? The goal of the present Stage I (CEBRA R21) application is to develop an experimental paradigm for integrated proteomic and functional analysis of defined subsets of sensory neurons. Subsets of sensory neurons will be identified and sorted using flow cytometry. The sorted neurons will be processed for largescale expression analysis using proteomic approaches or for functional analysis such as Ca2+ imaging. In a long-term Stage II project, this paradigm will be applied to studies of sensory neuron plasticity under conditions of chronic pain. The current project will use the cytokine Tumor Necrosis Factor alpha (TNFa) and its role in neuropathic pain as a model system for the development of this innovative experimental paradigm and its validation for the study of sensory neuron plasticity. The following specific aims will address the hypothesis that TNFa contributes to nerve injury-induced plasticity through TNF receptor-mediated effects on sensory neuron protein expression and function: 1) Examine the expression of TNF receptors in acutely dissociated sensory neurons under normal and neuropathic conditions using flow cytometry. These experiments will establish protocols for analysis and sorting of sensory neurons by flow cytometry. 2) In cells expressing TNF receptors, identify proteins whose expression is modulated by TNFa after nerve injury. These experiments will adapt cutting-edge proteomic approaches to analysis of flow cytometry-sorted sensory neurons. 3) Determine if TNFa treatment of sensory neurons expressing TNF receptors modulates pronociceptive and analgesic signaling. These experiments will develop procedures for functional analysis in flow cytometrysorted sensory neurons. In summary, the studies in this application will extend our understanding of the role of TNFa in neuropathic pain, while developing an innovative approach to the analysis of sensory neuron plasticity. In the long term, characterization of the plasticity associated with specific conditions of chronic pain in specific subsets of sensory neurons will lead to development of more selective therapeutic interventions that avoid the unwanted side effects of commonly used analgesics such as opiates. ? ? ? ?