@article {70284, title = {Astrocyte-induced internal state transitions reshape brainwide sensory, integrative, and motor computations}, journal = {bioRxiv}, year = {2026}, month = {2026 Feb 05}, abstract = {

Animals rapidly adapt to changing circumstances by shifting how they perceive, integrate, and act. Such flexibility is often attributed to transitions between internal states that exert widespread influence across the brain. Yet the mechanisms that drive state transitions and how they reconfigure brainwide computation remain unclear. Larval zebrafish, when actions are rendered futile by decoupling visual flow feedback from swimming in virtual reality, enter a temporary passive, energy-preserving state. In this state, astrocyte calcium levels are elevated, and swim reinitiation requires greater accumulated visual motion. Using whole-brain, cellular-resolution activity imaging, we observed widespread circuit alterations underlying this disengaged state: neuronal visual responses weakened, visual motion integration over time became dramatically leakier, motor inhibition increased, and motor preparation slowed, together suppressing conversion of sensory evidence into action. Astrocyte calcium rose during futile swimming, tracked the emergence and resolution of these brainwide changes, and was both necessary and sufficient to drive them. Thus, astrocytes orchestrate internal states that profoundly reshape neural computations, most powerfully at intermediate integrative processing stages, to meet changing demands.

}, doi = {10.64898/2026.02.05.704034}, url = {https://www.biorxiv.org/content/early/2026/02/05/2026.02.05.704034}, author = {Lim, Jing-Xuan and Wei, Ziqiang and Narayan, Sujatha and Zhang, Yan and Hasseman, Jeremy P. and Kolb, Ilya and Zheng, Jihong and Sheikhattar, Alireza and Mi, Xuelong and Zheng, Wei and Yang, Xueying and Beriashvili, Mariam and Fleishman, Greg and Wee, Caroline L. and de Zeeuw, Chris and Yu, Guoqiang and Babadi, Behtash and Rubinov, Mikail and Looger, Loren L. and Bergles, Dwight E. and Fitzgerald, James E. and Ahrens, Misha B.} } @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 {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 {69993, title = {RubyACRs enable red-shifted optogenetic inhibition in freely behaving Drosophila}, journal = {eLife}, year = {2025}, month = {2025 Sep 05}, abstract = {

Optogenetic activators with red-shifted excitation spectra, such as Chrimson, have significantly advanced Drosophila neuroscience. However, until recently, available optogenetic inhibitors required shorter activation wavelengths, which don{\textquoteright}t penetrate tissue as effectively and are stronger visual stimuli to the animal, potentially confounding behavioral results. Here, we assess the efficacy of two newly identified anion-conducting channelrhodopsins with spectral sensitivities similar to Chrimson: A1ACR and HfACR (RubyACRs). Electrophysiology and functional imaging confirmed that RubyACRs effectively hyperpolarize neurons, with stronger and faster effects than the widely used inhibitor GtACR1. Activation of RubyACRs led to circuit-specific behavioral changes in three different neuronal groups. In glutamatergic motor neurons, activating RubyACRs suppressed adult locomotor activity. In PPL1-γ1pedc dopaminergic neurons, pairing odors with RubyACR activation during learning produced odor responses consistent with synaptic silencing. Finally, activation of RubyACRs in the pIP10 neuron suppressed pulse song during courtship. Together, these results demonstrate that RubyACRs are effective and reliable tools for neuronal inhibition in Drosophila, expanding the optogenetic toolkit for circuit dissection in freely behaving animals.

Preprint: https://www.biorxiv.org/content/early/2025/06/15/2025.06.13.659144

}, doi = {10.7554/eLife.108210.1}, url = {https://doi.org/10.7554/eLife.108210.1}, author = {Bushey, Daniel and Shiozaki, Hiroshi and Shuai, Yichun and Zheng, Jihong and Jayaraman, Vivek and Hasseman, Jeremy P. and Kolb, Ilya and GENIE Project Team and Turner, Glenn C.} } @article {69231, title = {Recording physiological history of cells with chemical labeling.}, journal = {Science}, volume = {383}, year = {2024}, month = {2024 Feb 23}, pages = {890-897}, abstract = {

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

}, keywords = {Animals, Calcium, Cell Physiological Phenomena, Cells, Coloring Agents, Gene Expression Profiling, Protein Interaction Domains and Motifs, Staining and Labeling, Zebrafish}, issn = {1095-9203}, doi = {10.1126/science.adg0812}, author = {Huppertz, Magnus-Carsten and Wilhelm, Jonas and Grenier, Vincent and Schneider, Martin W and Falt, Tjalda and Porzberg, Nicola and Hausmann, David and Hoffmann, Dirk C and Hai, Ling and Tarnawski, Miroslaw and Pino, Gabriela and Slanchev, Krasimir and Kolb, Ilya and Acuna, Claudio and Fenk, Lisa M and Baier, Herwig and Hiblot, Julien and Johnsson, Kai} } @article {69021, title = {Extracellular glutamate and GABA transients at the transition from interictal spiking to seizures.}, journal = {Brain}, year = {2023}, month = {2023 Oct 03}, abstract = {

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal \~{}1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.

}, issn = {1460-2156}, doi = {10.1093/brain/awad336}, author = {Shimoda, Yoshiteru and Leite, Marco and Graham, Robert T and Marvin, Jonathan S and Hasseman, Jeremy and Kolb, Ilya and Looger, Loren L and Magloire, Vincent and Kullmann, Dimitri M} } @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 {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 {65821, title = {Robotic Multi-Probe-Single-Actuator Inchworm Neural Microdrive}, journal = {eLife}, year = {2022}, month = {2022 Nov 10}, abstract = {

Electrophysiology is one of the major experimental techniques used in neuroscience. The favorable spatial and temporal resolution as well as the increasingly larger site counts of brain recording electrodes contribute to the popularity and importance of electrophysiology in neuroscience. Such electrodes are typically mechanically placed in the brain to perform acute or chronic freely moving animal measurements. The micro positioners currently used for such tasks employ a single translator per independent probe being placed into the targeted brain region, leading to significant size and weight restrictions. To overcome this limitation, we have developed a miniature robotic multi-probe neural microdrive that utilizes novel phase-change-material-filled resistive heater micro-grippers. The microscopic dimensions, gentle gripping action, independent electronic actuation control, and high packing density of the grippers allow for micrometer-precision independent positioning of multiple arbitrarily shaped parallel neural electrodes with only a single piezo actuator in an inchworm motor configuration. This multi-probe-single-actuator design allows for significant size and weight reduction, as well as remote control and potential automation of the microdrive. We demonstrate accurate placement of multiple independent recording electrodes into the CA1 region of the rat hippocampus\ in vivo\ in acute and chronic settings. Thus, our robotic neural microdrive technology is applicable towards basic neuroscience and clinical studies, as well as other multi-probe or multi-sensor micro-positioning applications.

}, doi = {10.7554/eLife.71876}, author = {Smith, R.D. and Kolb, Ilya and Tanaka, Shinsuke and Lee, A.K. and Harris, T.D. and Barbic, M.} } @article {67695, title = {Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts.}, journal = {Bio Protocol}, volume = {11}, year = {2021}, month = {2021 Jul 20}, pages = {e4085}, abstract = {

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly (\< 30 s), resulting in a high yield of whole-cell recording success rate (\> 90\%) for over 100 reuses of a single pipette. For most patch-clamp experiments (\< 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract: Rapid enzymatic cleaning for reuse of patch-clamp pipettes.

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.4085}, author = {Landry, Corey R and Yip, Mighten C and Kolb, Ilya and Stoy, William A and Gonzalez, Mercedes M and Forest, Craig R} } @article {65650, title = {Bright and high-performance genetically encoded Ca indicator based on mNeonGreen fluorescent protein.}, journal = {ACS Sensors}, year = {2020}, month = {2020 Jul 08}, abstract = {

Genetically encodable calcium ion (Ca) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60\% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

}, issn = {2379-3694}, doi = {10.1021/acssensors.0c00279}, author = {Zarowny, Landon and Abhi Aggarwal and Rutten, Virginia M S and Kolb, Ilya and Patel, Ronak and Huang, Hsin-Yi and Chang, Yu-Fen and Phan, Tiffany and Kanyo, Richard and Ahrens, Misha B and Allison, W Ted and Podgorski, Kaspar and Campbell, Robert E} } @article {65056, title = {A genetically encoded fluorescent sensor for in vivo imaging of GABA.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Jul 15}, pages = {763-770}, abstract = {

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0471-2}, author = {Marvin, Jonathan S and Shimoda, Yoshiteru and Magloire, Vincent and Leite, Marco and Kawashima, Takashi and Jensen, Thomas P and Kolb, Ilya and Knott, Erika L and Novak, Ondrej and Podgorski, Kaspar and Leidenheimer, Nancy J and Rusakov, Dmitri A and Ahrens, Misha B and Kullmann, Dimitri M and Looger, Loren L} } @article {65204, title = {High-yield, automated intracellular electrophysiology in retinal pigment epithelia.}, journal = {Journal of Neuroscience Methods}, volume = {328}, year = {2019}, month = {2019 Dec 01}, pages = {108442}, abstract = {

BACKGROUND: Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput.

NEW METHOD: A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques.

RESULTS: The automated algorithm improves yield per attempt from 17\% (manually, n = 23) to 22\% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 μm and electroporating (buzzing) just prior to this depth (5 μm) maximized yield.

COMPARISON WITH EXISTING METHOD: Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods.

CONCLUSIONS: This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.

}, issn = {1872-678X}, doi = {10.1016/j.jneumeth.2019.108442}, author = {Lewallen, Colby F and Wan, Qin and Maminishkis, Arvydas and Stoy, William and Kolb, Ilya and Hotaling, Nathan and Bharti, Kapil and Forest, Craig R} }