@article {68665, title = {Fast and sensitive GCaMP calcium indicators for imaging neural populations.}, journal = {Nature}, year = {2023}, month = {2023 Mar 15}, abstract = {

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting {\textquoteright}jGCaMP8{\textquoteright} sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

}, issn = {1476-4687}, doi = {10.1038/s41586-023-05828-9}, author = {Zhang, Yan and Rozsa, Marton and Liang, Yajie and Bushey, Daniel and Wei, Ziqiang and Zheng, Jihong and Reep, Daniel and Broussard, Gerard Joey and Tsang, Arthur and Tsegaye, Getahun and Narayan, Sujatha and Obara, Christopher J and Lim, Jing-Xuan and Patel, Ronak and Zhang, Rongwei and Ahrens, Misha B and Turner, Glenn C and Wang, Samuel S-H and Korff, Wyatt L and Schreiter, Eric R and Svoboda, Karel and Hasseman, Jeremy P and Kolb, Ilya and Looger, Loren L} } @article {68630, title = {Fast and sensitive GCaMP calcium indicators for neuronal imaging.}, journal = {The Journal of Physiology}, year = {2023}, month = {2023 Feb 22}, abstract = {

We review the principles of development and deployment of genetically encoded calcium indicators (GECIs) for the detection of neural activity. Our focus is on the popular GCaMP family of green GECIs, culminating in the recent release of the jGCaMP8 sensors, with dramatically improved kinetics relative to previous generations. We summarize the properties of GECIs in multiple color channels (blue, cyan, green, yellow, red, far-red) and highlight areas for further improvement. With their low-millisecond rise-times, the jGCaMP8 indicators allow new classes of experiments following neural activity in timeframes approaching the underlying computations. Abstract legend: GCaMP calcium sensors are widely used to report neuronal activity via fluorescence readout. This article is protected by copyright. All rights reserved.

}, issn = {1469-7793}, doi = {10.1113/JP283832}, author = {Zhang, Yan and Looger, Loren L} } @article {67848, title = {Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.}, journal = {eLife}, volume = {11}, year = {2022}, month = {2022 Jan 04}, abstract = {

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ({\textquoteright}iDrugSnFRs{\textquoteright}) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by \> 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

}, issn = {2050-084X}, doi = {10.7554/eLife.74648}, author = {Nichols, Aaron L and Blumenfeld, Zack and Fan, ChengCheng and Luebbert, Laura and Blom, Annet Em and Cohen, Bruce N and Marvin, Jonathan S and Borden, Philip M and Kim, Charlene H and Muthusamy, Anand K and Shivange, Amol V and Knox, Hailey J and Campello, Hugo Rego and Wang, Jonathan H and Dougherty, Dennis A and Looger, Loren L and Gallagher, Timothy and Rees, Douglas C and Lester, Henry A} } @article {68558, title = {Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics.}, journal = {Bio-Protocol}, volume = {12}, year = {2022}, month = {2022 Nov 20}, abstract = {

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.4551}, author = {Beatty, Zoe G and Muthusamy, Anand K and Unger, Elizabeth K and Dougherty, Dennis A and Tian, Lin and Looger, Loren L and Shivange, Amol V and Bera, Kallol and Lester, Henry A and Nichols, Aaron L} } @article {67311, title = {The functional organization of excitatory synaptic input to place cells.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {2021 Jun 11}, pages = {3558}, abstract = {

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 {\textmu}m. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-23829-y}, author = {Adoff, Michael D and Climer, Jason R and Davoudi, Heydar and Marvin, Jonathan S and Looger, Loren L and Dombeck, Daniel A} } @article {49142, title = {Functional Imaging and Optogenetics in Drosophila.}, journal = {Genetics}, volume = {208}, year = {2018}, month = {2018 Apr}, pages = {1291-1309}, abstract = {

Understanding how activity patterns in specific neural circuits coordinate an animal{\textquoteright}s behavior remains a key area of neuroscience research. Genetic tools and a brain of tractable complexity make a premier model organism for these studies. Here, we review the wealth of reagents available to map and manipulate neuronal activity with light.

}, issn = {1943-2631}, doi = {10.1534/genetics.117.300228}, author = {Simpson, Julie H and Looger, Loren L} } @article {47601, title = {Falling apart.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016}, abstract = {

Destabilized nanobodies can be used to deliver fluorescent proteins and enzymes to specific targets inside cells.

}, issn = {2050-084X}, doi = {10.7554/eLife.18203}, author = {Marvin, Jonathan S and Looger, Loren L} } @article {45844, title = {Fixation-resistant photoactivatable fluorescent proteins for CLEM.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Mar}, pages = {215-8}, abstract = {

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.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3225}, author = {Paez-Segala, Maria G and Sun, Mei G and Shtengel, Gleb and Viswanathan, Sarada and Baird, Michelle A and Macklin, John J and Patel, Ronak and Allen, John R and Howe, Elizabeth S and Piszczek, Grzegorz and Hess, Harald F and Davidson, Michael W and Wang, Yalin and Looger, Loren L} } @article {6122, title = {Fine time-course expression analysis identifies cascades of activation and repression and maps a regulator of mammalian sex determination.}, journal = {PLoS Genetics}, volume = {9}, year = {2013}, month = {2013 Jul}, pages = {e1003630}, abstract = {

In vertebrates, primary sex determination refers to the decision within a bipotential organ precursor to differentiate as a testis or ovary. Bifurcation of organ fate begins between embryonic day (E) 11.0{\textendash}E12.0 in mice and likely involves a dynamic transcription network that is poorly understood. To elucidate the first steps of sexual fate specification, we profiled the XX and XY gonad transcriptomes at fine granularity during this period and resolved cascades of gene activation and repression. C57BL/6J (B6) XY gonads showed a consistent \ 5-hour delay in the activation of most male pathway genes and repression of female pathway genes relative to 129S1/SvImJ, which likely explains the sensitivity of the B6 strain to male-to-female sex reversal. Using this fine time course data, we predicted novel regulatory genes underlying expression QTLs (eQTLs) mapped in a previous study. To test predictions, we developed an in vitro gonad primary cell assay and optimized a lentivirus-based shRNA delivery method to silence candidate genes and quantify effects on putative targets. We provide strong evidence that Lmo4 (Lim-domain only 4) is a novel regulator of sex determination upstream of SF1 (Nr5a1), Sox9, Fgf9, and Col9a3. This approach can be readily applied to identify regulatory interactions in other systems.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003630}, author = {Munger, Steven C. and Natarajan, Anirudh and Looger, Loren L and Ohler, Uwe and Capel, Blanche} } @article {3931, title = {Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.}, journal = {Nature Neuroscience}, volume = {13}, year = {2010}, month = {2010 Nov}, pages = {1433-40}, abstract = {

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.

}, keywords = {Action Potentials, Animals, Calcium, Cerebral Cortex, Computer-Assisted, Dendrites, Genetic, Hippocampus, Image Processing, Inbred C57BL, Male, Membrane Potentials, Mice, Neurons, Nonlinear Dynamics, Patch-Clamp Techniques, Space Perception, Spatial Behavior, Synapsins, Transduction, User-Computer Interface}, issn = {1546-1726}, doi = {10.1038/nn.2648}, author = {Dombeck, Daniel A and Harvey, Christopher D and Tian, Lin and Looger, Loren L and Tank, David W} }