The authors would like to thank Bob Freeman for training and the Kirschner lab for the use of the frog facility, and members of the Van Vactor lab for support. We thank the Nikon Imaging Center at Harvard Medical School for assistance with light microscopy for the images in Figure 1. This work was funded by the following: NRSA NIH fellowship and NIH K99 fellowship to L.A.L, Basic Science Partnership funding (<a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a>) to AEF, and NIH RO1 NS035909 to D.V.V.

Note: We describe the steps in the first two sections only in brief, as excellent protocols with detailed information have been published elsewhere that focus more specifically on these steps (e.g.8-12). Additionally, the general protocol of working with Xenopus spinal neurons in live cell culture has been previously published in a detailed methods article8. We highly recommend reviewing that article as a complement to this video, although here we do provide enough information to s...

<ol>
	<li>Lowery, L. A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+understanding+the+growth+cone+machinery.">The trip of the tip: understanding the growth cone machinery.</a> Nat. Rev. Mol. Cell Biol. 10, 332-343 (2009).</li><li>Buck, K. B., Zheng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+turning+induced+by+direct+local+modification+of+microtubule+dynamics.">Growth cone turning induced by direct local modification of microtubule dynamics.</a> J. Neurosci. 22, 9358-9367 (2002).</li><li>Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microtubule+plus+end+tracking+protein+Orbit/MAST/CLASP+acts+downstream+of+the+tyrosine+kinase+Abl+in+mediating+axon+guidance.">The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.</a> Neuron. 42, 913-926 (2004).</li><li>Santiago-Medina, M., Myers, J. P., Gomez, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+adhesion+and+signaling+dynamics+in+Xenopus+laevis+growth+cones.">Imaging adhesion and signaling dynamics in Xenopus laevis growth cones.</a> Dev. Neurobiol. , (2011).</li><li>Tanaka, E. M., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+behavior+in+the+growth+cones+of+living+neurons+during+axon+elongation.">Microtubule behavior in the growth cones of living neurons during axon elongation.</a> J. Cell Biol. 115, 345-363 (1991).</li><li>Tanaka, E., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microtubules+in+growth+cone+turning+at+substrate+boundaries.">The role of microtubules in growth cone turning at substrate boundaries.</a> J. Cell Biol. 128, 127-137 (1995).</li><li>Tanaka, E., Ho, T., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microtubule+dynamics+in+growth+cone+motility+and+axonal+growth.">The role of microtubule dynamics in growth cone motility and axonal growth.</a> J. Cell Biol. 128, 139-155 (1995).</li><li>Gomez, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+Xenopus+spinal+neurons+in+live+cell+culture.">Working with Xenopus spinal neurons in live cell culture.</a> Methods Cell Biol. 71, 129-156 (2003).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis: A Laboratory Manual 2010. , (2010).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+Eggs+from+Xenopus+laevis+Females.">Obtaining Eggs from Xenopus laevis Females.</a> J. Vis. Exp. (18), e890 (2008).</li><li>Mimoto, M. S., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+gene+function+in+Xenopus+laevis.">Manipulation of gene function in Xenopus laevis.</a> Methods Mol. Biol. 770, 55-75 (2011).</li><li>Lavery, D. L., Hoppler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+and+loss-of-function+strategies+in+Xenopus.">Gain-of-function and loss-of-function strategies in Xenopus.</a> Methods Mol. Biol. 469, 401-415 (2008).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus Laevis (Daudin). , (1994).</li><li>Guirland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cAMP+signaling+through+G-protein-coupled+receptors+mediates+growth+cone+attraction+induced+by+pituitary+adenylate+cyclase-activating+polypeptide.">Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide.</a> J. Neurosci. 23, 2274-2283 (2003).</li><li>Spitzer, N. C., Lamborghini, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+action+potential+mechanism+of+amphibian+neurons+isolated+in+culture.">The development of the action potential mechanism of amphibian neurons isolated in culture.</a> Proc. Natl. Acad. Sci. U.S.A. 73, 1641-1645 (1976).</li><li>Turney, S. G., Bridgman, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+stimulates+and+guides+axonal+outgrowth+via+growth+cone+myosin+II+activity.">Laminin stimulates and guides axonal outgrowth via growth cone myosin II activity.</a> Nat. Neurosci. 8, 717-719 (2005).</li><li>Weinl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+turning+of+Xenopus+retinal+axons+induced+by+ephrin-A5.">On the turning of Xenopus retinal axons induced by ephrin-A5.</a> Development. 130, 1635-1643 (2003).</li><li>Knoll, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nat. Protoc. 2, 1216-1224 (2007).</li><li>Wheeler, G. N., Hamilton, F. S., Hoppler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+expression+in+transgenic+Xenopus+embryos.">Inducible gene expression in transgenic Xenopus embryos.</a> Curr. Biol. 10, 849-852 (2000).</li></ol>

No conflicts of interest declared.

Xenopus laevis neural explants send out neurites in a very robust manner by 24 hr after plating on the laminin/poly-lysine substrate if conditions are appropriate. With this substrate, growth cones are highly motile and can achieve axon lengths of up to 1 mm, extending in all directions outward from the explant, although typical lengths are 100 &mu;m or more. If neurites do not grow out, there are a limited number of reasons for this to be the case. One possibility is that the neural explants did not properly a...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Chorionic Gonadotropin</td>
 <td>Argent Chemical Laboratories</td>
 <td>C-HCG-ON-10</td>
 </tr>
 <tr>
 <td>Cysteine</td>
 <td>Sigma-Aldrich</td>
 <td>52-90-4</td>
 </tr>
 <tr>
 <td>mMessage mMachine kit</td>
 <td>Ambion</td>
 <td>AM1340</td>
 </tr>
 <tr>
 <td>Capillary Borosil Needles 1.2 mm (OD) x 0.9 mm (ID)</td>
 <td>FHC</td>
 <td>30-31-0</td>
 </tr>
 <tr>
 <td>Ficoll</td>
 <td>Sigma</td>
 <td>F2637</td>
 </tr>
 <tr>
 <td>Dumont #5 Biologie Inox Forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-20</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>&nbsp;Sigma-Aldrich</td>
 <td>&nbsp;9001-12-1</td>
 </tr>
 <tr>
 <td>Mattek dishes</td>
 <td>Mat Tek Corporation</td>
 <td>P35G-1.5-14-C</td>
 </tr>
 <tr>
 <td>L-15</td>
 <td>Invitrogen</td>
 <td>21083-027</td>
 </tr>
 <tr>
 <td>Poly-l-lysine</td>
 <td>Sigma</td>
 <td>P-1399</td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Sigma</td>
 <td>L2020</td>
 </tr>
 <tr>
 <td>NT3</td>
 <td>Sigma</td>
 <td>N1905</td>
 </tr>
 <tr>
 <td>BDNF</td>
 <td>Sigma</td>
 <td>B3795</td>
 </tr>
 </tbody>
</table>

neural explant cultures from xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

neural-explant-cultures-from-xenopus-laevis

Research

JoVE Journal

Neuroscience

12.4K visualizações.  Harvard Medical School. Explantes neurais de dissecados Xenopus laevis Embriões que expressam proteínas de fusão fluorescentes permite a imagiologia da dinâmica de crescimento do cone do citoesqueleto.