Cerebral Cortex Electroporation to Study Projection Neuron Migration

Emilie Pacary1, François Guillemot2

1 Université de Bordeaux, Bordeaux, 2 The Francis Crick Institute, Mill Hill Laboratory, London
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.26
DOI:  10.1002/cpns.13
Online Posting Date:  October, 2016
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Abstract

Brain electroporation is a rapid and powerful approach to study neuronal development. In particular, this technique has become a method of choice for studying the process of radial migration of projection neurons in the embryonic cerebral cortex. This method has considerably helped to describe in detail the different steps of radial migration and to characterize the molecular mechanisms controlling this process. Delineating the complexities of neuronal migration is critical to our understanding not only of normal cerebral cortex formation but also of neurodevelopmental disorders resulting from neuronal migration defects. Here, we describe in detail the protocols to perform in utero or ex vivo electroporation of progenitor cells in the ventricular zone of the cerebral cortex with the aim of studying the process of radial migration of projection neurons during embryonic development. © 2016 by John Wiley & Sons, Inc.

Keywords: electroporation; migration; neuron; cerebral cortex

     
 
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Table of Contents

  • Introduction
  • Significance Statement
  • Basic Protocol 1: In Utero Electroporation of the Cerebral Cortex to Study Radial Migration of Projection Neurons
  • Alternate Protocol 1: Ex Vivo Electroporation of the Cerebral Cortex to Study Radial Migration of Projection Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Utero Electroporation of the Cerebral Cortex to Study Radial Migration of Projection Neurons

  Materials
  • Buprenorphine, 30 µg/ml (see recipe)
  • 0.9% sodium chloride (NaCl; Cooper)
  • Pregnant mice
  • DNA solution (see recipe)
  • DNAse/RNAse free water (Sigma‐Aldrich, cat. no. W4502)
  • 2% Fast Green (see recipe)
  • 2.5% isoflurane
  • Vetasept Chlorhexidine Surgical Scrub (Animalcare, cat. no. XHG007)
  • 70% ethanol
  • 4% paraformaldehyde (PFA) in PBS
  • 20% sucrose in PBS (see recipe)
  • OCT embedding matrix (Cell Path, cat. no. KMA‐0100‐00 A)
  • 0.01% Triton in PBS
  • 0.01% Triton/10% serum (v/v) in PBS (permeabilization‐blocking solution)
  • Anti‐GFP primary antibody
  • Secondary anti‐GFP antibody conjugated to Alexa Fluor 488
  • TOTO‐3 iodide (1 mM in DMSO; Thermo Fisher Scientific, cat. no. T3604)
  • Aqua Poly/mount (mounting medium; Polysciences, cat. no. 18606)
  • Endofree Plasmid Maxi kit (Qiagen, cat. no. 12362)
  • 25‐G needles (0.5 × 16 mm)
  • 1‐ml syringes
  • Heating pad
  • Recovery chamber
  • Sterile drapes
  • Scalpel
  • Sterile swabs, 10 × 10 cm
  • Extra thin Iris scissors (Fine Science Tools, cat. no. 14088‐10)
  • Curved forceps (Fine Science Tools, cat. no. 91197‐00)
  • 2‐ring forceps (Fine Science Tools, cat. no. 11103‐09)
  • Needle holder (Fine Science Tools, cat. no. 12002‐12)
  • Graefe forceps (Fine Science Tools, cat. no. 11050‐10)
  • Absorbable sutures (Péters Surgical, cat. no. 18S15B)
  • Platinum Tweezertrode, 5‐mm electrode diameter (BTX Harvard Apparatus, cat. no. 45‐0489)
  • Electroporator (BTX Harvard Apparatus, cat. no. ECM830)
  • Borosilicate glass capillaries (1.0 mm O.D. × 0.58 mm I.D.; Harvard Apparatus, cat. no. 30‐0016)
  • Micropipet puller
  • Microloader tips (Eppendorf, cat. no. 5242956003)
  • 0.5‐ to 10‐µl pipet and tips
  • Capillary holder (Eppendorf, cat. no. 5176190002)
  • Mask
  • Anesthetic induction chamber
  • Surgery mask
  • Electric razor
  • 20‐ml syringes
  • Femtojet microinjector (Eppendorf, cat. no. 5247000013)
  • Foot control for microinjector (Eppendorf, cat. no. 5247623002)
  • 9‐cm petri dishes
  • Straight forceps
  • Fluorescent binocular microscope
  • 0.22‐µm vacuum‐driven filtration system (with 250‐ml receiver bottle; for preparation of solutions)
  • Histomold trays, 6 × 8 mm (Leica, cat. no. 14702218311)
  • Flat spatula
  • −80°C freezer
  • Cryostat
  • Anti‐roll plate
  • Superfrost Plus microscope slides (Thermo Fisher Scientific, cat. no. J1800AMNZ)
  • 5 place slide mailers
  • Rotating shaker
  • Humidified box
  • Coverslips
  • Cover glass forceps (Fine Science Tools, cat. no. 11073‐10)
  • Fluorescent microscope
  • Confocal microscope
  • Analysis software (ImageJ or Metamorph)

Alternate Protocol 1: Ex Vivo Electroporation of the Cerebral Cortex to Study Radial Migration of Projection Neurons

  Additional Materials (also see protocol 1Basic Protocol)
  • L15 complete culture medium (see recipe)
  • Neurobasal complete culture medium (see recipe)
  • Agarose type VII (Sigma‐Aldrich, cat. no. A9045)
  • 0.3% Triton/3% BSA/10% serum (v/w/v) in PBS (permeabilization‐blocking solution)
  • Glue
  • Patafix adhesive
  • Microwave
  • 56°C water bath
  • 24‐well plates
  • Perforated spoon
  • Vibratome
  • 6‐well plates
  • Tissue culture hood
  • Millicell cell culture inserts (Millipore, cat. no. PICMORG50)
  • 1‐ml pipets and 200‐µl tips
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Figures

Videos

Literature Cited

Literature Cited
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  McManus, M.F. and Golden, J.A. 2005. Neuronal migration in developmental disorders. J. Child Neurol. 20:280‐286. doi: 10.1177/08830738050200040301.
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  Ohtaka‐Maruyama, C. and Okado, H. 2015. Molecular pathways underlying projection neuron production and migration during cerebral cortical development. Front. Neurosci. 9:447. doi: 10.3389/fnins.2015.00447.
  Pacary, E. and Guillemot, F. 2014. In utero electroporation to study mouse brain development. Methods Mol. Biol. 1082:285‐293. doi: 10.1007/978‐1‐62703‐655‐9_19.
  Pacary, E., Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel‐Potter, M., Parras, C., Bell, D.M., Ridley, A.J., Parsons, M., and Guillemot, F. 2011. Proneural transcription factors regulate different steps of cortical neuron migration through Rnd‐mediated inhibition of RhoA signaling. Neuron 69:1069‐1084. doi: 10.1016/j.neuron.2011.02.018.
  Pacary, E., Haas, M.A., Wildner, H., Azzarelli, R., Bell, D.M., Abrous, D.N., and Guillemot, F. 2012. Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation. J. Vis. Exp. 65:e4163. doi: 10.3791/4163.
  Saito, T. and Nakatsuji, N. 2001. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240:237‐246. doi: 10.1006/dbio.2001.0439.
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  Tielens, S., Godin, J.D., and Nguyen, L. 2016. Real‐time recordings of migrating cortical neurons from GFP and Cre recombinase expressing mice. Curr. Protoc. Neurosci. 73:3.29.1‐3.29.23. doi: 10.1002/0471142301.ns0329s74.
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