Project SummaryAccidental overdoses fatalities define the opioid epidemic despite research efforts for alternative means toaddress Opioid Use Disorder [1] and alternative chronic pain therapies [2]. The Centers for Disease Controlreport over 400000 lives lost since 1999 and more than 130 people lose their life to an accidental overdose daily[3]. This number will continue to climb with excessive utilization of prescription opioids and the presence ofstronger synthetic opioids in the illicit market [4-6]. A potentially fatal opioid overdose can be successfullyreversed with naloxone mu opioid receptor antagonist [7] but with the number of pain patients rising aboveheart disease and diabetes [2] and the increased presence of fentanyl in the illicit opioid market adequatedistribution of naloxone to reverse an overdose before oxygen deprivation occurs remains difficult [8]. Attemptsto reduce opioid overdoses with novel opioid-like compounds have been largely unsuccessful [9]. Therefore itis essential that unexplored therapeutic strategies to prevent opioid-induced respiratory depression bediscovered.Fatal opioid overdoses are typically attributed to respiratory depression during which neurons within thepreBtzinger complex (pBc) in the brainstem that control reflexive inspiration are inhibited. Our pilot data suggestthat 1) the pBc contains the components of the endocannabinoid system including the cannabinoid receptor 2(CB2R) 2) CB2R activation by endogenous cannabinoid system (ECBS) lipids is critical to normal respirationcontrol and 3) exogenous application of a CB2R agonist mitigates morphine induced respiratory depression.The current proposal will build upon these findings using behavioral pharmacology whole bodyplethysmography imaging molecular biology analytical chemistry and gene-editing to test the hypothesis thatendogenous cannabinoid levels in the pBc are reduced during opioid induced respiratory depression(OIRD) and that administration of a brain penetrant CB2R agonist will mitigate OIRD. Aim 1 will test CB2Ragonism as a strategy to mitigate to OIRD. Each aim contains rationally designed studies that include sexdifferences through inclusion of male and female mice application to both acute and chronic use of medicinaland recreational opioids (fentanyl oxycodone and heroin) and multiple chemical classes of CB2R agonists toprevent and reverse OIRD. Aim 2 will determine levels of endocannabinoid lipids enzymes and receptors inthe preBotzinger complex (pBc) during OIRD. Successful completion of proposed studies will serve toenhance our knowledge of the role of ECBS within the pBc in during normal respiration and during OIRDand validate targeting the CB2R as a safe treatment therapeutic to reduce opioid overdoses.