University of Kentucky View Institution's Website 49 articles published in JoVE Biology Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens Renata Belisário1, Maria F. Torres1,2, Ester A. S. Buiate1,3, Katia V. Xavier1,4, Etta M. Nuckles1, Lisa J. Vaillancourt1 1Department of Plant Pathology, University of Kentucky, 2Department of Biological Sciences, University of Cincinnati, 3Bayer Crop Science, 4Everglades Research and Education Center, University of Florida This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues. Bioengineering Equipment Setup and Artifact Removal for Simultaneous Electroencephalogram and Functional Magnetic Resonance Imaging for Clinical Review in Epilepsy Jihye Bae1, Jordan L. Clay2, Bhoj Raj Thapa1, David Powell3, Heidi Turpin3, Saghi Tasori Partovi3, Rachel Ward-Mitchell3, Balu Krishnan4, Andreas Koupparis5, Meriem Bensalem Owen2, Flavius D. Raslau2,6,7 1Department of Electrical and Computer Engineering, University of Kentucky, 2Department of Neurology, University of Kentucky, 3Department of Neuroscience, University of Kentucky, 4Cleveland Clinic, 5Cyprus Institute of Neurology and Genetics, 6Department of Radiology, University of Kentucky, 7Department of Neurosurgery, University of Kentucky This article details simultaneous electroencephalogram and functional magnetic resonance imaging (EEG-fMRI) recording procedures that can be used in both clinical and research settings. EEG processing procedures to remove imaging artifacts for clinical review are also included. This study focuses on the example of epilepsy during the interictal period. Medicine Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis Smita Joshi*1, Alexis N. Smith1, Kanakanagavalli Shravani Prakhya1, Hammodah R. Alfar1, Joshua Lykins1, Ming Zhang1, Irina Pokrovskaya2, Maria Aronova3, Richard D. Leapman3, Brian Storrie2, Sidney W. Whiteheart*1 1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis. Neuroscience Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice Teresa Macheda1, Kelly Roberts1, Adam D. Bachstetter1,2,3 1Spinal Cord & Brain Injury Research Center, University of Kentucky, 2Department of Neuroscience, University of Kentucky, 3Sanders-Brown Center on Aging, University of Kentucky The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained. Neuroscience Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin Brandon A. Miller1,2, Shelei Pan3, Peter H. Yang3, Catherine Wang1, Amanda L. Trout1, Dakota DeFreitas3, Sruthi Ramagiri3, Scott D. Olson2, Jennifer M. Strahle3,4,5 1Department of Neurosurgery, University of Kentucky, 2Department of Pediatric Surgery, University of Texas, 3Department of Neurological Surgery, Washington University in St. Louis School of Medicine, 4Department of Orthopedic Surgery, Washington University in St. Louis School of Medicine, 5Department of Pediatrics, Washington University in St. Louis School of Medicine We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans. Biology Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda Xien Chen1, Subba Reddy Palli1 1Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase. Developmental Biology Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish Rebecca A. Petersen1, Ann C. Morris1 1Department of Biology, University of Kentucky Here, a protocol is provided for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. Biology In vivo Measurement of Knee Extensor Muscle Function in Mice Camille R. Brightwell1,2, Ted G. Graber3, Benjamin D. Brightwell4,5, Matthew Borkowski6, Brian Noehren5,7, Christopher S. Fry1,2 1Department of Athletic Training and Clinical Nutrition, University of Kentucky, 2Center for Muscle Biology, University of Kentucky, 3Department of Physical Therapy, East Carolina University, 4Kinesiology and Health Promotion Graduate Program, University of Kentucky, 5Biomotion Lab, College of Health Sciences, University of Kentucky, 6Aurora Scientific, 7Department of Physical Therapy, College of Health Sciences, University of Kentucky Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque. Neuroscience Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells Carolina Galeano-Naranjo1,2, A. Catalina Veléz-Ortega1, Gregory I Frolenkov1 1Department of Physiology, College of Medicine, University of Kentucky, 2Universidad Nacional de Colombia Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells. Bioengineering Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells Joree N. Sandin1, Surya P. Aryal2, Thomas Wilkop3,4, Christopher I. Richards2,4, Martha E. Grady1 1Department of Mechanical Engineering, University of Kentucky, 2Department of Chemistry, University of Kentucky, 3Department of Physiology, University of Kentucky, 4UK Light Microscopy Core, University of Kentucky Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples. Environment Simulating Impacts of Ice Storms on Forest Ecosystems John L. Campbell1, Lindsey E. Rustad1, Charles T. Driscoll2, Ian Halm3, Timothy J. Fahey4, Habibollah Fakhraei5, Peter M. Groffman6,7, Gary J. Hawley8, Wendy Leuenberger9, Paul G. Schaberg10 1Northern Research Station, U.S. Forest Service, Durham, NH, 2Department of Civil and Environmental Engineering, Syracuse University, 3Northern Research Station, U.S. Forest Service, North Woodstock, NH, 4Department of Natural Resources, Cornell University, 5Department of Civil and Environmental Engineering, Southern Illinois University, 6Advanced Science Research Center at the Graduate Center, City University of New York, 7Cary Institute of Ecosystem Studies, 8Rubenstein School of Environment and Natural Resources, University of Vermont, 9Department of Forestry and Natural Resources, University of Kentucky, 10Northern Research Station, U.S. Forest Service, Burlington, VT Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions. Neuroscience Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia Bei Zhang1, Binoy Joseph2, Kathryn E. Saatman2, Lei Chen2 1College of Public Health, Shaanxi University of Chinese Medicine, 2Spinal Cord and Brain Injury Research Center, Department of Physiology, University of Kentucky A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery. Medicine Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease Jonathan H. Chung1, Christopher M. Walker2, Stephen Hobbs3 1Department of Radiology, University of Chicago Medicine, 2Department of Radiology, University of Kansas Medical Center, 3Department of Radiology, University of Kentucky Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease. Biology High-Throughput Assays of Critical Thermal Limits in Insects David N. Awde1, Tatum E. Fowler1, Fernan Pérez-Gálvez1, Mark J. Garcia1, Nicholas M. Teets1 1Department of Entomology, University of Kentucky Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays. Cancer Research Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish Meghan G. Haney1,2, L. Henry Moore1, Jessica S. Blackburn1,2 1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Markey Cancer Center, University of Kentucky Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit. Biochemistry Use of Alu Element Containing Minigenes to Analyze Circular RNAs Justin R. Welden1, Anna Pawluchin1, Jacob van Doorn1, Stefan Stamm1 1Institute for Molecular and Cellular Biochemistry, University of Kentucky We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA. Immunology and Infection Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis Gabrielle Keb1, Kenneth A. Fields1 1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM. Bioengineering Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier Wanzhu Zhao1, Linjiang Han1, Younsoo Bae2, Devika S. Manickam1 1Department of Pharmaceutical, Administrative and Social Sciences, Duquesne University, 2Department of Pharmaceutical Sciences, University of Kentucky We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells. Bioengineering Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes Joseph S. Najem1,2, Graham J. Taylor2,3, Nick Armendarez4, Ryan J. Weiss5, Md Sakib Hasan5, Garrett S. Rose5, Catherine D. Schuman6, Alex Belianinov7, Stephen A. Sarles2, C. Patrick Collier2,3,7 1Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, 3Bredesen Center for Interdisciplinary Research, University of Tennessee, 4Department of Biosystems and Agriculture Engineering, University of Kentucky, 5Department of Electrical Engineering and Computer Science, University of Tennessee, 6Computer Science and Mathematics Division, Oak Ridge National Laboratory, 7Center for Nanophase Materials Sciences, Oak Ridge National Laboratory Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane. Medicine Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions Hisashi Sawada1, Jeff Z. Chen2, Bradley C. Wright1, Jessica J. Moorleghen1, Hong S. Lu1,2, Alan Daugherty1,2 1Saha Cardiovascular Research Center, University of Kentucky, 2Department of Physiology, University of Kentucky Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice. Biochemistry Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies K. Martin Chow1, Sidney W. Whiteheart1, Jeffrey R. Smiley2, Savita Sharma1, Kathy Boaz2, Meggie J. Coleman2, Alvina Maynard3, Louis B. Hersh1, Craig W. Vander Kooi1 1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Division of Laboratory Animal Resources, University of Kentucky, 3River Hill Ranch A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described. Biology Bovine Mammary Gland Biopsy Techniques Veridiana L. Daley1,4, Charles Dye2, Sophie H. Bogers3, R. Michael Akers4, Francisco C. Rodriguez3, John P. Cant5, John Doelman5, Peter Yoder4, Karan Kumar2, Dane Webster6, Mark D. Hanigan4 1National Animal Nutrition Program, a National Research Support Project (NRSP-9), Department of Animal and Food Sciences, University of Kentucky, 2School of Performing Arts, Virginia Tech, 3Department of Large Animal Clinical Sciences, The Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 4Department of Dairy Science, Virginia Tech, 5Department of Animal Biosciences, University of Guelph, 6School of Visual Arts, Virginia Tech This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations. Neuroscience Isolation of Cerebral Capillaries from Fresh Human Brain Tissue Anika M.S. Hartz1, Julia A. Schulz2, Brent S. Sokola2, Stephanie E. Edelmann1, Andrew N. Shen1, Ralf G. Rempe2, Yu Zhong1, Nader El Seblani3, Bjoern Bauer2 1Sanders-Brown Center on Aging, Department of Pharmacology and Nutritional Sciences, University of Kentucky, 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 3Department of Neuroscience, University of Kentucky Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue. Medicine Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans Vedant A. Gupta1, Steve W. Leung1, Michael A. Winkler2, Vincent L. Sorrell1 1Gill Heart Institute, Division of Cardiovascular Medicine, University of Kentucky, 2Department of Radiology, University of Kentucky Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes. Environment Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar Carrie A. Deans1,2, Gregory A. Sword1, Paul A. Lenhart3, Eric Burkness2, William D. Hutchison2, Spencer T. Behmer1 1Department of Entomology, Texas A&M University, 2Department of Entomology, University of Minnesota, 3Department of Entomology, University of Kentucky The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology. Bioengineering Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana B. K. Amos1, Victoria G. Pook1, Seth Debolt1 1Department of Horticulture, University of Kentucky A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology. Developmental Biology Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles Richard D. Andreatta1, Joseph C. Stemple1, Tanya S. Seward2, Colleen A. McMullen2 1Department of Rehabilitation Sciences, University of Kentucky, 2Department of Physiology, University of Kentucky Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles. Biology Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking Ashley M. Fox-Loe1, Brandon J. Henderson2, Christopher I. Richards1 1Department of Chemistry, University of Kentucky, 2Department of Biomedical Sciences, Marshall University Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane. Medicine Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice Hong Lu1, Deborah A. Howatt1, Anju Balakrishnan1, Jessica J. Moorleghen1, Debra L. Rateri1, Lisa A. Cassis2, Alan Daugherty1 1Saha Cardiovascular Research Center, University of Kentucky, 2Department of Pharmacology and Nutritional Sciences, University of Kentucky Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice. Biology Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng1, Paul M.L. Janssen2, Robert W. Grange3, Lin Yang4, Alan H. Beggs5, Lindsay C. Swanson5, Stacy A. Cossette1,6, Alison Frase7, Martin K. Childers8, Henk Granzier9, Emanuela Gussoni5, Michael W. Lawlor1 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5 The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies. Biology A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits Rekha Kushwaha1, Kim R. Schäfermeyer1, A. Bruce Downie1 1Department of Horticulture, University of Kentucky Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors. Neuroscience Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis Josh Titlow1, Zana R. Majeed1,2, John G Nicholls3, Robin L. Cooper1 1Department of Biology, University of Kentucky, 2Department of Biology, College of Science, University of Salahaddin, Iraq, 3Department of Neurobiology and Cognitive Neuroscience, SISSA, Italy This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields. Neuroscience Neural Circuit Recording from an Intact Cockroach Nervous System Josh S. Titlow1, Zana R. Majeed1,2, H. Bernard Hartman3, Ellen Burns1, Robin L. Cooper1 1Department of Biology, University of Kentucky, 2Department of Biology, University of Salahaddin, 3Oregon Institute of Marine Biology, University of Oregon This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci. Neuroscience Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises Zana R. Majeed1,2, Josh Titlow1,2, H. Bernard Hartman3, Robin Cooper1,2 1Department of Biology, University of Kentucky, 2Center of Muscle Biology, University of Kentucky, 3Oregon Institute of Marine Biology, University of Oregon Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs. Medicine Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model Alexandra Amaro-Ortiz1,2, Jillian C. Vanover1,3, Timothy L. Scott1,2, John A. D'Orazio1,2,3,4 1The Markey Cancer Center, University of Kentucky College of Medicine, 2Graduate Center for Toxicology, University of Kentucky College of Medicine, 3Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 4Department of Pediatrics, University of Kentucky College of Medicine Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay. Immunology and Infection Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food Elsa N. Bou Ghanem1, Tanya Myers-Morales1, Grant S. Jones1, Sarah E.F. D'Orazio1 1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens. Immunology and Infection Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells Gavin I. Ellis1, Mary Catherine Reneer1, Alejandra Catalina Vélez-Ortega1, Andrea McCool1, Francesc Martí1 1Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here. Neuroscience Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice Janet Alder1, Wendy Fujioka1, Jonathan Lifshitz2,3, David P. Crockett1, Smita Thakker-Varia1 1Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2Spinal Cord and Brain Injury Research Center, 3Department of Anatomy and Neurobiology, Department of Physical Medicine and Rehabilitation, University of Kentucky Chandler Medical Center Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described. Biology Mitochondrial Isolation from Skeletal Muscle Mary L. Garcia-Cazarin*1, Natalie N. Snider*1, Francisco H. Andrade1 1Department of Physiology, University of Kentucky This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour. Neuroscience Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology Diana M. Mathis1, Jennifer L. Furman2, Christopher M. Norris2,3 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. Biology Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises Martha M. Robinson1, Jonathan M. Martin1, Harold L. Atwood2, Robin L. Cooper1 1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories. Neuroscience Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise Ann S. Cooper*1, Bonnie Leksrisawat*1, Allison B. Gilberts*1, A. Joffre Mercier*2, Robin L. Cooper*1 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. Neuroscience Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises Brittany Baierlein*1, Alison L. Thurow*1, Harold L. Atwood*2, Robin L. Cooper*1 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. Neuroscience Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception Bonnie Leksrisawat*1, Ann S. Cooper*1, Allison B. Gilberts*1, Robin L. Cooper*1 1Department of Biology, University of Kentucky The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope. Neuroscience Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle Wen Hui Wu*1, Robin L. Cooper*1 1Department of Biology, University of Kentucky This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals. Biology Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies Ann S. Cooper1, Kylah E. Rymond1, Matthew A. Ward1, Easter L. Bocook1, Robin L. Cooper1 1Department of Biology, University of Kentucky, Lexington We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies. Biology Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle Ann S. Cooper1, Robin L. Cooper1 1Department of Biology, University of Kentucky The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity. Biology Measures of Heart and Ventilatory Rates in Freely Moving Crayfish Sonya M. Bierbower1, Robin L. Cooper1 1Department of Biology, University of Kentucky Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state. Biology Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method Alan Daugherty1, Debra Rateri1, Lu Hong1, Anju Balakrishnan1 1Cardiovascular Research Center, University of Kentucky The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.