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Tissues were rinsed with PBS and mounted with fluorsave reagent (Calbiochem; Millipore). Images were captured in an optical microscope (Nikon E-800; Izasa, Werfen Group, Valencia, Spain) with 40�� lens for the cerebellum and 20�� lens for the prefrontal cortex. We considered cFos positive (cFos+) peroxidase staining those cells showing a brown labelling in the nucleus (see Fig.?1a). We counted the first plane of three sagittal sections at the granule cell layer of the vermis cerebellum (L ?0.04 to 0.72?mm) (Paxinos & Franklin 2008) in selected regions of interest (ROIs) of 20?000?��m2 at the apical (external surface of the internal granular layer) selleck inhibitor and medial zones (deep portions of lobule) of each cerebellar lobule, for a total area of 40?000?��m2 per lobule and section. Purkinje neurons were counted in an area of 20?000?��m2 in the apical and medial regions and they were considered cFos+ when exhibiting a uniform and constant staining in the soma (see Fig.?1a). For the prefrontal cortex, we counted cFos+ neurons in ROIs of 20?000?��m2 of the cingulate, prelimbic, infralimbic and orbitofrontal medial cortex (from bregma 2.22?mm to bregma 1.94?mm) (Fig.?7). Cell count was performed automatically with ImageJ (now FIJI; NIH sponsored image analysis program) software. RVX-208 Fluorescent microphotographs were taken with an Olympus FV1000 confocal microscope (Olympus Europa Holding GMBH, Hamburg, Germany) with 60?��?oil lens (Fig.?1b). All statistical analyses were conducted BMS-777607 mw using the Statistica 6.0 software package (Statsoft, Inc., Tulsa, OK, USA). Behavioural data were analysed by means of one-way analysis of variance (ANOVA), followed by Tukey's honestly significant difference (HSD) post hoc tests and by means of Kruskal�CWallis ANOVA by ranks and chi-squared tests for dyadic comparisons. Differences between groups on cFos staining at different brain regions were analysed using separate one-way (group) multi-variate analyses of variance (MANOVAs) followed by univariate ANOVAs and Tukey's HSD tests, when possible. In all these analyses, the number of pairings at the training phase was used as a covariate. Finally, Pearson's r correlation index was used to ascertain the degree of correlation between preference for the CS+ preference and cFos staining in particular brain regions. The level of significance was set at P?