Glutamate Receptor Modulation of Calcium Signaling in Neuropathic Pain

Understanding the mechanisms underlying chronic pain is crucial to developing novel therapeu-tic strategies that will reduce the burden of chronic pain in humans. The objective of the present application is to understand the role of AMPA receptor function in the setting of chronic neuro-pathic pain. The central hypothesis is that nerve injury increases GluR2 AMPA receptor trafficking in the dorsal horn (Aim 2), thus potentiating neuronal activity and Ca2+ mobili-zation (Aim 1), and that this mechanism of central sensitization is tonically inhibited by endogenous opioids (Aim 3) and thereby leading to central sensitization and ultimately neuropathic pain. Our approach uses Ca2+ imaging, electrophysiological, biochemical and be-havioral methods to determine: if nerve injury induces AMPA receptor mediated increases in Ca2+ mobilization and AP frequency (Aim 1), the mechanisms underlying injury-induced chang-es in Ca2+ levels in dorsal horn neurons (Aim 2); and the temporal relationship between potenti-ated responses at different stimulus intensities and the development of neuropathic pain-like behavior. Preliminary data suggests that peripheral nerve injury increases glutamate-evoked Ca2+ mobilization. While AMPA receptor regulation has been implicated in inflammatory pain, the mechanism that mediates injury-induced potentiation of Ca2+ levels is not known. Studies in this proposal will use pharmacological methods to isolate the AMPA-mediated current and Ca2+ imaging to determine if nerve injury produces an increase in AMPA-mediated Ca2+ mobilization. In order to determine if nerve injury induces phosphorylation and internalization of Ca2+ imper-meable AMPA GluA2 receptors subunits, total and pGluA2 levels in dorsal horn will be deter-mined using Western blotting techniques. Subcellular fractionation and Western blotting will be employed to determine if nerve injury causes internalization of GluR2 receptor subunits. Based on my new data indicating that opioids tonically inhibit glutamate receptor function after nerve injury and thus masks neuropathic pain, Aim 3 will determine whether these signs of plasticity are tonically inhibited by endogenous opioids. These studies will evaluate the effects of opioid receptor antagonists on: 1) DRS-stimulated Ca2+ transients and neuronal activity using simulta-neous Ca2+ imaging and whole-cell electrophysiology and 2) GluR2 phosphorylation and subcel-lular localization using Western blotting techniques. The training described in this proposal will provide the opportunity to acquire the technical research skills, expand my knowledge of the pain field, establish professional networks, and generate publications and preliminary data needed to become a successful independent scientist.