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136 Janelia Publications

Showing 11-20 of 136 results
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    11/14/13 | Motor control of Drosophila courtship song.
    Shirangi TR, Stern DL, Truman JW
    Cell Reports. 2013 Nov 14;5:678-86. doi: 10.1016/j.celrep.2013.09.039

    Many animals utilize acoustic signals-or songs-to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D. melanogaster (hg1) that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a "modular" anatomical substrate for the evolution of diverse songs.

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    Svoboda LabHarris LabFetter Lab
    11/12/13 | Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography.
    Rah J, Bas E, Colonell J, Mishchenko Y, Karsh B, Fetter RD, Myers EW, Chklovskii DB, Svoboda K, Harris TD, Isaac JT
    Frontiers in Neural Circuits. 2013;7:177. doi: 10.3389/fncir.2013.00177

    The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

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    Magee Lab
    11/06/13 | Imaging neuronal populations in behaving rodents: paradigms for studying neural circuits underlying behavior in the mammalian cortex.
    Chen JL, Andermann ML, Keck T, Xu N, Ziv Y
    The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 2013 Nov 6;33(45):17631-40. doi: 10.1523/JNEUROSCI.3255-13.2013

    Understanding the neural correlates of behavior in the mammalian cortex requires measurements of activity in awake, behaving animals. Rodents have emerged as a powerful model for dissecting the cortical circuits underlying behavior attributable to the convergence of several methods. Genetically encoded calcium indicators combined with viral-mediated or transgenic tools enable chronic monitoring of calcium signals in neuronal populations and subcellular structures of identified cell types. Stable one- and two-photon imaging of neuronal activity in awake, behaving animals is now possible using new behavioral paradigms in head-fixed animals, or using novel miniature head-mounted microscopes in freely moving animals. This mini-symposium will highlight recent applications of these methods for studying sensorimotor integration, decision making, learning, and memory in cortical and subcortical brain areas. We will outline future prospects and challenges for identifying the neural underpinnings of task-dependent behavior using cellular imaging in rodents.

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    Gonen Lab
    11/05/13 | Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.
    Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T
    eLife. 2013 Nov 5;2:e01319. doi: 10.7554/eLife.01319

    Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an \~{}16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

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    Riddiford Lab
    11/05/13 | Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster.
    Bilen J, Atallah J, Azanchi R, Levine JD, Riddiford LM
    Proceedings of the National Academy of Sciences of the United States of America. 2013 Nov 5;110:18321-6. doi: 10.1073/pnas.1318119110

    Juvenile hormone (JH) coordinates timing of female reproductive maturation in most insects. In Drosophila melanogaster, JH plays roles in both mating and egg maturation. However, very little is known about the molecular pathways associated with mating. Our behavioral analysis of females genetically lacking the corpora allata, the glands that produce JH, showed that they were courted less by males and mated later than control females. Application of the JH mimic, methoprene, to the allatectomized females just after eclosion rescued both the male courtship and the mating delay. Our studies of the null mutants of the JH receptors, Methoprene tolerant (Met) and germ cell-expressed (gce), showed that lack of Met in Met(27) females delayed the onset of mating, whereas lack of Gce had little effect. The Met(27) females were shown to be more attractive but less behaviorally receptive to copulation attempts. The behavioral but not the attractiveness phenotype was rescued by the Met genomic transgene. Analysis of the female cuticular hydrocarbon profiles showed that corpora allata ablation caused a delay in production of the major female-specific sex pheromones (the 7,11-C27 and -C29 dienes) and a change in the cuticular hydrocarbon blend. In the Met(27) null mutant, by 48 h, the major C27 diene was greatly increased relative to wild type. In contrast, the gce(2.5k) null mutant females were courted similarly to control females despite changes in certain cuticular hydrocarbons. Our findings indicate that JH acts primarily via Met to modulate the timing of onset of female sex pheromone production and mating.

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    Ji Lab

    Inherent aberrations of gradient index (GRIN) lenses used in fluorescence endomicroscopes deteriorate imaging performance. Using adaptive optics, we characterized and corrected the on-axis and off-axis aberrations of a GRIN lens with NA 0.8 at multiple focal planes. We demonstrated a rotational-transformation-based correction procedure, which enlarged the imaging area with diffraction-limited resolution with only two aberration measurements. 204.8 × 204.8 µm2 images of fluorescent beads and brain slices before and after AO corrections were obtained, with evident improvements in both image sharpness and brightness after AO correction. These results show great promises of applying adaptive optical two-photon fluorescence endomicroscope to three-dimensional (3D) imaging.

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    11/01/13 | Caged naloxone reveals opioid signaling deactivation kinetics.
    Banghart MR, Williams JT, Shah RC, Lavis LD, Sabatini BL
    Molecular Pharmacology. 2013 Nov;84(5):687-95. doi: 10.1124/mol.113.088096

    The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis.

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    Svoboda Lab
    10/30/13 | Distinct balance of excitation and inhibition in an interareal feedforward and feedback circuit of mouse visual cortex.
    Yang W, Carrasquillo Y, Hooks BM, Nerbonne JM, Burkhalter A
    The Journal of Neuroscience. 2013 Oct 30;33(44):17373-84. doi: 10.1523/JNEUROSCI.2515-13.2013

    Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LM pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1 pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LM pathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1 inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LM and FBLM→V1 circuits are organized in a pathway- and lamina-specific fashion.

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    10/29/13 | Fast structural responses of gap junction membrane domains to AB5 toxins.
    Majoul IV, Gao L, Betzig E, Onichtchouk D, Butkevich E, Kozlov Y, Bukauskas F, Bennett MV, Lippincott-Schwartz J, Duden R
    Proceedings of the National Academy of Sciences of the United States of America. 2013 Oct 29;110(44):E4125-33. doi: 10.1073/pnas.1315850110

    Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast ( 500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

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    10/25/13 | Correlative photoactivated localization and scanning electron microscopy.
    Kopek BG, Shtengel G, Grimm JB, Clayton DA, Hess HF
    PLoS One. 2013 Oct 25;8(10):e77209. doi: 10.1371/journal.pone.0077209

    The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

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