1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health
The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.
Published January 22, 2015. Keywords: Biochemistry, Biomimetic model system, Giant Unilamellar Vesicle, reconstitution, ion channel, transmembrane protein, KvAP, electroformation, gel assisted swelling, agarose, inside-out patch clamp, electrophysiology, fluorescence microscopy
1Institute of Neuroscience and Medicine (INM-2), Research Centre Jülich, 2Department of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty, JARA, RWTH Aachen University
Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.
Published January 10, 2015. Keywords: Neuroscience, Patch-clamp, paired recordings, neurons, synaptic connections, gap junctions, biocytin labeling, structure-function correlations
1Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY, 2Department of Biochemistry, SMBS, University at Buffalo, SUNY, 3Molecular and Cellular Neuroscience Department, The Scripps Research Institute, 4Department of Biochemistry and Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY
Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.
Published June 9, 2014. Keywords: Neuroscience, biophysics, ion channels, single-channel recording, NMDA receptors, gating, electrophysiology, patch-clamp, kinetic analysis
1Biotactical Engineering, Faculty of Engineering and Industrial Science, Swinburne University of Technology, 2Department of Otolaryngology, The University of Melbourne
Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.
Published July 31, 2013. Keywords: Neuroscience, Biomedical Engineering, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Primary Cell Culture, Biophysics, Electrophysiology, fiber optics, infrared neural stimulation, patch clamp, in vitro models, spiral ganglion neurons, neurons, patch clamp recordings, cell culture
1Institute of Integrative Neuroanatomy, NeuroCure Cluster of Excellence, Charité Universitätmedizin
Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.
Published September 30, 2014. Keywords: Neuroscience, electrophysiology, acute slice, whole-cell patch-clamp recording, neuronal morphology, immunocytochemistry, parvalbumin, hippocampus, inhibition, GABAergic interneurons, synaptic transmission, IPSC, GABA-B receptor
1Laboratory of Neural Microcircuitry - Brain Mind Institute, Ecole Polytechnique Federale de Lausanne
Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.
Published October 18, 2013. Keywords: Neuroscience, Patch-clamp, automatic positioning, whole-cell, neuronal recording, in vitro, multi-electrode
1Anatomy and Cell Biology, Wayne State University School of Medicine, 2Ophthalmology, Wayne State University School of Medicine
We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.
Published February 11, 2015. Keywords: Neuroscience, Retina, Patch clamp recording, light response, mouse, dark adaptation, infrared
1Department of Biological Sciences, University of Alberta
We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.
Published September 10, 2013. Keywords: Neuroscience, Synapses, Zebrafish, Ligand-Gated Ion Channels, Neurosciences, Mauthner cells, reticulospinal neurons, Zebrafish, synapse, ion channels, AMPA receptors, NMDA receptors, action potentials, glycine receptors
1Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami
We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.
Published July 10, 2013. Keywords: Neuroscience, Neurobiology, Anatomy, Physiology, Cellular Biology, Molecular Biology, Environmental Sciences, Marine Biology, Receptors, Neurophysiology, Neurotransmitter, Neurotransmitter Agents, Patch Clamp Recordings, Primary Cell Culture, Electrophysiology, L-Glutamate, NMDA, D-Aspartate, dissection, ganglia, buccal ganglion, neurons, invertebrate, Aplysia californica, california sea slug, mollusk, animal model
1Department of Molecular Pharmacology & Therapeutics, Loyola University Chicago
Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.
Published September 14, 2012. Keywords: Physiology, Molecular Biology, Medicine, Anatomy, Vascular smooth muscle, mesenteric artery, patch clamp, Kv channel, vasoconstriction, electrophysiology
1Institute for Microstructural Sciences, National Research Council of Canada, 2Institute for Biological Sciences, National Research Council of Canada, 3Hotchkiss Brain Institute, University of Calgary
We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.
Published February 7, 2012. Keywords: Neuroscience, disease models, pharmaceutical screens, electrophysiological recordings, patch-clamp, silicon planar patch-clamp chip, cultured neurons
1The 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.
Published January 19, 2012. Keywords: Neuroscience, synaptic physiology, axon terminal, synaptic ribbon, retina microcircuits, goldfish, zebrafish, brain slices, patch-clamp, membrane capacitance measurements, calcium-imaging, exocytosis, transmitter release
1Natural Science Division, Pepperdine University
The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.
Published October 17, 2012. Keywords: Neuroscience, Physiology, Anatomy, Cellular Biology, zebrafish, Danio rerio, hair cells, electrophysiology, patch clamp, auditory, vestibular, inner ear
1Department of Physiology and Biophysics, University of Washington
Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.
Published June 21, 2013. Keywords: Physiology, Biophysics, Molecular Biology, Biochemistry, Genetics, Cellular Biology, Proteins, Membranes, Artificial, Lipid Bilayers, Liposomes, Phospholipids, biochemistry, Lipids, Giant Unilamellar Vesicles, liposome, electrophysiology, electroformation, reconstitution, patch clamp
1Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis
Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.
Published January 10, 2011. Keywords: Cellular Biology, patch clamp, ion channel, electrophysiology, biophysics, exogenous expression system, Xenopus oocyte, mRNA, transcription
1Department of Internal Medicine, Yale University
A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.
Published May 4, 2013. Keywords: Neuroscience, Medicine, Biomedical Engineering, Molecular Biology, Cellular Biology, Biochemistry, Neurobiology, Anatomy, Physiology, F1FO ATPase, mitochondria, patch clamp, electrophysiology, submitochondrial vesicles, Bcl-xL, cells, rat, animal model
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.
Published October 16, 2008. Keywords: Cellular Biology, Electrophysiology, Patch Clamp, Voltage Clamp, Oocytes, Biophysics, Gigaseal, Ion Channels
1Vollum Institute, Oregon Health and Sciences University
Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.
Published November 20, 2010. Keywords: Neuroscience, Zebrafish, synapse, electrophysiology, patch clamp, acetylcholine receptor, neuromuscular, cholinergic/action potential, myasthenic syndrome, motor control
1Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.
Published October 22, 2008. Keywords: Basic Protocols, electrophysiology, patch clamp, voltage clamp, biophysics, gigaseal, ion channels
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.
Published September 12, 2010. Keywords: Neuroscience, vision, mice, retina, photoreceptor, bipolar cell, slice preparation, patch clamp
1National Institute of Neurological Disorders and Stroke, National Institute of Health
We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.
Published July 29, 2007. Keywords: Neuroscience, membrane fusion, exocytosis, endocytosis
JoVE Application Notes
The IonFlux Automated Patch Clamp System provides high throughput, cost-effective ion channel screening for a wide range of electrophysiology applications. Fast compound exchange, low cost per data point, and convenient well plate formats make the system ideal for both ligand- and voltage-gated ion channel targets. The IonFlux HT provides an industry-leading 10,000 data points per day, while the IonFlux 16 provides true automated patch clamp performance for about the cost of a manual patch clamp rig.
Published September 15, 2010. Keywords: Ion channel, patch clamp, electrophysiology, Fluxion Biosciences, IonFlux, high throughput screening, cell based assay
1Department of Biomedical Engineering, Washington University in St. Louis
The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.
Published March 11, 2014. Keywords: Developmental Biology, Voltage clamp, Cut-open, Oocyte, Voltage Clamp Fluorometry, Sodium Channels, Ionic Currents, Xenopus laevis
1SISSA, International School for Advanced Studies, 2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 3SISSA Unit, Italian Institute of Technology
Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.
Published October 29, 2011. Keywords: Neuroscience, caged compounds, caged cAMP, caged Ca, olfactory sensory neuron, olfaction, whole-cell patch-clamp, flash photolysis, flash lampc
1Neuroscience Center, University of Helsinki, 2Neurotar LTD, 3A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 4Laboratory Animal Center, University of Helsinki
This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal’s head.
Published June 29, 2014. Keywords: Empty Value, awake, in vivo two-photon microscopy, blood vessels, dendrites, dendritic spines, Ca2+ imaging, intrinsic optical imaging, patch-clamp
1Cardiovascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Division of Cardiology, Sapienza University
Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.
Published September 24, 2013. Keywords: Cellular Biology, Medicine, Cardiology, Cellular Biology, Anatomy, Physiology, Mice, Ion Channels, Primary Cell Culture, Cardiac Electrophysiology, adult mouse cardiomyocytes, cell isolation, IonOptix, Cell Culture, adenoviral transfection, patch clamp, fluorescent nanosensor
1Department NeuroFarBa, Division of Pharmacology, University of Florence, 2Department of Clinical and Experimental Medicine, Division of Physiology, University of Florence
Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.
Published April 21, 2014. Keywords: Medicine, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
1School 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.
Published October 15, 2012. Keywords: Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Patch clamp, in situ patch clamp, Drosophila, electrophysiology, motoneuron, neuron, CNS
1Department 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.
Published July 17, 2011. Keywords: Neuroscience, brain, patch-clamp, ion channels, tonic current, slices, whole-cell current, single-channel current, GABAA, GABA
1Institute of Pharmacology, University of Duisburg-Essen, 2Division of Experimental Cardiology, University of Heidelberg
We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.
Published July 3, 2013. Keywords: Cellular Biology, Medicine, Molecular Biology, Physiology, Anatomy, Cardiology, Pharmacology, human atrial myocytes, cell isolation, collagenase, calcium transient, calcium current, patch-clamp, ion currents, isolation, cell culture, myocytes, cardiomyocytes, electrophysiology, patch clamp
1Biological Sciences, University of Illinois at Chicago
Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.
Published October 1, 2014. Keywords: Neuroscience, reticulospinal synapse, reticulospinal axons, presynaptic terminal, presynaptic calcium, voltage-gated calcium channels, vesicle fusion, synaptic transmission, neurotransmitter release, spinal cord, lamprey, synaptic vesicles, acute dissociation
1Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, 2Department of Behavioral Neuroscience, Oregon Health & Science University
Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.
Published December 8, 2013. Keywords: Neuroscience, Synaptic Transmission, Action Potentials, Circadian Rhythm, Excitatory Postsynaptic Potentials, Life Sciences (General), circadian rhythm, suprachiasmatic nucleus, membrane potential, patch clamp recording, fluorescent probe, intracellular calcium
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.
Published May 16, 2013. Keywords: Neuroscience, Neurobiology, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Cellular Biology, Neurons, Retinal Neurons, Retinal Ganglion Cells, Eye, Retina, Neurosciences, retina, ganglion cells, synaptic conductance, artificial conductance, tetrodotoxin (TTX), patch clamp, dynamic clamp, conductance clamp, electrophysiology, mouse, animal model
1NRW Research Group 'Dendritic integration in the CNS', Department of Epileptology, University of Bonn, 2Neuronal Networks Group, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE)
In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.
Published July 31, 2013. Keywords: Neuroscience, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Biomedical Engineering, Biophysics, Biochemistry, biology (general), animal biology, Nervous System, Life Sciences (General), Neurosciences, brain slices, dendrites, inhibition, excitation, glutamate, GABA, micro-iontophoresis, iontophoresis, neurons, patch clamp, whole cell recordings
1Graduate Program in Neuroscience, University of California Riverside, 2Department of Cell Biology and Neuroscience, University of California Riverside, 3Center for Glial-Neuronal Interactions, University of California Riverside
Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.
Published March 20, 2014. Keywords: Neuroscience, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
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.
Published November 26, 2012. Keywords: Neuroscience, Physiology, Anatomy, Medicine, hippocampus preparation, acute brain slice, electrophysiology, patch-clamp, neurons, astrocytes, astroglial, neuroglial interactions, glutamate transporter current, potassium current, paired recordings, synaptic activity, synaptically-evoked responses
1Institute of Neurobiology, Heinrich Heine University Düsseldorf
We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.
Published October 8, 2014. Keywords: Neuroscience, Neurosciences, two-photon microscopy, patch-clamp, UV-flash photolysis, mouse, hippocampus, caged compounds, glutamate, brain slice, dendrite, sodium signals
1Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health
We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.
Published August 7, 2013. Keywords: Neurobiology, Neuroscience, Biochemistry, Molecular Biology, Cellular Biology, Anatomy, Physiology, Biophysics, Astrocytes, Synapses, Glutamic Acid, Membrane Transport Proteins, Astrocytes, glutamate transporters, uptake, clearance, hippocampus, stratum radiatum, CA1, gene, brain, slice, animal model
1Department of Physiology and Biophysics, University of Miami
We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.
Published September 21, 2013. Keywords: Developmental Biology, Eukaryota, Biological Phenomena, Cell Physiological Phenomena, C. elegans, cell culture, embryonic cells
1Department of Biology, University of Waterloo
Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).
Published January 19, 2011. Keywords: Neuroscience, brain, invertebrate, calcium channel, electrophysiology, voltage-gated
JoVE Developmental Biology
1Neuroscience & Behavioral Disorders, Duke-NUS Graduate Medical School, 2Lee Kong Chian School of Medicine, Nanyang Technological University
A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.
Published February 17, 2015. Keywords: Developmental Biology, Neuroscience, Channelrhodopsin-2, Co-culture, Neurons, Astrocytes, induced Pluripotent Stem Cells, Neural progenitors, Differentiation, Cell culture, Cortex
1Laboratory of Cell and Molecular Biology, University of Wisconsin – Madison, 2Department of Genetics, University of Wisconsin – Madison
To complement commonly used methods to study TRPV4’s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.
Published December 31, 2013. Keywords: Basic Protocol, Eukaryota, Archaea, Bacteria, Life Sciences (General), Mechanosensation, Ion channels, Lipids, patch clamp, Xenopus Oocytes, yeast, luminometry, force sensing, voltage clamp, TRPV4, electrophysiology
1Department of Neurobiology, Hebrew University of Jerusalem
In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.
Published October 30, 2014. Keywords: Neuroscience, Acute brain slicing, electrophysiology, mice, rats, in vitro, cerebellum, adult, vibratome
1Department 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.
Published October 29, 2012. Keywords: Neuroscience, Nicotinic, acetylcholine, neurotransmitter, neuron, patch clamp, brain slice, picospritzer
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt
Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.
Published May 11, 2013. Keywords: Biochemistry, Biophysics, Molecular Biology, Cellular Biology, Physiology, Proteins, Membrane Lipids, Membrane Transport Proteins, Kinetics, Electrophysiology, solid supported membrane, SSM, membrane transporter, lactose permease, lacY, capacitive coupling, solution exchange, model membrane, membrane protein, transporter, kinetics, transport mechanism
1Department of Neuroscience, University of Florida, 2Department of Psychiatry, University of Florida
The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.
Published November 21, 2012. Keywords: Neuroscience, Cellular Biology, Physiology, Medicine, Simultaneous Patch Clamp and Voltametry, In Vitro Voltametry, Dopamine, Oxidation, Whole-cell Patch Clamp, Dopamine Transporter, Reverse transport, Efflux
1Department of Physiology and Membrance Biology, University of California, Davis
We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.
Published December 21, 2010. Keywords: Immunology, human T lymphocytes, CRAC channels, CRAC currents, patch-clamp
1Baker Laboratory of Pharmacology, Department of Pharmacology, Kirksville College of Osteopathic Medicine, AT Still University of Health Sciences
Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.
Published December 24, 2013. Keywords: Neuroscience, DiI, patch clamp, sensory neurons, muscle afferent neurons, immunocytochemistry, electrophysiology
1Department of Pharmacology and Physiology, Rutgers New Jersey Medical School, 2Center for Taste and Feeding Behavior, Universite de Bourgogne
The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.
Published November 27, 2013. Keywords: Neuroscience, membrane potential dye, ventromedial hypothalamus, adult neurons, glucose sensing, fluorescence imaging, arcuate nucleus
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.
Published November 19, 2012. Keywords: Neuroscience, Medicine, Anatomy, Physiology, hippocampus, traumatic brain injury, electrophysiology, patch clamp, voltage sensitive dye, extracellular recording, high-performance liquid chromatography, gas chromatography-mass spectrometry