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2041 Janelia Publications

Showing 1-10 of 2041 results
01/15/22 | Fluorescent chemigenetic actuators and indicators for use in living animals.
Farrants H, Tebo AG
Current Opinion in Pharmacology. 2022 Jan 15;62:159-167. doi: 10.1016/j.coph.2021.12.007

Fluorescent indicators and actuators provide a means to optically observe and perturb dynamic events in living animals. Although chemistry and protein engineering have contributed many useful tools to observe and perturb cells, an emerging strategy is to use chemigenetics: systems in which a small molecule dye interacts with a genetically encoded protein domain. Here we review chemigenetic strategies that have been successfully employed in living animals as photosensitizers for photoablation experiments, fluorescent cell cycle indicators, and fluorescent indicators for studying dynamic biological signals. Although these strategies at times suffer from challenges, e.g. delivery of the small molecule and assembly of the chemigenetic unit in living animals, the advantages of using small molecules with high brightness, low photobleaching, no chromophore maturation time and expanded color palette, combined with the ability to genetically target them to specific cell types, make chemigenetic fluorescent actuators and indicators an attractive strategy for use in living animals.

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01/12/22 | Toroidal topology of population activity in grid cells.
Gardner RJ, Hermansen E, Pachitariu M, Burak Y, Baas NA, Dunn BA, Moser M, Moser EI
Nature. 2022 Jan 12:. doi: 10.1038/s41586-021-04268-7

The medial entorhinal cortex is part of a neural system for mapping the position of an individual within a physical environment. Grid cells, a key component of this system, fire in a characteristic hexagonal pattern of locations, and are organized in modules that collectively form a population code for the animal's allocentric position. The invariance of the correlation structure of this population code across environments and behavioural states, independent of specific sensory inputs, has pointed to intrinsic, recurrently connected continuous attractor networks (CANs) as a possible substrate of the grid pattern. However, whether grid cell networks show continuous attractor dynamics, and how they interface with inputs from the environment, has remained unclear owing to the small samples of cells obtained so far. Here, using simultaneous recordings from many hundreds of grid cells and subsequent topological data analysis, we show that the joint activity of grid cells from an individual module resides on a toroidal manifold, as expected in a two-dimensional CAN. Positions on the torus correspond to positions of the moving animal in the environment. Individual cells are preferentially active at singular positions on the torus. Their positions are maintained between environments and from wakefulness to sleep, as predicted by CAN models for grid cells but not by alternative feedforward models. This demonstration of network dynamics on a toroidal manifold provides a population-level visualization of CAN dynamics in grid cells.

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01/10/21 | A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae.
Imambocus BN, Zhou F, Formozov A, Wittich A, Tenedini FM, Hu C, Sauter K, Macarenhas Varela E, Herédia F, Casimiro AP, Macedo A, Schlegel P, Yang C, Miguel-Aliaga I, Wiegert JS, Pankratz MJ, Gontijo AM, Cardona A, Soba P
Current Biology. 2022 Jan 10;32(1):149-63. doi: 10.1016/j.cub.2021.10.069

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

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01/07/22 | Characterization of ultrapotent chemogenetic ligands for research applications in non-human primates
Jessica Raper , Mark A. G. Eldridge , Scott. M. Sternson , Jalene Y. Shim , Grace P. Fomani , Barry J. Richmond , Thomas Wichmann , Adriana Galvan
bioRxiv. 2022 Jan 07:. doi: 10.1101/2022.01.06.475241

Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of “Pharmacologically Selective Actuator Modules” (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent “Pharmacologically Selective Effector Molecules” (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in in vitro studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus macaques received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817(0.064 mg/kg) or uPSEM792 (0.87 mg/kg) and plasma and CSF samples were collected over the course of 48 hours. Both compounds exhibited good brain penetrance, relatively slow washout and negligible conversion to potential metabolites - varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

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01/06/22 | The importance of accounting for movement when relating neuronal activity to sensory and cognitive processes.
Edward Zagha , Jeffrey C Erlich , Soohyun Lee , Gyorgy Lur , Daniel H O'Connor , Nicholas A Steinmetz , Carsen Stringer , Hongdian Yang
Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2022 Jun 06:. doi: 10.1523/JNEUROSCI.1919-21.2021

A surprising finding of recent studies in mouse is the dominance of widespread movement-related activity throughout the brain, including in early sensory areas. In awake subjects, failing to account for movement risks misattributing movement-related activity to other (e.g., sensory or cognitive) processes. In this article, we 1) review task designs for separating task-related and movement-related activity, 2) review three 'case studies' in which not considering movement would have resulted in critically different interpretations of neuronal function, and 3) discuss functional couplings that may prevent us from ever fully isolating sensory, motor, and cognitive-related activity. Our main thesis is that neural signals related to movement are ubiquitous, and therefore ought to be considered first and foremost when attempting to correlate neuronal activity with task-related processes.

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01/05/22 | Rapid synaptic plasticity contributes to a learned conjunctive code of position and choice-related information in the hippocampus.
Zhao X, Hsu C, Spruston N
Neuron. 2022 Jan 05;110(1):96-108.e4. doi: 10.1016/j.neuron.2021.10.003

To successfully perform goal-directed navigation, animals must know where they are and what they are doing-e.g., looking for water, bringing food back to the nest, or escaping from a predator. Hippocampal neurons code for these critical variables conjunctively, but little is known about how this "where/what" code is formed or flexibly routed to other brain regions. To address these questions, we performed intracellular whole-cell recordings in mouse CA1 during a cued, two-choice virtual navigation task. We demonstrate that plateau potentials in CA1 pyramidal neurons rapidly strengthen synaptic inputs carrying conjunctive information about position and choice. Plasticity-induced response fields were modulated by cues only in animals previously trained to collect rewards based on available cues. Thus, we reveal that gradual learning is required for the formation of a conjunctive population code, upstream of CA1, while plateau-potential-induced synaptic plasticity in CA1 enables flexible routing of the code to downstream brain regions.

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01/04/22 | Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.
Nichols AL, Blumenfeld Z, Fan C, Luebbert L, Blom AE, Cohen BN, Marvin JS, Borden PM, Kim CH, Muthusamy AK, Shivange AV, Knox HJ, Campello HR, Wang JH, Dougherty DA, Looger LL, Gallagher T, Rees DC, Lester HA
eLife. 2022 Jan 04;11:. doi: 10.7554/eLife.74648

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

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01/01/22 | ER proteins decipher the tubulin code to regulate organelle distribution.
Zheng P, Obara CJ, Szczesna E, Nixon-Abell J, Mahalingan KK, Roll-Mecak A, Lippincott-Schwartz J, Blackstone C
Nature. 2022 Jan 01;601(7891):132-138. doi: 10.1038/s41586-021-04204-9

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm, forming abundant contacts with other organelles. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

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12/23/21 | Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels.
Grimm JB, Lavis LD
Nature Methods. 2021 Dec 23:. doi: 10.1038/s41592-021-01338-6

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.

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12/23/20 | Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.
Unger EK, Keller JP, Altermatt M, Liang R, Matsui A, Dong C, Hon OJ, Yao Z, Sun J, Banala S, Flanigan ME, Jaffe DA, Hartanto S, Carlen J, Mizuno GO, Borden PM, Shivange AV, Cameron LP, Sinning S, Underhill SM, Olson DE, Amara SG, Temple Lang D, Rudnick G, Marvin JS, Lavis LD, Lester HA, Alvarez VA, Fisher AJ, Prescher JA, Kash TL, Yarov-Yarovoy V, Gradinaru V, Looger LL, Tian L
Cell. 2020 Dec 23;183(7):1986-2002.e26. doi: 10.1016/j.cell.2020.11.040

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

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