RESUMEN
Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.
Asunto(s)
Encéfalo/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Biosíntesis de Proteínas , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Células Cultivadas , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Alineación de Secuencia , SinapsisRESUMEN
New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits throughout adulthood. Although the addition of these new neurons may facilitate the formation of new memories, as they integrate, they provide additional excitatory drive to CA3 pyramidal neurons. During development, to maintain homeostasis, new neurons form preferential contacts with local inhibitory circuits. Using retroviral and transgenic approaches to label adult-generated granule cells, we first asked whether a comparable process occurs in the adult hippocampus in mice. Similar to development, we found that, during adulthood, new neurons form connections with inhibitory cells in the dentate gyrus, hilus, and CA3 regions as they integrate into hippocampal circuits. In particular, en passant bouton and filopodia connections with CA3 interneurons peak when adult-generated dentate granule cells (DGCs) are â¼4 weeks of age, a time point when these cells are most excitable. Consistent with this, optical stimulation of 4-week-old (but not 6- or 8-week-old) adult-generated DGCs strongly activated CA3 interneurons. Finally, we found that CA3 interneurons were activated robustly during learning and that their activity was strongly coupled with activity of 4-week-old (but not older) adult-generated DGCs. These data indicate that, as adult-generated neurons integrate into hippocampal circuits, they transiently form strong anatomical, effective, and functional connections with local inhibitory circuits in CA3. Significance statement: New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits throughout adulthood. Understanding how these cells integrate within well formed circuits will increase our knowledge about the basic principles governing circuit assembly in the adult hippocampus. This study uses a combined connectivity analysis (anatomical, functional, and effective) of the output connections of adult-born hippocampal cells to show that, as these cells integrate into hippocampal circuits, they transiently form strong connections with local inhibitory circuits. This transient increase of connectivity may represent an homeostatic process necessary to accommodate changes in the excitation/inhibition balance induced by the addition of these new excitatory cells to the preexisting excitatory hippocampal circuits.
Asunto(s)
Hipocampo/citología , Neurogénesis/fisiología , Neuronas/citología , Neuronas/fisiología , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Envejecimiento , Animales , Hipocampo/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , OptogenéticaRESUMEN
KIF1Bß is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bß isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bß (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bß localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bß is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bß forms RNA-protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bß and Calmodulin is Ca(+2)-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bß at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bß is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bß, its interaction with Ca(2+) sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.
Asunto(s)
Sistema Nervioso Central/metabolismo , Dendritas/metabolismo , Cinesinas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Neuronas Motoras/metabolismo , Ribonucleoproteínas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular Tumoral , Humanos , Cinesinas/genética , Ratones , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/etiología , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptores de Glutamato/metabolismo , Transducción de SeñalRESUMEN
Across systems, higher-order interactions between components govern emergent dynamics. Here we tested whether contextual threat memory retrieval in mice relies on higher-order interactions between dorsal CA1 hippocampal neurons requiring learning-induced dendritic spine plasticity. We compared population-level Ca2+ transients as wild-type mice (with intact learning-induced spine plasticity and memory) and amnestic mice (TgCRND8 mice with high levels of amyloid-ß and deficits in learning-induced spine plasticity and memory) were tested for memory. Using machine-learning classifiers with different capacities to use input data with complex interactions, our findings indicate complex neuronal interactions in the memory representation of wild-type, but not amnestic, mice. Moreover, a peptide that partially restored learning-induced spine plasticity also restored the statistical complexity of the memory representation and memory behavior in Tg mice. These findings provide a previously missing bridge between levels of analysis in memory research, linking receptors, spines, higher-order neuronal dynamics and behavior.
RESUMEN
Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011). Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms (Lee et al., 2004), disrupting the function of the transcription factor CREB impairs both processes (Kida et al., 2002; Mamiya et al., 2009). Phosphorylation of CREB at Ser133 recruits CREB binding protein (CBP)/p300 coactivators to activate transcription (Chrivia et al., 1993; Parker et al., 1996). In addition to this well known mechanism, CREB regulated transcription coactivators (CRTCs), previously called transducers of regulated CREB (TORC) activity, stimulate CREB-mediated transcription, even in the absence of CREB phosphorylation. Recently, CRTC1 has been shown to undergo activity-dependent trafficking from synapses and dendrites to the nucleus in excitatory hippocampal neurons (Ch'ng et al., 2012). Despite being a powerful and specific coactivator of CREB, the role of CRTC in memory is virtually unexplored. To examine the effects of increasing CRTC levels, we used viral vectors to locally and acutely increase CRTC1 in the dorsal hippocampus dentate gyrus region of mice before training or memory reactivation in context fear conditioning. Overexpressing CRTC1 enhanced both memory consolidation and reconsolidation; CRTC1-mediated memory facilitation was context specific (did not generalize to nontrained context) and long lasting (observed after virally expressed CRTC1 dissipated). CREB overexpression produced strikingly similar effects. Therefore, increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as established reactivated, memories without compromising memory quality.
Asunto(s)
Giro Dentado/fisiología , Memoria/fisiología , Factores de Transcripción/fisiología , Animales , Condicionamiento Psicológico/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Giro Dentado/metabolismo , Miedo/fisiología , Miedo/psicología , Femenino , Genes fos/fisiología , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Neuronas/fisiología , Cultivo Primario de Células , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección/métodosRESUMEN
The proteome of glutamatergic synapses is diverse across the mammalian brain and involved in neurodevelopmental disorders (NDDs). Among those is fragile X syndrome (FXS), an NDD caused by the absence of the functional RNA-binding protein FMRP. Here, we demonstrate how the brain region-specific composition of postsynaptic density (PSD) contributes to FXS. In the striatum, the FXS mouse model shows an altered association of the PSD with the actin cytoskeleton, reflecting immature dendritic spine morphology and reduced synaptic actin dynamics. Enhancing actin turnover with constitutively active RAC1 ameliorates these deficits. At the behavioral level, the FXS model displays striatal-driven inflexibility, a typical feature of FXS individuals, which is rescued by exogenous RAC1. Striatal ablation of Fmr1 is sufficient to recapitulate behavioral impairments observed in the FXS model. These results indicate that dysregulation of synaptic actin dynamics in the striatum, a region largely unexplored in FXS, contributes to the manifestation of FXS behavioral phenotypes.
Asunto(s)
Síndrome del Cromosoma X Frágil , Animales , Ratones , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Actinas/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Ratones Noqueados , Espinas Dendríticas/metabolismo , Mamíferos/metabolismoRESUMEN
Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Memoria a Largo Plazo/fisiología , Memoria a Corto Plazo/fisiología , Regulación hacia Arriba/fisiología , Análisis de Varianza , Animales , Proteínas Bacterianas/genética , Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteína de Unión a CREB/genética , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Carbazoles/farmacología , Línea Celular Transformada , Chlorocebus aethiops , Condicionamiento Clásico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Discriminación en Psicología , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Miedo , Transferencia Resonante de Energía de Fluorescencia , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Alcaloides Indólicos/farmacología , Potenciación a Largo Plazo/genética , Proteínas Luminiscentes/genética , Aprendizaje por Laberinto , Memoria a Largo Plazo/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Técnicas de Placa-Clamp , Fenilalanina/genética , ARN Mensajero/metabolismo , Ratas , Conducta Social , Transfección/métodos , Tirosina/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The pruning of dendritic spines during development requires autophagy. This process is facilitated by long-term depression (LTD)-like mechanisms, which has led to speculation that LTD, a fundamental form of synaptic plasticity, also requires autophagy. Here, we show that the induction of LTD via activation of NMDA receptors or metabotropic glutamate receptors initiates autophagy in the postsynaptic dendrites in mice. Dendritic autophagic vesicles (AVs) act in parallel with the endocytic machinery to remove AMPA receptor subunits from the membrane for degradation. During NMDAR-LTD, key postsynaptic proteins are sequestered for autophagic degradation, as revealed by quantitative proteomic profiling of purified AVs. Pharmacological inhibition of AV biogenesis, or conditional ablation of atg5 in pyramidal neurons abolishes LTD and triggers sustained potentiation in the hippocampus. These deficits in synaptic plasticity are recapitulated by knockdown of atg5 specifically in postsynaptic pyramidal neurons in the CA1 area. Conducive to the role of synaptic plasticity in behavioral flexibility, mice with autophagy deficiency in excitatory neurons exhibit altered response in reversal learning. Therefore, local assembly of the autophagic machinery in dendrites ensures the degradation of postsynaptic components and facilitates LTD expression.
Asunto(s)
Autofagia/fisiología , Espinas Dendríticas/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Proteoma/metabolismo , Proteómica/métodos , Potenciales Sinápticos/fisiología , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Fragile X syndrome (FXS) results from the loss of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD-95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3' untranslated region of the PSD-95 (also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD-95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Animales , Encéfalo/citología , Supervivencia Celular/fisiología , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión de Mamíferos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Guanilato-Quinasas , Inmunoprecipitación/métodos , Hibridación in Situ/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Biosíntesis de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección , Tubulina (Proteína)/metabolismoRESUMEN
Gastrin-releasing peptide (GRP) has been proposed as a peptidergic molecule for behavioral fear and itching. Immunohistochemistry and in situ hybridization studies have shown that GRP and GRP receptor are widely distributed in forebrain areas. Less information is available for the functional action for GRP in the prefrontal cortex including the anterior cingulate cortex (ACC). Here we used whole-cell patch-clamp recording technique to study the modulation of synaptic transmission by GRP in the ACC. We found that GRP increased the frequency of sIPSCs recorded while had no significant effect on sEPSCs in ACC pyramidal neurons. The facilitatory effect of GRP on sIPSCs was blocked by the GRP receptor antagonist, RC3095. In the presence of TTX, however, GRP had no effect on the mIPSCs. Therefore, activation of GRP receptor may facilitate the excitation of the interneurons and enhanced spontaneous GABAergic, but not glutamatergic neurotransmission. Similar results on GRP modulation of GABAergic transmission were observed in the insular cortex and amygdala, suggesting a general possible effect of GRP on cortical inhibitory transmission. Our results suggest that GRP receptor is an important regulator of inhibitory circuits in forebrain areas.
Asunto(s)
Péptido Liberador de Gastrina/metabolismo , Giro del Cíngulo/metabolismo , Inhibición Neural/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción/efectos de los fármacos , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Bombesina/análogos & derivados , Bombesina/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Glutamatos/metabolismo , Giro del Cíngulo/citología , Giro del Cíngulo/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interneuronas/citología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Ratones Endogámicos C57BL , Inhibición Neural/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Picrotoxina/farmacología , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores de Bombesina/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Fragile X syndrome (FXS), a common form of inherited mental retardation, is caused by the lack of fragile X mental retardation protein (FMRP). The animal model of FXS, Fmr1 knockout mice, have deficits in the Morris water maze and trace fear memory tests, showing impairment in hippocampus-dependent learning and memory. However, results for synaptic long-term potentiation (LTP), a key cellular model for learning and memory, remain inconclusive in the hippocampus of Fmr1 knockout mice. Here, we demonstrate that FMRP is required for glycine induced LTP (Gly-LTP) in the CA1 of hippocampus. This form of LTP requires activation of post-synaptic NMDA receptors and metabotropic glutamateric receptors, as well as the subsequent activation of extracellular signal-regulated kinase (ERK) 1/2. However, paired-pulse facilitation was not affected by glycine treatment. Genetic deletion of FMRP interrupted the phosphorylation of ERK1/2, suggesting the possible role of FMRP in the regulation of the activity of ERK1/2. Our study provide strong evidences that FMRP participates in Gly-LTP in the hippocampus by regulating the phosphorylation of ERK1/2, and that improper regulation of these signaling pathways may contribute to the learning and memory deficits observed in FXS.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Análisis de Varianza , Animales , Biofisica , Bromodesoxiuridina/metabolismo , Butadienos/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glicina/farmacología , Glicinérgicos/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/genética , Masculino , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Técnicas de Placa-Clamp/métodos , Picrotoxina/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiologíaRESUMEN
Copy-number variants of the CYFIP1 gene in humans have been linked to autism spectrum disorders (ASD) and schizophrenia (SCZ), two neuropsychiatric disorders characterized by defects in brain connectivity. Here, we show that CYFIP1 plays an important role in brain functional connectivity and callosal functions. We find that Cyfip1-heterozygous mice have reduced functional connectivity and defects in white matter architecture, similar to phenotypes found in patients with ASD, SCZ and other neuropsychiatric disorders. Cyfip1-deficient mice also present decreased myelination in the callosal axons, altered presynaptic function, and impaired bilateral connectivity. Finally, Cyfip1 deficiency leads to abnormalities in motor coordination, sensorimotor gating and sensory perception, which are also known neuropsychiatric disorder-related symptoms. These results show that Cyfip1 haploinsufficiency compromises brain connectivity and function, which might explain its genetic association to neuropsychiatric disorders.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Encéfalo/metabolismo , Predisposición Genética a la Enfermedad/genética , Proteínas del Tejido Nervioso/metabolismo , Esquizofrenia/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Trastorno del Espectro Autista/diagnóstico por imagen , Axones , Conducta Animal , Encéfalo/diagnóstico por imagen , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Haploinsuficiencia , Heterocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso/genética , Fenotipo , Desempeño Psicomotor , Esquizofrenia/diagnóstico por imagen , Esquizofrenia/genética , Filtrado Sensorial , Sustancia BlancaRESUMEN
Dopamine D(2) receptor (D(2)DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D(2) receptors in this brain area are essentially obscure. We have studied the physiological responses of the D(2)DR stimulations in mice lacking the brain cytoplasmic RNA BC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D(2)-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence on D(2) receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB(1) receptors was intact in the striatum of BC1 knock-out (KO) mice despite D(2) and CB(1) receptors mediated similar electrophysiological actions. Interestingly, the fragile X mental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D(2)-mediated transmission. Because D(2)DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D(2)DR indirectly, probably regulating translation of molecules involved in D(2)DR turnover and/or stability.
Asunto(s)
Cuerpo Estriado/citología , Neuronas/fisiología , Receptores de Dopamina D2/fisiología , Ribonucleoproteínas Citoplasmáticas Pequeñas/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Compuestos de Bifenilo/farmacología , Células Cultivadas , Antagonistas de los Receptores de Dopamina D2 , Glutamato Descarboxilasa/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Oligonucleótidos/farmacología , Técnicas de Placa-Clamp/métodos , Piperazinas/farmacología , ARN Largo no Codificante , ARN Mensajero/biosíntesis , ARN no Traducido , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ribonucleoproteínas Citoplasmáticas Pequeñas/deficiencia , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The formation of long-lasting memories requires coordinated changes in gene expression and protein synthesis. Although many studies implicate DNA modifications (DNA methylation, histone modifications) in memory formation, the contributions of RNA modifications remain largely unexplored. Here we investigated the role of mRNA methylation in hippocampal-dependent memory formation in mice. RNA modifications are highly dynamic and readily reversible. Methyltransferases add a methyl group to mRNA while demethylases remove methyl groups. Here we focused on examining the role of the best characterized RNA demethylase, FTO (fat mass and obesity-associated) in memory. We observed that FTO is expressed in the nuclei, dendrites and near dendritic spines of mouse dorsal hippocampal CA1 neurons. Next, we found that contextual fear conditioning transiently (0.5 h) decreased Fto levels in these neurons, with the largest decrease in FTO observed near synapses. The decrease in FTO observed shortly after contextual fear conditioning suggests that FTO normally constrains memory formation. To directly test this, we artificially decreased FTO levels in dorsal hippocampus of otherwise normal (wild-type) mice by microinjecting before training a single herpes simplex virus (HSV) vector expressing either CRISPR/Cas9 or shRNA targeted against Fto. Decreasing FTO using either method specifically enhanced contextual fear memory. Together, these results show the importance of FTO during memory formation and, furthermore, implicate mRNA modification and epi-transcriptomics as novel regulators of memory formation.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/fisiología , Región CA1 Hipocampal/metabolismo , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Memoria/fisiología , ARN Mensajero/metabolismo , Animales , Miedo/psicología , Masculino , Metilación , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BLRESUMEN
The brain cytoplasmic (BC1) RNA is a non-coding RNA (ncRNA) involved in neuronal translational control. Absence of BC1 is associated with altered glutamatergic transmission and maladaptive behavior. Here, we show that pyramidal neurons in the barrel cortex of BC1 knock out (KO) mice display larger excitatory postsynaptic currents and increased spontaneous activity in vivo. Furthermore, BC1 KO mice have enlarged spine heads and postsynaptic densities and increased synaptic levels of glutamate receptors and PSD-95. Of note, BC1 KO mice show aberrant structural plasticity in response to whisker deprivation, impaired texture novel object recognition and altered social behavior. Thus, our study highlights a role for BC1 RNA in experience-dependent plasticity and learning in the mammalian adult neocortex, and provides insight into the function of brain ncRNAs regulating synaptic transmission, plasticity and behavior, with potential relevance in the context of intellectual disabilities and psychiatric disorders.Brain cytoplasmic (BC1) RNA is a non-coding RNA that has been implicated in translational regulation, seizure, and anxiety. Here, the authors show that in the cortex, BC1 RNA is required for sensory deprivation-induced structural plasticity of dendritic spines, as well as for correct sensory learning and social behaviors.
Asunto(s)
Aprendizaje/fisiología , Neocórtex/fisiología , Plasticidad Neuronal/fisiología , Células Piramidales/fisiología , ARN Citoplasmático Pequeño/genética , Animales , Secuencia de Bases , Células Cultivadas , Espinas Dendríticas/metabolismo , Espinas Dendríticas/fisiología , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Hibridación Fluorescente in Situ , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Neocórtex/citología , Neocórtex/metabolismo , Plasticidad Neuronal/genética , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Privación Sensorial/fisiología , Homología de Secuencia de Ácido Nucleico , Conducta Social , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Vibrisas/metabolismo , Vibrisas/fisiologíaRESUMEN
Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X(2)R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X(2)R. Double immunohistochemistry showed that virtually all orexin-immunoreactive neurons are also P2X(2)R immunoreactive, whereas 80% of P2X(2)R-immunoreactive neurons are also orexin positive. Triple-labeling experiments, combining fluorescence in situ hybridization for P2X(2)R mRNA and P2X(2)R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X(2)R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X(2)R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal.
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Adenosina Trifosfato/metabolismo , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Animales , Nivel de Alerta/fisiología , Dendritas/metabolismo , Dendritas/ultraestructura , Conducta Alimentaria/fisiología , Técnica del Anticuerpo Fluorescente/métodos , Hipotálamo/citología , Inmunohistoquímica , Hibridación in Situ , Masculino , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Receptores de Orexina , Orexinas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Receptores Purinérgicos P2X2RESUMEN
The dentate gyrus (DG) is important for encoding contextual memories, but little is known about how a population of DG neurons comes to encode and support a particular memory. One possibility is that recruitment into an engram depends on a neuron's excitability. Here, we manipulated excitability by overexpressing CREB in a random population of DG neurons and examined whether this biased their recruitment to an engram supporting a contextual fear memory. To directly assess whether neurons overexpressing CREB at the time of training became critical components of the engram, we examined memory expression while the activity of these neurons was silenced. Chemogenetically (hM4Di, an inhibitory DREADD receptor) or optogenetically (iC++, a light-activated chloride channel) silencing the small number of CREB-overexpressing DG neurons attenuated memory expression, whereas silencing a similar number of random neurons not overexpressing CREB at the time of training did not. As post-encoding reactivation of the activity patterns present during initial experience is thought to be important in memory consolidation, we investigated whether post-training silencing of neurons allocated to an engram disrupted subsequent memory expression. We found that silencing neurons 5 min (but not 24 h) following training disrupted memory expression. Together these results indicate that the rules of neuronal allocation to an engram originally described in the lateral amygdala are followed in different brain regions including DG, and moreover, that disrupting the post-training activity pattern of these neurons prevents memory consolidation.
Asunto(s)
Hipocampo/citología , Neuronas/fisiología , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Miedo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Optogenética , Transducción GenéticaRESUMEN
Collections of cells called engrams are thought to represent memories. Although there has been progress in identifying and manipulating single engrams, little is known about how multiple engrams interact to influence memory. In lateral amygdala (LA), neurons with increased excitability during training outcompete their neighbors for allocation to an engram. We examined whether competition based on neuronal excitability also governs the interaction between engrams. Mice received two distinct fear conditioning events separated by different intervals. LA neuron excitability was optogenetically manipulated and revealed a transient competitive process that integrates memories for events occurring closely in time (coallocating overlapping populations of neurons to both engrams) and separates memories for events occurring at distal times (disallocating nonoverlapping populations to each engram).
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Amígdala del Cerebelo/fisiología , Miedo/fisiología , Consolidación de la Memoria/fisiología , Recuerdo Mental/fisiología , Neuronas/fisiología , Amígdala del Cerebelo/citología , Animales , Comunicación Celular , Condicionamiento Psicológico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , OptogenéticaRESUMEN
The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology-anatomical and functional-connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.
RESUMEN
Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.