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Dive into the research topics where Loren L. Looger is active.

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Featured researches published by Loren L. Looger.


Nature | 2013

Ultrasensitive fluorescent proteins for imaging neuronal activity

Tsai-Wen Chen; Trevor J. Wardill; Yi Sun; Stefan R. Pulver; Sabine L. Renninger; Amy Baohan; Eric R. Schreiter; Rex A. Kerr; Michael B. Orger; Vivek Jayaraman; Loren L. Looger; Karel Svoboda; Douglas S. Kim

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5–40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.


Nature Methods | 2009

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

Lin Tian; S. Andrew Hires; Tianyi Mao; Daniel Huber; M. Eugenia Chiappe; Sreekanth H. Chalasani; Leopoldo Petreanu; Jasper Akerboom; Sean A. McKinney; Eric R. Schreiter; Cornelia I. Bargmann; Vivek Jayaraman; Karel Svoboda; Loren L. Looger

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation–evoked fluorescence responses were significantly enhanced with GCaMP3 (4–6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.


The Journal of Neuroscience | 2012

Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging

Jasper Akerboom; Tsai-Wen Chen; Trevor J. Wardill; Lin Tian; Jonathan S. Marvin; Sevinç Mutlu; Nicole Carreras Calderón; Federico Esposti; Bart G. Borghuis; Xiaonan Richard Sun; Andrew Gordus; Michael B. Orger; Ruben Portugues; Florian Engert; John J. Macklin; Alessandro Filosa; Aman Aggarwal; Rex A. Kerr; Ryousuke Takagi; Sebastian Kracun; Eiji Shigetomi; Baljit S. Khakh; Herwig Baier; Leon Lagnado; Samuel S.-H. Wang; Cornelia I. Bargmann; Bruce E. Kimmel; Vivek Jayaraman; Karel Svoboda; Douglas S. Kim

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.


Nature | 2003

Computational design of receptor and sensor proteins with novel functions.

Loren L. Looger; Mary A. Dwyer; James Jefferson Smith; Homme W. Hellinga

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.


Nature Methods | 2009

A bright and photostable photoconvertible fluorescent protein

Sean A. McKinney; Christopher S. Murphy; Kristin L. Hazelwood; Michael W. Davidson; Loren L. Looger

Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the ∼10-nm localization precision of its parent.


Nature Neuroscience | 2010

Functional imaging of hippocampal place cells at cellular resolution during virtual navigation

Daniel A. Dombeck; Christopher D. Harvey; Lin Tian; Loren L. Looger; David W. Tank

Spatial navigation is often used as a behavioral task in studies of the neuronal circuits that underlie cognition, learning and memory in rodents. The combination of in vivo microscopy with genetically encoded indicators has provided an important new tool for studying neuronal circuits, but has been technically difficult to apply during navigation. Here we describe methods for imaging the activity of neurons in the CA1 region of the hippocampus with subcellular resolution in behaving mice. Neurons that expressed the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-restrained mice performed spatial behaviors in a setup combining a virtual reality system and a custom-built two-photon microscope. We optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit. The combination of virtual reality and high-resolution functional imaging should allow a new generation of studies to investigate neuronal circuit dynamics during behavior.


Frontiers in Molecular Neuroscience | 2013

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Jasper Akerboom; Nicole Carreras Calderón; Lin Tian; Sebastian Wabnig; Matthias Prigge; Johan Tolö; Andrew Gordus; Michael B. Orger; Kristen E. Severi; John J. Macklin; Ronak Patel; Stefan R. Pulver; Trevor J. Wardill; Elisabeth Fischer; Christina Schüler; Tsai-Wen Chen; Karen S. Sarkisyan; Jonathan S. Marvin; Cornelia I. Bargmann; Douglas S. Kim; Sebastian Kügler; Leon Lagnado; Peter Hegemann; Alexander Gottschalk; Eric R. Schreiter; Loren L. Looger

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.


Nature | 2012

Multiple dynamic representations in the motor cortex during sensorimotor learning

Daniel Huber; Diego A. Gutnisky; Simon Peron; Daniel H. O'Connor; J. S. Wiegert; Lin Tian; Thomas G. Oertner; Loren L. Looger; Karel Svoboda

The mechanisms linking sensation and action during learning are poorly understood. Layer 2/3 neurons in the motor cortex might participate in sensorimotor integration and learning; they receive input from sensory cortex and excite deep layer neurons, which control movement. Here we imaged activity in the same set of layer 2/3 neurons in the motor cortex over weeks, while mice learned to detect objects with their whiskers and report detection with licking. Spatially intermingled neurons represented sensory (touch) and motor behaviours (whisker movements and licking). With learning, the population-level representation of task-related licking strengthened. In trained mice, population-level representations were redundant and stable, despite dynamism of single-neuron representations. The activity of a subpopulation of neurons was consistent with touch driving licking behaviour. Our results suggest that ensembles of motor cortex neurons couple sensory input to multiple, related motor programs during learning.


Nature Methods | 2013

An optimized fluorescent probe for visualizing glutamate neurotransmission

Jonathan S. Marvin; Bart G. Borghuis; Lin Tian; Joseph Cichon; Mark T. Harnett; Jasper Akerboom; Andrew Gordus; Sabine L. Renninger; Tsai-Wen Chen; Cornelia I. Bargmann; Michael B. Orger; Eric R. Schreiter; Jonathan B. Demb; Wen-Biao Gan; S. Andrew Hires; Loren L. Looger

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.


Nature | 2010

Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall

Yang Xiang; Quan Yuan; Nina Vogt; Loren L. Looger; Lily Yeh Jan; Yuh Nung Jan

Photoreceptors for visual perception, phototaxis or light avoidance are typically clustered in eyes or related structures such as the Bolwig organ of Drosophila larvae. Unexpectedly, we found that the class IV dendritic arborization neurons of Drosophila melanogaster larvae respond to ultraviolet, violet and blue light, and are major mediators of light avoidance, particularly at high intensities. These class IV dendritic arborization neurons, which are present in every body segment, have dendrites tiling the larval body wall nearly completely without redundancy. Dendritic illumination activates class IV dendritic arborization neurons. These novel photoreceptors use phototransduction machinery distinct from other photoreceptors in Drosophila and enable larvae to sense light exposure over their entire bodies and move out of danger.

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Lin Tian

University of California

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Eric R. Schreiter

Howard Hughes Medical Institute

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Jonathan S. Marvin

Howard Hughes Medical Institute

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Karel Svoboda

Howard Hughes Medical Institute

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Jasper Akerboom

Howard Hughes Medical Institute

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Douglas S. Kim

Howard Hughes Medical Institute

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Sarada Viswanathan

Howard Hughes Medical Institute

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Vivek Jayaraman

Howard Hughes Medical Institute

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