University of Vermont COBRE 2 (P20 RR016435)
"Center for Neuroscience Excellence"
Research Project 2: "Development Regulation of GluR2 AMPA Receptor Subunit Expression"
Investigator: Miguel Martin-Caraballo, Ph.D.
Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors can be regulated by the insertion of one or more edited GluR2 subunits, which significantly reduces the permeability of Ca2+ through the channel. Our preliminary data indicates that ventral spinal cord neurons in the chicken embryo generate a significant intracellular Ca2+ signal following AMPA receptor stimulation at embryonic day (E) 5, an early stage of development. By E11, intracellular Ca2+ signals generated by AMPA receptor stimulation are significantly reduced. GluR2 protein expression is first detected by immunoblot analysis at E8, at the peak of programmed cell death in the chick spinal cord. Quantitative real time PCR analysis also indicates that GluR2 mRNA expression is low at E5 and increases several folds in E8 ventral spinal cord neurons. GluR2 mRNA is significantly reduced after chronic treatment of chick embryos with the GABA receptor agonist muscimol, an agent that reduces spontaneous motor activity in the chick spinal cord. Paralysis of the developing embryo with the nicotinic acetylcholine receptor blocker d-tubocurare, which blocks neuromuscular transmission, does not affect GluR2 mRNA expression. These data suggest that centrally generated electrical activity but not retrograde signals from the muscle regulates GluR2 subunit expression in developing motor neurons. The experiments outlined in this proposal are designed to test the hypotheses that electrical activity regulates the expression of GluR2 subunit and that the function of AMPA receptor-mediated Ca2+ flux during the development of spinal cord motor neurons is to promote programmed cell death or to inhibit dendritic outgrowth.
Here we propose to pursue the following specific aims: 1) To investigate whether genetic suppression of electrical activity prevents GluR2 subunit expression in the chick spinal cord in vivo, 2) To determine whether chronic depolarization or patterned electrical stimulation regulate the synthesis and insertion of GluR2 subunits in vitro, and 3) To determine the function of AMPA receptor mediated Ca2+ fluxes on motor neurons in vivo. This research will provide new insights into the cellular mechanisms that regulate receptor channel expression and its role in neuronal differentiation.