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 mL): 5 mL HBSS, 9 mL DMEM high glucose, 850 &mu;L of 30 % glucose, 5 mL horse serum (25 %). Add 200 &mu;L 100X Penicllin/Streptomycin. Store at 4&deg;C.</li>
	</ol>
	</li>
	<li>Prepare before starting the dissection
	<ol>
		<li>Prepare 100 mL of 4 % low melting agarose (LMP agarose) in 1X Krebs buffer: microwave the solution until the agarose is completely dissolved and then place the agarose in a 45 &deg;C water bath.</li>
		<li>Fill up the vibratome buffer tray with ice-cold 1X Krebs buffer and start the cooling element (keep at 4&deg;C). Fix a razor blade in the blade carrier and set-up the vibratome area with a scalpel, a fine paintbrush and a mini perforated spoon to pick up the slices. Prepare sterile Petri dishes (35 x 10 mm) with 1x Krebs buffer for collecting the slices. Keep on ice.</li>
		<li>Set-up the dissection area with sterile Petri dishes (100 x 15 mm) for dissection and smaller sterile Petri dishes (35 x 10 mm) for embedding, small scissors, two fine forceps (Dumont 5), a mini perforated spoon, a glass Pasteur pipette fire polished with a round closed tip and 1 L of 1X Krebs buffer on ice. Wipe all the dissection tools with 70 % ethanol.</li>
		<li>Add culture medium to the wells of a six-well plate (1.5 mL/well) and place it in a 37 &deg;C incubator.</li>
		<li>Prepare a six-well plate with 1.5 mL/well sterile 1X Krebs buffer and 15 &mu;L Penicilin/Streptomycin (100X)/well. Under sterile conditions, place Millicell Cell Culture inserts into the wells. Place the six-well plate next to the vibratome so that the brain slices can be transferred onto the filter membranes immediately after sectioning.</li>
	</ol>
	</li>
</ol>
2. Dissection and embedding of embryonic brains
<ol>
	<li>Anesthetize a pregnant female mouse using isoflurane and sacrifice the mouse by cervical dislocation (embryos should be at stage E12.5). Dissect out the uterus of the mouse by pulling up the uterus with forceps. Use other forceps to separate the mesometrium away from the uterus. Place the uterus in ice-cold 1X Krebs buffer. Use a fine forceps to separate the muscular wall of the uterus, Reichert's membrane and the visceral yolk sac from the embryo. Remove the embryos from the uterus. Place the dissected embryos into a separate Petri dish with sterile 1X Krebs buffer.</li>
	<li>Dissect the brain under a stereomicroscope. To dissect the brain, first cut off the head of the embryo. Fix the head by piercing fine forceps through the head (eye level). Use another pair of forceps to carefully remove the skin and skull. Use forceps to carefully lift the brain out and transfer it into a Petri dish with sterile 1X Krebs buffer. It is very important that the integrity of the whole brain is maintained during dissection, since damages to the brain tissue will create problems (such as tissue shredding) during sectioning on the vibratome.</li>
	<li>Wash the brains once in 4 % low-melting point (LMP) agarose. Embed 2-3 brains at a time in fresh 4 % LMP agarose. Place the embedding dishes on ice as even as possible. Use a Pasteur pipette with a fire-polished round tip to lift the brains until the bottom of the agarose is solidified. The brains should settle in a flat position horizontal to the bottom of the agarose block.</li>
	<li>After the agarose has fully solidified (after approximately 3 min), trim the agarose surrounding the brains and glue the agarose block onto the specimen stage of the vibratome. When gluing the blocks, make sure that the ventral side of the brain is parallel to the platform, since the brains should be cut in a horizontal section plane.</li>
</ol>
3. Vibratome sectioning
<ol>
	<li>Use a razor blade for sectioning. To obtain intact slices it is very important to maintain the temperature at 4&deg;C during the sectioning.</li>
	<li>Section 300 &mu;m thick horizontal slices at a frequency of 50 Hz, blade amplitude of 1.1 mm and a speed of 25 mm/sec.</li>
	<li>Use the fine paintbrush to push the slice in a mini perforated spoon to collect the brain slices and transfer them into a dish with sterile ice-cold 1X Krebs buffer. Choose the slice that contains ventral midbrain tissue (see Figure 1). In an E12.5 mouse brain there is just one 300 &mu;m horizontal slice that contains ventral midbrain tissue including dopaminergic neurons.</li>
</ol>
4. Slice culture
Steps 4.2-4.5 should be carried out under sterile conditions.
<ol>
	<li>Transfer the brain slices onto a Millicell cell culture membrane insert in a six-well plate with 1x Krebs buffer (see point 1.2.5). To transfer the slice use the mini perforated spoon (Fine Science Tools) and a fine paintbrush. Up to 3 slices can be placed on one membrane.</li>
	<li>Transfer the membrane with the slice to the six-well plate with culture medium (see point 1.2.5). The top of the membrane should not be covered by medium. The brain slice receives medium from below and air from above.</li>
	<li>Place the six-well plate in a incubator with 5 % CO2 at 37 &deg;C. It is very important that the slices are placed in the incubator within 2 hours after the initial step of the dissection. A more extended preparation time can result in poor survival of the slices.</li>
	<li>Slices can be maintained in vitro up to 3 days. Change 50% of the culture medium on the 2nd day.</li>
</ol>
5. Time-lapse imaging
<ol>
	<li>Let the slices recover in the incubator for 4-5 hours before starting time-lapse imaging.</li>
	<li>For time-lapse imaging, keep the slices on the membrane insert and transfer the insert into a 35 mm Ibidi &mu;-dish (the bottom of the dish consists of material with high optical quality).</li>
	<li>Add 1 mL of culture medium plus 1.5 &mu;L ascorbic acid (200 mM) to the dish. Ascorbic acid protects the slices against phototoxicity.</li>
	<li>Incubate slices in an environmental chamber at 37 &deg;C with 5 % CO2 during time-lapse imaging.</li>
</ol>
6. Representative Results
Figure 1 illustrates the preparation of organotypic slice cultures from E12.5 mouse brain. Figure 2 shows horizontal organotypic slices (acute and after several days in culture) obtained form E12.5 mouse brain. For comparison, frozen brain sections at the equivalent developmental stages are shown. Midbrain dopaminergic neurons are visualized with immunohistochemistry for tyrosine hydroxylase (TH). Midbrain dopaminergic neurons project to targets in the forebrain. These projections start to form at E12.5 and extend towards the forebrain during the subsequent days. We consider the development of forebrain projections as a good indication for the normal development of dopaminergic neurons in culture. In the horizontal slices midbrain dopaminergic neurons extend projections towards their proper forebrain target area. After 3 DIV (days in vitro) or when the forebrain target areas are damaged, aberrant projections extend towards the dorsal midbrain. Examples of coronal slices of E12.5 midbrain are shown in Figure 3. Dopaminergic neurons visualized with immunostaining for TH extend aberrant projections to the dorsal midbrain. Figure 4 shows an analysis of proliferating, necrotic and apoptotic cells in the organotypic slice cultures. BrdU immunostaining to visualize proliferating cells demonstrates that cells proliferate under culture conditions after 1 DIV. Proliferation is reduced after 4 DIV. After 3 DIV, many cells in the ventral midbrain undergo necrosis (propidium iodide staining) and apotosis (immunostaining for cleaved caspase-3). Figure 5 shows migratory paths of YFP-labeled neurons monitored in a time-lapse imaging experiment in an acute slice.
<img alt="Figure 1" src="/files/ftp_upload/3350/3350fig1.jpg" /> 
Figure 1. Schematic illustrating the preparation of organotypic slice cultures. 300 &mu;m horizontal brain slices are prepared by sectioning an E12.5 brain using a vibratome. (A) Schematic sagittal view of an E12.5 mouse brain. Levels of sections are indicated. The area containing dopaminergic neurons is depicted in pink, projections are indicated in blue. (B) Schematics of the three slices that can be obtained from dorsal to ventral and that contain both forebrain (Fb) and midbrain (Mb). Note that only one slice contains dopaminergic neurons (slice b). The appropriate slice can be identified based on the position of the ventricles and the continuity of midbrain and forebrain tissue (arrows, compare section b with section a and c). The area containing dopaminergic neurons is indicated in pink, the area containing dopaminergic precursors is depicted in green, blue arrows indicate developing projections. (C) The slice containing dopaminergic neurons is cultured on membrane inserts. Abbreviations: vMb, ventral midbrain; dMb, dorsal midbrain; Hyp, hypothalamus; Hb, hindbrain.
<img alt="Figure 2" src="/files/ftp_upload/3350/3350fig2.jpg" /> 
Figure 2. Projections of midbrain dopaminergic neurons in organotypic slice cultures are dependent on the integrity of the forebrain. Immunohistochemistry for tyrosine hydroxylase (TH) to label dopaminergic neurons. (A) Acute slice (0 DIV) showing the normal location of dopaminergic neurons in the ventral midbrain. The white arrowhead indicates the area that is shown in higher magnification in the inset. Projections to the forebrain have not yet developed. After 1 DIV, projections to the forebrain start to form. Projections extend into the forebrain at 2-3 DIV. White arrowheads indicate the location of the cell bodies shown in higher magnification in the insets (white frame). Yellow arrows highlight the normal projections in intact slices shown in higher magnification in the insets (yellow frame). Aberrant projections develop in slices with damaged forebrain. Red arrows indicate the aberrant projections to the dorsal midbrain shown in higher magnification in the insets (red frame). Damage is indicated with red asterisks. After 3 DIV, most slices (n=5/7) had aberrant projections towards the dorsal midbrain. (B) TH immunostaining on horizontal frozen brain sections at different developmental stages to show the development of dopaminergic projections in vivo. White arrowheads indicate the location of the cell bodies shown in higher magnification in the insets (white frame). Yellow arrows highlight the position of the projections shown in higher magnification in the insets (yellow frame).The level of the frozen sections was chosen to closely match the level of the organotypic slice cultures. Note that a single frozen sections (12 &mu;m) does not represent the entire organotypic slice (300 &mu;m). Therefore, projections shown at E13.5 and E14.5 were observed on sections 120 &mu;m more ventral than the section containing the cell bodies (yellow asterisks).
<img alt="Figure 3" src="/files/ftp_upload/3350/3350fig3.jpg" /> 
Figure 3. Midbrain coronal slice cultures stained for tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. (A-C) Slices after 1, 2 or 3 DIV. Dopaminergic neurons develop aberrant projections towards the dorsal midbrain (red arrows). Arrowheads show the location of the dopaminergic cell bodies.
<img alt="Figure 4" src="/files/ftp_upload/3350/3350fig4.jpg" /> 
Figure 4. Cell proliferation and cell viability in midbrain organotypic slice cultures. (A) Proliferating cells were labeled by the addition of BrdU (50 ng/mL, Sigma) to the culture medium for 18 h. Slices were subsequently immunostained for BrdU. After 1 DIV, BrdU labeled cells are located in the ventricular zones (red arrows). After 4 DIV, proliferating cells are more scattered and a distinct ventricular zone is no longer maintained. (B) Necrotic cells were labeled by addition of propidium iodide (1&mu;g/&mu;L, Sigma) to the culture medium for 2 hrs and visualized by fluorescence microscopy. After 1 DIV, the ventral midbrain (vMb) is not necrotic, but many propidium iodide labeled cells can be seen in the dorsal midbrain and forebrain. After 3 DIV the cell viability decreases in the ventral midbrain. Scale bar: 500 &mu;m (C) Immunostaing for cleaved caspase-3 to visualize apoptotic cells. At 1 or 2 DIV, only few dopaminergic neurons (TH) are apoptotic. After 3 DIV, dopaminergic neurons start to undergo apoptosis. Panels in the middle and on the right are higher magnifications of the boxed area in the left panels. The higher magnifications images are maximum intensity projections of z-stacks of 14-16 frames. Images were taken every 0.5 &mu;m with an Zeiss Apotome set-up.
<img alt="Figure 5" src="/files/ftp_upload/3350/3350fig5.jpg" /> 
Figure 5. Migratory route of YFP-labeled neurons in an acute slice. (A) Horizontal slice used for time lapse imaging of YFP-labeled neurons. The slice was incubated for 5 hrs prior to imaging. Cells were labeled using an inducible Cre/loxP system6. ShhCreER mice 8 and ROSAloxP-STOP-loxP-EYFP reporter mice 9 were used. Recombination of the ROSA reporter allele (and EYFP expression) is induced in cells that express CreER (Shh-expressing cells), but only upon administration of Tamoxifen (Sigma). In this example, Tamoxifen (3 mg/40 g body weight) was administered to pregnant mice at E8.5. This experimental set-up results primarily in the labeling of precursors of dopaminergic neurons and their descendants in the ventral midbrain10,11 . Scale bar: 500 &mu;m. (B) Tracing the migratory route of YFP-labeled neurons in an acute slice. Time-lapse images of YFP-labeled fate mapped neurons were acquired every 30 min for a total time of 5 hrs 30 min on a Zeiss Axio-Observer microscope (objective EC PlnN 10x /0.3). Slices were incubated in an environmental chamber (incubator XLS1 Pecon) at 37 &deg;C and supplied with 5 % CO2 during the imaging. The initial position of the cells is marked with red arrows; migration positions are marked with red asterisks.

<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

17.9K vistas.  University of Bonn. Un método para generar cortes organotípicos del cerebro medio E12.5 embrionarias murinas se describe. Las culturas rebanada organotípicos se puede utilizar para observar el comportamiento de las neuronas dopaminérgicas o neuronas del cerebro medio ventral.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...