We thank Martine Emond and Isabel Brachmann for their help in establishing the organotypic slice culture system and Wolfgang H&uuml;bner and Liviu Gabriel Bodea for critical reading of the manuscript. We would like to thank Frank Costantini for the R26 reporter mice and Cliff Tabin for the ShhCreER mice. This study was funded by a Research Award from the Ministry of Science and Research of North-Rhine Westphalia (Programm zur F&ouml;rderung der R&uuml;ckkehr des wissenschaftlichen Spitzennachwuchses aus dem Ausland).

Parts of this protocol are modified from Daza et al., 2007 7.
1. Preparations
<ol>
	<li>Can be prepared one day in advance
	<ol>
		<li>Prepare 1X Krebs buffer (1.5 L): 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4:H2O, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose, 25 mM NaHCO3; adjust pH to 7.4. Filter sterilize (0.22 &mu;m pore size) and store at 4&deg;C.</li>
		<li>Prepare the culture medium (20 m...

<ol>
	<li>Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nat. Neurosci. 7 (2), 136-136 (2004).</li><li>Martini, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+selection+of+leading+process+branches+mediates+chemotaxis+during+tangential+neuronal+migration.">Biased selection of leading process branches mediates chemotaxis during tangential neuronal migration.</a> Development. 136 (1), 41-41 (2009).</li><li>Marin, O., Valiente, M., Ge, X., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+neuronal+cell+migrations.">Guiding neuronal cell migrations.</a> Cold Spring Harb. Perspect. Biol. 2 (2), a001834-a001834 (2010).</li><li>Ayala, R., Shu, T., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trekking+across+the+brain:+the+journey+of+neuronal+migration.">Trekking across the brain: the journey of neuronal migration.</a> Cell. 128 (1), 29-29 (2007).</li><li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nat. Rev. Neurosci. 8 (1), 21-21 (2007).</li><li>Legue, E., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate+mapping+using+site-specific+recombinases.">Genetic fate mapping using site-specific recombinases.</a> Methods. Enzymol. 477, 153-153 (2010).</li><li>Daza, R. A., Englund, C., Hevner, R. F. <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+culture+of+embryonic+brain+tissue.">Organotypic slice culture of embryonic brain tissue.</a> CSH Protoc. , (2007).</li><li>Harfe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+expansion-based+temporal+Shh+gradient+in+specifying+vertebrate+digit+identities.">Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.</a> Cell. 118 (4), 517-517 (2004).</li><li>Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Dev. Biol. 1, 4-4 (2001).</li><li>Joksimovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+separable+Shh+domains+in+the+midbrain+define+distinct+dopaminergic+progenitor+pools.">Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (45), 19185-19185 (2009).</li><li>Blaess, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-spatial+changes+in+Sonic+Hedgehog+expression+and+signaling+reveal+different+potentials+of+ventral+mesencephalic+progenitors+to+populate+distinct+ventral+midbrain+nuclei.">Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.</a> Neural. Dev. 6 (1), 29-29 (2011).</li><li>Hippenmeyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3 (5), e159-e159 (2005).</li></ol>

We have nothing to disclose.

The organotypic slice culture method presented here provides a system for the short-term in vitro analysis of developing dopaminergic neurons and their migratory and projection routes in the embryonic ventral midbrain. We found that there are a number of critical steps in the protocol that should be carefully attended to in order to obtain slices that allow the normal development of ventral midbrain dopaminergic neurons. The most critical step is the dissection of the embryonic brain, which has to be both fast a...

Table of specific reagents and equipment

<table border="1">
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma-Aldrich</td>
 <td>D6429</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucose 30%</td>
 <td>Sigma-Aldrich</td>
 <td>G7528-250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Horse Serum</td>
 <td>Invitrogen</td>
 <td>26050-088 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM (4,5g/L Glc., with L-Gln, Na Pyr, NaHCO3) </td>
 <td>Sigma-Aldrich</td>
 <td>D6429-500 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin 100x</td>
 <td>Sigma-Aldrich</td>
 <td>P4333-20 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-ascorbic acid</td>
 <td>Sigma-Aldrich</td>
 <td>A4403</td>
 <td>prepare 200mM stock and store at -20&deg;C</td>
 </tr>
 <tr>
 <td>UltraPure LMP agarose</td>
 <td>Invitrogen </td>
 <td>15517-022 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Millicel inserts</td>
 <td>Millipore</td>
 <td>PICMORG50</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&mu;-dish 35 mm, low</td>
 <td>Ibidi</td>
 <td>80136 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vibratome</td>
 <td>Microm</td>
 <td>HM 650V </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Razor Blade</td>
 <td>Plano GmbH</td>
 <td>121-6</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Histoacryl &nbsp;glue</td>
 <td>BRAU9381104 </td>
 <td>Braun Aesculap </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perforated Spoon Dia 
 diameter 15 mm </td>
 <td>Fine Science Tools</td>
 <td>10370 -18</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps 5 Dumoxel</td>
 <td>Fine Science Tools</td>
 <td>11252 - 30</td>
 <td>&nbsp;</td>
 </tr>
</table>


Antibodies used for immunostainings:


<table border="1">
 <tr>
 <td >Name of the antibody</td>
 <td>Company</td>
 <td >Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Rabbit anti-tyrosine hydroxylase </td>
 <td>Millipore </td>
 <td>AB152</td>
 <td>Dilution 1:500</td>
 </tr>
 <tr>
 <td>Mouse anti-tyrosine hydroxylase </td>
 <td>Millipore</td>
 <td>MAB318 </td>
 <td>Dilution 1:500</td>
 </tr>
 <tr>
 <td>Mouse anti-BrdU </td>
 <td>BD Pharmingen</td>
 <td>555627</td>
 <td>Dilution 1:200</td>
 </tr>
 <tr>
 <td>Rabbit anti-cleaved caspase 3</td>
 <td>Cell Signaling Technology</td>
 <td>9661 </td>
 <td>Dilution 1:200 </td>
 </tr>
 <tr>
 <td>Donkey anti-rabbit IgG-Alexa 488 </td>
 <td>Invitrogen </td>
 <td>A21206</td>
 <td>Dilution 1:500</td>
 </tr>
 <tr>
 <td>Donkey anti-mouse IgG-Cy3</td>
 <td>Jackson ImmunoResearch </td>
 <td>715-165-150</td>
 <td>Dilution 1:200</td>
 </tr>
</table>

organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Watch this Scientific Journal Video about Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro at JoVE.com

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

organotypic-slice-cultures-embryonic-ventral-midbrain-system-to-study

Research

JoVE Journal

Neuroscience

17.8K visualizações.  University of Bonn. Um método para gerar fatias organotípicas da E12.5 mesencéfalo embrionário murino é descrito. As culturas fatia organotípicas pode ser usado para observar o comportamento de neurônios dopaminérgicos ou outros neurônios do mesencéfalo ventral.