Doshisha University

14 ARTICLES PUBLISHED IN JoVE

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

¹Disease Mechanism Research Core, RIKEN Brain Science Institute, ²Graduate School of Science and Engineering, Saitama University

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Cagri Yalgin^1,2, M. Rezaul Karim^1,2, Adrian W. Moore¹

¹Disease Mechanism Research Core, RIKEN Brain Science Institute, ²Graduate School of Science and Engineering, Saitama University

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Neuroscience

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

M. Rezaul Karim¹, Keita Endo², Adrian W Moore³, Hiroaki Taniguchi¹

¹Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha University, ²Laboratory for Circuit Mechanisms of Sensory Perception, RIKEN Brain Science Institute, ³Disease Mechanism Research Core, RIKEN Brain Science Institute

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Developmental Biology

An Efficient Method to Obtain Dedifferentiated Fat Cells

Hiroaki Taniguchi^*1, Tomohiko Kazama^*1, Kazuhiro Hagikura^*1, Chii Yamamoto¹, Minako Kazama¹, Yuki Nagaoka¹, Taro Matsumoto¹

¹Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Developmental Biology

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

M. Rezaul Karim¹, Tomohiro Haruta², Taro Matsumoto³, Hiroki Oda², Hiroaki Taniguchi^3,4,5

¹Biotechnology and Genetic Engineering Department, Jahangirnagar University, ²JT Biohistory Research Hall, ³Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, ⁴Graduate School of Life and Medical Sciences, Doshisha University, ⁵Institute of Genetics and Animal Breeding of the Polish Academy of Sciences

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Biochemistry

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Megumi Tsuchiya^*1, M. Rezaul Karim^*2, Taro Matsumoto³, Hidesato Ogawa¹, Hiroaki Taniguchi^3,4,5

¹Graduate School of Frontier Biosciences, Osaka University, ²Department of Biotechnology and Genetic Engineering, Jahangirnagar University, ³Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, ⁴Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, ⁵Graduate School of Life and Medical Sciences, Doshisha University

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Immunology and Infection

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Minas Nalbandian¹, Zsolt Radak², Masaki Takeda³

¹Graduate School of medicine, Kyoto University, ²Research Institute of Sports Science, University of Physical Education, ³Graduate School of Sports and Health Science, Doshisha University

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

Neuroscience

Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Masaya Ikegawa^*1, Takashi Nirasawa^*2, Nobuto Kakuda¹, Tomohiro Miyasaka¹, Yuki Kuzuhara¹, Shigeo Murayama³, Yasuo Ihara⁴

¹Department of Life and Medical Systems, Doshisha University, ²Bruker Daltonics K.K., ³The Brain Bank for Aging Research, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, ⁴Graduate School of Brain Science, Doshisha University

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Biochemistry

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Takehiro Yamazaki^*1, Hiroaki Taniguchi^*2, Shoto Tsuji¹, Shiho Sato¹, Takahiro Kenmotsu¹, Kenichi Yoshikawa¹, Koichiro Sadakane¹

¹Faculty of Life and Medical Sciences, Doshisha University, ²The Institute of Genetics and Animal Breeding, Polish Academy of Sciences

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Behavior

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Akira Masuda^1,2, Yuki Kobayashi¹, Shigeyoshi Itohara¹

¹Laboratory for Behavioral Genetics, Center for Brain Science, RIKEN, ²Organization for Research Initiatives and Development, Doshisha University

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer's disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Developmental Biology

Neurogenesis Using P19 Embryonal Carcinoma Cells

Paweł Leszczyński¹, Magdalena Śmiech¹, Aamir S. Teeli¹, Aleksandra Zołocińska², Anna Słysz², Zygmunt Pojda², Mariusz Pierzchała³, Hiroaki Taniguchi¹

¹Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, ²Department of Regenerative Medicine, Maria Skłodowska-Curie Institute - Oncology Center, ³Department of Genomics and Biodiversities, Institute of Genetics and Animal Breeding, Polish Academy of Sciences

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

Behavior

A Task for Assessing the Impact of a Partner on the Speed and Accuracy of Motor Performance in Rats

Yayoi Sekiguchi^1,2, Toshimichi Hata³

¹Graduate School of Psychology, Doshisha University, ²Japan Society for the Promotion of Science, ³Faculty of Psychology, Doshisha University

A procedure to measure the speed and accuracy of rats’ motor performance in a social condition is described. The protocol enables us to investigate the effect of the mere presence of others on speed and accuracy of motor performance in one experiment.

Biochemistry

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Ayaka Hagita-Tatsumoto^1,2, Tomohiro Miyasaka^1,2,3

¹Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, ²Center for Research in Neurodegenerative Diseases, Doshisha University, ³Laboratory of Physiology and Anatomy, School of Pharmacy, Nihon University

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

Behavior

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Fumiya Sawatani¹, Yuta Tamatsu¹, Kaoru Ide¹, Hirotsugu Azechi¹, Susumu Takahashi¹

¹Laboratory of Cognitive and Behavioral Neuroscience, Graduate School of Brain Science, Doshisha University

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.