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2 Publications

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    11/01/18 | Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR.
    Marvin JS, Scholl B, Wilson DE, Podgorski K, Kazemipour A, Mueller JA, Schoch-McGovern S, Wang SS, Quiroz FJ, Rebola N, Bao H, Little JP, Tkachuk AN, Hantman AW, Chapman ER, Dietrich D, DiGregorio DA, Fitzpatrick D, Looger LL
    Nature Methods. 2018 Nov;15(11):9386-9. doi: 10.1038/s41592-018-0171-3

    Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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    10/25/18 | Improved methods for marking active neuron populations.
    Moeyaert B, Holt G, Madangopal R, Perez-Alvarez A, Fearey BC, Trojanowski NF, Ledderose J, Zolnik TA, Das A, Patel D, Brown TA, Sachdev RN, Eickholt BJ, Larkum ME, Turrigiano GG, Dana H, Gee CE, Oertner TG, Hope BT, Schreiter ER
    Nature Communications. 2018 Oct 25;9(1):4440. doi: 10.1038/s41467-018-06935-2

    Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

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