Glutamate and aspartate mediate almost all of the fast excitatory synaptic responses in the mammalian CNS. In the normal brain, the intracellular concentrations of these excitatory amino acids are up to 1000-fold higher than the extracellular concentrations. These EAAs depolarize neurons by activating receptors coupled to ion channels. During the past few years, EAAs have also been shown to activate receptors that are coupled to second messenger systems through G-proteins. These receptors have been called metabotropic receptors. During acute insults to the CNS, the accumulation of GLU and ASP in the extracellular space in brain and the resulting excessive activation of EAA receptors coupled to ion channels may contribute to the neuronal death that occurs in several acute insults to the CNS, including stroke, head trauma, and hypoglycemia. The focus of this grant is to understand factors that regulate extracellular concentrations of EAAs. The investigator hypothesizes that the metabotropic EAA receptors serve as sensors of extracellular concentrations of EAAs and regulate the transport, release, and synthesis of excitatory neurotransmitters. It is proposed to use cell lines and cloned transporters expressed in oocytes to study regulation of transport activity by protein kinases and metabotropic EAA receptors. The investigator proposes to examine the regulation of release of EAAs by metabotropic receptors. Initially brain slices prepared from regions that express different subtypes of transporters and different subtypes of metabotropic receptors will be used followed by in vivo microdialysis to compliment these studies. It is proposed to study the regulation of metabolism of glu by metabotropic receptors using 15N- and 14C-labeled stable isotopes to follow both the nitrogen and carbon backbone used for biosynthesis of these neurotransmitters. Finally, the investigator proposes to study the regulation of excitotoxicity by metabotropic receptors and to study the contributions of altered transport, release, and metabolism to this regulation. Most approaches to limiting neurotoxicity of EAAs focus on preventing the excessive activation of the postsynaptic receptors. The goal of this laboratory is to define the role of other proteins in brain that may normally limit this toxicity. The long term goal of this research is to develop alternative strategies that will protect the brain from excitotoxicity.
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