JoVE Neuroscience
A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices
1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Precisionary Instruments Inc., 3Departments of Molecular & Human Genetics and Neuroscience, Baylor College of Medicine (BCM), 4Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital
Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.
JoVE Neuroscience
Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices
Department of Physiology, School of Medicine, University of Saarland, Homburg, Germany
In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.
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 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 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 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 Editorial
JoVE 8th Issue
JoVE Clinical and Translational Medicine
Mouse Model of Middle Cerebral Artery Occlusion
1Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, 2Department of Biological Sciences, Kent State University
We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.
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
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
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 Neuroscience
GABA-activated Single-channel and Tonic Currents in Rat Brain Slices
Department of Neuroscience, Uppsala University, Sweden
We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.
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 Neuroscience
Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses
1Department of Psychology, Randolph-Macon College, 2Department of Psychology, Marshall University
The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn’t require invasive techniques to manipulate the expression of the behavior.
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
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 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 Neuroscience
Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices
Department of Biology, University of Texas San Antonio - UTSA
Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.
JoVE Neuroscience
Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques
Department of Pharmacology, University of Tennessee College of Medicine
Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.
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
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
Functional Calcium Imaging in Developing Cortical Networks
Department of Integrative Neurophysiology, VU University, Amsterdam
Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.
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 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 General
Organotypic Slice Culture of E18 Rat Brains
Institute for Regeneration Medicine, University of California, San Francisco - UCSF
JoVE Neuroscience
Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique
Neurosciences Institute, University of Texas San Antonio - UTSA
In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.
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 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 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 Neuroscience
Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation
1Neuroscience Center, University of Helsinki, 2Faculty of Biological and Enviromental Sciences, University of Helsinki
This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.
JoVE Neuroscience
Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents
Partially isolated cortex (“undercut”) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.
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 Neuroscience
Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices
Department of Cellular and Molecular Physiology, Yale University School of Medicine
An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.
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 General
Monitoring Acupuncture Effects on Human Brain by fMRI
1Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2William Beaumont Hospital
FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.
JoVE Neuroscience
Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures
1Dept. of Anatomy, Physiology and Genetics, Uniformed Services University, 2Neuroscience Program, Uniformed Services University
Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.
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
Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury
1Division of Neurology, Children's Hospital of Philadelphia, 2Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 3Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania
A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.
JoVE Neuroscience
Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices
1Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Collège de France, 2Paris Diderot University
The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.
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
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
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
Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin
Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.
JoVE Neuroscience
Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 2Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.
JoVE Neuroscience
Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney, 2The MARCS Institute, University of Western Sydney, 3Discipline of Physiology, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney
This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.
JoVE 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 General
How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)
1Department of Neurology, Emory University School of Medicine, 2Coulter Department of Biomedical Engineering, Laboratory for Neuroengineering, Georgia Institute of Technology and Emory, University School of Medicine, 3Emory University School of Medicine
This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.
JoVE Clinical and Translational Medicine
Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 3Department of Physiology and Functional Genomics, University of Florida, 4Department of Neurology, University of Florida
Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.
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.
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