We thank Dr. Shirin Bonni for providing the pSil-GFP construct, Dr. Alper Uzun for the illustration of Figure 1, and the Leduc Bioimaging facility for confocal microscopy. EMM is supported by the Career Award for Medical Science from the Burroughs Wellcome Fund, a NARSAD Young Investigators Award, and NIH NCRR COBRE P20 RR018728-01. SBL is supported by NIH NCRR COBRE P20 RR018728-01, and has received support from PHS NRSA 5T32MH019118-20.

1. Preparing Culture Solutions and Media (not in video)
<ol>
 <li>Prepare 1 liter of Complete Hank's balanced salt solution (HBSS) containing 1x HBSS, 2.5 mM Hepes (pH7.4), 30 mM D-glucose, 1 mM CaCl2, 1 mM MgSO4 and 4 mM NaHCO3. Add double distilled water (ddH2O). Filter sterilize with a 0.2-&mu;m filter and store at 4 &deg;C.</li>
 <li>Prepare slice culture medium using 35 mL of Basal Medium Eagle media, 12.9 mL of Complete HBSS, 20 mM D-glucose (1.35 mL of 1 M solution), 1...

<ol>
	<li>Angevine, J. B., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+study+of+cell+migration+during+histogenesis+of+cerebral+cortex+in+the+mouse.">Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.</a> Nature. 192, 766-766 (1961).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-392 (2004).</li><li>Barnes, A. P., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+axon-dendrite+polarity+in+developing+neurons.">Establishment of axon-dendrite polarity in developing neurons.</a> Annu. Rev. Neurosci. 32, 347-347 (2009).</li><li>Haydar, T. F., Bambrick, L. L., Krueger, B. K., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+for+analysis+of+proliferation,+cell+death,+and+migration+in+the+embryonic+neocortex.">Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex.</a> Brain Res. Brain Res. Protoc. 4, 425-425 (1999).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. 2002, pl9-pl9 (2002).</li><li>Guerrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-BAR+domain+of+srGAP2+induces+membrane+protrusions+required+for+neuronal+migration+and+morphogenesis.">The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.</a> Cell. 138, 990-990 (2009).</li><li>Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+regulation+of+axonal+morphogenesis+by+the+Cdh1-APC+target+SnoN.">Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.</a> Neuron. 50, 389-389 (2006).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PLoS Genet. 4, e1000111-e1000111 (2008).</li><li>Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303, 1026-1026 (2004).</li><li>Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+paraffin-embedding+of+mouse+tissues+for+GFP+visualization.">Fixation and paraffin-embedding of mouse tissues for GFP visualization.</a> Cold Spring Harb Protoc. 2009, 5298-5298 (2009).</li><li>Hand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-45 (2005).</li><li>Taniguchi, Y., Young-Pearse, T., Sawa, A., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Electroporation+as+a+Tool+for+Genetic+Manipulation+in+Vivo+to+Study+Psychiatric+Disorders:+From+Genes+to+Circuits+and+Behaviors.">In Utero Electroporation as a Tool for Genetic Manipulation in Vivo to Study Psychiatric Disorders: From Genes to Circuits and Behaviors.</a> Neuroscientist. 18, 169-179 (2012).</li></ol>

These methods involving the ex vivo electroporation of plasmids encoding double stranded RNA hairpins 8 and culture of organotypic slices4 provide several distinct advantages. First, these methods allow for a rapid assessment of RNAi-derived phenotypes. The inclusion of an expression cassette encoding GFP in the same pSilencer vector that contains the U6 promoter that drives the double stranded RNA hairpin allows for a rapid identification and characterization of cells electroporated with t...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hamilton syringe </td>
 <td>HAMILTON </td>
 <td>80008</td>
 <td>31 gauge, 0.5 inches long,PT-4 (level of point beveling), 10&mu;l volume</td>
 </tr>
 <tr>
 <td>Platinum tweezertrodes</td>
 <td>BTX</td>
 <td>45-0489</td>
 <td> 5mm size</td>
 </tr>
 <tr>
 <td>ECM830 electroporator</td>
 <td>BTX</td>
 <td>45-0002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BTX Footswitch</td>
 <td>BTX</td>
 <td>45-0208</td>
 <td>For use with ECM830 electroporator</td>
 </tr>
 <tr>
 <td>Vibrating blade microtome</td>
 <td>LEICA</td>
 <td>VT1000 S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6- well dish to use with inserts</td>
 <td>FALCON</td>
 <td>353502 </td>
 <td>Contains notches to fit inserts</td>
 </tr>
 <tr>
 <td>Tissue culture inserts</td>
 <td>FALCON</td>
 <td>353090 </td>
 <td>0.4 micrometer </td>
 </tr>
 <tr>
 <td>Fast Green</td>
 <td>SIGMA</td>
 <td>F7252</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low Melting Agarose</td>
 <td>FISHER</td>
 <td>BP165-25</td>
 <td>DNA grade</td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Sigma-Aldrich</td>
 <td>L2020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma-Aldrich</td>
 <td>P5899</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Basal Medium Eagle</td>
 <td>Sigma Aldrich</td>
 <td>B-1522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HBSS 10x without Ca and Mg</td>
 <td>GIBCO</td>
 <td>14180-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES-free acid</td>
 <td>Sigma- Aldrich</td>
 <td>H4034</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

methods-for-study-neuronal-morphogenesis-ex-vivo-rnai-electroporation

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JoVE Journal

Neuroscience

11.7K 次观看.  Brown University. 我们描述涉及的方法进行快速评估的功能基因在大脑皮层的发展，体外电质粒共表达抑制核糖核酸（RNA干扰）和绿色荧光蛋白在小鼠胚胎皮层。该协议是适合研究神经发育的各个方面，如神经，神经细胞迁移和神经元的形态，包括树突和轴突生长。