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1.
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
2.
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
3.
Med Image Anal ; 22(1): 77-88, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25791436

RESUMO

Automated sample preparation and electron microscopy enables acquisition of very large image data sets. These technical advances are of special importance to the field of neuroanatomy, as 3D reconstructions of neuronal processes at the nm scale can provide new insight into the fine grained structure of the brain. Segmentation of large-scale electron microscopy data is the main bottleneck in the analysis of these data sets. In this paper we present a pipeline that provides state-of-the art reconstruction performance while scaling to data sets in the GB-TB range. First, we train a random forest classifier on interactive sparse user annotations. The classifier output is combined with an anisotropic smoothing prior in a Conditional Random Field framework to generate multiple segmentation hypotheses per image. These segmentations are then combined into geometrically consistent 3D objects by segmentation fusion. We provide qualitative and quantitative evaluation of the automatic segmentation and demonstrate large-scale 3D reconstructions of neuronal processes from a 27,000 µm(3) volume of brain tissue over a cube of 30 µm in each dimension corresponding to 1000 consecutive image sections. We also introduce Mojo, a proofreading tool including semi-automated correction of merge errors based on sparse user scribbles.


Assuntos
Encéfalo/ultraestrutura , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Neurônios/ultraestrutura , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Aumento da Imagem/métodos , Aprendizado de Máquina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Técnica de Subtração
4.
Development ; 141(20): 3994-4005, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25294943

RESUMO

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Animais , Proteínas de Bactérias/química , Cruzamentos Genéticos , Éxons , Feminino , Técnicas Genéticas , Genoma , Proteínas Luminescentes/química , Masculino , Ovário/metabolismo , Fatores Sexuais , Testículo/metabolismo , Transcrição Genética
5.
IEEE Trans Vis Comput Graph ; 20(12): 2466-75, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26356960

RESUMO

Proofreading refers to the manual correction of automatic segmentations of image data. In connectomics, electron microscopy data is acquired at nanometer-scale resolution and results in very large image volumes of brain tissue that require fully automatic segmentation algorithms to identify cell boundaries. However, these algorithms require hundreds of corrections per cubic micron of tissue. Even though this task is time consuming, it is fairly easy for humans to perform corrections through splitting, merging, and adjusting segments during proofreading. In this paper we present the design and implementation of Mojo, a fully-featured single-user desktop application for proofreading, and Dojo, a multi-user web-based application for collaborative proofreading. We evaluate the accuracy and speed of Mojo, Dojo, and Raveler, a proofreading tool from Janelia Farm, through a quantitative user study. We designed a between-subjects experiment and asked non-experts to proofread neurons in a publicly available connectomics dataset. Our results show a significant improvement of corrections using web-based Dojo, when given the same amount of time. In addition, all participants using Dojo reported better usability. We discuss our findings and provide an analysis of requirements for designing visual proofreading software.


Assuntos
Gráficos por Computador , Conectoma/métodos , Imageamento Tridimensional/métodos , Adulto , Algoritmos , Animais , Feminino , Humanos , Internet , Masculino , Camundongos , Software , Adulto Jovem
6.
Bioinformatics ; 27(16): 2216-23, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21742636

RESUMO

MOTIVATION: Three-dimensional reconstruction of consecutive serial-section transmission electron microscopy (ssTEM) images of neural tissue currently requires many hours of manual tracing and annotation. Several computational techniques have already been applied to ssTEM images to facilitate 3D reconstruction and ease this burden. RESULTS: Here, we present an alternative computational approach for ssTEM image analysis. We have used biologically inspired receptive fields as a basis for a ridge detection algorithm to identify cell membranes, synaptic contacts and mitochondria. Detected line segments are used to improve alignment between consecutive images and we have joined small segments of membrane into cell surfaces using a dynamic programming algorithm similar to the Needleman-Wunsch and Smith-Waterman DNA sequence alignment procedures. A shortest path-based approach has been used to close edges and achieve image segmentation. Partial reconstructions were automatically generated and used as a basis for semi-automatic reconstruction of neural tissue. The accuracy of partial reconstructions was evaluated and 96% of membrane could be identified at the cost of 13% false positive detections. AVAILABILITY: An open-source reference implementation is available in the Supplementary information. CONTACT: seymour.kb@ed.ac.uk; douglas.armstrong@ed.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Transmissão/métodos , Neurônios/ultraestrutura , Algoritmos , Animais , Membrana Celular/ultraestrutura , Drosophila/ultraestrutura
7.
PLoS One ; 5(8): e12062, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20706579

RESUMO

BACKGROUND: A complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed. METHODOLOGY/PRINCIPAL FINDINGS: Using automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep. CONCLUSIONS: We conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans.


Assuntos
Ração Animal , Comportamento Animal , Drosophila melanogaster/fisiologia , Sono , Vigília , Animais , Comportamento Animal/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Feminino , Masculino , Atividade Motora/efeitos dos fármacos , Fatores Sexuais , Sono/efeitos dos fármacos , Sacarose/farmacologia , Vigília/efeitos dos fármacos , Leveduras
8.
Database (Oxford) ; 2010: baq005, 2010 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-20624714

RESUMO

Protein-trap strains of Drosophila melanogaster provide a very useful tool for examining the 3D-expression patterns of proteins and purification of protein complexes. Here we present BrainTrap, available at http://fruitfly.inf.ed.ac.uk/braintrap, an online database of 3D confocal datasets showing reporter gene expression and protein localization in the adult brain of Drosophila. Full size images throughout the volume of the entire brain can be viewed interactively in a web browser. The database includes searchable annotations linked to the FlyBase Drosophila anatomy ontology. Anatomical search criteria can be specified using automatic completion and a hierarchical browser for the ontology. The provenance of all annotation is retained and the location where the annotator made the conclusion can be highlighted. Database URL: http://fruitfly.inf.ed.ac.uk/braintrap.


Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Bases de Dados de Proteínas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Animais , Drosophila melanogaster/anatomia & histologia , Perfilação da Expressão Gênica/estatística & dados numéricos , Genes de Insetos , Imageamento Tridimensional , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Distribuição Tecidual
9.
PLoS One ; 5(3): e9783, 2010 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-20339544

RESUMO

BACKGROUND: Diurnal variations in the incidence of events such as heart attack and stroke suggest a role for circadian rhythms in the etiology of cardiovascular disease. The aim of this study was to assess the influence of the suprachiasmatic nucleus (SCN) circadian clock on cardiovascular function. METHODOLOGY/PRINCIPAL FINDINGS: Heart rate (HR), blood pressure (BP) and locomotor activity (LA) were measured in circadian mutant (Vipr2(-/-)) mice and wild type littermates, using implanted radio-telemetry devices. Sleep and wakefulness were studied in similar mice implanted with electroencephalograph (EEG) electrodes. There was less diurnal variation in the frequency and duration of bouts of rest/activity and sleep/wake in Vipr2(-/-) mice than in wild type (WT) and short "ultradian" episodes of arousal were more prominent, especially in constant conditions (DD). Activity was an important determinant of circadian variation in BP and HR in animals of both genotypes; altered timing of episodes of activity and rest (as well as sleep and wakefulness) across the day accounted for most of the difference between Vipr2(-/-) mice and WT. However, there was also a modest circadian rhythm of resting HR and BP that was independent of LA. CONCLUSIONS/SIGNIFICANCE: If appropriate methods of analysis are used that take into account sleep and locomotor activity level, mice are a good model for understanding the contribution of circadian timing to cardiovascular function. Future studies of the influence of sleep and wakefulness on cardiovascular physiology may help to explain accumulating evidence linking disrupted sleep with cardiovascular disease in man.


Assuntos
Ritmo Circadiano , Frequência Cardíaca/fisiologia , Núcleo Supraquiasmático/metabolismo , Animais , Comportamento Animal , Pressão Sanguínea , Fenômenos Fisiológicos Cardiovasculares , Eletroencefalografia/métodos , Hemodinâmica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Movimento
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