102 research outputs found

    Mechanical and Electrophysiological Properties of the Sarcolemma of Muscle Fibers in Two Murine Models of Muscle Dystrophy: Col6a1−/− and Mdx

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    This study aimed to analyse the sarcolemma of Col6a1−/− fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1−/− and increased in mdx fibers. In the membrane of Col6a1−/− fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation

    A cellular-resolution atlas of the larval zebrafish brain

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    Understanding brain-wide neuronal dynamics requires a detailed map of the underlying circuit architecture. We built an interactive cellular-resolution atlas of the zebrafish brain at 6 days post-fertilization (dpf) based on the reconstructions of over 2,000 individually GFP-labeled neurons. We clustered our dataset in "morphotypes,'' establishing a unique database of quantitatively described neuronal morphologies together with their spatial coordinates in vivo. Over 100 transgene expression patterns were imaged separately and co-registered with the single-neuron atlas. By annotating 72 non-overlapping brain regions, we generated from our dataset an inter-areal wiring diagram of the larval brain, which serves as ground truth for synapse-scale, electron microscopic reconstructions. Interrogating our atlas by "virtual tract tracing'' has already revealed previously unknown wiring principles in the tectum and the cerebellum. In conclusion, we present here an evolving computational resource and visualization tool, which will be essential to map function to structure in a vertebrate brain

    A novel sensor for ion electron emission microscopy

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    Abstract An ion electron emission microscope (IEEM) to be installed at the SIRAD heavy ion irradiation facility at the 15 MV tandem accelerator of the INFN Legnaro laboratory (Italy) will be used to characterize the sensitivity of electronic devices to single event effects (SEE) to ion impacts with micrometric lateral resolutions. The secondary electrons emitted by ion impacts from the target surface are transported and focused by an electron microscope onto a micro-channel plate (MCP) detector coupled to a fast phosphor. The luminous signal is then detected by a position sensitive photon detector located outside the vacuum chamber. The high repetition rates and high spatial resolution, required to temporally distinguish ion impacts for SEE studies and avoid degrading of the initial resolution of the IEEM and MCP are met by the system, presented here for the first time, based on two orthogonal linear CCDs

    Ultra-fast quantum randomness generation by accelerated phase diffusion in a pulsed laser diode

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    We demonstrate a high bit-rate quantum random number generator by interferometric detection of phase diffusion in a gain-switched DFB laser diode. Gain switching at few-GHz frequencies produces a train of bright pulses with nearly equal amplitudes and random phases. An unbalanced Mach-Zehnder interferometer is used to interfere subsequent pulses and thereby generate strong random-amplitude pulses, which are detected and digitized to produce a high-rate random bit string. Using established models of semiconductor laser field dynamics, we predict a regime of high visibility interference and nearly complete vacuum-fluctuation-induced phase diffusion between pulses. These are confirmed by measurement of pulse power statistics at the output of the interferometer. Using a 5.825 GHz excitation rate and 14-bit digitization, we observe 43 Gbps quantum randomness generation.This work was supported by the ERC under project MAMBO (Proof of Concept of PER- CENT) and project AQUMET, MINECO under projects FIS2011-23520, TEC2010-14832, and Explora INTRINQRA, Galician Regional Government under projects CN2012/279 and CN2012/260 “Consolidation of research units: AtlantTIC” and FEDER under project Ref: UPVOV10-3E-492.Peer ReviewedPostprint (published version

    A Protective Role for ELR+ Chemokines during Acute Viral Encephalomyelitis

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    The functional role of ELR-positive CXC chemokines in host defense during acute viral-induced encephalomyelitis was determined. Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice resulted in the rapid mobilization of PMNs expressing the chemokine receptor CXCR2 into the blood. Migration of PMNs to the CNS coincided with increased expression of transcripts specific for the CXCR2 ELR-positive chemokine ligands CXCL1, CXCL2, and CXCL5 within the brain. Treatment of JHMV-infected mice with anti-CXCR2 blocking antibody reduced PMN trafficking into the CNS by >95%, dampened MMP-9 activity, and abrogated blood-brain-barrier (BBB) breakdown. Correspondingly, CXCR2 neutralization resulted in diminished infiltration of virus-specific T cells, an inability to control viral replication within the brain, and 100% mortality. Blocking CXCR2 signaling did not impair the generation of virus-specific T cells, indicating that CXCR2 is not required to tailor anti-JHMV T cell responses. Evaluation of mice in which CXCR2 is genetically silenced (CXCR2−/− mice) confirmed that PMNs neither expressed CXCR2 nor migrated in response to ligands CXCL1, CXCL2, or CXCL5 in an in vitro chemotaxis assay. Moreover, JHMV infection of CXCR2−/− mice resulted in an approximate 60% reduction of PMN migration into the CNS, yet these mice survived infection and controlled viral replication within the brain. Treatment of JHMV-infected CXCR2−/− mice with anti-CXCR2 antibody did not modulate PMN migration nor alter viral clearance or mortality, indicating the existence of compensatory mechanisms that facilitate sufficient migration of PMNs into the CNS in the absence of CXCR2. Collectively, these findings highlight a previously unappreciated role for ELR-positive chemokines in enhancing host defense during acute viral infections of the CNS

    In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes

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    Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells

    An Optical Illusion Pinpoints an Essential Circuit Node for Global Motion Processing

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    Direction-selective (DS) neurons compute the direction of motion in a visual scene. Brain-wide imaging in larval zebrafish has revealed hundreds of DS neurons scattered throughout the brain. However, the exact population that causally drives motion-dependent behaviors\u2014e.g., compensatory eye and body movements\u2014remains largely unknown. To identify the behaviorally relevant population of DS neurons, here we employ the motion aftereffect (MAE), which causes the well-known \u201cwaterfall illusion.\u201d Together with region-specific optogenetic manipulations and cellular-resolution functional imaging, we found that MAE-responsive neurons represent merely a fraction of the entire population of DS cells in larval zebrafish. They are spatially clustered in a nucleus in the ventral lateral pretectal area and are necessary and sufficient to steer the entire cycle of optokinetic eye movements. Thus, our illusion-based behavioral paradigm, combined with optical imaging and optogenetics, identified key circuit elements of global motion processing in the vertebrate brain

    Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging

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    We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane-GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution
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