PROJECT SUMMARY Since the mid-2010s, the US has experienced a sharp rise in drug overdose fatalities driven by the emergence of the ultra-potent synthetic opioid fentanyl within the illicit drug supply. Recently, evidence derived from drug seizures, epidemiological surveys, and clinical case reports show that fentanyl is being increasingly adulterated with xylazine, an alpha-2 adrenergic receptor agonist approved for veterinary use as a sedative and analgesic. According to anecdotal reports, xylazine is a desirable adulterant because it prolongs the otherwise brief euphoria produced by fentanyl. However, fentanyl-xylazine mixtures have also been associated with severe health risks as compared to fentanyl alone, including potentiated drug-induced hypoxia and enhanced resistance to the opioid receptor antagonist naloxone. Collectively, these interactive effects likely contribute to the greater overall risk for irreversible overdose and lethality recently observed in preclinical studies and clinical case reports. This has led the US Government to designate fentanyl-xylazine mixtures as a “highly toxic chemical of concern” and an “emerging threat” requiring rapid response. Alpha-2 receptor activation reduces central norepinephrine levels, resulting in a physiological toxidrome resembling opioid exposure that includes respiratory depression, bradycardia, and hypotension. However, the precise impact of fentanyl-xylazine mixtures on cardiorespiratory function has not been examined, leaving unresolved the specific manner by which lethality might be prevented in an emergency medical setting. Furthermore, xylazine-induced potentiation of fentanyl’s subjective effects has not yet been evaluated in animal models, and thus the pharmacological mechanisms underlying this interactive effect remain poorly understood. In this proposal, we will test the hypothesis that xylazine enhances both the cardiorespiratory-depressant and subjective effects of fentanyl, and that dual antagonism of opioid receptors and alpha-2 adrenergic receptors prevents the effects of combined fentanyl-xylazine exposure on these measures in rats. In Aim 1, we will use telemetry and plethysmography to comprehensively characterize xylazine’s impacts on fentanyl-induced changes in heart rate, blood pressure, core temperature, and respiration. In Aim 2, we will determine whether xylazine intensifies and/or prolongs the interoceptive-stimulus effects of fentanyl using drug discrimination procedures. Finally, in both Aims, we will test whether the effects of fentanyl-xylazine mixtures are fully antagonized by co-administration of naloxone and the highly-selective alpha-2 receptor antagonist atipamezole, an FDA-approved veterinary medication that safely and effectively reverses the sedative and cardiorespiratory-depressant effects of alpha-2 receptors agonists in humans. If successful, these studies will identify the receptor targets that mediate both the desirable and harmful effects of fentanyl-...