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186 Janelia Publications

Showing 51-60 of 186 results
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    Svoboda Lab
    08/23/17 | Circuits in the rodent brainstem that control whisking in concert with other orofacial motor actions.
    McElvain LE, Friedman B, Karten HJ, Svoboda K, Wang F, Deschênes M, Kleinfeld D
    Neuroscience. 2017 Aug 23;368:152-70. doi: 10.1016/j.neuroscience.2017.08.034

    The world view of rodents is largely determined by sensation on two length scales. One is within the animal's peri-personal space. Sensorimotor control on this scale involves active movements of the nose, tongue, head, and vibrissa, along with sniffing to determine olfactory clues. The second scale involves the detection of more distant space through vision and audition; these detection processes also impact repositioning of the head, eyes, and ears. Here we focus on orofacial motor actions, primarily vibrissa-based touch but including nose twitching, head bobbing, and licking, that control sensation at short, peri-personal distances. The orofacial nuclei for control of the motor plants, as well as primary and secondary sensory nuclei associated with these motor actions, lie within the hindbrain. The current data support three themes: First, the position of the sensors is determined by the summation of two drive signals, i.e., a fast rhythmic component and an evolving orienting component. Second, the rhythmic component is coordinated across all orofacial motor actions and is phase-locked to sniffing as the animal explores. Reverse engineering reveals that the preBötzinger inspiratory complex provides the reset to the relevant premotor oscillators. Third, direct feedback from somatosensory trigeminal nuclei can rapidly alter motion of the sensors. This feedback is disynaptic and can be tuned by high-level inputs. The elucidation of synergistic coordination of orofacial motor actions to form behaviors, beyond that of a common rhythmic component, represents a work in progress that encompasses feedback through the midbrain and forebrain as well as hindbrain areas.

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    08/18/17 | Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner.
    Hong J, Murugesan S, Betzig E, Hammer JA
    PLoS One. 2017;12(8):e0183174. doi: 10.1371/journal.pone.0183174

    Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS.

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    08/17/17 | Simulating extracted connectomes.
    Gornet J, Scheffer LK
    bioRxiv. 2017 Aug 17:. doi: 10.1101/177113

    Connectomes derived from volume EM imaging of the brain can generate detailed physical models of every neuron, and simulators such as NEURON or GENESIS are designed to work with such models. In principal, combining these technologies, plus transmitter and channel models, should allow detailed and accurate simulation of real neural circuits. Here we experiment with this combination, using a well-studied system (motion detection in Drosophila. Since simulation requires both the physical geometry (which we have) and the models of the synapses (which are not currently available), we built approximate synapses corresponding to their known and estimated function. Once we did so, we reproduced direction selectivity in T4 cells, one of the main functions of this neural circuit. This verified the basic functionality of both extraction and simulations, and provided a biologically relevant computation we could use in further experiments. We then compared models with different degrees of physical realism, from full detailed models down to models consisting of a single node, to examine the tradeoff of simulation resources required versus accuracy achieved. Our results show that much simpler models may be adequate, at least in the case of medulla neurons in Drosophila. Such models can be easily derived from fully detailed models, and result in simulations that are much smaller, much faster, and accurate enough for many purposes. Biologically, we show that a lumped neuron model reproduces the main motion detector operation, confirming the result of Gruntman, that dendritic compution is not required for this function.

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    08/09/17 | General synthetic method for Si-Fluoresceins and Si-Rhodamines.
    Grimm JB, Brown TA, Tkachuk AN, Lavis LD
    ACS Central Science. 2017 Aug 09;3(9):975-85. doi: 10.1021/acscentsci.7b00247

    The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compounds. Extensive exploration of these xanthene dyes has yielded general structure–activity relationships where the development of new probes is limited only by imagination and organic chemistry. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging experiments. Nevertheless, efforts to tune the chemical and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient preparation of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3–5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in solution, and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biological imaging.

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    Fetter LabTruman LabZlatic LabCardona Lab
    08/09/17 | The complete connectome of a learning and memory centre in an insect brain.
    Eichler K, Li F, Litwin-Kumar A, Park Y, Andrade I, Schneider-Mizell CM, Saumweber T, Huser A, Eschbach C, Gerber B, Fetter RD, Truman JW, Priebe CE, Abbott LF, Thum AS, Zlatic M, Cardona A
    Nature. 2017 Aug 09;548(7666):175-182. doi: 10.1038/nature23455

    Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.

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    Fetter LabTruman LabZlatic LabCardona Lab
    08/08/17 | Organization of the drosophila larval visual circuit.
    Larderet I, Fritsch PM, Gendre N, Neagu-Maier GL, Fetter RD, Schneider-Mizell CM, Truman JW, Zlatic M, Cardona A, Sprecher SG
    eLife. 2017 Aug 8:e28387. doi: 10.7554/eLife.28387

    Visual systems transduce, process and transmit light-dependent environmental cues. Computation of visual features depends on photoreceptor neuron types (PR) present, organization of the eye and wiring of the underlying neural circuit. Here, we describe the circuit architecture of the visual system of Drosophila larvae by mapping the synaptic wiring diagram and neurotransmitters. By contacting different targets, the two larval PR-subtypes create two converging pathways potentially underlying the computation of ambient light intensity and temporal light changes already within this first visual processing center. Locally processed visual information then signals via dedicated projection interneurons to higher brain areas including the lateral horn and mushroom body. The stratified structure of the larval optic neuropil (LON) suggests common organizational principles with the adult fly and vertebrate visual systems. The complete synaptic wiring diagram of the LON paves the way to understanding how circuits with reduced numerical complexity control wide ranges of behaviors.

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    08/07/17 | Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis.
    Zeytuni N, Hong C, Flanagan KA, Worrall LJ, Theiltges KA, Vuckovic M, Huang RK, Massoni SC, Camp AH, Yu Z, Strynadka NC
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Aug 07:. doi: 10.1073/pnas.1704310114

    Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure.

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    08/04/17 | Best practices for managing large CryoEM facilities.
    Alewijnse B, Ashton AW, Chambers MG, Chen S, Cheng A, Ebrahim M, Eng ET, Hagen WJ, Koster AJ, Lopez CS, Lukoyanova N, Ortega J, Renault L, Reyntjens S, Rice WJ, Scapin G, Schrijver R, Siebert A, Stagg SM, et al
    Journal of Structural Biology. 2017-08-04;199(3):225-36. doi: 10.1016/j.jsb.2017.07.011

    This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6–7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

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    08/03/17 | Multi-scale approaches for high-speed imaging and analysis of large neural populations.
    Friedrich J, Yang W, Soudry D, Mu Y, Ahrens MB, Yuste R, Peterka DS, Paninski L
    PLoS Computational Biology. 2017 Aug 03;13(8):e1005685. doi: 10.1371/journal.pcbi.1005685

    Progress in modern neuroscience critically depends on our ability to observe the activity of large neuronal populations with cellular spatial and high temporal resolution. However, two bottlenecks constrain efforts towards fast imaging of large populations. First, the resulting large video data is challenging to analyze. Second, there is an explicit tradeoff between imaging speed, signal-to-noise, and field of view: with current recording technology we cannot image very large neuronal populations with simultaneously high spatial and temporal resolution. Here we describe multi-scale approaches for alleviating both of these bottlenecks. First, we show that spatial and temporal decimation techniques based on simple local averaging provide order-of-magnitude speedups in spatiotemporally demixing calcium video data into estimates of single-cell neural activity. Second, once the shapes of individual neurons have been identified at fine scale (e.g., after an initial phase of conventional imaging with standard temporal and spatial resolution), we find that the spatial/temporal resolution tradeoff shifts dramatically: after demixing we can accurately recover denoised fluorescence traces and deconvolved neural activity of each individual neuron from coarse scale data that has been spatially decimated by an order of magnitude. This offers a cheap method for compressing this large video data, and also implies that it is possible to either speed up imaging significantly, or to "zoom out" by a corresponding factor to image order-of-magnitude larger neuronal populations with minimal loss in accuracy or temporal resolution.

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    Ji Lab
    08/01/17 | Adaptive optical versus spherical aberration corrections for in vivo brain imaging.
    Biomedical Optics Express. 2017 Aug;8(8):3891-902. doi: 10.1364/BOE.8.003891

    Adjusting the objective correction collar is a widely used approach to correct spherical aberrations (SA) in optical microscopy. In this work, we characterized and compared its performance with adaptive optics in the context of in vivo brain imaging with two-photon fluorescence microscopy. We found that the presence of sample tilt had a deleterious effect on the performance of SA-only correction. At large tilt angles, adjusting the correction collar even worsened image quality. In contrast, adaptive optical correction always recovered optimal imaging performance regardless of sample tilt. The extent of improvement with adaptive optics was dependent on object size, with smaller objects having larger relative gains in signal intensity and image sharpness. These observations translate into a superior performance of adaptive optics for structural and functional brain imaging applications in vivo, as we confirmed experimentally.

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