RESUMEN
An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.
Asunto(s)
Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Caenorhabditis elegans/citología , Conectoma , Modelos Neurológicos , Vías Nerviosas , Sinapsis/fisiología , Envejecimiento/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/ultraestructura , Caenorhabditis elegans/anatomía & histología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/ultraestructura , Individualidad , Interneuronas/citología , Microscopía Electrónica , Neuronas/citología , Conducta EstereotipadaRESUMEN
Maps of the nervous system that identify individual cells along with their type, subcellular components and connectivity have the potential to elucidate fundamental organizational principles of neural circuits. Nanometer-resolution imaging of brain tissue provides the necessary raw data, but inferring cellular and subcellular annotation layers is challenging. We present segmentation-guided contrastive learning of representations (SegCLR), a self-supervised machine learning technique that produces representations of cells directly from 3D imagery and segmentations. When applied to volumes of human and mouse cortex, SegCLR enables accurate classification of cellular subcompartments and achieves performance equivalent to a supervised approach while requiring 400-fold fewer labeled examples. SegCLR also enables inference of cell types from fragments as small as 10 µm, which enhances the utility of volumes in which many neurites are truncated at boundaries. Finally, SegCLR enables exploration of layer 5 pyramidal cell subtypes and automated large-scale analysis of synaptic partners in mouse visual cortex.
Asunto(s)
Neurópilo , Corteza Visual , Humanos , Animales , Ratones , Neuritas , Células Piramidales , Aprendizaje Automático Supervisado , Procesamiento de Imagen Asistido por ComputadorRESUMEN
In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.
Asunto(s)
Encéfalo/citología , Vías Nerviosas/fisiología , Neurogénesis , Organoides/citología , Organoides/efectos de la radiación , Línea Celular , Separación Celular , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dendritas , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Luz , Red Nerviosa/citología , Red Nerviosa/efectos de la radiación , Vías Nerviosas/citología , Vías Nerviosas/efectos de la radiación , Especificidad de Órganos , Organoides/crecimiento & desarrollo , Células Fotorreceptoras de Vertebrados/citología , Células Madre Pluripotentes/citología , Retina/citología , Retina/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Tiempo , TranscriptomaRESUMEN
The hypothalamic suprachiasmatic nucleus (SCN) is the central pacemaker for mammalian circadian rhythms. As such, this ensemble of cell-autonomous neuronal oscillators with divergent periods must maintain coordinated oscillations. To investigate ultrastructural features enabling such synchronization, 805 coronal ultrathin sections of mouse SCN tissue were imaged with electron microscopy and aligned into a volumetric stack, from which selected neurons within the SCN core were reconstructed in silico. We found that clustered SCN core neurons were physically connected to each other via multiple large soma-to-soma plate-like contacts. In some cases, a sliver of a glial process was interleaved. These contacts were large, covering on average â¼21% of apposing neuronal somata. It is possible that contacts may be the electrophysiological substrate for synchronization between SCN neurons. Such plate-like contacts may explain why the synchronization of SCN neurons is maintained even when chemical synaptic transmission or electrical synaptic transmission via gap junctions is blocked. Such ephaptic contact-mediated synchronization among nearby neurons may therefore contribute to the wave-like oscillations of circadian core clock genes and calcium signals observed in the SCN.
Threedimensional reconstruction of SCN tissue via serial electron microscopy revealed a novel structural feature of SCN neurons that may account for interneuronal synchronization that persists even when the predominant mechanisms of neuronal communication are blocked. We found that SCN core neurons are connected by multiple somasoma contact specializations, ultrastructural elements that could enable synchronization of tightly packed neurons organized in clustered networks. This extensive network of platelike somasoma contacts among clustered SCN neurons may provide insight into how â¼20,000 autonomous neuronal oscillators with a broad range of intrinsic periods remain synchronized in the absence of ordinary communication modalities, thereby conferring the resilience required for the SCN to function as the mammalian circadian pacemaker.
Asunto(s)
Ratones Endogámicos C57BL , Animales , Ratones , Neuronas del Núcleo Supraquiasmático/fisiología , Masculino , Núcleo Supraquiasmático/fisiología , Núcleo Supraquiasmático/citología , Neuronas/fisiologíaRESUMEN
Connectomics provides essential nanometer-resolution, synapse-level maps of neural circuits to understand brain activity and behavior. However, few researchers have access to the high-throughput electron microscopes necessary to generate enough data for whole circuit or brain reconstruction. To date, machine-learning methods have been used after the collection of images by electron microscopy (EM) to accelerate and improve neuronal segmentation, synapse reconstruction and other data analysis. With the computational improvements in processing EM images, acquiring EM images has now become the rate-limiting step. Here, in order to speed up EM imaging, we integrate machine-learning into real-time image acquisition in a singlebeam scanning electron microscope. This SmartEM approach allows an electron microscope to perform intelligent, data-aware imaging of specimens. SmartEM allocates the proper imaging time for each region of interest - scanning all pixels equally rapidly, then re-scanning small subareas more slowly where a higher quality signal is required to achieve accurate segmentability, in significantly less time. We demonstrate that this pipeline achieves a 7-fold acceleration of image acquisition time for connectomics using a commercial single-beam SEM. We apply SmartEM to reconstruct a portion of mouse cortex with the same accuracy as traditional microscopy but in less time.
RESUMEN
Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.
Asunto(s)
Corteza Cerebelosa , Animales , Femenino , Ratones , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/citología , Corteza Cerebelosa/ultraestructura , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Conectoma/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Colorantes Fluorescentes/química , Ratones Endogámicos C57BL , CitologíaRESUMEN
Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1 mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.
Asunto(s)
Cilios , Conectoma , Humanos , Neuronas/fisiología , Corteza Cerebral , Neuroglía/fisiologíaRESUMEN
To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.
Asunto(s)
Corteza Cerebral , Humanos , Axones/fisiología , Axones/ultraestructura , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/ultraestructura , Dendritas/fisiología , Neuronas/ultraestructura , Oligodendroglía/ultraestructura , Sinapsis/fisiología , Sinapsis/ultraestructura , Lóbulo Temporal/ultraestructura , MicroscopíaRESUMEN
Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.
RESUMEN
During development, animals can maintain behavioral output even as underlying circuitry structurally remodels. After hatching, C. elegans undergoes substantial motor neuron expansion and synapse rewiring while the animal continuously moves with an undulatory pattern. To understand how the circuit transitions from its juvenile to mature configuration without interrupting functional output, we reconstructed the C. elegans motor circuit by electron microscopy across larval development. We observed the following: First, embryonic motor neurons transiently interact with the developing post-embryonic motor neurons prior to remodeling of their juvenile wiring. Second, post-embryonic neurons initiate synapse development with their future partners as their neurites navigate through the juvenile nerve cords. Third, embryonic and post-embryonic neurons sequentially build structural machinery needed for the adult circuit before the embryonic neurons relinquish their roles to post-embryonic neurons. Fourth, this transition is repeated region by region along the body in an anterior-to-posterior sequence, following the birth order of neurons. Through this orchestrated and programmed rewiring, the motor circuit gradually transforms from asymmetric to symmetric wiring. These maturation strategies support the continuous maintenance of motor patterns as the juvenile circuit develops into the adult configuration.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/fisiología , Neuronas Motoras/fisiología , Sinapsis/fisiología , Neuritas , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
Dendritic spines are membranous protrusions that receive essentially all excitatory inputs in most mammalian neurons. Spines, with a bulbous head connected to the dendrite by a thin neck, have a variety of morphologies that likely impact their functional properties. Nevertheless, the question of whether spines belong to distinct morphological subtypes is still open. Addressing this quantitatively requires clear identification and measurements of spine necks. Recent advances in electron microscopy enable large-scale systematic reconstructions of spines with nanometer precision in 3D. Analyzing ultrastructural reconstructions from mouse neocortical neurons with computer vision algorithms, we demonstrate that the vast majority of spine structures can be rigorously separated into heads and necks, enabling morphological measurements of spine necks. We then used a database of spine morphological parameters to explore the potential existence of different spine classes. Without exception, our analysis revealed unimodal distributions of individual morphological parameters of spine heads and necks, without evidence for subtypes of spines. The postsynaptic density size was strongly correlated with the spine head volume. The spine neck diameter, but not the neck length, was also correlated with the head volume. Spines with larger head volumes often had a spine apparatus and pairs of spines in a post-synaptic cell contacted by the same axon had similar head volumes. Our data reveal a lack of morphological subtypes of spines and indicate that the spine neck length and head volume must be independently regulated. These results have repercussions for our understanding of the function of dendritic spines in neuronal circuits.
Asunto(s)
Espinas Dendríticas , Neuronas , Animales , Axones/ultraestructura , Dendritas/fisiología , Espinas Dendríticas/fisiología , Mamíferos , Ratones , Microscopía Electrónica , Neuronas/fisiología , SinapsisRESUMEN
The extent to which attending to one stimulus while ignoring another influences the integration of visual and inertial (vestibular, somatosensory, proprioceptive) stimuli is currently unknown. It is also unclear how cue integration is affected by an awareness of cue conflicts. We investigated these questions using a turn-reproduction paradigm, where participants were seated on a motion platform equipped with a projection screen and were asked to actively return a combined visual and inertial whole-body rotation around an earth-vertical axis. By introducing cue conflicts during the active return and asking the participants whether they had noticed a cue conflict, we measured the influence of each cue on the response. We found that the task instruction had a significant effect on cue weighting in the response, with a higher weight assigned to the attended modality, only when participants noticed the cue conflict. This suggests that participants used task-induced attention to reduce the influence of stimuli that conflict with the task instructions.
Asunto(s)
Atención , Señales (Psicología) , Propiocepción , Percepción Visual , Adulto , Análisis de Varianza , Femenino , Humanos , Masculino , Estimulación Luminosa , Estimulación Física , Psicofísica , Rotación , Adulto JovenRESUMEN
Recent developments in serial-section electron microscopy allow the efficient generation of very large image data sets but analyzing such data poses challenges for software tools. Here we introduce Volume Annotation and Segmentation Tool (VAST), a freely available utility program for generating and editing annotations and segmentations of large volumetric image (voxel) data sets. It provides a simple yet powerful user interface for real-time exploration and analysis of large data sets even in the Petabyte range.
Asunto(s)
Conectoma/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Neuronas/fisiología , Programas Informáticos , Algoritmos , Bases de Datos Factuales , Humanos , Microscopía Electrónica/métodosRESUMEN
To stimulate progress in automating the reconstruction of neural circuits, we organized the first international challenge on 2D segmentation of electron microscopic (EM) images of the brain. Participants submitted boundary maps predicted for a test set of images, and were scored based on their agreement with a consensus of human expert annotations. The winning team had no prior experience with EM images, and employed a convolutional network. This "deep learning" approach has since become accepted as a standard for segmentation of EM images. The challenge has continued to accept submissions, and the best so far has resulted from cooperation between two teams. The challenge has probably saturated, as algorithms cannot progress beyond limits set by ambiguities inherent in 2D scoring and the size of the test dataset. Retrospective evaluation of the challenge scoring system reveals that it was not sufficiently robust to variations in the widths of neurite borders. We propose a solution to this problem, which should be useful for a future 3D segmentation challenge.
RESUMEN
Myelin is a defining feature of the vertebrate nervous system. Variability in the thickness of the myelin envelope is a structural feature affecting the conduction of neuronal signals. Conversely, the distribution of myelinated tracts along the length of axons has been assumed to be uniform. Here, we traced high-throughput electron microscopy reconstructions of single axons of pyramidal neurons in the mouse neocortex and built high-resolution maps of myelination. We find that individual neurons have distinct longitudinal distribution of myelin. Neurons in the superficial layers displayed the most diversified profiles, including a new pattern where myelinated segments are interspersed with long, unmyelinated tracts. Our data indicate that the profile of longitudinal distribution of myelin is an integral feature of neuronal identity and may have evolved as a strategy to modulate long-distance communication in the neocortex.
Asunto(s)
Vaina de Mielina/fisiología , Neocórtex/citología , Células Piramidales/fisiología , Corteza Somatosensorial/citología , Corteza Visual/citología , Animales , Axones/fisiología , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Neocórtex/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Células Piramidales/citología , Corteza Somatosensorial/fisiología , Corteza Visual/fisiologíaRESUMEN
The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly-the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments.
Asunto(s)
Mapeo Encefálico/métodos , Encéfalo/anatomía & histología , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Electrónica/métodos , Animales , Microtomía/métodos , Programas Informáticos , Coloración y Etiquetado , Adhesión del Tejido/métodosRESUMEN
We describe a scalable database cluster for the spatial analysis and annotation of high-throughput brain imaging data, initially for 3-d electron microscopy image stacks, but for time-series and multi-channel data as well. The system was designed primarily for workloads that build connectomes- neural connectivity maps of the brain-using the parallel execution of computer vision algorithms on high-performance compute clusters. These services and open-science data sets are publicly available at openconnecto.me. The system design inherits much from NoSQL scale-out and data-intensive computing architectures. We distribute data to cluster nodes by partitioning a spatial index. We direct I/O to different systems-reads to parallel disk arrays and writes to solid-state storage-to avoid I/O interference and maximize throughput. All programming interfaces are RESTful Web services, which are simple and stateless, improving scalability and usability. We include a performance evaluation of the production system, highlighting the effec-tiveness of spatial data organization.
RESUMEN
VIDEO ABSTRACT: Using light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate â¼10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS.
Asunto(s)
Axones/fisiología , Neuronas Motoras/fisiología , Desarrollo de Músculos/fisiología , Unión Neuromuscular/crecimiento & desarrollo , Factores de Edad , Animales , Animales Recién Nacidos , Axones/ultraestructura , Bungarotoxinas/metabolismo , Toxina del Cólera/metabolismo , Embrión de Mamíferos , Imagenología Tridimensional , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Modelos Biológicos , Neuronas Motoras/ultraestructura , Unión Neuromuscular/embriología , Unión Neuromuscular/ultraestructuraRESUMEN
The otoliths are stimulated in the same fashion by gravitational and inertial forces, so otolith signals are ambiguous indicators of self-orientation. The ambiguity can be resolved with added visual information indicating orientation and acceleration with respect to the earth. Here we present a Bayesian model of the statistically optimal combination of noisy vestibular and visual signals. Likelihoods associated with sensory measurements are represented in an orientation/acceleration space. The likelihood function associated with the otolith signal illustrates the ambiguity; there is no unique solution for self-orientation or acceleration. Likelihood functions associated with other sensory signals can resolve this ambiguity. In addition, we propose two priors, each acting on a dimension in the orientation/acceleration space: the idiotropic prior and the no-acceleration prior. We conducted experiments using a motion platform and attached visual display to examine the influence of visual signals on the interpretation of the otolith signal. Subjects made pitch and acceleration judgments as the vestibular and visual signals were manipulated independently. Predictions of the model were confirmed: (1) visual signals affected the interpretation of the otolith signal, (2) less variable signals had more influence on perceived orientation and acceleration than more variable ones, and (3) combined estimates were more precise than single-cue estimates. We also show that the model can explain some well-known phenomena including the perception of upright in zero gravity, the Aubert effect, and the somatogravic illusion.