The long term goal of this K01 career award is to understand the mechanisms that lead to injury-induced central sensitization and to establish ameliorative therapeutic targets for chronic pain. My preliminary data suggests that AMPA receptors drive glutamate-evoked Ca2+ transients in spinal cord neurons and that glutamate-evoked Ca2+ transients are potentiated following nerve injury. The central hypothesis is that nerve injury potentiates neuronal activity and Ca2+ mobilization (Aim 1), via increased GluR2 AMPA receptor phosphorylation and trafficking in the dorsal horn (Aim 2), and that this mechanism of neuropathic pain is tonically inhibited by endogenous opioids (Aim 3). This hypothesis will be tested using innovative simultaneous Ca2+ imaging and whole-cell electrophysiology to evaluate dorsal root stimulated (DRS) Ca2+ transients and neuronal activity in dorsal horn of adult spinal cord slices.
Aim 1. Experiment 1a will evaluate the effect f sham or spared nerve injury (SNI) on isolated AMPA receptor Ca2+ signals and membrane currents. Experiment 1b will evaluate Ca2+ mobilization and membrane currents DRS at A, A? and c-fiber activation strength.
Aim 2 While AMPA GluR2 phosphorylation and trafficking have been implicated in inflammatory pain, the mechanisms that mediate nerve injury-induced potentiation of Ca2+ levels are not known. To address this question, Experiment 2a will determine levels and subcellular localization of total and phosphorylated GluR2 in dorsal horn. Based on studies indicating inflammation-induced GluR2 internalization is NMDA receptor-dependent, Experiment 2b will test the hypothesis that intrathecal administration of NMDA receptor antagonists will reduce nerve-injury induced phosphorylation and internalization of GluR2.
Aim 3. My new data suggest that opioids tonically inhibit glutamate receptor function after inflammation and thus mask neuropathic pain.
Aim 3 seeks to extend this discovery to our model of peripheral neuropathic pain. Experiment 3a will attempt to correlate Ca2+ mobilization with pain-like behavior following traditional SNI and a modified nerve injury model that resolves over 4 weeks. Establishing a decrease in Ca2+ mobilization in association with behavioral resolution will allow us to test our hypothesis in Experiment 3b. Experiment 3b will evaluate the effects of opioid receptor antagonists on: DRS-stimulated Ca2+ transients and neuronal activity, and GluR2 phosphorylation and subcellular localization. We expect that opioid receptor antagonists will increase Ca2+ mobilization, neuronal activity, GluR2 phosphorylation and shift Ca2+-impermeable GluR2 expression away from the cell surface.
Chronic pain management is a major scientific and health care challenge, as current analgesic drugs rarely provide sufficient efficacy in the absence of serious side effects. This project is relevant to NIDA's mission because it will 1) determine mechanisms that lead to injury-induced central sensitization and 2) establish clinically relevant therapeutic targets for alleviating chronic pain and 3) target mechanisms of endogenous opioids on long-lasting pain vulnerability following injury. This Research Plan employs highly innovative wide-field calcium imaging from numerous cells in a single spinal cord slice from adult mice.
|Doolen, Suzanne; Iannitti, Tommaso; Donahue, Renee R et al. (2018) Fingolimod reduces neuropathic pain behaviors in a mouse model of multiple sclerosis by a sphingosine-1 phosphate receptor 1-dependent inhibition of central sensitization in the dorsal horn. Pain 159:224-238|
|Doolen, Suzanne; Cook, Jennifer; Riedl, Maureen et al. (2017) Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling. Glia 65:1976-1989|