Xylazine

All animals were anesthetized with ketamine (60 mg kg⁻¹, i.p. Fort Dodge, Fort Dodge, Iowa) and xylazine (5 mg kg⁻¹; i.p.; Boehringer Ingelheim, St. Joseph, MO) before the surgical procedures. Splenectomy: Splenectomy was performed as we described in J Exp Med^{11 (link)}. Anesthetized animals were subjected to an abdominal incision on the epigastrium and mesogastrium. The spleen was exposed by gentle retraction of the stomach to the side. The three main branches of the spleen artery were stabilized with nylon thread, ligated and cut. The spleen was removed and the wound was closed with sutures; catgut for the abdominal wall, and nylon thread for the skin. Animals were splenectomized 3 days before the experimental procedure. Adrenalectomy: Adrenalectomy was performed as described²⁹ . A dorsal incision from the first to the third lumbar vertebrae was performed on anesthetized animals. The latissimus dorsi muscle was dissected and pulled away on both sides until the kidneys were visible. Both adrenal glands and their adipose tissue were removed. Adrenalectomized animals were given drinking water with 0.9% NaCl for 10 days before the experimental procedure.

On PND 55 or 56, rats underwent jugular catheter implantation surgery. Rats were treated with carprofen (5 mg/kg, s.c.) the day before surgery and for 2 days after surgery. Male rats were anesthetized with a combination of ketamine (Butler Schein, Dublin, OH, USA), xylazine (Akorn, Inc., Decatur, IL, USA) and acepromazine (Boehringer Ingelheim, St. Joseph, MO, USA), which were mixed together to yield a single cocktail (75/7.5/0.75 mg/kg; 0.15 ml/100 g body weight; i.p.); female rats received a ketamine and xylazine cocktail diluted with sterile water (55/7.5 mg/kg; i.p.), but they did not receive acepromazine based on veterinary consultation. During surgery, a silastic catheter was inserted into the right jugular vein and threaded under the skin to an incision on the scalp. A cannula was connected to the end of the catheter and was secured to the skull with dental acrylic and jeweler’s screws. Following surgery, rats stayed in individual polycarbonate cages overnight, then returned to their original housing assignment and recovered for 7 days before the start of self-administration training.