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1431 Janelia Publications

Showing 1-10 of 1431 results
10/26/18 | A practical guide to adaptive light-sheet microscopy.
Royer LA, Lemon WC, Chhetri RK, Keller PJ
Nature Protocols. 2018 Oct 26;13(11):2462–2500. doi: 10.1038/s41596-018-0043-4

We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1-2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.

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Druckmann LabMagee Lab
10/22/18 | Active dendritic integration and mixed neocortical network representations during an adaptive sensing behavior.
Ranganathan GN, Apostolides PF, Harnett MT, Xu N, Druckmann S, Magee JC
Nature Neuroscience. 2018 Oct 22;21(11):1583-90. doi: 10.1038/s41593-018-0254-6

Animals strategically scan the environment to form an accurate perception of their surroundings. Here we investigated the neuronal representations that mediate this behavior. Ca imaging and selective optogenetic manipulation during an active sensing task reveals that layer 5 pyramidal neurons in the vibrissae cortex produce a diverse and distributed representation that is required for mice to adapt their whisking motor strategy to changing sensory cues. The optogenetic perturbation degraded single-neuron selectivity and network population encoding through a selective inhibition of active dendritic integration. Together the data indicate that active dendritic integration in pyramidal neurons produces a nonlinearly mixed network representation of joint sensorimotor parameters that is used to transform sensory information into motor commands during adaptive behavior. The prevalence of the layer 5 cortical circuit motif suggests that this is a general circuit computation.

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10/18/18 | Astrocytes integrate and drive action potential firing in inhibitory subnetworks.
Deemyad T, Lüthi J, Spruston N
Nature Communications. 2018 Oct 18;9(1):4336. doi: 10.1038/s41467-018-06338-3

Many brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons and through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials (barrage firing) following repeated depolarizing stimuli. Here we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibits barrage firing. Thus, networks of astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving barrages of action potentials in some populations of inhibitory interneurons.

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10/18/18 | In toto imaging and reconstruction of post-implantation mouse development at the single-cell level.
McDole K, Guignard L, Amat F, Berger A, Malandain G, Royer LA, Turaga SC, Branson K, Keller PJ
Cell. 2018 Oct 10;175(3):859-876. doi: 10.1016/j.cell.2018.09.031

The mouse embryo has long been central to the study of mammalian development; however, elucidating the cell behaviors governing gastrulation and the formation of tissues and organs remains a fundamental challenge. A major obstacle is the lack of live imaging and image analysis technologies capable of systematically following cellular dynamics across the developing embryo. We developed a light-sheet microscope that adapts itself to the dramatic changes in size, shape, and optical properties of the post-implantation mouse embryo and captures its development from gastrulation to early organogenesis at the cellular level. We furthermore developed a computational framework for reconstructing long-term cell tracks, cell divisions, dynamic fate maps, and maps of tissue morphogenesis across the entire embryo. By jointly analyzing cellular dynamics in multiple embryos registered in space and time, we built a dynamic atlas of post-implantation mouse development that, together with our microscopy and computational methods, is provided as a resource.

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10/17/18 | A cortico-cerebellar loop for motor planning.
Gao Z, Davis C, Thomas AM, Economo MN, Abrego AM, Svoboda K, De Zeeuw CI, Li N
Nature. 2018 Oct 17:. doi: 10.1038/s41586-018-0633-x

Persistent and ramping neural activity in the frontal cortex anticipates specific movements. Preparatory activity is distributed across several brain regions, but it is unclear which brain areas are involved and how this activity is mediated by multi-regional interactions. The cerebellum is thought to be primarily involved in the short-timescale control of movement; however, roles for this structure in cognitive processes have also been proposed. In humans, cerebellar damage can cause defects in planning and working memory. Here we show that persistent representation of information in the frontal cortex during motor planning is dependent on the cerebellum. Mice performed a sensory discrimination task in which they used short-term memory to plan a future directional movement. A transient perturbation in the medial deep cerebellar nucleus (fastigial nucleus) disrupted subsequent correct responses without hampering movement execution. Preparatory activity was observed in both the frontal cortex and the cerebellar nuclei, seconds before the onset of movement. The silencing of frontal cortex activity abolished preparatory activity in the cerebellar nuclei, and fastigial activity was necessary to maintain cortical preparatory activity. Fastigial output selectively targeted the behaviourally relevant part of the frontal cortex through the thalamus, thus closing a cortico-cerebellar loop. Our results support the view that persistent neural dynamics during motor planning is maintained by neural circuits that span multiple brain regions, and that cerebellar computations extend beyond online motor control.

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10/16/18 | Expanding the optogenetics toolkit by topological inversion of rhodopsins.
Brown J, Behnam R, Coddington L, Tervo DG, Martin K, Proskurin M, Kuleshova E, Park J, Phillips J, Bergs AC, Gottschalk A, Dudman JT, Karpova AY
Cell. 2018 Oct 16:. doi: 10.1016/j.cell.2018.09.026

Targeted manipulation of activity in specific populations of neurons is important for investigating the neural circuit basis of behavior. Optogenetic approaches using light-sensitive microbial rhodopsins have permitted manipulations to reach a level of temporal precision that is enabling functional circuit dissection. As demand for more precise perturbations to serve specific experimental goals increases, a palette of opsins with diverse selectivity, kinetics, and spectral properties will be needed. Here, we introduce a novel approach of "topological engineering"-inversion of opsins in the plasma membrane-and demonstrate that it can produce variants with unique functional properties of interest for circuit neuroscience. In one striking example, inversion of a Channelrhodopsin variant converted it from a potent activator into a fast-acting inhibitor that operates as a cation pump. Our findings argue that membrane topology provides a useful orthogonal dimension of protein engineering that immediately permits as much as a doubling of the available toolkit.

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10/15/18 | The timing of action determines reward prediction signals in identified midbrain dopamine neurons.
Coddington LT, Dudman JT
Nature Neuroscience. 2018 Oct 15;21(11):1563-73. doi: 10.1038/s41593-018-0245-7

Animals adapt their behavior in response to informative sensory cues using multiple brain circuits. The activity of midbrain dopaminergic neurons is thought to convey a critical teaching signal: reward-prediction error. Although reward-prediction error signals are thought to be essential to learning, little is known about the dynamic changes in the activity of midbrain dopaminergic neurons as animals learn about novel sensory cues and appetitive rewards. Here we describe a large dataset of cell-attached recordings of identified dopaminergic neurons as naive mice learned a novel cue-reward association. During learning midbrain dopaminergic neuron activity results from the summation of sensory cue-related and movement initiation-related response components. These components are both a function of reward expectation yet they are dissociable. Learning produces an increasingly precise coordination of action initiation following sensory cues that results in apparent reward-prediction error correlates. Our data thus provide new insights into the circuit mechanisms that underlie a critical computation in a highly conserved learning circuit.

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10/14/18 | Social isolation-induced epigenetic and transcriptional changes in Drosophila dopaminergic neurons.
Agrawal P, Chung P, Heberlein U, Kent CF
bioRxiv. 2018 Oct 14:. doi: 10.1101/443226

Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. Here we report first use of a modified INTACT method in behavioral epigenetics of Drosophila: a method we call mini-INTACT. Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, we identified epigenetic signatures in socially-isolated and socially-enriched Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations were predicted by expression changes of four transcription factors and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep. Our work enables the use of Drosophila as a model for cell-type-specific behavioral epigenetics.

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10/11/18 | Development of 2-colour and 3D SMLM data analysis methods for fibrous spatial point patterns.
Peters R, Griffié J, Williamson D, Aaron J, Khuon S, Owen D
Journal of Physics D: Applied Physics. 2018 Oct 11:. doi: 10.1088/1361-6463/aae7ac

Abstract ingle molecule localisation microscopy (SMLM), experimentally achieved over a decade ago, has become a routinely used analytical tool across the life sciences. Synergistic advances in probe chemistry, optical physics and data analysis has propelled SMLM into the quantitative realm, enabling unprecedented access to the cellular machinery at the nanoscale. In its early years, SMLM primarily served as a platform for impressive rendered images of sub diffraction scale structures, however more recently a shift towards interrogating SMLM point pattern data in a robust mathematical framework has occurred. A prevalent theme in the SMLM field is the need for quantitative analytical methods, to better understand the underlying processes on which SMLM reports and to extract statistically valid biological insights. Whilst some forms of post processing analytics, for example cluster analysis, have been widely studied, others such as fibre analysis remain in their infancy. Here, we review the current state of the art of cluster analysis and fibre analysis and present new methods for their implementation in both 3D SMLM data sets and multi-colour data.

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10/11/18 | Thy1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a for neuronal population imaging in vivo.
Dana H, Novak O, Guardado-Montesino M, Fransen JW, Hu A, Borghuis BG, Guo C, Kim DS, Svoboda K
PloS One. 2018;13(10):e0205444. doi: 10.1371/journal.pone.0205444

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

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