Cerebellum (2012) 11:246–258 Purkinje cell axons in cerebellar white matter depending on their efferent targets [30, 31] and the non-uniform distribu- tion of Purkinje cell degeneration (Fig. 4e). Neurodegenerative Changes in Cerebellar Nuclei The loss of Purkinje cells in PCα1KO may not only have an effect on upstream neurons in the cerebellar cortex, but also on downstream neurons in the cerebellar nuclei. To test for loss of Purkinje cell target neurons, we calculated the volume (in mm3) and surface density (in number of cells/ mm2) of the MCN, AIN, PIN, and LCN in PCα1KO and WT mice at age P200–250. The volume quantification revealed pronounced volume reductions in all cerebellar nuclei of PCα1KO mice compared to those of WT mice, (LCN: t (10) = 8.63, p < 0.001; AIN: t (10) = 8.86, p < 0.001; PIN: U = 3.00, p < 0.02; MCN: U = 0.00, p < 0.01; total volume: U = 0.00, p < 0.01) (Table 1) (Fig. 7a–c). The surface density was significantly increased in all cerebellar nuclei of PCα1KO mice compared to those of WT mice (LCN: t (6)=−3.43, p<0.02; AIN: t (6)=−4.99, p<0.01; PIN: t (6) = −2.94, p < 0.03; MCN: t (3.354) = −4.19, p < 0.03) (Table 1) (Fig. 7d). The increased surface density also appeared in the total density of “small cells” (nucleus, <8.5 μm in diameter) averaged for all nuclei (M=262.5± 47.8 for WT and 509.9±111.3 for PCα1KO mice, t (6)= −4.09, p< 0.01) (Fig. 7e) but not for the “large cells” (nucleus, >8.5 μm in diameter; M=178.3±21.1 for WT and 198.0± 11.4 for PCα1KO mice, t (6)=−1.65, p=0.151) (Fig. 7f). Thus, a general decrease in volume and an increase in cell density can be seen with the exception of an unchanged “large cell” density. Discussion Here we used conditional gene targeting to ablate CaV2.1 calcium channels selectively in Purkinje cells in vivo in mice. This approach allowed us to study the specific contribution of these channels in Purkinje cells to the development of ataxia that is associated with mutations in the Cacna1a gene. Our cell type-specific knockout strategy 255 using conditional Cacna1a-knockout mice [21] succeeded in circumventing early postnatal lethality that is a very prominent feature in Cacna1a-knockout mice that lack CaV2.1 channels in all neurons [4–7]. In line with the well- defined expression of Cre recombinase in Purkinje cells in Pcp2Cre mice [22], Cre recombination affected α1A expres- sion in almost all Purkinje neurons, as is evident by the lack of anti-α1A staining in these cells at P30 while α1A expression seemed preserved in the rest of the brain (data not shown). We could demonstrate that lack of CaV2.1 channels in Purkinje cells is sufficient to cause an ataxic phenotype since the ataxia occurred prior to structural abnormalities. This finding adds to existing lines of evidence [8–11], which indicate that the cerebellar ataxia associated with abnormal CaV2.1 channel function can be caused by Purkinje cell dysfunction. Interestingly, our PCα1ΚΟ mutants exhibited a predom- inantly ataxic phenotype without episodic motor attacks during the first months of life. Notably, recently developed Cacna1apurk(−/−) mice, which lack functional CaV2.1 channels due to a Purkinje cell specific deletion of exon 1 of the Cacna1a gene, were reported to have dyskinesia attacks from 3 weeks on [32]. Intrinsically generated low- frequency oscillations in the cerebellum of tottering mice are thought to be the source of the episodic motor attacks in these mutants [16]. Further evidence for this hypothesis came from microinjections of the non-selective voltage- sensitive K+-channel blocker 3,4 DiAP in the cerebellum of tottering mice that abolished the episodic attacks of dyskinesia [33]. Future experiments will have to show whether similar mechanisms in the cerebellum of PCα1KO underlie the episodic nature of dyskinesia. Visual inspection of the mice revealed that the ataxia in PCα1KO mice started at P10–12, around the same age as in conventional α1KO mice that lack CaV2.1 channels in all neurons [4–7]. Likewise, loss of CaV2.1 function in Purkinje cells did not affect normal development of the cerebellar cortex, similar to what was shown for conventional CaV2.1KO mice [4, 5] and Cacna1apurk(−/−) mice [32]. These findings are in line with the fact that during the first postnatal week CaV1 (L-type) channels are the dominant voltage-gated Ca2+ channel type in Purkinje cell somata [34]. Table 1 Volume and surface density in the cerebellar nuclei (CN) The volume (mm3) and surface density (cells/mm2) are repre- sented for the lateral CN (LCN), anterior interposed CN (AIN), posterior interposed CN (PIN), and medial CN (MCN), as well as for the total CN Nucleus WT LCN AIN PIN MCN Total 0.125±0.016 0.196±0.026 0.089±0.030 0.198±0.004 0.608±0.071 Volume (mm3) Density (cells/mm2) 494.3± 83.4 370.0± 81.1 504.8± 41.1 394.1± 26.0 PCα1KO Volume (mm3) 0.057± 0.011 0.087± 0.015 0.046± 0.008 0.089± 0.015 0.280± 0.039 Density (cells/mm2) 842.6± 185.2 696.5± 102.9 668.0± 103.0 624.5± 106.8