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The Journal of Neuroscience, August 15, 2000, 20(16):6218-6224
Modulation of Absence Seizures by the GABAA Receptor:
A Critical Role for Metabotropic Glutamate Receptor 4 (mGluR4)
O. Carter
Snead III1, 4, 5, 6,
P. K.
Banerjee1, 5, 6,
McIntyre
Burnham2, 4, and
David
Hampson2, 3
Departments of 1 Pediatrics and
2 Pharmacology, 3 Faculty of Pharmacy, and
4 Bloorview Epilepsy Program, University of Toronto,
Toronto, Ontario, Canada, and 5 Division of Neurology and
the 6 Program in Brain and Behavior, Hospital for Sick
Children, Toronto, Ontario, Canada
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ABSTRACT |
Experimental absence seizures are associated with perturbations in
the presynaptic release of GABA and glutamate within
thalamocortical circuitry. The release of both glutamate and GABA is
regulated by group III metabotropic glutamate receptors (mGluRs).
Therefore, we examined the susceptibility of mice lacking the mGluR4
subtype of mGluR (mGluR4 /) versus their
wild-type controls (mGluR4+/+) to absence seizures
induced either by  -hydroxybutyrate (GHB) or the GABAB
agonist ( ) baclofen or by low doses of the GABAA receptor
(GABAAR) antagonists pentylenetetrazole, bicuculline, or
picrotoxin. There was no difference between
mGluR4/ and mGluR4+/+ mice in
threshold to absence seizures induced by either GHB or () baclofen.
In contrast, the mGluR4/ mice were markedly
resistant to absence seizures induced by low doses of
GABAAR antagonists. No differences were observed between mGluR4/ and mGluR4+/+ mice in
threshold to clonic or tonic seizures induced by higher doses of
GABAAR antagonists, strychnine, or electroshock, indicating that seizure resistance in the mGluR4/ mice was
restricted solely to absence seizures. The resistance of
mGluR4/ mice to absence seizures induced by
GABAAR antagonists was mimicked by bilateral administration
of a mGluR4 antagonist into the nucleus reticularis thalami (nRT) of
mGluR4+/+ mice. Conversely, intra-nRT administration
of a mGluR4 agonist in mGluR4+/+ mice exacerbated
GABAAR-induced absence seizures. These data indicate that
the presence of mGluR4 within nRT is critical to GABAergic modulation
of thalamocortical synchronization in normal and pathological states,
such as generalized absence epilepsy.
Key words:
group III metabotropic glutamate receptors; thalamus; thalamocortical; mGluR4; absence seizures; GABA
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INTRODUCTION |
Burst firing between reciprocally
interconnected glutamatergic thalamic relay neurons in the ventral
basal thalamus and neocortical pyramidal neurons is synchronized by
GABAergic neurons in the nucleus reticularis thalami (nRT) (Steriade et
al., 1993 ; Oh et al., 1995; Cox et al., 1997; Kim et al., 1997;
McCormick and Bal, 1997). The nRT-driven synchronization that drives
the phasic oscillatory activity within this circuitry generates normal
rhythms, such as sleep spindles, and pathological phenomena, such as
bilaterally synchronous spike-and-wave discharges (SWD), that
characterize generalized absence seizures (Snead, 1995; McCormick and
Bal, 1997; Danober et al., 1998; Snead et al., 1999).
Perturbations in the presynaptic release of glutamate and GABA within
thalamocortical circuitry have been demonstrated in several animal
models of absence seizures (Banerjee and Snead, 1995; Lin et al., 1995;
Richards et al., 1995). The release of glutamate and GABA in
thalamocortical circuitry is modulated by presynaptic metabotropic
glutamate receptors (mGluRs) (East et al., 1995; Salt et al., 1996;
Cochilla and Alford, 1998; Schaffhauser et al., 1998), giving rise to
the hypothesis that mGluRs also may play a role in the pathogenesis of
absence seizures. The eight mGluR subtypes (mGluR1-mGluR8) have been
classified into three groups on the basis of sequence homology, signal
transduction mechanisms, and pharmacological profiles (Nakanishi, 1994;
Pin and Duvoisin, 1995; Conn and Pin, 1997). Activation of group III mGluRs inhibits the release of glutamate and GABA from nerve terminals (East et al., 1995; Pin and Duvoisin, 1995; Neugebauer et al., 1997;
Schaffhauser et al., 1998). This subgroup of mGluRs is activated selectively by the glutamate analog
L-amino-4-phosphonobutyrate (L-AP4).
Among the group III mGluRs ultrastructural studies using
mGluR4-specific antibodies consistently have shown a presynaptic localization for this receptor (Kinoshita et al., 1996; Shigemoto et
al., 1997; Bradley et al., 1999). mGluR4 is highly expressed in the
rodent (Thomsen and Hampson, 1999) and human thalamus (Makoff et al.,
1996). The presence of mGluR4 mRNA in the thalamic relay neurons
(Ohishi et al., 1995) indicates that the receptor protein is expressed
on the terminals of these neurons, which make synaptic contacts with
neocortical pyramidal neurons and the GABAergic neurons in the nRT. On
the basis of the strategic position of mGluR4 in thalamocortical
circuitry, together with evidence for the involvement of GABA and
glutamate release in the mechanisms of absence seizures, we sought to
test the hypothesis that animals lacking the mGluR4 subtype of
metabotropic glutamate receptor have an altered sensitivity to absence seizures.
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MATERIALS AND METHODS |
Production, maintenance, and phenotype of mGluR4 knock-out mice.
CD-1 and 129svj mice were obtained from
Jackson Laboratories (Bar Harbor, ME). The mGluR4 knock-out mice are a
hybrid between these two strains of mice. The
mGluR4+/+ mouse line is also a hybrid
between CD-1 and 129svj mice. Gene targeting was
performed by inserting a pgk-1/neoexpression cassette into an
EcoRV site in an exon located in the first one-third of the
putative extracellular N-terminal domain of the mouse mGluR4 gene
(Pekhletski et al., 1996). Homozygous
(mGluR4/) knock-out mice are devoid of
mGluR4 protein (Pekhletski et al., 1996; Shigemoto et al., 1997;
Bradley et al., 1999). mGluR4/ mice
and their wild-type controls (mGluR4+/+)
were housed in a pathogen-free facility on a 12 hr light/dark schedule.
All analyses were conducted on animals 2-3 months of age. Although
mGluR4/ mice show deficits in motor
learning (Pekhletski et al., 1996) and spatial learning and memory
(Gerlai et al., 1998), they do not differ from wild-type littermates on
measures of general motor activity (open field test) or motor
coordination (bar cross test), and they show no detectable
abnormalities in neuroanatomy (Pekhletski et al., 1996).
Drugs. -Butyrolactone (GBL), pentylenetetrazole (PTZ),
bicuculline methiodide (BMI), picrotoxin (PXN), and strychnine were obtained from Sigma (St. Louis, MO).
L-2-Amino-4-phosphonobutyric acid (L-AP4) and
(RS)- -cyclopropyl-4-phosphonophenylglycine (CPPG) were
obtained from Tocris Cookson (Ballwin, MO). All other chemicals were
obtained from standard commercial sources and were of the highest
available purity.
Surgeries and recordings. Permanent epidural electrodes were
implanted in mGluR4/ and
mGluR4+/+ mice under halothane anesthesia
to allow for continuous recording of the electrocorticogram (ECoG).
Then 7 d were allowed for recovery before commencement of the
experiments. All ECoG recordings were made with animals in the freely
moving state within shielded, heated, clear Plexiglas containers so
that the behavioral response to drug could be observed and correlated
with any drug-induced ECoG event. The ECoG was recorded continuously
for 60 min before and for 3 hr after the administration of any drug.
Seizure models. Absence seizures were induced by
administering either -butyrolactone (Snead, 1988), () baclofen
(Aizawa et al., 1997), or one of the GABAAR
antagonists PTZ, bicuculline, or picrotoxin (Depaulis et al., 1989;
Matejovska et al., 1998). -Butyrolactone was given in a dose of 100 mg/kg as the pure drug. In previous work standardizing the
-hydroxybutyric acid (GHB) model of generalized seizures,
-butyrolactone has been shown to be biologically inactive; however,
after parenteral administration -butyrolactone is converted rapidly
and irreversibly to its active metabolite, GHB, by a circulating
lactonase (Lettieri and Fung, 1978; Snead, 1991). -Butyrolactone has
been used to induce the absence-like seizure because -butyrolactone
produces the same progression of EEG and behavioral events in the rat
as GHB (Snead et al., 1980) but with a more rapid onset of action and
predictable dose-response (Bearden et al., 1980). () Baclofen was
given in a dose of 20 mg/kg (Aizawa et al., 1997).
Low doses of GABAAR antagonists in rodents induce
absence-like seizures that are similar to those induced by
-butyrolactone (GBL) (Depaulis et al., 1989; Matejovska et al.,
1998). The absence seizures induced by both GBL and low doses of
GABAAR antagonists are characterized by 7-9 Hz
bilaterally synchronous SWD. These epileptiform discharges may be
recorded from depth, cortical, or epidural electrodes in freely moving
animals and are associated with behavioral arrest, facial myoclonus,
and vibrissal twitching. The SWD originate in, and are restricted to,
thalamocortical circuitry (Snead et al., 1999). Rats and mice are
unable to generate the 3 Hz SWD seen in human absence seizures;
however, the electrographic and behavioral seizures in both
-butyrolactone-treated and low-dose GABAAR
antagonist-treated rodents are abolished by antiepileptic drugs
specific for absence epilepsy, such as ethosuximide, as well as by
GABAB antagonists (Snead, 1988, 1992; Snead et
al., 1999).
A dose-response curve was constructed to determine the dose of PTX,
bicuculline, and picrotoxin required to induce absence-like seizures in
mGluR+/+ and
mGluR/ mice.
CD100 was defined as the minimal dose to induce
absence seizures in 100% of the mGluR+/+
mice tested. The dose range used to define the
CD100 of these GABAAR
antagonists for absence seizures was 15-35 mg/kg of PTZ, 2-4 mg/kg of
bicuculline, and 1-1.5 mg/kg of picrotoxin. All drugs were given
intraperitoneally. After -butyrolactone, () baclofen, PTZ,
bicuculline, or picrotoxin administration the experimental absence
seizures were quantitated as described below. Paired trials with
mGluR4/and
mGluR4+/+ mice were used in all experiments.
Seizure model experiments. Chemoconvulsive. A dose-response
curve was constructed in mGluR+/+ and
mGluR/ mice to determine the dose of
PTX, bicuculline, and picrotoxin needed to induce clonic and tonic
seizures. CD95 was defined as the dose of
chemoconvulsant that resulted in clonic or tonic seizures in 95% of
the animals that were tested. The dose range used to determine the
CD95 for clonic seizures was 35-60 mg/kg of PTZ, 3.5-5 mg/kg of bicuculline, and 1.5-2 mg/kg of picrotoxin. The dose
range used to determine the CD95 for tonic
seizures was 55-80 mg/kg of PTZ, 5-6 mg/kg of bicuculline, and 2-2.5
mg/kg of picrotoxin. In addition, the susceptibility of
mGluR4+/+ and
mGluR4/ mice to tonic seizures induced
by the glycine antagonist strychnine was tested by using a dose range
of 1-1.75 mg/kg to determine the CD95.
Strychnine was chosen as a non-GABAAR antagonist
chemoconvulsant in this series of experiments to determine whether
there was a generic or specific GABAAR-mediated
alteration in chemoconvulsive threshold. Paired trials with
mGluR4/ and
mGluR4+/+ mice were used in all experiments.
Seizure model experiments. Electroconvulsive shock (ECS).
ECS was administered via corneal electrodes by using a
purpose-built stimulator. Sixty Hertz sine-wave constant current was
used with a train duration of 0.2 sec. Five different intensities,
ranging from 5 to 50 mA, were tested in descending order, with a
minimum of 48 hr between tests. Before each stimulation the electrodes were dipped in 0.9% saline solution to improve electrical contact. The
CD95 was defined as the dose, in mA, that
resulted in seizures in 95% of the animals that were tested.
Intracerebral microinjection experiments. Microinjection
cannulae were implanted in the nRT bilaterally along with epidural electrodes in mGluR4/ and
mGluR4+/+ mice under halothane anesthesia,
using the atlas of Franklin and Paxinos (1997). The coordinates were
1.82 mm from bregma and 1.86 mm interaural. At 7 d after
surgery, 5 nmol of the mGluR4 antagonist CPPG (Jane et al., 1996), the
mGluR4 agonist L-AP4, or vehicle was infused in a volume of
0.5 µl into the nRT bilaterally 10 min before the intraperitoneal
administration of either -butyrolactone or PTZ. The dose of PTZ used
in the microinjection experiments was the CD100
from the absence seizure dose-response curves. The accuracy of
injection was determined by lesioning, using stimulator-generated (Grass Instruments, Quincy, MA) cathodal current (2mA/2 sec) through the indwelling cannulae to produce a localized lesion at the cannulae tips. The placement of cannulae was verified with reference to Franklin
and Paxinos (1997), and only those animals with proven cannulae
placement in nRT were used for analysis.
Data analysis. The experimental absence seizures induced by
-butyrolactone, PTZ, bicuculline, or picrotoxin were quantitated objectively by measuring the duration of the bilaterally synchronous SWD induced by these compounds. Latency was defined as the time in
minutes from administration of drug to the onset of SWD as recorded on
ECoG. SWD duration was measured and expressed as seconds per 20 min
epoch of time (Depaulis et al., 1989). -Butyrolactone, PTZ,
bicuculline, or picrotoxin-induced SWD duration was compared in
mGluR4/ and
mGluR4+/+ mice. Means and SE were
calculated for all SWD data. The data were expressed in latency of SWD
onset (in min). The data were subjected to analysis by two-way ANOVA,
with time treated as a repeated measure. In the absence model
experiments there were 10 mGluR4/ and
10 mGluR4+/+ mice in each group. For the
chemoconvulsive and ECS experiments there were 20 mGluR4/ and 20 mGluR4+/+ mice in each group.
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RESULTS |
mGluR4 knock-out mice were resistant to absence seizures induced by
low doses of GABAA receptor antagonists, but not to absence
seizures induced by GHB or () baclofen
There was no difference between
mGluR4+/+ and
mGluR4/ mice in the baseline ECoG
recordings (Fig. 1A).
Administration of -butyrolactone resulted in absence seizures in all
mGluR 4+/+ (Fig. 1B),
mGluR4/ (Fig. 1C),
CD-1, and 129svj mice (data not shown) that were
tested. These absence seizures were characterized by bilaterally
synchronous SWD on the ECoG (Fig. 1B,C). The behavior
associated with these epileptiform discharges consisted of facial
myoclonus, vibrissal twitching, and arrest of motor activity.
Administration of the low doses of GABAAR
antagonists (Fig. 1D) and () baclofen (data not
shown) resulted in ECoG and behavioral changes similar to those induced
by -butyrolactone. There were no significant differences between the
mGluR4+/+ and
mGluR4/ mice in the latencies from
administration of any of the drugs used to the onset of SWD. Similarly,
there were no significant differences between the
mGluR4+/+ and
mGluR4/ mice in SWD duration induced
by GBL and () baclofen (Fig. 2).
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Figure 1.
Electrocorticography (ECoG) of
mGluR4/ and mGluR4+/+ mice
given either GBL or PTZ. In all ECoG figures
LFR-PR and
RFR-PR represent the ECoG recording from
the left and right frontoparietal electrodes, respectively.
A, Baseline ECoG in a mGluR4/ mouse.
There was no difference between mGluR4+/+ and
mGluR4/ mice in baseline recordings.
B, ECoG in a mGluR4+/+ mouse 20 min
after 100 mg/kg of GBL. C, ECoG in a
mGluR4/ mouse 20 min after 100 mg/kg of GBL.
D, ECoG in an mGluR4+/+ mouse 20 min
after 30 mg/kg of PTZ. E, ECoG in an
mGluR4/ mouse 20 min after 30 mg/kg of PTZ. The
dose of PTZ was the CD100 in the absence seizure
dose-response studies. All drugs were given intraperitoneally.
Administration of PTZ and GBL to mGluR4+/+ mice and
of GBL to mGluR4/ mice resulted in the
bilaterally synchronous SWD shown. These paroxysms were associated with
absence-like behavior in all of the mGluR4/ mice
that were tested, namely facial myoclonus, vibrissal twitching, and
arrest of motor activity. Administration of () baclofen (20 mg/kg,
i.p.) produced EEG and behavioral findings similar to those shown for
PTZ and GBL in both mGluR4/ and
mGluR4+/+ mice. In addition to being refractory to
the PTZ-induced absence seizures as shown in E,
mGluR4/ mice also were resistant to bicuculline-
and picrotoxin-induced absence seizures.
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Figure 2.
Mean SWD duration ± SEM of
mGluR4/ and mGluR4+/+ mice
given either GHB or () baclofen. A, Mean SWD
duration ± SEM in mGluR4/ and
mGluR4+/+ mice (n = 10 for each
group) that received 100 mg/kg of GBL, the prodrug of GHB. There
was no significant difference between mGluR4/
and mGluR4+/+ mice in the duration of GHB-induced
absence seizures (p > 0.1, ANOVA).
B, Mean SWD duration ± SEM in
mGluR4/ and mGluR4+/+ mice
(n = 10 for each group) that received 20 mg/kg of
() baclofen. There was no significant difference between
mGluR4/ and mGluR4+/+ mice in
the duration of () baclofen-induced absence seizures
(p > 0.1, ANOVA).
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The dose-response curve for GABAAR
antagonist-induced absence seizures indicated that the
CD100 in mGluR4+/+
mice was 30 mg/kg for PTZ, 3 mg/kg for bicuculline, and 1.5 mg/kg for
picrotoxin. However, the mGluR4/ mice
were resistant to absence seizures induced by low doses of the
GABAAR antagonists (Fig.
3A).
mGluR4/ mice treated with low-dose PTZ
were completely resistant to GABAAR antagonist-induced absence seizures (Figs. 1E,
3B). Furthermore, the SWD induced by the bicuculline (Fig.
3C) and picrotoxin (Fig. 3D) in
mGluR4/ mice were uniformly and
markedly decreased in duration as compared with the SWD induced in
mGluR4+/+ mice (p < 0.001, ANOVA).
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Figure 3.
Dose-response curves and mean SWD duration ± SEM for GABAAR antagonist-induced absence seizures in
mGluR4/ and mGluR4+/+ mice.
A, The dose-response curve for GABAAR
antagonist-induced absence seizures in mGluR4/
and mGluR4+/+ mice (n = 10 for
each group). The CD100 for pentylenetetrazole (PTZ),
bicuculline (BMI), and picrotoxin (PXN) was 30, 3, and 1.5 mg/kg,
respectively, in the mGluR4+/+ mice. The
CD100 of PTZ for mGluR4+/+ mice failed
to induce absence seizures in any mGluR4/ mice.
The CD100 of bicuculline and picrotoxin for
mGluR4+/+ mice induced short-lived absence seizures
in only 10% of mGluR4/ mice that were tested.
SEM was <10% for all data points and is not shown.  ,
mGluR4+/+ (PXN);  , mGluR4+/+
(BMI);  , mGluR4+/+ (PTZ);  ,
mGluR4/ (PXN);  ,
mGluR4/ (BMI);  ,
mGluR4/ (PTZ). B, Mean SWD
duration ± SEM for PTZ-induced absence seizures in
mGluR4/ and mGluR4+/+ mice
(n = 10 for each group) that received 30 mg/kg of
PTZ, the CD100 for mGluR4+/+ mice. The
mGluR4/ mice were completely resistant to
absence seizures induced by low doses of this GABAAR
antagonist. C, Mean SWD duration ± SEM in
mGluR4/ and mGluR4+/+ mice
(n = 10 for each group) that received 3 mg/kg of
bicuculline, the CD100 for mGluR4+/+
mice. The SWD duration in the absence seizure induced by low-dose
GABAAR antagonists was significantly
(p < 0.001, ANOVA) shorter in the
mGluR4/ than in the mGluR4+/+
mice. D, Mean SWD duration ± SEM in
mGluR4/ and mGluR4+/+ mice
(n = 10 for each group) that received 1.5 mg/kg of
picrotoxin, the CD100 for mGluR4+/+
mice. The SWD duration in the absence seizures induced by low-dose
GABAAR antagonists was significantly
(p < 0.001, ANOVA) shorter in the
mGluR4/ than in the mGluR4+/+
mice.
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Because mGluR 4/ mice are a hybrid
between CD-1 and 129svj mice, it is conceivable
that any observed alteration in sensitivity of the mGluR
4/ mice to absence seizures could be
caused by genetic differences in the two strains of mice. To test this
possibility, we repeated the GABAAR
antagonist-induced absence seizure experiments on the two parent
strains of mice, using the CD100 for
mGluR4+/+ mice (Fig. 3A). There
were no significant differences between CD-1 and
129svj mice in SWD duration in absence seizures induced by
PTZ (Fig. 4), bicuculline, or picrotoxin
(data not shown). These data indicate that differences in the genetic
makeup of the two strains are unlikely to account for the resistance of the mGluR4/ mice to
GABAAR antagonist-induced absence seizures.
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Figure 4.
Mean SWD duration ± SEM of
mGluR4+/+, CD-1, or
129svj mice treated with 30 mg/kg of PTZ, the
CD100 for mGluR4+/+ mice. This dose of
PTZ induced absence-like seizures in all three strains. There were no
significant differences among the three groups in latency or SWD.
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mGluR4 knock-out mice and wild-type mice do not differ in threshold
to convulsive seizures
The dose-response curves indicated that the EEG and behavior
changes induced by GABAAR antagonists in mice
represent a highly dose-specific continuum (Fig.
5A). Low doses induce
absence-like seizures as described above. As the dosage of
GABAAR antagonists is increased, clonic seizures
appear. With a further increase in dosage tonic seizures emerge. There
was no temporal progression from one seizure type to another with a
specific dose of GABAAR antagonist. The
CD95 for the induction of clonic seizures with PTZ, bicuculline, and picrotoxin in the
mGluR4+/+ mice was 45, 4.5, and 1.8 mg/kg,
respectively. By comparison, the CD95 for the
induction of clonic seizures with PTZ, bicuculline, and picrotoxin in
the mGluR4/ mice was 40, 5.0, and 1.9 mg/kg, respectively (Fig. 5B). This was not significantly
different (p > 0.1, ANOVA; n = 20/group). There was no significant difference in the
CD95 for the induction of tonic seizures with
PTZ, bicuculline, picrotoxin (Fig. 5C), and strychnine (data
not shown). In the mGluR4+/+ mice the
CD95 was 74, 5.5, 2.2, and 1.5 mg/kg,
respectively, whereas the CD95 for the induction
of tonic seizures with PTZ, bicuculline, picrotoxin, PTZ, and
strychnine in the mGluR4/ mice was 70, 5.5, 2.4, and 1.5 mg/kg, respectively (p > 0.1, ANOVA; n = 20/group). There also was no significant
difference between mGluR4+/+ and
mGluR4/ mice in the ECS seizure model
experiments. The CD95 was 10 mA for both
(dose-response curve not shown).
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Figure 5.
Dose-response curves for GABAAR
antagonist-induced convulsive seizures in
mGluR4/ and mGluR4+/+ mice.
A, Dose specificity of seizures induced by
GABAAR antagonists in mGluR4+/+ mice.
Seizure Grade refers to the type of seizure that was
induced. One is absence, two is clonic, and three is tonic seizure. The
dosage shown for PTZ is the CD100 rather than
CD95 because only 10 animals were used to generate the
absence seizure dose-response curve. B, The
dose-response curve for GABAAR antagonist-induced clonic
seizures in mGluR4/ and
mGluR4+/+ mice (n = 20 for each
group). There was no significant difference between the
mGluR4/ and mGluR4+/+ mice in
the CD95 of the GABAAR antagonists
(p > 0.1, ANOVA; n = 20/group). The CD95 for the induction of clonic seizures
with PTZ, bicuculline (BMI), and picrotoxin (PXN) in the
mGluR4+/+ mice was 45, 4.5, and 1.8 mg/kg,
respectively. The CD95 for the induction of clonic seizures
with PTZ, bicuculline, and picrotoxin in the
mGluR4/ mice was 40, 5.0, and 1.9 mg/kg,
respectively. SEM was <10% for all data points and is not shown. ,
mGluR4+/+ (PXN); , mGluR4+/+
(BMI); , mGluR4+/+ (PTZ); ,
mGluR4/ (PXN); ,
mGluR4/ (BMI); ,
mGluR4/ (PTZ). C, The
dose-response curve for GABAAR antagonist-induced tonic
seizures in mGluR4/ and
mGluR4+/+ mice (n = 20 for each
group). There was no significant difference between the
mGluR4/ and mGluR4+/+ mice in
the CD95 of the GABAAR antagonists
(p > 0.1, ANOVA; n = 20/group). In the mGluR4+/+ mice the
CD95 for PTZ, bicuculline, and picrotoxin was 74, 5.5, and
2.2 mg/kg, respectively, whereas the CD95 for the induction
of tonic seizures with PTZ, bicuculline, and picrotoxin in the
mGluR4/ mice was 70, 5.5, and 2.4, respectively.
SEM was <10% for all data points and is not shown.
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Microinjection of an mGluR4 antagonist into the nRT of
mGluR4+/+ mice mimicked the phenotype of the
mGluR4/ mice
Bilateral microinjection of the mGluR4 antagonist CPPG into the
nRT had no effect on baseline ECoG and induced no ictal behaviors in
the mGluR4+/+ mice. In addition, bilateral
intra-nRT infusion of CPPG had no effect on GBL-induced absence
seizures in mGluR4+/+ mice (Fig.
6A). However, bilateral
intra-nRT infusion of CPPG in mGluR4+/+
mice resulted in a marked reduction in the duration of absence seizures
induced by low doses of PTZ (Fig. 6B). Thus,
microinjection of the mGluR4 antagonist CPPG into the nRT of
mGluR4+/+ mice conferred resistance to
PTZ-induced absence seizures and mimicked the phenotype of the
mGluR4/ mice.
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Figure 6.
Mean SWD duration ± SEM of
mGluR4+/+ mice treated with CPPG or
L-AP4 given intra-nRT before induction of absence seizures
with either PTZ or GHB. A, Mean SWD duration ± SEM
for mGluR4+/+ mice (n = 10 for
each group) that received 100 mg/kg of GBL 10 min after bilateral
intra-nRT infusion of 5 nmol of the mGluR4 antagonist CPPG, the mGluR4
agonist L-AP4, or vehicle control. There was no significant
(p > 0.1, ANOVA) difference in SWD duration
among any of the nRT treatment groups. B, Mean SWD
duration ± SEM for mGluR4+/+ mice
(n = 10 for each group) that received 30 mg/kg of
PTZ 10 min after bilateral intra-nRT infusion of 5 nmol of the mGluR4
antagonist CPPG or vehicle control. Intra-nRT infusion of CPPG
conferred significant (p < 0.001, ANOVA)
resistance to absence seizures induced by low doses of PTZ.
C, Mean SWD duration ± SEM for
mGluR4+/+ mice (n = 10 for each
group) that received 30 mg/kg of PTZ 10 min after bilateral intra-nRT
infusion of 5 nmol of the mGluR4 agonist L-AP4 or vehicle
control. L-AP4 administration into the nRT significantly
(p < 0.001, ANOVA) exacerbated absence
seizures induced by low doses of PTZ.
|
|
Bilateral microinjection of the mGluR4 agonist L-AP4 into
the nRT had no effect on baseline ECoG and induced no ictal behaviors in the mGluR4+/+ mice. Bilateral intra-nRT
infusion of L-AP4 in mGluR4+/+
before the intraperitoneal administration of PTZ mice had an effect
opposite to that of CPPG. Bilateral intra-nRT infusion of CPPG resulted
in resistance to PTZ-induced absence seizures, but bilateral intra-nRT
infusion of L-AP4 was associated with a significant
(p < 0.001, ANOVA) prolongation of PTZ-induced
SWD duration (Fig. 6C) (i.e., exacerbation of PTZ-induced
absence seizures). However, neither CPPG nor
L-AP4 given into the nRT affected the ability of
GBL to induce SWD in mGluR4+/+ mice (Fig.
6A).
| |
DISCUSSION |
We have hypothesized that mice lacking the mGluR4 gene would have
an altered sensitivity to pharmacologically induced absence seizures.
The experimental observations that prompted the formulation of this
hypothesis were based on two findings. First, group III mGluRs are
located presynaptically and modulate both GABA and glutamate release
(East et al., 1995; Pin and Duvoisin, 1995; Salt et al., 1996;
Neugebauer et al., 1997). Second, glutamate and GABA release are
altered in thalamocortical circuitry in absence seizures (Banerjee and
Snead, 1995; Lin et al., 1995; Richards et al., 1995; Hu et al., 2000).
Therefore, we compared the sensitivity of
mGluR4/ and
mGluR4+/+ mice to absence seizures induced
by GBL, the GABABR agonist () baclofen, or low
doses of the three GABAAR antagonists PTZ,
bicuculline, and picrotoxin. In these experiments there was no
significant difference between the
mGluR4+/+ and
mGluR4/ mice in the sensitivity to
absence seizures induced by GHB or () baclofen. However,
mGluR4/ mice were highly resistant to
absence seizures induced by low doses of GABAAR antagonists.
GHB is a naturally occurring metabolite of GABA and has the ability to
induce absence seizures in a number of animal species, including rats
and mice (Snead et al., 1999). The precise mechanism by which GHB
induces absence seizures is not known, but there is some evidence that
GHB may be a weak GABABR agonist (Bernasconi et
al., 1999; Lingenhoehl et al., 1999). However, other studies suggest
that GHB exerts its effect at unique GHB-specific binding sites (Snead,
1996; Maitre, 1997). Therefore, to be certain that we were examining
GABABR-mediated absence seizures in the
mGluR4/ mice, we also induced absence
seizures with the specific GABABR agonist ()
baclofen. GABABR agonists cause absence seizures
by acting on thalamocortical relay neurons in the ventrobasal thalamus (Liu et al., 1992; McCormick and Bal, 1997; Snead et al., 1999). The
observation that both GHB-induced and GABABR
agonist-induced absence seizures are equally robust in
mGluR4/ and
mGluR4+/+ mice suggests that
GABAB-mediated inhibition within the
thalamocortical circuitry is unaffected by the absence of mGluR4.
We then determined whether the resistance of
mGluR4/ mice to absence seizures
induced by low doses of GABAAR antagonists
extended to convulsive seizures. Convulsive seizures were induced by
both chemoconvulsant drugs and ECS. No significant differences were observed between mGluR4/ and
mGluR4+/+ mice in any of the models that
were used for convulsive seizures. The threshold of
mGluR4/ mice to chemoconvulsant- and
electroconvulsant-induced clonic and tonic seizures is an important
issue because there is increasing interest in the therapeutic potential
of mGluR modulation in epileptogenesis (Thomsen and Dalby, 1998;
Meldrum et al., 1999; Lie et al., 2000). Activation of group I mGluRs
appears to produce convulsive seizures, whereas some group I
antagonists are reported to be anticonvulsant in rodent models of
limbic or convulsive seizures (Tizzano et al., 1995; Chapman et al.,
1999). However, the effect of group III mGluR agonists and antagonists
on seizures is less clear. Selective activation of group III mGluRs has
been reported to decrease epileptiform activity in rat neocortex or
hippocampus (Burke and Hablitz, 1994; Tizzano et al., 1995; Abdul-Ghani
et al., 1997), but administration of L-AP4
intracerebroventricularly reportedly causes seizures and exacerbates
sound-induced seizures in mice (Ghauri et al., 1996). The proconvulsant
activity of L-AP4 in some studies could be the result of
activation of NMDA receptors at higher concentrations of this amino
acid (Thomsen and Dalby, 1998). In our experiments no alterations in
EEG were seen, nor were any ictal behaviors induced in
mGluR4+/+ mice after bilateral intra-nRT
infusion of either L-AP4 or CPPG.
If the hypothesis that selective activation of mGluR4 results in an
elevated threshold for limbic and convulsive seizure activity (Burke
and Hablitz, 1994; Tizzano et al., 1995; Abdul-Ghani et al., 1997) is
correct, the convulsive threshold for clonic and tonic seizures would
be predicted to be lower in animals devoid of mGluR4. However, in our
experiments the CD95 for clonic and tonic
seizures induced by higher doses of GABAAR
antagonists, the glycine antagonist strychnine, or ECS in the
mGluR4/ mice was not significantly
different from that observed for the mGluR4+/+ mice. Given the evidence for the
exclusive involvement of thalamus and cortex in the genesis of absence
seizures (Snead, 1995; McCormick and Bal, 1997; Danober et al., 1998;
Snead et al., 1999), these findings implicate thalamocortical circuitry
as the neuronal network involved in the resistance of
mGluR4/ to absence seizures induced by
GABAAR antagonists and indicate the
circuitry-dependent nature of any pro- or anticonvulsant action that
might result from mGluR4 activation. An explanation for the dramatic
resistance to absence seizures in the mGluR4 knock-out mice may be that
other group III mGluRs are unable to compensate for the lack of mGluR4
in the thalamus. mGluR6 is expressed only in the retina, and within the
thalamus the expression of mGluR8 is restricted to the reticular
neurons (Saugstad et al., 1997; Corti et al., 1998). mGluR7, which has
very low affinity for glutamate and L-AP4, is expressed at
low levels in several thalamic relay nuclei of the rat (Ohishi et al.,
1995; Neto et al., 2000). However, a preliminary report has indicated
that mGluR7 knock-out mice are not resistant to seizures but instead
develop convulsant seizures several weeks after birth (Sansig et al.,
1999).
Physiological activation of group III mGluR is reported to depress
glutamate-mediated excitation in corticothalamic neurons and, thus, to
decrease corticothalamic input to thalamus (Turner and Salt, 1999).
However, a different part of the thalamocortical circuitry is involved
in the mechanism of GABAAR antagonist-induced absence seizures. Low doses of GABAAR antagonists
induce absence seizures by blocking intra-nRT
GABAAR-mediated inhibition (Huguenard and Prince,
1994; Ulrich and Huguenard, 1997) rather than acting on corticothalamic
pathways. Therefore, we tested and confirmed the hypothesis that the
nRT is critical to the resistance of
mGluR4/ mice to
GABAAR antagonist-induced absence seizures. In
the low-dose PTZ model of absence seizures, microinjection of the
mGluR4 antagonist CPPG into the nRT of wild-type
mGluR4+/+ mice mimicked the phenotype of
the mGluR4/ mice. Conversely,
bilateral intra-nRT administration of the mGluR4 agonist
L-AP4 in mGluR4/ mice
resulted in exacerbation of absence seizures induced by PTZ, but not by GHB.
Recurrent GABAergic collateral connections provide intranuclear
GABAAR-mediated inhibition within nRT that
regulates nRT output during thalamic oscillations. The intra-nRT
recurrent GABAergic inhibitory activity acts as a desynchronizer to
prevent hypersynchrony and the induction of absence seizures (Huguenard
and Prince, 1994; Ulrich and Huguenard, 1997; Huntsman and Huguenard,
1999). Suppression of intra-nRT GABAAR-mediated
inhibition, either by disruption of the gene coding for the
 3 subunit of the GABAAR
(Homanics et al., 1997) or by administration of low doses of
GABAAR antagonists, results in a marked increase
in oscillatory synchrony that is characteristic of absence seizures
(Huntsman et al., 1999; Sohal et al., 2000). Therefore, intra-nRT
GABAAR-mediated inhibition appears to be required
for preventing the pathological hypersynchrony of absence seizures. We
suggest that in mGluR4/ mice the
absence of mGluR4 in presynaptic glutamatergic terminals that project
from thalamic relay neurons to nRT may cause an increase in glutamate
release (East et al., 1995) on GABAergic neurons within the nRT. This
results in an enhancement of intra-nRT
GABAAR-mediated inhibition leading to increased
desynchronization within the thalamocortical circuitry and a resistance
to absence seizures induced by low doses of
GABAAR antagonists. This effect is opposite to
that seen in mice devoid of the 3 subunit of
the postsynaptic GABAAR
(3/). This mutant
is of particular relevance to the question of the role of the nRT in
modulation of neuronal synchrony and absence seizures because the
3 subunit is restricted mainly to the nRT. In
the 3/ mice,
GABAAR-mediated inhibition was nearly abolished
in nRT but was unaffected in thalamocortical relay nuclei. Further, in the 3/ mice
oscillatory synchrony was intensified dramatically, leading to the
conclusion that recurrent inhibitory connections within nRT act as
desynchronizers (i.e., decreased intra-nRT
GABAAR-mediated inhibition causes hypersynchrony)
(Huntsman et al., 1999).
Recent computer modeling experiments have extended these findings by
showing that intra-nRT inhibition restricts intrathalamic oscillatory
activity to particular spatiotemporal patterns to allow for focal
recurrent activity that is relevant for normal thalamocortical function
while preventing the pathological synchronization that results in
absence seizures (Sohal et al., 2000). The selective resistance of
mGluR4/ mice to absence seizures
induced by GABAAR antagonists indicates that the
absence of mGluR4 reinforces the intra-nRT
GABAAR-mediated control of thalamocortical
synchrony. These findings suggest that there is a tight regulation
between mGluR4 and GABAAR within intra-nRT inhibitory circuitry. Presynaptic glutamatergic mechanisms at the level
of the nRT may play a key role in the maintenance of the fine balance
between synchronization and desynchronization within thalamocortical
circuitry and, therefore, in the pathogenesis of absence seizures. Our
data indicate that mGluR4-mediated perturbation of GABAergic function
within the nRT of mGluR4/ mice causes
a shift of the synchronization/desynchronization equilibrium toward
desynchronization. Thus, an increase in desynchronization in this
circuit produces resistance to absence seizures induced by
GABAAR antagonists.
The findings that mGluR4/ mice are
resistant to absence seizures induced by low doses of
GABAAR antagonists and that this phenotype is
mimicked by the intra-nRT administration of an mGluR4 antagonist suggest that mGluR4 antagonist drugs may be potentially useful in the
treatment of absence epilepsy. There is precedent for drugs that act in
a similar circuitry-specific manner having clinical utility in this
disorder. The therapeutic efficacy of benzodiazepines in generalized
absence seizures may be attributed to the fact that these compounds
enhance GABAAR-mediated inhibition within the nRT
and enhance desynchronization, with a resultant decrease in
hypersynchrony and absence seizures (Huguenard and Prince, 1994; Gibbs
et al., 1996; Snead et al., 1999).
| |
FOOTNOTES |
Received March 29, 2000; revised May 5, 2000; accepted May 23, 2000.
This work was supported in part by Medical Research Council Canada and
by the Bloorview Children's Hospital Foundation. We are grateful to
Mr. Chun Che Liu for superb technical support.
Correspondence should be addressed to Dr. O. Carter Snead III,
University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8,
Canada. E-mail: csnead{at}sickkids.on.ca.
| |
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TOP
ABSTRACT
INTRODUCTION
