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