The collagen matrix assay has been developed and improved by the work of many different research groups during the past two to three decades. The approaches described here for dopaminergic and striatal explants greatly benefit from these studies. In addition, the authors would like to thank Asheeta Prasad for her help in setting up striatal explant cultures. Work in the lab was funded by the Human frontier Science Program Organization (Career Development Award), the Netherlands Organization of Health Research and Development (ZonMW-VIDI and ZonMW-TOP), the Europanian Union (mdDA-NeuroDev, FP7/2007-2011, Grant 222999) (to RJP), and the Netherlands Organization for Scientific Research (TopTalent; to ERES).

1. Preparation of Rat Tail Collagen
<ol>
 <li>Collect 6-10 adult rat tails (it is possible to store the tails at -20 &deg;C until use).</li>
 <li>Soak the tails in 95% ethanol overnight at room temperature (RT).</li>
</ol>
Dissection of rat tails (in tissue culture hood): 
(keep tools in 70% ethanol when not using them and make sure that all solutions, tools and glassware used throughout this procedure are sterile) 
<ol start="3">
 <li>To collect tendons from the tails, cut off...

<ol>
	<li>van den Heuvel, D. M., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+connected+in+the+dopamine+system.">Getting connected in the dopamine system.</a> Prog. Neurobiol. 85, 75-93 (2008).</li><li>Lobo, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+striatonigral+and+striatopallidal+medium+spiny+neurons+past,+present,+and+future.">Molecular profiling of striatonigral and striatopallidal medium spiny neurons past, present, and future.</a> Int. Rev. Neurobiol. 89, 1-35 (2009).</li><li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298, 1959-1964 (2002).</li><li>Chilton, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+mechanisms+of+axon+guidance.">Molecules mechanisms of axon guidance.</a> Dev. Biol. 292, 13-24 (2006).</li><li>Lumsden, A. G., Davies, 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=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J., Jessel, 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=Chemotropic+guidance+of+developing+axons+in+the+mammalian+central+nervous+system.">Chemotropic guidance of developing axons in the mammalian central nervous system.</a> Nature. 336, 775-778 (1988).</li><li>Kolk, S. M., Gunput, R. A., Tran, T. S., van den Heuvel, D. M., Prasad, A. A., Hellemons, A. J., Adolfs, Y., Ginty, D. D., Kolodkin, A. L., Burbach, J. P., Smidt, M. P., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+3F+is+a+bifunctional+guidance+cue+for+dopaminergic+axons+and+controls+their+fasciculation,+channeling,+rostral+growth,+and+intracortical+targeting.">Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channeling, rostral growth, and intracortical targeting.</a> J. Neurosci. 29, 12542-12557 (2009).</li><li>Fenstermaker, A. G., Prasad, A. A., Bechara, A., Adolfs, Y., Tissir, F., Goffinet, A., Zou, Y., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/planar+cell+polarity+signaling+controls+the+anterior-posterior+organization+of+monoaminergic+axons+in+the+brainstem.">Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem.</a> J. Neurosci. 30, 16053-16064 (2010).</li><li>Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip2+controls+the+differentiation+of+medium+spiny+neurons+and+the+establishment+of+the+cellular+architecture+of+the+striatum.">Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.</a> J. Neurosci. 28, 622-632 (2008).</li><li>De Wit, J., Toonen, R. F., Verhage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+local+retention+of+secretory+vesicle+cargo+in+cortical+neurons.">Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.</a> J. Neurosci. 29, 23-37 (2009).</li><li>G&#228;hwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. <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:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends Neurosci. 20, 471-477 (1997).</li></ol>

The collagen matrix assay described here has been used and improved by many different labs in the past decades to investigate a variety of axon guidance molecules and neuronal systems (e.g. see references 5-8). These studies have shown that this assay is a powerful tool for studying the effects and regulation of axon guidance molecules secreted by different (intermediate) target tissues.
However, it should be noted that the collagen matrix is a substitute for the extracellular environment (EC...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>BioWhittaker</td>
 <td>14-801F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glutamine (200mM)</td>
 <td>PAA</td>
 <td>M11-004</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>VWR International</td>
 <td>441476L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Merck</td>
 <td>444203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Minimum Essential Media (MEM)</td>
 <td>Gibco</td>
 <td>61100-087</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>Gibco</td>
 <td>21103-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium</td>
 <td>Gibco</td>
 <td>11415-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15070-063</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Prolong Gold Antifade Reagent</td>
 <td>Invitrogen</td>
 <td>P36930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dialysis tubing</td>
 <td>Spectrum Labs</td>
 <td>132660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rabbit anti-Tyrosine Hydroxylase</td>
 <td>Pel-Freez</td>
 <td>P40101-0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rabbit anti-Darpp32 (H-62)</td>
 <td>Santa-Cruz</td>
 <td>Sc-11365</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse anti-&beta;III tubulin</td>
 <td>Sigma</td>
 <td>T8660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor labeled secondary antibodies</td>
 <td>Invitrogen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

dissection-culture-mouse-dopaminergic-striatal-explants-three

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Neuroscience

25.5K 조회수.  University Medical Center Utrecht. midbrain의 도파민 시스템과 striatum에서 Explants가를위한 콜라겐 매트릭스 분석에 사용됩니다 체외에서 분석. 본 분석에서는 axonal 파생물 및지도는 조작과 계량 수 있습니다. 그것은 또한 다른 지역이나 분자 단서를 평가에 대해 수정할 수 있습니다.