Rats in the 6-OHDA + DCS group underwent two separate surgical procedures. First, they were implanted with spinal stimulation electrodes under anesthesia induced with 5% halothane, ketamine (100 mg/kg), xylazine (10 mg/kg) and atropine (0.05 ml). Postoperative weight was monitored daily. The implantation procedure was adapted from previous studies5 (link)36 (link). The electrodes were inserted in the epidural space under T2 (thoracic vertebra) and tied to it with surgical suture. This prevented electrode migration and facilitated stimulation over a long period. One week later, after recovery of initial weight, rats were anesthetized for a second surgery, with 5% halothane, followed by intramuscular injections of ketamine (100 mg/kg), xylazine(10 mg/kg) and atropine (0.05 ml). A total of 52.5 ug 6-OHDA hydrobromide (Sigma Company, USA - 3.5 mg/ml in 0.05% ascorbate saline) was injected bilaterally into the striatum, at 3 locations on each side, using a needle, driven by a syringe pump (Sage, Model 361, Firstenberg Machinery Co Inc., USA) via 10 uL Hamilton syringe, at 1 uL/min. The needle was left in situ for 5 minutes and withdrawn slowly, to prevent backtracking of the drug. Anteroposterior, mediolateral and dorsoventral coordinates for the injections were: +1.0, +/−3.0, −5.0; −0.1, +/−3.7, −5.0 and −1.2, +/−4.5, −5.037 (link) from bregma. Destruction of noradrenergic fibers and terminals was prevented by 1,3-Dimethyl-2-imidazolidinone (DMI, Sigma Company, 25 mg/kg), administered IP, 30 minutes prior to 6-OHDA treatment38 (link).
Rats belonging to the 6-OHDA lesion and sham control groups underwent only the surgical procedure required to perform the lesion; animals in the 6-OHDA lesion group received bilateral injections of 6-OHDA, while rats in the sham control group received only vehicle solution (0.05% ascorbate saline). Time course of the entire experiment is shown in Fig. 1a. The lesion procedures were performed by the same individuals throughout all the experiment and all groups were run in parallel. Extreme care was taken to consistently maintain the timing of the various methods and conditions during the lesion procedure.