Dynamic changes in AMPA receptor (AMPAr) density and phosphorylation as well as number of Ca2+ permeable AMPAr in plasma membrane contribute to long term potentiation and synaptic strengthening. Tumor necrosis factor (TNF), a glial product, has been shown to elicit insertion of Ca2+ permeable AMPAr in plasma membrane of hippocampal neurons. In spinal cord, TNF contributes to chronic pain following a variety of injuries. The mechanism by which TNF enhances dorsal horn neuronal activity is unknown. I propose that following peripheral inflammation, glial TNF, working through a PI3K/Akt pathway, phosphorylates GluR1 subunits at 2 distinct sites, causing the AMPAr containing them to be inserted into plasma membranes. The increased AMPAr contain a disproportionate number that are Ca2+permeable, further contributing to synaptic strengthening. I will measure P-GluR1, insertion of AMPAr into the membrane and number of dorsal horn cells with functional Ca2+permeable AMPAr as a result of paw inflammation. I will use a combination of Western blots, subcellular fractionation, kainite induced cobalt loading and confocal microscopy. Spinal administration of specific inhibitors to every step in the proposed pathway will demonstrate that these elements are necessary for pain behavior and GluR1 insertion into neuronal plasma membranes as well as the sequence of events. Finally we will use 2 strains of knock in mice that do not phosphorylated GluR1 at individual serine residues. Use of these mice will further delineate the pathway and specify the necessary point of action of multi-purpose kinases. These experiments will further our understanding of fundamental spinal cord mechanisms. Ultimately, it will allow us to more logically and successfully design selective agents to treat chronic pain.
Tissue inflammation induces spinal cord release of TNF, sensitization of spinal neurons, and pain behavior. The mechanism by which TNF sensitizes pain transmission neurons is unknown. The proposal explores the possibility that TNF, via several identified steps, causes insertion of more and more active glutamate receptors into neuronal membranes to make them more excitable.
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