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62 Janelia Publications

Showing 1-10 of 62 results
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    Looger Lab
    12/16/10 | Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.
    Xiang Y, Yuan Q, Vogt N, Looger LL, Jan LY, Jan YN
    Nature. 2010 Dec 16;468(7326):921-6. doi: 10.1038/nature09576

    Photoreceptors for visual perception, phototaxis or light avoidance are typically clustered in eyes or related structures such as the Bolwig organ of Drosophila larvae. Unexpectedly, we found that the class IV dendritic arborization neurons of Drosophila melanogaster larvae respond to ultraviolet, violet and blue light, and are major mediators of light avoidance, particularly at high intensities. These class IV dendritic arborization neurons, which are present in every body segment, have dendrites tiling the larval body wall nearly completely without redundancy. Dendritic illumination activates class IV dendritic arborization neurons. These novel photoreceptors use phototransduction machinery distinct from other photoreceptors in Drosophila and enable larvae to sense light exposure over their entire bodies and move out of danger.

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    12/07/10 | Reconstructing embryonic development.
    Khairy K, Keller PJ
    Genesis. 2010 Dec 7;49(7):488-513. doi: 10.1002/dvg.20698

    Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources.

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    Looger Lab
    12/02/10 | A dimorphic pheromone circuit in Drosophila from sensory input to descending output.
    Ruta V, Datta SR, Vasconcelos ML, Freeland J, Looger LL, Axel R
    Nature. 2010 Dec 2;468(7324):686-90. doi: 10.1038/nature09554

    Drosophila show innate olfactory-driven behaviours that are observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically programmed. Despite the numerical simplicity of the fly nervous system, features of the anatomical organization of the fly brain often confound the delineation of these circuits. Here we identify a neural circuit responsive to cVA, a pheromone that elicits sexually dimorphic behaviours. We have combined neural tracing using an improved photoactivatable green fluorescent protein (PA-GFP) with electrophysiology, optical imaging and laser-mediated microlesioning to map this circuit from the activation of sensory neurons in the antennae to the excitation of descending neurons in the ventral nerve cord. This circuit is concise and minimally comprises four neurons, connected by three synapses. Three of these neurons are overtly dimorphic and identify a male-specific neuropil that integrates inputs from multiple sensory systems and sends outputs to the ventral nerve cord. This neural pathway suggests a means by which a single pheromone can elicit different behaviours in the two sexes.

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    11/25/10 | Nanoscale architecture of integrin-based cell adhesions.
    Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM
    Nature. 2010 Nov 25;468(7323):580-4. doi: 10.1038/nature09621

    Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity, and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signaling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a \~{}40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signaling layer containing integrin cytoplasmic tails, focal adhesion kinase, and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

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    Looger Lab
    11/10/10 | Toward the second generation of optogenetic tools.
    Knöpfel T, Lin MZ, Levskaya A, Tian L, Lin JY, Boyden ES
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2010 Nov 10;30(45):14998-5004. doi: 10.1523/JNEUROSCI.4190-10.2010

    This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.

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    Looger LabSvoboda Lab
    11/01/10 | Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.
    Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW
    Nature Neuroscience. 2010 Nov;13(11):1433-40. doi: 10.1038/nn.2648

    Spatial navigation is often used as a behavioral task in studies of the neuronal circuits that underlie cognition, learning and memory in rodents. The combination of in vivo microscopy with genetically encoded indicators has provided an important new tool for studying neuronal circuits, but has been technically difficult to apply during navigation. Here we describe methods for imaging the activity of neurons in the CA1 region of the hippocampus with subcellular resolution in behaving mice. Neurons that expressed the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-restrained mice performed spatial behaviors in a setup combining a virtual reality system and a custom-built two-photon microscope. We optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit. The combination of virtual reality and high-resolution functional imaging should allow a new generation of studies to investigate neuronal circuit dynamics during behavior.

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    Svoboda Lab
    11/01/10 | The functional asymmetry of auditory cortex is reflected in the organization of local cortical circuits.
    Oviedo HV, Bureau I, Svoboda K, Zador AM
    Nature Neuroscience. 2010 Nov;13(11):1413-20. doi: 10.1038/nn.2659

    The primary auditory cortex (A1) is organized tonotopically, with neurons sensitive to high and low frequencies arranged in a rostro-caudal gradient. We used laser scanning photostimulation in acute slices to study the organization of local excitatory connections onto layers 2 and 3 (L2/3) of the mouse A1. Consistent with the organization of other cortical regions, synaptic inputs along the isofrequency axis (orthogonal to the tonotopic axis) arose predominantly within a column. By contrast, we found that local connections along the tonotopic axis differed from those along the isofrequency axis: some input pathways to L3 (but not L2) arose predominantly out-of-column. In vivo cell-attached recordings revealed differences between the sound-responsiveness of neurons in L2 and L3. Our results are consistent with the hypothesis that auditory cortical microcircuitry is specialized to the one-dimensional representation of frequency in the auditory cortex.

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    Looger Lab
    10/29/10 | Filtering of visual information in the tectum by an identified neural circuit.
    Del Bene F, Wyart C, Robles E, Tran A, Looger L, Scott EK, Isacoff EY, Baier H
    Science. 2010 Oct 29;330(6004):669-73. doi: 10.1126/science.1192949

    The optic tectum of zebrafish is involved in behavioral responses that require the detection of small objects. The superficial layers of the tectal neuropil receive input from retinal axons, while its deeper layers convey the processed information to premotor areas. Imaging with a genetically encoded calcium indicator revealed that the deep layers, as well as the dendrites of single tectal neurons, are preferentially activated by small visual stimuli. This spatial filtering relies on GABAergic interneurons (using the neurotransmitter γ-aminobutyric acid) that are located in the superficial input layer and respond only to large visual stimuli. Photo-ablation of these cells with KillerRed, or silencing of their synaptic transmission, eliminates the size tuning of deeper layers and impairs the capture of prey.

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    Riddiford Lab
    10/01/10 | Developmental expression of mRNAs for epidermal and fat body proteins and hormonally regulated transcription factors in the tobacco hornworm, Manduca sexta.
    Hiruma K, Riddiford LM
    Journal of Insect Physiology. 2010 Oct;56(10):1390-5. doi: 10.1016/j.jinsphys.2010.03.029

    This paper provides a compilation of diagrammatic representations of the expression profiles of epidermal and fat body mRNAs during the last two larval instars and metamorphosis of the tobacco hornworm, Manduca sexta. Included are those encoding insecticyanin, three larval cuticular proteins, dopa decarboxylase, moling, and the juvenile hormone-binding protein JP29 produced by the dorsal abdominal epidermis, and arylphorin and the methionine-rich storage proteins made by the fat body. The mRNA profiles of the ecdysteroid-regulated cascade of transcription factors in the epidermis during the larval molt and the onset of metamorphosis and in the pupal wing during the onset of adult development are also shown. These profiles are accompanied by a brief summary of the current knowledge about the regulation of these mRNAs by ecdysteroids and juvenile hormone based on experimental manipulations, both in vivo and in vitro.

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    10/01/10 | Error tolerant indexing and alignment of short reads with covering template families.
    Giladi E, Healy J, Myers G, Hart C, Kapranov P, Lipson D, Roels S, Thayer E, Letovsky S
    Journal of Computational Biology: A Journal of Computational Molecular Cell Biology. 2010 Oct;17(10):1397-1411. doi: 10.1089/cmb.2010.0005

    The rapid adoption of high-throughput next generation sequence data in biological research is presenting a major challenge for sequence alignment tools—specifically, the efficient alignment of vast amounts of short reads to large references in the presence of differences arising from sequencing errors and biological sequence variations. To address this challenge, we developed a short read aligner for high-throughput sequencer data that is tolerant of errors or mutations of all types—namely, substitutions, deletions, and insertions. The aligner utilizes a multi-stage approach in which template-based indexing is used to identify candidate regions for alignment with dynamic programming. A template is a pair of gapped seeds, with one used with the read and one used with the reference. In this article, we focus on the development of template families that yield error-tolerant indexing up to a given error-budget. A general algorithm for finding those families is presented, and a recursive construction that creates families with higher error tolerance from ones with a lower error tolerance is developed.

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