Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Quasar1
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UNIVERSITY of CALIFORNIA, SAN DIEGO The
UNIVERSITY OF CALIFORNIA, SAN DIEGO The Transporter-Opsin-G protein-coupled receptor (TOG) Superfamily A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Daniel Choi Yee Committee in charge: Professor Milton H. Saier Jr., Chair Professor Yunde Zhao Professor Lin Chao 2014 The Thesis of Daniel Yee is approved and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Chair University of California, San Diego 2014 iii DEDICATION This thesis is dedicated to my parents, my family, and my mentor, Dr. Saier. It is only with their help and perseverance that I have been able to complete it. iv TABLE OF CONTENTS Signature Page ............................................................................................................... iii Dedication ...................................................................................................................... iv Table of Contents ........................................................................................................... v List of Abbreviations ..................................................................................................... vi List of Supplemental Files ............................................................................................ vii List of -
Optical Inhibition of Larval Zebrafish Behaviour with Anion
Mohamed et al. BMC Biology (2017) 15:103 DOI 10.1186/s12915-017-0430-2 METHODOLOGY ARTICLE Open Access Optical inhibition of larval zebrafish behaviour with anion channelrhodopsins Gadisti Aisha Mohamed1, Ruey-Kuang Cheng1, Joses Ho2, Seetha Krishnan3, Farhan Mohammad4, Adam Claridge-Chang2,4 and Suresh Jesuthasan1,2,4* Abstract Background: Optical silencing of activity provides a way to test the necessity of neurons in behaviour. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown to potently inhibit activity in cultured mammalian neurons and in Drosophila. Here, we test the usefulness of these channels in larval zebrafish, using spontaneous coiling behaviour as the assay. Results: When the GtACRs were expressed in spinal neurons of embryonic zebrafish and actuated with blue or green light, spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 μW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required slightly stronger illumination. No inhibition was seen in non-expressing siblings. After light offset, the movement of GtACR-expressing fish increased, which suggested that termination of light- induced neural inhibition may lead to activation. Consistent with this, two-photon imaging of spinal neurons showed that blue light inhibited spontaneous activity in spinal neurons of GtACR1-expressing fish, and that the level of intracellular calcium increased following light offset. Conclusions: These results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in larval zebrafish with high efficiency. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property can provide insight into the effects of transiently stimulating a circuit. -
Imaging Thoughts - an Investigation of Neural Circuits Encoding Changes in Behavior in Zebrafish By
Imaging thoughts - an investigation of neural circuits encoding changes in behavior in zebrafish by Claire Oldfield A dissertation submitted in partial satisfaction of the requirement for the degree of Doctor of Philosophy in Neuroscience In the Graduate Division of the University of California, Berkeley Committee in charge: Professor Ehud Y. Isacoff, Chair Professor Dan E. Feldman Professor Marla Feller Professor Craig Miller Summer 2016 Abstract Imaging thoughts - an investigation of neural circuits encoding changes in behavior in zebrafish By Claire Oldfield Doctor of Philosophy in Neuroscience University of California, Berkeley Professor Ehud Isacoff, Chair Experience influences how we perceive the world, how we interact with our environment, and how we develop. In fact, almost all animals can modify their behavior as a result of experience. Psychologists have long distinguished between different forms of learning and memory, and later determined that they are encoded in distinct brain areas. Neuroscientists dating back to Santiago Ramón y Cajal suggested that learning and memory might be encoded as changes in synaptic connections between neurons, but it wasn’t until the second half of the 20th century that experimental evidence corroborated this idea. Specific firing patterns of neurons during learning results in strengthening or weakening of synapses, and morphological modifications that can lead to long lasting changes in neural circuits. The molecular mechanisms that drive these changes are remarkably conserved across vertebrates. However, understanding how synaptic plasticity is integrated at a network level remains a big challenge in neuroscience. Relatively few studies have focused on how neural circuits encode changes in behavior in a natural context. -
A Voltage-Dependent Fluorescent Indicator for Optogenetic Applications, Archaerhodopsin-3: Structure and Optical Properties
F1000Research 2017, 6:33 Last updated: 30 JUL 2021 RESEARCH NOTE A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling [version 3; peer review: 3 approved] Dmitrii M. Nikolaev1, Anton Emelyanov1, Vitaly M. Boitsov1, Maxim S Panov2, Mikhail N. Ryazantsev 2,3 1Saint-Petersburg National Research Academic University of the Russian Academy of Science, St. Petersburg, Russian Federation 2Saint-Petersburg State University, St. Petersburg, Russian Federation 3Saint-Petersburg Scientific Center of the Russian Academy of Sciences, St. Petersburg, Russian Federation v3 First published: 11 Jan 2017, 6:33 Open Peer Review https://doi.org/10.12688/f1000research.10541.1 Second version: 17 Jan 2017, 6:33 https://doi.org/10.12688/f1000research.10541.2 Reviewer Status Latest published: 15 Nov 2017, 6:33 https://doi.org/10.12688/f1000research.10541.3 Invited Reviewers 1 2 3 Abstract It was demonstrated in recent studies that some rhodopsins can be version 3 used in optogenetics as fluorescent indicators of membrane voltage. (revision) report report One of the promising candidates for these applications is 15 Nov 2017 archaerhodopsin-3. While it has already shown encouraging results, there is still a large room for improvement. One of possible directions version 2 is increasing the intensity of the protein's fluorescent signal. Rational (revision) report report report design of mutants with an improved signal is an important task, which 17 Jan 2017 requires both experimental and theoretical studies. Herein, we used a homology-based computational approach to predict the three- version 1 dimensional structure of archaerhodopsin-3, and a Quantum 11 Jan 2017 Mechanics/Molecular Mechanics (QM/MM) hybrid approach with high- level multireference ab initio methodology (SORCI+Q/AMBER) to 1. -
Optogenetic Neuronal Silencing in Drosophila During Visual Processing Alex S
www.nature.com/scientificreports OPEN Optogenetic Neuronal Silencing in Drosophila during Visual Processing Alex S. Mauss , Christian Busch & Alexander Borst Optogenetic channels and ion pumps have become indispensable tools in neuroscience to manipulate Received: 26 April 2017 neuronal activity and thus to establish synaptic connectivity and behavioral causality. Inhibitory Accepted: 6 October 2017 channels are particularly advantageous to explore signal processing in neural circuits since they permit Published: xx xx xxxx the functional removal of selected neurons on a trial-by-trial basis. However, applying these tools to study the visual system poses a considerable challenge because the illumination required for their activation usually also stimulates photoreceptors substantially, precluding the simultaneous probing of visual responses. Here, we explore the utility of the recently discovered anion channelrhodopsins GtACR1 and GtACR2 for application in the visual system of Drosophila. We frst characterized their properties using a larval crawling assay. We further obtained whole-cell recordings from cells expressing GtACR1, which mediated strong and light-sensitive photocurrents. Finally, using physiological recordings and a behavioral readout, we demonstrate that GtACR1 enables the fast and reversible silencing of genetically targeted neurons within circuits engaged in visual processing. Genetically expressed optogenetic ion channels and pumps confer light sensitivity to neurons of interest, allow- ing to control their activity on demand1,2. Such techniques have become powerful means to establish neuronal connectivity as well as causal relationships between neuronal activity and behavior. Remote control of neuronal activity by light has many advantages: it is fast, reversible, easy to parameterize and applicable in intact behaving animals. However, it poses challenges for studies in visual systems, since here endogenous light-sensing cells, the photoreceptors, are also activated by light required for optogenetic control. -
A Striatal Interneuron Circuit for Continuous Target Pursuit
ARTICLE https://doi.org/10.1038/s41467-019-10716-w OPEN A striatal interneuron circuit for continuous target pursuit Namsoo Kim1, Haofang E. Li1, Ryan N. Hughes1, Glenn D.R. Watson1, David Gallegos2, Anne E. West 2, Il Hwan Kim3 & Henry H. Yin1,2 Most adaptive behaviors require precise tracking of targets in space. In pursuit behavior with a moving target, mice use distance to target to guide their own movement continuously. 1234567890():,; Here, we show that in the sensorimotor striatum, parvalbumin-positive fast-spiking inter- neurons (FSIs) can represent the distance between self and target during pursuit behavior, while striatal projection neurons (SPNs), which receive FSI projections, can represent self- velocity. FSIs are shown to regulate velocity-related SPN activity during pursuit, so that movement velocity is continuously modulated by distance to target. Moreover, bidirectional manipulation of FSI activity can selectively disrupt performance by increasing or decreasing the self-target distance. Our results reveal a key role of the FSI-SPN interneuron circuit in pursuit behavior and elucidate how this circuit implements distance to velocity transforma- tion required for the critical underlying computation. 1 Department of Psychology and Neuroscience, Duke University, Durham, NC 27708, USA. 2 Department of Neurobiology, Duke University, Durham, NC 27708, USA. 3 Department of Anatomy and Neurobiology, University of Tennessee Health and Science Center, Memphis, TN 27708, USA. Correspondence and requests for materials should be addressed to H.H.Y. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2715 | https://doi.org/10.1038/s41467-019-10716-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10716-w hether pursuing a prey or approaching a mate, natural pursuit performance: the worse the pursuit performance, the Wbehaviors often involve continuous tracking of targets more self-velocity lags self-target distance, as expected if distance in space. -
Wide. Fast. Deep. Recent Advances in Multi-Photon Microscopy of in Vivo Neuronal Activity
TechSights Wide. Fast. Deep. Recent Advances in Multi- Photon Microscopy of in vivo Neuronal Activity https://doi.org/10.1523/JNEUROSCI.1527-18.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1527-18.2019 Received: 2 March 2019 Revised: 27 September 2019 Accepted: 27 September 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Wide. Fast. Deep. Recent Advances in Multi-Photon Microscopy of in vivo Neuronal Activity. 2 Abbreviated title: Recent Advances of in vivo Multi-Photon Microscopy 3 Jérôme Lecoq1, Natalia Orlova1, Benjamin F. Grewe2,3,4 4 1 Allen Institute for Brain Science, Seattle, USA 5 2 Institute of Neuroinformatics, UZH and ETH Zurich, Switzerland 6 3 Dept. of Electrical Engineering and Information Technology, ETH Zurich, Switzerland 7 4 Faculty of Sciences, University of Zurich, Switzerland 8 9 Corresponding author: Jérôme Lecoq, [email protected] 10 Number of pages: 24 11 Number of figures: 6 12 Number of tables: 1 13 Number of words for: 14 ● abstract: 196 15 ● introduction: 474 16 ● main text: 5066 17 Conflict of interest statement: The authors declare no competing financial interests. 18 Acknowledgments: We thank Kevin Takasaki and Peter Saggau (Allen Institute for Brain Science) for providing helpful 19 comments on the manuscript; we thank Bénédicte Rossi for providing scientific illustrations. -
Han-Optogenetic-Review-ACS-2012.Pdf
Review pubs.acs.org/chemneuro In Vivo Application of Optogenetics for Neural Circuit Analysis Xue Han Biomedical Engineering Department, Boston University, Boston, Massachusetts, United States ABSTRACT: Optogenetics combines optical and genetic methods to rapidly and reversibly control neural activities or other cellular functions. Using genetic methods, specific cells or anatomical pathways can be sensitized to light through exogenous expression of microbial light activated opsin proteins. Using optical methods, opsin expressing cells can be rapidly and reversibly controlled by pulses of light of specific wavelength. With the high spatial temporal precision, optogenetic tools have enabled new ways to probe the causal role of specific cells in neural computation and behavior. Here, we overview the current state of the technology, and provide a brief introduction to the practical considerations in applying optogenetics in vivo to analyze neural circuit functions. KEYWORDS: Channelrhodopsin, archaerhodopsin, halorhodopsin, cell type specificity ptogenetics is a new field being rapidly established upon algae), and others that mediate a variety of cellular functions O the first demonstration of precise activation of neurons (for reviews on opsin structure and function, see refs 9 and 10). expressing a light-activated microbial opsin, channelrhodopsin- Most sensory rhodopsins function through recruiting intra- 2, with pulses of blue light in 2005.1 Microbial (type I) opsins cellular signaling molecules without direct ion transport are classes of monolithic light activated proteins, encoded by function. However, channelrhodopsins can mediate phototaxis small genes of under a kilobase long. Three major classes of as a light-gated cation channel at high light intensity and as a microbial opsins have been adapted to optogenetically control calcium channel at low light intensity.11 cellular functions, channelrhodopsins, halorhodopsins, and Microbial opsins share sequence homology and are archaerhodopsins (Figure 1). -
Optical Inhibition of Zebrafish Behavior with Anion Channelrhodopsins
bioRxiv preprint doi: https://doi.org/10.1101/158899; this version posted July 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Optical inhibition of zebrafish behavior with anion channelrhodopsins Gadisti Aisha Mohamed1, Ruey-Kuang Cheng1, Joses Ho2, Seetha Krishnan3, Farhan Mohammad4, Adam Claridge-Chang2, 4 and Suresh Jesuthasan1, 2 1. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore. 2. Institute of Molecular and Cell Biology, Singapore. 3. Graduate School for Integrative Sciences and Engineering, National University of Singapore. 4. Duke-NUS Medical School, Singapore Abstract In behavioral analysis, optical electrical silencing provides a way to test neu- ronal necessity. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown—in neuronal culture and in Drosophila—to inhibit neu- rons potently. Here, we test the usefulness of these channels in zebrafish. When the GtACRs were expressed in motor neurons and actuated with blue or green light, fish spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 µW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required stronger illumination. After light offset, GtACR- expressing fish movement increased; this suggested that termination of light- induced neural inhibition may lead to depolarization. Consistent with this, two-photon imaging of spinal neurons showed that intracellular calcium also increased following light offset. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property may help provide insight into the effects of stimulating a circuit transiently. -
High-Performance Genetically Targetable Optical Neural Silencing by Light-Driven Proton Pumps
Vol 463 | 7 January 2010 | doi:10.1038/nature08652 LETTERS High-performance genetically targetable optical neural silencing by light-driven proton pumps Brian Y. Chow1,2*, Xue Han1,2*, Allison S. Dobry1,2, Xiaofeng Qian1,2, Amy S. Chuong1,2, Mingjie Li1,2, Michael A. Henninger1,2, Gabriel M. Belfort2, Yingxi Lin2, Patrick E. Monahan1,2 & Edward S. Boyden1,2 The ability to silence the activity of genetically specified neurons in screen, as did two other proton pumps, the Leptosphaeria maculans a temporally precise fashion would provide the opportunity to opsin (Mac/LR/Ops)4 and cruxrhodopsin-1 (ref. 10) (albeit less than investigate the causal role of specific cell classes in neural compu- that of Arch; Fig. 1a). All light-driven chloride pumps assessed had tations, behaviours and pathologies. Here we show that members lower screen photocurrents than these light-driven proton pumps. of the class of light-driven outward proton pumps can mediate Arch is a yellow–green light-sensitive (Fig. 1b) opsin that seems to powerful, safe, multiple-colour silencing of neural activity. The express well on the neural plasma membrane (Fig. 1c; see Sup- gene archaerhodopsin-3 (Arch)1 from Halorubrum sodomense plementary Notes on Arch expression levels and enhancing Arch enables near-100% silencing of neurons in the awake brain when membrane trafficking). Arch-mediated currents exhibited excellent virally expressed in the mouse cortex and illuminated with yellow kinetics of light-activation and post-light recovery. After illumination, light. Arch mediates currents of several hundred picoamps at low Arch currents rose with a 15–85% onset time of 8.8 6 1.8 ms light powers, and supports neural silencing currents approaching (mean 6 standard error (s.e.) reported throughout, unless otherwise 900 pA at light powers easily achievable in vivo. -
Low-Cost Calcium Fluorometry for Long-Term Nanoparticle Studies In
www.nature.com/scientificreports OPEN Low‑cost calcium fuorometry for long‑term nanoparticle studies in living cells Connor L. Beck1, Clark J. Hickman1,2 & Anja Kunze 1* Calcium fuorometry is critical to determine cell homeostasis or to reveal communication patterns in neuronal networks. Recently, characterizing calcium signalling in neurons related to interactions with nanomaterials has become of interest due to its therapeutic potential. However, imaging of neuronal cell activity under stable physiological conditions can be either very expensive or limited in its long‑term capability. Here, we present a low‑cost, portable imaging system for long‑term, fast‑ scale calcium fuorometry in neurons. Using the imaging system, we revealed temperature‑dependent changes in long‑term calcium signalling in kidney cells and primary cortical neurons. Furthermore, we introduce fast‑scale monitoring of synchronous calcium activity in neuronal cultures in response to nanomaterials. Through graph network analysis, we found that calcium dynamics in neurons are temperature‑dependent when exposed to chitosan‑coated nanoparticles. These results give new insights into nanomaterial‑interaction in living cultures and tissues based on calcium fuorometry and graph network analysis. Imaging calcium dynamics in and between neurons is essential to analyse neural signalling and to better under- stand how drugs, metabolites, and neural treatments impact the plasticity of signalling in neural networks. Commercially available techniques to record calcium activity rely on the induction of a calcium-dependent fuorescent sensor and fuorescent-based high-resolution microscopy for both in vivo and in vitro applications 1. High-resolution optical microscopy, e.g., two-photon or confocal microscopy, has revealed valuable knowledge about subcellular calcium signalling2. -
Xenorhodopsins, an Enigmatic New Class of Microbial Rhodopsins Horizontally Transferred Between Archaea and Bacteria
UC San Diego UC San Diego Previously Published Works Title Xenorhodopsins, an enigmatic new class of microbial rhodopsins horizontally transferred between Archaea and Bacteria Permalink https://escholarship.org/uc/item/8rg5f54b Journal Biology Direct, 6(1) ISSN 1745-6150 Authors Ugalde, Juan A Podell, Sheila Narasingarao, Priya et al. Publication Date 2011-10-10 DOI http://dx.doi.org/10.1186/1745-6150-6-52 Supplemental Material https://escholarship.org/uc/item/8rg5f54b#supplemental Peer reviewed eScholarship.org Powered by the California Digital Library University of California Ugalde et al. Biology Direct 2011, 6:52 http://www.biology-direct.com/content/6/1/52 DISCOVERYNOTES Open Access Xenorhodopsins, an enigmatic new class of microbial rhodopsins horizontally transferred between archaea and bacteria Juan A Ugalde1, Sheila Podell1, Priya Narasingarao1 and Eric E Allen1,2* Abstract Based on unique, coherent properties of phylogenetic analysis, key amino acid substitutions and structural modeling, we have identified a new class of unusual microbial rhodopsins related to the Anabaena sensory rhodopsin (ASR) protein, including multiple homologs not previously recognized. We propose the name xenorhodopsin for this class, reflecting a taxonomically diverse membership spanning five different Bacterial phyla as well as the Euryarchaeotal class Nanohaloarchaea. The patchy phylogenetic distribution of xenorhodopsin homologs is consistent with historical dissemination through horizontal gene transfer. Shared characteristics of xenorhodopsin-containing microbes include the absence of flagellar motility and isolation from high light habitats. Reviewers: This article was reviewed by Dr. Michael Galperin and Dr. Rob Knight. Findings disseminated photoreceptor and photosensory activities Microbial rhodopsins are a widespread family of photo- across large evolutionary distances [1].