The hippocampus plays an important role in memory formation and is a primary site of pathology in several neurological disorders including Alzheimer's disease (AD) and epilepsy. Cholinergic projections to the hippocampus provide a major neuromodulatory input to glutamatergic neurons and synapses in the hippocampus and are critical for memory and attention mechanisms. Cholinergic transmission in the hippocampus is mediated primarily by muscarinic acetylcholine receptors (mAChRs), which have been classified into m1-m5 subtypes. One of the most prominent effects of mAChR activation in the hippocampus is a marked potentiation of currents through the NMDA subtype of glutamate receptor. This is likely to enhance NMDA receptor-dependent LTP and could play an important role in cholinergic regulation of cognitive function. Development of a complete understanding of the mechanisms involved in cholinergic modulation of NMDA receptor currents could be useful for development of agents that are effective in ameliorating the loss of cognitive function in patients with AD and other memory disorders. Recent studies and preliminary data from this laboratory suggest that mAChR-induced activation of tyrosine kinases in hippocampal slices may be necessary for potentiation of NMDA receptor currents. A series of studies is proposed to rigorously test the hypothesis that mAChR activation results in tyrosine phosphorylation of NMDA subunits and that tyrosine phosphorylation of NMDA receptor subunits is necessary for mAChR-induced potentiation of NMDA-evoked currents. Specific inhibitors of tyrosine kinases and mutant mice lacking major non-receptor tyrosine kinases will be used in a series of electrophysiology studies to determine whether activation of tyrosine kinases is required for potentiation of NMDA receptor currents. Biochemical and molecular approaches will then be used to identify tyrosine phosphorylation sites on NMDA receptor subunits involved in tyrosine kinase-induced potentiation of NMDA-evoked currents and determine whether mAChR activation increases phosphorylation of these sites on NMDA receptor subunits in hippocampal slices. Finally, mAChR subtype-specific toxins and other subtype-specific agents will be used to determine which mAChR subtypes mediate mAChR-induced phosphorylation of NMDA receptor subunits. Immunocytochemistry with double labeling and confocal microscopy will then be used to verify that these mAChR subtypes are colocalized on hippocampal neurons with the NMDA receptor subunits that are phosphorylated by mAChR activation.
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