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Vol. 55, Issue 1, 23-31, January 1999
Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey, Pennsylvania
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Tottering mice inherit a recessive mutation of the calcium channel
1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel 1A
subunit gene encodes the pore-forming protein of P/Q-type
voltage-dependent calcium channels and is predominantly expressed in
cerebellar granule and Purkinje neurons with moderate expression in
hippocampus and inferior colliculus. Because calcium misregulation
likely underlies the tottering mouse phenotype, calcium channel
blockers were tested for their ability to block the motor episodes.
Pharmacologic agents that specifically block L-type
voltage-dependent calcium channels, but not P/Q-type calcium
channels, prevented the inducible dystonia of tottering mutant mice.
Specifically, the dihydropyridines nimodipine, nifedipine, and
nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine
verapamil all prevented restraint-induced tottering mouse motor
episodes. Conversely, the L-type calcium channel agonist
Bay K8644 induced stereotypic tottering mouse dystonic at
concentrations significantly below those required to induce seizures in
control mice. In situ hybridization demonstrated that
L-type calcium channel 1C subunit mRNA
expression was up-regulated in the Purkinje cells of tottering mice.
Radioligand binding with [3H]nitrendipine also revealed a
significant increase in the density of L-type calcium
channels in tottering mouse cerebellum. These data suggest that
although a P/Q-type calcium channel mutation is the primary defect in
tottering mice, L-type calcium channels may contribute to
the generation of the intermittent dystonia observed in these mice. The
susceptibility of L-type calcium channels to
voltage-dependent facilitation may promote this abnormal motor phenotype.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Neurological mutants of the mouse are important tools for studying central nervous system development and provide models of human disease. For many of these mutants, the mutation has been identified but the mechanism by which the mutation causes the abnormal phenotype is unclear. Defining the cellular and neural systems affected by the mutation is necessary for understanding the mutant phenotype in the mouse and for applying the model to human disease states.
The neurological mouse mutation tottering (gene symbol: tg)
causes spike and wave discharges (Noebels and Sidman, 1979
), ataxia, and abnormal motor episodes characteristic of intermittent dystonia (Green and Sidman, 1962). The most extensively studied of these phenotypes is the spike and wave discharge, nonconvulsive bilaterally synchronous epileptic bursts recorded by electroencephalogram. This
phenotype is associated with hyperarborization of locus ceruleus axons
in tottering mutants (Levitt and Noebels, 1981) as selective lesion of
these axons with 6-hydroxydopamine dramatically reduced the spike and
wave discharges (Noebels, 1984). By contrast, the intermittent dystonic
episodes of tottering mice are neither associated with an abnormal
electroencephalographic pattern (Kaplan et al., 1979; Noebels and
Sidman, 1979) nor prevented by 6-hydroxydopamine lesion (Noebels,
1984). Although this abnormal motor phenotype of tottering mice is the
most behaviorally obvious, the intermittent dystonic episodes have not
been well characterized.
Tottering dystonic episodes are highly stereotyped, with the first
observable characteristic being extension of the hindlimbs. This
initial phase is followed by abduction at the hip and extension at the
knee, ankle, and paw with a stiffly arched back that presses the
perineum against the cage bottom. The motor dysfunction then spreads to
involve the forelimbs and head, with severe flexion of the neck. In the
final phase, the mice regain control of the hindlimbs, often rearing,
while forepaw and facial muscles continue contractions (Green and
Sidman, 1962). The entire episode lasts 30 to 60 min without loss of consciousness.
The molecular basis of the tottering mouse phenotypes was identified
recently as a point mutation within the voltage-dependent calcium
channel 1A subunit (Fletcher et al., 1996).
Simultaneous with the identification of the tottering gene defect, the
mutations underlying the human disorders familial hemiplegic migraine
and episodic ataxia type-2 were identified as mutations in the human voltage-dependent calcium channel 1A subunit
(Ophoff et al., 1996). Voltage-dependent calcium channels consist of
four subunits: 1, 2,
, and 
. The 1 subunit is the
pore-forming protein, conferring ion selectivity and voltage
sensitivity; the other subunits influence the kinetic properties of the
channel. Six types of voltage-dependent calcium channels have been
distinguished by electrophysiology and pharmacology: L, P, Q, N, R, and
T; a multiplicity of genes and alternative splice sites encode the 1 subunits of the functionally defined
channels. All the voltage-dependent calcium channel
1 subunits have a similar membrane topography with four repetitive domains (I-IV), each consisting of six
transmembrane segments (S1-S6) (for reviews see Catterall, 1995;
Wheeler et al., 1995). The 1A subunit gene
encodes P- and/or Q-type calcium channels, which are distinguished from
other types of calcium channels by their sensitivity to blockade by
-agatoxin-IVA and -agatoxin-TK. The tottering
missense mutation results in a proline-to-leucine substitution at amino
acid 601, within the extracellular loop between S5 and S6 in repetitive
domain II of the calcium channel (Fletcher et al., 1996). Although the
functional significance of this mutation is not yet known, the region
in which the mutation lies is thought to be important in ion
selectivity (Catterall, 1995).
Calcium misregulation resulting from the calcium channel mutation is a
likely cellular mechanism underlying the abnormal phenotypes of
tottering mice. Therefore, calcium channel blockers were tested for
their ability to block the intermittent dystonic episodes in tottering
mice. The only specific P/Q-type calcium channel antagonists currently
available are the spider toxins -agatoxin IVA and -agatoxin TK,
peptides with 71% identity at the amino acid level (Mintz et al.,
1992; Teramoto et al., 1993). These peptides have been studied
extensively in vitro but are not commonly used in vivo as they do not
cross the blood-brain barrier. In contrast, several L-type
calcium channel blockers readily cross the blood-brain barrier to exert
central effects. Dihydropyridines (e.g., nimodipine, nifedipine,
nitrendipine), benzothiazepines (e.g., diltiazem), and
phenylalkylamines (e.g., verapamil) have been demonstrated to protect
against electrically and chemically induced seizures (Larkin et al.,
1992; Palmer et al., 1993; Wurpel and Iyer, 1994). Therefore
L-type calcium channel blockers were tested for their
ability to prevent dystonia in tottering mutant mice.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Mouse Mutants.
C57BL/6J-+/tg mice were
obtained from The Jackson Laboratories and bred at the Pennsylvania
State University College of Medicine to produce tottering mutants.
Adult tottering mutant mice (2-12 months of age) were identified
either by analysis of polymerase chain reaction (PCR) amplification of
tightly linked simple-sequence-length polymorphisms in
C57BL/6J-+/tg x C57BL/6J-+/tg cross
progeny (Campbell and Hess, 1996, 1997) or by absence of
oligosyndactylism in Os +/+ tg x
Os +/+ tg cross-progeny (Campbell and
Hess, 1998). Genotype was confirmed by observation of stereotyped
dystonic episodes. Age- and gender-matched C57BL/6J-+/+ mice were used
as controls where appropriate. All animals were drug-naive at the
beginning of the experiments. All animal procedures were in accordance
with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
In Situ Hybridization.
Tottering mutant
(tg/tg), heterozygous (+/tg), and normal
(+/+) gender-matched littermates (2-3 months of age) were deeply anesthetized with carbon dioxide and sacrificed by decapitation. The
brains were rapidly removed, frozen in isopentane at 
20 to 40°C,
and stored at 70°C. Twenty-micrometer sagittal sections were
cut using a cryostat and thaw mounted on Superfrost Plus glass slides
(Fisher) with each slide bearing control and mutant sections. After
drying, the slide-mounted sections were again stored at 70°C until
use in in situ hybridization experiments.
1A
subunit gene, but not other calcium channel 1
subunit genes, was generated by reverse transcription-PCR. A 554-bp DNA
segment corresponding to base pairs 5736 to 6289 of the mouse calcium
channel 1A subunit cDNA (GenBank U76716) was
amplified and inserted into pBluescript II SK(). A 418-bp
EcoRI fragment corresponding to base pairs 8596 to 9014 of
the 1C subunit 3' untranslated region was
subcloned into pBluescript II SK(); this sequence is unique to the
1C subunit; the original clone was obtained
from the I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory,
Livermore, CA) cDNA clone ID#AA462894 (Lennon et al., 1996). The
1D subunit cDNA (residues 2803-3246)
subcloned in pGEM was a generous gift from Dr. Hemin Chin (National
Institutes of Health). In vitro transcription was performed at 37°C
for 2 h in a 25-µl volume containing 40 mM Tris, pH 7.9; 6 mM
MgCl2; 2 mM dithiothreitol (DTT); 40 U of RNase
inhibitor (Promega, Madison, WI); 400 µM each of ATP, GTP, and UTP;
10 µM [35S]CTP (800 Ci/mmol, Amersham Corp.,
Arlington Heights, IL); 1 µg of linearized plasmid; and 20 U of RNA
polymerase (Promega). After transcription, the DNA template was removed
by digestion with RNase-free DNase (Promega Biotec) for 30 min at
37°C, and the transcripts were size reduced to 150 to 250 nucleotides
by alkalai treatment with 0.2 N NaOH for 45 min on ice. The probes then
were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and
the unincorporated nucleotides were removed by fractionation on a G50
Sephadex Nick column (Pharmacia, Piscataway, NJ).
Pretreatment of the slide-mounted sections included fixation in
buffered 4% formaldehyde for 15 min at room temperature followed by a
5-min rinse in 0.1 M phosphate-buffered saline. Slides then were
treated with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl/0.15 M NaCl (pH 8.0) for 10 min and rinsed in 2× standard saline citrate (SSC; 0.15 M NaCl, 0.015 M sodium citrate). Sections then were dehydrated in graded ethanols followed by two 5-min incubations in
chloroform. One-minute incubations in 100 and 95% ethanol were followed by air drying.
Slides were hybridized with 100 µl of buffer containing 7.5 ng of
cRNA probe in 50% formamide, 0.75 M NaCl, 20 mM 1,4-piperazine diethane sulfonic acid, pH 6.8, 10 mM EDTA, 10% dextran sulfate, 5×
Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 50 mM DTT, 0.2% sodium dodecyl sulfate, and 100 µg/ml each salmon sperm DNA and yeast tRNA. Slides were coverslipped, sealed with Royalbond Grip contact cement, and hybridized for 16 h at 56°C.
After hybridization, coverslips were removed in 4× SSC plus 300 mM
2-mercaptoethanol at room temperature, incubated in this solution for
15 min, and incubated in 4× SSC without 2-mercaptoethanol for 15 min
at room temperature. Slides were treated with 1:1 formamide/buffer (0.6 M NaCl, 40 mM Tris base, 2 µM EDTA, 20 mM HCl) at 60°C for 20 min
followed by a 5-min rinse in room temperature 2× SSC. Sections then
were treated with 50 µg/ml pancreatic RNase A in 0.5 M NaCl, 50 mM
Tris, pH 8.0, and 5 mM EDTA for 30 min at 37°C, washed in graded salt
solutions (2×, 1×, and 0.5× SSC each for 5 min at room temperature),
with a final wash in 0.1× SSC at 65°C for 30 min. Slides then were
cooled to room temperature in 0.1× SSC, dipped in 60% ethanol with
0.33 M ammonium acetate, and air dried. Sections were exposed to X-ray
film (DuPont Cronex) for 8 to 96 h and subsequently dipped in
Kodak NTB-2 photographic emulsion (diluted 1:2 with
dH2O), exposed at 4°C, and developed in Kodak D-19 developer.
Quantitative analyses were performed using NIH Image software on
captured darkfield images of the emulsion-dipped sections. Images of
representative areas from each brain were captured at 40×
magnification. Silver grain densities in the Purkinje cell were
determined in boxes of 900 µm2; cerebellar
granule cells were assessed in 8100-µm2 boxes;
nucleus of the brachium of the inferior colliculus and thalamic nuclei
were assessed in 112,000-µm2 boxes; all other
regions were assessed in 225,000-µm2 boxes.
Data were analyzed by two-way analysis of variance (ANOVA) or by
Student's t test where appropriate.
Induction of Dystonia in Tottering Mutant Mice. Intermittent dystonic episodes in tottering mutant mice are inducible. For all experiments assessing the effects of calcium channel blockers on tottering mouse motor episodes, adult mice were transported daily from the vivarium to the laboratory and acclimated for at least 4 h. The extensive acclimation period provided sufficient recovery time for tottering mutants, which routinely express dystonia upon transport. Mice then were injected with vehicle or drug and placed back in the home cage for 30 min. To induce dystonic episodes after drug treatment, mice were restrained in a 60-ml syringe for 10 min, released into a novel plastic cage for 30 min, then returned to the home cage. The mice were scored for the presence or absence of dystonia from the time of injection until 10 min after return to the home cage, a total of 80 min.
Intracerebroventricular Injections of L-Type Calcium Channel Blockers. At least 4 h after transport to the laboratory, tottering mutant mice were anesthetized with methoxyflurane (Pittman-Moore, Inc., Mundelein, IL). After anesthetization (3-3.5 min in methoxyflurane), a small slit was made at the base of the skull and a 27-gauge syringe needle, blunted to a length of 5 mm, was inserted at the midline between the skull and first vertebra. Saline vehicle or drug was injected directly into the fourth ventricle in a volume of 5 µl. Drug masses tested were diltiazem (20 µg) and verapamil (10 µg). All drugs were dissolved in saline. Dihydropyridines were not used i.c.v. because they are relatively insoluble; the vehicle in which dihydropyridines could be solubilized for s.c. administration caused massive intracerebral bleeding, prohibiting the use of these drugs i.c.v. Mice recovered from the anesthesia within 15 min. Thirty minutes after injection, the mice were subjected to the dystonia-induction paradigm described above. All mice were sacrificed immediately after the observation period. Data were analyzed by one-way ANOVA followed by Dunnett t test.
Subcutaneous Injections of L-Type Calcium Channel Blockers and Activators. The L-type calcium channel blockers nimodipine, nifedipine, nitrendipine, diltiazem, and verapamil were injected s.c. into tottering mutant mice in a volume of 5 ml/kg. Groups of five to six tottering mice were used for each dose-response analysis with five trials at each drug concentration. Mice were injected and challenged in the dystonia induction paradigm (see above) once daily for 5 consecutive days at each dose. The order of drug doses was randomized for each mouse. The mice were allowed 2 days of recovery between drug doses when progressing to a higher dose; 9 days elapsed before treatment with a lower dose of drug. Data were analyzed by one-way factorial ANOVA followed by Dunnett t tests comparing the response at each drug dose with vehicle.
To assay the effects of s.c. injection of the L-type calcium channel activator Bay K8644 on tottering mouse dystonia, five tottering mutant females and five age-matched C57BL/6J-+/+ control females (5-7 months of age) were transported to the laboratory daily. After a period of acclimation of at least 4 h, Bay K8644 or vehicle at a volume of 5 ml/kg was injected s.c. The mice were returned directly to the home cage and observed for 60 min with no attempts to induce the motor episodes environmentally. The mice were injected with vehicle on the first day followed by increasing doses of Bay K8644 on subsequent consecutive days. Data were analyzed by one-way factorial ANOVA followed by Dunnett t tests comparing the response at each drug concentration with vehicle.[3H]Nitrendipine-Binding Assays.
Tottering
(tg/tg) mutant mice and gender-matched normal (+/+)
controls 2 to 3 months of age were deeply anesthetized with carbon
dioxide and decapitated. The brains were removed rapidly, dissected
into cerebellum and forebrain discarding brainstem and olfactory bulbs,
and frozen at 70°C until use. Tissues were homogenized in 100 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) with a Tekmar tissue
homogenizer (Cincinnati, OH) at setting 70. The homogenates were
centrifuged at 30,000g for 10 min, and the supernatant was discarded. Pellets were resuspended in ice-cold buffer and centrifuged again at 30,000g for 10 min. Pellets were
resuspended to 25 mg wet weight tissue/ml buffer (forebrain) or 50 mg/ml buffer (cerebellum). Protein concentrations were determined with
the Pierce (Rockford, IL) BCA protein assay.
). Each
reaction contained either 5 mg/ml (cerebellum) or 2.5 mg/ml (forebrain)
tissue. Six concentrations of [3H]nitrendipine
(78.5 Ci/mmol) ranging from 20 to 1000 pM defined total binding;
nonspecific binding was determined in the presence of 1 µM
nifedipine. Reactions were terminated by rapid filtration over Whatman
GF/C filters, which were washed rapidly three times with 3 ml of
ice-cold buffer. Filters were allowed to dry and then were placed in
ScintiVerse (Fisher) scintillation fluid. Radioactivity was determined
by liquid scintillation spectroscopy at an efficiency of 35 to 40%.
Data were analyzed using GraphPad Prism 2.0 software.
Drugs. [3H]nitrendipine was purchased from NEN (Boston, MA). All L-type calcium channel blockers and activators were obtained from Research Biochemicals International (Natick, MA). Verapamil was dissolved in 0.9% saline. Nimodipine and nitrendipine were dissolved in 14.5% ethanol/4.5% Tween 80 (Sigma)/81% 0.9% saline. Nifedipine and Bay K8644 were dissolved in 24% ethanol/4% Tween 80/72% 0.9% saline.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Effect of Bay K8644 on Tottering Mouse Dystonic Episodes.
Tottering mouse dystonia and the seizures induced by the
L-type calcium channel activator Bay K8644 in normal mice
are behaviorally distinct. In contrast to the stereotypic spastic
contractions and extensions that characterize the tottering mouse
phenotype, Bay K8644-induced seizures in normal mice are characterized
by a stiff tail, arched back, squeaking, forelimb and hindlimb clonus, flexion and extension of hindlimbs, jumping, catatonia, and loss of
righting reflex (Palmer et al., 1993).
Effect of Subcutaneous Injection of L-Type Calcium Channel Blockers on Tottering Mouse Dystonic Episodes. The dihydropyridines nimodipine, nitrendipine, and nifedipine each prevented restraint-induced tottering mouse dystonia in a dose-dependent manner (Fig. 1). Nimodipine significantly reduced the occurrence of these episodes at 1, 5, 10, and 20 mg/kg and was completely protective against tottering mouse dystonia at 10 and 20 mg/kg. Nifedipine and nitrendipine each significantly prevented the expression of the movement disorder at 5, 10, and 20 mg/kg. Nitrendipine was completely protective at 20 mg/kg. Even the highest doses of dihydropyridines tested caused no observable side-effects: the mice were active and the ataxia of tottering mice was not noticeably altered.
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Effect of Intracerebroventricular Injection of L-Type Calcium Channel Blockers on Tottering Mouse Dystonic Episodes. Tottering mutant mice injected i.c.v. with saline vehicle exhibited dystonic events in 64% of trials (Table 2). The L-type calcium channel blocker diltiazem eliminated the motor episodes when injected i.c.v., similar to s.c. injection. The phenylalkylamine verapamil, which does not cross the blood-brain barrier and failed to prevent tottering mouse dystonia upon s.c. injection, significantly reduced the frequency of the episodes in tottering mutant mice to 20% when administered i.c.v. (Table 2). Thus, injection of L-type calcium channel blockers directly into the central nervous system effectively prevented the intermittent dystonic episodes exhibited by tottering mutant mice.
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L-Type Calcium Channel 1C and
1D Subunit mRNA Expression.
Consistent with
previous reports (Tanaka et al., 1995), the calcium channel
1C subunit mRNA was expressed throughout the brain
including cerebral cortex, thalamus, hippocampus, and cerebellum (Fig.
4). Quantitative analyses of grain
density in parasagittal brain sections from control (+/+) and tottering
(tg/tg) mice (n = 3 per genotype)
revealed a significant increase in the expression of calcium channel
1C subunit mRNA in Purkinje cells of the cerebellum in
tottering mice (Table 3). Quantitation
was performed on Purkinje cells from both Lobule V and Lobule VIII; in
both lobules, expression of the 1C subunit was increased
significantly in tottering mice. In fact, 1C subunit
mRNA is expressed at extremely low levels in Purkinje cells of control
mice, but expressed abundantly in Purkinje cells of tottering mice
(Fig. 4). However, 1C subunit mRNA was not increased
equally in all Purkinje cells whereby a dramatic increase in expression
was observed in some cells but little increase in expression was
observed in others (Fig. 4). In contrast, calcium channel
1C subunit mRNA expression was comparable in cerebellar
granule cells and deep cerebellar nuclei in control and tottering mice
(Table 3), indicating a cell-type-specific regulation of the
1C subunit resulting from the calcium channel 1A subunit mutation. A significant increase in
1C subunit mRNA also was observed in the nucleus of the
brachium of the inferior colliculus in tottering mice whereas thalamic
nuclei, hippocampus, and olfactory bulb expression did not
significantly differ from control mice (Table 3). Although an increase
in 1C subunit expression was observed in the cortex,
this trend did not reach significance (P < .1).
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1D subunit was expressed
throughout brain, similar to the 1C subunit,
but with abundant Purkinje cell expression observed in both control and
tottering mice (data not shown); 1D subunit
mRNA distribution was as described in previous reports (Tanaka et al.,
1995). The 1D subunit was not expressed in the
nucleus of the brachium of the inferior colliculus where a significant
increase in 1C subunit mRNA expression was
observed. Quantitative analyses of grain density in parasagittal brain
sections demonstrated that 1D subunit
expression was similar in control (+/+) and tottering
(tg/tg) mice (n = 3 per genotype; Table
4).
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P/Q-Type Calcium Channel 1A Subunit mRNA
Expression.
The calcium channel 1A subunit mRNA was
expressed in the cerebellum, inferior colliculus, and hippocampus as
described in previous reports (Tanaka et al., 1995; Fletcher et al.,
1996; data not shown). Hippocampal expression appeared uniform
throughout the dentate gyrus and the cornu ammonus subdivisions. The
calcium channel 1A subunit mRNA expression was most
abundant in cerebellar cortex with high levels in cerebellar granule
cells and Purkinje cells.
1C subunit mRNA expression was observed. Grain
densities in four to six representative high-power section images from
individual cerebellar lobules (III-X) of each genotype were analyzed.
A two-way factorial ANOVA indicated no significant differences among
the three genotypes in either the Purkinje cell (ANOVA,
P > 0.24) or granule cell (ANOVA, P > 0.86) layers. Similarly, no significant differences among lobules were
observed in Purkinje cells (ANOVA, P > 0.25) and no
genotype-by-lobule interaction was observed in either the Purkinje cell
(ANOVA, P > 0.95) or granule cell (ANOVA,
P > 0.62) layers. Although a significant effect of
lobule in the granule cell layer was suggested by the two-way ANOVA
(P < .02), a posthoc Scheffe F-test revealed no significant differences among the mean grain densities
(P > .05). Thus, the tottering mutation does not
affect expression of the calcium channel 1A
subunit mRNA within cerebellar lobules III through X.
[3H]Nitrendipine Saturation Binding.
[3H]nitrendipine saturation-binding assays were performed
in control and tottering mouse forebrain and cerebellum. Saturation analyses demonstrated specific and saturable binding of
[3H]nitrendipine to sites in mouse forebrain and
cerebellum homogenates; representative saturation-binding curves are
shown in Fig. 5. Scatchard plots of
saturation data demonstrated that [3H]nitrendipine
binding was increased in the forebrain of tg/tg mice;
however, this trend did not reach significance, likely because the
increase observed in 1C subunit mRNA expression occurred in only very limited regions of the forebrain whereas 1D
subunit mRNA expression was not altered by the mutation. In contrast, [3H]nitrendipine binding was significantly increased
(+75%) in tottering mouse cerebellum compared with control
mouse cerebellum (P < .05; Table
5); these data are consistent with the
increase in 1C subunit mRNA expression in tottering
mouse Purkinje cells. The KD values did not
differ significantly among the four tissues (Table 5). Scatchard plots
were linear, indicating a single binding site and suggesting that the
tg mutation had not created a new dihydropyridine-binding site.
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Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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These data suggest that L-type calcium channels are
up-regulated in response to the mutation in the P/Q-type calcium
channel in tottering mice. Furthermore, L-type calcium
channels appear to influence the expression of the intermittent
dystonic episodes in tottering mice. The tg mutation
directly alters the mouse calcium channel 1A
subunit gene, which encodes the pore-forming protein of P/Q-type
calcium channels. Diffusable P/Q-type calcium channel blockers
currently are unavailable, and, as such, the role of the mutant
P/Q-type calcium channel in the expression of the motor phenotype is
unclear at this time. However, pharmacologic blockade of
L-type calcium channels with several distinct
agents prevented tottering mouse dystonia. Additionally,
L-type calcium channel mRNA expression and
binding was increased in tottering mice. The increase in
L-type calcium channels coupled with the ability
of L-type channel blockers to prevent the
intermittent dystonic episodes suggest that
L-type calcium channels play a role in the
abnormal motor phenotype expressed by tottering mutant mice.
Several agents that specifically alter L-type calcium
channel activity affected the behavioral expression of the tottering mouse dystonic episodes. L-type calcium channel antagonists
of the dihydropyridine class, nimodipine, nifedipine, and nitrendipine, prevented restraint-induced dystonia in a dose-dependent manner. Diltiazem, an L-type calcium channel blocker of the
benzothiazepine class, prevented restraint-induced dystonia when
injected either s.c. or i.c.v. The dihydropyridines and diltiazem cross
the blood-brain barrier efficiently (Naito et al., 1986; Larkin et al.,
1992), and both drug types bind extracellular sites on
L-type calcium channels involving the S6 segments of
repetitive domains III and IV (Kraus et al., 1996; Peterson et al.,
1996). The binding sites appear allosterically linked but distinct
because diltiazem enhances the binding of dihydropyridines (Yamamura et
al., 1982). The phenylalkylamine verapamil also prevented dystonia when
injected i.c.v.; the phenylalkylamine-binding site on
L-type calcium channels is clearly distinct from the
dihydropyridine and benzothiazepine sites, involving an intracellular
site between S6 of repetitive domain IV and the C-terminal tail
(Striessnig et al., 1990). Thus, five different L-type
calcium channel blockers from three different drug classes all
prevented the expression of the dystonic phenotype attesting to the
specificity of the effect.
The ability of verapamil to prevent tottering mouse dystonia when
injected i.c.v. underscores the role of central nervous system
L-type calcium channel blockade in the prevention of this phenotype. The same drug injected peripherally failed to prevent dystonia in tottering mice at sublethal concentrations. Verapamil, a
potent and specific L-type calcium channel antagonist, does not cross the blood-brain barrier in appreciable amounts (Hamann et
al., 1983). The differential effects of the drug injected via two
different routes indicates that blockade of peripheral
L-type calcium channels in cardiac and skeletal muscle,
where L-type calcium channels are the predominant carriers
of calcium current, is insufficient to prevent dystonia. These data
suggest that L-type calcium channel blockers prevent the
tottering mouse motor abnormalities by acting within the central
nervous system rather than on cardiac or skeletal muscle.
Activation of L-type calcium channels with the
dihydropyridine Bay K8644 induced stereotypical tottering mouse
dystonia at low doses and seizures more typical of this drug at higher
doses. The two different disorders induced by Bay K8644 in tottering mice presumably result from regionally distinct calcium channel activation. In a tottering mouse, the dystonia may be induced at low
doses through up-regulated L-type calcium channels.
Specific increases in calcium channel 1C
subunit mRNA were observed in cerebellar Purkinje cells and the nucleus
of the brachium of the inferior colliculus. As the dose of Bay K8644 is
increased, the "classical" Bay K8644 seizures likely were induced
through normosensitive L-type calcium channels located
elsewhere in brain. This also would explain why wild-type mice injected
with Bay K8644 do not exhibit tottering-like motor abnormalities as the
up-regulated expression of 1C subunits is
required for expression of dystonia.
P/Q-type calcium channels are expressed throughout brain with the
highest expression levels corresponding to regions of high cell density
including cerebellar cortex, hippocampus, and olfactory bulb (Stea et
al., 1994; Tanaka et al., 1995; Volsen et al., 1995). Thus, any
compensatory changes in calcium channel expression may be more evident
in cerebellar cortex, but it is not clear why L-type
calcium channels are not increased in other brain regions that express
high levels of P/Q-type calcium channels. Consistent with the mRNA
expression patterns, P/Q-type calcium channel currents predominate in
cultured rat cerebellar cortex neurons, carrying 85 to 95% of the
total calcium current in Purkinje cells (Mintz et al., 1992) and nearly
half the calcium current in granule cells (Randall and Tsien, 1995). In
contrast, L-type currents account for a small fraction of
the total current (<20%) in both cell types. Thus, an increase in
L-type calcium channels could lead to a dramatic change in
the conductance of calcium ions in these neurons. The potential for
dramatic functional effects is particularly evident in Purkinje cells
where L-type channels are restricted to the soma and
proximal dendrites (Hell et al., 1993) and thus are positioned to have
greater influence on the axon hillock than would be expected by a
random distribution.
Although the functional significance of the tottering mutation on
P/Q-type calcium channels is as yet unknown, functional differences
between L-type and P/Q-type calcium channels are well characterized. In experiments directly comparing
1A (P/Q-type) and 1C
(L-type) calcium channels under standard conditions, Sather et al. (1993) found that the single-channel conductance of
1C channels is measurably greater than that of
1A channels; the same is true of native
L-type channels compared with native P/Q-type channels.
Both L-type and P/Q-type calcium channel currents can be
facilitated by a prepulse (Bourinet et al., 1994), with P/Q-type currents facilitated by relief of G-protein-mediated inhibition (Currie
and Fox, 1997). The facilitated L-type currents can be very
large (more than three times the control currents) and are inducible by
either a single strong depolarization or a short train of pulses
(Bourinet et al., 1994), whereas the facilitation of P/Q-type currents
is more subtle, with only ~18% of the inhibition being
voltage-sensitive (Currie and Fox, 1997). This facilitation of
L-type calcium currents may explain how L-type
calcium channel expression promotes the expression of intermittent
dystonia in tottering mice. It is possible that temporal summation of
calcium influx through voltage-dependent calcium channels, blunted in normal mice by a paucity of facilitation-susceptible L-type
calcium channels, may be augmented by the increased density of
L-type channels in tottering mutants.
Mutations of the calcium channel 1A subunit
are associated with three distinct human diseases and two distinct
mouse neurologic phenotypes. The human spinocerebellar ataxia type 6 (SCA6) and mouse leaner (tgla) mutations
both disrupt the intracellular carboxyl tail of the calcium channel
(Fletcher et al., 1996; Zhuchenko et al., 1997) and cause a persistent
debilitating ataxia associated with severe degeneration of cerebellar
neurons (Herrup and Wilczynski, 1982; Zhuchenko et al., 1997). In
contrast, the human disorders familial hemiplegic migraine (FHM) and
episodic ataxia type-2 (EA-2) mutations as well as the mouse tottering
(tg) mutation alter the transmembrane and/or pore regions of
the channel (Fletcher et al., 1996; Ophoff et al., 1996) and cause
episodic attacks of neurologic dysfunction with little or no obvious
changes in cerebellar morphology (Isaacs and Abbott, 1994; von
Brederlow et al., 1995; Terwindt et al., 1996). FHM is characterized by
brief periods of hemiparesis or weakness accompanied by migraine
headaches. EA-2 patients exhibit intermittent attacks of ataxia
precipitated by stress or exercise. The genotypic and phenotypic
similarities between these human diseases and the intermittent motor
phenotype of tottering mutant mice suggest the tottering mouse is a
useful model for determining how mutations of the calcium channel
1A subunit can generate episodic neurologic
dysfunction in humans. Furthermore, tottering mice are an important
tool for determining cell-type-specific regulation of calcium channel
subtype expression and function in mammalian brain.
| |
Acknowledgments |
|---|
We thank Jesse B. North and Hehui Li for technical assistance and Dr. H. A. Jinnah and Brandy E. Fureman for helpful discussion.
| |
Footnotes |
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Received June 19, 1998; Accepted October 2, 1998
Financial support for this research was provided by Public Health Service Grants NIH NS33592 and NIH NS34845, the Klingenstein Foundation, and the Epilepsy Foundation of America. Portions of this work were presented previously in abstract form (Society for Neuroscience Abstracts 23:2418).
Send reprint requests to: Ellen J. Hess, Ph.D., Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: ehess{at}psu.edu
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Abbreviations |
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SSC, standard saline citrate; DTT, dithiothreitol; ANOVA, analysis of variance.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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) and
tg/tg (
) forebrain (A) and cerebellum
(B). Insets, Scatchard plot of the same data.