For the last 10-15 years our laboratory has had a major role in the development and use of new spectroscopy and imaging techniques in vivo to study the regulation of pathways of brain glucose metabolism. Over the last 4 years of this grant we reported a number of important findings which indicate that release of glutamate and GABA from neurons and their cycling between neurons and glia has a major influence on brain energy metabolism and that the isoforms of a key enzyme of GABA synthesis, glutamic acid decarboxylase (GAD) mediate different proportions of total GABA synthesis. Our central hypothesis is that specific GAD isoforms and supply routes of glutamate carbon play key roles in regulating GABA synthesis and GABA neurotransmitter cycling. We propose the following specific aims: 1a) Determine the relationship between the rates of GABA synthesis, GABA neurotrasmitter cycling, and glutamate/glutamine cycle over a wide range of cortical metabolic activity. 1b) Assess the role of cofactor interaction and phosphorylation of the GAD isoforms in the regulation of GABA synthesis and GABA/glutamine cycling flux. 2) Investigate the role of GAD isoforms in regulation of GABA cycling flux through the two glutamate precursor pathways using GABA-transaminase inhibition and GABA elevation to selectively alter GAD isoform composition. 3) Quantitate the key metabolic pathway fluxes that supply glutamate precursors for GABA synthesis and GABA/glutamine cycling. 4) Determine the relationship between vesicular and non-vesicular GABA, precursor glutamate pathways, and GAD isoforms using diffusion-sensitized MRS in vivo. Using Magnetic Resonance spectroscopy (MRS) and 13C-labeled isotopes in vivo, and enzymatic assays in vitro with pharmacological and molecular biological interventions, we will examine the role of GAD isoforms and glutamate precursors in GABA synthesis and GABA neurotransmitter cycling The uniqueness of this project derives from i) the combination of the these techniques, ii) the track record of the investigator in addressing such questions using MRS, and iii) it complements ongoing clinical investigations of GABA and glutamate metabolism in epilepsy and neuropsychiatric disorders.
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