JoVE Neuroscience
Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS
1Department of Zoology, University of Oklahoma - Norman, 2Department of Biology, Brandeis University
We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.
JoVE General
Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations
1Department of Biology and Biochemistry, University of Bath, 2Randall Division of Cell and Molecular Biophysics, King's College London
Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.
JoVE General
Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
Dipartimento di Biologia Strutturale e Funzionale, University of Naples
Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.
JoVE Neuroscience
Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae
Epigenetics and Progenitor Cells Keystone, Fox Chase Cancer Center
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
JoVE General
Purification of Transcripts and Metabolites from Drosophila Heads
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.
JoVE Neuroscience
Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster
Department of Neurobiology, Stanford University
Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.
JoVE General
Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute
Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.
JoVE General
Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana
1Department of Energy - Plant Research Laboratory, Michigan State University (MSU), 2Department of Biochemistry and Molecular Biology, Michigan State University (MSU)
The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.
JoVE Neuroscience
Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines
1Department of Biology, Pace University, 2Cellular and Molecular Medicine, University of California, San Diego, 3Division of Cell Biology and Cell Physiology, Zoological Institute, Braunschweig University of Technology
The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.
JoVE Neuroscience
Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics
1Disease Mechanism Research Core, RIKEN Brain Science Institute, 2Graduate School of Science and Engineering, Saitama University
The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.
JoVE General
Primary Cell Cultures from Drosophila Gastrula Embryos
1Department of Genetics, Harvard Medical School, 2 , Howard Hughes Medical Institute
We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.
JoVE General
Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions
Department of Biology, Brandeis
This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.
JoVE General
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine
We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.
JoVE Neuroscience
Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining
Department of Biomolecular Genetics, University of Rochester Medical Center
Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.
JoVE Neuroscience
Laser Capture Microdissection of Drosophila Peripheral Neurons
1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.
JoVE General
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Molecular, Cellular and Developmental Biology, University of Michigan
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
JoVE Neuroscience
Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae
1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 2Scholars Academy/MARC Scholar, University of Houston-Downtown, 3Genes and Development Graduate Program, University of Texas Graduate School of Biomedical Sciences, 4Neuroscience Graduate Program, University of Texas Graduate School of Biomedical Sciences
In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
JoVE General
Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting
1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.
JoVE Neuroscience
Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster
1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne
We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.
JoVE Neuroscience
Methods to Assay Drosophila Behavior
1Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 2Department of Genetics, Louisiana State University Health Sciences Center
Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.
JoVE General
Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc
Department of Zoology, University of British Columbia - UBC
Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.
JoVE General
Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging
This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.
JoVE General
Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation
This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.
JoVE Neuroscience
In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons
Department of Biological Sciences, SUNY-University at Buffalo
This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.
JoVE Neuroscience
Single Drosophila Ommatidium Dissection and Imaging
MRC Centre for Developmental Neurobiology, King's College London
The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.
JoVE Neuroscience
Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall
1Disease Mechanism Research Core, RIKEN Brain Science Institute, 2Graduate School of Science and Engineering, Saitama University
To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.
JoVE Neuroscience
Preparation of Drosophila Central Neurons for in situ Patch Clamping
School of Life Sciences, Arizona State University
In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
JoVE General
Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining
Division of Biology, California Institute of Technology
We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.
JoVE Immunology and Infection
Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
JoVE Neuroscience
Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster
1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent
The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.
JoVE General
Dissection of Oenocytes from Adult Drosophila melanogaster
Department of Biology, University of Toronto
In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.
JoVE Neuroscience
Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain
The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.
JoVE General
Drosophila Pupal Abdomen Immunohistochemistry
Department of Biological Sciences, University of Alabama
Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.
JoVE Neuroscience
Appetitive Associative Olfactory Learning in Drosophila Larvae
1Department of Biology, University of Konstanz, 2Department of Biology, University of Fribourg
Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.
JoVE General
Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo
Department of Molecular and Cellular Biology, University of California, Davis
This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.
JoVE Neuroscience
Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila
1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Current Address: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, 3Department of Molecular Biology and Biochemistry, Rutgers University
We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.
JoVE Neuroscience
An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila
Neurodevelopment Group, School of Biosciences, University of Birmingham
An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.
JoVE General
Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation
Department of Cellular Biology and Anatomy, Medical College of Georgia
In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.
JoVE Neuroscience
Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe
1Center for Integrative Genomics, University of Lausanne, 2Department of Biology, University of Konstanz
We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.
JoVE Neuroscience
Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles
Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.
JoVE General
Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting
1School of Life Sciences, University of Warwick, 2Warwick Systems Biology, University of Warwick
A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.
JoVE General
Whole Cell Recordings from Brain of Adult Drosophila
This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.
JoVE General
Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae
This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.
JoVE General
Cell Tracking Using Photoconvertible Proteins During Zebrafish Development
Max Delbrück Center for Molecular Medicine
Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.
JoVE General
Electrophysiological Recording in the Drosophila Embryo
1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University
Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.
JoVE Immunology and Infection
An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response
1Biology Department, The City College of New York, CUNY, 2The Graduate Center, The City University of New York
Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.
JoVE General
Measurement of Lifespan in Drosophila melanogaster
1Department of Molecular and Integrative Physiology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan
Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.
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