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2721 Janelia Publications
Showing 161-170 of 2721 resultsTo support cognitive function, the CA3 region of the hippocampus performs computations involving attractor dynamics. Understanding how cellular and ensemble activities of CA3 neurons enable computation is critical for elucidating the neural correlates of cognition. Here we show that CA3 comprises not only classically described pyramid cells with thorny excrescences, but also includes previously unidentified 'athorny' pyramid cells that lack mossy-fiber input. Moreover, the two neuron types have distinct morphological and physiological phenotypes and are differentially modulated by acetylcholine. To understand the contribution of these athorny pyramid neurons to circuit function, we measured cell-type-specific firing patterns during sharp-wave synchronization events in vivo and recapitulated these dynamics with an attractor network model comprising two principal cell types. Our data and simulations reveal a key role for athorny cell bursting in the initiation of sharp waves: transient network attractor states that signify the execution of pattern completion computations vital to cognitive function.
Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.
Zika virus (ZIKV) is an emerging flavivirus that caused thousands of human infections in recent years. Compared to other human flaviviruses, ZIKV replication is not well understood. Using fluorescent, transmission electron, and focused ion beam-scanning electron microscopy, we examined ZIKV replication dynamics in Vero 76 cells and in the brains of infected laboratory mice. We observed the progressive development of a perinuclear flaviviral replication factory both in vitro and in vivo. In vitro, we illustrated the ZIKV lifecycle from particle cell entry to egress. ZIKV particles assembled and aggregated in an induced convoluted membrane structure and ZIKV strain-specific membranous vesicles. While most mature virus particles egressed via membrane budding, some particles also likely trafficked through late endosomes and egressed through membrane abscission. Interestingly, we consistently observed a novel sheet-like virus particle array consisting of a single layer of ZIKV particles. Our study further defines ZIKV replication and identifies a novel hallmark of ZIKV infection.
Chronic stress could induce severe cognitive impairments. Despite extensive investigations in mammalian models, the underlying mechanisms remain obscure. Here, we show that chronic stress could induce dramatic learning and memory deficits in The chronic stress-induced learning deficit (CSLD) is long lasting and associated with other depression-like behaviors. We demonstrated that excessive dopaminergic activity provokes susceptibility to CSLD. Remarkably, a pair of PPL1-γ1pedc dopaminergic neurons that project to the mushroom body (MB) γ1pedc compartment play a key role in regulating susceptibility to CSLD so that stress-induced PPL1-γ1pedc hyperactivity facilitates the development of CSLD. Consistently, the mushroom body output neurons (MBON) of the γ1pedc compartment, MBON-γ1pedc>α/β neurons, are important for modulating susceptibility to CSLD. Imaging studies showed that dopaminergic activity is necessary to provoke the development of chronic stress-induced maladaptations in the MB network. Together, our data support that PPL1-γ1pedc mediates chronic stress signals to drive allostatic maladaptations in the MB network that lead to CSLD.
The application of microscopy in biomedical research has come a long way since Antonie van Leeuwenhoek discovered unicellular organisms. Countless innovations have positioned light microscopy as a cornerstone of modern biology and a method of choice for connecting omics datasets to their biological and clinical correlates. Still, regardless of how convincing published imaging data looks, it does not always convey meaningful information about the conditions in which it was acquired, processed, and analyzed. Adequate record-keeping, reporting, and quality control are therefore essential to ensure experimental rigor and data fidelity, allow experiments to be reproducibly repeated, and promote the proper evaluation, interpretation, comparison, and re-use. To this end, microscopy images should be accompanied by complete descriptions detailing experimental procedures, biological samples, microscope hardware specifications, image acquisition parameters, and image analysis procedures, as well as metrics accounting for instrument performance and calibration. However, universal, community-accepted Microscopy Metadata standards and reporting specifications that would result in Findable Accessible Interoperable and Reproducible (FAIR) microscopy data have not yet been established. To understand this shortcoming and to propose a way forward, here we provide an overview of the nature of microscopy metadata and its importance for fostering data quality, reproducibility, scientific rigor, and sharing value in light microscopy. The proposal for tiered Microscopy Metadata Specifications that extend the OME Data Model put forth by the 4D Nucleome Initiative and by Bioimaging North America [1-3] as well as a suite of three complementary and interoperable tools are being developed to facilitate the process of image data documentation and are presented in related manuscripts [4-6].
Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.
The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.
Similar to many insect species, Drosophila melanogaster is capable of maintaining a stable flight trajectory for periods lasting up to several hours. Because aerodynamic torque is roughly proportional to the fifth power of wing length, even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to both the damaged and intact wings. Thus, the neural network responsible for stable flight must be capable of sustaining fine-scaled control over wing motion across a large dynamic range. In this study, we describe an unusual type of descending neuron (DNg02) that projects directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike many descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, we demonstrate that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using two-photon functional imaging, we show that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics. Collectively, we have identified a critical set of descending neurons that provides the sensitivity and dynamic range required for flight control.
The human pathogen targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nano-architectural and dynamic changes of focal adhesions. The consequence of -stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, inserts TarP within focal adhesions to alter their organization and stability.
We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1-2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.