Name: Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord (Video) | JoVE Journal
Uploaded: 2010-05-25T17:15:00.000+00:00
Duration: 12 min 23 s
Description: Learn to dissect and culture commissural neurons from E13 rat embryonic spinal cord using trypsin dissociation. This method enables detailed study of axon growth and guidance mechanisms in neural development.

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5 minutes

This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Peter Lougheed Medical Research Foundation, the McGill Program in Neuroengineering, the Fonds de Recherche en Sant&eacute; du Qu&eacute;bec (FRSQ), and the Canada Foundation for Innovation (CFI). S&eacute;bastien D. Langlois was supported by a Master's Training Award from the Fonds de la recherch&eacute; en sant&eacute; du Qu&eacute;bec (FRSQ) and by a Frederick Banting and Charles Best Canada Graduate Scholarships Master's Award from the Canadian Institutes of Health Research (CIHR). We are thankful to Jessica M.T. Pham for assistance with figures.

1. Dissection of embryonic rat dorsal spinal cord
General recommendations
Keep L-15 medium on ice and frequently change the medium in the dissection dish to keep the embryos cool. This helps preserve tissue integrity. All steps are performed with two pairs of Dumont #5 forceps unless stated. To avoid contamination, spray all tools and working surfaces with 70% ethanol and keep dissection medium bottle closed. To transfer embryos between dishes, use a cut plastic pipette or a perforated spoon. It is critical not to damage the spinal cord (nicking, stretching) to successfully complete the dissection.
Preparation
<ul>
	<li>Cold L-15 medium</li>
	<li>50 mL of L-15 + 10% heat-inactivated horse serum (HiHS). Keep on ice.</li>
</ul>
Spinal cord dissection
<ol>
	<li>Euthanize an E13 pregnancy-staged rat (E0 = first day following mating day) with a CO2 chamber according to institutional guidelines.</li>
	<li>Spray 70% ethanol on the abdomen. Pinch and pull up the skin of the lower abdominal region with forceps and cut with surgical scissors. Repeat to cut through the muscle and peritoneal layers to reach the abdominal cavity. Create a V-shape incision by cutting tissue along the sides of the abdomen, up to the thorax. Lift and pull back the tissue to expose the abdominal cavity.</li>
	<li>The uterus is attached at three locations: at the lower center abdomen, and at both upper lateral corners. Lift the uterus by grabbing on tissue between embryonic sacs. Cut the connective tissue to remove the uterus and place in a Petri dish filled with L-15 on ice.</li>
	<li>The next steps are done under a dissection microscope. To separate an embryo from extraembryonic tissues and embryonic membranes, grab the tissue between uterine sacs with one pair of forceps,. With the other pair, pinch the more transparent side of the sac to cut through the superficial membranes (the darker side is the placenta). Squeeze out the embryo by gently pressing the placenta side of the sac. Remove all embryos and place in a Petri dish filled with L-15 on ice.</li>
	<li>Place one embryo in a 10 cm Petri dish filled with ice-cold L-15. Using microscissors, cut away the head and posterior parts at angles shown in figure 1a. Cutting at these angles will help position the embryo when placed "ventral face down". </li>
	<li>Position the embryo "ventral face down" (anterior pointing away, posterior pointing towards the experimenter) (fig. 1b).</li>
	<li>Firmly hold the embryo from one side, using the forceps to "pin down" the embryo (fig. 1c). Do not move the forceps, as it will tear the tissue. With the other pair of forceps, peel off the skin covering the back of the embryo starting from the region in between the "holding" forceps (fig 1d-e). This will expose the spinal cord which, at this point, is wrapped by membranes (the meninges). Grab the skin from the sides rather than from above the spinal cord to avoid nicking the spinal cord. </li>
	<li>Partially detach the tissue to the right side of the spinal cord (fig. 1f). Starting at the level of the holding forceps, poke the tissue with closed forceps, as close as possible to the spinal cord. Then, slowly open the forceps to tear the tissue. This should detach the dorsal root ganglia (DRGs) from the spinal cord, and should also disrupt ventral organs. Leave some tissue attached at the anterior and posterior ends. Leaving tissue attached adds weight to the embryo, thus preventing it from being pulled upward during the next step. In addition, that tissue is used to hold on to the embryo in later steps.</li>
 <li>Starting from the anterior end, use the hook-shaped tungsten needle to cut the meninges and open the spinal cord along the roofplate (fig. 1g).</li>
	<li>Rotate the embryo 180 degrees (posterior pointing away, anterior pointing towards the experimenter) (fig. 1h). This allows the experimenter to use the same hand to detach the tissue from the other side of the embryo. </li>
	<li>Hold the embryo by grabbing onto the previously detached side, and disrupt the tissue from the remaining side using the same method (see 1.8.). When done, completely detach the tissue on both sides of the spinal cord. Alternative: Completely detach the tissue remaining on the left side, but leave some tissue attached at the anterior and posterior end on the right side. This can sometimes help maintain the spinal cord in position in step 1.12.</li>
	<li>Place the spinal cord on its side and remove most of the remaining mesenchymal tissue and DRGs (fig. 1i).</li>
	<li>At this step, the meninges and the spinal cord are two "sheets" of tissue apposed onto each other. Pin down the larger, anterior-most portion of the spinal cord, and peel off a short segment of meninges from the spinal cord (fig. 1j, left). Now, hold the two separated segments by grabbing across with forceps, and peel off the meninges with a smooth, constant movement (fig. 1j). Uneven "peeling" will lead to breakage of the spinal cord and/or the meninges layer. It is usually not possible to recover the portion of the spinal cord still attached to meninges.</li>
	<li>Using a plastic pipette, transfer the isolated spinal cord to a Petri dish containing L-15 + 10% HiHS and leave on ice. Typically, spinal cords of all embryos are collected before dissecting the dorsal portions.</li>
</ol>
Dorsal spinal cord dissection
<ol start="15">
	<li>Place one spinal cord in a Petri dish containing L-15+10% HiHS, lying flat in an "open-book" configuration. Cut away the wider, anterior portion (which includes part of the hindbrain) (fig. 1k).</li>
	<li>The dorsal tissue is located in the lateral-most parts of the spinal cord (fig 1l, left). While pinning the cord with a straight tungsten needle, use the L-shaped tungsten needle to cut out a strip that is 1/5th the width of half the spinal cord. (fig. 1l, right). Place the dorsal strips in a 15 mL plastic tube containing L-15 + 10% HiHS on ice. Dorsal neural tube sections of ~12 E13 embryos will yield ~3.5-4 million cells for plating after dissociation. More cells can be obtained by performing a coarse dissection of the dorsal spinal cord (cutting wider dorsal strips), but commissural neuron purity will be reduced.</li>
</ol>
2. Commissural neuron culture
General recommendations
All steps should be performed under sterile conditions in a tissue culture hood unless otherwise stated. Use fresh medium and freshly-thawed supplements and reagents. The dissociation and trituration steps are performed in Ca2+/Mg2+-free HBSS to minimize Ca2+/Mg2+-dependent adhesion.
Preparation
<ul>
	<li>coated coverslips (use German Desag glass) or tissue culture plates (see coating procedure below).</li>
	<li>warm Neurobasal Plating Media (see below), in tissue culture dishes and CO2 equilibrated in the tissue culture incubator for at least 1.5 h before plating.</li>
	<li>2.5% trypsin in the 37 °C waterbath</li>
	<li>2 bottles of HBSS Ca2+/Mg2+-free, one at 4 °C, one at 37 °C.</li>
	<li>two fire-polished glass Pasteur pipettes one with a diameter half the usual size, and one with a diameter slightly smaller than half. Use sterilized Pasteur pipettes. To fire-polish the pipettes, use a Bunsen burner (the top of the light blue flame) to melt the tip to slightly decrease the diameter. Because this step is performed outside the tissue culture hood, spray the pipettes with 70% ethanol before placing under the tissue culture hood. For more efficient recovery of neurons, coat the Pasteur pipettes with media containing serum before the beginning of the dissociation or just before the trituration step (fill pipettes with media and keep for 30 seconds). This will prevent cells from sticking to the inside of the Pasteur pipette during the trituration.</li>
	<li>sterile milliQ water for washing PLL-coated dishes or coverslips</li>
	<li>12.5% MgSO4 solution in HBSS</li>
</ul>
Poly-L-lysine coating
If using glass coverslips, acid-wash for 24 hours and sterilize prior to plating (see Kaech and Banker, 2006). Use German Desag glass coverslips.
To coat poly-L-lysine on glass coverslips or plastic tissue culture dishes:
<ul>
	<li>under a tissue culture hood, cover surfaces with a small dome of 100 ug/ml PLL solution for 1.75-2 hours.</li>
	<li>wash twice with milliQ water, at least 5 minutes per wash (can be performed during the cell dissociation step below).</li>
	<li>store in water until use. Do not let the PLL-coated surface dry. To reduce PLL wastage for coverslips coating, place the coverslips in sterile bacterial Petri dishes. Coat and wash the coverslips by placing a dome of liquid on the coverslips. The bacterial Petri dishes are hydrophobic, so the liquid should remain on the glass coverslips. Transfer the coverslips to tissue culture dishes just before plating the cells. For neurons plated on plastic tissue culture dishes, adhesion is normally higher, thus neurite elongation might be diminished.</li>
</ul>
Dissociation and plating
<ol>
	<li>Verify that the dorsal strips have settled down to the bottom of the tube. Remove most of the L-15+10% HiHS with a Pasteur pipette. Quickly wash the dorsal neural tube strips once by adding 3 ml of cold (4 °C) HBSS.</li>
	<li>Let the dorsal neural tube strips settle for 2 minutes, then remove the HBSS with a Pasteur pipette.</li>
	<li>Add warm (37 °C) HBSS to a volume of 4.7 ml. Then add 0.3 ml of 2.5 % trypsin to give a final concentration of 0.15% trypsin.</li>
	<li>Incubate at 37 °C in the waterbath for 7 min. Mix once gently half-way through the incubation.</li>
 <li>Add 30 μl DNAse (25 000 U/mL) for a final concentration of 150 U/mL. Add 60 μl of MgSO4 and mix briefly, for a final concentration of 0.15%. For tissue from coarse dissections, incubate for an extra 1 minute at 37 °C in the waterbath. At this stage, the dorsal neural tube sections should be fragmented. If the dorsal neural tube sections have not begun to fragment, it usually means that the 2.5% trypsin stock is old, and new stock should be thawed, or the wash with cold HBSS did not effectively remove the HiHS from the sample.</li>
	<li>Centrifuge the tissue fragments at 200 g for 4 min.</li>
	<li>Remove the supernatant with a Pasteur pipette, leaving ~50-100 μl of liquid at the bottom of the tube.</li>
	<li>Flick the tube gently to loosen the pellet, then wash the cells by adding 5 ml of warm HBSS. Let settle at room temperature for 2 min.</li>
	<li>Centrifuge at 200 g for 5 min.</li>
	<li>Remove the supernatant with a Pasteur pipette, leaving ~50-100 μl of liquid at the bottom of the tube.</li>
	<li>Flick the tube gently to loosen the pellet and partially resuspend the cells. Then add 2 ml of warm HBSS.</li>
	<li>Use the small (half-diameter) fire-polished glass Pasteur pipette to dissociate the cells by slowly pipetting up-and-down 4-6 times. Avoid making bubbles, and pipette the liquid against the side of the tube. Do not over-triturate.</li>
	<li>Use the smallest fire-polished glass Pasteur pipette to further dissociate the cells by slowly pipetting up-and-down 3-4 times. Avoid making bubbles, and pipette the liquid against the side of the tube. Do not over-triturate. For tissue from coarse dissections, add an extra 1 ml of warm HBSS to the tube at the end of the dissociation. When dissociating the cells, it is not necessary to dissociate all the cell clumps and aggregates. Stop pipetting up-and-down or change to a Pasteur pipette with a smaller diameter if you see no further decrease in the size of the cell aggregates with further pipetting.</li>
	<li>Let any remaining tissue fragments settle in the tube for 1 min. It is not necessary to transfer the cells to a new tube.</li>
	<li>Take 20 μl of cell suspension and add 5 μl of trypan blue. Count cells on a haemocytometer. Neurons should be ≥95% viable by trypan blue exclusion.</li>
	<li>Plate the neurons in Neurobasal Plating Media (see Recipes below).
<ul>
	<li>Suggested plating densities for obtaining isolated neurons (low-density cultures to avoid neurons clumping or overlapping each other):</li>
	<li>120 000 - 180 000 cells/well for a 6 well plate</li>
	<li>60 000 - 75 000 cells/well for a 12 well plate</li>
</ul>
</li>
	<li>16-18 hours later, change the media to Neurobasal Growth Media (see Recipes below) Do not use a vacuum pump to aspirate the media from the culture dish when changing media; gently use a pipette. This avoids dislodging the neurons.</li>
</ol>
Representative Results:
Four hours after plating, neurons should have adhered to the poly-L-lysine (PLL)-coated surface. Under phase contrast illumination, adhered cell bodies are typically relatively flat and oval-shaped (fig. 2a). Cells that have not well adhered appear as spheres that move slightly when the dish is very gently tapped on the side. Many factors can potentially impede the adhesion of cells (see discussion). 
After 30 hours in vitro, most neurons have extended an axon with a visible growth cone (fig 2c,d). If poor axonal growth is observed, verify that the Neurobasal Growth Media has been made with fresh medium and supplements. Neurons remain healthy for at least 6 days in these conditions. This procedure has proven to reliably yield preparations highly enriched in commissural neurons, with ~90% of neurons expressing DCC (Yam et al. 2009). The width of the dorsal spinal cord strip that is used for preparing the cell suspension will affect the purity of the culture, with greater purity when thinner strips are used. An example application is shown in figure 3 (immunofluorescence). See the article by Yam et al. (2009) for more examples.
<img src="/files/ftp_upload/1773/1773fig1tmb.jpg" border="0" alt="Embryo dissection diagram, showing anterior-posterior orientation and stepwise incision method." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Schematics of spinal cord dissection steps. D = Dorsal, V =Ventral. <a href="https://www.jove.com/files/ftp_upload/1773/1773fig1.jpg">Click here to see a larger figure.</a>
<img src="/files/ftp_upload/1773/1773fig2.jpg" alt="Microscope image, cell growth over time, showing morphology changes at 4h and 30h in experiment." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2.Representative result of isolated commissural neurons plated on a PLL-coated glass coverslip. a,b) 4 hours after plating, neurons have adhered to the surface. Bar = 20 μm. c,d) 30 hours after plating, most neurons have extended an axon with a visible growth cone. Bar = 20 μm.
<img src="/files/ftp_upload/1773/1773fig3.jpg" alt="Fluorescent microscopy image, neuron structure, axon and dendrite visualization, scale 15µm." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. A commissural neuron immunostained for gamma-tubulin (green), with F-actin labeled by phalloidin (F-actin, red) and the nucleus by DAPI (blue).
Recipes and comments
Neurobasal Plating Media
<ul>
	<li>Neurobasal</li>
	<li>10% Heat-inactivated FBS (HiFBS)</li>
	<li>2 mM L-glutamine (from 200 mM stock solution) Optional: Penicillin/Streptomycin antibiotics (use half the normal concentration)</li>
</ul>
Neurobasal Growth Media
<ul>
	<li>Neurobasal</li>
	<li>B27 (1/50 dilution from stock)</li>
	<li>2 mM L-glutamine (from 200 mM stock solution)</li>
	<li>Optional: Penicillin/Streptomycin antibiotics (use half the normal concentration) Once the media is made, it can be kept at 4 °C for two weeks. To equilibrate the temperature and the pH of media before plating the cells, place media in tissue culture Petri dishes and place in a tissue culture incubator for at least 1.5 hours.</li>
</ul>
Neurobasal
After a bottle of Neurobasal medium has been opened, it can be kept for one month at 4 °C in the dark. Dispose of Neurobasal which has been opened for more than one month otherwise cell survival will be lower.
Heat-inactivated fetal bovine serum (HiFBS) or horse serum (HiHS)
To heat-inactivate FBS or HS, heat at 56 °C in a water bath for 30 minutes. Swirl the bottle approximately every 10 minutes or so. (For accuracy use a bottle of similar size filled with water. Place a thermometer in the water bottle to see when 56 °C is reached. Begin timing at this point.) Heat-inactivated FBS may need to be centrifuged to clear precipitates, and can be aliquoted and re-frozen at -20 °C.
L-Glutamine
Always thaw a fresh aliquot of L-glutamine for each experiment.
B27
Aliquots of B27 can be kept at 20 °C for long term storage, or at 4 °C for up to one month.

<ol><li>Charron, F., Stein, E., Jeong, J., McMahon, A. P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. , 113-1111 (2003).</li>
<li>Fabre, P., Shimogori, T., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Segregation+of+ipsilateral+retinal+ganglion+cell+axons+at+the+optic+chiasm+requires+the+Shh+Receptor+Boc%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh Receptor Boc.</a> Journal of Neuroscience. 30, 266-275 (2010).</li>
<li>Helms, A. W., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progenitors+of+dorsal+commissural+interneurons+are+defined+by+MATH1+expression%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Progenitors of dorsal commissural interneurons are defined by MATH1 expression.</a> Development. 125, 919-928 (1998).</li>
<li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+hippocampal+neurons%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Culturing hippocampal neurons.</a> Nature Protocols. 1, 2406-2415 (2006).</li>
<li>Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier-Lavigne, M., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boc+is+a+receptor+for+sonic+hedgehog+in+the+guidance+of+commissural+axons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Boc is a receptor for sonic hedgehog in the guidance of commissural axons.</a> Nature. 444, 369-373 (2006).</li>
<li>Yam, P. T., Langlois, S. D., Morin, S., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonic+hedgehog+guides+axons+through+a+noncanonical%2C+Src-family-kinase-dependent+signaling+pathway%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway.</a> Neuron. 62, 349-362 (2009).</li>
</ol>

We have described a method to dissect and culture commissural neurons from embryonic rat spinal cord. This procedure has been routinely used in our lab to reliably prepare neurons to study the cellular and molecular mechanisms of axon guidance. For cell biology and immunochemistry experiments, dissection of one litter produces sufficient neurons. When more cells are needed, such as in many biochemistry experiments, dissection of two litters may be required. For a trained person, dissection and dissociation of ~20 embryos...

<table><tbody><tr><td>Neurobasal medium, liquid</td><td>Invitrogen</td><td>21103-049</td><td>See Recipes and Comments</td></tr><tr><td>B27 supplement 50X</td><td>Invitrogen</td><td>17504</td><td>See Recipes and Comments</td></tr><tr><td>Poly-L-lysine 0.01% solution</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>L-15 medium, powder</td><td>Invitrogen</td><td>41300-070</td><td></td></tr><tr><td>Trypsin 2.5% (10X)</td><td>Invitrogen</td><td>15090-046</td><td></td></tr><tr><td>DNAse I, 25000 U/mL</td><td>Worthington Biochemical</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2643</td><td></td></tr><tr><td>HBSS, Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;-free</td><td>Invitrogen</td><td>14170-112</td><td></td></tr><tr><td>L-glutamine 200mM, liquid</td><td>Invitrogen</td><td>25030-081</td><td>See Recipes and Comments</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Dissection of embryonic rat dorsal spinal cord (see also Table I)&lt;/p&gt;&lt;ul&gt;&lt;li&gt;E13 pregnancy-staged rat&lt;/li&gt;&lt;li&gt;Ethanol 70%&lt;/li&gt;&lt;li&gt;Surgical scissors, Fine Science Tools&lt;/li&gt;&lt;li&gt;Forceps, Dumont #5, Fine Science Tools&lt;/li&gt;&lt;li&gt;Petri dishes&lt;/li&gt;&lt;li&gt;L-15 medium&lt;/li&gt;&lt;li&gt;Dissection microscope, Leica&lt;/li&gt;&lt;li&gt;Plastic transfer pipettes&lt;/li&gt;&lt;li&gt;Microscissors, Fine Science Tools&lt;/li&gt;&lt;li&gt;Tungsten needles and pin holders (one hook-shaped needle, one L-shapes needle, one straight needle), Fine Science Tools&lt;/li&gt;&lt;li&gt;Heat-inactivated Horse Serum (HiHS)&lt;/li&gt;&lt;li&gt;15 ml plastic tubes&lt;/li&gt;&lt;/ul&gt;&lt;p class=&quot;jove_step&quot;&gt;Commissural neuron culture (see also Table I)&lt;/p&gt;&lt;ul&gt;&lt;li&gt;Tissue culture incubators (37&amp;#x00B0;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, humidity controlled)&lt;/li&gt;&lt;li&gt;German Desag glass coverslips and/or plastic tissue culture dishes&lt;/li&gt;&lt;li&gt;Poly-L-lysine, Sigma&lt;/li&gt;&lt;li&gt;Sterile milliQ H&lt;sub&gt;2&lt;/sub&gt;O&lt;/li&gt;&lt;li&gt;Neurobasal, Invitrogen&lt;/li&gt;&lt;li&gt;Heat-inactivated Fetal Bovine Serum (HiFBS)&lt;/li&gt;&lt;li&gt;L-Glutamine&lt;/li&gt;&lt;li&gt;B27, Invitrogen&lt;/li&gt;&lt;li&gt;Penicillin/Streptomycin antibiotics&lt;/li&gt;&lt;li&gt;37&amp;#x00B0;C waterbath&lt;/li&gt;&lt;li&gt;Sterile Pasteur Pipettes&lt;/li&gt;&lt;li&gt;Bunsen gas burner&lt;/li&gt;&lt;li&gt;HBSS, Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;-free, Invitrogen&lt;/li&gt;&lt;li&gt;DNAse, Worthington&lt;/li&gt;&lt;li&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;/li&gt;&lt;li&gt;Centrifuge&lt;/li&gt;&lt;li&gt;Hemacytometer&lt;/li&gt;&lt;li&gt;Trypan Blue Solution&lt;/li&gt;&lt;/ul&gt;</td></tr></tbody></table>

dissection and culture of commissural neurons from embryonic spinal cord

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

This video demonstrates a method to dissect and culture comm choral neurons from embryonic spinal cord. The procedure begins by harvesting E 13 rat embryos and then dissecting out the spinal cords. During the dissection, spinal cords are opened along the roof plate and prepared in the open book configuration.Dorsal tissue strips are dissected and collected in a tube. The dorsal tissue is dissociated into a single cell suspension by trypsin and mechanical disruption. Commissural neurons are then cultured on polylysine coated glass cover slips or tissue culture dishes.They can then be used to study the cellular and molecular mechanisms of axon growth and guidance. Hi, I'm Sebastian from the Laboratory of Molecular Biology of neural Development at the Clinical Research Institute of Montreal. Hi, I'm Steve Moen, also from the Molecular Biology of Neural Development Laboratory.Today we will show you a procedure for dissecting and culturing commissural neurons from embryonic spinal cord. We use this procedure in our laboratory to study the cellular and molecular mechanisms of accent guidance. So let's get started.Today, spinal cords will be dissected from freshly harvested E 13 rat embryos placed in a dish of L 15 medium throughout the dissection. It is important to keep embryos and solutions on ice. Remember that for all dissection steps, it is crucial not to nickel stretch the spinal cord as this can impede the isolation of a fully intact spinal cord.Under a dissecting microscope, separate the embryo from all extra embryonic tissues and embryonic membranes. Then place one embryo in the dissection dish. Use micro scissors to cut away the head and posterior parts of the angles shown here.This will create a flat surface that will help position the embryo for easy access to the spinal cord. Position the embryo ventral side down with the anterior pointing away. Use forceps to firmly pin down the embryo on one side.Do not pinch or release the forceps or the tissue will tear with the second pair of forceps. Grasp the skin to the side of the spinal cord. Twe the tips of the holding forceps.Peel away the skin covering the back of the embryo to expose the spinal cord wrapped in its meninges to partially detach the tissue to the right of the spinal cord. Use closed forceps to poke the tissue starting at the level of the holding forceps. Get as close as possible to the spinal cord without touching it.Repeat several times along the spinal cord. This would attach some dorsal root ganglia and disrupt ventral organs. Leave some tissue attached at the anterior and posterior ends starting from the anterior end.Use a hook shaped tungsten needle to cut the meninges and open the spinal cord along the roof plate. Rotate the embryo 180 degrees and disrupt the tissue from the remaining side. Using the same method.Then completely detach the tissue on the left side of the spinal cord, the side that was disrupted earlier originally on the right place the spinal cord on its side and use forceps to remove most of the remaining mesenchymal tissue and dorsal root ganglia. Use forceps to peel open a short flap of meninges. At this point, the meninges and spinal cord are two sheets of sandwich tissue.Use the forceps to pin down the larger portion of the spinal cord at the anterior end. Grasp the two separated segments with forceps and peel off the meninges. With a smooth constant movement, uneven peeling can break the meninges layer and or the spinal cord.If the meninges layer breaks during pulling, the portion of the spinal cord that remains attached to the meninges is usually unrecoverable. Although it is sometimes possible. Use a plastic pipee to gently transfer the isolated spinal cord to a dish of L 15 medium with 10%heat inactivated horse serum on ice before processing more embryos.Remember to change the medium in the dissection dish with chilled L 15 to keep the embryo cool and help preserve tissue integrity. Harvest spinal cords from all the embryos before proceeding to the dorsal spinal cord dissections. To begin the dorsal cord dissection, place one freshly isolated spinal cord in a Petri dish containing L 15 with 10%heat and activated horse serum.Cut away the wider anterior portion, which includes part of the hind brain. Then lay the spinal cord flat and spread out like an open book. The dorsal tissue is located in the most lateral parts of the open book spinal cord while pinning the cord with a straight tungsten needle using L-shaped tungsten needle to cut out a strip approximately one fifth, the width of one half of the spinal cord cutting wider strips will increase yield, but reduce the purity of the culture.Transfer multiple dorsal strips to a 15 milliliter conical tube containing L 15 medium with 10%horse serum and place the tube on ice. The rest of the procedure will take place near or inside the tissue culture hood. For these cultures, it is important to prepare solutions and equipment in advance, usually before dissection.The tation step uses fire polished pasta pipes of two sizes, one of a diameter, which is half the original size and the other slightly smaller. Before starting coat the two fire polished pipes with serum containing medium by aspirating medium into the pipes and letting sit for 30 seconds. This will reduce the number of cells that stick to the glass during ation.Begin by verifying that the dorsal strips have settled to the bottom of the tube. Under lamina flow hood, remove most of the SUP natant with a plain pasta per pet. Quickly wash with three milliliters of cold magnesium and calcium free HBSS.Let the dorsal neural tube strip settle for two minutes. Then remove the HBSS with the pasta. Bring the volume up to 4.7 milliliters.With warm HBSS, add 0.3 milliliters of 2.5%trypsin to give a final concentration of 0.15%Tryin and mix gently incubate in a 37 degree Celsius water bath for seven minutes. Mixing once gently halfway through the incubation. Once the incubation is up, add DNAs for a final concentration of 150 units per milliliter at magnesium sulfate for a final concentration of 0.15%and mix briefly.The dorsal neural tube sections should now be fragmented. Centrifuge the tissue fragments at 200 G for four minutes. Remove the SUP supernatant with a pasta perpe leaving about 50 to 100 microliters of liquid at the bottom of the tube.Flick the tube gently to loosen the pellet. Then wash the cells by adding five milliliters of warm HBSS. Let the cells settle at room temperature for two minutes.Spin down and wash the cells again. This time, resus suspending in two milliliters of warm HBSS. Use a half diameter fire polish pipet to triturate the cells by slowly pipetting up and down four to six times, pipee the liquid against the side of the tube to break up larger clumps to reduce cell death.Avoid making bubbles and do not overate. Use the smallest fire polish glass pasta perpe to further dissociate the cells by slowly pipetting up and down three to four times in the same manner. It is not necessary to dissociate all the cell clumps as aggregates tend to form of dead cells in debris.Let any tissue fragments settle for one minute. Then remove 20 microliters of the SUP natant, which should contain suspended cells for counting in a hemo soter, the neurons should be at least 95%viable by trian blue ion plate. The neurons in neuro basal plating media at a concentration that is appropriate for your application.16 to 18 hours later, change the medium to neural basal growth media to avoid dislodging. The neurons gently remove the media with a pipet rather than aspirating with a vacuum pump. Here are some representative results of isolated commissural neurons plated on PLL Coated glass cover slips four hours after plating neurons have adhered to the surface 30 hours following plating.Most neurons have extended axons with a visible growth cone. These neurons can be easily used in immunofluorescence experiments. In this example, microtubules are labeled in green f acton in red and NA in blue.We've just shown you how to dissect and culture commissural neurons from embryonic spinal cords. When doing this procedure, it's important to remember that the purity of the commissural neurons depends on the thickness of the dorsal strip that are dissected out. So that's it.Thanks for watching and good luck with your experiments.

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Research

JoVE Journal

Neuroscience

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons.  An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors.  Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

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

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

The video demonstrates the isolation of oculomotor, trochlear, and spinal motor neurons from a transgenic mouse embryo. The embryo expresses motor neurons labeled with a green fluorescent protein (GFP) for easy visualization and identification. The motor neurons are surgically isolated under a fluorescence microscope and maintained in a culture medium.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Chicken Embryo Spinal Cord Slice Culture Protocol

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord.  In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

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.

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

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Learn to dissect and culture commissural neurons from E13 rat embryonic spinal cord using trypsin dissociation. This method enables detailed study of axon growth and guidance mechanisms in neural development.

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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