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1457 Janelia Publications

Showing 1301-1310 of 1457 results
08/01/10 | Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy.
Keller PJ, Schmidt AD, Santella A, Khairy K, Zhirong Bao , Wittbrodt J, Stelzer EH
Nature Methods. 08/2010;7(8):637-42. doi: 10.1038/nmeth.1476

Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality, and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.

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Magee Lab
08/01/10 | Network mechanisms of theta related neuronal activity in hippocampal CA1 pyramidal neurons.
Losonczy A, Zemelman BV, Vaziri A, Magee JC
Nature Neuroscience. 2010 Aug;13(8):967-72. doi: 10.1038/nn.2597

Although hippocampal theta oscillations represent a prime example of temporal coding in the mammalian brain, little is known about the specific biophysical mechanisms. Intracellular recordings support a particular abstract oscillatory interference model of hippocampal theta activity, the soma-dendrite interference model. To gain insight into the cellular and circuit level mechanisms of theta activity, we implemented a similar form of interference using the actual hippocampal network in mice in vitro. We found that pairing increasing levels of phasic dendritic excitation with phasic stimulation of perisomatic projecting inhibitory interneurons induced a somatic polarization and action potential timing profile that reproduced most common features. Alterations in the temporal profile of inhibition were required to fully capture all features. These data suggest that theta-related place cell activity is generated through an interaction between a phasic dendritic excitation and a phasic perisomatic shunting inhibition delivered by interneurons, a subset of which undergo activity-dependent presynaptic modulation.

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07/15/10 | Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. (With commentary)
Frost NA, Shroff H, Kong H, Betzig E, Blanpied TA
Neuron. 2010 Jul 15;67(1):86-99. doi: 10.1016/j.neuron.2010.05.026

Within dendritic spines, actin is presumed to anchor receptors in the postsynaptic density and play numerous roles regulating synaptic transmission. However, the submicron dimensions of spines have hindered examination of actin dynamics within them and prevented live-cell discrimination of perisynaptic actin filaments. Using photoactivated localization microscopy, we measured movement of individual actin molecules within living spines. Velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

Commentary: A nice application of single particle tracking PALM (sptPALM), showing the flow of actin in the spines of live cultured neurons. Since 2008, the PALM in our lab has largely become a user facility, available to outside users as well as Janelians. Grad student Nick Frost in Tom Blanpied’s group at the U. of Maryland Med School visited on a number of occasions to use the PALM, with training and assistance from Hari.

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07/01/10 | Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior.
Seelig JD, Chiappe ME, Lott GK, Dutta A, Osborne JE, Reiser MB, Jayaraman V
Nature Methods. 2010 Jul;7:535-40. doi: 10.1038/nmeth.1468

Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball’s motion is tracked at high resolution and can be treated as a proxy for the fly’s own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.

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Magee Lab
06/29/10 | Two-photon single-cell optogenetic control of neuronal activity by sculpted light.
Andrasfalvy BK, Zemelman BV, Tang J, Vaziri A
Proceedings of the National Academy of Sciences of the United States of America. 2010 Jun 29;107(26):11981-6. doi: 10.1073/pnas.1006620107

Recent advances in optogenetic techniques have generated new tools for controlling neuronal activity, with a wide range of neuroscience applications. The most commonly used approach has been the optical activation of the light-gated ion channel channelrhodopsin-2 (ChR2). However, targeted single-cell-level optogenetic activation with temporal precessions comparable to the spike timing remained challenging. Here we report fast (< or = 1 ms), selective, and targeted control of neuronal activity with single-cell resolution in hippocampal slices. Using temporally focused laser pulses (TEFO) for which the axial beam profile can be controlled independently of its lateral distribution, large numbers of channels on individual neurons can be excited simultaneously, leading to strong (up to 15 mV) and fast (< or = 1 ms) depolarizations. Furthermore, we demonstrated selective activation of cellular compartments, such as dendrites and large presynaptic terminals, at depths up to 150 microm. The demonstrated spatiotemporal resolution and the selectivity provided by TEFO allow manipulation of neuronal activity, with a large number of applications in studies of neuronal microcircuit function in vitro and in vivo.

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06/15/10 | Automated tracking and analysis of centrosomes in early Caenorhabditis elegans embryos.
Jaensch S, Decker M, Hyman AA, Myers EW
Bioinformatics. 2010 Jun 15;26(12):i13-20. doi: 10.1093/bioinformatics/btq190

The centrosome is a dynamic structure in animal cells that serves as a microtubule organizing center during mitosis and also regulates cell-cycle progression and sets polarity cues. Automated and reliable tracking of centrosomes is essential for genetic screens that study the process of centrosome assembly and maturation in the nematode Caenorhabditis elegans.

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06/15/10 | Automatic reconstruction of 3D neuron structures using a graph-augmented deformable model.
Peng H, Ruan Z, Atasoy D, Sternson S
Bioinformatics. 2010 Jun 15;26:i38-46. doi: 10.1093/bioinformatics/btq212

Digital reconstruction of 3D neuron structures is an important step toward reverse engineering the wiring and functions of a brain. However, despite a number of existing studies, this task is still challenging, especially when a 3D microscopic image has low single-to-noise ratio and discontinued segments of neurite patterns.

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06/02/10 | Identifying neuronal lineages of Drosophila by sequence analysis of axon tracts.
Cardona A, Saalfeld S, Arganda I, Pereanu W, Schindelin J, Hartenstein V
The Journal of Neuroscience. 2010 Jun 2;30(22):7538-53. doi: 10.1523/JNEUROSCI.0186-10.2010

The Drosophila brain is formed by an invariant set of lineages, each of which is derived from a unique neural stem cell (neuroblast) and forms a genetic and structural unit of the brain. The task of reconstructing brain circuitry at the level of individual neurons can be made significantly easier by assigning neurons to their respective lineages. In this article we address the automation of neuron and lineage identification. We focused on the Drosophila brain lineages at the larval stage when they form easily recognizable secondary axon tracts (SATs) that were previously partially characterized. We now generated an annotated digital database containing all lineage tracts reconstructed from five registered wild-type brains, at higher resolution and including some that were previously not characterized. We developed a method for SAT structural comparisons based on a dynamic programming approach akin to nucleotide sequence alignment and a machine learning classifier trained on the annotated database of reference SATs. We quantified the stereotypy of SATs by measuring the residual variability of aligned wild-type SATs. Next, we used our method for the identification of SATs within wild-type larval brains, and found it highly accurate (93-99%). The method proved highly robust for the identification of lineages in mutant brains and in brains that differed in developmental time or labeling. We describe for the first time an algorithm that quantifies neuronal projection stereotypy in the Drosophila brain and use the algorithm for automatic neuron and lineage recognition.

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Magee LabHarris Lab
06/01/10 | Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal.
Royer S, Zemelman BV, Barbic M, Losonczy A, Buzsáki G, Magee JC
The European Journal of Neuroscience. 2010 Jun;31:2279-91. doi: 10.1002/cbic.201000254

Recordings of large neuronal ensembles and neural stimulation of high spatial and temporal precision are important requisites for studying the real-time dynamics of neural networks. Multiple-shank silicon probes enable large-scale monitoring of individual neurons. Optical stimulation of genetically targeted neurons expressing light-sensitive channels or other fast (milliseconds) actuators offers the means for controlled perturbation of local circuits. Here we describe a method to equip the shanks of silicon probes with micron-scale light guides for allowing the simultaneous use of the two approaches. We then show illustrative examples of how these compact hybrid electrodes can be used in probing local circuits in behaving rats and mice. A key advantage of these devices is the enhanced spatial precision of stimulation that is achieved by delivering light close to the recording sites of the probe. When paired with the expression of light-sensitive actuators within genetically specified neuronal populations, these devices allow the relatively straightforward and interpretable manipulation of network activity.

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06/01/10 | Near-isotropic 3D optical nanoscopy with photon-limited chromophores.
Tang J, Akerboom J, Vaziri A, Looger LL, Shank CV
Proceedings of the National Academy of Sciences of the United States of America. 2010 Jun 1;107(22):10068-73. doi: 10.1073/pnas.1004899107

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.

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