250 P30 and P200–250 (n=5 per genotype, per age group) were trained on the first day (by placing the mice on the rod that rotated with a constant speed of 5 rpm for 5 min) and then subjected to two trials separated by 30 min. On days 2 to 4, the P30 and P200–250 mice were directly subjected to two trials without pre-training. The results are presented as maximal rotating speed with mean±SD of five mice each with two trials per day over 5 days. 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. rotarod performance) with three (for ∼P18 mice) or five levels (for P30 and P200–250 mice). Rotarod performance within the groups was subse- quently tested for significant changes over time by use of separate repeated measures ANOVAs for each genotype with one within-subjects factor with again three or five levels. The assumption of normality of the distributions (as tested by Kolmogorov–Smirnov tests), sphericity (as tested by Mauchly’s tests) and equality of error variances (as tested by Levene’s tests) were not violated. Motor function was also assessed in P30 and P200–250 WT (n= 4 and 5, respectively) and PCα1KO (n =4 and 5, respectively) mice using a dyskinesia rating scale that was previously described and validated in several mouse mutants [28, 29], including mice carrying a Cacna1a mutation. At the start of the experiment, each mouse was transferred to a new cage. Mice were scored for 1 min at 10-min intervals over a 60-min period. Abnormal motor behavior within the head/neck, trunk, and limbs were recorded in terms of type, including tremor (trunk, limbs, neck, and face), tonic flexion, extension (trunk, limbs, neck, and face), or twisting (trunk and neck) and clonus (limbs and face). Uncoordinated or ataxic motor behavior not attributable to a single-body part was also recorded, including falling, listing, and stumbling. Assessments of the abnormal motor behavior were then calculated by summing the dyskinesia scores during the 60-min observation period. Mice were observed on at least two separate occasions and mean dyskinesia scores were generated for each mouse. Statistical differences were determined by Mann–Whitney U tests, because the data were non-parametric in nature. Results Generation of Mice That Lack CaV2.1 Channels in Purkinje Cells Homozygous PCα1KO mice were generated by crossing Cacna1aflox/flox mice with transgenic mice expressing Cre recombinase driven by the Purkinje-specific Pcp2 gene promoter (i.e. Pcp2Cre mice—see “Materials and Methods”) (Fig. 1a). Successful genomic deletion of exon 4 of the Cerebellum (2012) 11:246–258 Cacna1a gene was assessed at the RNA level by RT-PCR on cerebral and cerebellar total RNA (Fig. 1b). Whereas in the cerebrum only non-recombined PCR product of 580 bps was observed, in the cerebellum both non-recombined and recombined (480 bps) products were present. This indicated that Cre recombination only had occurred in part of the cerebellar neuron population. Immunohistochemical staining for α1A protein showed that Cre recombination was Purkinje cell-specific as no protein signal was present in the soma of virtually any Purkinje neurons of PCα1KO mice (Fig. 1d, f). However, α1A protein expression in other cerebellar struc- tures, e.g. the CN, appeared not different between PCα1KO and WT mice. These findings are in line with the reported Purkinje cell specificity of the Pcp2Cre mice [22]. Motor Coordination Is Severely Affected in Purkinje Cell CaV2.1-Knockout Mice PCα1KO mice, unlike conventional α1KO mice [4–7], have a normal life span. PCα1KO mice appear normal until P10 to P12 when they start showing an abnormal motor phenotype with a loss of balance during walking and problems righting themselves (see Supplementary video). Young PCα1KO ∼P18 mice (n = 3 per genotype) performed significantly worse on the rotarod than did their WT littermates, (F (1, 4) = 184.90, p < 0.001) (Fig. 2a). However, there was no significant genotype X times change in performance over time interaction effect (F (2, 8) = 1.00, p = 0.41) and neither PCα1KO mice nor WT mice showed an improvement over time (F (2, 4) = 2.00, p = 0.25 and F (2, 4) = 7.11, p = 0.54, respectively). This indicates that although WT mice performed significantly better on the rotarod, the young age and/or the intensity of the training (more repeats during this 1-day protocol induced fatigue in the ∼P18 WT mice as also seen with a P40 WT mouse (data not shown)) prevented a significant improvement. Rotarod experiments of PCα1KO mice tested at P30 (n = 5 per genotype) consisted of two trials per day for five consecutive days and revealed that these mice were severely impaired, in that they were not able to stay on the accelerating rod for longer than 60 ± 7.8 s on the fifth day (Fig. 2b), which corresponded to a rotating speed of 8 rpm. This maximum rotating speed was significantly slower than that in WT mice, which stayed on the accelerating rod for 272 ± 19.1 s on the fifth day, which corresponds to a rotating speed of 40 rpm (F (1, 8) = 1297.32, p < 0.001) (Fig. 2b). Additionally, there was a significant genotype X change in performance over time interaction effect (F (4, 32) = 4.89, p < 0.01), indicat- ing that there was a significant difference in change in performance over time between the two groups. More specifically, whereas the WT mice significantly improved their performance over time (F (4, 16) = 7.14, p < 0.01), the