Cerebellum (2012) 11:246–258 CaV2.1 channels exhibit a severe phenotype of ataxia and dystonia with a strong cerebellar component that starts around postnatal day (P)12, and die, if not given very special care, around P20 [4–7]. Histological analysis in the few “survivors” indicated that a progressive gradual loss of Purkinje cells started between P45 and P100 [5]. Cerebellar ataxia and dystonia can also be part of the phenotype of certain naturally occurring Cacna1a mouse mutants. For instance, leaner, tottering, rolling Nagoya, but not rocker, are characterized to different extent by granule and/or Purkinje cell death and chronic ataxia [8–11]. For tottering and leaner mice, it was shown that the chronic ataxia is related to irregular Purkinje cell simple spike firing caused by the loss of precision in intrinsic activity of Purkinje cells and aberrant synaptic input [12–15]. In addition to the cerebellar ataxia phenotype, tottering mice also exhibit episodic dystonia, which is related to transient low-frequency oscillations in the cerebellar cortex [16]. Notably, when tottering mutants lose their Purkinje cells on a Purkinje cell degeneration mutant background, the dystonic phenotype disappears [17]. Together these findings indicate that mutations that cause a reduction of CaV2.1- mediated Ca2+-influx affect the information processing in the cerebellar cortex and, thereby, induce chronic ataxia and episodic dystonia. Apart from the aberrant activity in the cerebellar cortex in Cacna1a mutants, also the activity of cerebellar nuclear (CN) neurons is subject to abnormal firing patterns, as was recently shown in tottering mice [18]. Both Purkinje cells and CN neurons receive input (direct and indirect through local interneurons and/or granule cells) from mossy fibers and climbing fibers. While granule cell transmission to Purkinje cells in α1KO, tottering, leaner, and rolling Nagoya mice is abnormal, the response to climbing fiber activation is seemingly normal in these mutants [15, 19, 20]. Besides the excitatory input from climbing and mossy fibers, the neurons in the CN receive input from local interneurons and from the cerebellar cortex through Purkinje cells. Apart from the impact of these inputs, the firing pattern of CN neurons is determined by their intrinsic activities. Since Purkinje cells and CN neurons, as well as the neurons and fibers that innervate them, express CaV2.1 channels [3], the exact origin of the aberrant cerebellar activity in CaV2.1 mutants that causes cerebellar ataxia could not be resolved using the existing Cacna1a mouse mutants. Therefore, we used the Cre-lox system to ablate CaV2.1 channels exclusively in Purkinje cells by crossing condi- tional Cacna1a mice that carry a “floxed” allele [21] with transgenic mice that express Cre recombinase under the control of the Purkinje cell-specific Pcp2 promoter [22]. Hereby, we were not only able to rescue the early lethality seen in α1KO mice, but also to demonstrate that lack of 247 CaV2.1 channel function in Purkinje cells is sufficient to cause ataxia and that progressive Purkinje cell loss starts well after the onset of the ataxia. Materials and Methods Animals All animal experiments were performed in accordance with guidelines of the respective universities and the national legislation. Previously, we generated conditional Cacna1a mice using a gene-targeting approach [21]. The Cacna1a allele in these mice was “floxed” by introducing loxP sites flanking exon 4 (i.e. Cacna1aflox mice) (Fig. 1a). A genomic deletion of exon 4 is achieved by crossing the conditional mice with Pcp2Cre transgenic mice that express Cre recombinase exclusively in Purkinje cells [22]. Homozygous Cacna1aflox/flox mice were first crossed with homozygous Pcp2Cre mice generating double heterozygous transgenic offspring. Female double trans- genic mice were then crossed with Cacna1aflox/wt males to generate mice that are Cacna1a deficient (and thereby CaV2.1 channel deficient) in Purkinje cells (i.e. Purkinje cell CaV2.1-α1AKO (PCα1KO)). The same cross also produced mice in which Cacna1a was not affected (wild type (WT)). In total, we used 29 PCα1KO and 29 WT littermates. Genotyping of the Cacna1aflox allele was performed by PCR using primers P1 (5′-acctacagtctgccag gag-3′) and P2 (5′-tgaagcccagacatccttgg-3′) and genomic DNA as a template. Genomic deletion of exon 4 after Cre recombination was confirmed by PCR using primers P3 (5′-agtttctattggacagtgctggt-3′) and P4 (5′-ttgcttagcatgcaca gagg-3′). For genotyping of the Cre transgene primers P5 (5′-acttagcctgggggtaactaaact-3′) and P6 (5′-ggtatctctgacca gagtcatcct-3′) were used. Reverse Transcription PCR Total RNA was isolated from freshly dissected cerebellum and cerebrum of mice. For RT-PCR, first-strand cDNA was synthesized using random primers. Subsequently, Cacna1a- specific PCRs were performed using primers P7 (5′- gatgacacggaaccatac-3′) and P8 (5′-attgtagaggagatcagtcc-3′) that are located in exon 3 and 6, respectively. Histology Mouse brains (20 PCα1KO and 20 WT) were obtained at various ages after cardiac perfusion with PBS followed by 4% buffered paraformaldehyde (PFA). Post-fixation was per- formed for 1 h in 4% PFA followed by overnight incubation in 10% sucrose in 0.1 M phosphate buffer at 4°C. Subse-