We thank Karine Kindbeiter, Muriel Bozon, and Florie Reynaud for their help. The work is supported by CNRS, ANR YODA program and Labex DevWeCan.

DAY 1
1. Dissection of the Spinal Cord Floor Plate (FP) from E12.5 Mouse Embryos
Note: The entire procedure requires the use of sterile conditions. It is preferable to perform the dissection under a dissection hood to avoid contamination. The surface of the hood should be cleaned with ethanol. All dissection instruments must be sterilized and kept in a sterile Petri dish. The liquids (medium, dissection medium) must be kept closed&#160;and put in an ice bath. Each embryo should be collected in individual fresh drop. This is important for tissue preservation. The dissection is performed with Dumont #5 forceps. To transfer the embryos between dishes, grip the umbilical cord or the head without any damage to the hindbrain. It is critical not to damage the spinal cord to successfully complete the dissection. Prepare cold Phosphate Buffered Saline (PBS), cold Hank&#39;s Balanced Salt Solution (HBSS)-6.5% glucose and room temperature neurobasal medium.
1.1 Spinal cord dissection
Animals are treated according to the animal care guidelines of the Centre National de la Recherche Scientifique following European directives. The method of euthanasia is cervical dislocation, it consist of applying pressure to the neck and dislocating the spinal column from the brain. This method is recommended for mouse animal models, and can be performed in a fast and efficient way.
<ol>
	<li>Euthanize a pregnant mouse at E12.5 of gestation (E0.5= first day following matting). Place the mouse ventral side up.</li>
	<li>Soak the abdomen with 70% ethanol. Pinch the skin of the abdomen with Adson forceps and cut through the skin and the peritoneal layers with surgical scissors. Make an incision and use it to cut on both side of the mouse to expose the abdominal cavity.</li>
	<li>A string of embryos is present on each side of the mouse. Grasp one horn of the uterus in the tissue between two embryos and dissect it out starting from the exterior part. Transfer the intact uterus to a 100 mm Petri dish with cold PBS on ice.</li>
	<li>Extract the embryos from extra-embryonic tissues. Grasp the placenta tissue (in dark red) with two pairs of forceps and pull softly to tear the tissue and remove the embryos from the uterine sac.</li>
</ol>
Note: For a better conservation, collect the embryos from their uterine sac one by one.
<ol start="5">
	<li>The next steps are done under binocular magnifying glass and dissection hood. Place one embryo in a drop of cold HBSS-6.5% glucose.</li>
	<li>Using forceps, decapitate the embryo.</li>
</ol>
Note: Cut between the lower jaw and the hindbrain, the posterior part of the hindbrain is important for the next steps.
<ol start="7">
	<li>Place the embryo ventral face down with the anterior part facing the experimenter.</li>
	<li>Pinch the skin of the embryo with one pair of forceps and with the other pair pull away the skin.</li>
</ol>
Note: This step has to be repeated until the skin is completely removed from the anterior part of the embryo.
<ol start="9">
	<li>Turn the embryo anterior side to the left. With one forceps &#34;pin down&#34; the embryo in the anterior part. With the other forceps grab the skin and pull towards the rostral side of the embryo. The spinal cord is then accessible.</li>
	<li>Use one side of the forceps to cut the meninges that still wrap the spinal cord. The spinal cord is now open.</li>
	<li>Turn the embryo anterior side to the right.</li>
	<li>Detach the tissue from the spinal cord starting from the cervical side of the embryo. Slide the forceps under the spinal cord (the forceps should be held closed to avoid spinal cord damage). The forceps have to be visible under the spinal cord. Small rotatory movements under the spinal cord are sufficient to detach the surrounding tissue and the dorsal root ganglia (DRG).</li>
</ol>
Note: Leaving attached surrounding tissue add weight to the spinal cord, thus increasing the probability of breaking it during the next step.
<ol start="13">
	<li>Place the isolated spinal cord in a fresh drop of cold HBSS-6.5% glucose.</li>
	<li>Place the spinal cord in a flat position, meninges on the top and anterior side away from the experimenter.</li>
	<li>&#34;Pin down&#34; the spinal cord using the remaining hindbrain with one forceps, and with the second forceps grab the meninges. Peel off the meninges starting from the rostral side of the spinal cord.</li>
</ol>
Note: The movement should be slow and constant to avoid any breaking of the spinal cord. Vibratory movements can facilitate the detachment of the meninges.
1.2 FP dissection
<ol>
	<li>Pinch the hindbrain with one forceps.</li>
	<li>The FP is the central transparent part of the tissue. Using a scalpel cut out the floor plate.</li>
</ol>
Note: To cut both sides with the same quality, start from the rostral side and alternately cut the right side and the left side of the floor plate.
<ol start="3">
	<li>Conserve the dissected FP in Neurobasal medium at room temperature.</li>
</ol>
2. FP Culture
Note: All steps should be performed under sterile conditions in a tissue culture hood. Use fresh medium and freshly thawed supplements and reagents. Prepare sterile glass coverslips, warm neurobasal+B27, plasma, and HBSS-thrombin.
2.1 FP culture
<ol>
	<li>Place several sterile coverslips in a 100 mm Petri dish and pipette 20 &#956;l plasma in the center of each coverslip.</li>
	<li>Transfer two to four FP in each plasma drop and add 20 &#956;l HBSS-thrombin next to the plasma drop and carefully mix. Keep the coverslips for a minimum of ten minutes at room temperature to allow coagulation of the plasma.</li>
</ol>
Note: Transfer two FPs in case of complete dissection and more in case of partial dissection. Mix slowly with a pipette tip and avoid contact with the FP explants.
The plasma clot retracts a few &#956;m as a sign of the coagulation. The coagulation must not be stopped prematurely, to avoid detachment of the plasma clot.
<ol start="3">
	<li>Transfer each coverslip in a 24-well plate and add 500 &#956;l of Neurobasal+B27.&#160;Incubate 48 hr at 37 &#176;C.</li>
</ol>
DAY 3
3. FP Conditioned Medium (FPcm) Harvesting
Note: Harvesting should be performed under sterile conditions in a tissue culture hood. If the supernatant is contaminated or if floor plates are floating (meaning they might have died during the culture period) then do not harvest the FPcm from this well.
3.1 FPcm harvesting
<ol>
	<li>Harvest carefully the medium from each well.</li>
</ol>
Note: Do not pipette the polymerized plasma-thrombin mix or any FP fragments.
<ol start="2">
	<li>Store 100 &#956;l aliquots at -80 &#176;C.</li>
</ol>
DAY 4
4. Dissection of Spinal Cord Commissural Neurons from E12.5 Mouse Embryos
Note: This preparation does not need to be performed under sterile conditions. Follow the procedure described above to collect the embryos. Prepare cold PBS, cold HBSS-6.5% glucose and cold neurobasal.
4.1 Spinal cord dissection
<ol>
	<li>Using forceps, decapitate the embryo.</li>
</ol>
Note: Cut between the lower jaw and the hindbrain, the posterior part of the hindbrain is important for the next steps.
<ol start="2">
	<li>Place the embryo ventral face down in a fresh drop with the anterior part facing the experimenter.</li>
	<li>Pinch the skin of the embryo with one forceps and with the other one pull away the skin.</li>
</ol>
Note: This step has to be repeated until the skin is completely removed from the anterior part of the embryo.
<ol start="4">
	<li>Turn the embryo anterior side to the left. With one forceps &#34;pin down&#34; the embryo in the anterior part. With the other forceps, grasp the skin and pull towards the rostral side of the embryo, to expose the spinal cord.</li>
	<li>Use one side of the forceps to cut the meninges that still wrap the spinal cord. The spinal cord is now exposed and open.</li>
	<li>Position the embryo anterior side to the right.</li>
	<li>Detach the tissue from the spinal cord starting from the cervical side of the embryo. Slide the forceps under the spinal cord (keep the forceps closed). Forceps must visible under the spinal cord. Small rotatory movements under the spinal cord are sufficient to detach the adjacent tissue and the dorsal root ganglia (DRG).</li>
	<li>Transfer the isolated spinal cord in a new drop of cold HBSS-6.5% glucose.</li>
	<li>Place the spinal cord in a flat position, meninges on the top and anterior side away from the experimenter.</li>
	<li>&#34;Pin down&#34; the spinal cord using the remaining hindbrain with one forceps, with the second forceps grab the meninges. Peel off the meninges starting from the rostral side of the spinal cord.</li>
</ol>
4.2 Commissural Neuron dissection
<ol>
	<li>Pinch the hindbrain with one forceps.</li>
	<li>The Commissural neurons are located in the most lateral part of the spinal cord (the dorsal side). Cut out this lateral part with a fine scalpel.</li>
</ol>
Note: The dorsal part of the spinal cord can be distinguished from the ventral part by difference of cell density, the ventral part being more opaque.
Note: To cut both sides with the same quality, start from the rostral side and alternately cut a small length of the right side and of the left side.
<ol start="3">
	<li>Conserve the dissected tissue in Neurobasal medium on ice for immediate use.</li>
</ol>
5. Treatment of Commissural Tissue with the FPcm
Note: This procedure does not require sterile conditions. The tissue should be collected in individual 1.5 ml tubes. The frozen FPcm should be used only once. Avoid multiple refreezing. Prepare warm FPcm (37 &#176;C).
5.1 Treatments of commissural neurons
<ol>
	<li>Dilacerate the commissural tissue with small scissors.</li>
</ol>
Note: Cut the tissue as small as possible, to potentiate the accessibility of the cells.
<ol start="2">
	<li>Distribute fairly the tissue between the different experimental conditions control treatment and FPcm treatment.</li>
	<li>Centrifuge the tissue fragments at 800 x g for 1 min at 4 &#176;C.</li>
	<li>Check the repartition between tubes.</li>
</ol>
Note: If necessary redistribute and centrifuge again until fragments are equally distributed.
<ol start="5">
	<li>Remove the supernatant.</li>
	<li>Add the warm FPcm and control treatments to the tissues.</li>
</ol>
Note: The volume must be adjusted to the amount of the pellet and should be at least twice the volume of the pellet.
<ol start="7">
	<li>Incubate at 37 &#176;C for 30 min.</li>
</ol>
Note: Gently shake every 10-15 min
<ol start="8">
	<li>Centrifuge at 800 x g for 1 min and remove the treatment.</li>
	<li>Add the lysis buffer</li>
</ol>
Note: The volume must be adjusted to the amount of the pellet and should be at least twice the volume of the pellet.
<ol start="10">
	<li>Pipette up and down to resuspend the pellet.</li>
</ol>
Note: A vortex can also be used.
<ol start="11">
	<li>Incubate 15 min on ice.</li>
	<li>Centrifuge at 14,000 x g for 15 min at 4 &#176;C.</li>
	<li>Keep the supernatant and measure the amount of protein with a Bradford assay9.</li>
	<li>Add 6x&#160;Laemmli Buffer.</li>
	<li>Incubate at 95 &#176;C for 5 min to denature the protein content.</li>
</ol>
Note: Samples can be used immediately or can be stored at -80 &#176;C.
<ol start="16">
	<li>Assess the effects of the treatment by Western blot10.</li>
</ol>
Note: At least 50 &#956;g of total proteins should be loaded for each lane.

<ol>
	<li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell. Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Placzek, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+floor+plate:+multiple+cells,+multiple+signals.">The floor plate: multiple cells, multiple signals.</a> Nat. Rev. Neurosci. 6, 230-240 (2005).</li><li>Chedotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Curr. Opin. Neurobiol. 21, 68-75 (1016).</li><li>Nawabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Ruiz de Almodovar, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+mediates+commissural+axon+chemoattraction+through+its+receptor+Flk1.">VEGF mediates commissural axon chemoattraction through its receptor Flk1.</a> Neuron. 70, 966-978 (2011).</li><li>Charoy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gdnf+activates+midline+repulsion+by+Semaphorin3B+via+NCAM+during+commissural+axon+guidance.">gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance.</a> Neuron. 75, 1051-1066 (2012).</li><li>Kiecker, C., Lumsden, A. <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+organizers+in+patterning+the+nervous+system.">The role of organizers in patterning the nervous system.</a> Annu. Rev. Neurosci. 35, 347-367 (2012).</li><li>Le Dreau, G., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+of+the+neural+tube:+a+tale+of+three+signals.">Dorsal-ventral patterning of the neural tube: a tale of three signals.</a> Dev. Neurobiol. 72, 1471-1481 (2012).</li><li>Bradford, M. 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J. Vis. Exp. , (2010).</li><li>Salinas, 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=The+morphogen+sonic+hedgehog+collaborates+with+netrin-1+to+guide+axons+in+the+spinal+cord.">The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord.</a> Trends Neurosci. 26, 641-643 (2003).</li><li>Bechara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK-MAPK-dependent+adhesion+disassembly+downstream+of+L1+contributes+to+semaphorin3A-induced+collapse.">FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.</a> EMBO J. 27, 1549-1562 (2008).</li></ol>

The authors have nothing to disclose

The production of floor-plate conditioned medium provides an efficient and easy way for assessing the biological properties of floor plate released signals.
The plasma-thrombin matrix provides excellent condition for tissue survival. Nevertheless, this enriched environment might be a limitation for some types of experiments. Thus, the floor plate tissue can also be cultivated in agarose matrix. The quality of the floor plate conditioned medium can be assessed by the detection of known floor-pl...

The floor-plate is a well-known patterning center of the developing spinal cord, playing key roles in the specification of progenitors and postmitotic cell lineages and controlling axon navigation7,8. The experimental procedure described here to produce FPcm allows investigators to assess the functional properties of floor plate-derived signals in a various contexts of developmental processes, from cell patterning and survival to cell and axon migration.
To illustrate the use of such FPcm, dorsal spinal cord tissues containing commissural neurons are dissected, dilacerated and stimulated with the FPcm. The tissues can then be processed for Western blot analysis. This allows investigating regulations of the axon guidance machinery by floor-plate released signals. The method of treatment of dilacerated fresh tissue holds the great advantage of preserving the microenvironment of cells within the tissue. Thus the consequences of the treatment by FPcm or any types of treatment are assessed in a more physiological way than in cell and tissue culture conditions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS REQUIRED</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, Ca2+/Mg2+- free</td>
			<td>Invitrogen</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Sigma</td>
			<td>P-3266</td>
			<td>1mg/ml (Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T-4648</td>
			<td>10mg/ml(Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Hepes (260g/mol)</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5513</td>
			<td>0,5M pH8</td>
		</tr>
		<tr>
			<td>MgCL2</td>
			<td>Euromedex</td>
			<td>2189</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>S-6508</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Reagents required</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TOOLS AND MATERIAL</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Tips forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro knives</td>
			<td>Fine Science Tools</td>
			<td>10136-14</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Tools and material</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Dissection and FP culture</td>
					</tr>
					<tr>
						<td>Ethanol 70%</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection hood</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection microscope</td>
						<td></td>
					</tr>
					<tr>
						<td>Petri dishes</td>
						<td></td>
					</tr>
					<tr>
						<td>glass coverslips</td>
						<td>18mm x 18mm</td>
					</tr>
					<tr>
						<td>Bunsen gas burner</td>
						<td></td>
					</tr>
					<tr>
						<td>24 well plate</td>
						<td></td>
					</tr>
					<tr>
						<td>tissue culture incubator (37&#176;C, 5% CO2, humidity controlled)</td>
						<td></td>
					</tr>
					<tr>
						<td>Centrifuge</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">RECIPES</td>
					</tr>
					<tr>
						<td colspan="2">FP culture media</td>
					</tr>
					<tr>
						<td>Neurobasal</td>
						<td></td>
					</tr>
					<tr>
						<td>B27</td>
						<td>1/50e dilution from stock</td>
					</tr>
					<tr>
						<td colspan="2">HBSS - Thrombin</td>
					</tr>
					<tr>
						<td>HBSS</td>
						<td>1 ml</td>
					</tr>
					<tr>
						<td>Thrombin 10mg/ml</td>
						<td>30 &#956;l</td>
					</tr>
					<tr>
						<td colspan="2">Lysis Buffer pH7,4</td>
					</tr>
					<tr>
						<td>Hepes (260 g/mol)</td>
						<td>25mM</td>
					</tr>
					<tr>
						<td>EDTA (pH8)</td>
						<td>5mM</td>
					</tr>
					<tr>
						<td>MgCL2</td>
						<td>1mM</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>10%</td>
					</tr>
					<tr>
						<td>IGEPAL (NP40)</td>
						<td>0.10%</td>
					</tr>
					<tr>
						<td>Protease Inhibitor Cocktail</td>
						<td>1X</td>
					</tr>
					<tr>
						<td>Sodium Orthovanadate</td>
						<td>0,2M</td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">Laemmli Buffer</td>
					</tr>
					<tr>
						<td>Tris 0,5M, pH 6,8</td>
						<td>375 mM</td>
					</tr>
					<tr>
						<td>SDS</td>
						<td>6%</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>60%</td>
					</tr>
					<tr>
						<td>DTT</td>
						<td>0,6M</td>
					</tr>
					<tr>
						<td>Bromophenol blue</td>
						<td></td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

culture of isolated floor plate tissue and production of conditioned medium to assess functional properties of floor plate-released signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons1. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FPcm)4-6. We then exemplify the use of this FPcm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.

Commissural tissues were treated by the FPcm and the samples were processed for analysis of receptor levels in Western blot. Application of the FPcm was shown to increase the levels of a guidance receptor, PlexinA1, which mediates the sensitivity of commissural axons to the midline repellent Semaphorin3B after crossing (Figure 2). This revealed that signals released by the FP regulate PlexinA1 levels4,6.
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Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

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Research

JoVE Journal

Neuroscience

7.9K Views.  University of Lyon 1.  Плиты перекрытия является одним из важнейших структура развивающихся спинного мозга, обеспечивающих к диффузии сигналы предшественников и дифференцированных нейронов. Мы описываем способ получения от пола пластины кондиционированной среды, чтобы примени...

Video: Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse Formation Assay Using Presynapse Organizer Beads and &amp;ldquo;Neuron Ball&amp;rdquo; Culture

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...