Nigrostriatal 6-OHDA lesion

Lesioning with 6-OHDA (6-OHDA hydrochloride, Sigma-Aldrich, St. Louis, MO, USA) was performed as previously described (Meng et al., 2015), with minor modifications. Briefly, rats were anesthetized with 100 mg/kg chloral hydrate through intraperitoneal injection before surgery, and were immobilized in a stereotaxic frame to target the right medial forebrain bundle at the following coordinates relative to the bregma, according to the Paxinos and Watson (2006) rat brain atlas: anterior–posterior: –4.4 mm, medial–lateral: –1.4 mm, and dorsal–ventral: +8.5 mm. To reduce brain damage and allow precise targeting into the medial forebrain bundle, a modified injection procedure was used according to a previous study (Gonzalez-Perez et al., 2010). Briefly, the solution was delivered by a glass capillary needle with a diameter of 100 μm, which was mounted on a microinjection pump connected to a 5 μL Hamilton syringe via polyethylene tubing with a diameter of 1 mm. The 2, 4, or 8 μg of 6-OHDA in a total of 2 μL in 0.02% ascorbic acid was infused with a glass capillary needle at a rate of 1 μL/min. The needle was left in place for an additional 10 minutes for maximal diffusion. Lesions were not induced in the control group; however, the vehicle groups were infused with 2 μL of saline containing 0.02% ascorbic acid in an identical manner to the 2, 4, or 8 μg 6-OHDA groups.