Excitatory amino acid (glutamate) receptors comprise a class of metabotropic glutamate receptors (mGluRs) coupled through G proteins to the formation of intracellular second messengers. The coupling of these receptors to phospholipase C (PLC) results in the hydrolysis of membrane phosphoinositides (PI) and generation of two second messenger molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DG). Evidence is now accumulating which points to the existence of multiple subtypes of PI-coupled mGluRs. This heterogeneity has been postulated based on the pharmacology of agonist and antagonist action in different brain regions and selected neuronal populations, as well as during development, and on the basis of molecular cloning. Numerous studies suggest that mGluRs coupled to PI hydrolysis play a crucial role in brain function related to synaptic transmission, development and neuronal injury. For this reason, and in view of the possible therapeutic implications, it is essential to understand the functional heterogeneity of these receptors, the pharmacology of their recognition sites and the role that second messengers generated by their activation play in intracellular neuronal signalling. The long term objective of this research project is to explore in vitro, using primary cultures of neurons, the heterogeneity of PI-coupled mGluRs in terms of the molecular mechanisms associated with the receptor- mediated activation of PI hydrolysis, the significance of mGluR- stimulated PI hydrolysis in neuronal function, and the pharmacology and regulatory properties of mGluR-induced PI responses. Accordingly, primary cultures of cerebellar granule cells will be used to: 1. Evaluate the functional expression of PI-coupled mGluRs by determining the developmental pattern of expression of mRNA and the appearance of the PI response in neurons cultured in media enhancing receptor expression and by establishing the agonist and antagonist pharmacology of these receptors. 2. Determine the role of mGluRs in PI metabolism by identifying the specific substrates and products of PLCs coupled to mGluRs and establishing the relative contribution of mGluRs to the formation of two second messengers IP3 and DG. 3. Determine the role of PI-coupled mGluRs in intracellular Ca2+ homeostasis by using Fura-2 imaging to evaluate the magnitude of IP3-sensitive Ca2+ stores and the contribution of mGluR agonists and antagonists to the regulation of intracellular Ca2+ concentrations. This approach, combined with mRNA measurements, will be extended to identify the functional expression of PI-coupled mGluRs present in neuronal cultures prepared from other brain areas. 4. Determine the role of ionotropic glutamate receptors in the regulation of PI hydrolysis including the role of NMDA receptors in the stimulation and inhibition of PI hydrolysis and the molecular nature of interactions between mGluRs and AMPA receptors. These studies will provide information fundamental for the understanding of the pharmacological and neurochemical properties of PI-coupled mGluRs, their role in neuronal function, and for development of future therapeutic strategies.
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