Time‐Lapse Imaging and Cell Tracking of Migrating Cells in Slices and Flattened Telencephalic Vesicles

Verónica Murcia‐Belmonte1, Giovanna Expósito1, Eloísa Herrera1

1 Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Científicas‐Universidad Miguel Hernández, CSIC‐UMH, Alicante
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.31
DOI:  10.1002/cpns.24
Online Posting Date:  April, 2017
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Abstract

Neuronal migration is a vital process needed for subsequent assembly and function of neural circuitry during embryonic development. The vast majority of neural progenitors are generated far from their final destination and need to migrate considerable distances to reach their specific cortical layer. Innovations in cell culture techniques and fluorescence microscopy now facilitate the direct visualization of cell movements during cortical development. Here, a detailed protocol to record and analyze a particular type of early migrating neurons, the Cajal Retzius Cells, during the development of the telencephalic vesicles in mammals is described. This method applied to other reporter mouse lines or to electroporated mouse embryos can be also used to analyze the migration of different types of moving neurons during cortical development. © 2017 by John Wiley & Sons, Inc.

Keywords: telencephalic vesicle; Cajal Retzius Cells; neuronal migration; time‐lapse imaging; brain development

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of CRC in Prethalamic Slices
  • Alternate Protocol 1: Flattened Telencephalic Vesicle Preparation
  • Support Protocol 1: Time‐Lapse Imaging and Cell Tracking
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of CRC in Prethalamic Slices

  Materials
  • Pregnant Tg(Zic2‐EGFP)HT146Gsat line (Escalante et al., ; Murillo et al., ) obtained from the Mutant Mouse Regional Resource Center (RRID, cat. no. MMRRC_030037_UCD)
  • 10× Krebs (see recipe), ice cold
  • 3% low‐melting agarose (see recipe) in 0.01 M PBS (50 ml), maintain in 42ºC water bath
  • Sterile‐filtered 1× Krebs/HEPES/P/S (see recipe)
  • MEM medium (see recipe)
  • Neurobasal medium (see recipe)
  • Immersol W (2010) (Zeiss)
  • 35‐mm plastic Petri dishes
  • Dissection scope and cold‐light source (Leica Microsystems, model no. MZ6 and KL 1500 LCD)
  • Small, extra‐sharp scissors and forceps (3‐mm straight blade, Vannas Spring Scissors, Fine Science Tools, cat. no. 15000‐00; Dumont no. 5SF forceps, Inox super‐fine tip, Fine Science Tools, cat. no. 11252‐00)
  • Plastic embedding molds (Peel‐A‐Way Disposable Embedding Molds, 22 × 22 × 20–mm deep, Polyscience, cat. no. 18646A)
  • Vibratome (Leica Microsystems, cat. no. VT1000S)
  • Razor (e.g., Gillette Platinum C/5)
  • Small spatula (0.3‐mm wide, straight Iris spatula, Fine Science Tools, cat. no. 10094‐13)
  • 35‐mm glass‐bottom dishes (MatTek Corporation, cat. no. P35G‐1.5‐14‐C)
  • Fluorescence microscope
  • Millicell cell culture inserts (30‐mm, hydrophilic PTFE membranes, 0.4‐μm, Millipore, cat. no. PICM0RG50)
  • 95% O 2, 5% CO 2, 37ºC humidified incubator
  • Laser scanning spectral confocal microscope TCS SP2 AOBS (Leica Microsystems) attached to a Leica DMIRE2 inverted microscope with a long working distance, water‐immersion objective (Leica HCX APO U‐V‐I 20×/NA 0.5 W)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Alternate Protocol 1: Flattened Telencephalic Vesicle Preparation

  Additional Materials (also see protocol 1Basic Protocol)
  • Sterile PBS
  • Ultra‐fine‐tipped curved forceps, 0.01 × 0.005–mm (Moria, cat. no. 11370‐42)

Support Protocol 1: Time‐Lapse Imaging and Cell Tracking

  Materials
  • Immersol W 2010 (Zeiss)
  • 35‐mm glass‐bottom dish containing culture (see protocol 1Basic Protocol or protocol 2Alternate Protocol)
  • Laser scanning confocal microscope (Leica SP2 AOBS) attached to an inverted microscope (Leica DMIRE2) equipped with microscope chamber, temperature controller, and gas mixer
  • Open source Java image processing software (FIJI/ImageJ, https://fiji.sc/)
  • Scientific software for advanced image visualization and analysis (Imaris software available from Bitplane AG Oxford Instruments Company, www.bitplane.com)
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Figures

Videos

Literature Cited

Literature Cited
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