Real‐time Recordings of Migrating Cortical Neurons from GFP and Cre Recombinase Expressing Mice

Sylvia Tielens1, Juliette D. Godin1, Laurent Nguyen2

1 Interdisciplinary Cluster for Applied Genoproteomics (GIGA‐R), University of Liège, Liège, 2 Walloon Excellence in Life Sciences and Biotechnology (WELBIO), University of Liège, Liège
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.29
DOI:  10.1002/0471142301.ns0329s74
Online Posting Date:  January, 2016
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Abstract

The cerebral cortex is one of the most intricate regions of the brain that requires elaborate cell migration patterns for its development. Experimental observations show that projection neurons migrate radially within the cortical wall, whereas interneurons migrate along multiple tangential paths to reach the developing cortex. Tight regulation of the cell migration processes ensures proper positioning and functional integration of neurons to specific cerebral cortical circuits. Disruption of neuronal migration often leads to cortical dysfunction and/or malformation associated with neurological disorders. Unveiling the molecular control of neuron migration is thus fundamental to understanding the physiological or pathological development of the cerebral cortex. In this unit, protocols allowing detailed analysis of patterns of migration of both interneurons and projection neurons under different experimental conditions (i.e., loss or gain of function) are presented. © 2016 by John Wiley & Sons, Inc.

Keywords: live imaging; migration; neuron; organotypic slices; focal electroporation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Recording of Cortical Interneuron Migration on Organotypic Slices
  • Alternate Protocol 1: Recordings of Projection Neurons on Organotypic Slices
  • Support Protocol 1: Focal Electroporation of Organotypic Slices
  • Basic Protocol 2: Recordings of Morphological Events During Cortical Interneuron Migration
  • Support Protocol 2: Ex Vivo Electroporation of the Medial Ganglionic Eminences
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Recording of Cortical Interneuron Migration on Organotypic Slices

  Materials
  • DMEM/F12 culture medium (see recipe)
  • Neurobasal culture medium (see recipe)
  • Low‐melting‐point agarose
  • Hanks balanced salt solution with Mg2+ and Ca2+ (HBSS; Lonza, cat. no. 10‐508F)
  • E12.5 pregnant mouse
  • 100% ethanol
  • 37°C water bath
  • Tissue culture hood
  • 35‐mm MatTek glass bottom dish (MatTek, cat. no. P35G‐0‐20‐C)
  • 30‐mm Millicell cell culture insert (Millipore, cat. no. PICM0RG50)
  • Forceps
  • 37°C cell culture incubator (5% CO 2)
  • Small scissors
  • 35 × 10–mm tissue culture dish
  • Ice
  • 24‐well tissue culture plate
  • Fluorescence microscope
  • Perforated spoon
  • Glue
  • Vibratome (e.g., Leica VT100S) with appropriate blade
  • Razor blade or scalpel (to cut solid agarose)
  • Flat spatula
  • Bright‐field microscope
  • Confocal microscope (e.g., Nikon Inverted Eclipse Ti) with temperature‐controlled incubation chamber and acquisition software (e.g., Nikon NIS‐Elements)
  • Computer running ImageJ analysis software with MTrackJ and Bio‐Formats Importer plugins

Alternate Protocol 1: Recordings of Projection Neurons on Organotypic Slices

  Additional Materials (see also protocol 1)
  • Plasmids expressing NeuroD‐CRE‐IRES‐GFP or NeuroD‐X‐IRES‐GFP (where X is the gene of interest) at 3 μg/μl.
  • E14.5 floxed embryos or wild‐type embryos
  • Reagents and equipment for in utero electroporation (see Pacary and Guillemot, ; Saito, )

Support Protocol 1: Focal Electroporation of Organotypic Slices

  Additional Materials (see also protocol 1)
  • Low‐melting‐point agarose
  • PBS (Lonza, cat. no. BE17‐512F)
  • Plasmid solution (see recipe)
  • 50‐ml conical tube
  • Microwave
  • 60 × 15–mm tissue culture dish
  • Ice
  • 35 × 10–mm tissue culture dish
  • Petri dish electrode (electroporation device; Nepa Gene, cat. no. CUY701P7E)
  • Glass Pasteur pipet
  • Glass capillary (1.0‐mm o.d. × 0.58‐mm i.d. × 100‐mm length; Harvard Apparatus, cat. no. 3000016)
  • Flaming/Brown micropipet puller (Sutter Instruments P‐97)
  • Small scissors
  • Dissection microscope
  • Microloader tips (e.g., Eppendorf)
  • Square wave electroporator (Harvard Apparatus ECM830)
  • Electroporation injector (Eppendorf FemtoJet)

Basic Protocol 2: Recordings of Morphological Events During Cortical Interneuron Migration

  Materials
  • DMEM/F12 culture medium (see recipe)
  • Poly‐L‐ornithine (Sigma, cat. no. P4638)
  • Laminin (Sigma, cat. no. L2020)
  • PBS (Lonza, cat. no. BE17‐512F)
  • E13.5 pregnant mouse
  • 100% ethanol
  • Hanks balanced salt solution with Mg2+ and Ca2+ (HBSS; Lonza, cat. no. 10‐508F)
  • Neurobasal culture medium (see recipe)
  • 37°C water bath
  • 35‐mm MatTek glass bottom dish (MatTek, cat. no. P35G‐0‐20‐C)
  • 37°C incubator with adjustable CO 2 controller
  • Forceps (at least two pair)
  • Small scissors
  • 10‐cm cell culture dish
  • Ice
  • Tissue culture hood
  • 1.5‐ml microcentrifuge tube
  • Fire‐polished glass Pasteur pipet (MLS, cat. no. HC0020)
  • 40‐μm nylon mesh (Corning, cat. no. 431750)
  • Scepter 2.0 handheld cell counter (Millipore)
  • Fluorescence microscope
  • Confocal microscope (e.g., Nikon Inverted Eclipse Ti) with temperature‐controlled incubation chamber and acquisition software (e.g., Nikon NIS‐Elements)
  • Computer running ImageJ analysis software with MTrackJ and Bio‐Formats Importer plugins

Support Protocol 2: Ex Vivo Electroporation of the Medial Ganglionic Eminences

  Additional Materials (see also protocol 4)
  • Plasmid solution (see recipe)
  • E13.5 pregnant mouse
  • Hanks balanced salt solution with Mg2+ and Ca2+ (HBSS; Lonza, cat. no. 10‐508F)
  • PBS (Lonza, cat. no. BE17‐512F)
  • Neurobasal culture medium (see recipe)
  • Glass capillary (1.0‐mm o.d. × 0.58‐mm i.d. × 100‐mm l; Harvard Apparatus, cat. no. 3000016)
  • Flaming/Brown micropipet puller (Sutter Instruments P‐97)
  • Dissection microscope
  • Microloader tips (e.g., Eppendorf)
  • Square wave electroporator (Harvard Apparatus ECM830)
  • Electroporation injector (Eppendorf, Femtojet)
  • Petri dish electrode (electroporation device; Nepa Gene, cat. no. CUY701P7E)
  • 50‐ml conical tube
  • Ice
  • Small scissors
  • Sylgard dissection dish (Living Systems Instrumentation, cat. no. DD‐90‐S‐BLK)
  • Dissection pins, size 6 (FST, cat. no. 26001‐65)
  • 3‐mm electrode forceps (Nepa Gene, cat. no. CUY650‐P3)
  • 37°C incubator with adjustable CO 2 controller
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Figures

Videos

Literature Cited

Literature Cited
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  Saito, T. 2015. In Utero Electroporation of the Mouse Embryo. In Electroporation Methods in Neuroscience, Vol. 102, pp. 1‐20. Springer, New York.
  Stenman, J., Toresson, H., and Campbell, K. 2003. Identification of two distinct progenitor populations in the lateral ganglionic eminence: Implications for striatal and olfactory bulb neurogenesis. J. Neurosci. 23:167‐174.
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  van den Berghe, V., Stappers, E., Vandesande, B., Dimidschstein, J., Kroes, R., Francis, A., Conidi, A., Lesage, F., Dries, R., Cazzola, S., Berx, G., Kessaris, N., Vanderhaeghen, P., van Ijcken, W., Grosveld, F.G., Goossens, S., Haigh, J.J., Fishell, G., Goffinet, A., Aerts, S., Huylebroeck, D., and Seuntjens, E. 2013. Directed migration of cortical interneurons depends on the cell‐autonomous action of Sip1. Neuron 77:70‐82. doi: 10.1016/j.neuron.2012.11.009.
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