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      Hyperpolarization by activation of halorhodopsin results in enhanced synaptic transmission: Neuromuscular junction and CNS circuit

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          Abstract

          Optogenetics offers a unique method to regulate the activity of select neural circuits. However, the electrophysiological consequences of targeted optogenetic manipulation upon the entire circuit remain poorly understood. Analysis of the sensory-CNS-motor circuit in Drosophila larvae expressing eHpHR and ChR2-XXL revealed unexpected patterns of excitability. Optical stimulation of motor neurons targeted to express eNpHR resulted in inhibition followed by excitation of body wall contraction with repetitive stimulation in intact larvae. In situ preparations with direct electrophysiological measures showed an increased responsiveness to excitatory synaptic activity induced by sensory stimulation within a functional neural circuit. To ensure proper function of eNpHR and ChR2-XXL they were expressed in body wall muscle and direct electrophysiological measurements were obtained. Under eNpHR induced hyperpolarization the muscle remained excitable with increased amplitude of excitatory postsynaptic synaptic potentials. Theoretical models to explain the observations are presented. This study aids in increasing the understanding of the varied possible influences with light activated proteins within intact neural circuits.

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          Most cited references67

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          Ultrafast optogenetic control.

          Channelrhodopsins such as channelrhodopsin-2 (ChR2) can drive spiking with millisecond precision in a wide variety of cells, tissues and animal species. However, several properties of this protein have limited the precision of optogenetic control. First, when ChR2 is expressed at high levels, extra spikes (for example, doublets) can occur in response to a single light pulse, with potential implications as doublets may be important for neural coding. Second, many cells cannot follow ChR2-driven spiking above the gamma (approximately 40 Hz) range in sustained trains, preventing temporally stationary optogenetic access to a broad and important neural signaling band. Finally, rapid optically driven spike trains can result in plateau potentials of 10 mV or more, causing incidental upstates with information-processing implications. We designed and validated an engineered opsin gene (ChETA) that addresses all of these limitations (profoundly reducing extra spikes, eliminating plateau potentials and allowing temporally stationary, sustained spike trains up to at least 200 Hz).
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            Drosophila, the golden bug, emerges as a tool for human genetics.

            Ethan Bier (2004)
            Drosophila melanogaster is emerging as one of the most effective tools for analyzing the function of human disease genes, including those responsible for developmental and neurological disorders, cancer, cardiovascular disease, metabolic and storage diseases, and genes required for the function of the visual, auditory and immune systems. Flies have several experimental advantages, including their rapid life cycle and the large numbers of individuals that can be generated, which make them ideal for sophisticated genetic screens, and in future should aid the analysis of complex multigenic disorders. The general principles by which D. melanogaster can be used to understand human disease, together with several specific examples, are considered in this review.
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              Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution

              The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: SupervisionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 July 2018
                2018
                : 13
                : 7
                : e0200107
                Affiliations
                [1 ] Department of Biology and Center for Muscle Biology, University of Kentucky, Lexington, Kentucky, United States of America
                [2 ] Goethe University Frankfurt am Main, Frankfurt, Germany
                EPFL, SWITZERLAND
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-7234-4361
                Article
                PONE-D-18-00342
                10.1371/journal.pone.0200107
                6029800
                29969493
                6b772993-f5ba-4172-a584-a1b73d405b1e
                © 2018 Mattingly et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 January 2018
                : 19 June 2018
                Page count
                Figures: 6, Tables: 0, Pages: 20
                Funding
                Funded by: Deutscher Akademischer Austausch Dienst (DAAD) German Academic Exchange Service, RISE - Program (Research Internships in Science and Engineering)
                Award Recipient :
                Funded by: Kentucky Science and Engineering Foundation (KSEF-3712-RDE-019) at the Kentucky Science and Technology Corporation
                Award Recipient :
                KW had a summer fellowship from DADD: German Academic Exchange Service, RISE - Program (Research Internships in Science and Engineering) to work in the USA on this project. RLC received funding from the Kentucky Science and Engineering Foundation (KSEF-3712-RDE-019) at the Kentucky Science and Technology Corporation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Neurons
                Motor Neurons
                Biology and Life Sciences
                Neuroscience
                Cellular Neuroscience
                Neurons
                Motor Neurons
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Larvae
                Physical Sciences
                Physics
                Electromagnetic Radiation
                Light
                Light Pulses
                Research and Analysis Methods
                Bioassays and Physiological Analysis
                Electrophysiological Techniques
                Muscle Electrophysiology
                Biology and Life Sciences
                Anatomy
                Nervous System
                Neuroanatomy
                Neural Pathways
                Medicine and Health Sciences
                Anatomy
                Nervous System
                Neuroanatomy
                Neural Pathways
                Biology and Life Sciences
                Neuroscience
                Neuroanatomy
                Neural Pathways
                Biology and Life Sciences
                Physiology
                Electrophysiology
                Membrane Potential
                Medicine and Health Sciences
                Physiology
                Electrophysiology
                Membrane Potential
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Neurons
                Nerve Fibers
                Biology and Life Sciences
                Neuroscience
                Cellular Neuroscience
                Neurons
                Nerve Fibers
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Muscle Fibers
                Biology and Life Sciences
                Anatomy
                Musculoskeletal System
                Muscles
                Muscle Fibers
                Medicine and Health Sciences
                Anatomy
                Musculoskeletal System
                Muscles
                Muscle Fibers
                Custom metadata
                Data for this study are from the following previous study: Cooper, Robin L. (2018, April 30). Hyperpolarization by activation of halorhodopsin results in enhanced synaptic transmission: Neuromuscular junction and CNS circuit. All relevant data are available on the Open Science Framework repository at the following: osf.io/esx2v.

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