RESPIRATORY, NEUROINFLAMMATORY, AND MOTOR-IMPAIRING EFFECTS OF ACUTE AND CHRONIC FENTANYL AND XYLAZINE EXPOSURE IN MICE - PROJECT SUMMARY OPIOID MISUSE REMAINS A LEADING CAUSE OF PREVENTABLE DEATH IN THE UNITED STATES, WITH SYNTHETIC OPIOIDS LIKE FENTANYL ACCOUNTING FOR MOST OVERDOSE FATALITIES. FENTANYL'S HIGH POTENCY AND ABILITY TO DEPRESS BRAINSTEM RESPIRATORY CENTERS VIA M-OPIOID RECEPTOR ACTIVATION MAKE IT EXCEPTIONALLY LETHAL. COMPOUNDING THIS CRISIS IS THE INCREASING ADULTERATION OF FENTANYL WITH XYLAZINE, AN A2-ADRENERGIC RECEPTOR (A2AR) AGONIST USED IN VETERINARY MEDICINE, WHICH CONTRIBUTES TO PROFOUND SEDATION AND LETHALITY DUE TO INHIBITION OF CENTRAL NORADRENERGIC SIGNALING. THIS PROBLEM IS EXACERBATED BY THE WIDE VARIABILITY IN FENTANYL-XYLAZINE RATIOS FOUND IN THE ILLICIT DRUG SUPPLY. PRELIMINARY DATA FROM OUR LABORATORY DEMONSTRATE THAT CO-ADMINISTRATION OF FENTANYL AND XYLAZINE IN MICE PRODUCES RESPIRATORY DEPRESSION THAT CAN BE REVERSED BY NALOXONE, BUT MORE RAPIDLY SO WHEN COMBINED WITH AN A2AR ANTAGONIST, UNDERSCORING THE CRITICAL NEED FOR ADJUNCTIVE REVERSAL STRATEGIES. BEYOND ACUTE LETHALITY, REPEATED POLYSUBSTANCE-INDUCED RESPIRATORY DEPRESSION AND HYPOXIA MAY LEAD TO LASTING MOTOR AND NEUROIMMUNE DYSFUNCTION IN SURVIVORS. DESPITE THE RAPID EMERGENCE OF FENTANYL-XYLAZINE MIXTURES IN THE ILLICIT DRUG SUPPLY, THE PHYSIOLOGICAL AND NEUROINFLAMMATORY EFFECTS OF CHRONIC EXPOSURE AND STRATEGIES TO ENHANCE OVERDOSE REVERSAL REMAIN LARGELY UNKNOWN. THIS PROJECT AIMS TO CHARACTERIZE THE ACUTE AND CHRONIC RESPIRATORY, MOTOR, AND NEUROINFLAMMATORY CONSEQUENCES OF FENTANYL AND XYLAZINE CO-USE AND TO DETERMINE WHETHER A2AR ANTAGONISM CAN AUGMENT NALOXONE-MEDIATED REVERSAL. IN AIM 1, I WILL EVALUATE WHETHER SYSTEMATICALLY INCREASING THE PROPORTION OF XYLAZINE WITH A FIXED DOSE OF FENTANYL WORSENS RESPIRATORY DEPRESSION, HYPOXIA, BRADYCARDIA, MOTOR FUNCTION, AND NEUROINFLAMMATION USING WHOLE-BODY PLETHYSMOGRAPHY, PULSE OXIMETRY, A BATTERY OF FUNCTIONAL MOTOR ASSAYS, AND MULTIPLEX CYTOKINE/CHEMOKINE ASSAYS PERFORMED IN BRAINSTEM REGIONS THAT REGULATE BREATHING AS WELL AS THE DORSAL STRIATUM INVOLVED IN MOTOR FUNCTION. IN AIM 2, I WILL ASSESS THE ROLE OF A2AR SELECTIVITY AND CELLULAR LOCALIZATION IN PHYSIOLOGICAL DEPRESSION CAUSED BY FENTANYL AND XYLAZINE BY USING PHARMACOLOGICAL ANTAGONISTS WITH VARYING SUBTYPE SPECIFICITY (ATIPAMEZOLE, BRL-44408) AND CONDITIONAL KNOCKOUT MICE LACKING A2AAR IN ASTROGLIA. COLLECTIVELY, THESE STUDIES WILL ELUCIDATE THE PHYSIOLOGICAL AND NEUROIMMUNE CONSEQUENCES OF FENTANYL-XYLAZINE CO-USE, IDENTIFY RECEPTOR- AND CELL-SPECIFIC MECHANISMS UNDERLYING OVERDOSE-RELATED PHYSIOLOGY, AND ASSESS WHETHER A2AR ANTAGONISM MAY SERVE AS A POTENTIAL ADJUNCTIVE THERAPEUTIC STRATEGY TO IMPROVE OVERDOSE REVERSAL. THIS WORK WILL ALSO PROVIDE RIGOROUS MULTIDISCIPLINARY TRAINING IN RESPIRATORY PHYSIOLOGY, PHARMACOLOGY, AND NEUROIMMUNOLOGY, SUPPORTING MY DEVELOPMENT AS AN INDEPENDENT SCIENTIST FOCUSED ON TRANSLATIONAL APPROACHES TO ADDRESS SUBSTANCE USE AND ITS IMPACT ON PUBLIC HEALTH.