Name: dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays
Uploaded: 2011-12-20T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

This work was funded by National Institutes of Health Grant RO1DC002756 and the Purdue Research Foundation. We thank Doris Wu and Wiese Chang for advice with experiments and Rodney McPhail for help with figures.

Recipes
Chick Ringers
<table border="1">
 <tbody>
 <tr>
 <td>7.2 g</td>
 <td>NaCl</td>
 </tr>
 <tr>
 <td>0.23 g</td>
 <td>CaCl2 + 2H2O</td>
 </tr>
 <tr>
 <td>0.37 g</td>
 <td>KCl</td>
 </tr>
 <tr>
 <td>0.115 g</td>
 <td>Na2HPO4 </td>
 </tr>
 <tr>
 <td>900 ml</td>
 <td>Water (Tissue culture grade)</td>
 </tr>
 </tbody>
</table>
Note: Adjust pH to 7.4. Bring final volume to 1000 ml with water....

<ol>
	<li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+in+the+inner+ear.">Axon guidance in the inner ear.</a> Int. J. Dev. Biol. 51 (6-7), 549-549 (2007).</li><li>Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+inner+ear+afferent+connections:+forming+primary+neurons+and+connecting+them+to+the+developing+sensory+epithelia.">Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia.</a> Brain Res. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+and+neurotrophin-3+support+the+survival+and+neuritogenesis+response+of+developing+cochleovestibular+ganglion+neurons.">Brain-derived neurotrophic factor and neurotrophin-3 support the survival and neuritogenesis response of developing cochleovestibular ganglion neurons.</a> Dev. Biol. 159 (1), 266-266 (1993).</li><li>Bianchi, L. M., Cohan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+neurotrophins+and+CNTF+on+developing+statoacoustic+neurons:+comparison+with+an+otocyst-derived+factor.">Effects of the neurotrophins and CNTF on developing statoacoustic neurons: comparison with an otocyst-derived factor.</a> Dev. Biol. 159 (1), 353-353 (1993).</li><li>Juurlink, B. e. r. n. h. a. r. d. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Spinal+Somatic+Motoneurons+in+Culture.">Chick Spinal Somatic Motoneurons in Culture.</a> Protocols for Neural Cell Culture.. 59, (2001).</li><li>Fantetti, K. N., Zou, Y., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnts+and+Wnt+inhibitors+do+not+influence+axon+outgrowth+from+chicken+statoacoustic+ganglion+neurons.">Wnts and Wnt inhibitors do not influence axon outgrowth from chicken statoacoustic ganglion neurons.</a> Hear. Res. , (2011).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-49 (1951).</li><li>Bourikas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+guides+commissural+axons+along+the+longitudinal+axis+of+the+spinal+cord.">Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord.</a> Nat. Neurosci. 8 (3), 297-297 (2005).</li><li>Charron, F., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+brain+wiring+functions+for+classical+morphogens:+a+role+as+graded+positional+cues+in+axon+guidance.">Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance.</a> Development. 132 (10), 2251-2251 (2005).</li><li>Augsburger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24 (1), 127-127 (1999).</li><li>Lyuksyutova, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior-posterior+guidance+of+commissural+axons+by+Wnt-frizzled+signaling.">Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling.</a> Science. 302 (5652), 1984-1984 (2003).</li></ol>

We present a method to dissect and culture E4 SAG and E6 spinal cord explants, from chick, under serum-free conditions. This procedure is currently used in our lab to study the effects of various secreted ligands on SAG neuron survival and neurite outgrowth. Novel aspects of this protocol include the use of a serum-free system for culturing explants and the use of beads soaked in growth factors to study effects on SAG neurite outgrowth. A bead method similar to ours has been described for the chick spinal cord10</su...

<table border="1">
<tbody>
<tr>
<td>Reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comment</td>
</tr>
<tr>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We use a Leica M80 stereomicroscope with brightfield base illumination</td>
</tr>
<tr>
<td>Slide warmer</td>
<td>C.S. &amp; E.</td>
<td>&nbsp;</td>
<td>Collagen polymerization</td>
</tr>
<tr>
<td>Chicken egg incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>37&deg;C/5&#37; CO2 humidified cell culture incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dissection Materials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sylgard&copy; coated petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24-well cell culture plate</td>
<td>Corning</td>
<td>3526</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&#35;5 Dumont forceps</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&#35;55 Dumont forceps</td>
<td>Fine Science Tools</td>
<td>11295-51</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Stainless steel dissecting pins </td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>Embryo pinning, fine dissection</td>
</tr>
<tr>
<td>Moria perforated spoon</td>
<td>Fine Science Tools</td>
<td>10370-17</td>
<td>Embryo harvest/transfer</td>
</tr>
<tr>
<td>200 &micro;l wide mouth pipet tips</td>
<td>Dot Scientific Inc</td>
<td>118-96R</td>
<td>Explant transfer</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>E4 and E6 chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Chick Ringer&rsquo;s solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Embryo harvest</td>
</tr>
<tr>
<td>HBSS</td>
<td>Sigma</td>
<td>H8264</td>
<td>SAG dissection</td>
</tr>
<tr>
<td>L15 medium </td>
<td>Sigma</td>
<td>L5520</td>
<td>Spinal cord dissection medium</td>
</tr>
<tr>
<td>Fetal calf serum</td>
<td>Atlanta Biologicals</td>
<td>S11150</td>
<td>Spinal cord dissection medium</td>
</tr>
<tr>
<td>Vannas Scissors</td>
<td>World Precision Instruments</td>
<td>501778</td>
<td>Spinal cord dissection</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Collagen preparation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Rat-tail collagen Type I</td>
<td>BD Biosciences</td>
<td>354249</td>
<td>Explant culture</td>
</tr>
<tr>
<td>10X PBS (Sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To neutralize collagen</td>
</tr>
<tr>
<td>1N NaOH (Sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To neutralize collagen</td>
</tr>
<tr>
<td>Distilled water (Sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To neutralize collagen</td>
</tr>
<tr>
<td>pH indicator papers (6.0-8.1)</td>
<td>Whatman</td>
<td>2629-990</td>
<td>To check collagen pH</td>
</tr>
<tr>
<td>15 ml conical tubes</td>
<td>Dot Scientific Inc</td>
<td>818-PG</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>500 &micro;l wide mouth pipet tips</td>
<td>Dot Scientific Inc</td>
<td>119-R100</td>
<td>To pipet collagen</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Explant culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM/F12 medium</td>
<td>Sigma</td>
<td>D8437</td>
<td>Explant culture medium</td>
</tr>
<tr>
<td>ITS+1</td>
<td>Sigma</td>
<td>I2521</td>
<td>Medium supplement</td>
</tr>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>Medium supplement</td>
</tr>
<tr>
<td>CNTF (rat)</td>
<td>Sigma</td>
<td>C3835</td>
<td>Medium supplement</td>
</tr>
<tr>
<td>NT-3 (human)</td>
<td>Sigma</td>
<td>N1905</td>
<td>Medium supplement</td>
</tr>
<tr>
<td>Purified mouse Wnt5a </td>
<td>R&amp;D Systems</td>
<td>645-WN-010</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Affi-gel Blue gel beads</td>
<td>Bio-Rad</td>
<td>153-7302</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Immunohistochemistry</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Paraformaldehyde </td>
<td>Sigma</td>
<td>P6148</td>
<td>Fixation</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Rinses</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>Sigma</td>
<td>T9284</td>
<td>Blocking solution </td>
</tr>
<tr>
<td>Sodium azide </td>
<td>Sigma</td>
<td>S8032</td>
<td>Blocking solution</td>
</tr>
<tr>
<td>Calf serum</td>
<td>Invitrogen</td>
<td>16170-078</td>
<td>Blocking solution</td>
</tr>
<tr>
<td>Mouse monoclonal IgG2b anti-&beta; Tubulin 1+II antibody</td>
<td>Sigma</td>
<td>T8535</td>
<td>Labels cell bodies and neurites</td>
</tr>
<tr>
<td>Alexa fluor 488 goat anti &ndash;mouse IgG2b antibody</td>
<td>Invitrogen</td>
<td>A21141</td>
<td>Detects &beta; Tubulin antibody</td>
</tr>
<tr>
<td>Teflon micro spatula</td>
<td>VWR</td>
<td>57949-033</td>
<td>Release collagen gels from well</td>
</tr>
</tbody>
</table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

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