Project Summary Opioid medications are highly prescribed for pain management. However, these drugs have considerable abuse liability. As users increase their opioid intake, they build tolerance against their therapeutic effects. Despite well- described molecular and cellular mechanisms, tolerance can be reversed when the drug is taken in another context, a phenomenon known as associative analgesic tolerance. Since associative analgesic tolerance can lead to escalation of use, it increases the risk of opioid use disorder and fatal overdose. Uncovering neuronal mechanisms underlying associative analgesic tolerance will inform the development of new treatment options to mitigate pain while decreasing opioid use and overdoses. Using a novel behavioral training paradigm in mice, I identified brain regions activated by tolerance. The anterior cingulate cortex (ACC) plays a crucial role in contextual learning, nociception, and opioid analgesia, which are aspects of associative tolerance. My preliminary data indicate that associative tolerance induces the formation of a tolerance-active neuronal ensemble in the ACC (ACCtol-active). Preliminary studies using fiber photometry and chemogenetic approaches suggest that ACC activity tracks tolerance state and is necessary for associative tolerance expression. These data indicate that the ACC may be an orchestrator of associative tolerance. The ACC projects directly to the spinal cord (SC), and activation of this projection increases nociception. I hypothesize that ACCtol-active neurons produce associative tolerance by integrating context and drug signals to increase SC excitability in the presence of opioids. In the K99 phase, I will determine the role of the ACCtol-active ensemble in associative tolerance using a combination of transgenic tools for ensemble tagging, fiber photometry, and optogenetics. I anticipate that ACCtol-active activity will be upregulated during tolerance and that the bi-directional modulation of this ensemble will induce or prevent tolerance. In the R00 phase, I will use viral-mediated tracing and chemogenetic approaches to determine if ACCtol-active forms functional connections onto the SC and if modulating this pathway can alter associative tolerance. These experiments will help define the role of specific ACC ensembles and the ACC to SC circuit in associative analgesic tolerance. In addition to significant scientific advances in understanding the neural substrates driving contextual control of analgesic tolerance, this K99 proposal will provide me with training and mentorship to increase my technical and academic skills, building a foundation for my career as an independent investigator with my research laboratory. During the training phase, I will acquire extensive training in optical techniques, corticospinal circuit manipulations in vivo, and career development. This training, combined with my expertise in behavioral pharmacology and addiction models, will equip me to transition ...