@article {69840, title = {EPSILON: a method for pulse-chase labeling to probe synaptic AMPAR exocytosis during memory formation.}, journal = {Nat Neurosci}, year = {2025}, month = {2025 Mar 31}, abstract = {

A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms of learning and memory. Here we developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) exocytosis in vivo by sequential pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach yields synaptic-resolution maps of AMPAR exocytosis, a proxy for synaptic potentiation, in genetically targeted neurons during memory formation. In mice undergoing contextual fear conditioning, we investigated the relationship between synapse-level AMPAR exocytosis in CA1 pyramidal neurons and cell-level expression of the immediate early gene product cFos, a frequently used marker of engram neurons. We observed a strong correlation between AMPAR exocytosis and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins.

}, issn = {1546-1726}, doi = {10.1038/s41593-025-01922-5}, url = {https://doi.org/10.1038/s41593-025-01922-5}, author = {Kim, Doyeon and Park, Pojeong and Li, Xiuyuan and Wong-Campos, J David and Tian, He and Moult, Eric M and Grimm, Jonathan B and Lavis, Luke D and Cohen, Adam E} } @article {69846, title = {Far-red fluorescent genetically encoded calcium ion indicators.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Apr 07}, pages = {3318}, abstract = {

Genetically encoded calcium ion (Ca) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm, respectively, display large responses to Ca in vitro (ΔF/F = 6 for FR-GECO1a, 18 for FR-GECO1c), are bright under both one-photon and two-photon illumination, and have high affinities (apparent K = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca. FR-GECOs offer sensitive and fast detection of single action potentials in neurons, and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators.

Preprint:\ https://doi.org/10.1101/2020.11.12.380089

}, keywords = {Action Potentials, Animals, Calcium, Fluorescent Dyes, HEK293 Cells, Humans, Luminescent Proteins, Mice, Neurons, Optogenetics, Red Fluorescent Protein}, issn = {2041-1723}, doi = {10.1038/s41467-025-58485-z}, url = {https://doi.org/10.1038/s41467-025-58485-z}, author = {Dalangin, Rochelin and Jia, Bill Z and Qi, Yitong and Aggarwal, Abhi and Sakoi, Kenryo and Drobizhev, Mikhail and Molina, Rosana S and Patel, Ronak and Abdelfattah, Ahmed S and Zheng, Jihong and Reep, Daniel and Hasseman, Jeremy P and Zhao, Yufeng and Wu, Jiahui and Podgorski, Kaspar and Tebo, Alison G and Schreiter, Eric R and Hughes, Thomas E and Terai, Takuya and Paquet, Marie-Eve and Megason, Sean G and Cohen, Adam E and Shen, Yi and Campbell, Robert E} } @article {69703, title = {Ketamine induces plasticity in a norepinephrine-astroglial circuit to promote behavioral perseverance.}, journal = {Neuron}, year = {2024}, month = {2024 Dec 16}, pages = {1-15}, abstract = {

Transient exposure to ketamine can trigger lasting changes in behavior and mood. We found that brief ketamine exposure causes long-term suppression of futility-induced passivity in larval zebrafish, reversing the "giving-up" response that normally occurs when swimming fails to cause forward movement. Whole-brain imaging revealed that ketamine hyperactivates the norepinephrine-astroglia circuit responsible for passivity. After ketamine washout, this circuit exhibits hyposensitivity to futility, leading to long-term increased perseverance. Pharmacological, chemogenetic, and optogenetic manipulations show that norepinephrine and astrocytes are necessary and sufficient for ketamine{\textquoteright}s long-term perseverance-enhancing aftereffects. In\ vivo calcium imaging revealed that astrocytes in adult mouse cortex are similarly activated during futility in the tail suspension test and that acute ketamine exposure also induces astrocyte hyperactivation. The cross-species conservation of ketamine{\textquoteright}s modulation of noradrenergic-astroglial circuits and evidence that plasticity in this pathway can alter the behavioral response to futility hold promise for identifying new strategies to treat affective disorders.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2024.11.011}, url = {https://doi.org/10.1016/j.neuron.2024.11.011}, author = {Duque, Marc and Chen, Alex B and Hsu, Eric and Narayan, Sujatha and Rymbek, Altyn and Begum, Shahinoor and Saher, Gesine and Cohen, Adam E and Olson, David E and Li, Yulong and Prober, David A and Bergles, Dwight E and Fishman, Mark C and Engert, Florian and Ahrens, Misha B} } @article {68561, title = {Time-tagged ticker tapes for intracellular recordings.}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {2023 May 01}, pages = {631-9}, abstract = {

Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01524-7}, author = {Lin, Dingchang and Li, Xiuyuan and Moult, Eric and Park, Pojeong and Tang, Benjamin and Shen, Hao and Grimm, Jonathan B and Falco, Natalie and Jia, Bill Z and Baker, David and Lavis, Luke D and Cohen, Adam E} } @article {67890, title = {Voltage imaging identifies spinal circuits that modulate locomotor adaptation in zebrafish.}, journal = {Neuron}, year = {2022}, month = {2022 Jan 24}, abstract = {

Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.01.001}, author = {B{\"o}hm, Urs L and Kimura, Yukiko and Kawashima, Takashi and Ahrens, Misha B and Higashijima, Shin-Ichi and Engert, Florian and Cohen, Adam E} } @article {67991, title = {Voltage imaging identifies spinal circuits that modulate locomotor adaptation in zebrafish.}, journal = {Neuron}, volume = {110}, year = {2022}, month = {2022 Apr 06}, pages = {1211-1222.e4}, abstract = {

Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.01.001}, author = {B{\"o}hm, Urs L and Kimura, Yukiko and Kawashima, Takashi and Ahrens, Misha B and Higashijima, Shin-Ichi and Engert, Florian and Cohen, Adam E} } @article {67211, title = {High-fidelity estimates of spikes and subthreshold waveforms from 1-photon voltage imaging in~vivo.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Apr 06}, pages = {108954}, abstract = {

The ability to probe the membrane potential of multiple genetically defined neurons simultaneously would have a profound impact on neuroscience research. Genetically encoded voltage indicators are a promising tool for this purpose, and recent developments have achieved a high signal-to-noise ratio in\ vivo with 1-photon fluorescence imaging. However, these recordings exhibit several sources of noise and signal extraction remains a challenge. We present an improved signal extraction pipeline, spike-guided penalized matrix decomposition-nonnegative matrix factorization (SGPMD-NMF), which resolves supra- and subthreshold voltages in\ vivo. The method incorporates biophysical and optical constraints. We validate the pipeline with simultaneous patch-clamp and optical recordings from mouse layer 1 in\ vivo and with simulated and composite datasets with realistic noise. We demonstrate applications to mouse hippocampus expressing paQuasAr3-s or SomArchon1, mouse cortex expressing SomArchon1 or Voltron, and zebrafish spines expressing zArchon1.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.108954}, author = {Xie, Michael E and Adam, Yoav and Fan, Linlin Z and B{\"o}hm, Urs L and Kinsella, Ian and Zhou, Ding and Rozsa, Marton and Singh, Amrita and Svoboda, Karel and Paninski, Liam and Cohen, Adam E} } @article {47144, title = {A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.}, journal = {PloS one}, volume = {10}, year = {2015}, month = {2015 Oct 09}, pages = {e0139273}, abstract = {

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0139273}, author = {Henderson, Mark J and Baldwin, Heather A and Werley, Christopher A and Boccardo, Stefano and Whitaker, Leslie R and Yan, Xiaokang and Holt, Graham T and Schreiter, Eric R and Looger, Loren L and Cohen, Adam E and Kim, Douglas S and Harvey, Brandon K} }