Fentanyl

Pregnant embryonic day 16 (E16) C57BL/6N and FBV/N mice were purchased from Charles River. Pups were born 5–6 days later at the animal facility of the Max Planck Institute for Medical Research. On the day of treatment, the postnatal day 0–2 (P0–P2) pups were first removed from the home cages and their dams. Pups were immediately anesthetized with a mixture of Medetomidine (M-Domitor), Midazolam (M-Dormicum) and Fentanyl (F-Fentanyl) [MMF mixture], which was prepared by 50 μl Medetomidine (1 mg/ml, Pfizer), 100 μl Midazolam (5 mg/ml, Pfizer) and 100 μl Fentanyl (0.05 mg/ml, Pfizer). Except controls, each pup was injected subcutaneously in the area of the left hip with 2.5 μl MMF/g body weight (dosage/kg of body weight: Medetomidine 0.5 mg/kg, Midazolam 5 mg/kg and Fentanyl 0.05 mg/kg). For the P2 pups, 5–10 min after MMF application, the deeply anesthetized animals were injected with serotype 1/2 rAAV-SYN-Venus. Up to 30 min after MMF treatment, the AFN antagonists [50 μl Atipamezol (5 mg/ml, Pfizer) + 500 μl Flumazenil (0.1 mg/ml, Pfizer) + 300 μl Naloxon (0.4 mg/m, Pfizer)] were injected subcutaneously into the right hip (8.5 μl/g body weight). The pups recovered about 3 min after the application of the AFN mixture.

Anesthesia was induced with 2 mg/kg propofol (Fresenius Kabi, Lake Zurich, IL, USA), or 5 mcg/kg fentanyl (Hospira, Lake Forest, IL, USA) if the patient had an indwelling intravenous catheter. Otherwise, inhalational induction with sevoflurane 5% (Abbott Laboratories, Abbott Park, IL, USA) was performed. Anesthesia was maintained with sevoflurane and dexmedetomidine (Pfizer, New York, NY, USA; 1 mcg/kg/h). Following anesthetization and intubation, a Foley catheter was placed along with an arterial line and a double lumen central venous line.