Retina:
The ten-layered nervous tissue membrane of the eye. It is continuous with the Optic nerve and receives images of external objects and transmits visual impulses to the brain. Its outer surface is in contact with the Choroid and the inner surface with the Vitreous body. The outer-most layer is pigmented, whereas the inner nine layers are transparent.
JoVE General
Horizontal Slice Preparation of the Retina
1Dpt of Physiology and Biophysics, Dalhousie University, 2Massachusetts General Hospital, Harvard Medical School
Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.
JoVE Neuroscience
An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells
Department of Neuroscience, University of Minnesota
This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.
JoVE General
Organotypic Culture of Adult Rabbit Retina
Havard Medical School, MGH - Massachusetts General Hospital
This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.
JoVE General
The Gateway to the Brain: Dissecting the Primate Eye
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Departement de chimie-biologie, Universite du Quebec a Trois-Rivieres
The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.
JoVE General
In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells
1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons
Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.
JoVE General
Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons
The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.
JoVE Neuroscience
Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas
Department of Biology, New York University
The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.
JoVE Neuroscience
Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation
Department of Pathology and Immunology, Washington University School of Medicine
This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.
JoVE General
Microdissection of Zebrafish Embryonic Eye Tissues
Department of Biological Sciences, Purdue University
This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.
JoVE Neuroscience
Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation
1Neurosciences Graduate Program, University of Southern California, 2Zilkha Neurogenetic Institute, Department of Physiology and Biophysics, University of Southern California Keck School of Medicine
Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.
JoVE Neuroscience
In vivo Electroporation of Developing Mouse Retina
1Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, 2Department of Neurology, Johns Hopkins School of Medicine, 3Department of Ophthalmology, Johns Hopkins School of Medicine, 4Center for High-Throughput Biology, Johns Hopkins School of Medicine, 5Institute for Cell Engineering, Johns Hopkins School of Medicine
A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.
JoVE Neuroscience
Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine
We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.
JoVE Neuroscience
Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging
Storm Eye Institute, Medical University of South Carolina
A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.
JoVE Neuroscience
Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons
1Department of Neuroscience, Ohio State University College of Medicine, 2Department of Ophthalmology and Visual Science, University of Texas Medical School
An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.
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
Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina
Department of Neurobiology, University of Oldenburg
This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.
JoVE Neuroscience
Organotypic Culture of Full-thickness Adult Porcine Retina
1Neurology and Neurosciences, University of Medicine and Dentistry of New Jersey - UMDNJ, 2Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey - UMDNJ
Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.
JoVE General
In Ovo Electroporation in Embryonic Chick Retina
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2Department of Biomedical Engineering, Rutgers University
The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.
JoVE General
In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina
1Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine, 2Department of Biological Sciences, University of Notre Dame, 3Center for Zebrafish Research, University of Notre Dame
A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina.
JoVE Neuroscience
Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.
JoVE General
In vivo-like Organotypic Murine Retinal Wholemount Culture
Institute for Anatomy, Department of Neuroanatomy, University of Duisburg-Essen
This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.
JoVE General
Single-cell Suction Recordings from Mouse Cone Photoreceptors
We will show how to record flash responses from single mouse cones using a suction electrode.
JoVE Neuroscience
Single-cell Profiling of Developing and Mature Retinal Neurons
Department of Genetics, Development and Cell Biology, Neuroscience Program, Iowa State University
A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.
JoVE Clinical and Translational Medicine
A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe
1Fondazione Banca Degli Occhi del Veneto O.N.L.U.S., 2Telethon Institute for Genetics & Medicine (T.I.G.E.M.)
The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.
JoVE Clinical and Translational Medicine
Dissection of Human Vitreous Body Elements for Proteomic Analysis
Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa
This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.
JoVE Neuroscience
Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney, 2The MARCS Institute, University of Western Sydney, 3Discipline of Physiology, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney
This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.
JoVE General
Using Laser Tweezers For Manipulating Isolated Neurons In Vitro
This video describes the manipulation of cultured neurons using laser tweezers in vitro.
JoVE Neuroscience
Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices
The Vollum Institute, Oregon Health and Science University
Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.
JoVE Neuroscience
Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation
1Department of Neurobiology, Yale University, 2Program in Developmental Biology, Baylor College of Medicine
We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.
JoVE General
Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice
1Program in Neuroscience, Boston University, 2Department of Biology, Boston University, 3Department of Biomedical Engineering, Boston University
This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.
JoVE Bioengineering
Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography
1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University
Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.
JoVE Neuroscience
An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
1National Eye Institute, NIH, 2Ophthalmology Department, The Second Hospital of Harbin Medical University
This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.
JoVE Clinical and Translational Medicine
Multifocal Electroretinograms
John A. Moran Eye Center, University of Utah
The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.
JoVE General
Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy
Cellular and Molecular Physiology, Yale University School of Medicine
In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.
JoVE Neuroscience
Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS
Department of Surgery, University of Toronto
Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.
JoVE Neuroscience
Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System
Department of Surgery, University of Toronto
Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.
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 Clinical and Translational Medicine
Mouse Eye Enucleation for Remote High-throughput Phenotyping
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University
The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.
JoVE General
Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants
1The Second Teaching Hospital of Jilin University, 2Department of Ophthalmology, Harvard Medical School
Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.
JoVE Clinical and Translational Medicine
Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University, 3Omics Laboratory, University of Iowa, 4Department of Ophthalmology and Visual Sciences, University of Iowa
This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.
JoVE Neuroscience
Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue
Department of Biology, College of William and Mary
Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.
JoVE Neuroscience
How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow
Biomedical Engineering Department, University of Texas at Austin
This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.
JoVE Neuroscience
Morphometric Analyses of Retinal Sections
1Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, 2Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, 3State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong
This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.
JoVE Neuroscience
Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation
1Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, 2Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary
In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.
JoVE Neuroscience
Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology
National Eye Institute, National Institutes of Health
We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.
JoVE Neuroscience
Viral Tracing of Genetically Defined Neural Circuitry
1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School
A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.
JoVE General
Intravitreous Injection for Establishing Ocular Diseases Model
The University of Hong Kong - HKU
Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.
JoVE Editorial
September 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.
JoVE General
Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
The University of Hong Kong - HKU
This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.
JoVE General
Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos
1Department of Ophthalmology, University of Pittsburgh, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh
We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.
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