Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

JoVE 1133 3/16/2009

Nicholas J. Hornstein, Stefan R. Pulver, Leslie C. Griffith

Department of Biology, Brandeis

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

JoVE 4093 10/06/2012

Benedetta Artegiani*, Christian Lange*, Federico Calegari

DFG - Research Center and Cluster of Excellence for Regenerative Therapies Dresden, Germany

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

JoVE 4312 12/05/2012

Marin Manuel, C.J. Heckman

Department of Physiology, Northwestern University Feinberg School of Medicine

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

JoVE 3312 2/29/2012

Xiangrui Li¹, Zhong-Lin Lu^2,3,4,5

¹Center for Cognitive and Behavioral Brain Imaging, The Ohio State University, ²Department of Psychology, University of Southern California, ³Biomedical Engineering, University of Southern California, ⁴Neuroscience Graduate Program, University of Southern California, ⁵Department of Psychology, The Ohio State University

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

JoVE 3143 11/11/2011

Giselle Cheung, Michael A. Cousin

Membrane Biology Group, Centre for integrative Physiology, University of Edinburgh

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

JoVE 4390 1/15/2013

Wei Song*^1,2, Qing Wei*¹, Shuliang Jiao³, Hao F. Zhang^1,4

¹Department of Biomedical Engineering, Northwestern University, ²Department of Physics, Harbin Institute of Technology, ³Department of Ophthalmology, University of Southern California, ⁴Department of Ophthalmology, Northwestern University

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

JoVE 50022 2/04/2013

Shivendra Pandey¹, Evin Gultepe¹, David H. Gracias^1,2

¹Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, ²Department of Chemistry, The Johns Hopkins University

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

In Vivo Canine Muscle Function Assay

JoVE 2623 4/05/2011

Martin K. Childers¹, Robert W. Grange², Joe N. Kornegay³

¹Department of Neurology and Wake Forest Institute for Regenerative Medicine, Wake Forest University, ²Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, ³Departments of Pathology and Laboratory Medicine and Neurology and the Gene Therapy Center , University of North Carolina-Chapel Hill

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

High Density Event-related Potential Data Acquisition in Cognitive Neuroscience

JoVE 1945 4/16/2010

Scott D. Slotnick

Department of Psychology, Boston College

Event-related potential (ERP) recording is under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and this method often has poor spatial resolution. To foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

JoVE 2773 6/29/2011

Adam J. Kole¹, Elizabeth R.W. Knight², Mohanish Deshmukh¹

¹Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina, ²Curriculum in Neurobiology, Neuroscience Center, University of North Carolina

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

In vivo Neuronal Calcium Imaging in C. elegans

JoVE 50357 4/10/2013

Samuel H. Chung*^1,2, Lin Sun*^1,2, Christopher V. Gabel^1,2

¹Department of Physiology and Biophysics, Boston University School of Medicine, ²Boston University Photonics Center

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

JoVE 3807 2/08/2012

Bradley I. Coleman, Marc-Jan Gubbels

Department of Biology, Boston College

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Three-dimensional Optical-resolution Photoacoustic Microscopy

JoVE 2729 5/03/2011

Song Hu, Konstantin Maslov, Lihong V. Wang

Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. Louis

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Shape Memory Polymers for Active Cell Culture

JoVE 2903 7/04/2011

Kevin A. Davis, Xiaofan Luo, Patrick T. Mather, James H. Henderson

Department of Biomedical and Chemical Engineering, Syracuse Biomaterials Institute

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

JoVE 3128 9/20/2011

Xuehua Xu, Tian Jin

Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

JoVE 3754 5/03/2012

Sandeep Sharma, Cecile Hryhorczuk, Stephanie Fulton

CRCHUM and the Montreal Diabetes Research Center, University of Montreal

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

JoVE 4449 11/05/2012

Jeremy Willis*, Darla DeStephanis*, Yogin Patel, Vrushab Gowda, Shan Yan

Department of Biology, University of North Carolina at Charlotte

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

JoVE 3893 8/24/2012

Daniela Schiller¹, Candace M. Raio², Elizabeth A. Phelps³

¹Departments of Psychiatry and Neuroscience, and Friedman Brain Institute, Mt. Sinai School of Medicine, ²Department of Psychology, New York University, ³Department of Psychology and Center for Neural Science, New York University

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

JoVE 2900 6/10/2011

Pooneh Bagher¹, Luis Polo-Parada^1,2, Steven S. Segal^1,2

¹Department of Medical Pharmacology and Physiology, University of Missouri, ²Dalton Cardiovascular Research Center, University of Missouri

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

January 2012: This Month in JoVE

JoVE 4194 1/02/2012

Aaron Kolski-Andreaco

Here are some highlights from the January 2012 Issue of Journal of Visualized Experiments (JoVE).

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

JoVE 3237 6/20/2012

Joseph F.X. DeSouza^1,2, Shima Ovaysikia¹, Laura K. Pynn¹

¹Department of Psychology, Centre for Vision Research, York University, ²Department of Biology, Centre for Vision Research, York University

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

JoVE 50018 2/12/2013

Shivanjali Joshi-Barr¹, Jerome V. Karpiak², Yogesh Ner¹, Jessica H. Wen³, Adam J. Engler³, Adah Almutairi^1,2

¹Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, ²Biomedical Sciences Program, University of California, San Diego, ³Department of Bioengineering, University of California, San Diego

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

JoVE 4418 11/26/2012

Ulrike Pannasch*¹, Jérémie Sibille*^1,2, Nathalie Rouach¹

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.

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

JoVE 1142 4/26/2009

Eiji Shigetomi, Baljit S. Khakh

Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

MISSION esiRNA for RNAi Screening in Mammalian Cells

JoVE 2008 5/12/2010

Mirko Theis, Frank Buchholz

Max Planck Institute of Molecular Cell Biology and Genetics

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Visualization of Vascular Ca²⁺ Signaling Triggered by Paracrine Derived ROS

JoVE 3511 12/21/2011

Karthik Mallilankaraman¹, Rajesh Kumar Gandhirajan¹, Brian J. Hawkins², Muniswamy Madesh¹

¹Department of Biochemistry, Temple University, ²Department of Anesthesiology and Pain Medicine, University of Washington

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

CryoStor Cryopreservation Protocol - ADVERTISEMENT

JoVE 2206 10/07/2010

Aby Mathew

BioLife Solutions, Inc.

CryoStor cryopreservation solutions are used to prepare and preserve cells in ultra low temperature environments, without the need for serum, proteins, or high levels of cytotoxic agents.

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

JoVE 2785 6/02/2011

Maria Muccioli*¹, Michelle Pate*², Omowaleola Omosebi², Fabian Benencia^1,2,3

¹Molecular and Cell Biology Program, Ohio University, ²Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, ³Department of Biomedical Engineering, Russ College of Engineering and Technology, Ohio University

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

JoVE 4197 9/21/2012

Serendipity Zapanta Rinonos^1,2, Jeremy Saks¹, Jonida Toska³, Chun-Lun Ni^1,2, Alan M. Tartakoff^1,2

¹Department of Pathology, Case Western Reserve University School of Medicine, ²Cell Biology Program, Case Western Reserve University School of Medicine, ³Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Loading Drosophila Nerve Terminals with Calcium Indicators

JoVE 250 7/30/2007

Adam J. Rossano, Gregory T. Macleod

Department of Physiology, University of Texas Health Science Center at San Antonio (UTHSCSA)

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca²⁺-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

JoVE 3954 5/20/2012

Ming Yang¹, Peter C. Stapor¹, Shayn M. Peirce², Aline M. Betancourt³, Walter L. Murfee¹

¹Department of Biomedical Engineering, Tulane University, ²Department of Biomedical Engineering, University of Virginia, ³Center for Stem Cell Research and Regenerative Medicine, Tulane University

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.