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Washington State University

18 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers
Michael A. Muniak 1,2, Zachary M. Mayko 3, David K. Ryugo 2,4, Christine V. Portfors 3
1Department of Neuroscience, Johns Hopkins University, 2Garvan Institute of Medical Research, 3School of Biological Sciences, Washington State University, 4Department of Otolaryngology-HNS, Johns Hopkins University

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

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Neuroscience

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex
William C. Clegern 1, Michele E. Moore 1, Michelle A. Schmidt 1, Jonathan Wisor 1
1Department of Veterinary & Comparative Anatomy, Pharmacology and Physiology, Sleep and Performance Research Center, WWAMI Medical Education Program, Washington State University

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

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Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans
Marshall L. Deline 1,2, Tracy L. Vrablik 1,2, Jennifer L. Watts 1,2
1School of Molecular Biosciences, Washington State University, 2Center for Reproductive Biology, Washington State University

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

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Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells
Ryan L. Sontag 1, Cosmin Mihai 2, Galya Orr 2, Alexei Savchenko 3, Tatiana Skarina 3, Hong Cui 3, John R. Cort 1, Joshua N. Adkins 1, Roslyn N. Brown 4
1Biological Sciences Division, Pacific Northwest National Laboratory, 2Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, 3Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, 4Center for Bioproducts and Bioenergy, Washington State University

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

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Developmental Biology

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin
Lucia Salz 1,2, Ryan R. Driskell 1,2
1Centre for Stem Cells and Regenerative Medicine, King's College London, 2School of Molecular Biosciences, Washington State University

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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Developmental Biology

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice
Shuai Li 1, Wipawee Winuthayanon 1
1School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

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Environment

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System
Joseph B. DeShields 1, Rachel A. Bomberger 1, James W. Woodhall 2, David L. Wheeler 1, Natalia Moroz 1, Dennis A. Johnson 1, Kiwamu Tanaka 1
1Department of Plant Pathology, Washington State University, 2Parma Research and Extension Center, University of Idaho

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

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Immunology and Infection

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis
Aoi Hiroyasu 1, David C. DeWitt 2, Alan G. Goodman 1
1School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, 2Integrative Physiology and Neuroscience, College of Veterinary Medicine, Washington State University

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

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Behavior

Tickling, a Technique for Inducing Positive Affect When Handling Rats
Sylvie Cloutier 1,2, Megan R. LaFollette 3, Brianna N. Gaskill 3, Jaak Panksepp 1, Ruth C. Newberry 4
1Center for the Study of Animal Well-being, Department of Integrative Physiology and Neuroscience, Washington State University, 2Canadian Council on Animal Care, 3Department of Animal Sciences, Center for Animal Welfare Science, College of Agriculture, Purdue University, 4Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

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Environment

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils
Ricky W. Lewis *1, Anjuman A. Islam *1, Christine Jade Dilla-Ermita 2, Scot H. Hulbert 2, Tarah S. Sullivan 1
1Department of Crop and Soil Sciences, Washington State University, 2Department of Plant Pathology, Washington State University

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

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Immunology and Infection

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
Devanjali Dutta 1, Inha Heo 1,3, Roberta O'Connor 2
1Hubrecht Institute, Oncode Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), UMC Utrecht, 2Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, 3Janssen Pharmaceutica

We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.

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Immunology and Infection

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein
Vikramsingh Gujar *1, Michael B. Anderson *1, Kenneth E. Miller *1, Radhika D. Pande 1, Pranav Nawani 2, Subhas Das 1
1Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, 2Institute for Shock Physics, Washington State University

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 °C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

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JoVE Core

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
Nicholas R. Jaegers 1, Wenda Hu 2, Yong Wang 1, Jian Zhi Hu 1
1Pacific Northwest National Laboratory, 2Washington State University

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

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Chemistry

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping
Elias Nakouzi 1, Sakshi Yadav 1, Benjamin A. Legg 1, Shuai Zhang 1,2, Jinhui Tao 1, Christopher J. Mundy 1,3, Gregory K. Schenter 1,4, Jaehun Chun 1,5, James J. De Yoreo 1,2
1Physical and Computational Sciences Directorate, Pacific Northwest National Laboratory, 2Department of Materials Science and Engineering, University of Washington, 3Department of Chemical Engineering, University of Washington, 4Department of Chemistry, Washington State University, 5CUNY City College of New York

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

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Biochemistry

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
Matthew J. Gaffrey 1, Nicholas J. Day 1, Xiaolu Li 2, Wei-Jun Qian 1
1Integrative Omics, Biological Sciences Division, Pacific Northwest National Laboratory, 2Bioproducts Sciences and Engineering Laboratory, Department of Biological Systems Engineering, Washington State University

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

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Biochemistry

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription
Phillip Wibisono 1, Jingru Sun 1
1Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine, Washington State University

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

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Medicine

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients
Itsuma Nagao 1,2, Meg Nakazawa 1, Yoko M. Ambrosini 1
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, 2Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

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Medicine

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces
Gerald D. Dykstra *1, Minae Kawasaki *2, Yoko M. Ambrosini 2
1Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

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