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 Previous Article
The Journal of Neuroscience, November 15, 2000, 20(22):8643-8649
Neuroprotective Role of Dopamine Against Hippocampal Cell
Death
Yuri
Bozzi,
Daniela
Vallone, and
Emiliana
Borrelli
Institut de Génétique et de Biologie Moléculaire
et Cellulaire, Centre National de la Recherche Scientifique/Institut
National de la Santé et de la Recherche
Médicale/Université Louis Pasteur, 67404 Illkirch Cedex,
Communauté Urbaine de Strasbourg, France
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ABSTRACT |
Glutamate excitotoxicity plays a key role in the induction of
neuronal cell death occurring in many neuropathologies, including epilepsy. Systemic administration of the glutamatergic agonist kainic
acid (KA) is a well characterized model to study epilepsy-induced brain
damage. KA-evoked seizures in mice result in hippocampal cell death,
with the exception of some strains that are resistant to KA
excitotoxicity. Little is known about the factors that prevent epilepsy-related neurodegeneration. Here we show that dopamine has such
a function through the activation of the D2 receptor (D2R). D2R gene
inactivation confers susceptibility to KA excitotoxicity in two mouse
strains known to be resistant to KA-induced neurodegeneration. D2R /
mice develop seizures when administered KA doses that are not
epileptogenic for wild-type (WT) littermates. The spatiotemporal pattern of c-fos and c-jun mRNA induction
well correlates with the occurrence of seizures in D2R/ mice.
Moreover, KA-induced seizures result in extensive hippocampal cell
death in D2R/ but not WT mice. In KA-treated D2R/ mice,
hippocampal neurons die by apoptosis, as indicated by the presence of
fragmented DNA and the induction of the proapoptotic protein BAX. These
results reveal a central role of D2Rs in the inhibitory control of
glutamate neurotransmission and excitotoxicity.
Key words:
epilepsy; excitotoxicity; apoptosis; dopamine D2
receptors; glutamate receptors; kainic acid
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INTRODUCTION |
Dysfunctions of glutamatergic
neurotransmission are thought to play a key role in the induction of
neuronal cell death occurring after brain trauma and in neurological
disorders (Choi, 1988 ; Coyle and Puttfarcken, 1993; Meldrum 1995).
Sustained activation of glutamate receptor (GluR) signaling
pathways by extensive release of glutamate in susceptible areas leads
to neuronal damage through apoptosis (Choi, 1994; Pollard et al.,
1994).
Epilepsy is one pathological condition characterized by localized
bursts of electrical overactivity (seizures) in the cerebral hemispheres. Outbursts of electrical activity, commonly observed in
cortical and subcortical areas, can result in extensive neuronal cell
death in different brain regions. A glutamate-dopamine interaction has
been proposed to explain individual susceptibility to epilepsy, based
on the effect of antipsychotics (i.e., dopaminergic D2-like antagonists) (Starr, 1996). These compounds, despite their positive effects in the treatment of disorders such as schizophrenia, have been
shown to lower the seizure threshold in epileptic patients or even to
promote seizures in patients with no previous history of the disease.
Conversely, seizure inhibition has been observed in patients
administered antiparkinsonian drugs such as pergolide and
bromocriptine, both D2-like agonists (Starr, 1996). The limbic system
is crucially involved in the dopaminergic control of epileptic seizures. Indeed, limbic areas of the brain receive dopaminergic innervation (Verney et al., 1985) and express different types of
dopamine receptors (Jackson and Westlind-Danielsson, 1994). Among
these, D1 and D2 receptors seem to play opposite roles in regulating
the threshold for seizures (Starr, 1993).
We investigated the effect of glutamate-induced seizures and
neurotoxicity in dopamine D2 receptor knock-out (D2R/) mice (Baik
et al., 1995). Glutamate-induced seizures were evoked by the systemic
administration of kainic acid (KA), a potent agonist of the
AMPA/kainate class of glutamate receptors (Hollmann and Heinemann,
1994). Systemic administration of KA in rodents has been widely used
and characterized as a model to study the behavioral, anatomical,
cellular, and genetic bases of glutamate neurotoxicity (Schauwecker and Steward, 1997, and references therein). KA
treatment determines a well defined pattern of activation of specific
brain areas, which is correlated with the appearance of limbic seizures (Lothman and Collins, 1981; Lothman et al., 1981; Willoughby et al.,
1997). KA-evoked seizures result in neuronal cell loss in restricted
subfields of the hippocampal formation (Ben-Ari, 1985), with the
exception of some mouse strains that are resistant to KA excitotoxicity
(Schauwecker and Steward, 1997).
In the present study, we show that the absence of D2R in two mouse
strains known to be resistant to KA-induced neurodegeneration (C57BL/6
and mixed 129/Sv × C57BL/6) (Schauwecker and Steward, 1997)
confers susceptibility to KA excitotoxicity. D2R/ mice develop
seizures when administered KA doses that are not epileptogenic for
wild-type (WT) littermates. Moreover, KA-induced seizures result in
extensive hippocampal cell death in D2R/ but not WT mice. These
results suggest a neuroprotective role of dopamine D2 receptors against
glutamate-induced excitotoxicity.
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MATERIALS AND METHODS |
Animals. D2R/ mice were generated previously in
our laboratory (Baik et al., 1995). All mice used for this study were
male and female F2 hybrids (C57BL/6 × 129/Sv mixed background) obtained from heterozygote intermatings. For
the evaluation of KA-induced hippocampal cell death, we also used a
congenic C57BL/6 strain carrying the mutated D2R allele. This strain
was established by backcrossing D2R+/ mice (F2
hybrids) to C57BL/6 mice for five generations (Silva et al., 1997).
After the fifth backcross, the colony was expanded by inbreeding pairs
of nonsibling +/ male and female mice. Animals were housed in a 12 hr
light/dark cycle with food and water available ad libitum.
Experiments were conducted in conformity with the European Communities
Council Directive of November 24, 1986 (86/609/EEC).
Behavioral analyses. Adult (F2
hybrids; 3- to 5-month-old; weight, 20-30 gm) D2R+/+, D2R+/,
D2R/, 129/Sv, and C57BL/6 mice of both sexes were used
(n = 10 mice per genotype and treatment group). In all
of the experiments, the experimenter was blind to the genotype and
treatment of the animals. KA (Sigma, St. Louis, MO) was
dissolved in saline and administered intraperitoneally at 10, 20, and
35 mg/kg body weight. Saline-injected animals were used as controls.
Seizure severity was determined according to a previously defined
rating scale (Schauwecker and Steward, 1997): stage 0, normal behavior;
stage 1, immobility; stage 2, forelimb and/or tail extension, rigid
posture; stage 3, repetitive movements, head bobbing; stage 4, rearing
and falling; stage 5, continuous rearing and falling; stage 6, severe
whole-body convulsions; and stage 7, death. For each animal, the rating
scale value was scored every 20 min for a maximum of 2 hr. The maximum
rating scale values reached by each animal over the whole observation
period were used to calculate the rating scale value (±SEM) for each
treatment group. Statistical analysis was performed by Mann-Whitney
U test. Data from male and female animals were initially
analyzed separately. Because no difference in seizure rating scale
values was observed between the two genders in each genotype and
treatment group, data were pooled together.
Receptor autoradiography. Brain cryostat sections (10 µm)
from WT and D2R/ mice (3- to 5-month-old; n = 3 per
genotype) were used for autoradiographic binding. D2R binding was
performed with 125I-iodosulpride (2000 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, UK) as
described previously (Martres et al., 1985). For high-affinity kainate
binding (Berger and Ben-Ari, 1983), sections were preincubated for 15 min at 37°C in 50 mM Tris-acetate buffer and
incubated for 1 hr at 4°C with 20 nM
vinilydene-3H-kainic acid (30-60
Ci/mmol; NEN, Boston, MA). Low-affinity binding sites were displaced by
incubating the sections for 2 min at 4°C in the presence of 10 µM cold kainic acid in the same buffer. Sections were washed in ice-cold buffer, rinsed in
H2O, dried, and exposed for 1-3 months to Kodak
Biomax autoradiographic films (Eastman Kodak, Rochester, NY). Pictures
were taken directly from film autoradiograms.
Immunoblotting. Hippocampi from WT and D2R/ mice (3- to
5-month-old; n = 3 per genotype) were rapidly
dissected, frozen, and homogenized in 500 µl of lysis buffer (50 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 10%
glycerol, 0.1% SDS, 1 mM PMSF, 1 mg/ml
aprotinin, 1 mg/ml leupeptin, 1 mM sodium
orthovanadate, and 50 mM NaF). Same amounts (20 µg/sample) of hippocampal total protein were resolved by SDS-PAGE and
probed with GluR1, GluR2/3, GluR6/7, and KA2 polyclonal antibodies
(Upstate Biotechnology, Lake Placid, NY). Signals were revealed by
enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and
quantified by a Bio-Rad (Hercules, CA) GS-700 Imaging Densitometer.
In situ hybridization. WT and D2R/ mice (3- to
5-month-old; n = 3 per genotype and treatment group)
were killed at different times after saline or KA injection (1, 3, 6, and 12 hr). Brains were rapidly removed and frozen on dry ice.
In situ hybridizations on cryostat sections were performed
with 35S-labeled antisense
c-fos (Halazonetis et al., 1988) and c-jun (Mellström et al., 1991) riboprobes as described previously
(Bozzi and Borrelli, 1999). After hybridization, slides were exposed for 1-4 d at room temperature to Kodak BIOMAX x-ray films. Pictures were taken directly from film autoradiograms. The specificity of the
results was confirmed by the use of sense riboprobes, which gave no
detectable signal (data not shown). The extension and subdivision of
brain areas was determined according to Franklin and Paxinos
(1997).
Immunohistochemistry, terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling, and histology.
In situ terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end labeling (TUNEL) of fragmented DNA and
BAX immunohistochemistry were performed on brain cryostat sections from
male and female WT and D2R/ mice (F2 hybrids;
3- to 5-month-old) killed 16 hr after KA injection (20 or 35 mg/kg,
i.p.; n = 5 animals per genotype and treatment group).
TUNEL staining was detected by the ApopTag kit (Oncor). After
diaminobenzidine (DAB) reaction, sections were counterstained with
methyl green. For BAX immunostaining, adjacent sections were fixed in
acetone/methanol (1:1 mixture), incubated for 1 hr in blocking solution
(5% normal goat serum, 0.05% Tween 20, and 1× PBS) and probed
overnight at 4°C with an anti-BAX polyclonal antibody (1:100 dilution
in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA).
Sections were then washed, and signal was revealed with a fluorescent
(Cy3-conjugated) goat anti-rabbit secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Cresyl violet staining (Nissl
coloration) and glial fibrillary acidic protein (GFAP)
immunohistochemistry were performed on paraffin sections from WT and
D2R/ mice (F2 hybrids; 3- to 5-month-old)
killed 5 d after KA injection (20 or 35 mg/kg, i.p.;
n = 5 per genotype and treatment group). GFAP
immunohistochemistry was performed using an anti-GFAP polyclonal
antibody (1:800 dilution in blocking solution; Sigma), followed by
incubation with a Cy3-conjugated secondary antibody (Jackson ImmunoResearch).
Statistical analysis of TUNEL staining and morphological
evaluation of hippocampal cell damage. For the quantification of TUNEL experiments, five animals of each genotype and treatment were
used. For each animal, five different coronal sections were collected
(one every 150 µm), spanning through the whole dorsal hippocampus.
After TUNEL reaction followed by histological counterstaining (see
above), sections were observed at 10× primary magnification under a
light microscope. For each section, two measurements were taken (one
for each side) by counting the TUNEL-positive CA3 cells present in a
400 × 400 µm sample area. The extension of this area was
appropriately chosen to contain the whole CA3 region of each side. Only
those CA3 cells that exhibited a DAB nuclear staining were considered
TUNEL-positive. The different measurements taken from each animal were
initially used to calculate the average number of TUNEL-positive CA3
cells per area for each animal. The average values per animal
(n = 5 per genotype and treatment) were then used for
statistical analysis. Mann-Whitney U test was used to
compare the results obtained in the different genotypes and treatment
groups. Results are shown as the mean ± SEM value of TUNEL-positive CA3 cells per area.
To evaluate the degree of KA-induced hippocampal cell damage,
Nissl-stained sections from five animals of each genotype and treatment
were used. Sections from each mouse were assigned the following score
according to Morrison et al. (1996): little damage, occasional
single-cell degeneration in CA3 area; mild damage, small area with
degenerated CA3 pyramidal cells; and severe damage, extended area of
neuronal degeneration, neuronal cell loss and tissue sclerosis,
frequently including both dorsal and ventral hippocampal CA3 regions.
No difference in TUNEL or Nissl staining was observed between male and
female mice of the same genotype. Therefore, data were pooled together,
and all of the results shown in this study include both genders.
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RESULTS |
The distribution of D2R binding sites in the brain of WT and
D2R/ mice was evaluated by autoradiographic in situ
binding with 125I-iodosulpiride on brain
sections. D2Rs were present in the stratum lacunosum moleculare of the
dorsal hippocampus (CA1 and CA3 regions), as well as in the hilum of
dentate gyrus in WT mice (Fig.
1a) (Martres et al., 1985;
Bouthenet et al., 1987). As expected, D2R binding sites were absent in
the D2R/ hippocampus (Fig. 1a). Persistent
125I-iodosulpiride binding was detected in
the habenula of D2R/ mice, likely because of D3 dopamine
receptors present in this area (Bouthenet et al., 1991). Previous
studies from our laboratory demonstrated that there is no evidence for
any residual D2R binding in the D2R/ mouse brain (Baik et al.,
1995). Glutamate receptor channels of the AMPA/KA subclass are mainly
expressed in limbic areas and are involved in epileptogenesis
(Dingledine et al., 1990; Seeburg, 1993; Hollmann and Heinemann, 1994).
Autoradiographic in situ binding with
3H-KA revealed a comparable pattern of KA
high-affinity binding sites in the hippocampus (Fig. 1b),
cerebral cortex (Fig. 1b), and striatum (data not shown) of
WT and D2R/ mice. Immunoblotting analysis also revealed comparable
levels of KA (KA2 and GluR6/7) and AMPA (GluR1 and GluR2/3) receptor
subunits in the hippocampus of WT and D2R/ mice (Fig.
1c).
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Figure 1.
Expression of D2 and glutamate receptors in WT and
D2R/ mice. a, D2R binding sites in the hippocampus
of WT and D2R/ mice. b, High-affinity KA binding
sites in the cerebral cortex and hippocampus of WT and D2R/ mice.
ctx, Cerebral cortex; CA3, CA3
hippocampal subfield; DG, dentate gyrus;
slm, stratum lacunosum moleculare. Scale bar, 1.3 mm.
c, Immunoblotting of glutamate receptor subunits (as
indicated) on protein extracts from WT (+/+) and D2R/ (/)
hippocampi.
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The behavioral response of WT, D2R+/ and D2R/ male and female
mice to increasing doses of the epileptogenic drug KA (10, 20, and 35 mg/kg, i.p.) was then evaluated over a period of 2 hr after
administration. KA treatment had a clear dose-dependent effect in all
genotypes. At a dose of 10 mg/kg, WT and D2R+/ animals remained
immobile, whereas D2R/ showed preconvulsive behaviors (rigid
posture, tail stiffening, and forelimb extension) (Fig.
2). At 20 mg/kg, D2R/ mice showed
clear symptoms of limbic epileptic activity (repeated episodes of
rearing with forelimb clonus, alternating with rigid posture and head
bobbing) (Fig. 2), whereas WT and D2R+/ mice exhibited only
preconvulsive signs (Fig. 2). Finally, the high dose of 35 mg/kg
induced repeated limbic seizures of the same severity in the three
genotypes during the whole period of observation (Fig. 2). In each
treatment group, no difference was observed between male and
female mice of the same genotype. Saline-treated animals of all
genotypes showed no sign of epileptic activity during the entire period
of observation (data not shown). In parallel with these experiments, KA
effects were also evaluated in 129/Sv and C57BL/6 mice, the two strains of mice from which D2R/ mutants are derived (Baik et al., 1995). Both of these strains exhibited a similar behavioral KA dose-response profile as D2R+/ and WT siblings (Fig. 2).
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Figure 2.
Severity of KA-induced seizures in WT, D2R+/,
D2R/, C57BL/6, and 129/Sv mice. Dose-response effect of KA
treatments in the different genotypes tested, as indicated
(n = 10 mice per genotype). Columns represent the
maximum seizure rating scale value scored by each genotype over a
period of 2 hr after KA administration. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; Mann-Whitney U test between D2R/ and each of
the other genotypes.
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We next used immediate early gene (IEG) induction (Morgan and Curran,
1991; Willoughby et al., 1997) to study the spatiotemporal pattern of
neuronal activation in WT and D2R/ mice after 20 mg/kg KA
treatment. One hour after KA injection, a strong c-fos mRNA
induction was detected in the hippocampus and cerebral cortex in both
genotypes compared with saline-treated mice (Fig.
3a). Three hours after KA
administration, a strong and widespread c-fos expression was
detected throughout the entire brain in D2R/ mice (Fig.
3a), whereas WT animals showed a reduced labeling, restricted mainly to the hippocampus. A sustained c-fos
expression was still present in the hippocampus and cerebral cortex in
D2R/ mice 6 hr after KA treatment (Fig. 3a), whereas no
labeling was detected in WT siblings (except a weak labeling in
scattered cells of the hippocampus and cerebral cortex) (Fig.
3a). A robust c-jun induction was also observed
in the dentate gyrus, CA1, and CA3 regions 6 hr after 20 mg/kg KA
administration in D2R/ but not WT animals (Fig. 3b).
Thus, IEG expression correlates with the occurrence of limbic seizures
in mutant mice.
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Figure 3.
Spatiotemporal pattern of IEGs induction in the
brain of WT and D2R/ mice treated with 20 mg/kg KA.
c-fos (a) and c-jun
(b) mRNA in situ hybridizations.
Genotypes, treatments, and relevant brain areas are as indicated.
CA1, CA3, Pyramidal cell layers of the
hippocampus; ctx, cerebral cortex; DG,
dentate gyrus; thal, thalamus. Scale bars, 1.8 mm.
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c-jun induction is considered as one of the first steps in
the execution of neuronal programmed cell death (Estus et al., 1994).
Thus, we investigated two well documented parameters of this process:
DNA fragmentation (Pollard et al., 1994) and upregulation of the
proapoptotic protein BAX (Gillardon et al., 1995; Krajewski et al.,
1995; Deckwerth et al., 1996). WT and D2R/ mice of both sexes were
killed 16 hr after 20 or 35 mg/kg KA administration. This time point
was chosen according to previous studies, which investigated the time
course of KA-induced hippocampal programmed cell death (Sakhi et al.,
1994). TUNEL staining of brain sections revealed the presence of
fragmented DNA in CA3 cells in D2R/ but not WT mice (Figs.
4a,b,
5). In D2R/ mice, the number of TUNEL-positive cells was significantly increased in a dose-dependent manner (Fig. 5). In each treatment group, no difference was observed between male and female mice of the same genotype. A strong BAX immunoreactivity was also observed in the CA3 region of D2R/ but
not WT animals 16 hr after KA (20 and 35 mg/kg) administration (Fig.
4c). Basal levels of BAX protein were almost undetectable and did not differ in the hippocampus of WT and D2R/ mice (data not
shown). In D2R/ mice, the extension of BAX labeling was larger than
that of TUNEL staining (Fig. 4, compare b, c),
suggesting that a residual number of BAX-positive CA3 neurons are not
yet degenerated 16 hr after KA treatment. Nissl coloration of brain sections from animals killed 5 d after KA (20 and 35 mg/kg)
injection revealed a clear breach of staining in the CA3 subfield of
D2R/ but not WT mice (Fig.
6a,b). Cell loss in
D2R/ mice was present only in the CA3 subfield but not in other
regions of the hippocampus (Figs. 4, 6). The extension of tissue damage
in the CA3 subfield, as revealed by the sclerotic zone and by the
presence of densely stained pyknotic cells outside this zone (Fig.
6b and data not shown), was larger than the TUNEL-positive
region observed 16 hr after KA treatment (compare Figs. 4b,
6b). Consistent with brain tissue damage, an extensive
gliosis was observed in the CA3 subfield of D2R/ mice by GFAP
immunohistochemistry (Dusart et al., 1991) (Fig. 6c).
Morphological analysis of Nissl-stained sections was also performed to
evaluate the degree of CA3 hippocampal cell damage in the different
genotypes and treatment groups. Table 1
shows the number of mice of each genotype and treatment that expressed
none, little, mild, or severe cell damage (see Materials and Methods).
The degree of CA3 cell damage (Table 1, Fig.
6a,b) in D2R/ mice treated with 35 mg/kg KA
was higher than in D2R/ mice treated with 20 mg/kg KA, indicating a
dose-dependent toxic effect. This was also confirmed by the higher
number of TUNEL-positive cells (Figs. 4a,b, 5)
and by the larger extension of reactive gliosis (Fig. 6c)
detected in D2R/ mice treated with 35 mg/kg KA compared with
D2R/ mice treated with 20 mg/kg KA. For each treatment, no
difference in the degree of CA3 cell damage was observed between male
and female mice of the same genotype. Saline-treated control animals of
both genotypes revealed no histological abnormalities or gliosis in the
hippocampus (data not shown).
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Figure 4.
KA-evoked seizures induce CA3 neuron apoptosis in
D2R/ but not WT mice. a, TUNEL staining of the whole
dorsal hippocampus from mice killed 16 hr after KA treatment.
b, High-power magnifications taken from the same
sections shown in a. Arrowheads in
a and b indicate the extension of TUNEL
in a restricted part of the CA3 region. c, BAX
immunoreactivity in CA3 pyramidal cells of WT and D2R/ mice killed
16 hr after KA treatment. The absence of BAX labeling in the CA3 region
of D2R/ mice treated with 35 mg/kg KA (between
arrowheads) corresponds to the region containing TUNEL-positive
cells. Genotypes and treatments are as indicated. Scale bars:
a, 300 µm; b, c, 70 µm.
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Figure 5.
Quantification of TUNEL-positive hippocampal CA3
cells in WT and D2R/ mice treated with KA. Genotypes and treatments
are as indicated. Values are expressed as the mean ± SEM of
TUNEL-positive CA3 cells per area, as described in Materials and
Methods. *p < 0.05, D2R/ 20 mg/kg KA
(KA20) treatment versus WT KA20 and D2R/
saline; ***p < 0.0001, D2R/ 35 mg/kg KA
(KA35) treatment versus WT KA35 and D2R/
KA20; Mann-Whitney U test;
n = 5 animals per treatment group.
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Figure 6.
KA-evoked seizures induce CA3 cell damage in
D2R/ but not WT mice. a, Nissl staining of the whole
dorsal hippocampus from mice killed 5 d after KA treatment.
Asterisks indicate cell loss in a restricted part of the
CA3 subfield in D2R/ mice. b, High-power
magnifications taken from the same sections shown in a.
c, GFAP immunostaining of the CA3 region. Genotypes and
treatments are as indicated. Scale bars: a, 270 µm;
b, c, 70 µm.
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Reults shown demonstrate that, despite seizure activity that was
similar in WT and D2R/ mice treated with 35 mg/kg KA (Fig. 2), at
this KA dose, hippocampal cell death was only observed in D2R/
mice. To investigate the relationship between IEG expression and
hippocampal cell death, we therefore analyzed c-fos and
c-jun mRNA induction in seizing WT and D2R/ mice treated
with 35 mg/kg KA. Three hours after KA administration, WT and D2R/
mice exhibited a comparable level of c-fos induction in the
hippocampus, cerebral cortex, and subcortical areas (Fig.
7a). Conversely, a stronger c-jun induction was observed in the hippocampus and cerebral
cortex of D2R/ mice 12 hr after KA administration compared with WT animals (Fig. 7b). Thus, c-jun but not
c-fos mRNA induction correlates with the occurrence of cell
death in D2R/ mice.
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Figure 7.
IEGs induction in the brain of WT and D2R/
mice treated with 35 mg/kg KA. c-fos
(a) and c-jun
(b) mRNA in situ hybridizations.
Genotypes are as indicated. Scale bar, 1.2 mm.
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KA-evoked seizures do not induce hippocampal cell damage in certain
mouse strains (i.e., pure C57BL/6 or mixed 129/Sv × C57BL/6) (Schauwecker and Steward, 1997). Thus, the absence of KA-induced brain
damage (despite the presence of limbic seizures) (Fig. 2) in WT mice
derived from the mixed 129/Sv × C57BL/6 genetic background (Figs.
4, 6) further supports our finding on a key role of D2R in the
prevention of hippocampal cell death. However, to exclude a possible
contribution of the 129/Sv genetic background to the observed effects
(Silva et al., 1997), congenic C57BL/6 D2R/ mice were also analyzed
for their susceptibility to KA-induced excitotoxicity. In agreement
with previous findings (Schauwecker and Steward, 1997), Nissl staining
of brain sections from animals killed 5 d after KA treatment (20 or 35 mg/kg) did not reveal hippocampal cell damage in WT congenic
C57BL/6 mice (Fig. 8a). Conversely, an extensive cell loss and tissue sclerosis in the CA3
region was observed in D2R/ mice of the same background (Fig.
8b).
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Figure 8.
KA-evoked seizures induce CA3 cell damage in
D2R/ but not WT congenic C56BL/6 mice. Nissl staining of the whole
dorsal hippocampus from WT (a) and D2R/
(b) mice killed 5 d after 35 mg/kg KA
treatment. Asterisks indicate cell loss in a restricted
part of the CA3 subfield in D2R/ mice. Scale bar, 150 µm.
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DISCUSSION |
The increased response of D2R/ mice to nonepileptogenic doses
of KA demonstrates that the absence of D2R lowers the threshold for
KA-induced limbic seizures. This suggests that dopamine, through D2R,
exerts an inhibitory control on the response to seizure-promoting stimuli. The increased susceptibility to KA seizures showed by D2R/
is not attributable to a genetic background effect, because the two
mouse strains from which D2R/ mice are derived (C57BL/6 and 129/Sv)
showed the same KA dose-response profile as the WT derived from the
D2R/ line. Interestingly, half dosage of D2R in D2R+/ mice is
sufficient to provide the inhibitory control on limbic seizures because
these animals showed no limbic seizures at 20 mg/kg KA.
The expression patterns of the immediate early genes c-fos
and c-jun also correlate with the occurrence of limbic
seizures in D2R/ mice. Our findings indicate that, in D2R/
mice, KA-induced epileptic activity arises in the hippocampus and then
spreads to other brain areas. Thus, a tonic D2R-mediated dopaminergic inhibitory control regulates the activation of limbic areas during seizures. Indeed, D2-sensitive seizure-regulating sites have been already identified in the hippocampus and other limbic areas (Starr, 1996). D2Rs, synthesized by pyramidal cells of CA1/CA3 fields and by
granule cells of the dentate gyrus (Mansour et al., 1990), are mainly
localized in the stratum lacunosum moleculare of the CA1/CA3 fields
(Fig. 1a) (Martres et al., 1985), which contains the
dendrites of pyramidal and granule cells (Amaral and Witter, 1995).
D2Rs located on these cells could therefore contribute to the
inhibition of hippocampal neuron excitability. Indeed, dopamine has
markedly inhibitory effects on hippocampal pyramidal cells, as
suggested by a series of electrophysiological studies (Stanzione et
al., 1984; Pockett, 1985; Malenka and Nicoll, 1986; Otmakhova and
Lisman, 1999). Increased excitatory glutamatergic neurotransmission in
hippocampal neurons of D2R/ mice is not attributable to altered
levels of glutamate receptors, because comparable levels of KA and AMPA
receptors were found in the brain of WT and D2R/ mice (Fig. 1). A
speculative hypothesis is that the absence of D2R might potentiate
glutamatergic transmission by increasing the phosphorylation of
glutamate receptors in the hippocampus. Indeed, glutamate receptor
responses are potentiated via phosphorylation mechanisms (Wang et al.,
1991; Roche et al., 1996), which are contributed to by dopamine
receptor signaling (Hatt et al., 1995; Greengard et al., 1999; Yan et
al., 1999).
A major finding of this study is that, in D2R/ mice, the
stimulation of glutamate neurotransmission by KA results in extensive hippocampal cell death. Induction of c-jun mRNA and the
proapoptotic factor BAX, as well as the presence of TUNEL-positive
cells, suggest that hippocampal cell death in KA-treated D2R/ mice
mainly occurs by apoptosis (Charriaut-Marlangue et al., 1996). The
extension of BAX labeling and tissue damage in the CA3 subfield of
D2R/ mice were larger than that of TUNEL staining (compare Figs.
4c, 6b with 4b), clearly indicating
the progressive nature of apoptotic hippocampal cell death after
KA-induced seizures (Ben-Ari, 1985; Charriaut-Marlangue et al., 1996).
Cell loss in D2R/ mice was present only in the CA3 subfield (Figs.
4, 6) but not in other regions of the hippocampus. It is well known
that CA3 pyramidal neurons are the most vulnerable to the excitatory
and neurotoxic effects of KA (Ben-Ari, 1985) because of the high
density of KA binding sites in this region (Fig. 1b) (Berger
and Ben-Ari, 1983).
Recent studies have shown that epileptogenic doses of KA induce
hippocampal cell death only in some mouse strains (such as 129/Sv) but
not in others (such as C57BL/6 or 129/Sv × C57BL/6 hybrids),
whereas all of these strains exhibit the same susceptibility to
KA-induced seizures (Schauwecker and Steward, 1997). It has been
proposed previously (Schauwecker and Steward, 1997) that (1) the
occurrence of KA-induced seizures in mice is not necessarily related to
the occurrence of hippocampal cell death, and (2) the protection and
susceptibility to KA-induced excitotoxicity are determined by specific
gene(s) whose expression and/or function differs among different mouse
strains. Our results suggest that the D2R could be one of these genes.
In fact, genetic ablation of D2R confers susceptibility to KA
excitotoxicity in two mouse strains (mixed 129/Sv × C57BL/6 or
congenic C57BL/6) that are known to be resistant to KA-induced neurodegeneration.
We reported previously that D2R ablation causes hormonal dysfunctions
(Saiardi et al., 1997), mainly in female animals. It is well known from
a large series of studies that steroid hormones can influence seizure
threshold and seizure-related brain damage (Joels, 1997; Green and
Simpkins, 2000). However, in the present study, we could not find
evidence of difference between genders. This suggests that, at least in
our experimental model, hormonal alterations are not crucially involved
in the regulation of seizures and seizure-related cell death.
To our knowledge, our data constitute the first in vivo
demonstration of a direct role of D2R signaling in the control of epilepsy-related brain damage. We speculate that dopamine signaling, through D2R, modulates the susceptibility of hippocampal neurons to
enter apoptotic cell death pathways. In this respect, it has been
proposed that dopamine D2 receptors can exert a protective role against
glutamate-induced neurotoxicity in vitro (Amano et al.,
1994; Sawada et al., 1998). Glutamate toxicity has also been implicated
in cerebral ischemia and Parkinson's disease (Meldrum, 1995; Blandini
et al., 1996; Lange et al., 1997). In vivo, dopamine D2-selective agonists can protect hippocampal neurons against degeneration after cerebral ischemia (O'Neill et al., 1998). Moreover, several authors have postulated a neuroprotective role of dopamine D2
receptors in different animal models of parkinsonism (Olanow et al.,
1998). Indeed, the D2-selective agonist bromocriptine is able to
protect nigral neurons against
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration (Lange et al., 1994; Muralikrishnan and Mohanakumar, 1998).
A large series of experimental and clinical studies strongly suggest
that dopamine, through D2 receptors, can exert an anti-epileptic action
(Starr, 1996). Interestingly, KA-induced seizures in rodents are
generally considered as a good model of human temporal lobe epilepsy
(TLE) (Ben-Ari, 1985). Indeed, TLE can be successfully treated with the
administration of D2-like selective agonists and is also associated, at
least in certain cases, to pituitary tumors (Clemens, 1988;
Gatterau et al., 1990) that resemble those exhibited by D2R/ mice
(Saiardi et al., 1997). Together with these findings, our data suggest
that TLE might be attributable to a reduced expression or function of
dopamine D2 receptors. Our results also support the notion that
dopamine, through D2Rs, can exert a protective role against
neuropathologies that involve glutamate-induced neurodegeneration,
including epilepsy, ischemia, schizophrenia (Csernansky and Bardgett,
1998), and Parkinson's disease.
| |
FOOTNOTES |
Received June 23, 2000; revised Aug. 24, 2000; accepted Sept. 6, 2000.
This work was supported by grants from the Institut National de la
Santé et de la Recherche Médicale, Centre National de la
Recherche Scientifique, Hôpital Universitaire de Strasbourg, and
Association de la Recherche sur le Cancer to E.B. Y.B. was supported by fellowships from the Ligue Nationale contre le Cancer and
Fondation pour la Recherche Médicale. D.V. was supported by a
European Community fellowship. We thank Y. Ben-Ari, H. Gozlan, P. Sassone-Corsi, N. Foulkes, and all the members of the laboratory for
discussions. We thank V. Giroult, B. Boulay, and J.-M. Lafontaine for
technical help.
Correspondence should be addressed to Emiliana Borrelli, Institut
de Génétique et de Biologie Moléculaire et
Cellulaire, Centre National de la Recherche Scientifique/Institut
National de la Santé et de la Recherche
Médicale/Université Louis Pasteur, BP163, 67404 Illkirch
Cedex, Communauté Urbaine de Strasbourg, France. E-mail:
eb{at}igbmc.u-strasbg.fr.
Dr. Bozzi's present address: Neurobiology Laboratory, Scuola Normale
Superiore, Consiglio Nazionale delle Ricerche, Institute of
Neurophysiology, Via Alfieri 1, 56010 Ghezzano, Pisa, Italy. E-mail:
yuri{at}in.pi.cnr.it.
| |
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ABSTRACT
INTRODUCTION
