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      The €100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans

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          Abstract

          Small, genetically tractable species such as larval zebrafish, Drosophila, or Caenorhabditis elegans have become key model organisms in modern neuroscience. In addition to their low maintenance costs and easy sharing of strains across labs, one key appeal is the possibility to monitor single or groups of animals in a behavioural arena while controlling the activity of select neurons using optogenetic or thermogenetic tools. However, the purchase of a commercial solution for these types of experiments, including an appropriate camera system as well as a controlled behavioural arena, can be costly. Here, we present a low-cost and modular open-source alternative called ‘FlyPi’. Our design is based on a 3D-printed mainframe, a Raspberry Pi computer, and high-definition camera system as well as Arduino-based optical and thermal control circuits. Depending on the configuration, FlyPi can be assembled for well under €100 and features optional modules for light-emitting diode (LED)-based fluorescence microscopy and optogenetic stimulation as well as a Peltier-based temperature stimulator for thermogenetics. The complete version with all modules costs approximately €200 or substantially less if the user is prepared to ‘shop around’. All functions of FlyPi can be controlled through a custom-written graphical user interface. To demonstrate FlyPi’s capabilities, we present its use in a series of state-of-the-art neurogenetics experiments. In addition, we demonstrate FlyPi’s utility as a medical diagnostic tool as well as a teaching aid at Neurogenetics courses held at several African universities. Taken together, the low cost and modular nature as well as fully open design of FlyPi make it a highly versatile tool in a range of applications, including the classroom, diagnostic centres, and research labs.

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            Optimization of a GCaMP calcium indicator for neural activity imaging.

            Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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              An optimized fluorescent probe for visualizing glutamate neurotransmission

              We describe an intensity-based glutamate-sensing fluorescent reporter (“iGluSnFR”) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted post-synaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.
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                Author and article information

                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                18 July 2017
                July 2017
                18 July 2017
                : 15
                : 7
                : e2002702
                Affiliations
                [1 ] Werner Reichardt Centre for Integrative Neuroscience, University of Tübingen, Tübingen, Germany
                [2 ] Graduate school for Neural and Behavioural Neuroscience, University of Tübingen, Tübingen, Germany
                [3 ] TReND in Africa gUG, Bonn, Germany
                [4 ] Institute of Ophthalmic Research, University of Tübingen, Tübingen, Germany
                [5 ] Center of Integrative Genomics, University of Lausanne, Lausanne, Switzerland
                [6 ] Institute of Neurobiology, University of Tübingen, Tübingen, Germany
                [7 ] School of Life Sciences, University of Sussex, Brighton, United Kingdom
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-2808-4210
                Article
                pbio.2002702
                10.1371/journal.pbio.2002702
                5515398
                28719603
                6a0f08db-64d7-44a8-a5e0-580a994dce33
                © 2017 Maia Chagas 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
                Page count
                Figures: 6, Tables: 0, Pages: 21
                Funding
                H2020 ERC StG (grant number 677687). Support for TB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Baden Wuerttemberg Stiftung (grant number AZ 1.16101.09). Support for TB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The intramural fortüne program of the University of Tübingen (grant number 2125-0-0). Support for TB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Institute of Neurological Disorders and Stroke (grant number U01NS090562). Support for AMC. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number BA 5283/1-1). Support for TB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CIN-Werner Reichardt Centre for Integrative Neuroscience (grant number EXC307). DFG funding to ABA. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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