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1.
Curr Biol ; 32(21): 4645-4659.e3, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36283410

RESUMO

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.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Neurônios Motores/fisiologia , Sinapses/fisiologia , Neuritos , Proteínas de Caenorhabditis elegans/genética
2.
Curr Biol ; 32(1): 176-189.e5, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34822765

RESUMO

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animal's proximity, which need to be distinguishable from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how hair cells-the receptor cells of the lateral line-in zebrafish larvae discriminate between such reafferent and exafferent signals. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies (copies of the motor command that cancel out self-generated reafferent stimulation during locomotion) and that dopaminergic signals from the hypothalamus may have a role in threshold modulation, both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.


Assuntos
Sistema da Linha Lateral , Peixe-Zebra , Animais , Larva , Sistema da Linha Lateral/fisiologia , Locomoção/fisiologia , Rombencéfalo , Peixe-Zebra/fisiologia
3.
Nature ; 596(7871): 257-261, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349261

RESUMO

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.


Assuntos
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 Estereotipado
4.
Comput Vis ECCV ; 12363: 103-120, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33345257

RESUMO

For large-scale vision tasks in biomedical images, the labeled data is often limited to train effective deep models. Active learning is a common solution, where a query suggestion method selects representative unlabeled samples for annotation, and the new labels are used to improve the base model. However, most query suggestion models optimize their learnable parameters only on the limited labeled data and consequently become less effective for the more challenging unlabeled data. To tackle this, we propose a two-stream active query suggestion approach. In addition to the supervised feature extractor, we introduce an unsupervised one optimized on all raw images to capture diverse image features, which can later be improved by fine-tuning on new labels. As a use case, we build an end-to-end active learning framework with our query suggestion method for 3D synapse detection and mitochondria segmentation in connectomics. With the framework, we curate, to our best knowledge, the largest connectomics dataset with dense synapses and mitochondria annotation. On this new dataset, our method outperforms previous state-of-the-art methods by 3.1% for synapse and 3.8% for mitochondria in terms of region-of-interest proposal accuracy. We also apply our method to image classification, where it outperforms previous approaches on CIFAR-10 under the same limited annotation budget. The project page is https://zudi-lin.github.io/projects/#two_stream_active.

5.
Proc Natl Acad Sci U S A ; 117(31): 18780-18787, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32699144

RESUMO

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.


Assuntos
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/patologia
6.
Cell Rep ; 29(9): 2849-2861.e6, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31775050

RESUMO

During postnatal development, cerebellar climbing fibers alter their innervation strengths onto supernumerary Purkinje cell targets, generating a one-to-few connectivity pattern in adulthood. To get insight about the processes responsible for this remapping, we reconstructed serial electron microscopy datasets from mice during the first postnatal week. Between days 3 and 7, individual climbing fibers selectively add many synapses onto a subset of Purkinje targets in a positive-feedback manner, without pruning synapses from other targets. Active zone sizes of synapses associated with powerful versus weak inputs are indistinguishable. Changes in synapse number are thus the predominant form of early developmental plasticity. Finally, the numbers of climbing fibers and Purkinje cells in a local region nearly match. Initial over-innervation of Purkinje cells by climbing fibers is therefore economical: the number of axons entering a region is enough to assure that each ultimately retains a postsynaptic target and that none branched there in vain.


Assuntos
Cerebelo/fisiopatologia , Fibras Nervosas/metabolismo , Sinapses/metabolismo , Animais , Humanos , Camundongos
7.
Methods Cell Biol ; 152: 41-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31326026

RESUMO

The Automated Tape-Collecting Ultramicrotome (ATUM) is a tape-reeling device that is placed in a water-filled diamond knife boat to collect serial sections as they are cut by a conventional ultramicrotome. The ATUM can collect thousands of sections of many different shapes and sizes, which are subsequently imaged by a scanning electron microscope. This method has been used for large-scale connectomics projects of mouse brain, and is well suited for other smaller-scale studies of tissues, cells, and organisms. Here, we describe basic procedures for preparing a block for ATUM sectioning, handling of the ATUM, tape preparation, post-treatment of sections, and considerations for mapping, imaging, and aligning the serial sections.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Animais , Encéfalo/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Camundongos
8.
Front Neural Circuits ; 13: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31133819

RESUMO

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.


Assuntos
Encéfalo/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Neuroimagem/métodos , Animais , Callithrix , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos
9.
Nat Methods ; 15(12): 1029-1032, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397326

RESUMO

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.


Assuntos
Encéfalo/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Anticorpos de Domínio Único/imunologia , Animais , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
Small ; 13(22)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28417543

RESUMO

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.

11.
Elife ; 52016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27383271

RESUMO

Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Microtomia/métodos , Rede Nervosa/anatomia & histologia , Vias Neurais/anatomia & histologia , Retina/citologia , Coloração e Rotulagem/métodos , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL
12.
Cell ; 162(3): 648-61, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26232230

RESUMO

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.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Neocórtex/ultraestrutura , Neurônios/ultraestrutura , Animais , Automação , Axônios/ultraestrutura , Dendritos/ultraestrutura , Camundongos , Neocórtex/citologia , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestrutura
13.
Artigo em Inglês | MEDLINE | ID: mdl-25018701

RESUMO

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.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Animais , Microtomia/métodos , Software , Coloração e Rotulagem , Inclusão do Tecido/métodos
15.
Cell ; 154(2): 285-96, 2013 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-23870120

RESUMO

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.


Assuntos
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/metabolismo
16.
Neuron ; 74(5): 816-29, 2012 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-22681687

RESUMO

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 ∼10-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.


Assuntos
Axônios/fisiologia , Neurônios Motores/fisiologia , Desenvolvimento Muscular/fisiologia , Junção Neuromuscular/crescimento & desenvolvimento , Fatores Etários , Animais , Animais Recém-Nascidos , Axônios/ultraestrutura , Bungarotoxinas/metabolismo , Toxina da Cólera/metabolismo , Embrião de Mamíferos , Imageamento Tridimensional , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Modelos Biológicos , Neurônios Motores/ultraestrutura , Junção Neuromuscular/embriologia , Junção Neuromuscular/ultraestrutura
17.
Nat Protoc ; 7(2): 193-206, 2012 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-22240582

RESUMO

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Tecido Nervoso/ultraestrutura , Coloração e Rotulagem/métodos , Animais , Ácido Aspártico/química , Encéfalo/ultraestrutura , Membrana Celular/ultraestrutura , Ácido Cítrico/química , Sulfato de Cobre/química , Drosophila/ultraestrutura , Chumbo/química , Camundongos , Microtomia , Compostos Organometálicos/química , Osmio/química , Peixe-Zebra
18.
Adv Mater ; 24(3): 370-4, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22174186

RESUMO

Staining and etching is developed for the fabrication of two-dimensional arrays and three-dimensional foam-like metallic nanostructures from a piece of tissue using a combination of metal staining and oxygen plasma etching. This novel nanofabrication technique is repeatable and cost effective, which are very attractive features from the perspective of nanomanufacturing process.


Assuntos
Metais/química , Nanoestruturas/química , Nanotecnologia/métodos , Animais , Colágeno/química , Camundongos , Nanofibras/química , Tendões/química
19.
Langmuir ; 23(19): 9686-94, 2007 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-17696377

RESUMO

This paper describes the use of several methods of template stripping (TS) to produce ultraflat films of silver, gold, palladium, and platinum on both rigid and polymeric mechanical supports: a composite of glass and ultraviolet (UV)-curable adhesive (optical adhesive, OA), solder, a composite of poly(dimethyl siloxane) (PDMS) and OA, and bare OA. Silicon supporting its native oxide layer (Si/SiO2) serves as a template for both mechanical template stripping (mTS), in which the metal film is mechanically cleaved from the template, and chemical template stripping (cTS), in which the film-template composite is immersed in a solution of thiols, and the formation of the SAM on the metal film causes the film to separate from the template. Films formed on all supports have lower root-mean-square (rms) roughness (as measured by atomic force microscopy, AFM) than films used as-deposited (AS-DEP) by electron-beam evaporation. Monolayers of n-dodecanethiolate formed by the mTS and cTS methods are effectively indistinguishable by scanning tunneling microscopy (STM); molecularly resolved images could be obtained using both types of surfaces. The metal surfaces, before being cleaved, are completely protected from contact with the atmosphere. This protection allows metal surfaces intended to support SAMs to be prepared in large batch lots, stored, and then used as needed. Template stripping thus eliminates the requirement for evaporation of the film immediately before use and is a significant extension and simplification of the technology of SAMs and other areas of materials science requiring clean metal surfaces.

20.
Nat Methods ; 4(3): 215-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17277781

RESUMO

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.


Assuntos
Anatomia Transversal/métodos , Microscopia Crioeletrônica/métodos , Escherichia coli/ultraestrutura , Aumento da Imagem/métodos , Imageamento Tridimensional/métodos , Microtomia/métodos , Manejo de Espécimes/métodos , Liofilização/métodos , Prótons
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