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We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and mitochondria) are rendered and itemized in a database. We explore these data to study physical properties of brain tissue. For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters' rule). This online minable database provides general access to the intrinsic complexity of the neocortex and enables further data-driven inquiries.
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Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Neocórtex/ultraestrutura , Neurônios/ultraestrutura , Animais , Automação , Axônios/ultraestrutura , Dendritos/ultraestrutura , Camundongos , Neocórtex/citologia , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.
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Células Acinares/ultraestrutura , Encéfalo/citologia , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Neurônios/ultraestrutura , Glândula Parótida/citologia , Células Acinares/química , Células Acinares/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Modelos Biológicos , Neurônios/química , Neurônios/metabolismoRESUMO
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.
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Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento EstereotipadoRESUMO
Macular telangiectasia type 2 (MacTel), a late-onset macular degeneration, has been linked to a loss in the retina of Müller glial cells and the amino acid serine, synthesized by the Müller cells. The disease is confined mainly to a central retinal region called the MacTel zone. We have used electron microscopic connectomics techniques, optimized for disease analysis, to study the retina from a 48-y-old woman suffering from MacTel. The major observations made were specific changes in mitochondrial structure within and outside the MacTel zone that were present in all retinal cell types. We also identified an abrupt boundary of the MacTel zone that coincides with the loss of Müller cells and macular pigment. Since Müller cells synthesize retinal serine, we propose that a deficiency of serine, required for mitochondrial maintenance, causes mitochondrial changes that underlie MacTel development.
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Conectoma/métodos , Retina , Doenças Retinianas , Feminino , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Retina/citologia , Retina/diagnóstico por imagem , Retina/patologia , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologiaRESUMO
Morphological and molecular characteristics determine the function of biological tissues. Attempts to combine immunofluorescence and electron microscopy invariably compromise the quality of the ultrastructure of tissue sections. We developed NATIVE, a correlated light and electron microscopy approach that preserves ultrastructure while showing the locations of multiple molecular moieties, even deep within tissues. This technique allowed the large-scale 3D reconstruction of a volume of mouse hippocampal CA3 tissue at nanometer resolution.
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Encéfalo/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Anticorpos de Domínio Único/imunologia , Animais , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Correlated electron microscopy and cathodoluminescence (CL) imaging using functionalized nanoparticles is a promising nanoscale probe of biological structure and function. Nanodiamonds (NDs) that contain CL-emitting color centers are particularly well suited for such applications. The intensity of CL emission from NDs is determined by a combination of factors, including particle size, density of color centers, efficiency of energy deposition by electrons passing through the particle, and conversion efficiency from deposited energy to CL emission. This paper reports experiments and numerical simulations that investigate the relative importance of each of these factors in determining CL emission intensity from NDs containing nitrogen-vacancy (NV) color centers. In particular, it is found that CL can be detected from NV-doped NDs with dimensions as small as ≈40 nm, although CL emission decreases significantly for smaller NDs.
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Introduction: Macular Telangiectasia type 2 (MacTel), is an uncommon form of late-onset, slowly-progressive macular degeneration. Associated with regional Müller glial cell loss in the retina and the amino acid serine synthesized by Müller cells, the disease is functionally confined to a central retinal region - the MacTel zone. Methods: We have used high-throughput multi-resolution electron microscopy techniques, optimized for disease analysis, to study the retinas from two women, mother and daughter, aged 79 and 48 years respectively, suffering from MacTel. Results: In both eyes, the principal observations made were changes specific to mitochondrial structure both outside and within the MacTel zone in all retinal cell types, with the exception of those in the retinal pigment epithelium (RPE). The lesion areas, which are a hallmark of MacTel, extend from Bruch's membrane and the choriocapillaris, through all depths of the retina, and include cells from the RPE, retinal vascular elements, and extensive hypertrophic basement membrane material. Where the Müller glial cells are lost, we have identified a significant population of microglial cells, exclusively within the Henle fiber layer, which appear to ensheathe the Henle fibers, similar to that seen normally by Müller cells. Discussion: Since Müller cells synthesize retinal serine, whereas retinal neurons do not, we propose that serine deficiency, required for normal mitochondrial function, may relate to mitochondrial changes that underlie the development of MacTel. With mitochondrial changes occurring retina-wide, the question remains as to why the Müller cells are uniquely susceptible within the MacTel zone.
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Creating a high-resolution brain atlas in diverse species offers crucial insights into general principles underlying brain function and development. A volume electron microscopy approach to generate such neural maps has been gaining importance due to advances in imaging, data storage capabilities, and data analysis protocols. Sample preparation remains challenging and is a crucial step to accelerate the imaging and data processing pipeline. Here, we introduce several replicable methods for processing the brains of the gastropod mollusc, Berghia stephanieae for volume electron microscopy. Although high-pressure freezing is the most reliable method, the depth of cryopreservation is a severe limitation for large tissue samples. We introduce a BROPA-based method using pyrogallol and methods to rapidly process samples that can save hours at the bench. This is the first report on sample preparation and imaging pipeline for volume electron microscopy in a gastropod mollusc, opening up the potential for connectomic analysis and comparisons with other phyla.
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The fovea of the human retina, a specialization for acute and color vision, features a high concentration of cone photoreceptors. A pit on the inner retinal aspect is created by the centrifugal migration of post-receptoral neurons. Foveal cells are specified early in fetal life, but the fovea reaches its final configuration postnatally. Pre-term birth retards migration resulting in a small pit, a small avascular zone, and nearly continuous inner retinal layers. To explore the involvement of Müller glia, we used serial-section electron microscopic reconstructions to examine the morphology and neural contacts of Müller glia contacting a single foveal cone in a 28-year-old male organ donor born at 28 weeks of gestation. A small non-descript foveal avascular zone contained massed glial processes that included a novel class of 'inner' Müller glia. Similar to classic 'outer' Müller glia that span the retina, inner Müller glia have bodies in the inner nuclear layer (INL). These cells are densely packed with intermediate filaments and insert processes between neurons. Unlike 'outer' Müller glia, 'inner' Müller glia do not reach the external limiting membrane but instead terminate at the outer plexiform layer. One completely reconstructed inner cell ensheathed cone pedicles and a cone-driven circuit of midget bipolar and ganglion cells. Inner Müller glia outnumber foveal cones by 1.8-fold in the outer nuclear layer (221,448 vs. 123,026 cells/mm2). Cell bodies of inner Müller glia outnumber those of outer Müller glia by 1.7-fold in the INL (41,872 vs. 24,631 cells/ mm2). Müller glia account for 95 and 80% of the volume of the foveal floor and Henle fiber layer, respectively. Determining whether inner cells are anomalies solely resulting from retarded lateral migration of inner retinal neurons in pre-term birth requires further research.
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Large-scale electron microscopy (EM) has enabled the reconstruction of brain connectomes at the synaptic level by serially scanning over massive areas of sample sections. The acquired big EM data sets raise the great challenge of image mosaicking at high accuracy. Currently, it simply follows the conventional algorithms designed for natural images, which are usually composed of only a few tiles, using a single type of keypoint feature that would sacrifice speed for stronger performance. Even so, in the process of stitching hundreds of thousands of tiles for large EM data, errors are still inevitable and diverse. Moreover, there has not yet been an appropriate metric to quantitatively evaluate the stitching of biomedical EM images. Here we propose a two-stage error detection method to improve the EM image mosaicking. It firstly uses point-based error detection in combination with a hybrid feature framework to expedite the stitching computation while maintaining high accuracy. Following is the second detection of unresolved errors with a newly designed metric of EM stitched image quality assessment (EMSIQA). The novel detection-based mosaicking pipeline is tested on large EM data sets and proven to be more effective and as accurate when compared with existing methods.
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Algoritmos , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Encéfalo/diagnóstico por imagem , Humanos , Conectoma/métodos , AnimaisRESUMO
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.
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Camundongos Endogâmicos C57BL , Animais , Camundongos , Neurônios do Núcleo Supraquiasmático/fisiologia , Masculino , Núcleo Supraquiasmático/fisiologia , Núcleo Supraquiasmático/citologia , Neurônios/fisiologiaRESUMO
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.
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Córtex Cerebelar , Animais , Feminino , Camundongos , Córtex Cerebelar/metabolismo , Córtex Cerebelar/citologia , Córtex Cerebelar/ultraestrutura , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Conectoma/métodos , Neurônios/metabolismo , Neurônios/ultraestrutura , Corantes Fluorescentes/química , Camundongos Endogâmicos C57BL , CitologiaRESUMO
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.
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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.
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Córtex Cerebral , Humanos , Axônios/fisiologia , Axônios/ultraestrutura , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/ultraestrutura , Dendritos/fisiologia , Neurônios/ultraestrutura , Oligodendroglia/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Lobo Temporal/ultraestrutura , MicroscopiaRESUMO
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.
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Analysis of brain structure, connectivity, and molecular diversity relies on effective tissue fixation. Conventional tissue fixation causes extracellular space (ECS) loss, complicating the segmentation of cellular objects from electron microscopy datasets. Previous techniques for preserving ECS in mammalian brains utilizing high-pressure perfusion can give inconsistent results owing to variations in the hydrostatic pressure within the vasculature. A more reliable fixation protocol that uniformly preserves the ECS throughout whole brains would greatly benefit a wide range of neuroscience studies. Here, we report a straightforward transcardial perfusion strategy that preserves ECS throughout the whole rodent brain. No special setup is needed besides sequential solution changes, and the protocol offers excellent reproducibility. In addition to better capturing tissue ultrastructure, preservation of ECS has many downstream advantages such as accelerating heavy-metal staining for electron microscopy, improving detergent-free immunohistochemistry for correlated light and electron microscopy, and facilitating lipid removal for tissue clearing.
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Encéfalo , Espaço Extracelular , Animais , Reprodutibilidade dos Testes , Encéfalo/ultraestrutura , Microscopia Eletrônica , Fixação de Tecidos/métodos , MamíferosRESUMO
Connectomics allows mapping of cells and their circuits at the nanometer scale in volumes of approximately 1 mm3. Given that the human cerebral cortex can be 3 mm in thickness, larger volumes are required. Larger-volume circuit reconstructions of human brain are limited by 1) the availability of fresh biopsies; 2) the need for excellent preservation of ultrastructure, including extracellular space; and 3) the requirement of uniform staining throughout the sample, among other technical challenges. Cerebral cortical samples from neurosurgical patients are available owing to lead placement for deep brain stimulation. Described here is an immersion fixation, heavy metal staining, and tissue processing method that consistently provides excellent ultrastructure throughout human and rodent surgical brain samples of volumes 2 × 2 × 2 mm3 and up to 37 mm3 with one dimension ≤2 mm. This method should allow synapse-level circuit analysis in samples from patients with psychiatric and neurologic disorders.
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Conectoma , Humanos , Conectoma/métodos , Imersão , Microscopia Eletrônica , Coloração e Rotulagem , Encéfalo , BiópsiaRESUMO
Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.
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Here, we present the first analysis of the connectome of a small volume of the Octopus vulgaris vertical lobe (VL), a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via~1.8 × 106 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the~25 × 106VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only~12 ± 3.4SAMs. This synaptic site is likely a 'memory site' as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse 'memorizable' sensory representations to the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for 'sharpening' the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based on feedforward information flow.
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Conectoma , Octopodiformes , Animais , Octopodiformes/fisiologia , Memória/fisiologia , Neurônios/fisiologia , Encéfalo/fisiologiaRESUMO
Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, 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.