1,751 research outputs found
Image informatics strategies for deciphering neuronal network connectivity
Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies
Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images
A fundamental challenge in understanding how dendritic spine morphology controls learning and memory has been quantifying three-dimensional (3D) spine shapes with sufficient precision to distinguish morphologic types, and sufficient throughput for robust statistical analysis. The necessity to analyze large volumetric data sets accurately, efficiently, and in true 3D has been a major bottleneck in deriving reliable relationships between altered neuronal function and changes in spine morphology. We introduce a novel system for automated detection, shape analysis and classification of dendritic spines from laser scanning microscopy (LSM) images that directly addresses these limitations. The system is more accurate, and at least an order of magnitude faster, than existing technologies. By operating fully in 3D the algorithm resolves spines that are undetectable with standard two-dimensional (2D) tools. Adaptive local thresholding, voxel clustering and Rayburst Sampling generate a profile of diameter estimates used to classify spines into morphologic types, while minimizing optical smear and quantization artifacts. The technique opens new horizons on the objective evaluation of spine changes with synaptic plasticity, normal development and aging, and with neurodegenerative disorders that impair cognitive function
Modelo de arborización dendrÃtica basado en reconstrucciones de motoneuronas frénicas en ratas adultas
El área superficial de las dendritas en motoneuronas frénicas (PhrMNs) ha sido estimada anteriormente mediante
técnicas estereológicas basadas en suposiciones geométricas, y medida en tres dimensiones (3D) utilizando microscopÃa confocal.
Dado que el 97% del área receptora de una motoneurona corresponde a sus dendritas, la ramificación y extensión dendrÃtica
son fisiológicamente importantes para determinar la salida de sus campos receptivos. Sin embargo, limitaciones inherentes a las
estimaciones basadas en morfologÃa neuronal y la tinción incompleta de los árboles dendrÃticos mediante técnicas retrógradas
han dificultado los estudios sistemáticos de la morfologÃa dendrÃtica en PhrMNs. En este estudio, se utilizó una nueva técnica
que mejora la tinción dendrÃtica de las PhrMNs en preparaciones fijadas ligeramente. La reconstrucción dendrÃtica en 3D se logró
con gran precisión utilizando microscopÃa confocal en PhrMNs de ratas adultas. Luego de una etapa de pre-procesamiento, la
segmentación de los árboles dendrÃticos se realizó semi-automáticamente en 3D y usando mediciones directas del área superficial,
se derivó un modelo cuadrático para estimar dicha área partiendo del diámetro de la dendrita primaria (r2 = 0.932; p<0.0001).
Este método podrÃa mejorar la evaluación de la plasticidad neuronal en respuesta a trauma u otras enfermedades permitiendo la estimación de la arborización dendrÃtica en PhrMNs, ya que el diámetro de la dendrita primaria puede obtenerse confiablemente de
numerosas técnicas de tinción retrógrada.Stereological techniques that rely on morphological assumptions and direct three-dimensional (3D) confocal
measurements have been previously used to estimate the dendritic surface areas of phrenic motoneurons (PhrMNs). Given that
97% of a motoneuron’s receptive area is provided by dendrites, dendritic branching and overall extension are physiologically
important in determining the output of their synaptic receptive fields. However, limitations intrinsic to shape-based estimations
and incomplete labeling of dendritic trees by retrograde techniques have hindered systematic approaches to examine dendritic
morphology of PhrMNs. In this study, a novel method that improves dendritic filling of PhrMNs in lightly-fixed samples was used.
Confocal microscopy allowed accurate 3D reconstruction of dendritic arbors from adult rat PhrMNs. Following pre-processing,
segmentation was semi-automatically performed in 3D, and direct measurements of dendritic surface area were obtained. A
quadratic model for estimating dendritic tree surface area based on measurements of primary dendrite diameter was derived (r2 =
0.932; p<0.0001). This method may enhance interpretation of motoneuron plasticity in response to injury or disease by permitting
estimations of dendritic arborization of PhrMNs since measurements of primary dendrite diameter can be reliably obtained from a
number of retrograde labeling techniques
Gotta trace ‘em all: A mini-review on tools and procedures for segmenting single neurons toward deciphering the structural connectome
Decoding the morphology and physical connections of all the neurons populating a brain is necessary for predicting and studying the relationships between its form and function, as well as for documenting structural abnormalities in neuropathies. Digitizing a complete and high-fidelity map of the mammalian brain at the micro-scale will allow neuroscientists to understand disease, consciousness, and ultimately what it is that makes us humans. The critical obstacle for reaching this goal is the lack of robust and accurate tools able to deal with 3D datasets representing dense-packed cells in their native arrangement within the brain. This obliges neuroscientist to manually identify the neurons populating an acquired digital image stack, a notably time-consuming procedure prone to human bias. Here we review the automatic and semi-automatic algorithms and software for neuron segmentation available in the literature, as well as the metrics purposely designed for their validation, highlighting their strengths and limitations. In this direction, we also briefly introduce the recent advances in tissue clarification that enable significant improvements in both optical access of neural tissue and image stack quality, and which could enable more efficient segmentation approaches. Finally, we discuss new methods and tools for processing tissues and acquiring images at sub-cellular scales, which will require new robust algorithms for identifying neurons and their sub-structures (e.g., spines, thin neurites). This will lead to a more detailed structural map of the brain, taking twenty-first century cellular neuroscience to the next level, i.e., the Structural Connectome
Modelling Neuron Morphology: Automated Reconstruction from Microscopy Images
Understanding how the brain works is, beyond a shadow of doubt, one of the greatest challenges for modern science. Achieving a deep knowledge about the structure, function and development of the nervous system at the molecular, cellular and network levels is crucial in this attempt, as processes at all these scales are intrinsically linked with higher-order cognitive functions. The research in the various areas of neuroscience deals with advanced imaging techniques, collecting an increasing amounts of heterogeneous and complex data at different scales. Then, computational tools and neuroinformatics solutions are required in order to integrate and analyze the massive quantity of acquired information.
Within this context, the development of automaticmethods and tools for the study of neuronal anatomy has a central role. The morphological properties of the soma and of the axonal and dendritic arborizations constitute a key discriminant for the neuronal phenotype and play a determinant role in network connectivity. A quantitative analysis allows the study of possible factors influencing neuronal development, the neuropathological abnormalities related to specific syndromes, the relationships
between neuronal shape and function, the signal transmission and the network connectivity. Therefore, three-dimensional digital reconstructions of soma, axons and dendrites are indispensable for exploring neural networks. This thesis proposes a novel and completely automatic pipeline for neuron reconstruction with operations ranging from the detection and segmentation of the soma to the dendritic arborization tracing. The pipeline can deal with different datasets and acquisitions both at the network and at the single scale level without any user interventions or manual adjustment. We developed an ad hoc approach for the localization and segmentation of neuron bodies. Then, various methods and research lines have been investigated for the reconstruction of the whole dendritic arborization of each neuron, which is solved both in 2D and in 3D images
Microstimulation and multicellular analysis: A neural interfacing system for spatiotemporal stimulation
Willfully controlling the focus of an extracellular stimulus remains a significant challenge in the development of neural prosthetics and therapeutic devices. In part, this challenge is due to the vast set of complex interactions between the electric fields induced by the microelectrodes and the complex morphologies and dynamics of the neural tissue. Overcoming such issues to produce methodologies for targeted neural stimulation requires a system that is capable of (1) delivering precise, localized stimuli a function of the stimulating electrodes and (2) recording the locations and magnitudes of the resulting evoked responses a function of the cell geometry and membrane dynamics. In order to improve stimulus delivery, we developed microfabrication technologies that could specify the electrode geometry and electrical properties. Specifically, we developed a closed-loop electroplating strategy to monitor and control the morphology of surface coatings during deposition, and we implemented pulse-plating techniques as a means to produce robust, resilient microelectrodes that could withstand rigorous handling and harsh environments. In order to evaluate the responses evoked by these stimulating electrodes, we developed microscopy techniques and signal processing algorithms that could automatically identify and evaluate the electrical response of each individual neuron. Finally, by applying this simultaneous stimulation and optical recording system to the study of dissociated cortical cultures in multielectode arrays, we could evaluate the efficacy of excitatory and inhibitory waveforms. Although we found that the proximity of the electrode is a poor predictor of individual neural excitation thresholds, we have shown that it is possible to use inhibitory waveforms to globally reduce excitability in the vicinity of the electrode. Thus, the developed system was able to provide very high resolution insight into the complex set of interactions between the stimulating electrodes and populations of individual neurons.Ph.D.Committee Chair: Stephen P. DeWeerth; Committee Member: Bruce Wheeler; Committee Member: Michelle LaPlaca; Committee Member: Robert Lee; Committee Member: Steve Potte
Fast extraction of neuron morphologies from large-scale SBFSEM image stacks
Neuron morphology is frequently used to classify cell-types in the mammalian cortex. Apart from the shape of the soma and the axonal projections, morphological classification is largely defined by the dendrites of a neuron and their subcellular compartments, referred to as dendritic spines. The dimensions of a neuron’s dendritic compartment, including its spines, is also a major determinant of the passive and active electrical excitability of dendrites. Furthermore, the dimensions of dendritic branches and spines change during postnatal development and, possibly, following some types of neuronal activity patterns, changes depending on the activity of a neuron. Due to their small size, accurate quantitation of spine number and structure is difficult to achieve (Larkman, J Comp Neurol 306:332, 1991). Here we follow an analysis approach using high-resolution EM techniques. Serial block-face scanning electron microscopy (SBFSEM) enables automated imaging of large specimen volumes at high resolution. The large data sets generated by this technique make manual reconstruction of neuronal structure laborious. Here we present NeuroStruct, a reconstruction environment developed for fast and automated analysis of large SBFSEM data sets containing individual stained neurons using optimized algorithms for CPU and GPU hardware. NeuroStruct is based on 3D operators and integrates image information from image stacks of individual neurons filled with biocytin and stained with osmium tetroxide. The focus of the presented work is the reconstruction of dendritic branches with detailed representation of spines. NeuroStruct delivers both a 3D surface model of the reconstructed structures and a 1D geometrical model corresponding to the skeleton of the reconstructed structures. Both representations are a prerequisite for analysis of morphological characteristics and simulation signalling within a neuron that capture the influence of spines
Automatic dendritic spine detection using multiscale dot enhancement filters and sift features
Statistical characterization of morphological changes of dendritic spines is becoming of crucial interest in the field of neurobiology. Automatic detection and segmentation of dendritic spines promises significant reductions on the time spent by the scientists and reduces the subjectivity concerns. In this paper, we present two approaches for automated detection of dendritic spines in 2-photon laser scanning microscopy (2pLSM) images. The first method combines the
idea of dot enhancement filters with information from the dendritic skeleton. The second method learns an SVM classifier by utilizing some pre-labeled SIFT feature descriptors and uses the classifier to detect dendritic spines in new images. For the segmentation of detected spines, we employ a watershed-variational segmentation algorithm. We evaluate the proposed approaches by comparing with manual segmentations of domain experts and the results of a noncommercial software, NeuronIQ. Our methods produce promising detection rate with high segmentation accuracy thus can serve as a useful tool for spine analysis
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