David Geffen School of Medicine, University of California, Los Angeles

13 ARTICLES PUBLISHED IN JoVE

Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

¹Departments 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.

Biology

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Harpreet Singh¹, Sarah Warburton², Thomas M. Vondriska³, Baljit S. Khakh¹

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.

Biology

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Marino DiFranco¹, Marbella Quinonez¹, Joana Capote¹, Julio Vergara¹

¹Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Medicine

Quantitative Analysis of Chromatin Proteomes in Disease

Emma Monte¹, Haodong Chen¹, Maria Kolmakova¹, Michelle Parvatiyar¹, Thomas M. Vondriska^1,2,3, Sarah Franklin^1,4

¹Department of Anesthesiology, David Geffen School of Medicine at UCLA, ²Department of Medicine, David Geffen School of Medicine at UCLA, ³Department of Physiology, David Geffen School of Medicine at UCLA, ⁴Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Neuroscience

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Ruotian Jiang¹, Martin D. Haustein¹, Michael V. Sofroniew², Baljit S. Khakh^1,2

¹Department of Physiology, University of California Los Angeles, ²Department of Neurobiology, University of California Los Angeles

The properties and functions of astrocyte intracellular Ca²⁺ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca²⁺ signals within striatal astrocytes in situ.

Neuroscience

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

J. Christopher Octeau¹, Guido Faas², Istvan Mody^1,2, Baljit S. Khakh^1,3

¹Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, ²Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, ³Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles

Potassium ions contribute to the resting membrane potential of cells and extracellular K⁺ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K⁺-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K⁺ concentration dynamics in adult hippocampal slices.

Cancer Research

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Larry C. Cheng^*1,2, Zhen Li^*3, Thomas G. Graeber⁴, Nicholas A. Graham⁵, Justin M. Drake^1,2,3,6,7

¹Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, ²Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, ³Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, ⁴Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, ⁵Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, ⁶Pharmacology, Rutgers Robert Wood Johnson Medical School, ⁷Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Neuroscience

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Stefanie L. Moye¹, Blanca Diaz-Castro¹, Mohitkumar R. Gangwani¹, Baljit S. Khakh^1,2

¹Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, ²Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

Cancer Research

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Moe Ishihara^*1, Junhui Hu^*1, Xiaoyu Zhang², YongHyeon Choi³, Anthony Wong⁴, Celine Cano-Ruiz⁵, Rongwei Zhao⁶, Ping Tan⁷, Jonathan L. Tso¹, Lily Wu^1,8

¹Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, ²Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, ³Department of Bioengineering, Hanyang University, ⁴Department of Ecology and Evolutionary Biology, University of California, Los Angeles, ⁵Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, ⁶School of Life Sciences, Beijing Normal University, ⁷Department of Urology, West China Hospital, Sichuan University, ⁸Department of Urology, David Geffen School of Medicine, University of California, Los Angeles

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Cancer Research

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Allison C. Sharrow¹, Moe Ishihara¹, Junhui Hu¹, Il Hyun Kim¹, Lily Wu¹

¹Molecular and Medical Pharmacology, University of California Los Angeles

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Behavior

A Step-by-Step Implementation of DeepBehavior, Deep Learning Toolbox for Automated Behavior Analysis

Sanjay Shukla¹, Ahmet Arac¹

¹Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles

The purpose of this protocol is to utilize pre-built convolutional neural nets to automate behavior tracking and perform detailed behavior analysis. Behavior tracking can be applied to any video data or sequences of images and is generalizable to track any user-defined object.

Neuroscience

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Bijay Parajuli^1,2, Youichi Shinozaki^1,2, Eiji Shigetomi^1,2, Schuichi Koizumi^1,2

¹Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine, University of Yamanashi, ²GLIA Center, University of Yamanashi

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Neuroscience

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Satoshi Manita¹, Eiji Shigetomi^2,3, Haruhiko Bito⁴, Schuichi Koizumi^2,3, Kazuo Kitamura¹

¹Department of Neurophysiology, Faculty of Medicine, University of Yamanashi, ²Department of Neuropharmacology, Faculty of Medicine, University of Yamanashi, ³Yamanashi GLIA center, Interdisciplinary Graduate School of Medicine, University of Yamanashi, ⁴Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo

The present protocol describes making a large (6 x 3 mm²) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.