@article {6541, title = {Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.}, journal = {Frontiers in Molecular Neuroscience}, volume = {6}, year = {2013}, month = {2013 Mar 4}, pages = {2}, abstract = {

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 Ca(2+)-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 [Ca(2+)]) 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 Ca(2+) 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.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2013.00002}, author = {Akerboom, Jasper and Carreras Calder{\'o}n, Nicole and Tian, Lin and Wabnig, Sebastian and Prigge, Matthias and Tol{\"o}, Johan and Gordus, Andrew and Orger, Michael B. and Severi, Kristen E and Macklin, John J and Patel, Ronak and Pulver, Stefan R and Wardill, Trevor J. and Fischer, Elisabeth and Sch{\"u}ler, Christina and Chen, Tsai-Wen and Sarkisyan, Karen S and Marvin, Jonathan S and Bargmann, Cornelia I and Kim, Douglas S. and K{\"u}gler, Sebastian and Lagnado, Leon and Hegemann, Peter and Gottschalk, Alexander and Schreiter, Eric R and Looger, Loren L} } @article {6128, title = {An optimized fluorescent probe for visualizing glutamate neurotransmission.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013 Feb}, pages = {162-70}, abstract = {

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.

}, keywords = {Animals, Astrocytes, Biosensing Techniques, Caenorhabditis elegans, Calcium Signaling, Escherichia coli Proteins, Excitatory Postsynaptic Potentials, Fluorescent Dyes, Glutamic Acid, Green Fluorescent Proteins, Hippocampus, Mice, Motor Cortex, Neurons, Photic Stimulation, Pyramidal Cells, Recombinant Fusion Proteins, Retina, Signal-To-Noise Ratio, Synaptic Transmission, Zebrafish}, issn = {1548-7105}, doi = {10.1038/nmeth.2333}, author = {Marvin, Jonathan S and Borghuis, Bart G and Tian, Lin and Cichon, Joseph and Harnett, Mark T and Akerboom, Jasper and Gordus, Andrew and Renninger, Sabine L. and Chen, Tsai-Wen and Bargmann, Cornelia I and Orger, Michael B. and Schreiter, Eric R and Demb, Jonathan B and Gan, Wen-Biao and Hires, S Andrew and Looger, Loren L} } @article {6124, title = {Ultrasensitive fluorescent proteins for imaging neuronal activity.}, journal = {Nature}, volume = {499}, year = {2013}, month = {2013 Jul 18}, pages = {295-300}, abstract = {

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{\textendash}40-{\textmu}m long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

}, doi = {10.1038/nature12354}, author = {Chen, Tsai-Wen and Wardill, Trevor J. and Sun, Yi and Pulvar, Stefan R. and Renninger, Sabine L. and Baohan, Amy and Schreiter, Eric R and Kerr, Rex A and Orger, Michael B. and Jayaraman, Vivek and Looger, Loren L and Svoboda, Karel and Kim, Douglas S.} } @article {5937, title = {Whole-brain functional imaging at cellular resolution using light-sheet microscopy.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013 May}, pages = {413-20}, abstract = {

Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80\% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2434}, author = {Ahrens, Misha B and Orger, Michael B. and Robson, Drew N and Li, Jennifer M and Keller, Philipp J} } @article {5671, title = {Brain-wide neuronal dynamics during motor adaptation in zebrafish.}, journal = {Nature}, volume = {485}, year = {2012}, month = {2012 May 24}, pages = {471-7}, abstract = {

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

}, keywords = {Adaptation, Animals, Brain, Genetically Modified, Larva, Learning, Locomotion, Models, Nerve Net, Neurological, Neurons, Neuropil, Photic Stimulation, Physiological, Psychomotor Performance, Single-Cell Analysis, Zebrafish}, issn = {1476-4687}, doi = {10.1038/nature11057}, author = {Ahrens, Misha B and Li, Jennifer M and Orger, Michael B. and Robson, Drew N and Schier, Alexander F and Engert, Florian and Portugues, Ruben} } @article {5164, title = {Optimization of a GCaMP calcium indicator for neural activity imaging.}, journal = {The Journal of Neuroscience}, volume = {32}, year = {2012}, month = {2012 Oct 3}, pages = {13819-40}, chapter = {13819}, abstract = {

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 {\textquotedblleft}GCaMP5{\textquotedblright} 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.GCaMP5allows more sensitive detection of neural activity in vivo andmayfind widespread applications for cellular imaging in general.

}, doi = {10.1523/​JNEUROSCI.2601-12.2012}, author = {Akerboom, Jasper and Chen, Tsai-Wen and Wardill, Trevor J. and Marvin, Jonathan S and Mutlu, Sevinc and Carreras Caldero, Nicole and Esposti, Federico and Borghuis, Bart G and Sun, Xiaonan Richard and Gordus, Andrew and Orger, Michael B. and Portugues, Ruben and Engert, Florian and Macklin, John J and Filosa, Alessandro and Aggarwal, Aman and Kerr, Rex and Takagi, Ryousuke and Kracun, Sebastian and Shigetomi, Eiji and Khakh, Baljit S. and Baier, Herwig and Lagnado, Leon and Wang, Samuel S-H and Bargmann, Cornelia and Kimmel, Bruce and Jayaraman, Vivek and Svoboda, Karel and Kim, Douglas S. and Schreiter, Eric R and Looger, Loren L} }