The final data are presented as a mean value of the total number of each rat group. Brainstem sections were categorized according to their approximate rostrocaudal

location from the MDH subnucleus interpolaris junction. The pERK1/2-positive cells were counted under a 20× objective according to their location in the different laminae of the MDH from sections costained for PKCγ, a cellular marker that highlights the inner lamina II (Polgár et al. 1999). The delineation of the MDH was based on the Paxinos and Watson atlas. The data are expressed as the sum of the total number of labeled cells counted from all the sections analyzed in each animal. Tyrosine hydroxylase (TH) immunolabeling was performed using anti-TH primary antibody Inhibitors,research,lifescience,medical (Millipore, Molsheim, France), as described above. Quantification of the impact of the 6-OHDA lesion on the SNc was investigated as described previously (Paillé Inhibitors,research,lifescience,medical et al. 2007; Zengin-Toktas et al. 2013) using frozen coronal sections (40 μm) under a 20× objective. For each rat, TH-positive cells were counted at different rostrocaudal levels (every 0.12 mm; to Inhibitors,research,lifescience,medical −4.64 until −6.2 relative to bregma) using Image J software (ImageJ v1.41, National Institute of Health, Bethesda, MD). Split-cell counting errors were corrected using Abercrombie’s formula, where N = n [t/(t + d)] (N: total number of cells; n: number of cells counted; t: section thickness; and d: cell diameter). The total

number Inhibitors,research,lifescience,medical of cells in the SNc was calculated using Konigsmark’s formula, where Nt = Ns (St/Ss) (Nt total number of cells; Ns number of cells counted; St total number of sections through the SNc; Ss number of sections in which cells were counted (Paillé et al. 2007). Image analysis was Natural Product Library in vitro completed using ImageJ software. The point picker plugin available in ImageJ freeware can be used to mark cells with a colored cross by clicking with the mouse. The number of cells counted is displayed in a text box on completion. For each group Inhibitors,research,lifescience,medical of animals,

the data were the total number of cells in the section expressed as the percentage of the number of cell bodies measured for the shams (mean ± SD). Anatomical and behavioral analyses were performed by an experimenter blind to the treatments. Locomotor second impairment The rotarod test was performed 2 weeks after surgery. Locomotor impairment was determined on an accelerating rotarod treadmill (TSE systems GmbH, Bad Homburg, Germany). Before testing, the rats were habituated to the instrument at a constant (4 rpm, 5 min) and accelerated speed (40 rpm, 5 min, five times) for three consecutive half days. A testing session was performed on the fourth consecutive day. The rotarod was accelerated progressively from 4 rpm to 40 rpm over 5 min. The time that the rats remained balanced on the device was scored. The rotarod test results for each animal represent the average time (sec) spent on the rod for the last three sequences on the testing session (mean ± SD).