The hippocampus is critically involved in normal physiological processes such as learning and memory and in various pathological states such as temporal lobe epilepsy. The hippocampus has a well-defined trisynaptic circuit, and each synapse in this circuit uses glutamate as its neurotransmitter. Synaptically released glutamate exerts its actions on neurons via activation of ionotropic glutamate receptors (iGLuRs) and metabotropic glutamate receptors (mGluRs). iGluRs are ligand gated ion that mediate fast synaptic transmission. The mGluRs comprise a family of receptors coupled to intracellular second messengers through G proteins, and these receptors mediate intrinsic modulation in glutamatergic circuits. The mGluRs have a variety of neuromodulatory actions in hippocampus, including excitatory effects on neurons and modulation of synaptic transmission. 8 mGluRs have been cloned (mGlu1-8), and genetic studies have shown that one of these (mGlu5) is critical for hippocampus-dependent learning, indicating that mGlu5 is critical in hippocampal function. However, the cellular actions of mGIu5 have not been well defined. Because of the important role of mGIu5 in hippocampus-dependent learning, it is important to know how mGlu5 modulates the function of this structure, and further how mGlu5 may itself be modulated. We have shown that mGlu5 undergoes protein kinase C (PKC)-mediated desensitization and phosphorylation in expression systems. However, we have not yet unequivocally identified the sites of phosphorylation or determined whether this desensitization occurs in the brain. The studies proposed here will address the specific roles of mGlu5 in hippocampal function using genetically altered mice. Furthermore, we will test the hypothesis that mGIu5 undergoes PKC-mediated desensitization in response to physiological and pathological stimuli and that this desensitization requires PKC phosphorylation of specific sites in mGlu5. We will accomplish these goals using patch clamp physiology in wt and knockout mice and direct protein sequencing to identify sites of PKC phosphorylation. Finally, we will generate antibodies that recognize these sites of mGlu5 phosphorylation for use in determining whether these sites are phosphorylated in vivo. These studies will help identify the specific functions and mechanism of modulation of mGlu5 in hippocampus.
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