Animal care and

use procedures were conducted in accordance with NIH, USDA and institutional guidelines. An initial vector dose response study was carried out with a 1 month survival using 48 rats to determine the optimal dilution of AAV2/8-hSNCA and ratio of AAV2/8-hSNCA to silencing vector for use in efficacy experiments (Table S1). For the behavioral study, treatment groups were: AAV- hSNCA alone (n=16 at 1 month; n=11 at 2 months); AAV-hSNCA and AAV-NS (n=15 at 1 month and 10 at 2 months); AAV-hSNCA and AAV-mir30-SNCA (n=16 at 1 month and n=11 at 2 months). LBH589 cost At 1 month, rats were sacrificed for histological analysis of TH-IR fiber density in ST and Iba-1 in SN this website (n=5). The remaining rats were sacrificed at 2 month with half the rats prepared for histology and half for molecular analyses (n=5–6). A separate group of rats was injected for molecular analysis at 10 days (n=3). The skin overlying the skull of isoflurane-anesthetized rats was shaved. Stereotaxic surgeries were carried out using a Stoelting stereotaxic apparatus equipped with a Stoelting

quintessential stereotaxic injector holding a 10 µl Hamilton syringe with a 26 gauge needle. A hole was drilled in the skull over the appropriate injection site at stereotaxic coordinates, 5.5 mm posterior, −1.9 mm lateral and 7.4 mm ventral from Bregma (SN injection). The syringe was inserted into the brain at a speed of ~1 mm/min and then allowed to remain in place for 2 min before vector injection. 2 μl of each virus mixture was injected at a rate of 0.5 μl/min into one SN, and the needle was left in place for 5 min at the end of the injection in order to minimize diffusion up the needle track. The doses of each vector used for the dose response experiments are shown in Table S1. For the efficacy experiment, rats received 6.22×109 vg of AAV2/8-hSNCA alone or with either see more 3.51×1011 vg of AAV2/8-mir30-NS

or 3.26×1011 vg of AAV2/8-mir30-SNCA in a 2 μl volume. The syringe was withdrawn at a rate of ~1 mm/min. The drill hole was plugged with gel foam to control bleeding and Marcaine was spread around the surgical site. The skin was sutured and treated with topical antibiotic ointment and rats were returned to the vivarium upon recovery from anesthesia. Twenty-four hours after surgery, rats were transferred to a clean cage and the old bedding was autoclaved. Rats were placed inside of a plexiglass cylinder, partially surrounded by mirrors in a quiet, dark room. The activity of each rat was recorded on videotape under red light for a 10 min period. The number of times each paw was used for wall touches on the first 25 rearings of each rat was counted by an observer blinded to treatment group to determine forelimb use preference as previously published (Schallert et al.