Tracking Neuronal Migration in Adult Brain Slices

Karen Bakhshetyan1, Armen Saghatelyan2

1 Cellular Neurobiology Unit, Institut Universitaire en santé mentale de Québec, Quebec City, 2 Department of Psychiatry and Neuroscience, Université Laval, Quebec City
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.28
DOI:  10.1002/0471142301.ns0328s71
Online Posting Date:  April, 2015
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Abstract

Neuronal migration is one of the fundamental processes underlying the proper assembly and function of neural circuitry. The majority of neuronal precursors are generated far away from their sites of integration and need to migrate substantial distances to reach their final destination. Neuronal migration occurs not only in the embryonic brain but also in a few regions of the adult brain such as the olfactory bulb (OB). The mechanisms orchestrating cell migration in the adult brain are, however, poorly understood, despite their clinical relevance. Here we describe a method for time‐lapse imaging of cell migration in acute brain slices. This method, combined with genetic and/or pharmacological manipulations of different molecular pathways, makes it possible to determine the dynamics and molecular mechanisms of cell migration in the adult brain. In addition, time‐lapse imaging in acute brain slices makes it possible to monitor cell movement in a microenvironment that closely resembles in vivo conditions and to study neuroblast displacement along with other cellular elements such as astrocytes and blood vessels. © 2015 by John Wiley & Sons, Inc.

Keywords: cell migration; time‐lapse imaging; olfactory bulb; rostral migratory stream; blood vessels

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Time‐Lapse Video‐Imaging of Neuronal Migration in Adult Acute Brain Slices
  • Support Protocol 1: Stereotaxic Injection of Viral Vectors into the SVZ of the Adult Mouse
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Time‐Lapse Video‐Imaging of Neuronal Migration in Adult Acute Brain Slices

  Materials
  • Adult C57Bl/6 mice (Charles River)
  • Acute brain slices prepared from adult mice infected with viral particles 3 to 7 days (for monitoring tangential migration in the RMS) or 7 to 12 days (for monitoring radial migration in the OB and the RMS of the OB; see protocol 2Support Protocol)
  • Artificial cerebro‐spinal fluid (ACSF; see recipe)
  • Cutting solution (see recipe)
  • Dextran Texas Red (Molecular Probes)
  • 4% agar block (prepared using agar powder [Sigma‐Aldrich]; see recipe)
  • 95% O 2/5% CO 2
  • Surgical instruments for extracting the brain (e.g., scissors, scalpel, forceps)
  • Vibratome (Microm HM 650 V; Thermo Scientific)
  • Fluorescence wide‐field upright microscope, with motorized Z‐drive (BX61WI; Olympus)
  • 482/35 nm and 536/40 nm excitation and emission filters for imaging GFP‐labeled cells
  • 14‐bit cooled CCD camera with 1392 × 1040 imaging pixels (CoolSnap HQ2; Photometrics)
  • 40× water immersion objective lens with a 0.8 numerical aperture or higher (Olympus)
  • Illumination system, equipped with a 175 W xenon lamp (30 to 100 msec excitation time per z plane, Lambda DG‐4; Sutter Instruments)
  • Imaging chamber, mounted on a microscope stage (PH1 Series 20; Harvard Apparatus)
  • Automatic heating system (to maintain temperature in the imaging chamber at ∼32°C; Harvard Apparatus, cat. no. TC‐344B)
  • Multidimensional time‐lapse data acquisition software (MetaMorph, Molecular Devices)
  • Software for Z‐stack image acquisition, every 15 sec for 1 hr (usually 5 to 10 z‐planes at 3‐μm intervals; MetaMorph, Molecular Devices)
  • Custom‐made ACSF delivery system for continuous perfusion of slices with oxygenated ACSF at a 1 to 2 ml/min flow rate
  • Slice fixation mesh (nylon with 0.12‐mm diameter and ∼1‐mm2 holes)
NOTE: For standard animal techniques including stereotaxic surgery, anesthesia, and analgesia, see and .NOTE: The imaging and acquisition systems described in this unit are used in the authors’ laboratory and serve only as examples of the equipment required to perform time‐lapse imaging in acute slices. Other commercial and custom‐made systems can be used. However, we strongly recommend using imaging systems that can acquire time‐lapse images at least every 15 sec. This is important to ensure the unambiguous identification of the migratory and stationary phases of neuronal precursors, to better understand the dynamics of cell migration.

Support Protocol 1: Stereotaxic Injection of Viral Vectors into the SVZ of the Adult Mouse

  Materials
  • Adult C57Bl/6 mice (Charles River)
  • Proviodine (Rougier) or 70% ethanol (Sigma‐Aldrich)
  • Paraffin oil (Sigma‐Aldrich)
  • Solution containing viral particles (obtained from Vector Core Facilities, Institut Universitaire en santé mentale de Québec or University of North Carolina)
  • Ketamine (Bioniche)/xylazine (Bimeda) or isoflurane (PPC)
  • Lidocaine (AstraZeneca)
  • Anafen (Merial)
  • Micropipet puller
  • Stereotaxic injection apparatus equipped with a digital stereotaxic coordinate read‐out system (WPI)
  • Mouse adaptor mounted on a stereotaxic apparatus (WPI)
  • Sterile surgical instruments (e.g., scissors, scalpels, forceps)
  • Microdrill system
  • Heating pad
  • Nanoliter injector with nanoliter injector controller (WPI)
  • Binocular microscope
NOTE: For standard animal techniques including stereotaxic surgery, anesthesia, and analgesia, see and .
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Figures

Literature Cited

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