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22 Janelia Publications

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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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    08/20/18 | In vivo measurement of afferent activity with axon-specific calcium imaging.
    Broussard GJ, Liang Y, Fridman M, Unger EK, Meng G, Xiao X, Ji N, Petreanu L, Tian L
    Nature Neuroscience. 2018 Aug 20:. doi: 10.1038/s41593-018-0211-4

    In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity-both electrically and optically-has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions.

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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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    07/05/17 | Ion Channels: History, Diversity, and Impact.
    Brenowitz S, Duguid I, Kammermeier PJ
    Cold Spring Harbor Protocols. 2017 Jul 05;2017(7):pdb.top092288. doi: 10.1101/pdb.top092288

    From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.

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    06/12/17 | Neural signatures of dynamic stimulus selection in Drosophila.
    Sun Y, Nern A, Franconville R, Dana H, Schreiter ER, Looger LL, Svoboda K, Kim DS, Hermundstad AM, Jayaraman V
    Nature Neuroscience. 2017 Jun 12;20(8):1104-13. doi: 10.1038/nn.4581

    Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.

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    01/30/17 | Axonal Endoplasmic Reticulum Ca(2+) Content Controls Release Probability in CNS Nerve Terminals.
    de Juan-Sanz J, Holt GT, Schreiter ER, de Juan F, Kim DS, Ryan TA
    Neuron. 2017 Jan 30;93(4):867-81. doi: 10.1016/j.neuron.2017.01.010

    Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically encoded ER-targeted low-affinity Ca(2+) indicators optimized for examining axonal ER Ca(2+). Our experiments revealed that presynaptic function is tightly controlled by ER Ca(2+) content. We found that neuronal activity drives net Ca(2+) uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca(2+) except during prolonged repetitive firing. In contrast, we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca(2+)]ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca(2+) entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

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    05/30/16 | A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.
    Chu J, Oh Y, Sens A, Ataie N, Dana H, Macklin JJ, Laviv T, Welf ES, Dean KM, Zhang F, Kim BB, Tang CT, Hu M, Baird MA, Davidson MW, Kay MA, Fiolka R, Yasuda R, Kim DS, Ng H, Lin MZ
    Nature Biotechnology. 2016 May 30;34(7):760-7. doi: 10.1038/nbt.3550

    Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

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    03/24/16 | Sensitive red protein calcium indicators for imaging neural activity.
    Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS
    eLife. 2016 Mar 24;5:. doi: 10.7554/eLife.12727

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

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    10/09/15 | A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
    Henderson MJ, Baldwin HA, Werley CA, Boccardo S, Whitaker LR, Yan X, Holt GT, Schreiter ER, Looger LL, Cohen AE, Kim DS, Harvey BK
    PloS one. 2015 Oct 09;10(10):e0139273. doi: 10.1371/journal.pone.0139273

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

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    07/29/15 | Neuronal representation of ultraviolet visual stimuli in mouse primary visual cortex.
    Tan Z, Sun W, Chen T, Kim D, Ji N
    Scientific Reports. 2015 Jul 29;5:12597. doi: 10.1038/srep12597

    The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision.

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