Molecular Determinants of Synaptic Plasticity in Chronic Pain The long-term goal of our project is to identify the molecular and signaling mechanisms that govern synaptic plasticity under chronic pain conditions. Neuropathic pain remains a major therapeutic challenge, and neuronal plasticity at the spinal cord level is fundamentally important to the development of chronic neuropathic pain. N- methyl-D-aspartate receptors (NMDARs) are expressed in primary sensory neurons and their central terminals in the spinal dorsal horn. However, they are functionally inactive under normal conditions and become tonically activated to potentiate glutamatergic input to spinal dorsal horn neurons after nerve injury and chemotherapy- induced neuropathy. The molecular mechanisms regulating the synaptic activity and trafficking of NMDARs in the spinal dorsal horn remain poorly understood. Volume-regulated anion channels, which are formed by multiple different leucine-rich repeat-containing protein 8 (LRRC8) family members, are crucial to the regulation of cell volume. In our pilot studies, we found that LRRC8A was highly expressed in dorsal root ganglion (DRG) neurons. Also, traumatic nerve injury selectively downregulated LRRC8A, but not LRRC8B-LRRC8D, in the DRG. Furthermore, LRRC8A downregulation or conditional knockout in DRG neurons induces NMDAR-dependent pain hypersensitivity. Importantly, we discovered that LRRC8A physically interacted with NMDARs to control synaptic trafficking and activity of NMDARs. In this renewal application, we will specifically determine the roles of LRRC8A in the regulation of nociception and synaptic NMDARs at the spinal cord level in two neuropathic pain models. On the basis of our intriguing preliminary data, we propose to test the overall hypothesis that LRRC8A protein directly interacts with NMDARs and normally restrains the synaptic trafficking of NMDARs at the spinal cord level; nerve injury or chemotherapy diminishes LRRC8A expression and augments the synaptic expression and activity of NMDARs, leading to increased glutamatergic input to spinal dorsal horn neurons and chronic pain. We will apply several innovative and complementary approaches, including biochemical and cellular analyses, transgenic mice, and synaptic recordings to study how LRRC8A controls NMDARs and nociception at molecular, cellular, and behavioral levels. Our project will generate fundamental new information about the molecular basis of NMDAR-mediated synaptic plasticity in neuropathic pain. Findings from our project are expected to advance our knowledge of molecular mechanisms of nociceptive regulation and to guide the development of new strategies for treating chronic neuropathic pain.