252 Fig. 3 Overall cerebellar and Purkinje cell morphology in young Cacna1a Purkinje cell- specific knockout mice. Haema- toxilin and eosin staining reveals similar gross cerebellar mor- phology in PCα1KO (a) and WT (b) mice at P30. G granule cell layer, PC Purkinje cell layer, M molecular layer, WM white matter. Roman numbers indicate the cerebellar lobules. Golgi- Cox-stained cerebellar sections of PCα1KO (c) and WT (d) mice at P30 reveal normal morphology of the Purkinje cells in the two genotypes; e PCα1KO mice show similar level of dendrite arborization (linear Sholl analysis, for details see “Materials and Methods”) as their WT littermates Cerebellum (2012) 11:246–258 To study whether the severe ataxia in young PCα1KO mice come about due to structural abnormalities, we studied the cytoarchitecture at P30 in more detail (Fig. 3). Both a standard HE staining and a more detailed Golgi-Cox staining did not reveal qualitative differences between PCα1KO and WT littermates. Topological analysis of the dendritic branching of Purkinje cells from two WT mice (i.e. 25 cells) and three PCα1KO mice (i.e. 30 cells) showed no significant difference (F (1, 40) = 0.00, p = 0.98) between groups, nor was there a significant genotype X change in the dendritic branching with an increasing distance interaction effect (F (3.385, 135.397) = 1.03, p = 0.39). In both the WT and PCα1KO mice, the amount of dendritic branching did significantly change with increas- ing distance (F (3.281, 82.024) = 42.13, p < 0.001 and F (3.002, 45.036) = 20.09, p < 0.001, respectively). These data indicate that, although PCα1KO mutants are severely ataxic already at P18, their Purkinje cells did not reveal structural abnormalities. Progressive Cell Death of Purkinje Cells in the Absence of CaV2.1 Channels As CaV2.1 channels gate more than 90% of the high- voltage-gated Ca2+-influx in Purkinje cells, the morphology and survival of Purkinje cells in the PCα1KO mutants may be affected at later stages in their life. Therefore, we compared calbindin D28K-stained cerebellar sections of PCα1KO mice at various ages (i.e. P30, P60, P100, and P200–250) (Fig. 4). In line with the topological analysis described above, the immunostaining of the cerebellar cortex of PCα1KO mutants at P30 did not show overt differences with those of WT littermates (Fig. 4a). At later ages, however, we observed substantial Purkinje cell degen- eration in the cerebellum of PCα1KO mice (Fig. 4b–d). Starting at P60, Purkinje cells showed degenerative structural abnormalities, such as somatic sprouting and axonal swel- lings (Fig. 4b–d). These changes progressed until P200–250, at which PCα1KO mice (n=2) showed a significant decrease in Purkinje cell number compared with WT controls (n=3) (lobule 2, U=0.00, p<0.001; lobule 6, U=28.00, p<0.001; lobule 9, U =1.00, p<0.001; paramedical lobule, U= 0.00, p<0.001; paraflocculus, U= 179.00, p<0.001; flocculus, U= 0.00, p < 0.001) (Fig. 4e). Although the extent of the degeneration of Purkinje cells is evident, also the variability between various lobules is apparent. Given that we did not find any anti-CaV2.1-α1A staining in the Purkinje cells of PCα1KO mice (see Fig. 1c–f), this variability in the Purkinje cell death could not be explained by inter-cellular variations in the Cre expression or the recombination efficacy of the LoxP/Cre system. Neurodegenerative Changes in Cerebellar Cortex In order to visualize dying neurons, we used silver impregnation on tissue from mice at various ages (Fig. 5). At P30, PCα1KO mice showed very little argyrophylic staining (Fig. 5a, d), indicating that there are no overt degenerative changes in their cerebellum, despite their ataxia. However, at P45 the level of argyrophylic staining in PCα1KO mice dramatically increased. Many silver- stained somata, axons, and dendrites of dying Purkinje cells