University of Vermont COBRE (P20 RR016435)
"Center for Neuroscience Excellence"

Pilot Project 2: "Expression of the AMPA Receptor Subunit GluR2 in Chick Lumbar Motoneurons"
Investigator: Miguel Martin-Caraballo, Ph.D.

The calcium-permeability of AMPA receptors is determined by the insertion of a GluR2 subunit in the native channel: insertion of at least one GluR2 subunit results in calcium-impermeable AMPA receptors. Calcium-permeable AMPA receptors can be found during early embryonic development but the functional significance of this event is poorly understood. To gain a better understanding of the functional role of calcium-permeable AMPA receptors during neuronal development, we have explored GluR2 expression in the chick lumbar spinal cord. Fura-2 recordings of intracellular calcium indicate that AMPA receptor stimulation generates a significant calcium signal in spinal neurons at embryonic day (E) 6. Between E8 and E11, calcium signals generated by AMPA stimulation decrease in a population of large spinal neurons (putatively identified as motoneurons by DiI labeling). At all ages tested, calcium signals generated by AMPA are specific and can be eliminated by the broad AMPA receptor blocker CNQX. Real time PCR and western blot analysis indicate GluR2 mRNA and protein expression are absent in ventral spinal cords between E5 and E7. GluR2 expression becomes noticeable at E8 and remains high throughout E13. These findings suggest that there is a critical period for GluR2 expression in the chick spinal cord that coincides with important developmental processes like programmed cell death, and morphological and electrical differentiation of ventral spinal neurons. Inhibition of spontaneous electrical activity in the spinal cord with muscimol (a GABA receptor agonist that induces paralysis of the developing embryos by disrupting spinal cord network activity) results in a significant reduction in GluR2 mRNA expression in ventral spinal neurons. No change in GluR2 mRNA expression was observed by blocking neuromuscular activity with tubocurare. These findings suggest that ongoing electrical activity is required for proper GluR2 expression in the chick ventral spinal cord.