scholarly journals Petascale neural circuit reconstruction: automated methods

2021 ◽  
Author(s):  
Thomas Macrina ◽  
Kisuk Lee ◽  
Ran Lu ◽  
Nicholas L. Turner ◽  
Jingpeng Wu ◽  
...  
Keyword(s):  
Neural Circuit ◽  
Scale Up ◽  
Semantic Categories ◽  
Reconstructed Volume ◽  
Size Constraints ◽  
3D Electron Microscopy ◽  
Image Volume ◽  
Visual Cortical ◽  
3D Em ◽  
Automated Methods

3D electron microscopy (EM) has been successful at mapping invertebrate nervous systems, but the approach has been limited to small chunks of mammalian brains. To scale up to larger volumes, we have built a computational pipeline for processing petascale image datasets acquired by serial section EM, a popular form of 3D EM. The pipeline employs convolutional nets to compute the nonsmooth transformations required to align images of serial sections containing numerous cracks and folds, detect neuronal boundaries, label voxels as axon, dendrite, soma, and other semantic categories, and detect synapses and assign them to presynaptic and postsynaptic segments. The output of neuronal boundary detection is segmented by mean affinity agglomeration with semantic and size constraints. Pipeline operations are implemented by leveraging distributed and cloud computing. Intermediate results of the pipeline are held in cloud storage, and can be effortlessly viewed as images, which aids debugging. We applied the pipeline to create an automated reconstruction of an EM image volume spanning four visual cortical areas of a mouse brain. Code for the pipeline is publicly available, as is the reconstructed volume.

scholarly journals Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey A. Polilov ◽  
Anastasia A. Makarova ◽  
Song Pang ◽  
C. Shan Xu ◽  
Harald Hess
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Entire Volume ◽  
Simple Protocol ◽  
3D Electron Microscopy ◽  
3D Em

AbstractModern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

scholarly journals Progressive maturation of silent synapses governs the duration of a critical period

2015 ◽  
Vol 112 (24) ◽  
pp. E3131-E3140 ◽  
Author(s):  
Xiaojie Huang ◽  
Sophia K. Stodieck ◽  
Bianka Goetze ◽  
Lei Cui ◽  
Man Ho Wong ◽  
...  
Keyword(s):  
Visual Cortex ◽  
Critical Period ◽  
Neural Circuit ◽  
Ocular Dominance ◽  
Critical Periods ◽  
Ocular Dominance Plasticity ◽  
Silent Synapses ◽  
Synapse Maturation ◽  
Silent Synapse ◽  
Visual Cortical

During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95–dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.

Three-dimensional ultrastructure of the brain pericyte-endothelial interface

2021 ◽  
pp. 0271678X2110128
Author(s):  
Sharon Ornelas ◽  
Andrée-Anne Berthiaume ◽  
Stephanie K Bonney ◽  
Vanessa Coelho-Santos ◽  
Robert G Underly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Large Scale ◽  
Three Dimensional ◽  
Morphological Evidence ◽  
Data Set ◽  
Biochemical Interaction ◽  
3D Electron Microscopy ◽  
3D Em ◽  
Microscopy Techniques ◽  
The Brain

Pericytes and endothelial cells share membranous interdigitations called “peg-and-socket” interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

scholarly journals Extracellular space preservation aids the connectomic analysis of neural circuits

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Marta Pallotto ◽  
Paul V Watkins ◽  
Boma Fubara ◽  
Joshua H Singer ◽  
Kevin L Briggman
Keyword(s):  
Extracellular Space ◽  
Neural Circuits ◽  
Error Rates ◽  
Machine Learning Algorithms ◽  
Tissue Fixation ◽  
Electrical Synapses ◽  
3D Electron Microscopy ◽  
Substantial Error ◽  
Time Required ◽  
3D Em

Dense connectomic mapping of neuronal circuits is limited by the time and effort required to analyze 3D electron microscopy (EM) datasets. Algorithms designed to automate image segmentation suffer from substantial error rates and require significant manual error correction. Any improvement in segmentation error rates would therefore directly reduce the time required to analyze 3D EM data. We explored preserving extracellular space (ECS) during chemical tissue fixation to improve the ability to segment neurites and to identify synaptic contacts. ECS preserved tissue is easier to segment using machine learning algorithms, leading to significantly reduced error rates. In addition, we observed that electrical synapses are readily identified in ECS preserved tissue. Finally, we determined that antibodies penetrate deep into ECS preserved tissue with only minimal permeabilization, thereby enabling correlated light microscopy (LM) and EM studies. We conclude that preservation of ECS benefits multiple aspects of the connectomic analysis of neural circuits.

scholarly journals A semi-automated approach to dense segmentation of 3D white matter electron microscopy

2020 ◽  
Author(s):  
Michiel Kleinnijenhuis ◽  
Errin Johnson ◽  
Jeroen Mollink ◽  
Saad Jbabdi ◽  
Karla L. Miller
Keyword(s):  
Electron Microscopy ◽  
White Matter ◽  
Ground Truth ◽  
Outer Diameter ◽  
Myelinated Axons ◽  
Unmyelinated Axons ◽  
White Matter Microstructure ◽  
3D Electron Microscopy ◽  
3D Em ◽  
G Ratio

ABSTRACTPurposeNeuroscience methods working on widely different scales can complement and inform each other. At the macroscopic scale, magnetic resonance imaging methods that estimate microstructural measures have much to gain from ground truth validation and models based on accurate measurement of that microstructure. We present an approach to generate rich and accurate geometric models of white matter microstructure through dense segmentation of 3D electron microscopy (EM).MethodsVolumetric data of the white matter of the genu of the corpus callosum of the adult mouse brain were acquired using serial blockface scanning electron microscopy (SBF-SEM). A segmentation pipeline was developed to separate the 3D EM data into compartments and individual cellular and subcellular constituents, making use of established tools as well as newly developed algorithms to achieve accurate segmentation of various compartments.ResultsThe volume was segmented into six compartments comprising myelinated axons (axon, myelin sheath, nodes of Ranvier), oligodendrocytes, blood vessels, mitochondria, and unmyelinated axons. The myelinated axons had an average inner diameter of 0.56 μm and an average outer diameter of 0.87 μm. The diameter of unmyelinated axons was 0.43 μm. A mean g-ratio of 0.61 was found for myelinated axons, but the g-ratio was highly variable between as well as within axons.ConclusionThe approach for segmentation of 3D EM data yielded a dense annotation of a range of white matter compartments that can be interrogated for their properties and used for in silico experiments of brain structure. We provide the resulting dense annotation as a resource to the neuroscience community.

scholarly journals Cortical-like dynamics in recurrent circuits optimized for sampling-based probabilistic inference

2019 ◽  
Author(s):  
Rodrigo Echeveste ◽  
Laurence Aitchison ◽  
Guillaume Hennequin ◽  
Máté Lengyel
Keyword(s):  
Neural Circuit ◽  
Probabilistic Inference ◽  
Gamma Oscillations ◽  
Biological Properties ◽  
Stimulus Onset ◽  
Cortical Dynamics ◽  
Functional Roles ◽  
Sensory Cortices ◽  
Dynamical Features ◽  
Visual Cortical

Sensory cortices display a suite of ubiquitous dynamical features, such as ongoing noise variability, transient overshoots, and oscillations, that have so far escaped a common, principled theoretical account. We developed a unifying model for these phenomena by training a recurrent excitatory–inhibitory neural circuit model of a visual cortical hypercolumn to perform sampling-based probabilistic inference. The optimized network displayed several key biological properties, including divisive normalization, as well as stimulus-modulated noise variability, inhibition-dominated transients at stimulus onset, and strong gamma oscillations. These dynamical features had distinct functional roles in speeding up inferences and made predictions that we confirmed in novel analyses of awake monkey recordings. Our results suggest that the basic motifs of cortical dynamics emerge as a consequence of the efficient implementation of the same computational function—fast sampling-based inference—and predict further properties of these motifs that can be tested in future experiments.

scholarly journals Permeabilization-free en bloc immunohistochemistry for correlative microscopy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kara A Fulton ◽  
Kevin L Briggman
Keyword(s):  
Neuronal Cell ◽  
Cell Types ◽  
Brain Regions ◽  
Synaptic Proteins ◽  
En Bloc ◽  
Two Photon ◽  
Tissue Sections ◽  
3D Electron Microscopy ◽  
3D Em ◽  
Major Impediment

A dense reconstruction of neuronal synaptic connectivity typically requires high-resolution 3D electron microscopy (EM) data, but EM data alone lacks functional information about neurons and synapses. One approach to augment structural EM datasets is with the fluorescent immunohistochemical (IHC) localization of functionally relevant proteins. We describe a protocol that obviates the requirement of tissue permeabilization in thick tissue sections, a major impediment for correlative pre-embedding IHC and EM. We demonstrate the permeabilization-free labeling of neuronal cell types, intracellular enzymes, and synaptic proteins in tissue sections hundreds of microns thick in multiple brain regions from mice while simultaneously retaining the ultrastructural integrity of the tissue. Finally, we explore the utility of this protocol by performing proof-of-principle correlative experiments combining two-photon imaging of protein distributions and 3D EM.

scholarly journals Isotropic 3D electron microscopy reference library of whole cells and tissues

2020 ◽  
Author(s):  
C. Shan Xu ◽  
Song Pang ◽  
Gleb Shtengel ◽  
Andreas Müller ◽  
Alex T. Ritter ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Ion Beam ◽  
Reference Library ◽  
Whole Cells ◽  
3D Electron Microscopy ◽  
Mouse Pancreatic Islets ◽  
Focus Ion Beam ◽  
3D Em ◽  
Em Tomography

SummaryUnderstanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structure with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Here, we overcome these limitations by imaging whole cells and tissues with enhanced Focus Ion Beam Scanning Electron Microscopy (FIB-SEM) in high resolution with month-long acquisition duration. We use this approach to generate reference 3D image datasets at 4-nm isotropic voxels for ten different examples, including cultured cells (cancer, macrophages, and T-cells) as well as tissues (mouse pancreatic islets and the Drosophila fan-shaped body). We open access to all datasets in OpenOrganelle, an interactive web platform that allows accessing both the original 3D EM data, and subsequent organelle segmentation. Together, these data will serve as a reference library to explore comprehensive quantification of whole cells and their constituents, thus addressing questions related to cell identities, cell morphologies, cell-cell interactions, as well as intracellular organelle organization and structure.

NiAl precipitation in Fe-12w/o Cr-5w/o Ni-4w/o Al

1983 ◽  
Vol 41 ◽  
pp. 202-203
Author(s):  
L.E. Murr ◽  
J.S. Dunning ◽  
S. Shankar
Keyword(s):  
Solid Solution ◽  
Stainless Steel ◽  
Stainless Steels ◽  
Alloy Composition ◽  
Chromium Content ◽  
Scale Up ◽  
Optical Metallography ◽  
Property Evaluation ◽  
310 Stainless Steel ◽  
Microstructural Studies

Aluminum additions to conventional 18Cr-8Ni austenitic stainless steel compositions impart excellent resistance to high sulfur environments. However, problems are typically encountered with aluminum additions above about 1% due to embrittlement caused by aluminum in solid solution and the precipitation of NiAl. Consequently, little use has been made of aluminum alloy additions to stainless steels for use in sulfur or H2S environments in the chemical industry, energy conversion or generation, and mineral processing, for example.A research program at the Albany Research Center has concentrated on the development of a wrought alloy composition with as low a chromium content as possible, with the idea of developing a low-chromium substitute for 310 stainless steel (25Cr-20Ni) which is often used in high-sulfur environments. On the basis of workability and microstructural studies involving optical metallography on 100g button ingots soaked at 700°C and air-cooled, a low-alloy composition Fe-12Cr-5Ni-4Al (in wt %) was selected for scale up and property evaluation.

Ultrastructure of developing visual cortical synapses in the cat superior colliculus

1991 ◽  
Vol 49 ◽  
pp. 156-157
Author(s):  
Caroline A. Miller ◽  
Laura L. Bruce
Keyword(s):  
Superior Colliculus ◽  
Phosphate Buffer ◽  
Receptive Fields ◽  
Visual Cortical Area ◽  
Cortical Synapses ◽  
Visual Cortical ◽  
And Function ◽  
Phosphate Buffered Saline ◽  
The Brain ◽  
Cat Superior Colliculus

The first visual cortical axons arrive in the cat superior colliculus by the time of birth. Adultlike receptive fields develop slowly over several weeks following birth. The developing cortical axons go through a sequence of changes before acquiring their adultlike morphology and function. To determine how these axons interact with neurons in the colliculus, cortico-collicular axons were labeled with biocytin (an anterograde neuronal tracer) and studied with electron microscopy.Deeply anesthetized animals received 200-500 nl injections of biocytin (Sigma; 5% in phosphate buffer) in the lateral suprasylvian visual cortical area. After a 24 hr survival time, the animals were deeply anesthetized and perfused with 0.9% phosphate buffered saline followed by fixation with a solution of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer. The brain was sectioned transversely on a vibratome at 50 μm. The tissue was processed immediately to visualize the biocytin.

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