6 ohda hbr salt

Intracerebral injections were performed under general anesthesia using Isoflurane (Attane vet 1000 mg/g, VM Pharma AB) in a 2% air mixture. All working solutions containing viral vector and/or PFFs were prepared so that the final concentrations of viral vector and PFFs were 6x10¹² gc/mL and 2.5μg/μL, respectively. Animals were head-fixed in a stereotaxic frame with the incisor bar adjusted to the flathead position (–4.5 mm below the interaural line). Each solution was infused unilaterally into the midbrain using a pulled glass capillary attached to a 10μL Hamilton syringe. The following coordinates (from Bregma, in mm) and volumes were utilized: 2μL at AP = –5.3, ML = –0.8, DV = –7.5 and 2 uL at AP = –5.6, ML = –2.3, DV = –7.5 with an infusion rate of 0.5μL/min. After injection, the syringe was left in place for additional 3 min and thereafter slowly retracted. 6-OHDA was injected as described previously [45 (link)]. In brief, 5.41μg/μL 6-OHDA (calculated from free-base 6-OHDA-HBr salt, Sigma) was dissolved in 0.2 mg/ml ascorbic acid in 0.9% sterile saline and a volume of 3μL was injected at the following coordinates AP = –4.0, ML = –1.3, DV = –7.0. The injection speed was set to 1μL/min and the needle was left in place for an additional 3 min to allow for diffusion of the toxin.