Piribedil

Levodopa, bromocriptine, carbidopa, L-685,458, TAPI-1, Y-27632, and Forskolin (FSK) were purchased from Selleck Chemicals (Houston, TX, USA). Piribedil was from Tocris Bioscience (Bristol, UK). β-Secretase Inhibitor IV (BSI IV) was from Calbiochem (Hayward, CA, USA). Recombinant human BDNF, GNDF, IGF-I were from Peprotech (Rocky Hill, NJ, USA). cAMP and L-Ascorbic acid were from Sigma (St Louis, MO, USA). CellTiter-Glo was from Promega (Madison, WI, USA) and Effectene Transfection Reagent was purchased from QIAGEN (Hilden, Germany). Immunoblotting was performed with the following antibodies: anti-PS1 N-terminus (1–65) (PRB-354P, Covance, Davis, CA, USA); anti-APH1aL C-terminus (245–265) (PRB-550P, Covance); anti-NCT (N1660, Sigma); anti-Pen2 (P5622, Sigma); anti-BACE1 N-termimus (AP7774b, Abgent, Suzhou, China); anti-APP-CTF (A8717, Sigma); anti-sAPPα (secreted Amyloid Precursor Protein-α) (11088, IBL, Hokkaido, Japan); anti-HA (H6908, Sigma); anti-actin (A2066, Sigma); anti-D₂R (sc-9913, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunostaining was performed with the following antibodies: anti-nestin (MAB353, Milipore, Darmstadt, Germany); anti-Sox2 (sc-17320, Santa Cruz); anti-Ki67 (556003, BD Transduction Laboratories, San Jose, CA, USA), and anti-Doublecortin (DCX) (sc-8066, Santa Cruz).

Rats were sedated with intraperitoneal xylazine (10 mg/kg; Syntec do Brasil Ltda, Brazil) and anesthetized with intraperitoneal ketamine (90 mg/kg; Syntec do Brasil Ltda, Brazil). The following coordinates were used to the bilateral injury, bregma as a reference: SNpc (AP) = −5,0 mm, (ML) = ± 2,1 mm e (DV) = −8,0 mm (Paxinos and Watson, 2005 ). Needles were guided to the region of interest for a bilateral infusion of 1 μL of rotenone (12 μg/μL) or of dimethylsulfoxide—DMSO (Sigma-Aldrich®, United States) using an electronic infusion pump (Insight Instruments, Ribeirão Preto, Brazil) at a rate of 0.33 μL/min for 3 min (Saravanan et al., 2005 (link); Moreira et al., 2012 (link); Dos Santos et al., 2013 (link)). Complementarily, a guide cannula was implanted in the olfactory bulb of each rat allowing a subsequent 1 μL infusion of piribedil (3 μg/μL) (Tocris Bioscience®, United Kingdom), raclopride (10 μg/μL) (Sigma-Aldrich®, United States) or vehicle (DMSO) at a rate of 0.33 μL/min for 3 min, in their respective groups. Coordinates with reference to bregma for implantation of guide cannula were: (AP) = +7.08 mm (ML) = 0.0 mm and (DV) = −3.6 mm (Paxinos and Watson, 2005 ).