Nguyen, Kayla PROJECT SUMMARY/ABSTRACT Neuropathic Pain (NP) afflicts over 50% of patients with multiple sclerosis (MS), yet current treatments for pain relief are inadequate, in part because mechanisms that drive MS pain are poorly understood. In a widely-used animal model of MS, experimental autoimmune encephalomyelitis (EAE), recent electrophysiological recordings of primary afferent neurons (PANs) in the dorsal root ganglion (DRG) revealed: 1) hyperexcitability of medium- to-large diameter neurons and 2) an increase in afterhyperpolarization of small-diameter fibers. These data provide the premise for my long-term goal to investigate the sensitization mechanisms at PANs that drive NP in the EAE model. With behavioral, transgenic, and ablation techniques, I propose to assess the contribution of A and C-fibers to MS pain upon testing the overall hypothesis that EAE produces a persistent hyperexcitability of A and/or C-nociceptors (Aim 1,2) that is associated with increased activity of dorsal horn (DH) neurons (Aim 3) that then drives NP. Aim 1 uses neuronal ablation strategies to identify which subpopulations of primary afferent neurons contribute to NP in EAE. I will use simple conventional neuronal ablation strategies (neonatal capsaicin, intrathecal capsaicin, intrathecal IB4-saporin, intraplantar flagellin/QX-314), followed by a more labor-intensive but powerful conditional knockout approach to eliminate the activity of increasingly precise PAN subsets. I predict that the proposed interventions will reduce key indicators of EAE-induced pain: mechanical and cold hypersensitivity (reflexive pain) and conditioned place aversion (affective pain). Aim 2 uses chemogenetics and optogenetics to test the hypothesis that either Mrgprd- or TrkC-expressing PANs are necessary for NP in EAE. I predict that CreER selective manipulations to inhibit either the MrgprD+ subset of IB4 neurons or the TrkC+ subset of myelinated neurons (decision driven by the results of Aim 1a-b) will reduce EAE mechanical and cold hypersensitivity. Aim 3 will test the hypothesis that EAE sensitizes the somatosensory stimulus-induced activation of spinal interneurons and projection neurons. Our laboratory reported that EAE increases the activity of dorsal horn neurons as measured by the expression of pERK. I propose to extend these studies with a more powerful measure of neuronal activity using spinal cord slice Fura-2 calcium imaging, and by assessing the activity not only of interneurons, but also of projection neurons. The Pittsburgh Center for Pain Research will provide guidance, support, and learning opportunities that will promote the development of my scientific and investigative career aspirations.