We would like to thank the &#34;Association pour le d&#233;veloppement de la neurog&#233;n&#233;tique&#34; for Dr. Jacquier&#39;s fellowship and AFM-Telethon for its support through MyoNeurAlp strategic plan. We would also like to thank Dr. Chris Henderson, Dr. William Camu, Dr. Brigitte Pettmann, Dr. Cedric Raoul, and Dr. Georg Haase, who participated in developing and improving the technique and spread their knowledge.

All procedures involving animals were accepted by the ethical committee of the institution.
1. Solution Preparation
<ol>
	<li>Prepare 10 mL of 4% BSA dialyzed in L-15 medium.
	<ol>
		<li>Dissolve bovine serum albumin powder at 4% (w/v) in 10 mL of L-15 medium.</li>
		<li>Add the solution to a 20 mL dialysis cassette with a 20 kDa cut-off. Dialyze against 500 mL of L-15 medium for 3 days under agitation at 4 &#176;C. Change the L-15 medium every day.</li>
		<li>Filter the solution through a 0.22 &#181;m membrane and aliquot in 15 mL tubes. Store the tubes at -20 &#176;C. 
		NOTE: This protocol uses the following references: unmodified raw L-15 medium = L-15 medium, acidic L-15 medium = pH L-15, and basic L-15 medium = Bicarbonate L-15.</li>
	</ol>
	</li>
	<li>Prepare 200 mL of pH L-15.
	<ol>
		<li>Adjust the pH of the L-15 medium: First, fill a wash-bottle with some pieces of dry ice. Inject gas (CO2) into the L-15 medium until the color turns orange (~pH 6.4 and ~40 pressures). The pH can be also adjusted with a standard pH meter.</li>
		<li>Filter the medium through a 0.22 &#181;m membrane and store at 4 &#176;C for one month.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of Bicarbonate L-15.
	<ol>
		<li>Add 2.5 mL of 7% sodium bicarbonate to 97.5 mL of L-15 medium and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the IPCS (Insulin Putrescin Conalbumin Selenite) supplements.
	<ol>
		<li>Dissolve 2.5 mg of insulin in 0.5 mL of 0.1 M HCl. Add 4.5 mL of double-distilled water.</li>
		<li>Dissolve 8 mg of putrescine in 5 mL of phosphate-buffered saline (PBS).</li>
		<li>Dissolve 100 mg of conalbumin in 10 mL of PBS.</li>
		<li>Dissolve 1 mg of sodium selenite in 19.3 mL of water, adjust the pH to 7.4, and dilute the solution 10-fold.</li>
		<li>Combine 1 mL of insulin solution, 1 mL of putrescine solution, 1 mL of conalbumin, and 0.1 mL of sodium selenite solution to obtain 3.1 mL of IPCS supplement solution. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a 0.02 mM progesterone solution.
	<ol>
		<li>Dissolve 1 mg of progesterone in 1.6 mL of 80% ethanol.</li>
		<li>Dilute the solution 100-fold in 100% ethanol and store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of complete L-15 medium.
	<ol>
		<li>Add 1.8 mL of D-glucose solution (200 mg/mL) to 92 mL of pH L-15 to obtain a final concentration of 3.5 mg/mL glucose.</li>
		<li>Combine 1 mL of 100x Penicillin-Streptomycin (10,000 U/mL), 2 mL of heat-inactivated horse serum, 3.1 mL of IPCS mix, and 0.1 mL of progesterone solution (2 x 10-5 M).</li>
		<li>Filter the medium on a 0.22 &#181;m membrane and store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 5 mL of density gradient solution (final concentration of 5.5%) per experiment.
	<ol>
		<li>Add 476 &#181;L of the commercial 60% density gradient medium to 4524 &#181;L of L-15 (if possible, without red phenol to increase the visual contrast at the interphase; dilution ratio of 1:10.5).</li>
		<li>Store the solution at 4 &#176;C for 2 weeks or freeze it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of culture media.
	<ol>
		<li>Add 200 &#181;L of supplement medium to 9.6 mL of neuron cell culture medium.</li>
		<li>Add 200 &#181;L of heat-inactivated horse serum (which tends to differentiate glial cell precursors and works against proliferation) to 25 &#181;L of glutamine and 10 &#181;L of 2-mercaptoethanol.</li>
		<li>Filter the media through a 0.22 &#181;m filter.</li>
		<li>Add the following neurotrophic factors: ciliary neurotrophic factor (CNTF) at a final concentration of 10 ng/mL, and brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) at final concentrations of 1 ng/mL.</li>
		<li>Store the media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of dissection media.
	<ol>
		<li>Add 2.25 mL of glucose (200 mg/mL) to 47.05 mL of HBSS. Add 700 &#181;L of 1 M HEPES with pH 7.4. Store the media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a 4% PFA solution.
	<ol>
		<li>Dissolve 20 g of PFA in 500 mL of PBS.</li>
		<li>Under a chemical hood, heat the solution to 60 &#176;C and add a few drops of 1 M NaOH until the solution becomes clear.</li>
		<li>Filter the solution through a filter paper and adjust the pH to 7.2. Store it at -20 &#176;C. 
		NOTE: Alternatively, other commercial PFA solutions can be used and diluted to 4% in PBS.</li>
	</ol>
	</li>
	<li>Clean the dissection tools including the forceps, scissors, and silicone dishes with 70% ethanol before use, and with soap and clear water after use.</li>
</ol>
2. Poly-ornithine/Laminin (Po/L) Dish Coating
NOTE: Volumes are adapted to coat a 24-well plate.
<ol>
	<li>If needed, put a sterile 12 mm coverslip in the well.</li>
	<li>Coat the wells with 500 &#181;L of 10 &#181;g/mL Poly-L-Ornithine solution dissolved in water.</li>
	<li>Let the wells settle at room temperature for 1 h.</li>
	<li>Remove the Poly-L-Ornithine solution and air-dry for 30 min.</li>
	<li>Coat the wells overnight at 37 &#176;C with 500 &#181;L of 3 &#181;g/mL laminin in bicarbonate L-15. 
	NOTE: Wells can be stored at 37 &#176;C for 7 days in the incubator. For long incubations, adding sterile PBS between the wells can help to avoid medium evaporation.</li>
</ol>
3. Dissection
<ol>
	<li>Sacrifice a pregnant mouse when the embryos are at embryonic stage E12.5. Clean the belly with 70% ethanol.</li>
	<li>Remove the womb by cutting the two oviducts and the vagina and put it in a Petri dish filled with cold PBS (without Ca2+ Mg2+).</li>
	<li>Collect all the embryos and transfer them to fresh, cold PBS (without Ca2+ Mg2+) in a Petri dish.</li>
	<li>Put one embryo in a dish coated with silicone and full of dissection media (with HBSS, 4.5 g/L glucose, and 7 mM HEPES pH 7.4). 
	NOTE: Embryo dissection is performed under a microscope using clean fine forceps and scissors. Alternatively, a regular Petri dish without silicone can be used for dissection.</li>
	<li>Remove the head, tail, and viscera, using forceps as scissors.</li>
	<li>Maintain the embryo with the belly against the silicone with forceps.</li>
	<li>With a second pair of forceps, insert one of the tips into the central canal of the rostral spinal cord.</li>
	<li>Close the forceps to tear the dorsal tissue a few millimeters.</li>
	<li>Repeat this operation toward the caudal side until the entire spinal cord is open (Figure 1B).</li>
	<li>Detach the rostral part of the spinal cord (cerebral trunk) from the body.</li>
	<li>Turn the embryo to maintain the open spinal cord against the silicone.</li>
	<li>Pinch the rostral part of the spinal cord and pull the embryo by the head to extract the spinal cord. 
	NOTE: Meninges and dorsal root ganglia (DRGs) should remain attached to the embryo. Otherwise, carefully pull away any remaining meninges and DRGs still attached to the spinal cord. At this step, DRGs could also be collected for sensitive neuron culture (see Discussion).</li>
	<li>Flatten the spinal cord on the silicone and remove the dorsal half of the cord by cutting along the middle of each side using a scalpel. Cut the middle part into 10 pieces using a scalpel, and transfer the pieces to a new tube.</li>
</ol>
4. Spinal Cord Cell Suspension
<ol>
	<li>Repeat this dissection procedure for 6 to 8 spinal cords.</li>
	<li>Let the spinal cord pieces collect at the bottom of the tube and replace the HBSS with 1 mL of Ham-F10 medium.</li>
	<li>Add 10 &#181;L of trypsin (2.5% w/v; final concentration 0.025%). Incubate the tube for 10 min at 37 &#176;C.</li>
	<li>To stop the enzymatic digestion, transfer the fragments to a new 15 mL tube containing 800 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate twice by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Collect the supernatant in a fresh 15 mL tube.</li>
	<li>To the fragments, add 900 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate 6 times by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Add the supernatant to the 15 mL tube obtained in step 4.5.</li>
	<li>To the fragments, add 900 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate 10 times by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Add the supernatant to the 15 mL tube obtained in step 4.5. Proceed with the supernatant.</li>
	<li>Using a P1000 pipet, gently place a 2 mL BSA 4% (w/v) cushion at the bottom of supernatant tube. Centrifuge at 470 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet with 2 mL of complete L-15 medium.</li>
</ol>
5. MN Enrichment by Gradient Density
<ol>
	<li>Split the cell suspension into two 15 mL tubes (1 mL each). Then, add 2 mL of complete L-15 medium. If starting with 6 embryos, use the equivalent of 3 embryos per tube in 3 mL of medium.</li>
	<li>Add 2 mL of density gradient solution by slowly adding the solution to the bottom of each tube to obtain a sharp interface.</li>
	<li>Centrifuge at 830 x g for 15 min at room temperature. After this step, small cells should be at the bottom of the tube, whereas large cells such as MNs should be at the density gradient solution/medium interface.</li>
	<li>Collect the cells at the interface by aspirating 1 or 2 mL and transfer them to a fresh 15 mL tube. Adjust the volume&#160;to 10 mL with L-15 pH medium.</li>
	<li>Add 2 mL of the 4% BSA cushion to the bottoms of the tube&#160;and centrifuge at 470 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and resuspend the pellet in 3 mL of complete L-15 medium.</li>
	<li>Repeat step 5.5.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 2 mL of complete culture medium. Count the cell number and viability under a phase contrast microscope with a hemocytometer. 
	NOTE: For 6 spinal cords, the cell number is expected to be around 1 x 105 MNs.</li>
</ol>
6. MN Culture
<ol>
	<li>Dilute the MNs to the appropriate density, usually 5,000 to 10,000 cells in 500 &#181;L of culture medium per well in a 24-well plate. 
	NOTE: Seeding density is around 30,000 and 1,500 cells for 6- and 96-well plates, respectively.</li>
	<li>Remove the laminin solution with a P1000.</li>
	<li>Immediately transfer the MNs diluted in culture medium into the coating plates to avoid drying.</li>
	<li>Incubate the culture for 2 days at 37 &#176;C.</li>
</ol>
7. Magnetofection of MNs
NOTE: In the following steps, the quantities used are meant for the transfection of one well of a 24-well plate. Please refer to the manufacturer&#39;s protocol for another format.
<ol>
	<li>For the DNA preparation, resuspend 1 &#181;g of DNA in 50 &#181;L of neuronal culture medium and vortex for 5 s.</li>
	<li>For bead tube preparation, resuspend 1.5 &#181;L of beads in 50 &#181;L of neuronal culture medium.</li>
	<li>Add the 50 &#181;L bead solution to the 50 &#181;L DNA solution and incubate for 20 min at room temperature (total volume = 100 &#181;L).</li>
	<li>During this incubation, withdraw 100 &#181;L of culture medium from the well that will be transfected. 
	NOTE: Put the magnetic plate in the incubator to warm it to 37 &#176;C.</li>
	<li>Transfer 100 &#181;L of the DNA/bead mix to the well, and incubate the plate 20&#8211;30 min on the magnetic plate at 37 &#176;C.</li>
	<li>Wait at least 24 h before detection of cDNA expression.</li>
</ol>
8. Fixation and Staining
<ol>
	<li>Wash the culture 2 times with PBS.</li>
	<li>Incubate the culture for 20 min at room temperature in 4% PFA/PBS.</li>
	<li>Wash the culture 2 times with PBS.</li>
	<li>Incubate the culture in 500 &#181;L of blocking buffer (PBS, 4% BSA, 2% normal serum, 0.3% Triton X-100, 0.1 M glycine) for 1 h at room temperature. 
	NOTE: Normal serum should be derived from the same species as the secondary antibody (e.g., Normal Goat Serum).</li>
	<li>Incubate the culture overnight at 4 &#176;C with primary antibody diluted in 250 &#181;L of blocking buffer. 
	NOTE: For example, TUJ1 is used at 1/1000, SMI32 is used at 1/1,000, and Lc3b is used at 1/200.</li>
	<li>Wash the culture 3 times with PBS.</li>
	<li>Incubate the culture with fluorophore-conjugated secondary antibodies diluted in 250 &#181;L of blocking buffer for 1 hour at room temperature.</li>
	<li>Wash the culture 3 times with PBS.</li>
	<li>Mount on glass slides using mounting medium.</li>
</ol>

<ol>
	<li>Gonzalez, M. 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=A+novel+mutation+in+VCP+causes+Charcot-Marie-Tooth+Type+2+disease.">A novel mutation in VCP causes Charcot-Marie-Tooth Type 2 disease.</a> Brain. 137 (11), 2897-2902 (2014).</li><li>Johnson, J. O., 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=Exome+sequencing+reveals+VCP+mutations+as+a+cause+of+familial+ALS.">Exome sequencing reveals VCP mutations as a cause of familial ALS.</a> Neuron. 68 (5), 857-864 (2010).</li><li>Watts, G. D. J., 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=Inclusion+body+myopathy+associated+with+Paget+disease+of+bone+and+frontotemporal+dementia+is+caused+by+mutant+valosin-containing+protein.">Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein.</a> Nature Genetics. 36 (4), 377-381 (2004).</li><li>Brown, R. H., Al-Chalabi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyotrophic+Lateral+Sclerosis.">Amyotrophic Lateral Sclerosis.</a> New England Journal of Medicine. 377 (2), 162-172 (2017).</li><li>Taylor, J. P., Brown, R. H., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+ALS:+from+genes+to+mechanism.">Decoding ALS: from genes to mechanism.</a> Nature. 539 (7628), 197-206 (2016).</li><li>Henderson, C. E., Bloch-Gallego, E., Camu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+embryonic+motoneurons.">Purified embryonic motoneurons.</a> Nerve Cell Culture: A Practical Approach. , 69-81 (1995).</li><li>Henderson, C. E., 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:+a+potent+survival+factor+for+motoneurons+present+in+peripheral+nerve+and+muscle.">GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.</a> Science. 266 (5187), 1062-1064 (1994).</li><li>Schaller, S., 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=Novel+combinatorial+screening+identifies+neurotrophic+factors+for+selective+classes+of+motor+neurons.">Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.</a> Proceedings of the National Academy of Sciences. 114 (12), E2486-E2493 (2017).</li><li>Jacquier, 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=Alsin/Rac1+signaling+controls+survival+and+growth+of+spinal+motoneurons.">Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.</a> Annals of Neurology. 60 (1), 105-117 (2006).</li><li>Raoul, 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=Chronic+activation+in+presymptomatic+amyotrophic+lateral+sclerosis+(ALS)+mice+of+a+feedback+loop+involving+Fas,+Daxx,+and+FasL.">Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL.</a> Proceedings of the National Academy of Sciences. 103 (15), 6007-6012 (2006).</li><li>Madji Hounoum, B., 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=Wildtype+motoneurons,+ALS-Linked+SOD1+mutation+and+glutamate+profoundly+modify+astrocyte+metabolism+and+lactate+shuttling.">Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.</a> Glia. 65 (4), 592-605 (2017).</li><li>Magrane, J., Sahawneh, M. A., Przedborski, S., Estevez, A. G., Manfredi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Dynamics+and+Bioenergetic+Dysfunction+Is+Associated+with+Synaptic+Alterations+in+Mutant+SOD1+Motor+Neurons.">Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons.</a> Journal of Neuroscience. 32 (1), 229-242 (2012).</li><li>Jacquier, 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=Cryptic+amyloidogenic+elements+in+mutant+NEFH+causing+Charcot-Marie-Tooth+2+trigger+aggresome+formation+and+neuronal+death.">Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death.</a> Acta Neuropathologica Communications. 5, (2017).</li><li>Aebischer, J., 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=IFN&#947;+triggers+a+LIGHT-dependent+selective+death+of+motoneurons+contributing+to+the+non-cell-autonomous+effects+of+mutant+SOD1.">IFN&#947; triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1.</a> Cell Death and Differentiation. 18 (5), 754-768 (2011).</li><li>Wiese, S., 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=Isolation+and+enrichment+of+embryonic+mouse+motoneurons+from+the+lumbar+spinal+cord+of+individual+mouse+embryos.">Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos.</a> Nature Protocols. 5 (1), 31-38 (2010).</li><li>Camu, W., Henderson, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+embryonic+rat+motoneurons+by+panning+on+a+monoclonal+antibody+to+the+low-affinity+NGF+receptor.">Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor.</a> Journal of Neuroscience Methods. 44 (1), 59-70 (1992).</li><li>Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+Isolation+and+Culture+of+Mouse+Embryonic+Motoneurons.">Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li><li>Francius, C., Clotman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+spinal+motor+neuron+diversity:+a+long+quest+for+neuronal+identity.">Generating spinal motor neuron diversity: a long quest for neuronal identity.</a> Cellular and Molecular Life Sciences. 71 (5), 813-829 (2014).</li><li>Neto, E., 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=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+In+Vitro+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.</a> The Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (1), 16-22 (2004).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Research Notes. 9, (2016).</li><li>Yu, L., 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=Highly+efficient+method+for+gene+delivery+into+mouse+dorsal+root+ganglia+neurons.">Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.</a> Frontiers in Molecular Neuroscience. 8 (2), (2015).</li><li>Malin, S. A., Davis, B. M., Molliver, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+dissociated+sensory+neuron+cultures+and+considerations+for+their+use+in+studying+neuronal+function+and+plasticity.">Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.</a> Nature Protocols. 2 (1), 152-160 (2007).</li></ol>

The authors have nothing to disclose.

One of the critical points in this protocol is that the mouse embryos are dissected at a precise time window during development (E12.5) to optimize the amount of MNs obtained at the end. In addition, for optimal yield, the dissection should be performed in the morning or early in the afternoon. Before E12.5 (e.g., at E11.5), dissection is difficult, especially regarding the elimination of the meninges. After E12.5, the number of obtained MNs drops significantly. To control the embryo stage of development, adult females and males are first placed together at the end of the day. The next morning, the adults are separated, and females are checked for the presence of a vaginal plug. If a plug is present, embryos are at this point considered to be in E0.5 day of development. For these experiments, we typically use a mouse line that is vigorous and productive. Progenitors originating from a colony bred in Missouri were used in this protocol, and the strain was named CF1 (Carworth Farms strain 1). This strain was introduced at Charles River Laboratories France in 1967, and it acquired the name OF1 (Oncins France 1).
A second critical point is the centrifugation steps that greatly influence the purity of the culture. MNs represent a low percentage of the spinal cord cells (around 1%), and the goal is to obtain a culture containing 40 to 50% MNs among other spinal neuronal cells and less than 5% glial cell progenitors. To monitor the efficacy of centrifugations, cell suspension samples acquired before and after each step can be plated in MNs medium for 24 hours, followed by fixing and staining for the expression of motor neuron markers Hb9 (81.5C10, DSHB) or Islet-1/Islet-2 (ISL1/2, 40.2D6 /39.4D5, DSHB), whose combinations define different MNs subpopulation19. On more mature MNs, other markers such as Choline Acetyl Transferase (CHAT, Ab144P) or Neurofilament H non-phosphorylated (SMI-32P) can be used to estimate the quality of the culture. A good alternative for quickly monitoring the efficacy of purification is to use transgenic mice that express the Green Fluorescent Protein (GFP) specifically in MNs. For example, Wichterle and colleagues previously developed transgenic mice expressing GFP under the control of the mouse Hb9 (also called Hlxb9 or Mnx1) promoter18. Hb9::GFP transgenic mice display distinct expression of GFP in dendrites, axons, and somas of spinal MNs from embryonic day 9.5 to postnatal day 10.
MNs require growth on a permissive substrate obtained by Poly-L-Ornithine and laminin coating. Depending on experimental conditions, a variety of plastic-, polymer-, and glass-based containers can be used (e.g., 6-, 24-, 96-well plates, glass coverslips, or chamber slides). Among these options, microfluidic devices may be of interest to address specific questions regarding synapse formation or axonal guidance and transport20. MNs are known to be sensitive to changes in their microenvironment involving patterns, concentration factors, mechanical changes in the substrate (e.g., stiffness), sheer stress, and spatiotemporal gradient cues (e.g., topographic features, patterning of surfaces with substances of different cellular affinities). Microfluidic platforms are systems that can integrate multifactorial conditions, allowing for the determination of optimal cellular microenvironments.
Since the discovery of GDNF&#39;s survival effects7, the number of neurotrophic factors involved in MN initial growth and survival, guidance and development of axons, and inducing of synaptic plasticity is growing. Interestingly, each factor acts on a specific subpopulation of MNs8. For example, CNTF has been described to protect the median motor column (MMC) which innervates axial muscles, whereas HGF acts on the lateral motor column (LMC) which innervates limb muscles. On the other hand, GDNF and BDNF both show the strongest pro-survival activity on whole populations of MNs. Finally, the combination of GDNF, BDNF, and CNTF is sufficient for protecting more than 70% of a MN population and is commonly used in laboratories.
In standard culture conditions, MNs can be kept in vitro up to 14 days by replacing half of the culture media with fresh media once every 3 days, starting from the fifth day of culture. During in vitro maturation, MNs can be transfected by magnetofection at any time using this protocol. Efficiency and toxicity of this technique may be modulated by the nature of the plasmids and inserts. In our case, when magnetofection was performed on MN culture after 2 days in vitro using a 9 kb plasmid with a CAGGS promotor (pCAGEN21) controlling neurofilament cDNA, we observed an average of 31.48% &#177; 9.94 (standard deviation) of transfected MNs 48 hours post-transfection. Nevertheless, for other constructs, a different ratio of DNA/beads should be tested as described in the manufacturer&#39;s protocol. Another parameter that may influence the transfection level efficiency is the maturation of MNs over time. Indeed, during the first 3 days of culture, growth of axons and enlargement of somas will occur, which increases the surface area where the beads will penetrate. This parameter may increase MN transfection efficiency over time during maturation.
Finally, in comparison with MN cultures, it would be interesting to cultivate sensory neurons22,23,24. In particular, Charcot-Marie-Tooth disease is a sensory motor neuropathy characterized by distal muscular atrophy related to the degeneration of both spinal MNs and sensory neurons of DRGs. Interestingly, DRGs are located on both sides of the spinal cord and can be collected during the same dissection procedure used to obtain MNs. In this regard, it is possible to compare the pathophysiological mechanisms at work in these two relevant cell types, motoneurons and DRG sensitive neurons.

Neuromuscular diseases encompass a variety of clinically and genetically distinct pathologies that are characterized by the alteration of muscle and/or the nervous system. Because of advances in sequencing technologies, hundreds of genes responsible for these rare disorders have been identified during the last decade (list available at the Neuromuscular Disease Center, http://neuromuscular.wustl.edu/index.html). The variety of identified mutations indicates that different mutations in a single gene can cause different phenotypes and diseases1,2,3 and that mutations in different genes can produce similar phenotypes4,5. In this context, there are efforts to develop cellular models that can become powerful tools for analyzing mutation consequences and pathological mechanisms.
Spinal MNs have large somas located in the ventral horns of the spinal cord, form long axons to target skeletal muscle fibers, and allow for voluntary movements through the release of acetylcholine at neuromuscular junctions. Since MNs are affected by neuromuscular diseases such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy, Dr. Henderson and colleagues developed the first protocol6 that allowed for cultivation of in vitro spinal MNs and the discovery of neurotrophic factor GDNF7 (glial cell derived neurotrophic factor). Technical refinements since then have allowed for more accurate purification of spinal MNs and their subtypes using FACS8, but enrichment by density gradient remains powerful and widely used in laboratories currently working with primary spinal MNs9,10,11,12,13,14. Subsequently, it is also possible to obtain a higher MN purification grade through immunopanning by taking advantage of surface marker p75(NTR) 15,16,17.
The spinal cord contains different subtypes of cervical, thoracic, and lumbar MNs, as well as median and lateral motor columns that differ among their location in the anterior horn on a dorso-ventral axis and among the targets they innervate8,18. Primary MN cultures can recover all of these MN subtypes in physiological proportions. The main limitation of this technique is the low number of MNs obtained at the end of the procedure; in fact, it can be expected to obtain around 105 MNs from six embryos, which is suitable for microscopy but limiting for biochemistry experiments. To perform experiments with more standardized subtypes and abundant MNs (&#62;106 cells), embryonic stem cell-derived MNs should be considered18.
Transfection of wild-type/mutant transgenes or knockdown endogenous genes into primary MNs is a rapid and helpful tool for deciphering physiopathological pathways, especially when mouse models are unavailable. Magnetofection is one technique for transfecting primary neurons, similar to lipofection without the related neurotoxicity. Furthermore, transfection can be performed on mature neurons after several days in vitro, unlike techniques based on electroporation9. However, one disadvantage of this technique is that the beads bind nucleic acids in the culture, causing noise in DAPI labeling. Viral infection is likely the most efficient technique for transfecting MNs; however, magnetofection does not require certain safety procedures needed for viral production and cellular infection.

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone dissection dish</td>
			<td>Living systems instrumentation</td>
			<td>DD-90-S-BLK-3PK</td>
			<td>Sylgard</td>
		</tr>
		<tr>
			<td>round coverslip</td>
			<td>NeuVitro, Knittel glass</td>
			<td>GG-12-Pre</td>
			<td>12 mm</td>
		</tr>
		<tr>
			<td>Slide a Lyzer cassettes</td>
			<td>ThermoFisher Scientific</td>
			<td>66030</td>
			<td>20,000 MWCO ; 30 mL</td>
		</tr>
		<tr>
			<td>Filter unit</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>GP Sterile Syringe Filters</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>4 well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>167063</td>
			<td>Nunclon Delta treated plate</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST by Dumont</td>
			<td>11252-20</td>
			<td>#5 forceps</td>
		</tr>
		<tr>
			<td>scissor</td>
			<td>FST by Dumont</td>
			<td>14060-10</td>
			<td>fine scissors</td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>FST by Dumont</td>
			<td>10035-20</td>
			<td>curved blade</td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>FST by Dumont</td>
			<td>10316-14</td>
			<td>micro-knife scalpel</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>663102</td>
			<td>&#248; x h = 100 x 15 mm</td>
		</tr>
		<tr>
			<td>15 mL polypropylene tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>Watman</td>
			<td>1001125</td>
			<td>circle, 125 mm diameter</td>
		</tr>
		<tr>
			<td>glass chamber slide</td>
			<td>Lab-Tek</td>
			<td>154526</td>
			<td>4 chambers</td>
		</tr>
		<tr>
			<td>Plasmid pCAGEN</td>
			<td>Addgene</td>
			<td>#11160</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions and mediums</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11415056</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 medium, no red phenol</td>
			<td>ThermoFisher Scientific</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Conalbumin</td>
			<td>Sigma-Aldrich</td>
			<td>C7786</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-DL-Ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P8638</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin 2.5%, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca Mg</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td>without Mg2+ Ca2+</td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuron cell culture medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A3582901</td>
			<td>Neurobasal Plus medium</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma-Aldrich</td>
			<td>H6648-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer 1 M</td>
			<td>ThermoFisher Scientific</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradiant medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>Optiprep</td>
		</tr>
		<tr>
			<td>supplement medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A3582801</td>
			<td>B-27 Plus</td>
		</tr>
		<tr>
			<td>Horse serum heat inactivated</td>
			<td>ThermoFisher Scientific</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM</td>
			<td>ThermoFisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>ThermoFisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>penicilline/streptomycine</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immuno fluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>X0515</td>
			<td>without Mg2+ Ca2+</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Acetyl Transferase (CHAT)</td>
			<td>Chemicon</td>
			<td>Ab144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofilament H non phosphorylated (SMI32)</td>
			<td>Biolegends</td>
			<td>SMI-32P</td>
			<td>IF at 1/1000</td>
		</tr>
		<tr>
			<td>Islet-1</td>
			<td>DSHB</td>
			<td>40.2D6</td>
			<td></td>
		</tr>
		<tr>
			<td>Islet-2</td>
			<td>DSHB</td>
			<td>39.4D5</td>
			<td></td>
		</tr>
		<tr>
			<td>Hb9</td>
			<td>DSHB</td>
			<td>81.5C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Lab</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta3 tubulin (Tuj1 clone)</td>
			<td>Biolegends</td>
			<td>801201</td>
			<td>IF at 1/1000</td>
		</tr>
		<tr>
			<td>Lc3b</td>
			<td>Cell Signaling Technology</td>
			<td>#2775</td>
			<td>IF at 1/200</td>
		</tr>
	</tbody>
</table>

modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

After 24 hours in the culture, motoneurons (MNs) should already show significant axonal growth (at least 6 times longer than the soma size). In the following days, axons should continue to grow and display branching (Figure 2). There will be different morphologies due to subtype specificities. For example, median column MNs that innervate axial muscles have shorter and more branched axons than lateral motor column MNs that innervate limb muscles18.
These isolation and culturing techniques described above have recently been used to characterize a new mutation in the neurofilament gene NEFH that causes an autosomal dominant axonal form of CMT13. In this case, two days after plating, purified MNs were transfected with plasmids encoding either a mutant form of NEFH fused to eGFP or the wild-type form fused to eGFP. Two days later, mutant NEFH-eGFP formed aggregates along the cytoskeletal network. Four days after magnetofection, these aggregates evolved in a prominent perinuclear aggresome containing the LC3b autophagic marker (Figure 3). This approach allowed us to demonstrate that the mutant form of NEFH induced the formation of aggregates that are associated with autophagic pathways in primary MNs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57988/57988fig1.jpg" /> 
Figure 1: Overview of procedure main steps.&#160;(A) E12.5 mouse embryos are dissected to extract the ventral horn of the spinal cord. Then, neurons from the ventral horn cells are dissociated and MNs are purified by density gradient before plating. Finally, MNs are transfected by magnetofection and analyzed after a few days. (B)&#160;The first step in spinal cord dissection is when the roof plate of the spinal cord is opened along the rostro-caudal axis with forceps. Then, the spinal cord is detached from the body by pulling the cerebral trunk. At this point, meninges and dorsal root ganglia (DRG) remain on the embryonic body. The dorsal part of the spinal cord is cut longitudinally with a scalpel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57988/57988fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57988/57988fig2.jpg" /> 
Figure 2: Representative MN culture.&#160;(A)&#160;MNs morphology after 4 days of culture revealed by staining of endogenous non-phosphorylated neurofilament heavy chain (SMI32). Scale bar = 100 &#181;m. (B)&#160;MNs morphology after 4 days in vitro and transfected for eGFP transgene by magnetofection at day 2. MNs were counterstained with the marker SMI32 or the pan-neuronal marker Tuj1. Arrows show somas of the neurons. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57988/57988fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57988/57988fig3.jpg" /> 
Figure 3: Confocal images of magnetofected MNs expressing wild-type or mutant eGFP-NEFH constructs (in green) and stained for the autophagic marker LC3b (in red). Arrows show mutant eGFP-NEFH protein aggregates that are also LC3b positive. Scale bar = 10 &#181;m. Images are modified from Jacquier et al. 2017, Acta Neuropathologica Communication13.

Watch this Scientific Journal Video about Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons at JoVE.com

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

modeling-charcot-marie-tooth-disease-vitro-transfecting-mouse-primary

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K Views.  Université Lyon. The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Video: Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

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Chapters in this video

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro