4.3. Chondroitinase ABC (chABC) Treatment of PFC (Adult Animals)

Protease-free ChABC (chondroitinase and ChABC used interchangeably; Amsbio, Seikagaku, Japan) or penicillinase (p; Spectrum, TCI, USA) as a control enzyme (with no known substrate in the brain) was dissolved in 0.1% BSA to 50 U/mL of concentration and filtered through a 0.2-micron filter [71]. In these experiments, 8-week-old animals (n = 5 males and females each) were used. Four injections in the PFC (2 per hemisphere, 0.25 μL with a speed of 0.2 μL/min) were performed stereotaxically under xylazine-ketamine injectable anesthesia (at 30 mg/kg) with a 10 μL Hamilton syringe and a 33 gauge needle at the following sites (in mm from bregma): anterior-posterior (AP): +2.68 mm; lateral (ML): ±1 mm; ventral (V): −2 mm and −1 mm. The needle remained in place at the injection site for 3 min between injections and an additional 3 min before being slowly withdrawn over a 2-min period. An n = 6 was used for each group (with 3 males and 3 females). Animals were returned to their home cages and were observed for a week before behavioral testing.