Project Summary Chronic pain, a complex multidimensional disorder, remains a major health care issue and a therapeutic challenge. The development of new and improved therapeutic strategies requires the full understanding of mechanisms of chronic pain at all levels of the neuraxis and for all cell types, including non-neuronal elements. Neuroimmune signaling has emerged as a peripheral and spinal pain mechanism, but little is known about the role, regulation and therapeutic potential of molecular crosstalk between neuronal and glial cell types in the brain in the context of pain. To address this important knowledge gap we will build on our NIH-funded work (since 1999) that impacted the field by identifying neuroplasticity in the amygdala, a brain center for emotions, as a critical mechanism for emotional-affective aspects of pain and pain modulation. The proposed project will test the novel hypothesis that chronification of amygdala plasticity and neuropathic pain behaviors depends on a cascade of neuroimmune signaling that can be targeted to mitigate neuropathic pain. Specifically, enhanced synaptic drive of certain amygdala neuron types at the acute pain stage activates different types of glia, and glia-derived factors generate hyperexcitability in distinct amygdala neuron types at the chronic stage to maintain neuropathic pain. A comprehensive multidisciplinary approach will be used that integrates state-of-the-art transcriptomics, bioinformatics, behavioral assays, brain slice electrophysiology, pharmacology, chemogenetics, optogenetics, viral vector strategies, immunohistochemistry and molecular biology for the analysis of neuroimmune interactions within and between different types of neuronal and glial cells in the amygdala output region (central nucleus, CeA) in the well-established spinal nerve ligation (SNL) rat model of neuropathic pain. Male and female rats will be studied. A transgenic Crh-Cre rat model will be used to study the CeA corticotropin releasing factor (CRF) system, an important player in amygdala plasticity, and its role in neuroimmune signaling. Aim 1 will use a combination of cell-specific bulk and single-cell RNA sequencing (scRNA-Seq) to identify individual genes, molecular pathways and functional cellular states associated with pain chronification. A novel computational scRNA-Seq- based interactome analysis will determine neuron-glia interactions at the acute and chronic stages of neuropathic pain and identify drug-targetable molecular factors. Aim 2 will determine the behavioral significance of neuron-glia-neuron signaling in the CeA at different stages of neuropathic pain, using chemogenetic activation and inhibition of different cell types and pharmacological (or viral based) tools to modulate molecular factors. Sensory thresholds, emotional responses, non-evoked ongoing pain, and anxiety- and depression-like behaviors will be measured. Aim 3 will determine electrophysiological mechanisms of neuron-glia-neuron signa...