Laser Microdissection and Pressure Catapulting of Single Human Motor Neurons for RNA Editing Analysis

  • Hui Sun
  • Aruna Raja
  • Mary A. O’Connell
  • Valerie Mann
  • Brendon Noble
  • Liam P. KeeganEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 718)


Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. The properties of their ionotropic glutamate receptors largely determine how different neurons respond to glutamate. RNA editing in pre-mRNAs encoding subunits of glutamate receptors, particularly the GluR 2 subunit of AMPA receptors, controls calcium permeability, response time, and total ion flow in individual receptors as well as the density of AMPA receptors at synapses through effects on ER assembly, sorting, and plasma membrane insertion. When RNA editing fails in a neuron, calcium influx through AMPA receptors may cause neuron death by glutamate excitotoxicity, as in the case of vulnerable hippocampal CA1 pyramidal neurons that die after transient forebrain ischemia. Elevated cerebrospinal glutamate is common in ALS and loss of GluR 2 Q/R site RNA editing has been reported to occur selectively in lower motor neurons in a majority of Japanese sporadic ALS patients. We describe our methods for laser microdissection followed by RT-PCR analysis to study RNA editing in single motor neurons.

Key words

RNA editing ALS Neurodegeneration Brain bank Spinal motorneurons GluR-B GluR 2 Laser microdissection and pressure catapulting (LMPC) Cryosectioning Single cell analysis 



H.S. and A.R. were supported by Grant 6028 from the UK Motor Neurone Disease Association with additional support from the Scottish Motor Neurone Disease Association. L.K and M.O’C are supported by MRC Grant U.1275.01.005.00001.01. We thank Craig Nicol for assistance with figures and Paul Heath, Paul Ince, Pam Shaw and the Sheffield Brain and Spinal Cord Tissue bank for access to the human tissues being used in these studies.


  1. 1.
    Cleveland, D. W., and Rothstein, J. D. (2001) From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci 2, 806–819.PubMedCrossRefGoogle Scholar
  2. 2.
    Shaw, P. J., and Ince, P. G. (1997) Glutamate, excitotoxicity and amyotrophic lateral sclerosis. J Neurol 244 Suppl 2, S3–S14.PubMedCrossRefGoogle Scholar
  3. 3.
    Higuchi, M., Single, F. N., Köhler, M., Sommer, B., Sprengel, R., and Seeburg, P. H. (1993) RNA editing of AMPA receptor subunit GluR-B: A base-paired intron-exon structure determines position and efficiency. Cell 75, 1361–1370.PubMedCrossRefGoogle Scholar
  4. 4.
    Burnashev, N., Monyer, H., Seeburg, P. H., and Sakmann, B. (1992) Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron 8, 189–198.PubMedCrossRefGoogle Scholar
  5. 5.
    Peng, P. L., Zhong, X., Tu, W., Soundarapandian, M. M., Molner, P., Zhu, D., Lau, L., Liu, S., Liu, F., and Lu, Y. (2006) ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia. Neuron 49, 719–733.PubMedCrossRefGoogle Scholar
  6. 6.
    Kawahara, Y., Ito, K., Sun, H., Aizawa, H., Kanazawa, I., and Kwak, S. (2004) Glutamate receptors: RNA editing and death of motor neurons. Nature 427, 801.PubMedCrossRefGoogle Scholar
  7. 7.
    Kwak, S., and Kawahara, Y. (2005) Deficient RNA editing of GluR2 and neuronal death in amyotropic lateral sclerosis. J Mol Med 83, 110–120.PubMedCrossRefGoogle Scholar
  8. 8.
    Geiger, J. R. P., Melcher, T., Koh, D.-S., Sakmann, B., Seeburg, P. H., Jonas, P., and Monyer, H. (1995) Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15, 193–204.PubMedCrossRefGoogle Scholar
  9. 9.
    Kawahara, Y., Ito, K., Sun, H., Kanazawa, I., and Kwak, S. (2003) Low editing efficiency of GluR2 mRNA is associated with a low relative abundance of ADAR2 mRNA in white matter of normal human brain. Eur J Neurosci 18, 23–33.PubMedCrossRefGoogle Scholar
  10. 10.
    Vogel, A., Horneffer, V., Lorenz, K., Linz, N., Huttmann, G., and Gebert, A. (2007) Principles of laser microdissection and catapulting of histologic specimens and live cells. Methods Cell Biol 82, 153–205.PubMedCrossRefGoogle Scholar
  11. 11.
    Vogel, A., Lorenz, K., Horneffer, V., Huttmann, G., von Smolinski, D., and Gebert, A. (2007) Mechanisms of laser-induced dissection and transport of histologic specimens. Biophys J 93, 4481–4500.PubMedCrossRefGoogle Scholar
  12. 12.
    Schutze, K., and Lahr, G. (1998) Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol 16, 737–742.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hui Sun
    • 1
  • Aruna Raja
    • 1
  • Mary A. O’Connell
    • 1
  • Valerie Mann
    • 2
  • Brendon Noble
    • 2
  • Liam P. Keegan
    • 1
    Email author
  1. 1.MRC Human Genetics Unit, Institute of Genetics and Molecular MedicineWestern General HospitalEdinburghUK
  2. 2.MRC Centre for Regenerative MedicineUniversity of EdinburghEdinburghUK

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