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12 Janelia Publications

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    11/25/18 | Magnetocaloric materials as switchable high contrast ratio MRI labels.
    Barbic M, Dodd SJ, Morris HD, Dilley N, Marcheschi B, Huston A, Harris TD, Koretsky AP
    Magnetic Resonance in Medicine. 2018 Nov 25:. doi: 10.1002/mrm.27615

    PURPOSE: To develop switchable and tunable labels with high contrast ratio for MRI using magnetocaloric materials that have sharp first-order magnetic phase transitions at physiological temperatures and typical MRI magnetic field strengths.

    METHODS: A prototypical magnetocaloric material iron-rhodium (FeRh) was prepared by melt mixing, high-temperature annealing, and ice-water quenching. Temperature- and magnetic field-dependent magnetization measurements of wire-cut FeRh samples were performed on a vibrating sample magnetometer. Temperature-dependent MRI of FeRh samples was performed on a 4.7T MRI.

    RESULTS: Temperature-dependent MRI clearly demonstrated image contrast changes due to the sharp magnetic state transition of the FeRh samples in the MRI magnetic field (4.7T) and at a physiologically relevant temperature (~37°C).

    CONCLUSION: A magnetocaloric material, FeRh, was demonstrated to act as a high contrast ratio switchable MRI contrast agent due to its sharp first-order magnetic phase transition in the DC magnetic field of MRI and at physiologically relevant temperatures. A wide range of magnetocaloric materials are available that can be tuned by materials science techniques to optimize their response under MRI-appropriate conditions and be controllably switched in situ with temperature, magnetic field, or a combination of both.

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    10/03/18 | High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments.
    Dana H, Sun Y, Mohar B, Hulse B, Hasseman JP, Tsegaye G, Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman V, Looger LL, Schreiter ER, Svoboda K, Kim DS
    bioRxiv. 2018 Oct 3:. doi: 10.1101/434589

    Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics, and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

     

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    06/28/18 | Kilohertz frame-rate two-photon tomography.
    Kazemipour A, Novak O, Flickinger D, Marvin JS, King J, Borden P, Druckmann S, Svoboda K, Looger LL, Podgorski K
    bioRxiv. 2018 Jun 28:. doi: 10.1101/357269

    Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

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    03/26/18 | Photoactivatable drugs for nicotinic optopharmacology.
    Banala S, Arvin MC, Bannon NM, Jin X, Macklin JJ, Wang Y, Peng C, Zhao G, Marshall JJ, Gee KR, Wokosin DL, Kim VJ, McIntosh JM, Contractor A, Lester HA, Kozorovitskiy Y, Drenan RM, Lavis LD
    Nature Methods. 2018 Mar 26;15(5):347-50. doi: 10.1038/nmeth.4637

    Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

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    01/16/18 | A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.
    Shen Y, Dana H, Abdelfattah AS, Patel R, Shea J, Molina RS, Rawal B, Rancic V, Chang Y, Wu L, Chen Y, Qian Y, Wiens MD, Hambleton N, Ballanyi K, Hughes TE, Drobizhev M, Kim DS, Koyama M, Schreiter ER, Campbell RE
    BMC Biology. 2018 Jan 16;16(1):9. doi: 10.1186/s12915-018-0480-0

    BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores.

    RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

    CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

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    06/14/16 | A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging.
    Sofroniew NJ, Flickinger D, King J, Svoboda K
    eLife. 2016 Jun 14;5:e14472. doi: 10.7554/eLife.14472

    Imaging is used to map activity across populations of neurons. Microscopes with cellular resolution have small (<1 millimeter) fields of view and cannot simultaneously image activity distributed across multiple brain areas. Typical large field of view microscopes do not resolve single cells, especially in the axial dimension. We developed a 2-photon random access mesoscope (2p-RAM) that allows high-resolution imaging anywhere within a volume spanning multiple brain areas (∅ 5 mm x 1 mm cylinder). 2p-RAM resolution is near diffraction limited (lateral, 0.66 μm, axial 4.09 μm at the center; excitation wavelength = 970 nm; numerical aperture = 0.6) over a large range of excitation wavelengths. A fast three-dimensional scanning system allows efficient sampling of neural activity in arbitrary regions of interest across the entire imaging volume. We illustrate the use of the 2p-RAM by imaging neural activity in multiple, non-contiguous brain areas in transgenic mice expressing protein calcium sensors.

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    Gonen Lab
    05/01/16 | The collection of MicroED data for macromolecular crystallography.
    Shi D, Nannenga BL, de la Cruz MJ, Liu J, Sawtelle S, Calero G, Reyes FE, Hattne J, Gonen T
    Nature Protocols. 2016 May;11(5):895-904. doi: 10.1038/nprot.2016.046

    The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

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    Jayaraman LabDruckmann Lab
    06/16/15 | Dynamical feature extraction at the sensory periphery guides chemotaxis.
    Schulze A, Gomez-Marin A, Rajendran VG, Lott G, Musy M, Ahammad P, Deogade A, Sharpe J, Riedl J, Jarriault D, Trautman ET, Werner C, Venkadesan M, Druckmann S, Jayaraman V, Louis M
    eLife. 2015 Jun 16;4:. doi: 10.7554/eLife.06694

    Behavioral strategies employed for chemotaxis have been described across phyla, but the sensorimotor basis of this phenomenon has seldom been studied in naturalistic contexts. Here, we examine how signals experienced during free olfactory behaviors are processed by first-order olfactory sensory neurons (OSNs) of the Drosophila larva. We find that OSNs can act as differentiators that transiently normalize stimulus intensity-a property potentially derived from a combination of integral feedback and feed-forward regulation of olfactory transduction. In olfactory virtual reality experiments, we report that high activity levels of the OSN suppress turning, whereas low activity levels facilitate turning. Using a generalized linear model, we explain how peripheral encoding of olfactory stimuli modulates the probability of switching from a run to a turn. Our work clarifies the link between computations carried out at the sensory periphery and action selection underlying navigation in odor gradients.

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    05/14/15 | Behavioral responses to a repetitive visual threat stimulus express a persistent state of defensive arousal in Drosophila
    Gibson WT, Gonzalez CR, Fernandez C, Ramasamy L, Tabachnik T, Du RR, Felsen PD, Maire MM, Perona P, Anderson DJ
    Current Biology. 2015 May 14;25(11):1401-15. doi: 10.1016/j.cub.2015.03.058

    The neural circuit mechanisms underlying emotion states remain poorly understood. Drosophila offers powerful genetic approaches for dissecting neural circuit function, but whether flies exhibit emotion-like behaviors has not been clear. We recently proposed that model organisms may express internal states displaying “emotion primitives,” which are general characteristics common to different emotions, rather than specific anthropomorphic emotions such as “fear” or “anxiety.” These emotion primitives include scalability, persistence, valence, and generalization to multiple contexts. Here, we have applied this approach to determine whether flies’ defensive responses to moving overhead translational stimuli (“shadows”) are purely reflexive or may express underlying emotion states. We describe a new behavioral assay in which flies confined in an enclosed arena are repeatedly exposed to an overhead translational stimulus. Repetitive stimuli promoted graded (scalable) and persistent increases in locomotor velocity and hopping, and occasional freezing. The stimulus also dispersed feeding flies from a food resource, suggesting both negative valence and context generalization. Strikingly, there was a significant delay before the flies returned to the food following stimulus-induced dispersal, suggestive of a slowly decaying internal defensive state. The length of this delay was increased when more stimuli were delivered for initial dispersal. These responses can be mathematically modeled by assuming an internal state that behaves as a leaky integrator of stimulus exposure. Our results suggest that flies’ responses to repetitive visual threat stimuli express an internal state exhibiting canonical emotion primitives, possibly analogous to fear in mammals. The mechanistic basis of this state can now be investigated in a genetically tractable insect species.

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    11/06/14 | Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization.
    Lee D, Shtengel G, Osborne JE, Lee AK
    Nature Protocols. 2014 Nov 06;9(12):2784-95. doi: 10.1038/nprot.2014.190

    Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in ∼4-7 h (excluding histology) and the awake-patched version requires ∼1-4 h per day for ∼2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors.

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