RESUMEN
Memory engrams are formed through experience-dependent remodeling of neural circuits, but their detailed architectures have remained unresolved. Using 3D electron microscopy, we performed nanoscale reconstructions of the hippocampal CA3-CA1 pathway following chemogenetic labeling of cellular ensembles with a remote history of correlated excitation during associative learning. Projection neurons involved in memory acquisition expanded their connectomes via multi-synaptic boutons without altering the numbers and spatial arrangements of individual axonal terminals and dendritic spines. This expansion was driven by presynaptic activity elicited by specific negative valence stimuli, regardless of the co-activation state of postsynaptic partners. The rewiring of initial ensembles representing an engram coincided with local, input-specific changes in the shapes and organelle composition of glutamatergic synapses, reflecting their weights and potential for further modifications. Our findings challenge the view that the connectivity among neuronal substrates of memory traces is governed by Hebbian mechanisms, and offer a structural basis for representational drifts.
RESUMEN
Progressive cognitive decline in Alzheimer's disease could either be caused by a spreading molecular pathology or by an initially focal pathology that causes aberrant neuronal activity in a larger network. To distinguish between these possibilities, we generated a mouse model with expression of mutant human amyloid precursor protein (APP) in only hippocampal CA3 cells. We found that performance in a hippocampus-dependent memory task was impaired in young adult and aged mutant mice. In both age groups, we then recorded from the CA1 region, which receives inputs from APP-expressing CA3 cells. We observed that theta oscillation frequency in CA1 was reduced along with disrupted relative timing of principal cells. Highly localized pathology limited to the presynaptic CA3 cells is thus sufficient to cause aberrant firing patterns in postsynaptic neuronal networks, which indicates that disease progression is not only from spreading pathology but also mediated by progressively advancing physiological dysfunction.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Ratones , Humanos , Animales , Anciano , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/metabolismo , Neuronas/fisiología , Enfermedad de Alzheimer/metabolismo , Sinapsis/fisiología , Ratones TransgénicosRESUMEN
Chemical synapses of shared cellular origins have remarkably heterogeneous structures, but how this diversity is generated is unclear. Here, we use three-dimensional (3D) electron microscopy and artificial intelligence algorithms for image processing to reconstruct functional excitatory microcircuits in the mouse hippocampus and microcircuits in which neurotransmitter signaling is permanently suppressed with genetic tools throughout the lifespan. These nanoscale analyses reveal that experience is dispensable for morphogenesis of synapses with different geometric shapes and contents of membrane organelles and that arrangement of morphologically distinct connections in local networks is stochastic. Moreover, loss of activity increases the variability in sizes of opposed pre- and postsynaptic structures without disrupting their alignments, suggesting that inherently variable weights of naive connections become progressively matched with repetitive use. These results demonstrate that mechanisms for the structural diversity of neuronal synapses are intrinsic and provide insights into how circuits essential for memory storage assemble and integrate information.
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Imagenología Tridimensional , Microscopía Electrónica , Nanotecnología , Sinapsis/ultraestructura , Animales , Axones/metabolismo , Dendritas/metabolismo , Ratones , Modelos Neurológicos , Orgánulos/metabolismo , Orgánulos/ultraestructura , Procesos EstocásticosRESUMEN
Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neuron's chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block-face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.
Asunto(s)
Neuronas , Sinapsis , Animales , Ácido Glutámico , Ratones , Microscopía Electrónica , Vesículas Sinápticas , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de GlutamatoRESUMEN
Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/fisiología , Microglía/fisiología , Red Nerviosa/fisiología , Animales , Señalización del Calcio , Movimiento Celular , Convulsivantes , Electroencefalografía , Vigilancia Inmunológica , Ratones , Microglía/enzimología , Microglía/ultraestructura , Enfermedades del Sistema Nervioso/fisiopatología , Fenómenos Fisiológicos del Sistema Nervioso , Pilocarpina , Convulsiones/fisiopatología , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Early Alzheimer's disease (AD) affects the brain non-uniformly, causing hippocampal memory deficits long before wide-spread brain degeneration becomes evident. Here we addressed whether mossy fiber inputs from the dentate gyrus onto CA3 principal cells are affected in an AD mouse model before amyloid ß plaque deposition. We recorded from CA3 pyramidal cells in a slice preparation from 6-month-old male APP/PS1 mice, and studied synaptic properties and intrinsic excitability. In parallel we performed a morphometric analysis of mossy fiber synapses following viral based labeling and 3D-reconstruction. We found that the basal structural and functional properties as well as presynaptic short-term plasticity at mossy fiber synapses are unaltered at 6 months in APP/PS1 mice. However, transient potentiation of synaptic transmission mediated by activity-dependent release of lipids was abolished. Whereas the presynaptic form of mossy fiber long-term potentiation (LTP) was not affected, the postsynaptic LTP of NMDAR-EPSCs was reduced. In addition, we also report an impairment in feedforward inhibition in CA3 pyramidal cells. This study, together with our previous work describing deficits at CA3-CA3 synapses, provides evidence that early AD affects synapses in a projection-dependent manner at the level of a single neuronal population.SIGNIFICANCE STATEMENT Because loss of episodic memory is considered the cognitive hallmark of Alzheimer's disease (AD), it is important to study whether synaptic circuits involved in the encoding of episodic memory are compromised in AD mouse models. Here we probe alterations in the synaptic connections between the dentate gyrus and CA3, which are thought to be critical for enabling episodic memories to be formed and stored in CA3. We found that forms of synaptic plasticity specific to these synaptic connections are markedly impaired at an early stage in a mouse model of AD, before deposition of ß amyloid plaques. Together with previous work describing deficits at CA3-CA3 synapses, we provide evidence that early AD affects synapses in an input-dependent manner within a single neuronal population.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Región CA3 Hipocampal/fisiopatología , Fibras Musgosas del Hipocampo/fisiopatología , Células Piramidales/fisiología , Sinapsis/patología , Enfermedad de Alzheimer/patología , Animales , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/fisiología , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Sinapsis/fisiologíaRESUMEN
As biomedical imaging datasets expand, deep neural networks are considered vital for image processing, yet community access is still limited by setting up complex computational environments and availability of high-performance computing resources. We address these bottlenecks with CDeep3M, a ready-to-use image segmentation solution employing a cloud-based deep convolutional neural network. We benchmark CDeep3M on large and complex two-dimensional and three-dimensional imaging datasets from light, X-ray, and electron microscopy.
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Nube Computacional , Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Autism spectrum disorders (ASD) are a set of complex neurodevelopmental disorders for which there is currently no targeted therapeutic approach. It is thought that alterations of genes regulating migration and synapse formation during development affect neural circuit formation and result in aberrant connectivity within distinct circuits that underlie abnormal behaviors. However, it is unknown whether deviant developmental trajectories are circuit-specific for a given autism risk-gene. We used MRI to probe changes in functional and structural connectivity from childhood to adulthood in Fragile-X (Fmr1-/y) and contactin-associated (CNTNAP2-/-) knockout mice. Young Fmr1-/y mice (30 days postnatal) presented with a robust hypoconnectivity phenotype in corticocortico and corticostriatal circuits in areas associated with sensory information processing, which was maintained until adulthood. Conversely, only small differences in hippocampal and striatal areas were present during early postnatal development in CNTNAP2-/- mice, while major connectivity deficits in prefrontal and limbic pathways developed between adolescence and adulthood. These findings are supported by viral tracing and electron micrograph approaches and define 2 clearly distinct connectivity endophenotypes within the autism spectrum. We conclude that the genetic background of ASD strongly influences which circuits are most affected, the nature of the phenotype, and the developmental time course of the associated changes.
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Trastorno Autístico , Encéfalo/crecimiento & desarrollo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/patología , Factores de Edad , Animales , Animales Recién Nacidos , Trastorno Autístico/complicaciones , Trastorno Autístico/genética , Trastorno Autístico/patología , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/ultraestructura , Mapeo Encefálico , Conectoma , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen por Resonancia Magnética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/diagnóstico por imagen , Oxígeno/sangre , Transducción Genética , Proteína Fluorescente RojaRESUMEN
Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the "High" and "Low" scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.
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Axones/metabolismo , Hierro/análisis , Raíces Nerviosas Espinales/diagnóstico por imagen , Microtomografía por Rayos X/instrumentación , Animales , Metales Pesados , Ratones , Fantasmas de Imagen , Raíces Nerviosas Espinales/metabolismo , Coloración y EtiquetadoRESUMEN
Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/psicología , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Cognición , Modelos Animales de Enfermedad , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal , Receptor del Glutamato Metabotropico 5/genética , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/genéticaRESUMEN
Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV ∆G) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV ∆G particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features-including dendritic spines, axonal processes, and boutons-on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV ∆G variant together with a retrograde RABV ∆G for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV ∆G-mediated fluorescent marker expression. In conclusion RABV ∆G variants expressing a combination of markers and/or tools for stimulating/monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure-function relationship of neural circuits.
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Conectoma/métodos , Neuronas/fisiología , Neuronas/virología , Virus de la Rabia/fisiología , Sinapsis/fisiología , Sinapsis/virología , Animales , Transporte Biológico , Encéfalo/fisiología , Biología Computacional/métodos , Bases de Datos Factuales , Espinas Dendríticas/metabolismo , Vectores Genéticos , Procesamiento de Imagen Asistido por Computador , Ratones , Neuronas/citología , Transfección , Navegador WebRESUMEN
Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.
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Antagonistas del Receptor de Adenosina A2/farmacología , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Fármacos Neuroprotectores/farmacología , Presenilina-1/genética , Receptor de Adenosina A2A/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Potenciación a Largo Plazo , Memoria Episódica , Ratones , Ratones Transgénicos , Presenilina-1/metabolismo , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/ultraestructura , Triazinas/farmacología , Triazoles/farmacologíaRESUMEN
Fragile X syndrome (FXS), the most common inherited form of intellectual disability disorder and a frequent cause of autism spectrum disorder (ASD), is characterized by a high prevalence of sensory symptoms. Perturbations in the anatomical connectivity of neocortical circuits resulting in their functional defects have been hypothesized to contribute to the underlying etiology of these disorders. We tested this idea by probing alterations in the functional and structural connectivity of both local and long-ranging neocortical circuits in the Fmr1 (-/y) mouse model of FXS. To achieve this, we combined in vivo ultrahigh-field diffusion tensor magnetic resonance imaging (MRI), functional MRI, and viral tracing approaches in adult mice. Our results show an anatomical hyperconnectivity phenotype for the primary visual cortex (V1), but a disproportional low connectivity of V1 with other neocortical regions. These structural data are supported by defects in the structural integrity of the subcortical white matter in the anterior and posterior forebrain. These anatomical alterations might contribute to the observed functional decoupling across neocortical regions. We therefore identify FXS as a "connectopathy," providing a translational model for understanding sensory processing defects and functional decoupling of neocortical areas in FXS and ASD.
RESUMEN
Glycoprotein-deleted rabies virus (RABV ∆G) is a powerful tool for the analysis of neural circuits. Here, we demonstrate the utility of an anterograde RABV ∆G variant for novel neuroanatomical approaches involving either bulk or sparse neuronal populations. This technology exploits the unique features of RABV ∆G vectors, namely autonomous, rapid high-level expression of transgenes, and limited cytotoxicity. Our vector permits the unambiguous long-range and fine-scale tracing of the entire axonal arbor of individual neurons throughout the brain. Notably, this level of labeling can be achieved following infection with a single viral particle. The vector is effective over a range of ages (>14 months) aiding the studies of neurodegenerative disorders or aging, and infects numerous cell types in all brain regions tested. Lastly, it can also be readily combined with retrograde RABV ∆G variants. Together with other modern technologies, this tool provides new possibilities for the investigation of the anatomy and physiology of neural circuits.
Asunto(s)
Encéfalo/citología , Vectores Genéticos/metabolismo , Imagenología Tridimensional/métodos , Neuronas/citología , Virus de la Rabia/genética , Coloración y Etiquetado/métodos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Transporte Axonal/fisiología , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Glicoproteínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Virus de la Rabia/metabolismoRESUMEN
An understanding of how the brain processes information requires knowledge of the architecture of its underlying neuronal circuits, as well as insights into the relationship between architecture and physiological function. A range of sophisticated tools is needed to acquire this knowledge, and recombinant rabies virus (RABV) is becoming an increasingly important part of this essential toolbox. RABV has been recognized for years for its properties as a synapse-specific trans-neuronal tracer. A novel genetically modified variant now enables the investigation of specific monosynaptic connections. This technology, in combination with other genetic, physiological, optical, and computational tools, has enormous potential for the visualization of neuronal circuits, and for monitoring and manipulating their activity. Here we will summarize the latest developments in this fast moving field and provide a perspective for the use of this technology for the dissection of neuronal circuit structure and function in the normal and diseased brain.