Oregon Health and Science University

38 articles published in JoVE

• Medicine

  
  Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy Zhiqiang Hou¹, Yunpei Zhang¹, Lingling Neng¹, Jinhui Zhang, Xiaorui Shi^{1 1}Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

• Neuroscience

  
  Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy Suvithanandhini Loganathan*¹, Hannah E. Ball*¹, Ernesto Manzo^1,2, Daniela C. Zarnescu^{1,3 1}Department of Molecular and Cellular Biology, University of Arizona, Tucson, ²Vollum Institute, Oregon Health and Science University, ³Department of Neuroscience, University of Arizona, Tucson Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

• Immunology and Infection

  
  Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples Dario L. Frey*^1,2, Matteo Guerra*^1,2,3,4, Marcus A. Mall^1,2,5,6,7, Carsten Schultz^{1,3,8 1}Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), ²Dept. of Translational Pulmonology, University of Heidelberg, ³Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL), University of Heidelberg, ⁴Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University, University of Heidelberg, ⁵Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine, Charité – Universitätsmedizin Berlin, ⁶Berlin Institute of Health (BIH), ⁷German Center for Lung Research (DZL), Associated Partner Site, Berlin, ⁸Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

• Medicine

  
  Optical Clearing and Imaging of Immunolabeled Kidney Tissue Turgay Saritas¹, Victor G. Puelles^1,2,3, Xiao-Tong Su⁴, David H. Ellison^4,5,6, Rafael Kramann^{1,7 1}Division of Nephrology and Clinical Immunology, University Hospital RWTH Aachen, ²III. Department of Medicine, University Medical Center, Hamburg-Eppendorf, ³Department of Nephrology, Monash Health, ⁴Division of Nephrology and Hypertension, Oregon Health and Science University, ⁵Renal Section, Veterans Affairs Portland Health Care System, ⁶Fondation LeDucq Transatlantic Networks of Excellence, ⁷Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

• Neuroscience

  
  Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy Bart C. Jongbloets*¹, Lei Ma*¹, Tianyi Mao¹, Haining Zhong^{1 1}Vollum Institute, Oregon Health & Science University A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKARα, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

• Cancer Research

  
  Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer Rebecca Smith¹, Kaylyn Devlin¹, David Kilburn¹, Sean Gross¹, Damir Sudar², Elmar Bucher¹, Michel Nederlof², Mark Dane¹, Joe W. Gray¹, Laura Heiser¹, James E. Korkola^{1 1}Department of Biomedical Engineering, Knight Cancer Institute, Oregon Health and Science University, ²Quantitative Imaging Systems LLC The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

• Neuroscience

  
  Localization of the Locus Coeruleus in the Mouse Brain Katharina Schmidt¹, Bilal Bari², Martina Ralle³, Clorissa Washington-Hughes¹, Abigael Muchenditsi¹, Evan Maxey⁴, Svetlana Lutsenko^{1 1}Department of Physiology, Johns Hopkins University, School of Medicine, ²Department of Neuroscience, Johns Hopkins University, ³Department of Molecular and Medical Genetics, OHSU, ⁴X-ray science division, Advanced Photon Source, Argonne National Laboratory The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

• Neuroscience

  
  In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films Skyler L. Jackman¹, Christopher H. Chen², Wade G. Regehr^{2 1}Vollum Institute, Oregon Health and Science University, ²Harvard Medical School Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

• Biology

  
  Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors Maria M. Steele¹, Madeline J. Churchill², Alec P. Breazeale¹, Ryan S. Lane¹, Nicholas A. Nelson¹, Amanda W. Lund^{1,2,3,4 1}Department of Cell, Developmental, & Cancer Biology, Oregon Health and Science University, ²Department of Molecular Microbiology & Immunology, Oregon Health and Science University, ³Department of Dermatology, Oregon Health and Science University, ⁴Knight Cancer Institute, Oregon Health and Science University Here, we demonstrate the methods for in vivo quantification of leukocyte egress from naïve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

• Biology

  
  Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy Katsuyuki Matsushita¹, Kirsti Golgotiu¹, Daniel J. Orton², Richard D. Smith², Karin D. Rodland², Paul D. Piehowski², Michael P. Hutchens^{1,3 1}Anesthesiology & Perioperative Medicine, Oregon Health & Science University, ²Environmental and Biological Services Division, Pacific Northwest National Laboratory, ³Operative Care Division, Portland Veterans Affairs Medical Center We present use of 2-photon microscopy to place a micropipette within Bowman's urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

• Bioengineering

  
  Light-sheet Fluorescence Microscopy for the Study of the Murine Heart Yichen Ding¹, Zachary Bailey², Victoria Messerschmidt², Jun Nie³, Richard Bryant², Sandra Rugonyi⁴, Peng Fei³, Juhyun Lee^1,2, Tzung K. Hsiai^{1 1}Department of Bioengineering, University of California Los Angeles, ²Department of Bioengineering, University of Texas at Arlington, ³School of Optical and Electronic Information, Huazhong University of Science and Technology, ⁴Department of Biomedical Engineering, OSHU This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

• Behavior

  
  Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data Jeffrey Kaye¹, Christina Reynolds¹, Molly Bowman¹, Nicole Sharma¹, Thomas Riley¹, Ona Golonka¹, Jonathan Lee¹, Charlie Quinn¹, Zachary Beattie¹, Johanna Austin¹, Adriana Seelye¹, Katherine Wild¹, Nora Mattek^{1 1}Department of Neurology, ORCATECH - Oregon Center for Aging & Technology, Oregon Health & Science University Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use.

• Biology

  
  A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins John M. Powers¹, Xiao Lan Chang¹, Zhen Song¹, Hiroyuki Nakai^{1,2 1}Department of Molecular and Medical Genetics, Oregon Health & Science University, ²Department of Molecular Microbiology and Immunology, Oregon Health & Science University This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

• Medicine

  
  A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta Bailey Simon*¹, Matthew Bucher*², Alina Maloyan^{1 1}Knight Cardiovascular Institute, Oregon Health and Science University, ²Department of Obstetrics and Gynecology, Oregon Health and Science University Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNFα recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

• Biology

  
  An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus Shane C. McAllister¹, Ryan S. Hanson¹, Kyleen N. Grissom¹, Sara Botto², Ashlee V. Moses^{2 1}Division of Pediatric Infectious Diseases, University of Minnesota Medical School, ²Vaccine and Gene Therapy Institute, Oregon Health and Science University Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

• Cancer Research

  
  Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Charles E. Gast¹, Aubie K. Shaw^1,2, Melissa H. Wong^1,3, Lisa M. Coussens^{1,3 1}Cell, Developmental & Cancer Biology, Oregon Health & Science University, ²University of Minnesota, ³Knight Cancer Institute, Oregon Health & Science University Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

• Biology

  
  Visualizing the Early Stages of Phagocytosis Ali Rashidfarrokhi*², Veronica Richina*², Fikadu G. Tafesse^{1,2 1}Department of Molecular Microbiology & Immunology, Oregon Health & Science University, ²Ragon Institute of MGH, MIT and Harvard Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

• Cancer Research

  
  A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer Erica T. Goddard¹, Jacob Fischer¹, Pepper Schedin^{1 1}Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

• Neuroscience

  
  Mass Histology to Quantify Neurodegeneration in Drosophila Elizabeth R. Sunderhaus¹, Doris Kretzschmar^{1 1}Oregon Institute of Occupational Health Sciences, Oregon Health & Sciences University Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

• Biochemistry

  
  Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram Jonathan A. Coleman¹, Evan M. Green², Eric Gouaux^{1,3 1}Vollum Institute, Oregon Health & Science University, ²Graduate Group in Biophysics, University of California, San Francisco, ³Howard Hughes Medical Institute, Oregon Health & Science University This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

• Bioengineering

  
  Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) Andrew Nickerson^1,2,3, Tao Huang^1,2,3, Li-Jung Lin^1,2,3, Xioalin Nan^{1,2,3 1}Department of Biomedical Engineering, Oregon Health and Science University, ²Knight Cancer Institute, Oregon Health and Science University, ³OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

• Biology

  
  Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle William C.W. Chen¹, Arman Saparov^2,3, Mirko Corselli⁴, Mihaela Crisan⁵, Bo Zheng⁶, Bruno Péault^7,8, Johnny Huard^{9 1}Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, ²Department of Orthopedic Surgery, University of Pittsburgh, ³Nazarbayev University Research and Innovation System, Nazarbayev University, ⁴Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, ⁵Department of Cell Biology, Erasmus MC Stem Cell Institute, ⁶OHSU Center for Regenerative Medicine, Oregon Health & Science University, ⁷ Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

• Neuroscience

  
  Super-resolution Imaging of Neuronal Dense-core Vesicles Bethe A. Scalettar^1,2, Daniel Shaver², Stefanie Kaech³, Janis E. Lochner^{2,4 1}Department of Physics, Lewis & Clark College, ²Program in Biochemistry and Molecular Biology, Lewis & Clark College, ³Jungers Center for Neuroscience Research, Oregon Health & Science University, ⁴Department of Chemistry, Lewis & Clark College We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

• Bioengineering

  
  Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy Esra Güç*¹, Manuel Fankhauser*¹, Amanda W. Lund^1,2, Melody A. Swartz¹, Witold W. Kilarski^{1 1}Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, ²Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

• Bioengineering

  
  Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope Kevin G. Phillips^1,2, Sandra M. Baker-Groberg¹, Owen J.T. McCarty^{1,3 1}Department of Biomedical Engineering, Oregon Health & Science University, School of Medicine, ²Department of Dermatology, Oregon Health & Science University, School of Medicine, ³Department of Cell & Developmental Biology, Division of Hematology & Medical Oncology, Knight Cancer Institute, Oregon Health & Science University, School of Medicine We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

• Neuroscience

  
  Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways Stacy L. Fairbanks*¹, Rebekah Vest*¹, Saurabh Verma², Richard J. Traystman^1,3, Paco S. Herson^{1,3 1}Department of Anesthesiology, University of Colorado School of Medicine, ²Oregon National Primate Research Center, Oregon Health & Science University, ³Department of Pharmacology, University of Colorado School of Medicine Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

• Neuroscience

  
  Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons Robert P. Irwin¹, Charles N. Allen^{1,2 1}Center 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.

• Immunology and Infection

  
  Tractable Mammalian Cell Infections with Protozoan-primed Bacteria Samuel L. Drennan¹, Amrita Lama¹, Ben Doron¹, Eric D. Cambronne^{1 1}Department of Molecular Microbiology & Immunology, Oregon Health & Science University This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

• Medicine

  
  A Simple Method of Mouse Lung Intubation Sandhya Das¹, Kelvin MacDonald², Herng-Yu Sucie Chang¹, Wayne Mitzner^{1 1}Department of Environmental Health Sciences, Program in Respiratory Biology and Lung Disease, Johns Hopkins Bloomberg School of Public Health, ²Department of Pediatrics, Oregon Health Sciences University This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

• Biology

  
  Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays Katharina L. Dürr^1,2, Neslihan N. Tavraz¹, Susan Spiller¹, Thomas Friedrich^{1 1}Institute of Chemistry, Technical University of Berlin, ²The Vollum Institute, Oregon Health & Science University We describe a method to quantify the activity of K⁺-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb⁺ or Li⁺ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

• Biology

  
  Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization Leslie Smith¹, Mathew Thayer^{1 1}Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

• Medicine

  
  Doppler Optical Coherence Tomography of Retinal Circulation Ou Tan¹, Yimin Wang¹, Ranjith K. Konduru², Xinbo Zhang¹, SriniVas R. Sadda², David Huang^{1 1}Department of Ophthalmology, Oregon Health and Science University, ²Department of Ophthalmology, University of Southern California Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

• Neuroscience

  
  Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation Lingyan Wang¹, Han Jiang¹, John V. Brigande^{1 1}Oregon Hearing Research Center, Oregon Health & Science University The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

• Neuroscience

  
  Patch-clamp Capacitance Measurements and Ca²⁺ Imaging at Single Nerve Terminals in Retinal Slices Mean-Hwan Kim¹, Evan Vickers¹, Henrique von Gersdorff^{1 1}The Vollum Institute, Oregon Health and Science University Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca²⁺ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

• Medicine

  
  Method to Measure Tone of Axial and Proximal Muscle Victor S. Gurfinkel¹, Timothy W. Cacciatore², Paul J. Cordo¹, Fay B. Horak^{3 1}Department of Biomedical Engineering, Oregon Health and Science University, ²UCL Institute of Neurology, Queen Square, ³Department of Neurology, Oregon Health and Science University We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

• Medicine

  
  Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury Michael P. Hutchens¹, Richard J. Traystman², Tetsuhiro Fujiyoshi¹, Shin Nakayama¹, Paco S. Herson^{1 1}Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, ²Department of Pharmacology, University of Colorado Denver A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

• Neuroscience

  
  Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish Hua Wen¹, Paul Brehm^{1 1}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.

• Neuroscience

  
  DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices Gail K. Seabold¹, James B. Daunais², Andrew Rau³, Kathleen A. Grant³, Veronica A. Alvarez^{1 1}Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, ²Department Physiology and Pharmacology, Wake Forest University Health Sciences, ³Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.