Ganglia, Spinal:
Sensory ganglia located on the dorsal spinal roots within the vertebral column. The spinal ganglion cells are pseudounipolar. The single primary branch bifurcates sending a peripheral process to carry sensory information from the periphery and a central branch which relays that information to the spinal cord or brain.
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
Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University, 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences
Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.
JoVE Neuroscience
Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons
1Institute for Cellmorphology and molecular Neurobiology, Group for Cellbiology, Ruhr-University Bochum, 2Institute for Clinical Neurobiology, University of Wuerzburg
An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.
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
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
A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells
Biozentrum, University of Basel
Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.
JoVE Neuroscience
The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System
Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.
JoVE Clinical and Translational Medicine
The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice
1The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, 2Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen
The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.
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
Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center
Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.
JoVE Neuroscience
Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury
1Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen
We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.
JoVE Neuroscience
Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).
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
Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons
Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.
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
Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth
We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.
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 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 Neuroscience
Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise
1Department of Biology, University of Kentucky, 2Department of Biological Sciences, Brock University
In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.
JoVE Bioengineering
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
JoVE Neuroscience
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto
The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.
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
Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation
Department of Biochemistry and Cell Biology, Rice University
An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.
JoVE Immunology and Infection
A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
1Department of Microbiology, New York University School of Medicine, 2Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 3Department of Otolaryngology, New York University School of Medicine, 4Department of Cell Biology, New York University School of Medicine, 5Department of Physiology and Neuroscience, New York University School of Medicine, 6Department of Psychiatry, New York University School of Medicine, 7Center for Neural Science, New York University School of Medicine
The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.
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
Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification
1Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan
We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.
JoVE Neuroscience
Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones
1Department of Pharmacology, The University of Iowa, 2Department of Anesthesia, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa
Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.
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
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 Neuroscience
Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo
1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, 2Department of Medical Neurobiology, The Hebrew University of Jerusalem
How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.
JoVE Clinical and Translational Medicine
Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography
1Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 2Department of Biology, North Carolina Central University, Durham, 3Department of Physiology & Pharmacology and Hypertension & Vascular Research Center, Wake Forest University School of Medicine
An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.
JoVE General
Homarus Americanus Stomatogastric Nervous System Dissection
Volen Center for Complex Systems, Brandeis
We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).
JoVE General
Spinal Cord Electrophysiology
1The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, 2Biology Graduate Program, University of California San Diego - UCSD
A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.
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 Neuroscience
Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes
We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.
JoVE Clinical and Translational Medicine
Dorsal Column Steerability with Dual Parallel Leads using Dedicated Power Sources: A Computational Model
Boston Scientific , Neuromodulation
Using a mathematical model of spinal cord stimulation, we found that a multi-source system with independent power sources for each contact can target more central points of stimulation on the dorsal column (100 vs 3) and has 50-fold more field steering resolution (0.02mm vs 1mm) than a single-source system.
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
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
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 General
Cancer Borealis Stomatogastric Nervous System Dissection
Volen Center for Complex Systems, Brandeis
The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.
JoVE Neuroscience
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
1Department of Biology, Case Western Reserve University, 2Department of Neurosciences, Case Western Reserve University, 3Department of Biomedical Engineering, Case Western Reserve University
In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
JoVE Neuroscience
An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica
1Department of Biology, Case Western Reserve University, 2Department of Neurosciences, Case Western Reserve University, 3Department of Biomedical Engineering, Case Western Reserve University
We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.
JoVE Immunology and Infection
High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.
Department of Nematology, University of California, Riverside
Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.
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 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 General
Generation of Composite Plants in Medicago truncatula used for Nodulation Assays
Donald Danforth Plant Science Center, St. Louis, Missouri
We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.
JoVE Editorial
September 2011: This Month in JoVE
Here are some highlights from the September 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Gross Dissection of the Stomach of the Lobster, Homarus Americanus
Volen Center for Complex Systems, Brandeis
We describe the gross dissection of the stomach of the American lobster (Homarus americanus).
JoVE General
Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping
1Horticulture Department, University of Wisconsin-Madison, 2Department of Zoology, Oregon State University
The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.
JoVE Bioengineering
Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg
This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.
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