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174 Janelia Publications
Showing 131-140 of 174 resultsIn April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.
To successfully perform goal-directed navigation, animals must know where they are and what they are doing—e.g., looking for water, bringing food back to the nest, or escaping from a predator. Hippocampal neurons code for these critical variables conjunctively, but little is known about how this where/what code is formed or flexibly routed to other brain regions. To address these questions, we performed intracellular whole-cell recordings in mouse CA1 during a cued, two-choice virtual navigation task. We demonstrate that plateau potentials in CA1 pyramidal neurons rapidly strengthen synaptic inputs carrying conjunctive information about position and choice. Plasticity-induced response fields were modulated by cues only in animals previously trained to collect rewards based on these cues. Thus, we reveal that gradual learning is required for the formation of a conjunctive population code, upstream of CA1, while plateau-potential-induced synaptic plasticity in CA1 enables flexible routing of the code to downstream brain regions.
Many animals use coordinated limb movements to interact with and navigate through the environment. To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to map synaptic connectivity within a neuronal network that controls limb movements. We present a synapse-resolution EM dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we reconstructed 507 motor neurons, including all those that control the legs and wings. We show that a specific class of leg sensory neurons directly synapse onto the largest-caliber motor neuron axons on both sides of the body, representing a unique feedback pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM data acquisition more accessible and affordable to the scientific community.
Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.
Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes; including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 localizes to the apical plasma membrane as well as small, sub-apical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2 containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nano-scale size of these vesicles has limited analysis of their 3D organization. Using a cell system combined with 3D super resolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2 containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the sub-cortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association was enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis.
Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae. Canon neurons are cholinergic interneurons present in each abdominal neuromere and show wave-like activity during fictive backward locomotion. Optogenetic activation of Canon neurons induces relaxation of body wall muscles, whereas inhibition of these neurons disrupts timely muscle relaxation. Canon neurons provide excitatory outputs to inhibitory premotor interneurons. Canon neurons also connect with each other to form an intersegmental circuit and regulate their own wave-like activities. Thus, our results demonstrate how coordinated muscle relaxation can be realized by an intersegmental circuit that regulates its own patterned activity and sequentially terminates motor activities along the anterior-posterior axis.
Neurons decentralize protein synthesis from the cell body to support the active metabolism of remote dendritic and axonal compartments. The neuronal RNA transport apparatus, composed of cis-acting RNA regulatory elements, neuronal transport granule proteins, and motor adaptor complexes, drives the long-distance RNA trafficking required for local protein synthesis. Over the past decade, advances in human genetics, subcellular biochemistry, and high-resolution imaging have implicated each member of the apparatus in several neurodegenerative diseases, establishing failed RNA transport and associated processes as a unifying pathomechanism. In this review, we deconstruct the RNA transport apparatus, exploring each constituent's role in RNA localization and illuminating their unique contributions to neurodegeneration.
Probing the architecture, mechanism, and dynamics of genome folding is fundamental to our understanding of genome function in homeostasis and disease. Most chromosome conformation capture studies dissect the genome architecture with population- and time-averaged snapshots and thus have limited capabilities to reveal 3D nuclear organization and dynamics at the single-cell level. Here, we discuss emerging imaging techniques ranging from light microscopy to electron microscopy that enable investigation of genome folding and dynamics at high spatial and temporal resolution. Results from these studies complement genomic data, unveiling principles underlying the spatial arrangement of the genome and its potential functional links to diverse biological activities in the nucleus.
Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending 'tug-of-war' that controls a temporally shifting window of accessibility for the transcription initiation machinery.