways underlying synaptically activated gene transcription leading to long-lasting changes in synaptic efficacy and memory as well as the physiological mechanisms by which synaptic activity evokes nuclear calcium signals.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Sinapsis/metabolismo , Animales , Canales de Calcio Tipo L/fisiología , Núcleo Celular/genética , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potenciales de la Membrana , Memoria a Largo Plazo , Neuronas/citología , Neuronas/fisiología , Fosforilación , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sinapsis/genética , Transcripción GenéticaRESUMEN
Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.
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Células Madre Pluripotentes Inducidas , Receptores de N-Metil-D-Aspartato , Encéfalo/metabolismo , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Organoides , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo.
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Región CA1 Hipocampal/metabolismo , Señalización del Calcio , Núcleo Celular/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Células Piramidales/metabolismo , Sinapsis/metabolismo , Potenciales de Acción/fisiología , Animales , Región CA1 Hipocampal/citología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Estimulación Eléctrica , Indicadores y Reactivos , Células Piramidales/citología , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Umbral SensorialRESUMEN
Synaptic activity initiates many adaptive responses in neurons. Here we report a novel form of structural plasticity in dissociated hippocampal cultures and slice preparations. Using a recently developed algorithm for three-dimensional image reconstruction and quantitative measurements of cell organelles, we found that many nuclei from hippocampal neurons are highly infolded and form unequally sized nuclear compartments. Nuclear infoldings are dynamic structures, which can radically transform the geometry of the nucleus in response to neuronal activity. Action potential bursting causing synaptic NMDA receptor activation dramatically increases the number of infolded nuclei via a process that requires the ERK-MAP kinase pathway and new protein synthesis. In contrast, death-signaling pathways triggered by extrasynaptic NMDA receptors cause a rapid loss of nuclear infoldings. Compared with near-spherical nuclei, infolded nuclei have a larger surface and increased nuclear pore complex immunoreactivity. Nuclear calcium signals evoked by cytosolic calcium transients are larger in small nuclear compartments than in the large compartments of the same nucleus; moreover, small compartments are more efficient in temporally resolving calcium signals induced by trains of action potentials in the theta frequency range (5 Hz). Synaptic activity-induced phosphorylation of histone H3 on serine 10 was more robust in neurons with infolded nuclei compared with neurons with near-spherical nuclei, suggesting a functional link between nuclear geometry and transcriptional regulation. The translation of synaptic activity-induced signaling events into changes in nuclear geometry facilitates the relay of calcium signals to the nucleus, may lead to the formation of nuclear signaling microdomains, and could enhance signal-regulated transcription.
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Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Hipocampo/metabolismo , Histonas/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Compartimento Celular/fisiología , Núcleo Celular/ultraestructura , Forma de la Célula/fisiología , Células Cultivadas , Citosol/metabolismo , Hipocampo/ultraestructura , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/ultraestructura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Poro Nuclear/metabolismo , Técnicas de Cultivo de Órganos , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismoRESUMEN
In the adult subventricular zone (SVZ), astroglial stem cells generate transit-amplifying precursors (TAPs). Both stem cells and TAPs form clones in response to epidermal growth factor (EGF). However, in vivo, in the absence of sustained EGF receptor (EGFR) activation, TAPs divide a few times before differentiating into neuroblasts. The lack of suitable markers has hampered the analysis of stem cell lineage progression and associated functional changes in the neonatal germinal epithelium. Here we purified neuroblasts and clone-forming precursors from the neonatal SVZ using expression levels of EGFR and polysialylated neural cell adhesion molecule (PSANCAM). As in the adult SVZ, most neonatal clone-forming precursors did not express the neuroglia proteoglycan 2 (NG2) but displayed characteristics of TAPs, and only a subset exhibited antigenic characteristics of astroglial stem cells. Both precursors and neuroblasts were PSANCAM(+); however, neuroblasts also expressed doublecortin and functional voltage-dependent Ca(2+) channels. Neuroblasts and precursors had distinct outwardly rectifying K(+) current densities and passive membrane properties, particularly in precursors contacting each other, because of the contribution of gap junction coupling. Confirming the hypothesis that most are TAPs, cell tracing in brain slices revealed that within 2 days the majority of EGFR(+) cells had exited the cell cycle and differentiated into a progenitor displaying intermediate antigenic and functional properties between TAPs and neuroblasts. Thus, distinct functional and antigenic properties mark stem cell lineage progression in the neonatal SVZ.
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Encéfalo/citología , Linaje de la Célula , Neurogénesis/fisiología , Neuronas/citología , Células Madre/citología , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Encéfalo/metabolismo , Diferenciación Celular , Receptores ErbB/metabolismo , Citometría de Flujo , Inmunohistoquímica , Ratones , Neuronas/metabolismo , Técnicas de Placa-Clamp , Proteoglicanos/metabolismoRESUMEN
Excitotoxicity induced by NMDA receptors (NMDARs) is thought to be intimately linked to high intracellular calcium load. Unexpectedly, NMDAR-mediated toxicity can be eliminated without affecting NMDAR-induced calcium signals. Instead, excitotoxicity requires physical coupling of NMDARs to TRPM4. This interaction is mediated by intracellular domains located in the near-membrane portions of the receptors. Structure-based computational drug screening using the interaction interface of TRPM4 in complex with NMDARs identified small molecules that spare NMDAR-induced calcium signaling but disrupt the NMDAR/TRPM4 complex. These interaction interface inhibitors strongly reduce NMDA-triggered toxicity and mitochondrial dysfunction, abolish cyclic adenosine monophosphate-responsive element-binding protein (CREB) shutoff, boost gene induction, and reduce neuronal loss in mouse models of stroke and retinal degeneration. Recombinant or small-molecule NMDAR/TRPM4 interface inhibitors may mitigate currently untreatable human neurodegenerative diseases.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Descubrimiento de Drogas , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/química , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Señalización del Calcio , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Enfermedades Neurodegenerativas/tratamiento farmacológico , Dominios Proteicos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Canales Catiónicos TRPM/genética , Activación TranscripcionalRESUMEN
Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.
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Antivirales/farmacología , Aprobación de Drogas , Microscopía , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Quinolonas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , United States Food and Drug Administration , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Channelrhodopsins/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Optogenética , Receptores AMPA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Estados UnidosRESUMEN
The morphology of dendritic arbors determines the location, strength and interaction of synaptic inputs. It is therefore important to understand the factors regulating dendritic arborization both during development and in situations of physiological or pathological plasticity. We have recently shown that VEGF-D (Vascular Endothelial Growth Factor D) is required to maintain length and complexity of basal dendrites in mouse hippocampal pyramidal cells. Lack of VEGF-D resulted in long-term memory deficits, suggesting a link between dendritic morphology and cognitive function. Here, we compared the effect of VEGF-D expression on basal versus apical dendrites of CA1 pyramidal cells, as well as its importance for synaptic processing of network oscillations. We report opposing, layer-specific effects of VEGF-D knockdown which resulted in shrinkage of basal and increased complexity of apical dendrites. Synaptic potentials and layer-specific voltage gradients during network oscillations remained, however, unaltered. These findings reveal a high spatial selectivity of VEGF-D effects at the sub-cellular level, and strong homeostatic mechanisms which keep spatially segregated synaptic inputs in a balance.
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Células Piramidales , Factor D de Crecimiento Endotelial Vascular , Animales , Dendritas , Regulación hacia Abajo , Hipocampo , RatonesRESUMEN
BACKGROUND: The magnitude and longevity of synaptic activity-induced changes in synaptic efficacy is quantified by measuring evoked responses whose potentiation requires gene transcription to persist for more than 2-3 hours. While miniature EPSCs (mEPSCs) are also increased in amplitude and/or frequency during long-term potentiation (LTP), it is not known how long such changes persist or whether gene transcription is required. RESULTS: We use whole-cell patch clamp recordings from dissociated hippocampal cultures to characterise for the first time the persistence and transcription dependency of mEPSC upregulation during synaptic potentiation. The persistence of recurrent action potential bursting in these cultures is transcription-, translation- and NMDA receptor-dependent thus providing an accessible model for long-lasting plasticity. Blockade of GABAA-receptors with bicuculline for 15 minutes induced action potential bursting in all neurons and was maintained in 50-60% of neurons for more than 6 hours. Throughout this period, the frequency but neither the amplitude of mEPSCs nor whole-cell AMPA currents was markedly increased. The transcription blocker actinomycin D abrogated, within 2 hours of burst induction, both action potential bursting and the increase in mEPSCs. Reversible blockade of action potentials during, but not after this 2 hour transcription period suppressed the increase in mEPSC frequency and the recovery of burst activity at a time point 6 hours after induction. CONCLUSION: These results indicate that increased mEPSC frequency persists well beyond the 2 hour transcription-independent phase of plasticity in this model. This long-lasting mEPSC upregulation is transcription-dependent and requires ongoing action potential activity during the initial 2 hour period but not thereafter. Thus mEPSC upregulation may underlie the long term, transcription-dependent persistence of action potential bursting. This provides mechanistic insight to link gene candidates already identified by gene chip analysis to long lasting plasticity in this in vitro model.
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Potenciales Postsinápticos Excitadores/genética , Hipocampo/fisiología , Potenciales Postsinápticos Miniatura/genética , Plasticidad Neuronal/genética , Neuronas/fisiología , Transcripción Genética/fisiología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Potenciales Postsinápticos Miniatura/fisiología , Red Nerviosa/metabolismo , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Factores de TiempoRESUMEN
Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates gamma-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca2+ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons.
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Glutamato Descarboxilasa/biosíntesis , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Células Madre/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Potenciales de Acción/fisiología , Animales , Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Femenino , Glutamato Descarboxilasa/genética , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Embarazo , Células Madre/citología , Ácido gamma-Aminobutírico/genéticaRESUMEN
BACKGROUND: Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death. RESULTS: Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting. CONCLUSION: The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.