Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Published: May 25, 2010

doi:

Sébastien D. Langlois*^1,2, Steves Morin*¹, Patricia T. Yam^3,4, Frédéric Charron^2,5,6,7

¹Molecular Biology of Neural Development,Institut de Recherches Cliniques de Montréal, ²Division of Experimental Medicine and Program in Neuroengineering,McGill University, ³Program in Neuroengineering,McGill University, ⁴Montreal Neurological Institute, ⁵Department of Anatomy and Cell Biology,McGill University, ⁶Department of Biology,McGill University, ⁷Department of Medicine,Universite de Montreal – University of Montreal

Summary

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.

Abstract

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.

Protocol

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 pipett…

Discussion

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…

Acknowledgements

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 Santé du Québec (FRSQ), and the Canada Foundation for Innovation (CFI). Sébastien D. Langlois was supported by a Master’s Training Award from the Fonds de la recherché en santé du Québec (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.

Materials

Material Name	Company	Catalogue Number	Comment
Neurobasal medium, liquid	Invitrogen	21103-049	See Recipes and Comments
B27 supplement 50X	Invitrogen	17504	See Recipes and Comments
Poly-L-lysine 0.01% solution	Sigma	P4707
L-15 medium, powder	Invitrogen	41300-070
Trypsin 2.5% (10X)	Invitrogen	15090-046
DNAse I, 25000 U/mL	Worthington
MgSO₄	Sigma	M2643
HBSS, Ca²⁺/Mg²⁺-free	Invitrogen	14170-112
L-glutamine 200mM, liquid	Invitrogen	25030-081	See Recipes and Comments

Dissection of embryonic rat dorsal spinal cord (see also Table I)

E13 pregnancy-staged rat
Ethanol 70%
Surgical scissors, Fine Science Tools
Forceps, Dumont #5, Fine Science Tools
Petri dishes
L-15 medium
Dissection microscope, Leica
Plastic transfer pipettes
Microscissors, Fine Science Tools
Tungsten needles and pin holders (one hook-shaped needle, one L-shapes needle, one straight needle), Fine Science Tools
Heat-inactivated Horse Serum (HiHS)
15 ml plastic tubes

Commissural neuron culture (see also Table I)

Tissue culture incubators (37°C, 5% CO₂, humidity controlled)
German Desag glass coverslips and/or plastic tissue culture dishes
Poly-L-lysine, Sigma
Sterile milliQ H₂O
Neurobasal, Invitrogen
Heat-inactivated Fetal Bovine Serum (HiFBS)
L-Glutamine
B27, Invitrogen
Penicillin/Streptomycin antibiotics
37°C waterbath
Sterile Pasteur Pipettes
Bunsen gas burner
HBSS, Ca²⁺/Mg²⁺-free, Invitrogen
DNAse, Worthington
MgSO₄
Centrifuge
Hemacytometer
Trypan Blue Solution

Referências

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

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Langlois, S. D., Morin, S., Yam, P. T., Charron, F. Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord. J. Vis. Exp. (39), e1773, doi:10.3791/1773 (2010).