GABAergic neurons in the ventrolateral periaqueductal gray mediate fentanyl withdrawal and self-administration in mice

Xylazine is a pervasive adulterant in clandestine opioid supplies. This trend is troubling, as xylazine carries its own acute side effects and its long-term cognitive and motivational effects are not known. Thus, we developed and validated a model of oral xylazine self-administration that is conducive to polysubstance studies and can easily be implemented in animal research labs. Mice underwent 4-hour drinking sessions where their only source of drinking water was adulterated with xylazine (0.01-1mg/mL). Oral bioavailability and brain penetrance were validated using mass spectrometry on brain and plasma samples collected after a drinking session. Male and female mice decreased fluid intake at high concentrations of xylazine, with male mice consuming less than female mice at intermediate concentrations (0.1 and 0.3mg/mL). Female mice had decreased locomotion following drinking sessions at 0.1, 0.3, and 1mg/mL. After a 4-hour drinking session, all mice received brain xylazine concentrations that are above the Ki (affinity) and EC50 (potency) of several known binding targets of xylazine. We then performed a three-bottle choice experiment where mice had the option of drinking from water with xylazine, water with fentanyl, or plain water. In three-bottle choice, mice consumed less xylazine-containing water than fentanyl-containing or plain water. Our results indicate that xylazine is orally bioavailable in mice, that mice will readily drink xylazine to pharmacologically and behaviorally relevant doses, and that mice prefer consuming fentanyl and water over xylazine.