Reed C. Carroll - US grants

The overall goal of this research project is to investigate the role of protein phosphatases in long term depression (LTD) of synaptic transmission in the mammalian brain. LTD along with long term potentiation (LTP) are prominent candidates for molecular mechanisms underlying such important neuronal functions as learning and memory formation. In this project, rat hippocampal slices will be used to obtain whole cell recordings of excitatory synaptic responses in CA1 pyramidal cells. The bulk of proposed experiments involve the perfusion of purified protein phosphatases (PP1 and PPP2B) into CA1 pyramidal cells while evoked synaptic responses are monitored. Specifically the following experiments will be performed. (1) Changes in the synaptic strength of cells infused with phosphatases will investigate whether phosphatases are sufficient to induce synaptic depression. (2) Whether the phosphatase-induced synaptic depression shares some of the same mechanisms with LTD will be determined by performing occlusion experiments. (3) Whether infusion of phosphatases modify postsynaptic glutamate receptors will be determined by monitoring the responses to iontophoretically applied AMPA or (4) by monitoring spontaneous EPSCs. (5) The role of increased phosphatase activity in maintaining LTD will be examined by infusing phosphatase inhibitors at various times following the induction of LTD. The results of these experiments will provide important information about the biochemical mechanisms by which synaptic efficacy can be modified and is a prerequisite for a thorough understanding of many important nervous system functions.

[unreadable] DESCRIPTION (provided by applicant): During development, activity refines neural connections by modulating the number and relative strength of synaptic inputs onto neurons. Long-term depression (LTD) is a form of synaptic plasticity believed to play a critical role in neural circuit formation, perhaps as an initial step in synapse elimination. Multiple signaling pathways can trigger LTD in a single neuron, however, the relative functional impact of these different pathways on mediating synaptic input is unclear. Two forms of LTD have been identified in hippocampal CA1 pyramidal neurons, metabotropic glutamate receptor (mGluR) - and NMDA- type glutamate receptor (NMDAR)-mediated, which have been proposed to share regulated endocytosis of synaptic AMPA-type glutamate receptors as an expression mechanism. However, differences in downstream signaling pathways and the absence of functional interaction between synaptic depressions activated by these receptors imply that they trigger the internalization of AMPARs through distinct, as yet undefined, mechanisms. We have found that activation of the NMDAR- or mGluR- coupled signaling pathways in pyramidal neurons actually causes the internalization of different populations of AMPARs. Furthermore, our preliminary studies suggest that the colocalization of the AMPAR- interacting proteins receptors GRIP with receptors at a subset of surface sites likely plays a role in mediating this signaling pathway selective AMPAR endocytosis. In this proposal, we will test the hypothesis that NMDARs and mGluRs target AMPARs for internalization from completely distinct synaptic sites mediated by the localized expression of GRIP. Furthermore, we will test whether GRIP promotes selective AMPAR endocytosis by establishing a stabilized synaptic population of AMPARs specifically targeted for internalization by mGluR activation but resistant to the actions of NMDARs. Results from these studies should provide important insight into mechanisms of AMPAR trafficking. In addition they should provide novel evidence that two forms of depression can differentially shape the connectivity of developing neuronal circuitry by acting at distinct synaptic sites on a single neuron. [unreadable] [unreadable]