Glutamate-evoked calcium signaling in spinal cord after nerve injury

A fundamental gap exists in understanding the cellular mechanisms that initiate and maintain neuropathic pain. This gap represents an important problem because current analgesic drugs rarely provide sufficient efficacy without serious side effects. The long-term goal is to understand the mechanisms that lead to injury-induced central sensitization and establish clinically relevant therapeutic targets for chronic pain. The objective in this application is to evaluate the contribution of ligand-, voltage- and store-operated Ca2+ channels to glutamate-evoked Ca2+ transients in the dorsal horn, and correlate enhanced Ca2+ responses with the magnitude of pain-like behavior. Based on preliminary data suggesting that glutamate-evoked Ca2+ responses in mouse spinal cord slices are potentiated after nerve injury, the central hypothesis is that nerve injury increases AMPA receptor signaling in the dorsal horn, leading to increases in [Ca2+]i that results in central sensitization and neuropathic pain. The rationale for the proposed project is that [Ca2+]i in dorsal horn neurons is essential for central sensitization and pain hypersensitivity. The central hypothesis will be tested by pursuing two specific aims: AIM 1 tests the hypothesis that glutamate-mediated activation of neuronal ionotropic AMPA receptors triggers Ca2+ entry into the cell via voltage-gated calcium channels. Electrophysiological recordings and real-time fluorescent labeling of astrocytes will be used to evaluate the cellular type that respond to glutamate with a rise in [Ca2+]i. Next, the relative contribution of ligand-, voltage- and store-operated Ca2+ channels will be determined by quantifying glutamate-evoked [Ca2+]i transients in the presence of selective antagonists for each of these calcium sources. AIM 2 tests the hypothesis that peripheral nerve injury potentiates glutamate-evoked Ca2+ responses, and this will correlate with the magnitude of allodynic behavior. To allow for a correlation analysis between behavior and [Ca2+]i, a variant model of nerve injury has been developed that gradually elicits robust allodynia in 1 week and then resolves in 4 weeks. Behavioral allodynia will be evaluated and compared to glutamate-evoked [Ca2+]i in spinal cord slices from animals that are sacrificed at 7, 14 and 21 d after nerve injury. This project employs innovative wide-field calcium imaging simultaneously from numerous cells in spinal cord slices from adult mice. The proposed research is significant because it reveals the Ca2+ channels that regulate glutamate-evoked Ca2+ transients, and is a critical first step in understanding nerve injury-induced potentiation of neuronal Ca2+ influx. Ultimately, this knowledge will establish clinically relevant therapeutic targets for alleviating chronic pain.