Receptors, Cholinergic:
Cell surface proteins that bind acetylcholine with high affinity and trigger intracellular changes influencing the behavior of cells. Cholinergic receptors are divided into two major classes, muscarinic and nicotinic, based originally on their affinity for nicotine and muscarine. Each group is further subdivided based on pharmacology, location, mode of action, and/or molecular biology.
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
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
Department of Biology, University of Victoria
We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.
JoVE Neuroscience
Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice
1Biology Department, Arcadia University, 2Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine
We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.
JoVE Neuroscience
Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish
Vollum 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.
JoVE Clinical and Translational Medicine
In vitro Measurements of Tracheal Constriction Using Mice
Department of Physiology, UT Health Science Center, San Antonio
Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.
JoVE Neuroscience
Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System
1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.
JoVE Neuroscience
Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction
1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München
Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.
JoVE Neuroscience
Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis
Center for Neural Repair and Rehabilitation, Temple University
In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.
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 Bioengineering
Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish
1Neurobiology, NCBS-TIFR, 2Department of Biological Sciences, TIFR
A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.
JoVE Clinical and Translational Medicine
The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation
1Laboratory of Experimental Urology, Department of Development and Regeneration, KU Leuven, Belgium, 2Laboratory for Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Belgium, 3TRP Research Platform Leuven (TRPLe), KU Leuven, Belgium
Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.
JoVE General
Gastrointestinal Motility Monitor (GIMM)
Department of Anatomy and Neurobiology, The University of Vermont
Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.
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 Clinical and Translational Medicine
Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice
Department of Anesthesiology, University of Colorado
A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.
JoVE Neuroscience
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto
The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.
JoVE Clinical and Translational Medicine
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.
JoVE Neuroscience
Forebrain Electrophysiological Recording in Larval Zebrafish
A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.
JoVE General
Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes
1Department of Neuroscience, University of Pennsylvania-School of Medicine, 2Department of Physiology, University of Pennsylvania-School of Medicine
This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.
JoVE Immunology and Infection
Induction of Experimental Autoimmune Hypophysitis in SJL Mice
Department of Pathology, The Johns Hopkins University
This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.
JoVE General
Elevated Plus Maze for Mice
1Genetic Engineering and Functional Genomics Unit, Frontier Technology Center, Graduate School of Medicine, Kyoto University, 2Institute for Comprehensive Medical Science Division of Systems Medicine, Fujita Health University
The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.
JoVE Neuroscience
Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures
1Department of Physiology, David Geffen School of Medicine at UCLA, 2Natural Science Division, Pepperdine University
This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.
JoVE Neuroscience
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.
JoVE General
Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes
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.
JoVE Immunology and Infection
Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Department of Structural Biology, University of Pittsburgh School of Medicine
This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.
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 General
Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography
Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.
JoVE General
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Department of Physiology and Biophysics, Weill Cornell Medical College
We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
JoVE General
Introduction to Solid Supported Membrane Based Electrophysiology
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.
JoVE Clinical and Translational Medicine
Analytical Techniques for Assaying Nitric Oxide Bioactivity
1Texas Therapeutics Institute, University of Texas Health Science Center at Houston, 2Deptartment of Pediatrics, Baylor College of Medicine
The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.
JoVE General
The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity
An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.
JoVE Neuroscience
Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae
Department of Biology, Institute of Cell and Developmental Biology, University of Fribourg
Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.
JoVE Neuroscience
Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster
1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent
The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.
JoVE Immunology and Infection
Intravital Microscopy of the Inguinal Lymph Node
1Interdisciplinary Science, University of Northern British Columbia, 2Northern Medical Program, University of Northern British Columbia
A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.
JoVE General
Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants
Department of Biological Sciences, University of Alabama
This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.
JoVE Neuroscience
Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise
1Department of Biology, University of Kentucky, 2Department of Biological Sciences, Brock University
In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.
JoVE Neuroscience
Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations
Department of Experimental Neurobiology, The Neurosciences Institute
We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.
JoVE General
Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments
1Laboratory for Behavior and Dynamic Cognition, Brain Science Institute, RIKEN, 2Laboratory for Biolinguistics, Brain Science Institute, RIKEN
In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.
JoVE General
Quantifying Agonist Activity at G Protein-coupled Receptors
1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University
A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.
JoVE General
A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice
1Department of Medicine, Baylor College of Medicine (BCM), 2Millenium Premier Group, 3Department of Immunology, Baylor College of Medicine (BCM)
Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.
JoVE Immunology and Infection
A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
1Department of Biological Sciences, University of Alberta, 2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, 3Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México
Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei
JoVE General
Imaging Protein-protein Interactions in vivo
Biochemistry and Molecular Biology, Virginia Commonwealth University
This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.
JoVE Neuroscience
Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells
1Department of Molecular Genetics and Molecular Biology , Duke University, 2Department of Chemistry, Duke University
Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.
JoVE General
An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images
1Medications Development, Ernest Gallo Clinic and Research Center, University of California, San Francisco, 2Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, 3Translational Research Institute and the Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.
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 General
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.
JoVE Immunology and Infection
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU)
This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.
JoVE General
Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.
JoVE General
Imaging of Estrogen Receptor-α in Rat Pial Arterioles using a Digital Immunofluorescent Microscope
Graduate School of Nursing, Uniformed Services University of the Health Sciences
The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-α (ERα) in rat pial arterial slices using a digital immunofluorescent microscope.
JoVE Clinical and Translational Medicine
Non-invasive Assessment of Microvascular and Endothelial Function
1Department of Family and Community Medicine, Thomas Jefferson University, 2Department of Pharmacology and Experimental Therapeutics, Biostatistics Division, Thomas Jefferson University, 3Department of Internal Medicine, Thomas Jefferson University
Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.
JoVE General
Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
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