@article {70093, title = {iGABASnFR2: Improved genetically encoded protein sensors of GABA}, journal = {eLife}, year = {2026}, month = {2026 Feb 25}, abstract = {

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However, the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20\% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance when compared directly to the original sensor. iGABASnFR2 enabled the first measurements of direction-selective GABA release in the retina. In vivo imaging in somatosensory cortex revealed that iGABASnFR2 can report volume-transmitted GABA release following whisker stimulation. Overall, the improved sensitivity and kinetics of iGABASnFR2 make it a more effective tool for imaging GABAergic transmission in intact neural circuits.

}, doi = {10.7554/eLife.108319.2}, url = {https://doi.org/10.7554/eLife.108319.2}, author = {Kolb, Ilya and Hasseman, Jeremy P and Matsumoto, Akihiro and Jensen, Thomas P and Kopach, Olga and Arthur, Benjamin J and Zhang, Yan and Tsang, Arthur and Reep, Daniel and Tsegaye, Getahun and Zheng, Jihong and Patel, Ronak H and Looger, Loren L and Marvin, Jonathan S and Korff, Wyatt L and Rusakov, Dmitri A and Yonehara, Keisuke and Turner, Glenn C} } @article {69833, title = {Glutamate indicators with increased sensitivity and tailored deactivation rates}, journal = {Nat Methods}, year = {2025}, month = {2025 Dec 23}, abstract = {

Understanding how neurons integrate signals from thousands of input synapses requires methods to monitor neurotransmission across many sites simultaneously. The fluorescent protein glutamate indicator iGluSnFR enables visualization of synaptic signaling, but the sensitivity, scale and speed of such measurements are limited by existing variants. Here we developed two highly sensitive fourth-generation iGluSnFR variants with fast activation and tailored deactivation rates: iGluSnFR4f for tracking rapid dynamics, and iGluSnFR4s for recording from large populations of synapses. These indicators detect glutamate with high spatial specificity and single-vesicle sensitivity in vivo. We used them to record natural patterns of synaptic transmission across multiple experimental contexts in mice, including two-photon imaging in cortical layers 1{\textendash}4 and hippocampal CA1, and photometry in the midbrain. The iGluSnFR4 variants extend the speed, sensitivity and scalability of glutamate imaging, enabling direct observation of information flow through neural networks in the intact brain.

}, doi = {10.1038/s41592-025-02965-z}, url = {https://doi.org/10.1038/s41592-025-02965-z}, author = {Aggarwal, Abhi and Negrean, Adrian and Chen, Yang and Iyer, Rishyashring and Reep, Daniel and Liu, Anyi and Palutla, Anirudh and Xie, Michael E. and MacLennan, Bryan J. and Hagihara, Kenta M. and Kinsey, Lucas W. and Sun, Julianna L. and Yao, Pantong and Zheng, Jihong and Tsang, Arthur and Tsegaye, Getahun and Zhang, Yonghai and Patel, Ronak H. and Arthur, Benjamin J. and Hiblot, Julien and Leippe, Philipp and Tarnawski, Miroslaw and Marvin, Jonathan S. and Vevea, Jason D. and Turaga, Srinivas C. and Tebo, Alison G. and Carandini, Matteo and Rossi, L. F. and Kleinfeld, David and Konnerth, Arthur and Svoboda, Karel and Turner, Glenn C. and Hasseman, Jeremy and Podgorski, Kaspar} } @article {69856, title = {Glutamate indicators with increased sensitivity and tailored deactivation rates}, journal = {Research Square}, year = {2025}, month = {2025 Apr 8}, abstract = {

Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neuron{\textquoteright}s thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (\< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide range of naturalistic synaptic transmission, including in the vibrissal cortex layer 4 and in hippocampal CA1 dendrites. iGluSnFR4 increases the sensitivity and scale (4s) or speed (4f) of tracking information flow in neural networks in vivo.

}, doi = {10.21203/rs.3.rs-6257403/v1}, url = {https://doi.org/10.21203/rs.3.rs-6257403/v1}, author = {Podgorski, Kaspar and Aggarwal, Abhi and Negrean, Adrian and Chen, Yang and Iyer, Rishyashring and Reep, Daniel and Liu, Anyi and Palutla, Anirudh and Xie, Michael and Maclennan, Bryan and Hagihara, Kenta and Kinsey, Lucas and Sun, Julianna and Yao, Pantong and Zheng, Jihong and Tsang, Arthur and Tsegaye, Getahun and Zhang, Yonghai and Patel, Ronak and Hasseman, Jeremy} } @article {69831, title = {iGABASnFR2: Improved genetically encoded protein sensors of GABA}, journal = {bioRxiv}, year = {2025}, month = {2025 Mar 25}, abstract = {

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis, and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20\% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance, enabling us to make the first measurements of direction selective GABA release in the retina and confirm a longstanding hypothesis for how sensitivity to motion arises in the visual system.

}, doi = {10.1101/2025.03.25.644953}, url = {https://www.biorxiv.org/content/early/2025/03/26/2025.03.25.644953}, author = {Kolb, Ilya and Hasseman, Jeremy P. and Matsumoto, Akihiro and Arthur, Benjamin J. and Zhang, Yan and Tsang, Arthur and Reep, Daniel and Tsegaye, Getahun and Zheng, Jihong and Patel, Ronak and Looger, Loren L. and Marvin, Jonathan S. and Korff, Wyatt L. and Yonehara, Keisuke and Turner, Glenn C.} } @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 {68772, title = {Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.}, journal = {Nature Methods}, volume = {20}, year = {2023}, month = {2023 Jun 01}, abstract = {

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR{\textquoteright}s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

}, issn = {1548-7105}, doi = {10.1038/s41592-023-01863-6}, author = {Aggarwal, Abhi and Liu, Rui and Chen, Yang and Ralowicz, Amelia J and Bergerson, Samuel J and Tomaska, Filip and Mohar, Boaz and Hanson, Timothy L and Hasseman, Jeremy P and Reep, Daniel and Tsegaye, Getahun and Yao, Pantong and Ji, Xiang and Kloos, Marinus and Walpita, Deepika and Patel, Ronak and Mohr, Manuel A and Tillberg, Paul W and Looger, Loren L and Marvin, Jonathan S and Hoppa, Michael B and Konnerth, Arthur and Kleinfeld, David and Schreiter, Eric R and Podgorski, Kaspar} } @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 {65618, title = {jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 May 25}, pages = {694-97}, abstract = {

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0835-7}, author = {Mohr, Manuel Alexander and Bushey, Daniel and Abhi Aggarwal and Marvin, Jonathan S and Kim, Jeong Jun and Marquez, Emiliano Jimenez and Liang, Yajie and Patel, Ronak and Macklin, John J and Lee, Chi-Yu and Tsang, Arthur and Tsegaye, Getahun and Ahrens, Allison M and Chen, Jerry L and Kim, Douglas S and Wong, Allan M and Looger, Loren L and Schreiter, Eric R and Podgorski, Kaspar} } @article {64986, title = {High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Jun 17}, pages = {649-57}, abstract = {

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0435-6}, author = {Dana, Hod and Sun, Yi and Mohar, Boaz and Hulse, Brad K and Kerlin, Aaron M and Hasseman, Jeremy P and Tsegaye, Getahun and Tsang, Arthur and Wong, Allan and Patel, Ronak and Macklin, John J and Chen, Yang and Konnerth, Arthur and Jayaraman, Vivek and Looger, Loren L and Schreiter, Eric R and Svoboda, Karel and Kim, Douglas S} } @article {49813, title = {High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments.}, journal = {bioRxiv}, year = {2018}, month = {2018 Oct 3}, abstract = {

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics, and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

}, doi = {10.1101/434589}, author = {Dana, Hod and Sun, Yi and Mohar, Boaz and Hulse, Brad and Hasseman, Jeremy P and Tsegaye, Getahun and Tsang, Arthur and Wong, Allan and Patel, Ronak and Macklin, John J and Chen, Yang and Konnerth, Arthur and Jayaraman, Vivek and Looger, Loren L and Schreiter, Eric R and Svoboda, Karel and Kim, Douglas S} } @article {47396, title = {Sensitive red protein calcium indicators for imaging neural activity.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Mar 24}, abstract = {

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

}, issn = {2050-084X}, doi = {10.7554/eLife.12727}, author = {Dana, Hod and Mohar, Boaz and Sun, Yi and Narayan, Sujatha and Gordus, Andrew and Hasseman, Jeremy P and Tsegaye, Getahun and Holt, Graham T and Hu, Amy and Walpita, Deepika and Patel, Ronak and Macklin, John J and Bargmann, Cornelia I and Ahrens, Misha B and Schreiter, Eric R and Jayaraman, Vivek and Looger, Loren L and Svoboda, Karel and Kim, Douglas S} } @article {6579, title = {A neuron-based screening platform for optimizing genetically-encoded calcium indicators.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e77728}, abstract = {

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0077728}, author = {Wardill, Trevor J. and Chen, Tsai-Wen and Schreiter, Eric R and Hasseman, Jeremy P and Tsegaye, Getahun and Fosque, Benjamin F and Behnam, Reza and Shields, Brenda C and Ramirez, Melissa and Kimmel, Bruce E and Kerr, Rex A and Jayaraman, Vivek and Looger, Loren L and Svoboda, Karel and Kim, Douglas S.} }