Project Summary In a murine model, neonatal morphine exposure (NME) results in prolonged pain hypersensitivity in adolescence and adulthood; however, the underlying mechanisms are not fully understood. In adults, morphine exposure has been found to alter the gut microbiome leading to gut dysbiosis. Gut dysbiosis has been associated with microglial activation and neuroinflammation, which are critical in the initiation and persistence of several pathological pain states. Activation of the microglia in the midbrain periaqueductal gray (PAG) and spinal cord has been found to be heavily implicated in the development of the opioid-induced hypersensitivity to pain. I recently showed that NME resulted in long-term gut dysbiosis persisting in adolescence and into adulthood. However, the role of the gut microbiome, in particular NME-induced gut dysbiosis, have not yet been investigated in prolonged pain hypersensitivity following NME. Therefore, the goal in this study is to investigate the role of the gut microbiome in driving prolonged hypersensitivity to pain following NME. The overarching hypothesis is that NME results in prolonged pain hypersensitivity by inducing long-lasting alterations to the gut microbiome and driving persistent neuroinflammation in the microglia. To test this hypothesis, in specific aim 1, the causal role of the gut microbiome in driving prolonged hypersensitivity to pain will be investigated using Fecal Microbial Transplantation (FMT) of a dysbiotic microbiome from NME-mice (donor) into naïve age-matched mice (recipient) to test whether an NME-microbiome alone results in pain hypersensitivity. Furthermore, probiotics will be administered during the NME period to rescue the NME- induced dysbiosis and to investigate whether a probiotic intervention can attenuate pain hypersensitivity following NME. To elucidate the underlying molecular mechanisms, aim 2 will seek to establish that NME- induced dysbiosis modulates microglial activation in regions that are essential for the processing and modulation of pain, in particular the PAG and spinal cord, which drives pain hyper-sensitivity. Thus, using immunohistochemistry, the activation status of the microglia in the PAG and spinal cord of NME-off springs will be assessed. Additionally, the levels of released pro-inflammatory and nociceptive markers will be assessed using RT-qPCR for mRNA determination and a Multiplex bead array for protein determination. Tissues from the mice with manipulated microbiome generated in aim 1 will also be investigated to implicate the gut microbiome. Finally, to further establish the involvement of the microglia on prolonged pain hypersensitivity, the microglia of NME-off springs will be depleted during adolescence and adulthood with the CSF1R antagonist, (PLX) 5622. Pain sensitivity will be tested following the short-term microglial depletion. Overall, this study will advance our current understanding of the mechanisms driving hypersensitivity to pain follow...