JoVE General
Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies
Department of Veterinary Integrative Biosciences, Texas A&M University (TAMU)
A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.
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 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 Editorial
October 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the October 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Neuroscience
T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice
1Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, 2Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), 3Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences
This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.
JoVE General
A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity
Bioscience Division, High Content Analysis Research and Development, Millipore Inc
This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.
JoVE General
Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures
1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University
This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.
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 Neuroscience
Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.
JoVE Neuroscience
Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia
Department of Pharmacology and Physiology, Drexel University College of Medicine
Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.
JoVE General
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
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
Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development
Department of Neuroscience and Physiology, SUNY Upstate Medical University
This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.
JoVE Neuroscience
Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
Department of Anatomy, University of Wisconsin-Madison
This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.
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 Neuroscience
Axoplasm Isolation from Rat Sciatic Nerve
Department of Biological Chemistry, Weizmann Institute of Science
We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.
JoVE Neuroscience
Isolation and Culture of Mouse Cortical Astrocytes
1Institute of Anatomy and Cell Biology, University of Freiburg, 2Centre of Chronic Immunodeficiency (CCI), University Medical Centre Freiburg, University of Freiburg
Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.
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 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 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 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 General
Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes
We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.
JoVE Neuroscience
Intracranial Injection of Adeno-associated Viral Vectors
Neurobiology and Anatomy, University of Rochester
Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.
JoVE General
Neuronal Nuclei Isolation from Human Postmortem Brain Tissue
Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School
The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.
JoVE Neuroscience
Preparation of Acute Subventricular Zone Slices for Calcium Imaging
Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine
A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.
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 Neuroscience
Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number
1Department of Neurobiology, Duke University, 2Department of Cell Biology, Duke University
This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.
JoVE Neuroscience
An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine
An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.
JoVE Neuroscience
Neonatal Subventricular Zone Electroporation
Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine
We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells
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 General
Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)
1Department of Biology, Columbia University, 2Department of Pathology and Cell Biology, Columbia University
This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.
JoVE General
Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School
Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.
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 Immunology and Infection
Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
1Institute for Memory Impairments and Neurological Disorders, University of California, 2Physical Medicine & Rehabilitation, University of California, 3Anatomy & Neurobiology, University of California, 4Sue and Bill Gross Stem Cell Research Center, University of California, 5Section of Molecular Biology, University of California, 6Reeve-Irvine Research Center, University of California
Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.
JoVE Neuroscience
Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5
1College of Veterinary Medicine, Western University of Health Sciences, 2Graduate College of Biomedical Sciences, Western University of Health Sciences, 3ReadiSorb, Products
A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.
JoVE General
Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors
We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.
JoVE Neuroscience
The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow
1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University
The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.
JoVE Neuroscience
Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol
1LSU Health Sciences Center - New Orleans, 2Medical School and Stanley S. Scott Cancer Center
We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.
JoVE General
Preparation of Dissociated Mouse Cortical Neuron Cultures
Department of Anatomy and Neurobiology, University of California, Irvine (UCI)
This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.
JoVE Neuroscience
Inducing Dendritic Growth in Cultured Sympathetic Neurons
Department of Molecular Biosciences, University of California, Davis
We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.
JoVE Neuroscience
Derivation of Glial Restricted Precursors from E13 mice
1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland, 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine
This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.
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 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 General
Generation of Single-Cell Suspensions from Mouse Neural Tissue
Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.
JoVE Neuroscience
Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay
1 Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, University of Florida
This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.
JoVE Editorial
June 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.
JoVE Editorial
JoVE 7th Issue
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
Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures
Department of Neurobiology and Behavior, University of California, Irvine
Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.
JoVE General
Dissecting the Non-human Primate Brain in Stereotaxic Space
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Département de chimie-biologie , Université du Québes à Trois-Rivières
The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.
More Results...
