(A) Xylazine (red arrow) significantly induced dopamine release (n = 6/group; repeated measures [RM] 1-way ANOVA main effect of time F_(44,220) = 4.499, ****P < 0.0001) and (B) reduced locomotor activity (n = 6/group; RM 1-way ANOVA main effect of time F_(44,220) = 3.452, ****P < 0.0001). (C) Pretreatment with the α2 adrenergic receptor antagonist atipamezole (red arrow) significantly blocked the effects of xylazine (blue arrow) on dopamine release (n = 6/group; 2-way RM-ANOVA main effect of treatment F_{(1, 10)} = 21.79, ***P = 0.0009; treatment × time interaction F_{(34, 340)} = 12.19, ****P < 0.0001 analyzing 70 minutes after atipamezole and xylazine treatments). (D) Atipamezole partially attenuated the effects on locomotor activity (n = 6/group; 2-way RM-ANOVA main effect of treatment F_(1,10) = 18.60, **P = 0.0015; treatment × time interaction F_(34,340) = 1.872, **P = 0.0030). (E) Fentanyl treatment (red arrow) induced significant dopamine release (n = 6/group; 1-way RM-ANOVA main effect of time F_{(44, 220)} = 34.52, ****P < 0.0001) and locomotor activity (n = 6/group; RM 1-way ANOVA main effect of time F_(44,220) = 1.828, **P = 0.0026) (F). (G) Coadministration of fentanyl and xylazine (red arrow) significantly augmented the effects of fentanyl alone on dopamine release (n = 6/group;2-way RM-ANOVA main effect of treatment F_(1,10) = 14.01, **P = 0.0038, and a significant interaction of treatment × time F_{(44, 440)} = 9.751, ****P < 0.0001), while locomotor activity was reduced compared to fentanyl alone (n = 4 combo, n = 6 fentanyl; 2-way RM-ANOVA main effect of treatment F_(1,8) = 60.65, ****P < 0.0001; treatment × time interaction F_(44,352) = 2.343, ****P < 0.0001) (H). All error bars are SEM.