JoVE Neuroscience
Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine
An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.
JoVE Neuroscience
Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method
1Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, 2Basic Medicine School, Fourth Military Medical University, 3Department of Neurology, David Geffen School of Medicine, UCLA, 4Aerospace Medicine School, Fourth Military Medical Univeristy
In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.
JoVE Neuroscience
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.
JoVE Neuroscience
Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System
1Department of Neuroscience, Tufts University, 2Neuroscience Program, Tufts Sackler School of Graduate Biomedical Sciences
This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.
JoVE Neuroscience
Live Imaging of Dorsal Root Axons after Rhizotomy
1Temple University, Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, 2Medical Research Service, Department of Veterans Affairs Hospital, 3Department of Neurobiology and Anatomy, Drexel University College of Medicine, 4Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine
An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.
JoVE General
Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord
Department of Neurobiology and Anatomy, Drexel University College of Medicine
Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.
JoVE Neuroscience
Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve
Department of Neurobiology and Behavior, University of California, Irvine
Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.
JoVE Neuroscience
Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord
1Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal, 2Division of Experimental Medicine and Program in Neuroengineering, McGill University, 3Program in Neuroengineering, McGill University, 4Montreal Neurological Institute, 5Department of Anatomy and Cell Biology, McGill University, 6Department of Biology, McGill University, 7Department of Medicine, Universite de Montreal - University of Montreal
This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.
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 Neuroscience
Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.
JoVE General
Analysis of Schwann-astrocyte Interactions Using In Vitro Assays
Cambridge Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge
This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.
JoVE Neuroscience
In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations
Institute of Molecular Life Sciences, University of Zurich
A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.
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
Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis
Center for Neural Repair and Rehabilitation, Temple University
In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.
JoVE Neuroscience
Analyzing Murine Schwann Cell Development Along Growing Axons
1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg
Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.
JoVE General
Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons
1Department of Biological Sciences, University of Southern California, 2Neuroscience Graduate Program, University of Southern California
This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.
JoVE Bioengineering
Axon Stretch Growth: The Mechanotransduction of Neuronal Growth
1Departments of Biomedical Engineering, New Jersey Institute of Technology, 2Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey
A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.
JoVE Neuroscience
Mechanical Manipulation of Neurons to Control Axonal Development
Department of Zoology, Michigan State University, East Lansing
Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.
JoVE Neuroscience
DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
Developmental Neurobiology, Max Delbrück Center for Molecular Medicine
The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.
JoVE Neuroscience
Neural Explant Cultures from Xenopus laevis
Department of Cell Biology, Harvard Medical School
Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.
JoVE General
A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform
1Department of Electrical and Computer Engineering, Texas A&M University (TAMU), 2Department of Veterinary Integrative Biosciences, Texas A&M University (TAMU)
We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.
JoVE Neuroscience
Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays
Department of Biological Sciences, Purdue University
We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.
JoVE Bioengineering
Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans
Department of Biology, University of Utah
Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.
JoVE General
Preparation of Aplysia Sensory-motor Neuronal Cell Cultures
1Dept. of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Dept. of Biological Chemistry, University of California, Los Angeles, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles
Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.
JoVE Editorial
August 2011: This Month in JoVE
Here are some highlights from the August 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Neuroscience
Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones
Department of Neuroscience, University of Minnesota
A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.
JoVE Neuroscience
In vivo Laser Axotomy in C. elegans
A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.
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 General
Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.
JoVE General
A Craniotomy Surgery Procedure for Chronic Brain Imaging
Department of Neurology, University of California, Los Angeles
This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.
JoVE Bioengineering
Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study
Department of Engineering Mechanics, University of Nebraska-Lincoln
A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.
JoVE General
Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
Department of Biology, University of Waterloo
Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).
JoVE Neuroscience
Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations
Department of Experimental Neurobiology, The Neurosciences Institute
We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.
JoVE General
Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos
1Genetics Training Program, University of Wisconsin-Madison, 2Department of Anatomy, University of Wisconsin-Madison, 3Department of Zoology, University of Wisconsin-Madison, 4Cell and Molecular Biology Training Program, University of Wisconsin-Madison
This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.
JoVE General
Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research
Department of Biomedical Engineering, Boston University
In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.
JoVE Neuroscience
The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures
Anatomical Institute, Department of Biomedicine, University of Basel
We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.
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 Neuroscience
Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice
1Biology Department, Arcadia University, 2Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine
We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.
JoVE Clinical and Translational Medicine
In vitro Measurements of Tracheal Constriction Using Mice
Department of Physiology, UT Health Science Center, San Antonio
Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.
JoVE Neuroscience
Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex
1Department of Molecular, Cellular Biology and Biochemistry, Brown University, 2Institute for Brain Science, Brown University, 3Department of Psychiatry and Human Behavior, Warren Alpert School of Medicine, Brown University
To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.
JoVE Clinical and Translational Medicine
Controlled Cervical Laceration Injury in Mice
1Norton Neuroscience Institute, Norton Healthcare, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Medical Neuroscience Graduate Program, and Department of Anatomy and Cell Biology, Indiana University School of Medicine
A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm.
JoVE General
Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons
Department of Biological Sciences, Simon Fraser University
Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.
JoVE Neuroscience
Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception
Department of Biology, University of Kentucky
The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.
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 Bioengineering
Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening
1Department of Biomedical Engineering, University of Texas at San Antonio, 2Department of Biology, University of Texas at San Antonio
We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.
JoVE Bioengineering
Postproduction Processing of Electrospun Fibres for Tissue Engineering
1Materials Science and Engineering, University of Sheffield, 2Department of Biomedical Science, University of Sheffield, 3Department of Chemistry, University of Sheffield
Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.
JoVE General
Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle
Department of Biology, University of Kentucky
The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.
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
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies
We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.
JoVE General
Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons
Nemours Biomedical Research, Alfred I. duPont Hospital for Children
We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.
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