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The Journal of Neuroscience, February 15, 2001, 21(4):1169-1178
Rocker Is a New Variant of the Voltage-Dependent Calcium Channel
Gene Cacna1a
Theresa A.
Zwingman1,
Paul E.
Neumann3,
Jeffrey
L.
Noebels4, and
Karl
Herrup1, 2
1 Department of Neuroscience and
2 University Alzheimer Center, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106, 3 Department of Anatomy and Neurobiology, Faculty of
Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7,
and 4 Department of Neurology and Division of Neuroscience,
Baylor College of Medicine, Houston, Texas 77030
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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  1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with
the leaner mutant allele
(Cacna1ala) produced mutant animals,
thus identifying rocker as a new allele of
Cacna1a (Cacna1arkr).
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.
Key words:
P/Q-type calcium channel; tottering; dendrite
maintenance; cerebellar catecholamine metabolism; mouse mutant; Ca2+ channel subunit; cerebellar Purkinje cells; gene mapping
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INTRODUCTION |
In neurons, voltage-dependent
Ca2+ channels (VDCCs) are involved in
diverse functions, including excitability (Llinas, 1988 ), neurotransmitter release (Wheeler et al., 1994; Dunlap et al., 1995;
Scholz and Miller, 1995), and regulation of gene expression (Bading et
al., 1993). In maturing neurons, VDCCs and calcium entry have been
implicated in neuronal migration (Komuro and Rakic, 1992), neurite
outgrowth (Kater and Mills, 1991; Moorman and Hume, 1993; Manivannan
and Terakawa, 1994), axon and dendrite extension (Cohan et al., 1987;
McCobb et al., 1989), and establishment of early synaptic connections
(Llinas and Sugimori, 1979; Mills and Kater, 1990; Vigers and
Pfenninger, 1991; Komuro and Rakic, 1992; Johnson and Byerly, 1993;
Spitzer, 1994).
VDCCs in mammalian neurons have been classified into five groups: L, N,
P/Q, R, and T, on the basis of their electrophysiological and
pharmacological properties. They are multisubunit complexes, the
expression and targeting of which require the assembly of the
pore-forming subunit 1 with  , 2/ , and  subunits. The calcium channel 1a subunit gene (Cacna1a) encodes the
pore-forming protein of P/Q-type channels. It is diffusely localized in
brain, with high levels of expression in cerebellar granule and
Purkinje cells (Stea et al., 1994; Tanaka et al., 1995; Westenbroek et al., 1995). Mutations of Cacna1a have been shown to
cause human neurological diseases such as familial hemiplegic migraine,
episodic ataxia-2 (Ophoff et al., 1996), and spinocerebellar ataxia 6 (Zhuchenko et al., 1997). In mice, mutations in the gene are found in
the neurological mutants tottering
(Cacna1atg, formerly tg),
leaner (Cacna1atg-la, formerly
la and tgla) (Fletcher et al.,
1996; Doyle et al., 1997), and rolling mouse Nagoya
(Cacna1atg-rol, formerly rol
and tgrol) (Mori et al., 2000) and are
expected in the other known mutant allele, tottering-3J
(Cacna1atg-3J, formerly
tg3J) (Green et al., 1988). Mutations in
the auxiliary subunits also result in neurological dysfunction in mice.
The 4 subunit is defective in lethargic (Burgess et al.,
1997), and 2 is defective in stargazer (Letts et al.,
1998).
The Cacna1a mutants, although clearly allelic, exhibit
distinct phenotypes (Tsuji and Meier, 1971) ranging from the milder tottering (Green and Sidman, 1962) and rolling
(Oda, 1981) alleles to the more severe leaner (Sidman et
al., 1965; Meier and MacPike, 1971) and tottering-3J (Green
et al., 1988). Phenotypic features that may be absent in some alleles
or may vary in onset and severity include ataxia, paroxysmal
dyskinesia, absence seizures (Meier and MacPike, 1971),
electrocorticographic "spike-and-wave" activity pattern similar to
that observed in human petit mal epilepsy (Noebels and Sidman, 1979),
reduced current density (Dove et al., 1998; Wakamori et al., 1998; Mori
et al., 2000; Qian and Noebels, 2000), cerebellar cell death (Herrup
and Wilczynski, 1982), increased adrenergic terminals from locus
coeruleus (LC) neurons (Levitt and Noebels, 1981), increased tyrosine
hydroxylase (TH) expression in Purkinje cells (Hess and Wilson, 1991;
Austin et al., 1992; Sawada et al., 1999), and ectopic dendritic spines
and axonal torpedoes on Purkinje cells (Rhyu et al., 1999a,b) (Table
1). Here we describe a new
Cacna1a mutant allele, rocker
(Cacna1arkr), that presents a fifth
distinct phenotype in this allelomorphic series.
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MATERIALS AND METHODS |
Animal husbandry. Ataxic animals were discovered at
the Children's Hospital (Boston, MA) in the fourth generation of a
male mouse mutagenized with ethylnitrosourea (ENU) at Kansas State University (Bode, 1984; Bode et al., 1987). The original
ENU-mutagenized male was of mixed genetic background. To assist
in the genetic analysis, the rocker gene was backcrossed
onto the C57BL/6J strain five times by mating homozygous
rocker animals with C57BL/6J animals (obtained from The
Jackson Laboratory, Bar Harbor, ME). The mutant gene passed through
both males and females and continued to be crossed onto the C57BL/6J
strain. Generation of mutants was achieved through the mating of
homozygous rocker males with heterozygous females, as well
as through heterozygous intercrosses. All animals were maintained in
the Case Western Reserve University Medical School Animal Resource
Center, a facility fully accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care. The mice were provided a
commercial diet and water ad libitum under conventional
conditions with controlled temperature, humidity, and lighting.
cDNA cloning and sequence analysis. cDNAs encoding the 1a subunit were isolated through reverse transcriptase (RT)-PCR using the random primers DNA labeling system (Life Technologies, Grand Island, NY) and total RNA. Eighteen PCR primers were designed according
to the published sequence data (Fletcher et al., 1996) such that the
entire 6495 bp reported sequence was covered. cDNA RT-PCR products were
sequenced using an automated sequencer (ABI prism model 377; Applied
Biosystems, Foster City, CA). Sequence was also obtained manually using
an Amplicycle Sequencing Kit (PerkinElmer Life Sciences,
Norwalk, CT) with -32P incorporation
according to the manufacturer's instructions. Sequence reactions
were electrophoresed through a 6% acrylamide denaturing gel and
exposed overnight at  70°C on X-OMAT AR film (Eastman Kodak,
Rochester, NY). Sequence information was obtained separately from four
mutants and four background control mice.
Histology. After being deeply anesthetized with Avertin
(0.02 cc/gm), animals were perfused through the heart with 4%
paraformaldehyde in 0.1 M sodium phosphate buffer
(PB), pH 7.4. The brain was dissected free of the skull case and stored
overnight in fresh fix. The following day, the tissue was dehydrated
through graded alcohols, infiltrated with paraffin (Paraplast Plus;
Fisher, Pittsburgh, PA), bisected on the midline, and embedded in the
sagittal plane. A full set of serial 10 µm sections was collected for
each half cerebellum counted. Using standard histological methods,
every 20th section was stained with 0.2% cresyl violet and examined at
400×. Purkinje cells were counted by previously described
profile-based counting methods (Herrup and Sunter, 1987). Purkinje
cells were identified by their large size and position in the Purkinje
cell layer. No obvious Purkinje-like cells were present in the
molecular layer; Purkinje cells in the internal granule cell layer
might have escaped detection because they would have been mistaken for Golgi II cells. All identified Purkinje cells that had a portion of
their nucleus in the section were counted. The nucleus-positive Purkinje cell counts were graphed as a function of distance from the
midline. The areas under the curves are proportional to the total
number of Purkinje cells in the cerebellum. This estimate was corrected
for split-cell counting errors by the method of Hendry (1976).
Immunohistochemistry. Animals were anesthetized deeply with
Avertin and perfused intracardially with 4% paraformaldehyde in PB.
The brains were immediately removed from the cranium and fixed for an
additional 4 hr at 4°C. The brains were then cryoprotected by sinking
in 18% (w/v) sucrose in PBS at 4°C overnight. Most samples were
embedded in OCT, frozen in powdered dry ice for 5 min,
and then allowed to equilibrate to the cutting temperature of the
cryostat (20°C). Cryostat sections (10 µm) were cut and allowed
to air dry on gelatin-coated slides.
For single-label immunofluorescence, sections were first rinsed in 1×
PBS for 5 min, followed by a 1 hr wash at room temperature in PBS
containing 0.5% Tween 20 and 5% normal goat serum (NGS). All
antibodies were diluted in PBS containing 0.5% Tween 20 and 5% normal
goat serum (see Table 2); sections were coverslipped with parafilm and
incubated overnight at 4°C in a humid chamber. The sections were
rinsed for 20 min in PBS, incubated in goat anti-mouse IgG (or goat
anti-rabbit IgG; 1:200 dilution) for 1.5 hr, rinsed for 20 min in PBS,
and then coverslipped using a glycerol/PBS mix at 1:1 ratio. Secondary
antibodies were Cy3-conjugated goat anti-mouse IgG and
Cy2-conjugated goat anti-rat IgG (Jackson ImmunoResearch, West
Grove, PA). Control sections were incubated without primary antibody and used to determine the level of nonspecific staining. A
Leitz DMRB microscope was used to view the immunofluorescent sections using rhodamine or fluorescein filters.
Tissue processed for immunohistochemistry with the anti-tyrosine
hydroxylase antibody was frozen under powdered dry ice, and 30-µm-thick sections were cut into PBS with a sliding microtome. Some
sections were stored at 20°C in an antifreeze solution [10% (w/v), polyvinylpyrrolidone 30% (w/v) sucrose, 10% (w/v)
ethylene glycol, and 50 mM sodium phosphate, pH 7.4].
Before staining, sections were rinsed in 50 mM Tris, pH
7.4, 1.5% NaCl (TBS), and permeabilized by incubation in 0.3% Triton
X-100 in TBS for 20-30 min at room temperature with gentle agitation.
Sections were then blocked by incubation in 5% NGS in TBS for 30 min
at room temperature. The monoclonal antibody against TH was diluted in
1% NGS in TBS 1:10,000 (Pel-Freez Biologicals, Browndeer, WI), and
sections were incubated overnight at 4°C. Sections were then
processed with the Vectastain Elite avidin-biotinylated peroxidase
reagents (Vector Laboratories, Burlingame, CA). Sections were incubated for 1 hr with biotinylated goat anti-mouse secondary antibody (1:200)
in 1% NGS in TBS at room temperature, washed for 10 min in 1% NGS in
TBS, and then washed three times for 7 min each in TBS alone. Then they
were incubated with avidin-biotinyl peroxidase complex (1:50 dilution
of each reagent in TBS) for 1 hr at room temperature. After being
washed three times for 10 min each in TBS, sections were developed
using 0.5 µg/ml diaminobenzidine and 0.1%
H2O2 in TBS. Sections were
rinsed, mounted on glass slides, dehydrated in graded ethanol
solutions, cleared with xylene, and coverslipped.
Golgi-Cox. Animals were asphyxiated by carbon dioxide, and
their brains were removed and immersed in fixative (10 mg/ml potassium dichromate, 10 mg/ml mercuric chloride, and 4.5 mg/ml potassium chromate; precipitate was cleared with 0.1N HCl). Brains were stored in
the dark, undisturbed for 6-8 weeks. The tissue was dehydrated first
in equal parts of acetone and alcohol for 24 hr at room temperature.
This solution was replaced with ether alcohol (anhydrous ether/ethyl
alcohol, 1:1) for 24 hr at room temperature. The brains were then
infiltrated with low-viscosity nitrocellulose (LVN) celloidin/parlodion
(Fisher Scientific, Pittsburg, PA) at concentrations of 5% (2 d), 10% (1 d), and 12% (5 d). Brains were embedded in fresh 12% LVN
and hardened with chloroform vapors overnight. The blocks were stored
in 70% ethanol until being cut with a sliding microtome.
Sections (100 µm) were hydrated in distilled water for 15 min and
developed in 5% sodium sulfite for 20 min. Sections were then
dehydrated in 95% ethanol for 1 hr, cleared in terpineol overnight,
mounted on slides, and coverslipped with Permount.
Genetic mapping. To localize the rocker mutation
to a specific mouse chromosomal region, we began genetic mapping using
differences in simple sequence length polymorphisms (SSLP). We selected
MIT SSLP markers that amplified PCR products of different sizes
in the C3H/HeJ and C57BL/6J strains. We began by selecting markers that
allowed us to scan the entire mouse genome at a resolution of 20 centimorgans (cM). Homozygous rocker mutants on a C57BL/6J genetic background were crossed to C3H/HeJ mice producing
(C57BL/6JxC3H/HeJ rkr/+) F1s. These were then backcrossed to
homozygous C57BL/6J-rkr/rkr animals. To test for linkage to
different chromosomal regions, we used a pooled PCR method (Pacek et
al., 1993). Pools of DNA aliquots were made such that each pool
consisted of equal amounts of DNA from each of 30 ataxic
(rkr/rkr) or normally behaving (+/rkr) animals. The neurological phenotype of each mouse was determined by
observation of abnormal gait at 3-4 weeks of age. Tail biopsies were
taken and digested with 0.4 µg/µl proteinase K. Digests were spun
at 13,000 × g for 5 min at room temperature. Either
PCR was performed directly on diluted proteinase K supernatant (1:50) from individual animals, or DNA was extracted using phenol/chloroform (1:1) followed by ethanol precipitation. Genotyping was done on pooled samples containing equal amounts of DNA (1 µg per animal) from
each mouse (30 animals total). Samples from ataxic (rkr/rkr) animals were pooled separately from samples of normally behaving (+/rkr) backcross littermates. Aliquots of the pools were
then assayed by PCR for each of the selected SSLP loci. Primers for these loci were obtained from Research Genetics. PCR amplification was
done on an MJ Research (Watertown, MA) thermal cycler in a total
volume of 50 µl containing 100 ng of mouse genomic DNA. Amplification
conditions were as follows: denaturation at 94°C for 1 min, followed
by 30-35 cycles of (1) 95°C for 30 sec, (2) 55°C for 30 sec, and
(3) 72°C for 30 sec. In this paradigm, if a given SSLP is unlinked to
rocker, the ataxic and wild-type pools should each amplify
two bands (C3H/HeJ and C57BL/6J) of intensity ratio 1:3. By contrast,
if the SSLP locus is linked to rocker, then the ataxic pool
should predominantly amplify the C57BL/6J band, whereas the wild-type
pool should amplify the two bands in a ratio closer to 1:1. An example
of each result is shown in Figure 5A.
Electrocorticographic recordings. Silver wire electrodes
(0.005 inch diameter) soldered to a microminiature connector
were implanted bilaterally into the subdural space over the frontal and
parietal cortex of anesthetized mice several days before recording (Noebels and Sidman, 1979). Cortical activity was recorded using a
digital electroencephalograph (TECA) from mutants and controls moving freely in the test cage for prolonged periods. Seizure behavior
was observed directly and annotated on all recordings.
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RESULTS |
The neurological mouse mutant was initially observed in
descendents of an ENU-mutagenized male mouse. The ataxic phenotype proved to be heritable as a Mendelian autosomal recessive trait and was
given the provisional gene name rocker (gene symbol
rkr). Rocker mutants display an ataxic gait
initiated by an action tremor at 3-4 weeks of age, suggesting a
possible cerebellar dysfunction. Affected animals are first
recognizable by a splayed stance of their hindlimbs and their clumsy,
uncoordinated behavior, especially on uneven surfaces. Despite the
motor problems, they still exhibit exploratory behavior and are capable
of quick movements. The rocker mutant mouse is able to swim
and manages to keep its head above the surface. No paroxysmal
dyskinesia phenotype has been observed in the rocker
homozygotes. At weaning age, there may be a slight decrease in size of
the affected animals compared with their normal littermates, but the
homozygous mutants of both sexes are fertile and appear to have a
normal life span.
On initial analysis of the rocker brain [postnatal day (P)
60-120], the cytoarchitecture appeared normal. Given the spectrum of
the behavioral symptoms, we extended our examination of the cerebellum.
We found a normal cerebellar morphology and cytoarchitecture in
rocker homozygotes (Fig.
1B) compared with wild
type (Fig. 1A). Measurements of the molecular layer
width and total area revealed no significant difference between
affected and nonaffected littermates. We performed Purkinje cell counts
to investigate whether there was any loss of Purkinje neurons in
1-year-old animals (Fig. 2). No
significant difference was seen between the number of Purkinje cells in
rocker versus wild-type cerebella. To investigate the
possible regional difference, our cell counts were also analyzed by
folia. No Purkinje cell decrease was detected in any of the individual
folia or in the anterior portion of the cerebellum (data not shown). To
extend this structural analysis, rocker brains were
immunostained with antibodies against a number of different neuronal
and non-neuronal antigens. No abnormal staining pattern was observed in
rocker cerebellum compared with wild-type animals using
antibodies against either neuronal antigens (NeuN, NF160, and TrkB) or
non-neuronal antigens (MBP, GFAP, and SV2). These antibodies are
summarized in Table 2. Consistent with
the normal cell counts, calbindin immunohistochemistry showed no gaps
in the Purkinje cell monolayer.
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Figure 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.
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Figure 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.
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In contrast to the near absence of phenotype in the immunocytochemical
studies, Golgi-Cox staining revealed dendritic structural abnormalities
in the cerebellar Purkinje cells. During normal aging, the wild-type
controls in our study underwent a slight reduction in the branching
complexity of the Purkinje cell dendritic arbor (Fig.
3A,B).
By contrast, age-matched rocker animals aged  1 year
displayed a Purkinje cell arbor that was significantly reduced in
dendritic mass (Fig. 3C,D). In addition to the
overall reduction in the size and complexity of the dendritic arbor, we observed what appeared to be an abnormal secondary outgrowth of the
tips of the distal dendrites (Fig. 3D, arrow).
The ends of the secondary branches appeared to extend, often turning
and growing back toward the cell body. This resulted in a bent or
serpentine-like appearance at the ends of the dendrites, and these
extensions appeared to have grown without the usual branching that is
found during normal dendritic development. The shafts of main Purkinje dendrites also showed a thickening in the upper molecular layer. We
examined the hippocampus and the cerebral cortex and found that in
cresyl violet-stained sections as well as Golgi impregnations, both
areas were indistinguishable from wild-type animals (Figs. 1A,B,
4A,B).
The dendritic changes illustrated in the Purkinje cells are atrophic in
nature because the Purkinje cell dendrites in rocker animals
examined at 6 months of age exhibit a normal appearance (data not
shown).
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Figure 3.
Golgi impregnation of individual cerebellar
Purkinje cells shows dendritic abnormalities in the adult
rocker 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 rocker
mutants 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.
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Figure 4.
Golgi preparations were used to examine other
areas of the brain known to express the calcium channel 1a, such as
the cerebral cortex. No distinguishable differences are evident between
wild-type (A) and rocker
(B) mutants. Scale bar, 50 µm.
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Heritable changes in descendents of ENU-mutagenized males are usually
assumed to result from a point mutation. The offspring of the male that
produced the rocker mutation included a new myotonia allele
named Clcn1adr-K (Neumann and Weber, 1989;
Gronemeier et al., 1994). We eliminated this additional mutation as the
source of the ataxic phenotype by complementation studies that showed
no allelism between Clcn1adr-K and
rocker. Furthermore, sequence analysis of rocker
muscle RT-PCR products confirmed that rocker was a different
mutation because it did not have the single amino acid change, I553T,
observed in the chloride channel mutant,
Clcn1adr-K (data not shown).
To localize the rocker mutation to a specific mouse
chromosomal region, we began genetic mapping using differences in
SSLP. Substantial linkage disequilibrium was found in the
amplification products of the D8Mit162 primer pair (Fig.
5A). Analysis of other markers
in this region confirmed this observation, and thus we were able to
localize rocker to mouse chromosome 8. An expanded series of
MIT markers was then analyzed in DNA from 107 individual progeny (Fig.
5B). This allowed us to narrow the location of
rocker to a 2 cM region between D8Mit162 and
D8Mit45. Because of its location and mutant phenotypes,
Cacna1a emerged as a significant candidate gene (Fig.
5B). To test for complementation, mating pairs were set up
between heterozygous leaner animals
(Cacna1a+/la) (The Jackson Laboratory, Bar
Harbor, ME) and heterozygous rocker animals. The
leaner mutation is maintained at The Jackson Laboratory in
repulsion with the tightly linked semidominant mutation
oligosyndactylism (Os) (Sidman et al., 1965).
Heterozygous Os induces fusion of the second and third digit
on all four paws; homozygous Os/Os is lethal early in
embryogenesis. Therefore, only the progeny of Os+/+la × Os+/+la with normal paws are homozygous for
leaner. In a cross of heterozygous rocker and
heterozygous leaner, 50% of the pups would be expected to
have Os, and 50% would have normal paws. Of the
normal-pawed animals, half would be expected to have an affected
phenotype if there were no complementation. Of 65 progeny examined from
this mating, 16 ataxic animals were identified, all with normal paws.
The mating of heterozygous rocker and heterozygous leaner mice led to the production of offspring, one-fourth
of whom were ataxic. This is consistent with the assumption that the
rocker and leaner mutations are allelic.
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Figure 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. The
rocker 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.
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The rocker mutation was assumed to be a small missense
mutation consistent with its appearance in an ENU mutagenesis
experiment. The sequence change was identified by automated sequencing
of the mouse 1a subunit cDNA from the rocker mouse brain.
This analysis revealed several single-nucleotide polymorphisms that
distinguish C57BL/6J and C3H/HeJ (the current and original background
strains, respectively). Only one of these was unique to
rocker, a C-to-A change at nucleotide residue 3929 (Fig.
6A). Wild-type animals from both parental strains (C57BL/6J and C3H/H3J) that were used in the
outcross have C at position 3929. Both types of splice variation,
1A-a and 1A-b, were found at the three different sites (Bourinet
et al., 1999) in the isolated cDNA fragments, revealing no specific
link between particular splice variants and C3929A substitution.
Together with the failure of genetic complementation, this sequence
change firmly establishes rocker as a new Cacna1a
allele, Cacna1arkr.
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Figure 6.
Determination of rocker mutation.
A, The sequence alteration in the rocker
mutant. 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 1A subunit and positions of
tottering (tg), rolling (rol), leaner
(la), and rocker (rkr) (indicated
by arrows).
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This mutation predicts a nonconservative amino acid substitution,
changing threonine (T) to lysine (K) at position 1310. T1310 is
nine amino acids into the extracellular, pore-forming loop that
connects segments 5 and 6 of repeat III (Fig.
6A,B). This amino acid change
results in the uncharacteristic arrangement of a charged polar side
chain located on the extracellular membrane near the region that is
thought to form the ion pore that passes calcium ions through the cell membrane.
The identification of rocker as a Cacna1a allele
prompted us to reexamine several phenotypes of the new mutation.
Because of previous reports of axonal swellings in the Purkinje cells of Cacna1a mutants (Rhyu et al., 1999a,b), we carefully
examined the rocker mice. No swellings of the Purkinje cell
axons were observed in the internal granule cell layer of either the
wild-type or the rocker mutants (Fig.
7A,C).
The shafts of the Purkinje cell dendrite in tottering
animals have been shown to bear ectopic spines. Despite the unusual
structural features of the rocker dendrite, no ectopic
spines were observed on either the primary or the distal dendritic
branches (Figs. 7D, 3D,
respectively).
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Figure 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 or
rocker 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 rocker
animals. Scale bars: shown in A for A and
C, 25 µm; shown in B for
B and D, 25 µm.
|
|
Previous studies of tottering and leaner mutant
mice have shown abnormalities in the temporal regulation of TH
expression in posterior vermal cerebellar Purkinje cells. Wild-type
animals show a transient Purkinje cell expression of TH that disappears by P40 (Fig.
8A,B).
By contrast, in tottering and leaner mutants, this transient expression persists into adulthood. We examined normal
and rocker animals at 3 months of age and at P40. To our surprise, we observed no Purkinje cell expression of TH at these ages
in the rocker mutant (Fig. 8C,D). The
lack of persistence of TH staining marks a significant variance between
rocker and the previously studied alleles at the
Cacna1a locus.
View larger version (96K):
[in this window]
[in a new window]
|
Figure 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 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 for
B, D, and F, 50 µm.
PCL, Purkinje cell layer; ML, molecular
cell layer; IGL, internal granule cell layer.
|
|
Another irregularity associated with tottering mice is the
presence of a characteristic pattern of EEG abnormalities that coincide
with absence seizures. To determine whether rocker mice had
the spike-and-wave patterns, we performed chronic monitoring of
electroencephalograph activity from adult rocker
homozygotes. These studies revealed the spontaneous appearance of
bilaterally synchronous and symmetrical 6-7 Hz spike-wave discharges
while the mouse was awake. These episodes occurred at a mean frequency range of 41-65 episodes per hour and a mean burst duration of 1-1.7
sec (Fig. 9). Behavioral arrest and, on
occasion, myoclonic movements of the vibrissae or neck always
accompanied spike-wave episodes. Normal motor behavior resumed
immediately on termination of the cortical discharge. The general
morphology and temporal parameters of the cortical burst activity and
behavioral arrest seizures are essentially identical to those recorded
from tottering mutants.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 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.
|
|
The Cacna1a gene product forms a membrane pore without
multimerization (unlike the entire family of potassium channels that function as tetramers). The rocker/leaner compound
heterozygote (Cacna1arkr/la)
offers an unusual opportunity to study the effects of co-expression of
two different mutant channels in a single neuronal membrane. Depending
on the phenotype, the compound heterozygote might be expected to show
either additive effects or dominance of one or the other allele. The
severity of the ataxia appeared to be intermediate between that of the
leaner and rocker homozygotes (as is reported for
the tottering/leaner compound heterozygote). Thus the ataxia phenotype is additive. By contrast, the leaner allele is
dominant over rocker in the expression of the dystonic
movement phenotype. Although rocker does not have
dyskinesia, both the compound heterozygote (Cacna1arkr/la) and leaner
mouse do. Rocker is dominant over leaner with
respect to the phenotype of cell loss because no decrement in Purkinje cell counts is observed in the cerebellar cortex of the compound heterozygote (Fig. 1C) (cell counts not shown). In the
phenotype of TH persistence, leaner is dominant over
rocker; posterior Purkinje cells of the compound
heterozygote retained significant TH levels in their cytoplasm well
into adulthood (Fig. 8E,F).
Finally, Golgi analysis of the compound heterozygote revealed ectopic
spines appearing on secondary dendrites. This is a
leaner-like phenotype, illustrating dominance of the
leaner allele. No axonal swellings were observed (data not shown).
| |
DISCUSSION |
In this study, we identified the mouse mutant rocker
(rkr) as a new allele of the calcium channel 1A subunit
gene, Cacna1arkr, using mapping results,
the failure of complementation with leaner animals, and
sequencing to identify a point mutation that occurs only in ataxic
animals. Rocker is the fifth mutant mouse Cacna1a allele in an allelomorphic series in which each mutation presents a
distinct phenotypic syndrome.
Comparison with other alleles of mouse Cacna1a
Overall, the phenotype of this new mutation bears the greatest
similarity to the original tottering mutation.
Rocker mutant mice display an ataxic gait initiated by an
action tremor. The age of onset of the rocker behavioral
abnormalities (P21-28) is approximately the same as
tottering. Analysis of the overall cytoarchitecture of the
adult rocker brain (P60-120) reveals no gross cytological abnormalities. We observed no reduction in the size of any of the three
layers of cerebellar cortex of the rocker cerebellum, and we
found no evidence of cell death (pyknotic cell bodies) in the molecular
or internal granule cell layers of rocker mutants. In
1-year-old animals, there is no detectable decrease in the number of
rocker Purkinje cells (Fig. 2). This plus the absence of
major alterations in cerebellar morphology (Fig. 1B)
also identify rocker as similar to the tottering
brain, as opposed to leaner or rolling in which
significant cell loss is reported (Muramoto et al., 1981; Herrup and
Wilczynski, 1982). Furthermore, electroencephalographic recordings
reveal frequent, spontaneous seizures when generalized, bilateral
spike-and-wave discharges are observed, similar to those found in
tottering. The point mutation identified in
rocker also appears similar to tottering in its
structural location on the channel. Both mutations are extracellular
and located near the pore-forming region of the channel (Fig.
6B).
Although our initial observations suggest a close similarity to the
tottering phenotype, further examination of
rocker reveals interesting and informative differences. In
other Cacna1a alleles, an abnormal persistence of TH gene
expression into adulthood has been reported in posterior/medial
cerebellar Purkinje cells (Muramoto et al., 1981; Hess and Wilson,
1991; Austin et al., 1992; Sawada et al., 1999). In rocker,
however, TH expression is downregulated on a time course identical to
wild type. Although an occasional Purkinje cell was positive in
rocker (Fig. 8C, arrow), similar observations have been reported in wild-type animals (Austin et al.,
1992; Fujii et al., 1994; Abbott et al., 1996; Sawada et al., 1999).
Previous explanations for the persistence of TH expression took note of
the known effect of calcium on the regulation of immediate early gene
expression (Ghosh et al., 1994) and the responsiveness of the TH
promoter to c-fos, calcium, and neuronal activity
(Gizang-Ginsberg and Ziff, 1990; Kilbourne et al., 1992; Sawada et al.,
1999). The existence of TH-positive Purkinje cells in other ataxic
mutant mice has suggested that abnormal expression of TH in Purkinje cells may not be specific to the Cacna1a allelic group
(Muramoto et al., 1981; Sawada et al., 1999) and instead may be
indicative of a developmental arrest or a more general predictor of
neuronal dysfunction caused by alterations in cellular
Ca2+ currents. One possibility is that a
certain magnitude of change (or absolute concentration) of
Ca2+ must be achieved to initiate the
downregulation of TH. Electrophysiological examination of the calcium
flux in rocker Purkinje cells might help evaluate the
validity of this hypothesis. The lack of TH expression in
rocker, even in the presence of absence seizures, allows us
to rule out the spike-and-wave discharges as sufficient to result in
the sustained TH expression, and vice versa. Furthermore, unlike the
tottering phenotype, rocker animals display no
evidence of motor seizures. These important clinical distinctions will allow rocker to serve as a useful model to assist in the
determination of the different molecular or cellular mechanisms
responsible for each of these phenotypes.
Golgi-Cox staining reveals dendritic abnormalities in the aged
rocker cerebellar cortex. Our analysis of adult
rocker animals (>1 year of age) reveals a reduction of
branching in the Purkinje cell dendritic arbor and a weeping willow
appearance of the secondary branches. Age-matched wild-type controls
show no similar reduction in the Purkinje cell dendritic arbor. Our
examination of younger rocker animals revealed that the
Purkinje cell initially develops a fully branched dendritic arbor,
which then regresses. Although Golgi-Cox analysis has been done for the
other alleles at the Cacna1a locus, aged animals have not
been examined. Other mouse mutants, such as weaver and
reeler, have displayed cerebellar Purkinje cells with a
weeping dendritic phenotype, as have irradiation- or toxin-induced
agranular models. These insults were applied during cerebellar
development, however, and have been attributed to a disruption in the
afferent input and the formation, rather than the maintenance, of the
dendritic tree.
Unlike the other known alleles, rocker mutants do not have
ectopic Purkinje cell spines (Figs. 3D, 7D).
Recent analysis by Rhyu and colleagues (1999a,b) has shown ectopic
spines on the secondary branches of Purkinje cells in the
tottering, rolling, and leaner animals. Although
we see a dramatic reduction in the number of tertiary branches in the
adult rocker, no aberrant sprouting of spines is seen on the
primary dendrites. The structural changes seen in tottering
and leaner and the absence of ectopic spines in
rocker suggest that the calcium channel defect in
rocker may be sufficient to sustain initial dendritic
development but may be less efficient in the processes needed to
maintain dendritic structure. Whether this is a direct effect of
Ca2+ changes on the biochemistry of
dendritic maintenance or a secondary effect of altered synaptic
function remains to be determined.
The compound heterozygote
(Cacna1arkr/la)
Additional insights into the function of the Cacna1a
gene can be found in the phenotype of the rocker/leaner
compound heterozygote (Cacna1arkr/la). The severity
of the motor phenotype of the compound heterozygote is intermediate
between the rocker and leaner phenotype and is first observed at P15-20. Although previous studies in
leaner animals have shown a continuous cell loss of both
Purkinje and granule cells in the cerebellum beginning at about P30, we
observed no such loss in compound heterozygotes (data not shown)
(Herrup and Wilczynski, 1982; Heckroth and Abbott, 1994).
The rocker/leaner compound heterozygous mice maintained the
expression of TH in the cerebellar Purkinje cells although heterozygous leaner/wild-type mice have a wild-type phenotype (Fig.
8E,F). This suggests that
the net disruption in either rocker or heterozygous leaner animals is inadequate to produce a persistent TH
phenotype, whereas the combination of leaner and
rocker is sufficient. The dominance of leaner
over rocker, but wild type over leaner suggests a
threshold model in which a certain absolute level of
Ca2+ physiology disruption is required to
block the regression of TH staining. Leaner channels in the
presence of wild type are sufficiently normal to trigger a regression
but replace the wild-type channel with a rocker form, and
there is enough imbalance to block regression. Preliminary
studies of other phenotypes in the compound heterozygous mice also
produce differences in dominance of the alleles. The presence of
ectopic spines, as in leaner animals, and the lack of axonal
swellings, as in rocker animals, suggest that further
examination of the compound heterozygote will yield insights into
allelic interactions. It would also be of interest to examine these
phenotypes in a rocker/tottering compound heterozygote.
The contribution of VDCCs to neuronal function in the intact animal is
clearly complex. The many mutants that disrupt these channels not only
illustrate the importance of the VDCCs but also emphasize the value of
the allelic series of mutations in a channel gene. Structural analysis
of the different mutations will lead to a better understanding of the
different mechanisms responsible for the diversity of resulting
phenotypes. Identification of this allelic series comes at an opportune
time to complement the rapid advances being made in the biophysics of
voltage-gated calcium channels. Mutations that have different
phenotypes will allow us to separate the molecular and cellular
pathways responsible for these phenotypes. Rocker is of
increased value not for its similarity to the other alleles in this
series, but for the distinct differences it introduces.
Rocker is the first allele to present absence seizures
without the intermittent movement disorder. It is also the first to
have no sustained expression of TH in the Purkinje cells, and it is the
only allele to have no ectopic spines. Finally, it is the first
reported Cacna1a allele with structural abnormalities in the
mature Purkinje cell dendrite.
Multiple systems in the CNS develop abnormalities in both the murine
and human hereditary diseases at this locus. Previous studies that
compared properties of the native and recombinant mutant channels
suggest that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior that lead to neuronal death
and cerebellar atrophy (Wakamori et al., 1998; Qian and Noebels, 2000).
The phenotype of rocker mice emphasizes how many of these
traits are affected differently by the various alleles.
| |
FOOTNOTES |
Received June 27, 2000; revised Nov. 21, 2000; accepted Dec. 7, 2000.
This work was supported by National Institutes of Health Grant
NS20591 (K.H.), Medical Research Council Grant MT-15507 (P.E.N.), and
National Institutes of Health Grant MH61092 (J.L.N.) We thank Caleb
Davis for technical assistance and extend special thanks to Dr.
Aravinda Chakravarti for his gracious persistence in obtaining cDNA
sequence from the entire rocker gene.
Correspondence should be sent to Dr. Karl Herrup, Alzheimer Research
Laboratory, Case Western Reserve University, E 504 School of Medicine,
10900 Euclid Avenue, Cleveland, OH 44106. E-mail: kxh26{at}po.cwru.edu.
| |
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ABSTRACT
INTRODUCTION
