@article {70026, title = {CHLOK: a chemigenetic multicolor labeling system to visualize neuronal birthdate and circuit integration}, journal = {Research Square}, year = {2025}, month = {2025 Jul 08}, abstract = {

Understanding how neurons integrate into developing circuits and contribute to functional activity is essential for decoding brain development and plasticity. However, current methods to study neuronal integration often suffer from low throughput, limited spatiotemporal resolution, or invasive procedures that hinder in vivo functional analysis. To overcome these challenges, we present a birthdate-labeling strategy, named CHLOK, based on HaloTag technology and a broad palette of fluorescent synthetic dyes. This approach enables precise multicolor labeling of neurons according to their maturation stage and allows flexible integration into functional assays through compatibility with calcium imaging and optogenetics. We validated CHLOK by mapping birthdate-resolved neuronal activity in the developing visual and motor systems of zebrafish larvae. Our results reveal distinct functional contributions of early- versus late-born neurons, providing new insights into the temporal dynamics of circuit formation. Furthermore, we demonstrate the versatility of this approach, showcasing age-specific multicolor calcium and voltage imaging as well as optogenetic manipulation. By overcoming key limitations of existing techniques, CHLOK offers a powerful, versatile and non-invasive tool for studying neural integration, circuit development and function in vivo.

}, doi = {10.21203/rs.3.rs-7039578/v1}, url = {https://doi.org/10.21203/rs.3.rs-7039578/v1}, author = {Faini, Giulia and Tuffery, Matthieu and Saleem, Amna and Zhang, Lixia and Du, Felix and Le Bourdelles, Guillaume and Duroure, Karine and Schreiter, Eric R and Tanese, Dimitrii and Emiliani, Valentina and Del Bene, Filippo and Koyama, Minoru} } @article {69994, title = {A multimodal adaptive optical microscope for in vivo imaging from molecules to organisms}, journal = {bioRxiv}, year = {2025}, month = {2025 Jun 16}, abstract = {

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

Preprint:\ https://www.biorxiv.org/content/early/2025/06/13/2025.06.02.657494

}, doi = {10.1101/2025.06.02.657494}, url = {https://www.biorxiv.org/content/early/2025/06/13/2025.06.02.657494}, author = {Fu, Tian-Ming and Liu, Gaoxiang and Milkie, Daniel E. and Ruan, Xiongtao and G{\"o}rlitz, Frederik and Shi, Yu and Ferro, Valentina and Divekar, Nikita S. and Wang, Wei and York, Harrison M. and Kilic, Velat and Mueller, Matthew and Liang, Yajie and Daugird, Timothy A. and Gacha-Garay, Maria J. and Larkin, Kathryn A. and Adikes, Rebecca C. and Harrison, Nathaniel and Shirazinejad, Cyna and Williams, Samara and Nourse, Jamison L. and Sheu, Shu-Hsien and Gao, Liang and Li, Tongchao and Mondal, Chandrani and Achour, Kemal and Hercule, Wilmene and Stabley, Daniel and Emmerich, Kevin and Dong, Peng and Drubin, David and Liu, Zhe J. and Clapham, David and Mumm, Jeff S. and Koyama, Minoru and Killilea, Alison and Bravo-Cordero, Jose Javier and Keene, C. Dirk and Luo, Liqun and Kirchhausen, Tom and Pathak, Medha M. and Arumugam, Senthil and Nunez, James K. and Gao, Ruixuan and Matus, David Q. and Martin, Benjamin L. and Swinburne, Ian A. and Betzig, Eric and Legant, Wesley R. and Upadhyayula, Srigokul} } @article {68690, title = {Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator}, journal = {Neuron}, volume = {111}, year = {2023}, month = {2023 May 17}, pages = {1547-1563}, abstract = {

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a\ point mutation\ that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65\% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple\ in\ vitro\ and\ in\ vivo\ comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of\ interneuron\ synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

}, issn = {08966273}, doi = {10.1016/j.neuron.2023.03.009}, url = {https://linkinghub.elsevier.com/retrieve/pii/S0896627323002052}, author = {Abdelfattah, Ahmed S. and Zheng, Jihong and Singh, Amrita and Huang, Yi-Chieh and Reep, Daniel and Tsegaye, Getahun and Tsang, Arthur and Arthur, Benjamin J. and Rehorova, Monika and Olson, Carl V.L. and Shuai, Yichun and Zhang, Lixia and Fu, Tian-Ming and Milkie, Daniel E. and Moya, Maria V. and Weber, Timothy D. and Lemire, Andrew L. and Baker, Christopher A. and Falco, Natalie and Zheng, Qinsi and Grimm, Jonathan B. and Yip, Mighten C. and Walpita, Deepika and Chase, Martin and Campagnola, Luke and Murphy, Gabe J. and Wong, Allan M. and Forest, Craig R. and Mertz, Jerome and Economo, Michael N. and Turner, Glenn C. and Koyama, Minoru and Lin, Bei-Jung and Betzig, Eric and Novak, Ondrej and Lavis, Luke D. and Svoboda, Karel and Korff, Wyatt and Chen, Tsai-Wen and Schreiter, Eric R. and Hasseman, Jeremy P. and Kolb, Ilya} } @article {68606, title = {TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.}, journal = {Neuron}, volume = {111}, year = {2023}, month = {2023 Feb 01}, pages = {345-361.e10}, abstract = {

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

}, keywords = {Animals, Cell Differentiation, Cell Lineage, Gene Expression Regulation, Developmental, Mice, Neurogenesis, Neuroglia, Neurons, Zebrafish}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.10.035}, author = {Espinosa-Medina, Isabel and Feliciano, Daniel and Belmonte-Mateos, Carla and Linda Miyares, Rosa and Garcia-Marques, Jorge and Foster, Benjamin and Lindo, Sarah and Pujades, Cristina and Koyama, Minoru and Lee, Tzumin} } @article {68518, title = {TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.}, journal = {Neuron}, year = {2022}, month = {2022 Nov 18}, abstract = {

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.10.035}, author = {Espinosa-Medina, Isabel and Feliciano, Daniel and Belmonte-Mateos, Carla and Linda Miyares, Rosa and Garcia-Marques, Jorge and Foster, Benjamin and Lindo, Sarah and Pujades, Cristina and Koyama, Minoru and Lee, Tzumin} } @article {67627, title = {In~vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Jun 22}, pages = {109284}, abstract = {

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1\ μM to 10\ mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109284}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {68342, title = {TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages}, journal = {bioRxiv}, year = {2021}, month = {10/2021}, abstract = {

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.One-Sentence Summary Gaining sequential genetic access to vertebrate cell lineages.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2021.10.27.466134}, url = {https://www.biorxiv.org/content/early/2021/10/28/2021.10.27.466134}, author = {Espinosa-Medina, Isabel and Feliciano, Daniel and Belmonte-Mateos, Carla and Garcia-Marques, Jorge and Foster, Benjamin and Miyares, Rosa Linda and Pujades, Cristina and Koyama, Minoru and Lee, Tzumin} } @article {65300, title = {A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies.}, journal = {bioRxiv}, year = {2020}, month = {2020 Feb 8}, abstract = {

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and\ in vivo\ preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

}, doi = {https://doi.org/10.1101/2020.02.07.939504}, author = {Borden, Philip and Zhang, Peng and Shivange, Amol V and Marvin, Jonathan S and Cichon, Joseph and Dan, Chuntao and Podgorski, Kaspar and Figueiredo, Antonio and Novak, Ondrej and Tanimoto, Masashi and Shigetomi, Eiji and Lobas, Mark A and Kim, Hyuntae and Zhu, Paula and Zhang, Yajun and Zheng, W. Sharon and Fan, ChengCheng and Wang, Guangfu and Xiang, Bowen and Gan, Li and Zhang, Guang-Xian and Guo, Kaiming and Lin, Li and Cai, Yuan and Yee, Andrew G. and Aggarwal, Aman and Ford, Christopher P. and Rees, Douglas C. and Dietrich, Dirk and Khakh, Baljit S. and Dittman, Jeremy S. and Gan, Wen-Biao and Koyama, Minoru and Jayaraman, Vivek and Cheer, Joseph F. and Lester, Henry A. and Zhu, J. Julius and Looger, Loren L.} } @article {65378, title = {A general approach to engineer positive-going eFRET voltage indicators}, journal = {Nature Communications}, volume = {11}, year = {2020}, month = {2020 Jul 10}, abstract = {

We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with {\textquotedblleft}positive-going{\textquotedblright} fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

}, author = {Abdelfattah, Ahmed S and Valenti, Rosario and Zheng, Jihong and Wong, Allan and Podgorski, Kaspar and Koyama, Minoru and Kim, Douglas S. and Schreiter, Eric R and Project Team GENIE} } @article {65377, title = {High-throughput cellular-resolution synaptic connectivity mapping in vivo with concurrent two-photon optogenetics and volumetric Ca2+ imaging}, journal = {bioRxiv}, year = {2020}, month = {2020 Feb 25}, abstract = {

The ability to measure synaptic connectivity and properties is essential for understanding neuronal circuits. However, existing methods that allow such measurements at cellular resolution are laborious and technically demanding. Here, we describe a system that allows such measurements in a high-throughput way by combining two-photon optogenetics and volumetric Ca2+\ imaging with whole-cell recording. We reveal a circuit motif for generating fast undulatory locomotion in zebrafish.

}, doi = {https://doi.org/10.1101/2020.02.21.959650}, author = {McRaven, Christopher and Tanese, Dimitrii and Zhang, Lixia and Yang, Chao-Tsung and Ahrens, Misha B and Emiliani, Valentina and Koyama, Minoru} } @article {65669, title = {A programmable sequence of reporters for lineage analysis.}, journal = {Nature Neuroscience}, volume = {23}, year = {2020}, month = {2020 Dec 01}, pages = {1618-28}, abstract = {

We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

}, issn = {1546-1726}, doi = {10.1038/s41593-020-0676-9}, author = {Garcia-Marques, Jorge and Isabel Espinosa Medina and Ku, Kai-Yuan and Yang, Ching-Po and Koyama, Minoru and Yu, Hung-Hsiang and Lee, Tzumin} } @article {65668, title = {Rational design of bioavailable photosensitizers for manipulation and imaging of biological systems.}, journal = {Cell Chemical Biology}, volume = {27}, year = {2020}, month = {2020 Aug 20}, pages = {1063-72}, abstract = {

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in\ vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2020.07.001}, author = {Binns, Thomas C and Ayala, Anthony X and Grimm, Jonathan B and Tkachuk, Ariana N and Castillon, Guillaume A and Phan, Sebastien and Zhang, Lixia and Brown, Timothy A and Liu, Zhe and Adams, Stephen R and Ellisman, Mark H and Koyama, Minoru and Lavis, Luke D} } @article {65081, title = {Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.}, journal = {Science}, volume = {365}, year = {2019}, month = {2019 Aug 13}, pages = {699-704}, abstract = {

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

}, issn = {1095-9203}, doi = {10.1126/science.aav6416}, author = {Abdelfattah, Ahmed S and Kawashima, Takashi and Singh, Amrita and Novak, Ondrej and Liu, Hui and Shuai, Yichun and Huang, Yi-Chieh and Campagnola, Luke and Seeman, Stephanie C and Yu, Jianing and Zheng, Jihong and Grimm, Jonathan B and Patel, Ronak and Friedrich, Johannes and Mensh, Brett D and Paninski, Liam and Macklin, John J and Murphy, Gabe J and Podgorski, Kaspar and Lin, Bei-Jung and Chen, Tsai-Wen and Turner, Glenn C and Liu, Zhe and Koyama, Minoru and Svoboda, Karel and Ahrens, Misha B and Lavis, Luke D and Schreiter, Eric R} } @article {64814, title = {Chronology-based architecture of descending circuits that underlie the development of locomotor repertoire after birth.}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Feb 25}, abstract = {

The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system. Little is known, however, of how nascent connections are organized to support such new behaviors alongside existing ones. To understand this, in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion. We found that new projections are continually layered laterally to existing neuropil, and give rise to distinct pathways that function in parallel to existing pathways. Across these chronologically layered pathways, the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics. Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animal{\textquoteright}s behavioral repertoire.

}, issn = {2050-084X}, doi = {10.7554/eLife.42135}, author = {Pujala, Avinash and Koyama, Minoru} } @article {65379, title = {CLADES: a programmable sequence of reporters for lineage analysis}, journal = {bioRxiv}, year = {2019}, month = {2019 May 30}, abstract = {

We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth order with reporter expression. This gives us temporal resolution of lineage development that can be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, marking each generation with the corresponding combination of reporters. CLADES thus offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

}, doi = {https://doi.org/10.1101/655308}, author = {Garcia-Marques, Jorge and Yang, Ching-Po and Isabel Espinosa Medina and Koyama, Minoru and Lee, Tzumin} } @article {64889, title = {Dynamic super-resolution structured illumination imaging in the living brain.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, year = {2019}, month = {2019 Apr 26}, pages = {9586-91}, abstract = {

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

}, issn = {1091-6490}, doi = {10.1073/pnas.1819965116}, author = {Turcotte, Raphael and Liang, Yajie and Tanimoto, Masashi and Zhang, Qinrong and Li, Ziwei and Koyama, Minoru and Betzig, Eric and Ji, Na} } @article {64866, title = {In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {bioRxiv}, year = {2019}, month = {2019 Mar 8}, abstract = {Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {\textmu}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.}, doi = {10.1101/571422}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {65162, title = {Single-cell reconstruction of emerging population activity in an entire developing circuit.}, journal = {Cell}, volume = {179}, year = {2019}, month = {2019 Sep 23}, abstract = {

Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.08.039}, author = {Wan, Yinan and Wei, Ziqiang and Looger, Loren L and Koyama, Minoru and Druckmann, Shaul and Keller, Philipp J} } @article {65078, title = {Unlimited genetic switches for cell-type-specific manipulation.}, journal = {Neuron}, volume = {104}, year = {2019}, month = {2019 Oct 23}, pages = {227-38}, abstract = {

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

}, doi = {https://doi.org/10.1016/j.neuron.2019.07.005}, author = {Garcia-Marques, Jorge and Yang, Ching-Po and Isabel Espinosa Medina and Mok, Kent and Koyama, Minoru and Lee, Tzumin} } @article {49151, title = {50 Hz volumetric functional imaging with continuously adjustable depth of focus.}, journal = {Biomedical Optics Express}, volume = {9}, year = {2018}, month = {2018 Apr}, pages = {1964-76}, abstract = {

Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), that turns frame rate into volume rate by extending the excitation focus in the axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the axial length of the Bessel focus. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 {\textmu}m depth in larval zebrafish at 50 Hz volume rate with adjustable axial extent of the imaged volume.

}, doi = {10.1364/BOE.9.001964}, author = {Lu, Rongwen and Tanimoto, Masashi and Koyama, Minoru and Na, Ji} } @article {49535, title = {Brain-wide circuit interrogation at the cellular level guided by online analysis of neuronal function.}, journal = {Nature Methods}, volume = {15}, year = {2018}, month = {2018 Nov 30}, pages = {1117-1125}, abstract = {

Whole-brain imaging allows for comprehensive functional mapping of distributed neural pathways, but neuronal perturbation experiments are usually limited to targeting predefined regions or genetically identifiable cell types. To complement whole-brain measures of activity with brain-wide manipulations for testing causal interactions, we introduce a system that uses measuredactivity patterns to guide optical perturbations of any subset of neurons in the same fictively behaving larval zebrafish. First, a light-sheet microscope collects whole-brain data that are rapidly analyzed by a distributed computing system to generate functional brain maps. On the basis of these maps, the experimenter can then optically ablate neurons and image activity changes across the brain. We applied this method to characterize contributions of behaviorally tuned populations to the optomotor response. We extended the system to optogenetically stimulate arbitrary subsets of neurons during whole-brain imaging. These open-source methods enable delineating the contributions of neurons to brain-wide circuit dynamics and behavior in individual animals.

}, doi = {10.1038/s41592-018-0221-x}, author = {Vladimirov, Nikita and Wang, Chen and H{\"o}ckendorf, Burkhard and Pujala, Avinash and Tanimoto, Masashi and Mu, Yu and Yang, Chao-Tsung and Wittenbach, Jason and Freeman, Jeremy and Preibisch, Stephan and Koyama, Minoru and Keller, Philipp J and Ahrens, Misha B} } @article {49010, title = {A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.}, journal = {BMC Biology}, volume = {16}, year = {2018}, month = {2018 Jan 16}, pages = {9}, abstract = {

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.

}, issn = {1741-7007}, doi = {10.1186/s12915-018-0480-0}, author = {Shen, Yi and Dana, Hod and Abdelfattah, Ahmed S and Patel, Ronak and Shea, Jamien and Molina, Rosana S and Rawal, Bijal and Rancic, Vladimir and Chang, Yu-Fen and Wu, Lanshi and Chen, Yingche and Qian, Yong and Wiens, Matthew D and Hambleton, Nathan and Ballanyi, Klaus and Hughes, Thomas E and Drobizhev, Mikhail and Kim, Douglas S and Koyama, Minoru and Schreiter, Eric R and Campbell, Robert E} } @article {49011, title = {Mutual inhibition of lateral inhibition: a network motif for an elementary computation in the brain.}, journal = {Current Opinion in Neurobiology}, volume = {49}, year = {2018}, month = {2018 Jan 16}, pages = {69-74}, abstract = {

A series of classical studies in non-human primates has revealed the neuronal activity patterns underlying decision-making. However, the circuit mechanisms for such patterns remain largely unknown. Recent detailed circuit analyses in simpler neural systems have started to reveal the connectivity patterns underlying analogous processes. Here we review a few of these systems that share a particular connectivity pattern, namely mutual inhibition of lateral inhibition. Close examination of these systems suggests that this recurring connectivity pattern ({\textquoteright}network motif{\textquoteright}) is a building block to enforce particular dynamics, which can be used not only for simple behavioral choice but also for more complex choices and other brain functions. Thus, a network motif provides an elementary computation that is not specific to a particular brain function and serves as an elementary building block in the brain.

}, issn = {1873-6882}, doi = {10.1016/j.conb.2017.12.019}, author = {Koyama, Minoru and Pujala, Avinash} } @article {49173, title = {Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.}, journal = {Science (New York, N.Y.)}, volume = {360}, year = {2018}, month = {2018 Apr 20}, abstract = {

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

}, issn = {1095-9203}, doi = {10.1126/science.aaq1392}, author = {Liu, Tsung-Li and Upadhyayula, Srigokul and Milkie, Daniel E and Singh, Ved and Wang, Kai and Swinburne, Ian A and Mosaliganti, Kishore R and Collins, Zach M and Hiscock, Tom W and Shea, Jamien and Kohrman, Abraham Q and Medwig, Taylor N and Dambournet, Daphne and Forster, Ryan and Cunniff, Brian and Ruan, Yuan and Yashiro, Hanako and Scholpp, Steffen and Meyerowitz, Elliot M and Hockemeyer, Dirk and Drubin, David G and Martin, Benjamin L and Matus, David Q and Koyama, Minoru and Megason, Sean G and Kirchhausen, Tom and Betzig, Eric} } @article {48346, title = {Stochastic protein labeling enables long-term single molecule observation in vivo.}, journal = {bioRxiv}, year = {2017}, month = {2017 Mar 13}, abstract = {Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.}, doi = {10.1101/116186}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {48304, title = {Video-rate volumetric functional imaging of the brain at synaptic resolution.}, journal = {Nature Neuroscience}, volume = {20}, year = {2017}, month = {2017 Feb 27}, pages = {620-8}, abstract = {

Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

}, issn = {1546-1726}, doi = {10.1038/nn.4516}, author = {Lu, Rongwen and Sun, Wenzhi and Liang, Yajie and Kerlin, Aaron and Bierfeld, Jens and Seelig, Johannes D and Wilson, Daniel E and Scholl, Benjamin and Mohar, Boaz and Tanimoto, Masashi and Koyama, Minoru and Fitzpatrick, David and Orger, Michael B and Ji, Na} } @article {48752, title = {Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2017}, month = {2017 Jan 09}, pages = {343-8}, abstract = {

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of \>10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact\ zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics\ in vivo.

}, doi = {10.1073/pnas.1713895115}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {65008, title = {A circuit motif in the zebrafish hindbrain for a two alternative behavioral choice to turn left or right.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016 08 09}, abstract = {

Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies. Key inhibitory neurons in the circuit lie in a column of morphologically similar cells that is one of a series of such columns that form a developmental and functional ground plan for building hindbrain networks. Repetition within the columns of the network motif we defined may therefore lie at the foundation of other lateralized behavioral choices.

}, keywords = {Action Potentials, Animals, Animals, Genetically Modified, Behavior, Animal, Choice Behavior, Laser Therapy, Locomotion, Neural Pathways, Neurons, Patch-Clamp Techniques, Perception, Rhombencephalon, Zebrafish}, issn = {2050-084X}, doi = {10.7554/eLife.16808}, author = {Koyama, Minoru and Minale, Francesca and Shum, Jennifer and Nishimura, Nozomi and Schaffer, Chris B and Fetcho, Joseph R} } @article {6445, title = {Mapping a sensory-motor network onto a structural and functional ground plan in the hindbrain.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Jan 18}, pages = {1170-5}, abstract = {

The hindbrain of larval zebrafish contains a relatively simple ground plan in which the neurons throughout it are arranged into stripes that represent broad neuronal classes that differ in transmitter identity, morphology, and transcription factor expression. Within the stripes, neurons are stacked continuously according to age as well as structural and functional properties, such as axonal extent, input resistance, and the speed at which they are recruited during movements. Here we address the question of how particular networks among the many different sensory-motor networks in hindbrain arise from such an orderly plan. We use a combination of transgenic lines and pairwise patch recording to identify excitatory and inhibitory interneurons in the hindbrain network for escape behaviors initiated by the Mauthner cell. We map this network onto the ground plan to show that an individual hindbrain network is built by drawing components in predictable ways from the underlying broad patterning of cell types stacked within stripes according to their age and structural and functional properties. Many different specialized hindbrain networks may arise similarly from a simple early patterning.

}, keywords = {Animals, Body Patterning, Brain Mapping, Computer-Assisted, Electrophysiology, Escape Reaction, Genetically Modified, Image Processing, Immunohistochemistry, Interneurons, Larva, Nerve Net, Patch-Clamp Techniques, Psychomotor Performance, Rhombencephalon, Zebrafish}, issn = {1091-6490}, doi = {10.1073/pnas.1012189108}, author = {Koyama, Minoru and Kinkhabwala, Amina and Satou, Chie and Higashijima, Shin-Ichi and Fetcho, Joseph} } @article {6444, title = {A structural and functional ground plan for neurons in the hindbrain of zebrafish.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Jan 18}, pages = {1164-9}, abstract = {

The vertebrate hindbrain contains various sensory-motor networks controlling movements of the eyes, jaw, head, and body. Here we show that stripes of neurons with shared neurotransmitter phenotype that extend throughout the hindbrain of young zebrafish reflect a broad underlying structural and functional patterning. The neurotransmitter stripes contain cell types with shared gross morphologies and transcription factor markers. Neurons within a stripe are stacked systematically by extent and location of axonal projections, input resistance, and age, and are recruited along the axis of the stripe during behavior. The implication of this pattern is that the many networks in hindbrain are constructed from a series of neuronal components organized into stripes that are ordered from top to bottom according to a neuron{\textquoteright}s age, structural and functional properties, and behavioral roles. This simple organization probably forms a foundation for the construction of the networks underlying the many behaviors produced by the hindbrain.

}, keywords = {Age Factors, Animals, Body Patterning, Calcium, Confocal, Electrophysiology, Genetically Modified, Interneurons, Microscopy, Models, Nerve Net, Neurological, Neurotransmitter Agents, Rhombencephalon, Zebrafish}, issn = {1091-6490}, doi = {10.1073/pnas.1012185108}, author = {Kinkhabwala, Amina and Riley, Michael and Koyama, Minoru and Monen, Joost and Satou, Chie and Kimura, Yukiko and Higashijima, Shin-Ichi and Fetcho, Joseph} } @article {6443, title = {MRI-based localization of electrophysiological recording sites within the cerebral cortex at single-voxel accuracy.}, journal = {Nature Methods}, volume = {4}, year = {2007}, month = {2007 Feb}, pages = {161-8}, abstract = {

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 mum). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 mum within the cortex of 2-3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.

}, keywords = {Animal, Animals, Behavior, Cerebral Cortex, Electrophysiology, Imaging, Macaca, Magnetic Resonance Imaging, Microelectrodes, Phantoms}, issn = {1548-7091}, doi = {10.1038/nmeth987}, author = {Matsui, Teppei and Koyano, Kenji W and Koyama, Minoru and Nakahara, Kiyoshi and Takeda, Masaki and Ohashi, Yohei and Naya, Yuji and Miyashita, Yasushi} } @article {6442, title = {Functional magnetic resonance imaging of macaque monkeys performing visually guided saccade tasks: comparison of cortical eye fields with humans.}, journal = {Neuron}, volume = {41}, year = {2004}, month = {2004 Mar 4}, pages = {795-807}, abstract = {

The frontal and parietal eye fields serve as functional landmarks of the primate brain, although their correspondences between humans and macaque monkeys remain unclear. We conducted fMRI at 4.7 T in monkeys performing visually-guided saccade tasks and compared brain activations with those in humans using identical paradigms. Among multiple parietal activations, the dorsal lateral intraparietal area in monkeys and an area in the posterior superior parietal lobule in humans exhibited the highest selectivity to saccade directions. In the frontal cortex, the selectivity was highest at the junction of the precentral and superior frontal sulci in humans and in the frontal eye field (FEF) in monkeys. BOLD activation peaks were also found in premotor areas (BA6) in monkeys, which suggests that the apparent discrepancy in location between putative human FEF (BA6, suggested by imaging studies) and monkey FEF (BA8, identified by microstimulation studies) partly arose from methodological differences.

}, keywords = {Analysis of Variance, Animals, Cerebral Cortex, Female, Humans, Macaca, Magnetic Resonance Imaging, Male, Photic Stimulation, Psychomotor Performance, Rats, Saccades, Species Specificity, Visual Fields}, issn = {0896-6273}, author = {Koyama, Minoru and Hasegawa, Isao and Osada, Takahiro and Adachi, Yusuke and Nakahara, Kiyoshi and Miyashita, Yasushi} }