title: Simultaneous neuron- and astrocyte-specific fluorescent marking.
abstract: Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Schulze
authors.affiliation: Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hayata-Takano
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kamo
authors.affiliation: iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: takanobunakazawa-tky@umin.ac.jp.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Nakazawa
authors.affiliation: iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nagayasu
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kasai
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Seiriki
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Norihito
authors.initials: N
authors.lastname: Shintani
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ago
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Camille
authors.initials: C
authors.lastname: Farfan
authors.affiliation: Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Psychiatry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Hashimoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Hyogo University of Health Science, 1-3-6 Minatojima, Chuo-ku, Kobe, Hyogo 650-8530, Japan.
authors.forename: Akemichi
authors.initials: A
authors.lastname: Baba
authors.affiliation: Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: hasimoto@phs.osaka-u.ac.jp.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hashimoto
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006657:Histones / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005456:Fluorescent Dyes; D006657:Histones; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Astrocyte; Astrocyte-specific promoter; Fluorescent protein; Lentiviral vector; Neuron; Neuron-specific promoter