Atropine sulfate

Between day 13 and day 20 after the antibody injection, the rats were again anaesthetized by 4% isoflurane, followed by an application of 2% isoflurane through a tight mask. Atropine sulfate (B. Braun Melsungen AG, Melsungen, Germany, 0.5 mg/mL 1:10 with sodium chloride 0.9%) was injected subcutaneously to prevent salivation. The animals were tracheotomized in order to be artificially ventilated with a mixture of oxygen-enriched room air and 2% isoflurane. The depth of anaesthesia was routinely assessed and held at a level in which noxious stimuli (pinching of earlobes) failed to elicit motor reflexes or changes in systemic arterial pressure. The body temperature of the animals was recorded by a thermoprobe inserted into the rectum and was kept at 37–37.5 °C with a feedback-controlled heating pad. Systemic blood pressure was recorded with a pressure transducer connected to the catheter inserted into the right femoral artery. The expiratory CO₂ was continuously monitored (Artema MM 200, Karl Heyer, Bad Ems, Germany) and maintained at 3–3.5%. The head of the animal was fixed in a stereotaxic frame and held by ear bars and a snout clamp. The eyes were covered with dexpanthenol ointment (Bepanthen^®, Bayer Vital GmbH, Leverkusen, Germany) to prevent dehydration of the cornea.

For examination with ERG, mfERG, OCT, AF, FA, and ABR on 3‐week old animals were anesthetized by intramuscular injection of 2 mg/kg azaperone (Stresnil^®, Elanco, Germany), 0.02 mg/kg atropine sulfate (B. Braun, Germany) and 20 mg/kg ketamine (Ursotamin^®, Serumwerke Bernburg, Germany). Anesthesia was continued by intravenous injection of propofol (Fresenius, Germany) according to effect. After endotracheal intubation, pigs were mechanically ventilated and cardiovascular function was monitored throughout the procedures. To exclude eyeball movement during examinations, a peripheral muscle relaxant (Rocuronium, Inresa, Germany) was applied. For ABR on older animals, animals were starved overnight before intramuscular application of TKX (tiletamine 4 mg/kg, zolazepam 4 mg/kg (Zoletil 100; Virbac), ketamine 5 mg/kg (Narketan 10; Chassot), and xylazine 1 mg/kg (Rometar 2%; Spofa).

The hearing function of adult Bdnf^Pax2KO and controls was studied by measuring and analyzing auditory brainstem responses (ABR) and auditory steady-state responses (ASSR), as previously described (Zuccotti et al., 2012 (link); Rüttiger et al., 2013 (link); Wolter et al., 2018 (link)). Animals were exposed to enriching sound as described (Matt et al., 2018 (link)). Animals were anesthetized with intraperitoneal injections of fentanyl (0.05 mg/kg bodyweight, Fentadon; Albrecht GmbH, Aulendorf, Germany), midazolam (2.5 mg/kg body weight, Midazolam-hameln; Hameln Pharma plus GmbH, Hameln, Germany), medetomidin (0.5 mg/kg bodyweight, Sedator; Albrecht GmbH, Aulendorf, Germany) and atropine sulfate (0.2 mg/kg body weight, B. Braun, Melsungen, Germany). Additional doses of anesthetics were administered if needed.