In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts

Masato Sawada; Mami Matsumoto; Keishi Narita; Natsuko Kumamoto; Shinya Ugawa; Sen Takeda; Kazunobu Sawamoto

doi:10.21769/BioProtoc.3823

In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts

Bio Protoc. 2020 Nov 20;10(22):e3823. doi: 10.21769/BioProtoc.3823.

Authors

Masato Sawada^{1

2}, Mami Matsumoto^{1

2}, Keishi Narita³, Natsuko Kumamoto⁴, Shinya Ugawa⁴, Sen Takeda³, Kazunobu Sawamoto^{1

2}

Affiliations

¹ Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
² Division of Neural Development and Regeneration, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan.
³ Department of Anatomy and Cell Biology, Faculty of Medicine, University of Yamanashi, Chuo 409-3898, Japan.
⁴ Department of Anatomy and Neuroscience, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.

Abstract

Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then transduced with a plasmid(s) encoding a gene(s) of interest, aggregated by centrifugation, and cultured for 2 days in Matrigel. Time-lapse images of migratory behaviors of cultured neuroblasts and their ciliary structures, including the ciliary membrane and basal body, are acquired by confocal or superresolution laser-scanning microscopy. This method provides information about the spatiotemporal dynamics of neuroblasts' morphology and ciliary structures, and is widely applicable to various types of migrating neuronal and nonneuronal cells in various species.

Keywords: Confocal microscopy; Migration; Neuroblast; Postnatal neurogenesis; Primary cilium; Superresolution microscopy; Time-lapse imaging; Ventricular-subventricular zone.