PROJECT SUMMARY/ABSTRACT Prescription and illicit opioid use have risen to a public health crisis, with the landscape shifting to fentanyl use. Given that fentanyl is 100-fold more potent than morphine, its use is associated with a higher risk of fatal overdose. Naloxone (Narcan) reverses opioid overdose and precipitates withdrawal. However, recent reports indicate that xylazine, an anesthetic that is increasingly detected in accidental fentanyl overdose deaths, has become a highly sought-after adulterant that can prolong both the fentanyl “high” and the onset of fentanyl withdrawal, as well as precipitate resistance to naloxone in the reversal of overdose. Despite this, no systematic studies to date have evaluated the neurobehavioral mechanistic contributors to these effects. While opioid pharmacological mechanisms of action have been studied, it is not clear how the addition of xylazine to fentanyl inhibits the actions of naloxone, as either alone does not have this action. Excitingly, we have possibly discovered the mechanisms of how xylazine combined with fentanyl causes resistance to naloxone using our State-of-the- Art PamGene PamStation technology that measures hundreds of kinase activities in a single sample. We show preliminary data for the signaling pathways in the nucleus accumbens core (NAcore) that are hyper- or hypo- active, which gives us numerous potential targets for reversing naloxone resistance and xylazine’s effects on fentanyl self-administration (SA). We hypothesize that the xylazine-fentanyl combination activates non- canonical pathways that reduce consumption and prolong the onset of fentanyl withdrawal signs. Our objective here is to uncover these unknown mechanisms using our PamGene technology and determine how these impact neurocircuitry and behavior. Therefore, we will test these in our following Aims: 1) Determine how xylazine impacts the effects of fentanyl, 2) Determine viable therapeutic targets to resensitize fentanyl withdrawal following the escalation of xylazine-fentanyl SA, and 3) Determine neural circuits altering the NAcore and ventral tegmental area (VTA) kinomes following the escalation of xylazine-fentanyl SA and if these underlie naloxone resistance. We will map the active kinome of the NAcore and VTA following xylazine-fentanyl co-use and will test novel treatment targets that we find are changed via our extensive analysis. We will further test if these neural circuits contribute to co-use-induced alterations in targets that we have identified from our PamGene analysis, and thus we will determine if suppressing these circuits may enhance the actions of naloxone and restore neural targets altered by xylazine-fentanyl co-use (measured via kinome analysis). Together, these hypothesis-driven studies will markedly enhance our understanding of the neurobehavioral mechanisms underlying xylazine-fentanyl polysubstance use and withdrawal and will likely reveal novel treatment strategies for reversing the xylazine-fentanyl-induced naloxone inefficacy.

PROJECT NARRATIVE Prescription and illicit opioid use have been recognized as a public health crisis, and heroin is being rapidly replaced with illicitly-manufactured fentanyl. Xylazine, a veterinary anesthetic, is being used as an adulterant in fentanyl use, and this pattern of polysubstance use renders naloxone ineffective at precipitating withdrawal and thus reversing overdose. The proposed research has the potential to not only uncover novel neurobehavioral mechanisms by which xylazine and fentanyl polysubstance use uniquely alter the reward pathway, behavior, and render naloxone unable to precipitate withdrawal, but will also identify and target mechanisms to reverse these effects.