Rocker is a new variant of the voltage-dependent calcium channel gene Cacna1a

Abstract

Rocker (gene symbol rkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a "weeping willow" appearance of the secondary branches. Using simple sequence length polymorphism markers, the rocker locus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel alpha1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with the leaner mutant allele (Cacna1a(la)) produced mutant animals, thus identifying rocker as a new allele of Cacna1a (Cacna1a(rkr)). Sequence analysis of the cDNA revealed rocker to be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions between rocker and the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function.

Fig. 1.

Sagittal sections of wild-type, rocker (rkr/rkr), and compound heterozygote (rkr/la)mice stained with 0.2% cresyl violet. Rocker mutants show normal cytoarchitecture of whole mouse brains. Comparison of wild type (A) with homozygote rocker(B) or compound heterozygote (C) revealed no significant difference in cerebellar morphology.

Fig. 2.

Adult rocker mutants show no loss of cerebellar Purkinje cells. The total number of Purkinje cells is graphed as a function of the distance from the midline.Rocker mutants >1 year of age (black line with diamonds and black dashed line with triangles) showed no significant difference from the age-matched wild-type control (gray line with circles). Gaps at the midline reflect sections that could not be counted because of a technical artifact of sectioning.

Fig. 3.

Golgi impregnation of individual cerebellar Purkinje cells shows dendritic abnormalities in the adultrocker mutants. A, B, Branching in the wild-type Purkinje cell usually appears very dense, with an increasing number of branching points near the distal tips.C, D, Purkinje cells in older rockermutants show a decrease in branching and have downturned distal ends of their dendritic tree (D, arrow). Scale bars: A, C, 50 μm; B,D, 25 μm.

Fig. 4.

Golgi preparations were used to examine other areas of the brain known to express the calcium channel α 1a, such as the cerebral cortex. No distinguishable differences are evident between wild-type (A) and rocker(B) mutants. Scale bar, 50 μm.

Fig. 5.

Mapping of rocker phenotype to mouse chromosome 8. The second generation of backcrossed animals was separated into affected and wild-type groups based on the observed ataxic phenotype. DNAs from animals of known phenotype were mixed to create known ratios of B6 and C3H DNA as controls for the PCR amplification. Duplicate lanes from the affected pool (rkr pool) and wild-type pool (wt pool) were amplified with a number of MIT markers that recognize SSLPs in the mouse genome (two examples are shown in A).A, The first set of primers, D8Mit95, amplifies a 158 bp product from the B6 strain and a 154 bp product from the C3H strain. The second set of primers, D8Mit162, amplifies a 149 bp B6 band and a 131 bp C3H band. Therocker pool shows strong bands from the B6 and a much weaker band amplified from the C3H. The wild-type pool shows a more equal amplification of both the B6 and C3H PCR products. In each experiment, a control lane of B6 alone shows a single band, and a second control lane shows a heterozygote animal (F1, a known 1:1 ratio of B6/C3H) producing both the B6 and C3H PCR products. The negative control (no DNA lane) showed no nonspecific amplification. The estimated size of PCR products was determined using a 123 bp DNA marker (L).B, MIT markers on mouse chromosome 8 used to localize the candidate region in individual animals.

Fig. 6.

Determination of rocker mutation.A, The sequence alteration in the rockermutant. Rocker contains a cytosine (C) to adenosine (A) change at nucleotide position 3929 that results in a threonine (T) to lysine (K) alteration at amino acid position 1310. B, Proposed transmembrane topography of the α 1A subunit and positions oftottering (tg), rolling (rol), leaner (la), and rocker (rkr) (indicated by arrows).

Fig. 7.

Calbindin immunohistochemistry staining in adult wild-type (A, B) and rocker (C, D) cerebellum reveals no obvious structural abnormalities. No axonal swellings of the Purkinje cell axons are observed in the internal granule cell layer of either the wild-type orrocker mutants. No gaps in the Purkinje cell layer (PCL) are evident, and no decrease appears in the size of the molecular (ML) or internal granule (IGL) cell layers. The structure of the Purkinje cell dendritic arbor also appears normal. No ectopic spines are observed on proximal dendrites of either the wild-type or rockeranimals. Scale bars: shown in A for A andC, 25 μm; shown in B forB and D, 25 μm.

Fig. 8.

Immunostaining for tyrosine hydroxylase in the cerebellum of wild-type (A, B), rocker(C, D), and compound heterozygote (rkr/la) (E, F) adult animals reveal rocker to be unique among the α 1a mutants examined. A–D, Except for the occasional Purkinje cell TH staining (C, arrow), both wild type and rocker show the expected downregulation of TH expression. E, F, Large subpopulations of Purkinje cells show persistent TH expression in the compound heterozygote cerebellum. Scale bar: shown in F forB, D, and F, 50 μm.PCL, Purkinje cell layer; ML, molecular cell layer; IGL, internal granule cell layer.

Fig. 9.

Electrocorticographic activity recorded from awake adult rocker mutant. Traces from left and right hemispheres show bilateral, spontaneous cortical six to seven spike-wave synchronous discharge during behavioral arrest seizure. Electroencephalograph activity reverts to normal, low-amplitude, high-frequency rhythmic patterns immediately after the behavioral seizure episode. Calibration: 200 μV, 1 sec.