JoVE General
Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs
1Departments of Neurology, Columbia University, 2Departments of Psychiatry and Pharmacology, Columbia University, 3Department of Chemistry, Columbia University, 4eMolecules, Inc., 5Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, 6Division of Molecular Therapeutics, New York Psychiatric Institute
A new means to measure neurotransmission optically using fluorescent dopamine analogs.
JoVE Clinical and Translational Medicine
Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease
Centre for Neurobiology of Stress, Dept Biological Sciences, University of Toronto at Scarborough
A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing >95% loss of striatal dopamine and 90.63±-4.02 % ipsiversive rotational bias towards the side of the lesion.
JoVE Neuroscience
DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices
1Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, 2Department Physiology and Pharmacology, Wake Forest University Health Sciences, 3Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University
We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.
JoVE Neuroscience
Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry
1Department of Chemistry, Wayne State University,
Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.
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
Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures
Section on Critical Brain Dynamics, National Institute of Mental Health
A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.
JoVE General
The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia
Department of Neurology and Neurological Sciences, Stanford University School of Medicine
The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.
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 General
Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brasil
Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.
JoVE Neuroscience
Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue
1Weldon School of Biomedical Engineering, Purdue University, 2Department of Biological Sciences, Purdue University
Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.
JoVE Clinical and Translational Medicine
MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease
1Department of Pharmaceutical Biosciences, Uppsala University, 2Department of Chemical and Biological Engineering, Chalmers University of Technology
Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.
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.
JoVE General
Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber
1Dept. of Bioengineering, University of Illinois, Chicago, 2Department of Anatomy and Cell Biology, University of Illinois, Chicago
We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.
JoVE General
4D Imaging of Protein Aggregation in Live Cells
Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.
JoVE General
Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat
1Lacombe Research Centre, Agriculture and Agri-Food Canada, 2Grupo de investigación MERAGEM, Universidad de Córdoba, 3Department of Animal Science, University of Nebraska
Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.
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 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
Organotypic Hippocampal Slice Cultures
Department of Physiology and Biophysics, University of Washington School of Medicine
We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.
JoVE General
A Novel Ex vivo Culture Method for the Embryonic Mouse Heart
McAllister Heart Institute, University of North Carolina at Chapel Hill
Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.
JoVE Neuroscience
The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice
1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida
The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.
JoVE Neuroscience
Whole Cell Recording from an Organotypic Slice Preparation of Neocortex
Department of Anatomy and Neurobiology, University of Tennessee Health Science Center
This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.
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
The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury
Department of Neurology and Neurological Sciences, Stanford University School of Medicine
The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.
JoVE Neuroscience
Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection
1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies
This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.
JoVE Clinical and Translational Medicine
Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia
There is accumulating evidence, that ischemic preconditioning (PC) – a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.
JoVE Neuroscience
Chicken Embryo Spinal Cord Slice Culture Protocol
Research Department of Cell and Developmental Biology, University College London
Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.
JoVE Neuroscience
Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University
In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.
JoVE General
Organotypic Slice Culture of E18 Rat Brains
Institute for Regeneration Medicine, University of California, San Francisco - UCSF
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
Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro
Institute of Reconstructive Neurobiology, University of Bonn
A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.
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 Neuroscience
Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex
1Neuroinformatics DTC, University of Edinburgh, 2Centre for Integrative Physiology, University of Edinburgh
We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.
JoVE Neuroscience
Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals
1Section on Synaptic Pharmacology/Laboratory for Integrative Neuroscience, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, 2Department of Electronics Engineering, Ewha Womans University, 3Section on Transmitter Signaling/Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism
This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.
JoVE Immunology and Infection
Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France
This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.
JoVE Neuroscience
Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine
This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.
JoVE Bioengineering
Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences
We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.
JoVE Neuroscience
Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation
1Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.
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 General
Direct Tracheal Instillation of Solutes into Mouse Lung
1Department of Physiology, Emory University, 2Center for Respiratory Health, Emory University, 3Department of Medicine, Emory University
Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.
JoVE General
Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture
Center for Neuroscience, University of California, Davis
We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.
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
Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology
1Graduate Center for Gerontology, University of Kentucky College of Public Health, 2Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 3Sanders-Brown Center on Aging, University of Kentucky College of Medicine
This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer’s disease.
JoVE Neuroscience
Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet
The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.
JoVE Neuroscience
Preparation and Culture of Chicken Auditory Brainstem Slices
1Department of Otolaryngology-Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research Center, University of Washington, 2Department of Physiology and Biophysics, Virginia Merrill Bloedel Hearing Research Center, University of Washington
The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.
JoVE General
Thin Sectioning of Slice Preparations for Immunohistochemistry
1Department of Medicine, Yonsei University College of Medicine, Severance Hospital, 2Department of Psychiatry, Harvard Medical School
The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.
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 Neuroscience
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
1Department of Neurology and Committee on Neurobiology, The University of Chicago Medical Center, 2Department of Neurology, The University of Chicago Medical Center
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
JoVE General
Whole Cell Recordings from Brain of Adult Drosophila
This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.
JoVE Bioengineering
Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments
1School of Dentistry, Cardiff Institute of Tissue Engineering & Repair, Cardiff University, 2Shandong Qianfoshan Hospital, Shandong University School of Medicine, 3Dermatology and Ophthalmology Research, Institute for Regenerative Cures, University of California at Davis
This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).
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.
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