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Circular RNAs (circRNAs) are noncoding RNAs abundant in brain tissue, and many are derived from activity-dependent, linear mRNAs encoding for synaptic proteins, suggesting that circRNAs may directly or indirectly play a role in regulating synaptic development, plasticity, and function. However, it is unclear if the circular forms of these RNAs are similarly regulated by activity and what role these circRNAs play in developmental plasticity. Here, we employed transcriptome-wide analysis comparing differential expression of both mRNAs and circRNAs in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), a model of developmental plasticity. Among the differentially expressed mRNAs and circRNAs following 3-day MD, the circular and the activity-dependent linear forms of the Homer1 gene, circHomer1 and Homer1a respectively, were of interest as their expression changed in opposite directions: circHomer1 expression increased while the expression of Homer1a decreased following MD. Knockdown of circHomer1 prevented the depression of closed-eye responses normally observed after 3-day MD. circHomer1-knockdown led to a reduction in average dendritic spine size prior to MD, but critically there was no further reduction after 3-day MD, consistent with impaired structural plasticity. circHomer1-knockdown also prevented the reduction of surface AMPA receptors after 3-day MD. Synapse-localized puncta of the AMPA receptor endocytic protein Arc increased in volume after MD but were smaller in circHomer1-knockdown neurons, suggesting that circHomer1 regulates plasticity through mechanisms of activity-dependent AMPA receptor endocytosis. Thus, activity-dependent circRNAs regulate developmental synaptic plasticity, and our findings highlight the essential role of circHomer1 in V1 plasticity induced by short-term MD.
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Understanding cellular architectures and their connectivity is essential for interrogating system function and dysfunction. However, we lack technologies for mapping the multiscale details of individual cells and their connectivity in the human organ-scale system. We developed a platform that simultaneously extracts spatial, molecular, morphological, and connectivity information of individual cells from the same human brain. The platform includes three core elements: a vibrating microtome for ultraprecision slicing of large-scale tissues without losing cellular connectivity (MEGAtome), a polymer hydrogel-based tissue processing technology for multiplexed multiscale imaging of human organ-scale tissues (mELAST), and a computational pipeline for reconstructing three-dimensional connectivity across multiple brain slabs (UNSLICE). We applied this platform for analyzing human Alzheimer's disease pathology at multiple scales and demonstrating scalable neural connectivity mapping in the human brain.
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Enfermedad de Alzheimer , Encéfalo , Imagen Molecular , Humanos , Enfermedad de Alzheimer/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Imagen Molecular/métodos , Fenotipo , Hidrogeles/química , ConectomaRESUMEN
Micro-LEDs (µLEDs) have been explored for augmented and virtual reality display applications that require extremely high pixels per inch and luminance1,2. However, conventional manufacturing processes based on the lateral assembly of red, green and blue (RGB) µLEDs have limitations in enhancing pixel density3-6. Recent demonstrations of vertical µLED displays have attempted to address this issue by stacking freestanding RGB LED membranes and fabricating top-down7-14, but minimization of the lateral dimensions of stacked µLEDs has been difficult. Here we report full-colour, vertically stacked µLEDs that achieve, to our knowledge, the highest array density (5,100 pixels per inch) and the smallest size (4 µm) reported to date. This is enabled by a two-dimensional materials-based layer transfer technique15-18 that allows the growth of RGB LEDs of near-submicron thickness on two-dimensional material-coated substrates via remote or van der Waals epitaxy, mechanical release and stacking of LEDs, followed by top-down fabrication. The smallest-ever stack height of around 9 µm is the key enabler for record high µLED array density. We also demonstrate vertical integration of blue µLEDs with silicon membrane transistors for active matrix operation. These results establish routes to creating full-colour µLED displays for augmented and virtual reality, while also offering a generalizable platform for broader classes of three-dimensional integrated devices.
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The thalamus is the main gateway for sensory information from the periphery to the mammalian cerebral cortex. A major conundrum has been the discrepancy between the thalamus's central role as the primary feedforward projection system into the neocortex and the sparseness of thalamocortical synapses. Here we use new methods, combining genetic tools and scalable tissue expansion microscopy for whole-cell synaptic mapping, revealing the number, density and size of thalamic versus cortical excitatory synapses onto individual layer 2/3 (L2/3) pyramidal cells (PCs) of the mouse primary visual cortex. We find that thalamic inputs are not only sparse, but remarkably heterogeneous in number and density across individual dendrites and neurons. Most surprising, despite their sparseness, thalamic synapses onto L2/3 PCs are smaller than their cortical counterparts. Incorporating these findings into fine-scale, anatomically faithful biophysical models of L2/3 PCs reveals how individual neurons with sparse and weak thalamocortical synapses, embedded in small heterogeneous neuronal ensembles, may reliably 'read out' visually driven thalamic input.
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Neocórtex , Tálamo , Ratones , Animales , Tálamo/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Células Piramidales , MamíferosRESUMEN
Gibberellins (GAs) are a class of phytohormones, important for plant growth, and very difficult to distinguish because of their similarity in chemical structures. Herein, we develop the first nanosensors for GAs by designing and engineering polymer-wrapped single-walled carbon nanotubes (SWNTs) with unique corona phases that selectively bind to bioactive GAs, GA3 and GA4, triggering near-infrared (NIR) fluorescence intensity changes. Using a new coupled Raman/NIR fluorimeter that enables self-referencing of nanosensor NIR fluorescence with its Raman G-band, we demonstrated detection of cellular GA in Arabidopsis, lettuce, and basil roots. The nanosensors reported increased endogenous GA levels in transgenic Arabidopsis mutants that overexpress GA and in emerging lateral roots. Our approach allows rapid spatiotemporal detection of GA across species. The reversible sensor captured the decreasing GA levels in salt-treated lettuce roots, which correlated remarkably with fresh weight changes. This work demonstrates the potential for nanosensors to solve longstanding problems in plant biotechnology.
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Arabidopsis , Nanotubos de Carbono , Giberelinas/química , Giberelinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/metabolismo , Nanotubos de Carbono/química , Fluorescencia , ColorantesRESUMEN
Newly generated excitatory synapses in the mammalian cortex lack sufficient AMPA-type glutamate receptors to mediate neurotransmission, resulting in functionally silent synapses that require activity-dependent plasticity to mature. Silent synapses are abundant in early development, during which they mediate circuit formation and refinement, but they are thought to be scarce in adulthood1. However, adults retain a capacity for neural plasticity and flexible learning that suggests that the formation of new connections is still prevalent. Here we used super-resolution protein imaging to visualize synaptic proteins at 2,234 synapses from layer 5 pyramidal neurons in the primary visual cortex of adult mice. Unexpectedly, about 25% of these synapses lack AMPA receptors. These putative silent synapses were located at the tips of thin dendritic protrusions, known as filopodia, which were more abundant by an order of magnitude than previously believed (comprising about 30% of all dendritic protrusions). Physiological experiments revealed that filopodia do indeed lack AMPA-receptor-mediated transmission, but they exhibit NMDA-receptor-mediated synaptic transmission. We further showed that functionally silent synapses on filopodia can be unsilenced through Hebbian plasticity, recruiting new active connections into a neuron's input matrix. These results challenge the model that functional connectivity is largely fixed in the adult cortex and demonstrate a new mechanism for flexible control of synaptic wiring that expands the learning capabilities of the mature brain.
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Mamíferos , Registros , Animales , RatonesRESUMEN
As advances in microscopy imaging provide an ever clearer window into the human brain, accurate reconstruction of neural connectivity can yield valuable insight into the relationship between brain structure and function. However, human manual tracing is a slow and laborious task, and requires domain expertise. Automated methods are thus needed to enable rapid and accurate analysis at scale. In this paper, we explored deep neural networks for dense axon tracing and incorporated axon topological information into the loss function with a goal to improve the performance on both voxel-based segmentation and axon centerline detection. We evaluated three approaches using a modified 3D U-Net architecture trained on a mouse brain dataset imaged with light sheet microscopy and achieved a 10% increase in axon tracing accuracy over previous methods. Furthermore, the addition of centerline awareness in the loss function outperformed the baseline approach across all metrics, including a boost in Rand Index by 8%.
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Algoritmos , Imagenología Tridimensional , Animales , Axones , Encéfalo/diagnóstico por imagen , Humanos , Imagenología Tridimensional/métodos , Ratones , Redes Neurales de la ComputaciónRESUMEN
Neuronal ensembles that hold specific memory (memory engrams) have been identified in the hippocampus, amygdala, or cortex. However, it has been hypothesized that engrams of a specific memory are distributed among multiple brain regions that are functionally connected, referred to as a unified engram complex. Here, we report a partial map of the engram complex for contextual fear conditioning memory by characterizing encoding activated neuronal ensembles in 247 regions using tissue phenotyping in mice. The mapping was aided by an engram index, which identified 117 cFos+ brain regions holding engrams with high probability, and brain-wide reactivation of these neuronal ensembles by recall. Optogenetic manipulation experiments revealed engram ensembles, many of which were functionally connected to hippocampal or amygdala engrams. Simultaneous chemogenetic reactivation of multiple engram ensembles conferred a greater level of memory recall than reactivation of a single engram ensemble, reflecting the natural memory recall process. Overall, our study supports the unified engram complex hypothesis for memory storage.
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Mapeo Encefálico , Memoria , Animales , Encéfalo , Miedo/fisiología , Hipocampo/fisiología , Memoria/fisiología , RatonesRESUMEN
The complex connectivity of the mammalian brain underlies its function, but understanding how interconnected brain regions interact in neural processing remains a formidable challenge. Here we address this problem by introducing a genetic probe that permits selective functional imaging of distributed neural populations defined by viral labeling techniques. The probe is an engineered enzyme that transduces cytosolic calcium dynamics of probe-expressing cells into localized hemodynamic responses that can be specifically visualized by functional magnetic resonance imaging. Using a viral vector that undergoes retrograde transport, we apply the probe to characterize a brain-wide network of presynaptic inputs to the striatum activated in a deep brain stimulation paradigm in rats. The results reveal engagement of surprisingly diverse projection sources and inform an integrated model of striatal function relevant to reward behavior and therapeutic neurostimulation approaches. Our work thus establishes a strategy for mechanistic analysis of multiregional neural systems in the mammalian brain.
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Mapeo Encefálico , Imagen por Resonancia Magnética , Animales , Encéfalo/fisiología , Cuerpo Estriado , Imagen por Resonancia Magnética/métodos , Mamíferos , Ratas , RecompensaRESUMEN
Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.
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Trastorno del Espectro Autista , Predisposición Genética a la Enfermedad , Neuronas , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Corteza Cerebral/citología , Proteínas de Unión al ADN/genética , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Neuronas/clasificación , Neuronas/metabolismo , Neuronas/patología , Organoides/citología , Proteómica , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.
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Anticuerpos , Comunicación Celular , Diagnóstico por ImagenRESUMEN
Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.
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Emerging tissue transformation technologies provide an unprecedented opportunity to investigate system-level molecular and anatomical features in situ. Hydrogel-based methods engineer physicochemical tissue properties to render intact organs optically transparent and size and shape adjustable while preserving biomolecules at their physiological locations. When combined with advanced molecular tools, labeling, and imaging techniques, tissue transformation enables three-dimensional (3D) mapping of molecules, cells, and their interrelationships at increasing speeds and resolutions. In this review, we discuss the basic engineering principles of tissue transformation and labeling techniques as well as their broad applications, current challenges, and future potential.
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Hidrogeles/química , Ingeniería de Tejidos , Animales , Difusión , Humanos , Hidrogeles/síntesis química , Cinética , Permeabilidad , Transcriptoma/genéticaRESUMEN
The basal ganglia regulate a wide range of behaviors, including motor control and cognitive functions, and are profoundly affected in Parkinson's disease (PD). However, the functional organization of different basal ganglia nuclei has not been fully elucidated at the circuit level. In this study, we investigated the functional roles of distinct parvalbumin-expressing neuronal populations in the external globus pallidus (GPe-PV) and their contributions to different PD-related behaviors. We demonstrate that substantia nigra pars reticulata (SNr)-projecting GPe-PV neurons and parafascicular thalamus (PF)-projecting GPe-PV neurons are associated with locomotion and reversal learning, respectively. In a mouse model of PD, we found that selective manipulation of the SNr-projecting GPe-PV neurons alleviated locomotor deficit, whereas manipulation of the PF-projecting GPe-PV neurons rescued the impaired reversal learning. Our findings establish the behavioral importance of two distinct GPe-PV neuronal populations and, thereby, provide a new framework for understanding the circuit basis of different behavioral deficits in the Parkinsonian state.
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Globo Pálido/fisiopatología , Vías Nerviosas/fisiopatología , Neuronas/fisiología , Trastornos Parkinsonianos/fisiopatología , Animales , Femenino , Trastornos Neurológicos de la Marcha/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Parvalbúminas , Aprendizaje Inverso/fisiologíaRESUMEN
Tissue clearing of gross anatomical samples was first described over a century ago and has only recently found widespread use in the field of microscopy. This renaissance has been driven by the application of modern knowledge of optical physics and chemical engineering to the development of robust and reproducible clearing techniques, the arrival of new microscopes that can image large samples at cellular resolution and computing infrastructure able to store and analyze large data volumes. Many biological relationships between structure and function require investigation in three dimensions and tissue clearing therefore has the potential to enable broad discoveries in the biological sciences. Unfortunately, the current literature is complex and could confuse researchers looking to begin a clearing project. The goal of this Primer is to outline a modular approach to tissue clearing that allows a novice researcher to develop a customized clearing pipeline tailored to their tissue of interest. Further, the Primer outlines the required imaging and computational infrastructure needed to perform tissue clearing at scale, gives an overview of current applications, discusses limitations and provides an outlook on future advances in the field.
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Brain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and ~ 100 million cells reveals quantitative multiscale "phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.
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Corteza Cerebral/crecimiento & desarrollo , Organoides/citología , Organoides/crecimiento & desarrollo , Encéfalo/citología , Diferenciación Celular , Humanos , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/citología , Microscopía Fluorescente/métodos , Red Nerviosa/diagnóstico por imagen , Neuronas/citología , Fenotipo , Células Madre Pluripotentes/citologíaRESUMEN
Most deaths from cancer are explained by metastasis, and yet large-scale metastasis research has been impractical owing to the complexity of in vivo models. Here we introduce an in vivo barcoding strategy that is capable of determining the metastatic potential of human cancer cell lines in mouse xenografts at scale. We validated the robustness, scalability and reproducibility of the method and applied it to 500 cell lines1,2 spanning 21 types of solid tumour. We created a first-generation metastasis map (MetMap) that reveals organ-specific patterns of metastasis, enabling these patterns to be associated with clinical and genomic features. We demonstrate the utility of MetMap by investigating the molecular basis of breast cancers capable of metastasizing to the brain-a principal cause of death in patients with this type of cancer. Breast cancers capable of metastasizing to the brain showed evidence of altered lipid metabolism. Perturbation of lipid metabolism in these cells curbed brain metastasis development, suggesting a therapeutic strategy to combat the disease and demonstrating the utility of MetMap as a resource to support metastasis research.
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Neoplasias de la Mama/patología , Movimiento Celular , Metástasis de la Neoplasia/patología , Especificidad de Órganos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Procesamiento Automatizado de Datos , Femenino , Xenoinjertos , Humanos , Metabolismo de los Lípidos/genética , Ratones , Tipificación Molecular , Mutación , Metástasis de la Neoplasia/genética , Trasplante de Neoplasias , Proyectos PilotoRESUMEN
We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.
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Hidrogeles/química , Coloración y Etiquetado/métodos , Ingeniería de Tejidos/métodos , Acrilamida/química , Animales , Fenómenos Biomecánicos , Materiales Biomiméticos/química , Bioimpresión , Corteza Cerebral/química , Reactivos de Enlaces Cruzados/química , Módulo de Elasticidad , Hipocampo/química , Humanos , Ensayo de Materiales , Ratones , Estrés Mecánico , Resistencia a la TracciónRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.