Cerebellum (2012) 11:246–258 11 concentric circles increasing in size (with 15 μm intervals) were aligned with the Purkinje cell soma. The number of Purkinje cell dendrites intersecting with each circle was counted. Statistical differences between groups were determined by use of repeated measures ANOVAs with one “between-subjects” factor (genotype) and one “within-subjects” factor (i.e. number of Purkinje cell dendrites intersecting with the circle) with 11 levels. The change in the dendritic branching with increasing distance within the groups was subsequently tested by use of separate repeated measures ANOVAs for each genotype with one “within-subjects” factor with 11 levels. The assumption of normality of the distributions (as tested by Kolmogorov–Smirnov tests), and equality of error variances (as tested by Levene’s tests) were not violated. When the assumption of sphericity (as tested by Mauchly’s tests) was violated, Greenhouse–Geisser corrections were used. All statistical analyses were performed using SPSS 17.0 software (IBM corporation, Chicago, IL, USA). Quantification Number of Purkinje Cells and Degeneration of Purkinje Cell Axons Using 40-μm thick, calbindin-stained, transversally sectioned tissue from two PCα1KO mice and three WT littermates, we calculated the average number of Purkinje cells per 100 μm of cerebellar cortex. Five to ten pictures were taken at ×20 magnification from either the vermal parts of lobule 2, 6, or 9, or from the paramedian lobule, paraflocculus, or flocculus. In each picture, the number of Purkinje cells per 100 μm of Purkinje cell layer was determined using Metamorph (Universal Imaging, Downing Town, PA, USA). In absence of many Purkinje cells the difference in structure between the molecular and granular layers guided the observer to accurately estimate the location of the Purkinje cell layer. The extent of degeneration of Purkinje cell axons was qualitatively examined in sagittal cerebellar sections stained with silver impregnation as described elsewhere [23]. For each mouse, photographs of white matter were taken with a ×5 magnification for overview and a ×40 magnification for detailed examination of degenerating fibers. Neuronal Volume and Density Measurements Volume measurements were bilaterally taken from the medial cerebellar nuclei (MCN), anterior interposed nuclei (AIN), posterior interposed nuclei (PIN), and lateral cerebellar nuclei (LCN) using transversal, Nissl/calbindin- stained sections from three WT and three PCα1KO mice by the investigator blinded to the genotype. Contours were drawn around the four nuclei in every other section (e.g. 80-μm intervals) using NeuroLucida (MBF Bioscience, Williston, VT, USA) to construct a 3D image. Volumes (in 249 3 mm ) were then calculated using NeuroExplorer (Nex Technologies, Littleton, MA, USA). The dorsolateral hump was included in the AIN and the “interstitial cell group” was included in the PIN. Statistical differences were determined by an independent samples Student’s t test or an independent samples Mann–Whitney U test (depending on the normality of the distributions as tested by Kolmogorov– Smirnov tests). With regard to t tests, if the assumption of equality of variances was violated (as tested by Levene’s tests), corrected degrees of freedom were used. Neuronal surface density in the cerebellar nuclei was bilaterally determined in transversal paraffin-embedded, HE-stained cerebellar sections (4 μm) of two WT and two PCα1KO mice. For each CN, sections with a 60-μm interval were photographed at ×25 magnification (five to ten pictures per nucleus). Two independent investigators, each blinded to the genotype, counted the number of cells in each picture using Meta Imaging Series 4.6 (Molecular Devices Inc., Downingtown, PA, USA). Counting was done by drawing a contour around each CN and subsequently counting all somata with a clearly visible nucleolus by contouring the neuron’s nucleus. The surface density 2 (number of cells/mm ) was calculated for each section, after which the average density per CN was calculated. The total numbers of “small” and “large” cells were subse- quently determined using a threshold of 8.5-μm diameter (see also Ref. [27]). The 8.5-μm cut-off value was constructed using a scatter plot of the neuron nucleus’ diameter from which two groups could be distinguished. Statistical differences were determined by an independent samples Student’s t test or Mann–Whitney U test (depend- ing on the normality of the distributions as tested by Kolmogorov–Smirnov tests). With regard to t tests, if the assumption of equality of variances was violated (as tested by Levene’s tests), corrected degrees of freedom were used. Motor Behavior Analyses The accelerating Rotarod (UGO Basile S.R.L., Commerio VA, Italy) test was performed on a 3 cm diameter horizontal rotating rod. The rod initially rotated at a constant speed of 4 rpm and accelerated in a stepwise manner (i.e. 4 rpm every 30 s) to a maximum of 40 rpm after 270 s. The maximum time on the rod was 300 s. The latency to fall was recorded on the moment the mouse fell down or stopped walking (i.e. clinging to the rod) for three consecutive turns and subsequently converted to the “maximum rotating speed” by calculation of the rotating speed at the moment of the fall. The test was performed in a semi-dark room with a light source placed at the bottom to discourage the mice from jumping off the rod. Mice of ∼P18 (n=3 per genotype) were subjected to three trials separated by 90 min on 1 day without pre-training. Mice of