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7 Publications

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    12/03/15 | Cortex commands the performance of skilled movement.
    Guo J, Graves AR, Guo WW, Zheng J, Lee A, Rodríguez-González J, Li N, Macklin JJ, Phillips JW, Mensh BD, Branson K, Hantman AW
    eLife. 2015 Dec 3;4:. doi: 10.7554/eLife.10774

    Mammalian cerebral cortex is accepted as being critical for voluntary motor control, but what functions depend on cortex is still unclear. Here we used rapid, reversible optogenetic inhibition to test the role of cortex during a head-fixed task in which mice reach, grab, and eat a food pellet. Sudden cortical inhibition blocked initiation or froze execution of this skilled prehension behavior, but left untrained forelimb movements unaffected. Unexpectedly, kinematically normal prehension occurred immediately after cortical inhibition even during rest periods lacking cue and pellet. This 'rebound' prehension was only evoked in trained and food-deprived animals, suggesting that a motivation-gated motor engram sufficient to evoke prehension is activated at inhibition's end. These results demonstrate the necessity and sufficiency of cortical activity for enacting a learned skill.

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    Magee LabHarris Lab
    07/13/15 | Conjunctive input processing drives feature selectivity in hippocampal CA1 neurons.
    Bittner KC, Grienberger C, Vaidya SP, Milstein AD, Macklin JJ, Suh J, Tonegawa S, Magee JC
    Nature Neuroscience. 2015 Jul 13:. doi: 10.1038/nn.4062

    Feature-selective firing allows networks to produce representations of the external and internal environments. Despite its importance, the mechanisms generating neuronal feature selectivity are incompletely understood. In many cortical microcircuits the integration of two functionally distinct inputs occurs nonlinearly through generation of active dendritic signals that drive burst firing and robust plasticity. To examine the role of this processing in feature selectivity, we recorded CA1 pyramidal neuron membrane potential and local field potential in mice running on a linear treadmill. We found that dendritic plateau potentials were produced by an interaction between properly timed input from entorhinal cortex and hippocampal CA3. These conjunctive signals positively modulated the firing of previously established place fields and rapidly induced new place field formation to produce feature selectivity in CA1 that is a function of both entorhinal cortex and CA3 input. Such selectivity could allow mixed network level representations that support context-dependent spatial maps.

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    04/27/15 | High-performance probes for light and electron microscopy.
    Viswanathan S, Williams ME, Bloss EB, Stasevich TJ, Speer CM, Nern A, Pfeiffer BD, Hooks BM, Li W, English BP, Tian T, Henry GL, Macklin JJ, Patel R, Gerfen CR, Zhuang X, Wang Y, Rubin GM, Looger LL
    Nature Methods. 2015 Apr 27;12(6):568-76. doi: 10.1038/nmeth.3365

    We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

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    03/10/15 | Visualization and neuronal cell targeting during electrophysiological recordings facilitated by quantum dots.
    Field LD, Andrasfalvy BK, Galiñanes GL, Huber D, Barbic M, Macklin JJ, Susumu K, Delehanty JB, Huston AL, Makara JK, Medintz IL
    Proceedings of SPIE. 2015 Mar 10;9305:9305Y. doi: 10.1117/12.2076934

    The simultaneous visualization, identification and targeting of neurons during patch clamp-mediated electrophysiological recordings is a basic technique in neuroscience, yet it is often complicated by the inability to visualize the pipette tip, particularly in deep brain tissue. Here we demonstrate a novel approach in which fluorescent quantum dot probes are used to coat pipettes prior to their use. The strong two-photon absorption cross sections of the quantum dots afford robust contrast at significantly deeper penetration depths than current methods allow. We demonstrate the utility of this technique in multiple recording formats both in vitro and in vivo where imaging of the pipettes is achieved at remarkable depths (up to 800 microns). Notably, minimal perturbation of cellular physiology is observed over the hours-long time course of neuronal recordings. We discuss our results within the context of the role that quantum dot nanoprobes may play in understanding neuronal cell physiology.

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    03/01/15 | Fixation-resistant photoactivatable fluorescent proteins for CLEM.
    Paez-Segala MG, Sun MG, Shtengel G, Viswanathan S, Baird MA, Macklin JJ, Patel R, Allen JR, Howe ES, Piszczek G, Hess HF, Davidson MW, Wang Y, Looger LL
    Nature Methods. 2015 Mar;12(3):215-8. doi: 10.1038/nmeth.3225

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

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    02/13/15 | Labeling of active neural circuits in vivo with designed calcium integrators.
    Fosque BF, Sun Y, Dana H, Yang C, Ohyama T, Tadross MR, Patel R, Zlatic M, Kim DS, Ahrens MB, Jayaraman V, Looger LL, Schreiter ER
    Science. 2015 Feb 13;347(6223):755-60. doi: 10.1126/science.1260922

    The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

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    01/19/15 | A general method to improve fluorophores for live-cell and single-molecule microscopy.
    Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH, Lionnet T, Lavis LD
    Nature Methods. 2015 Jan 19;12(3):244-50. doi: 10.1038/nmeth.3256

    Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

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