Dissection and Culture of Embryonic Spinal Commissural Neurons

Simon W. Moore1, Timothy E. Kennedy1

1 McGill University, Montreal, Quebec, Canada
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.20
DOI:  10.1002/0471142301.ns0320s45
Online Posting Date:  October, 2008
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Abstract

Studies of spinal commissural neurons have provided substantial insight into the mechanisms that regulate axon guidance. Explants of embryonic spinal cords and isolated spinal commissural neurons have been important experimental tools for the identification and characterization of several guidance cues, including netrins, semaphorins, slits, sonic hedgehog, BMPs, and wnts. In this unit, protocols are provided for establishing these explant assays to assess the outgrowth and turning capacity of commissural axons. In addition, methods are included for preparing cultures highly enriched with embryonic commissural neurons, which have been used to probe the biochemical signaling mechanisms regulating axon guidance. Curr. Protoc. Neurosci. 45:3.20.1‐3.20.17. © 2008 by John Wiley & Sons, Inc.

Keywords: spinal commissural neurons; tungsten needle; hanging drop; collagen

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Spinal Commissural Neurons
  • Basic Protocol 2: Spinal Commissural Neuron Axon Outgrowth Assay
  • Basic Protocol 3: Commissural Neuron Axon Turning Assay
  • Support Protocol 1: Electrolytic Sharpening of Tungsten Wire
  • Support Protocol 2: Embedding Tissue in a Collagen Matrix
  • Support Protocol 3: Immunolabeling Commissural Axons within Explants
  • Support Protocol 4: Hanging Drop Aggregation of Adherent Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of Spinal Commissural Neurons

  Materials
  • Rat, pregnant at E14.5 (vaginal plug = E1)
  • 70% ethanol
  • L15 dissection medium (Invitrogen cat. no. 41300‐039), ice cold
  • Hanks’ balanced salt solution (HBSS) without Ca2+/Mg2+ ( appendix 2A)
  • Neurobasal/FBS culture medium (see recipe)
  • 0.4% trypan blue solution (e.g., Invitrogen cat. no. 15250‐061)
  • Poly‐D‐lysine (70 to 150 kDa, e.g., Sigma cat. no. P‐0899)
  • Neurobasal/B‐27 culture medium (see recipe)
  • Antibodies:
    • Mouse IgM anti‐Tag‐1 (4D7, Developmental Studies Hybridoma Bank, University of Iowa)
    • Mouse IgG anti‐DCC (BD Pharmingen cat. no. 554223)
    • Goat anti‐choline acetyltransferase (BD Pharmingen cat. no. 552623)
  • Dissection tools:
    • Dissecting microscope with at least 5× magnification and transmitted light base (e.g., Zeiss SV6 with a Diagnostic Instruments cat. no. TLB5006 base)
    • Forceps (Dumont no. 5; Fine Science Tools cat. no. 91150‐20)
    • Needle holder (insect pin holder; Fine Science Tools cat. no. 26015‐11)
    • Iris scissors (Fine Science Tools cat. no. 91460‐11)
    • Spring‐loaded scissors (Fine Science Tools cat. no. 91501‐09)
    • Surgical scissors (Fine Science Tools cat. no. 91402‐14)
    • Tungsten needles (see protocol 4)
  • Petri dishes (10‐cm and 60‐mm)
  • Plastic transfer pipets (e.g., Fisher cat. no. 13‐711‐7)
  • 1.5‐ml tubes
  • 15‐ml plastic conical tubes
  • 37°C water bath
  • Hemacytometer (e.g., Fisher Scientific cat. no. 02‐671‐54)
  • Tabletop centrifuge (e.g., IEC Clinical)
  • 37°C, 5% CO 2 humidified cell culture incubator

Basic Protocol 2: Spinal Commissural Neuron Axon Outgrowth Assay

  Materials
  • Rats (pregnant at E14.5; vaginal plug = E1)
  • L15 dissection medium (Invitrogen cat. no. 41300‐039) supplemented with 5% filtered horse or goat serum (L15/serum dissection medium)
  • Type I collagen: either bovine skin collagen (Inamed cat. no. 5409) or rat tail collagen (Sigma cat. no. C3867); preparation described elsewhere (Chandrakasan et al., ; Habermehl et al., )
  • Neurobasal/FBS culture medium (see recipe)
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • Mouth pipet with glass 100‐µl micropipets (e.g., VWR 53432‐921)
  • Methanol flame
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment for dissection (see protocol 1)

Basic Protocol 3: Commissural Neuron Axon Turning Assay

  Materials
  • Rats (pregnant at E12.5; vaginal plug = E1)
  • L15 dissection medium (Invitrogen cat. no. 41300‐039)
  • 2.5% trypsin (e.g., Invitrogen cat. no. 15090‐046)
  • L15 dissection medium (Invitrogen cat. no. 41300‐039) supplemented with 5% filtered horse or goat serum (L15/serum dissection medium)
  • Type I collagen: either bovine skin collagen (Inamed cat. no. 5409) or rat tail collagen (Sigma cat. no. C3867); preparation described elsewhere (Chandrakasan et al., ; Habermehl et al., )
  • Neurobasal/FBS culture medium (see recipe)
  • Dissection tools:
    • Dissecting microscope with at least 5× magnification and a transmitted light base (e.g., Zeiss SV6 with a Diagnostic Instruments cat. no. TLB5006 base)
    • Forceps (Dumont no. 5; Fine Science Tools cat. no. 91150‐20)
    • Iris scissors (Fine Science Tools cat. no. 91460‐11)
    • Needle holder (Fine Science Tools cat. no. 26015‐11)
    • Spring‐loaded scissors (Fine Science Tools cat. no. 91501‐09)
    • Tungsten needles (see protocol 4)
  • Plastic Pasteur pipets (e.g., Fisher cat. no. 13‐711‐7)
  • 35‐ and 60‐mm petri dishes
  • Mouth pipet with glass 100‐µl micropipets (e.g., VWR cat. no. 53432‐921)
  • Methanol flame
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment for embryo dissection (see protocol 1)

Support Protocol 1: Electrolytic Sharpening of Tungsten Wire

  Materials
  • 1 M sodium hydroxide (NaOH) in deionized water
  • Deionized water
  • Tungsten wire (0.5‐mm diameter; e.g., Omega Engineering cat. no. WW26020)
  • Needle‐nose pliers with a wire‐cutter portion (e.g., Radio Shack cat. no. 6429‐57)
  • Needle holder (e.g., Fine Science Tools cat. no. 26015‐11)
  • Power supply (regulated direct current capable of at least 2 amps; e.g., Fisher Scientific cat. no. S90163)
  • Electrodes (either a paper clip or a 10‐cm section of a metal hanger)
  • 125‐ml plastic jar (e.g., Fisher cat. no. 02‐912‐028)
  • Mini alligator clips (e.g., Radio Shack cat. no. 270‐378)
  • Stage micrometer (1‐mm with 10‐µm divisions; e.g., Fisher cat. no. 12‐561‐SM1)
  • Dissecting microscope with at least 5× magnification and a transmitted light base (e.g., Zeiss SV6 with a Diagnostic Instruments cat. no. TLB5006)

Support Protocol 2: Embedding Tissue in a Collagen Matrix

  Materials
  • Type I collagen: either bovine skin collagen (Inamed cat. no. 5409) or rat tail collagen (Sigma cat. no. C3867); preparation described elsewhere (Chandrakasan et al., ; Habermehl et al., )
  • 10× DMEM (see recipe)
  • 10× NaHCO 3 (see recipe)
  • 4‐well culture plates (e.g., NUNC cat. no. 176740)
  • 37°C, 5% CO 2 incubator
  • Glass pipets
  • Methanol flame
  • Mouth pipets (e.g., VWR cat. no. 53432‐921)

Support Protocol 3: Immunolabeling Commissural Axons within Explants

  Materials
  • Explants embedded in collagen
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • PBS ( appendix 2A)
  • Triton X‐100 (e.g., Fischer cat. no. BP151)
  • Serum (horse or goat; Invitrogen)
  • Antibodies:
    • Mouse IgM anti‐Tag‐1 (4D7, Developmental Studies Hybridoma Bank, University of Iowa)
    • Labeled secondary antibody against mouse IgM
  • Aqueous‐based mounting solution (e.g., Vectashield, Vector Labs., cat. no. H‐1000)
  • Nail polish
  • Tungsten needles (see protocol 4)
  • Glass slides
  • Coverslips

Support Protocol 4: Hanging Drop Aggregation of Adherent Cells

  Materials
  • Adherent cell line (such as HEK 293, COS) culture at 90% confluence in a 60‐mm tissue culture dish
  • Trypsin‐EDTA (e.g., Invitrogen cat. no. 25300)
  • L15 supplemented (Invitrogen cat. no. 41300‐039) with 5% filtered serum (L15/serum dissection medium), preferably fetal bovine serum
  • Dissecting microscope with at least 50× magnification and a transmitted light base (e.g., Zeiss SV6 with a Diagnostic Instruments cat. no. TLB5006)
  • Hemacytometer (e.g., Fisher Scientific cat. no. 02‐671‐54)
  • 30‐ and 35‐mm petri dishes
  • 37°C, 5% CO 2 incubator
  • Forceps
  • Mouth pipet with glass 100‐µl micropipets (e.g., VWR cat. no. 53432‐921)
  • Tungsten needle (see protocol 4)
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Figures

Videos

Literature Cited

   Augsburger, A., Schuchardt, A., Hoskins, S., Dodd, J., and Butler, S. 1999. BMPs as mediators of roof plate repulsion of commissural neurons. Neuron 24:127‐141.
   Bayer, S.A. and Altman, J. 1995. Neurogenesis and Neuronal Migration. In The Rat Nervous System (G. Paxinos, ed.) pp. 1041‐1078. Academic Press, San Diego, California.
   Bouchard, J.F., Moore, S.W., Tritsch, N.X., Roux, P.P., Shekarabi, M., Barker, P.A., and Kennedy, T.E. 2004. Protein kinase A activation promotes plasma membrane insertion of DCC from an intracellular pool: A novel mechanism regulating commissural axon extension. J. Neurosci. 24:3040‐3050.
   Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. 1993. Optimized survival of hippocampal neurons in B27‐supplemented Neurobasal, a new serum‐free medium combination. J. Neurosci. Res. 35:567‐576.
   Butler, S.J. and Dodd, J. 2003. A role for BMP heterodimers in roof plate–mediated repulsion of commissural axons. Neuron 38:389‐401.
   Chandrakasan, G., Torchia, D.A., and Piez, K.A. 1976. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution. J. Biol. Chem. 251:6062‐6067.
   Charron, F., Stein, E., Jeong, J., McMahon, A.P., and Tessier‐Lavigne, M. 2003. The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin‐1 in midline axon guidance. Cell 113:11‐23.
   Conrad, G.W., Bee, J.A., Roche, S.M., and Teillet, M.A. 1993. Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus. J. Neurosci. Methods 50:123‐127.
   Habermehl, J., Skopinska, J., Boccafoschi, F., Sionkowska, A., Kaczmarek, H., Laroche, G., and Mantovani, D. 2005. Preparation of ready‐to‐use, stockable and reconstituted collagen. Macromol. Biosci. 5:821‐828.
   Imondi, R. and Kaprielian, Z. 2001. Commissural axon pathfinding on the contralateral side of the floor plate: A role for B‐class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons. Development 128:4859‐4871.
   Kennedy, T.E., Serafini, T., de la Torre, J.R., and Tessier‐Lavigne, M. 1994. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78:425‐435.
   Long, H., Sabatier, C., Ma, L., Plump, A., Yuan, W., Ornitz, D.M., Tamada, A., Murakami, F., Goodman, C.S., and Tessier‐Lavigne, M. 2004. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42:213‐223.
   Lyuksyutova, A.I., Lu, C.C., Milanesio, N., King, L.A., Guo, N., Wang, Y., Nathans, J., Tessier‐Lavigne, M., and Zou, Y. 2003. Anterior‐posterior guidance of commissural axons by Wnt‐frizzled signaling. Science 302:1984‐1988.
   Ming, G.L., Song, H.J., Berninger, B., Holt, C.E., Tessier‐Lavigne, M., and Poo, M.M. 1997. cAMP‐dependent growth cone guidance by netrin‐1. Neuron 19:1225‐1235.
   O'Rahilly, R., Müller, F., and Streeter, G.L. 1987. Developmental Stages in Human Embryos Including a Revision of Streeter's “Horizons” and a Survey of the Carnegie Collection. Carnegie Institution of Washington, Washington, D.C.
   Placzek, M., Tessier‐Lavigne, M., Jessell, T., and Dodd, J. 1990. Orientation of commissural axons in vitro in response to a floor plate‐derived chemoattractant. Development 110:19‐30.
   Podratz, J.L., Rodriguez, E.H., DiNonno, E.S., and Windebank, A.J. 1998. Myelination by Schwann cells in the absence of extracellular matrix assembly. Glia 23:383‐388.
   Ramón y Cajal, S. 1999. Texture of the Nervous System of Man and the Vertebrates. Springer, Vienna/New York.
   Serafini, T., Kennedy, T.E., Galko, M.J., Mirzayan, C., Jessell, T.M., and Tessier‐Lavigne, M. 1994. The netrins define a family of axon outgrowth‐promoting proteins homologous to C. elegans UNC‐6. Cell 78:409‐424.
   Shekarabi, M., Moore, S.W., Tritsch, N.X., Morris, S.J., Bouchard, J.F., and Kennedy, T.E. 2005. Deleted in colorectal cancer binding netrin‐1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N‐WASP into an intracellular signaling complex that promotes growth cone expansion. J. Neurosci. 25:3132‐3141.
   Shirasaki, R., Katsumata, R., and Murakami, F. 1998. Change in chemoattractant responsiveness of developing axons at an intermediate target. Science 279:105‐107.
   Stoeckli, E.T., Sonderegger, P., Pollerberg, G.E., and Landmesser, L.T. 1997. Interference with axonin‐1 and NrCAM interactions unmasks a floor‐plate activity inhibitory for commissural axons. Neuron 18:209‐221.
   Tessier‐Lavigne, M., Placzek, M., Lumsden, A.G., Dodd, J., and Jessell, T.M. 1988. Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336:775‐778.
   Wang, H., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., and Tessier‐Lavigne, M. 1999. Netrin‐3, a mouse homolog of human NTN2L, is highly expressed in sensory ganglia and shows differential binding to netrin receptors. J. Neurosci. 19:4938‐4947.
   Zou, Y., Stoeckli, E., Chen, H., and Tessier‐Lavigne, M. 2000. Squeezing axons out of the gray matter: A role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 102:363‐375.
Key References
   Goslin, K. and Banker, G. 1998. Culturing Nerve Cells. MIT Press, Cambridge, Mass.
  Discusses both general and specific principles of neuronal culture.
   Moore, S.W. and Kennedy, T.E. 2006. Axon guidance during development and regeneration. In Textbook of Neural Repair and Rehabilitation (M. Selzer, S. Clarke, L. Cohen, P. Duncan, and F. Gage, eds.) pp 326‐345. Cambridge University Press, Cambridge.
  Provides an overview of axon guidance mechanisms and reviews spinal commissural axon guidance.
Internet Resources
  http://embryology.med.unsw.edu.au/embryo.htm
  This Website, created by Dr. Mark Hill of the University of South Wales in Sidney Australia, provides extensive information regarding the embryonic development of a variety of organisms.
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