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The Journal of Neuroscience, September 15, 2002, 22(18):7931-7940
Impaired D2 Dopamine Receptor Function in Mice Lacking Type 5 Adenylyl Cyclase
Ko-Woon
Lee1, 2,
Jang-Hee
Hong1, 3,
In Young
Choi1,
Yongzhe
Che4,
Ja-Kyeong
Lee4,
Sung-Don
Yang5,
Chang-Woo
Song5,
Ho Sung
Kang2,
Jae-Heun
Lee3,
Jai Sung
Noh6,
Hee-Sup
Shin7, and
Pyung-Lim
Han1
1 Department of Neuroscience and Ewha Institute of
Neuroscience, Ewha Womans University School of Medicine, Seoul,
110-783, Korea, 2 Department of Molecular Biology, Pusan
National University, Pusan, 609-735, Korea, 3 Department of
Pharmacology, College of Medicine, Chungnam National University,
Taejon, 301-747, Korea, 4 Department of Anatomy, Inha
University School of Medicine, Inchon, 400-712, Korea,
5 Toxicology Research Group, Korea Research Institute of
Chemistry and Technology, Taejon, 305-345, Korea,
6 Department of Psychiatry, Ajou University School of
Medicine, Suwon, 442-721, Korea, and 7 National Creative
Research Initiative Center for Calcium and Learning, Korea Institute of
Science and Technology, Seoul, 136-791, Korea
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ABSTRACT |
Dopamine receptor subtypes D1 and
D2, and many other seven-transmembrane receptors
including adenosine receptor A2A, are colocalized in
striatum of brain. These receptors stimulate or inhibit adenylyl cyclases (ACs) to produce distinct physiological and pharmacological responses and interact with each other synergistically or
antagonistically at various levels. The identity of the AC isoform that
is coupled to each of these receptors, however, remains unknown. To
investigate the in vivo role of the type 5 adenylyl
cyclase (AC5), which is preferentially expressed in striatum, mice
deficient for the AC5 gene were generated. The genetic
ablation of the AC5 gene eliminated >80% of
forskolin-induced AC activity and 85-90% of AC activity stimulated by
either D1 or A2A receptor agonists in striatum. However, D1- or A2A-specific pharmaco-behaviors
were basically preserved, whereas the signal cascade from
D2 to AC was completely abolished in
AC5
/, and motor
activity of AC5/ was
not suppressed by treatment of cataleptic doses of the antipsychotic drugs haloperidol and sulpiride. Interestingly, both haloperidol and
clozapine at low doses remarkably increased the locomotion of
AC5/ in the open field
test that was produced in part by a common mechanism that involved the
increased activation of D1 dopamine receptors. Together,
these results suggest that AC5 is the principal AC integrating signals
from multiple receptors including D1,
D2, and A2A in striatum and the cascade
involving AC5 among diverse D2 signaling pathways is
essential for neuroleptic effects of antipsychotic drugs.
Key words:
AC5; dopamine receptors; knock-out mice; antipsychotics; striatum; adenylyl cyclase; cAMP; adenosine
receptors
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INTRODUCTION |
Dopaminergic systems play central
roles not only in movement behaviors and motivated behaviors but also
in pathological states, including Parkinson's disease, drug
addictions, and schizophrenia (Ebadi and Srinivasan, 1995
; Missale et
al., 1998). Neuroanatomical substrates underlying these physiological
and pathological responses include the striatum, where dopamine
receptors are highly enriched (Levey et al., 1993; Surmeier et al.,
1996). Historically, all five dopamine receptor isoforms cloned in
mammals have been envisioned to stimulate or inhibit adenylyl cyclase
(AC) to generate their physiological and pharmacological responses.
After activation, D1-type dopamine receptors
(D1 and D5) stimulate AC,
whereas D2-type dopamine receptors
(D2, D3, and
D4) inhibit AC (Creese et al., 1983; Missale et
al., 1998; Sidhu and Niznik, 2000). Despite the long history of
dopamine receptor biology, the identity of the AC subtype that is
coupled to specific dopamine receptor isoforms in the brain has not
been verified.
To date, more than 10 different types of ACs, including cytosolic AC,
have been cloned and characterized in mammals (Buck et al., 1999;
Hanoune and Defer, 2001). Studies on the expression of ACs showed that
many AC isoforms were expressed at various levels in striatum (Glatt
and Snyder, 1993; Cali et al., 1994; Mons and Cooper, 1994; Lane-Ladd
et al., 1997; Matsuoka et al., 1997; Antoni et al., 1998; Liu et al.,
1998; Mons et al., 1998). The type 5 adenylyl cyclase (AC5) has been
thought to be a component of dopamine receptor signaling, primarily
because it was highly concentrated in striatum where
D1 and D2 were expressed
abundantly (Glatt and Snyder, 1993; Matsuoka et al., 1997).
Unfortunately, further progress in understanding the in vivo
role of ACs, including AC5, in dopamine receptor biology has been
hampered by the lack of AC subtype-specific inhibitors.
The A2A receptors are highly concentrated
in striatum, particularly in GABAergic striatopallidal
neurons where A2A and D2 receptors are colocalized (Fink et al., 1992; Augood and Emson, 1994).
Antagonistic interactions between A2A and
D2 receptors in the basal ganglia system
have been proposed to play a key role in the motor depressant effects
of adenosine receptor agonists and the motor stimulant effects of
adenosine receptor antagonists, such as caffeine (Ferre et al., 1997).
In addition, many other seven-transmembrane receptors, including opioid
receptors and muscarinic acetylcholine receptors, are also highly
concentrated in striatum. These receptors use AC as their effector and
interact with dopaminergic systems synergistically or antagonistically at various levels (Gomeza et al., 1999; Jang et al., 2000). However, the integrity of the effector system of these receptors in
vivo is basically undetermined.
One strategy to dissect the functional receptor-effector system is to
create mice lacking an effector candidate and to examine the signaling
pathway of receptors of interests with the mutant animals. Therefore,
the current study was undertaken to unravel the interaction of AC5 with
dopamine receptors and other receptors in striatum using AC5-deficient
mice. We demonstrated that AC5 interacted with many receptors
including dopamine receptors; consequently it acted as an effector
of integrating signals from multiple receptors.
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MATERIALS AND METHODS |
AC5 knock-out cassette, homologous
recombination of the AC5 gene, and genetics. The
N-terminal 369 bp cDNA fragment of the rat AC5 (GenBank
accession no. M96159) (Premont et al., 1992) was amplified by PCR using
two primers, 5'-GTCGAGGAAAAGGCCGAGCGGCCGAGG-3' and
5'-CAGCCATGATAAGGATCACGCCCACAG-3'. The fragment was used to screen for
mouse AC5 genomic DNA clones from a mouse 129SVJ genomic DNA
library (Stratagene). Two overlapping phage clones were isolated and
characterized to contain the 1.3 kb ApaI fragment that
covered the first half of predicted mouse AC5. The genomic DNA
sequences around the 1.3 kb ApaI fragment were determined,
and an exon identified in the fragment was designated as exon 2. A part
of the AC5 sequences was deposited into GenBank (AF417936).
The targeting cassette was constructed by subcloning the 2.2 kb
short-arm (BamHI-exon 2) and the 5.3 kb long-arm
(ApaI-XhoI) at each side of pGK-neo and TK in
front of the short-arm as depicted in Figure 1A. The pGK-neo clones were provided by P. Soriano (Fred Hutchison
Cancer Research Center, Seattle, WA) and D. S. Lim (Korea
University, Seoul, Korea). ES cell culture and embryo handlings
were conducted by following a standard procedure (Joyner, 1993; Kim et
al., 1997). Briefly, J1 embryonic stem (ES) cells were used for
homologous recombination, and seven independent chimeric mice were
generated. They were bred to C57BL/6J mice to obtain heterozygote F1
mice. Intercrossing between heterozygote F1 produced F2 hybrids of
homozygote (AC5/),
heterozygote (AC5+/), and
wild-type (AC5+/+) littermates, which were
used in this study.
Northern, Southern, and Western blot analyses. Northern blot
analysis was performed as described (Kim et al., 1999). Briefly, membrane blot carrying 30 µg of total RNA in each lane was prepared and hybridized with 32P-labeled probes
prepared from the PCR-amplified 529 bp fragment (4232-4760) of the
3'-end of rat AC5 cDNA (Premont et al., 1992). For genomic Southern
blots, a blot carrying XbaI-digested genomic DNA was
hybridized with the 32P-labeled 0.5 kb
SalI-HindIII fragment as indicated (see Fig. 1A). A PCR method was also used in genotyping. The
PCR primers were AC5-(A), 5'-ACCGTCGAGGATGGAGACGG-3' (971-990);
AC5-(A+), 5'-GTGGCTGTGGCAGC AACAGG-3' (1383-1402); and AC5-(pGK2r),
5'-CAGCGCGGCAGACGTGCGCT-3'. The PCR using the (A) and (A+) or (A) and
(pGK2r) combination generates 432 bp of wild-type allele and 665 bp of
mutant allele, respectively. For Western blot analyses, primary
antibodies for PKA C
(Santa Cruz
Biotechnology), PKA RI (Transduction Laboratories), PKA
RII (Santa Cruz Biotechnology),
Gs, Gi, Go,
and Gz (Santa Cruz
Biotechnology) were purchased.
Histological examinations. Tissue sections for
immunohistochemical staining, receptor binding study, or
hematoxylin-eosin staining were prepared as described (Kim et al.,
1999; Lee et al., 1999). Briefly, brain, heart, or kidney sections cut
at 40 µm by vibratome, at 14 µm by cryostat, or at 4 µm by
microtome after embedding with paraffin were fixed in 4%
paraformaldehyde for 15 min and placed for histological study. Animals
were handled in accordance with the guideline of animal care at Ewha
Womans University School of Medicine. The polyclonal anti-AC5 antibody was purchased from Santa Cruz Biotechnology. Primary antibodies for
substance P (Chemicon), dynorphin (Oncogene), neuropeptide Y
(Chemicon), enkephalin (Chemicon), tyrosine hydroxylase (Chemicon), GAD
(Chemicon), and parvalbumin (Santa Cruz Biotechnology) were purchased.
AC assay. Striatum, frontal cortex of cerebrum, and
cerebellum of adult brain were excised and subjected to AC assays. In preparations of the striatum, the dorsal striatum (caudate and putamen)
and ventral striatum (the nucleus accumbens and its surrounding regions) were included unless indicated otherwise. In preparations of
the frontal cortex, we included most cortical regions of the frontal
lobe but excluded olfactory bulbs, striatum, thalamus, and the cortical
regions posterior to the motor and sensory cortices. In preparations of
the cerebellum, anterior and posterior lobes were included.
AC assay was performed as described (Onali et al., 1985; Olianas et
al., 1997) with a modification using
125I-cAMP and anti-cAMP antibody (Amersham
Biosciences). Prepared brain tissues were homogenized separately using
a Teflon/glass homogenizer in 10 mM imidazole, pH 7.3, 2 mM EDTA, and 10% sucrose. After tissue debris was spun
down at 1000 rpm (23 × g), crude membrane fractions
were prepared by centrifugation at 25,000 × g at 4°C
for 30 min and suspended in 10 mM imidazole or 10 mM Tris-HCl (for D2 assay)
to a final concentration of 2-3 µg/µl. The resulting membrane
samples were aliquoted in 25-30 µl volume and stored at 
70°C
until use. For each AC reaction, 20 µg of total protein was used. All
reactions below were prepared in 0.1 ml volume, and membrane samples
were added last.
The forskolin-induced AC activation was produced in 100 mM
HEPES, pH 7.4, 100 mM NaCl, 4 mM
MgCl2, 2 mM EDTA, 0.5 mM
3-isobutyl-1-methylxantine (IBMX), 2 mM ATP, 20 mM phosphocreatine, and 5 U of creatine phosphokinase with
10 µM forskolin at 30°C for 15 min in
vitro.
For D1 and A2A activation
assays, reactions were performed in 10 mM imidazole, pH
7.3, 0.5 mM MgCl2, 0.5 mM
IBMX, 0.2 mM EGTA, 0.5 mM DTT, 0.01 mM pargyline, 1 µM GTP, 2 mM ATP,
20 mM phosphocreatine, and 5 U of creatine phosphokinase at
30°C for 15 min in vitro.
For the D2 activation assay, reactions were
incubated in 80 mM Tris/HCl, pH 7.4, 2 mM
MgSO4, 1 mM EGTA, 150 mM
NaCl, 0.5 mM IBMX, 0.5 mM DTT, 0.05 mM GTP, and 0.2 mM ATP at 30°C for 5 min in vitro. For the muscarinic acetylcholine receptor assay,
reactions were made in 80 mM Tris/HCl, pH 7.4, 2 mM MgCl2, 0.3 mM EGTA, 1 mM DTT, 0.5 mM IBMX, 0.1 mM GTP, and
0.2 mM ATP at 30°C for 5 min in
vitro.
Reactions were terminated by adding 0.5 ml of 0.1N HCl and centrifuged
at 22,000 × g for 5 min, and supernatant was taken. The amount of cAMP formed was determined by the
125I-cAMP assay system (Amersham
Biosciences). The assay was based on the competition between unlabeled
cAMP and a fixed quantity of 125I-cAMP and
anti-cAMP antibody. According to the manufacturer's instructions,
higher assay sensitivity was obtained by acetylation of protein
samples. Three microliters of the reaction sample were mixed with 100 µl of 0.05 M acetate buffer, pH 5.8. After
adding 8 µl of the mix of 1 vol of acetic anhydride and 2 vol of
triethylamine, reactions were vortexed vigorously for 7-8 min. Twenty
microliters of acetylated sample were mixed with 80 µl of assay
buffer (0.05 M acetate buffer, pH 5.8), followed
by adding 100 µl of anti-cAMP antibody. After incubation at 4°C for
3 hr, they were added with 100 µl of
125I-cAMP (30,000 cpm/ml) and incubated
further at 4°C overnight. Then they were mixed with 500 µl of
Amerlex-M secondary antibody conjugated with magnetic beads and
incubated at room temperature for 15 min. After centrifugation at
3000 × g for 10 min, pellet was used to determine the
bound radioactivity by Packard gamma counter. The radioactivity was
converted to picomoles of cAMP by comparison to the reference curve
that was constructed with standards. AC activities were presented by
averaging three to six independent measurements with duplicates, for
which protein samples were taken from more than three animals for each genotype.
Molecular cloning ACs present in the striatum of
AC5/. To identify AC responsible for
the AC activity in the striatum of
AC5/, RT-PCR analyses were
performed using a battery of primer sets: 5'-GACATTGTGGGCTTCAC-3'
and 5'-CTTCAGTAGCCTCAGCC-3' for AC1, 5'-CCTCGACACACTCTGGACGG-3' and
5'-GCTGGCAGTGCAGTAGCTC for AC2, 5'-GATGCAGCTGCTGAGGGAG-3' and
5'-CAGTCTTGGTCTTCTCCCGC for AC3, 5'-GGATTGCTGTCTTCTCTGG-3' and
5'-GTAGGTGATGATCAGAGCTG-3' for AC4, 5'-ACCGTCGAGGATGGAGACGG-3' and
5'-GTGGCTGTGGCAGCAACAGGC-3' for AC5, 5'-CCTGATACTCGGGATTTATG-3' and
5'-CCACAGCTGGGCAGTCCAG-3' for AC6, 5'-CAACATTGAATCACCTGGAC-3' and
5'-GATGGCCTGGAGTGTACTTC-3' for AC7, and 5'-CCGGCCTGGGCACATCTTTG-3' and
5'-CGGCGGGGCTCAGGCAGTC-3' for AC8. Undesired amplification of genomic
DNA in RT-PCR was monitored by using total RNA as template. The partial
sequences of mouse AC3 cDNA clone was deposited in GenBank
(AF253540).
D1 and D2
dopamine receptor binding assays. Striatum was homogenized
using a Teflon/glass homogenizer in 10 mM
Tris·HCl, pH 7.4, and crude membrane fractions were prepared.
Receptor-ligand binding experiments were performed using
3H-SCH23390 or
3H-spiperone for D1
and D2 receptors, respectively. Cold SCH23390 or
butaclamol was used for blocking of nonspecific binding. Binding reactions were performed for 1 hr at room temperature. For the 125I-sulpiride binding study, brain
sections mounted on a glass slide were overlaid with ligand binding
buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, and 0.1% BSA) containing 0.5 nM 125I-sulpiride
(2000 Ci/mmol). Nonspecific binding was blocked with cold sulpiride.
After incubation at room temperature for 60 min, the slides were washed
with ice-cold ligand binding buffer and ice-cold water, air dried, and autoradiographed.
Behavioral assessments. For the open field test, mice
(10-20 weeks) were placed individually in a test chamber and monitored on a TV screen after video recording. A test chamber consisted of a
60 × 60 cm2 floor with 40-cm-high
walls in which the floor was marked by lines to have 25 equal squares.
Animals were allowed to spend 15 min in the chamber for acclimation. At
the end of acclimation, each drug dissolved in 120 µl of 0.9% saline
was administered intraperitoneally. SKF38393, SCH23390, CGS21680,
quinpirole, oxotremorine, haloperidol, clozapine, sulpiride, and
dihydrexidine were purchased from Tocris. Horizontal locomotor activity
was judged by cumulative counts of line crossovers of animals for a 60 min period (15-75 min). Later, we established a computerized video
tracking system that was used to obtain the data for the open field
system in Figures 3D, 4D, and
8A,B. For this system, we used a
test chamber consisting of a 45 × 45 cm2 floor with 40-cm-high walls.
Horizontal locomotor activity was judged by the distance of the
animal's movement for 60 min after drug administration. In all
experiments, mice that were administered drugs were not used
further. For behavioral assessments, n = 4-9 for each genotype, unless indicated otherwise.
Catalepsy was measured by a bar test with a cutoff time of 3 min. The
forepaws of the mice were placed on a 1-cm-diameter bar held 5 cm above
the floor and 5 cm from the front wall, and the time spent in the given
posture was regarded as an indication of catalepsy.
Statistical analysis. Two-sample comparison was performed
using Student's t test, and multiple comparisons were made
using one-way ANOVA followed by the Newman-Keuls multiple range test. All data were presented as the mean ± SEM, and a statistical
difference was accepted at 5% level unless indicated otherwise.
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RESULTS |
Generation of AC5 knock-out mice
To elucidate the in vivo role of AC5, we generated mice
deficient for the AC5 by means of homologous recombination
(Fig.
1A,B). Homozygous males and females were viable and healthy. Northern analysis
revealed that the AC5 expression was totally abolished in
the striatum of AC5/ mice
(Fig. 1C). In agreement with these data, anti-AC5
immunoreactivity was absent in the brain of
AC5/ mice (Fig.
1D).
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Figure 1.
Targeted disruption of the AC5 gene
locus. A, Restriction map of 129SVJ genomic DNA clone
(wild type), targeting cassette
(pAC5-neo-TK), and homologous recombinant
(mutant). The 1.3 kb ApaI fragment in
wild type carried an exon covering most of the first half of AC5.
Predicted mouse AC5 cDNA at the top was drawn on the
basis of the published rat cDNA sequence, in which TM
indicates the transmembrane domains and C1 and C2
represent the catalytic domains located in the cytoplasmic side.
Drawing is not to scale. X, XbaI;
Xh, XhoI; S,
SalI; H, HindIII;
B, BamHI; A,
ApaI. B, Genomic Southern blot of F2
littermates. Arrows indicate wild-type (13 kb) and
mutant (9 kb) bands on the XbaI-digested genomic DNA
blot. C, Northern blot analysis shows no
AC5 messages in the striatum of
AC5/. The sizes of AC5
message were ~6 and 7.4 kb. D, Immunohistochemical
detection of AC5 expression in brain sections. Anti-AC5 antibody
detected a background level of immunoreactivity in the brain of
AC5/.
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Viability and development of
AC5/
mutant animals
The numbers of homozygotes, heterozygotes, and wild-type
littermates were 97 (23.7%), 210 (51.2%), and 103 (25.1%),
respectively, among the first 410 progenies generated from F1
intercrosses. There was no sex bias in F2 offspring. Both homozygous
males and females were fertile and bred normally. The
AC5/ mice were active in
their home cage and morphologically indistinguishable from their
wild-type littermates. Body weight was slightly reduced to 90-95% of
that of AC5+/+ during the first 20 weeks
after birth (data not shown). AC5 has been thought to be an important
component in the function of heart and kidney during development and in
adult life (Chabardes et al., 1999; Hanoune and Defer, 2001).
Histological examination, however, revealed no obvious difference
between AC5/ and wild-type
littermates in most respects, including the sizes and microscopic
structures of heart and kidney (data not shown). Together, these
results indicate that although the absence of the AC5 gene
results in slightly delayed growth after birth, the AC5 is
not essential for survival in mice.
Impairment of adenylyl cyclase activity in the brain of
AC5/ mice
To assess the AC activity abolished by the mutation of the
AC5, we compared AC activity in the brain of mutant animals
with that of wild-type littermates. In the striatum, the baseline AC activity of AC5/ in the
absence of exogenous activators was slightly reduced when compared with
that of AC5+/+. However, forskolin (10 µM) treatment enhanced AC activity >20-fold in
AC5/, which reached 18%
of that in AC5+/+ (Fig.
2A). Other AC subtypes
present in the striatum of
AC5/, for which we
identified AC1 through AC8, the expression of which was not altered as
determined by semiquantitative RT-PCR analyses (data not shown), may
account for the forskolin-induced AC activity in
AC5/. In the frontal
cortex of cerebrum and in the cerebellum, the forskolin (10 µM)-enhanced AC activity in
AC5/ was diminished to 73 and 60%, respectively, of that in wild-type littermates (Fig.
2B,C). Overall, these results
suggest that AC5 is the major AC in the striatum, whereas it
constitutes a minor AC in the frontal cortex and cerebellum of normal
mice.
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Figure 2.
Impairment of AC activity in
AC5/ mice. The
forskolin (10 µM)-stimulated AC activity in
AC5/ was reduced to
18% in the striatum (A), 73% in the cerebral
cortex (B), and 60% in the cerebellum
(C) of that in
AC5+/+. The baseline AC activity in
AC5/ was slightly
reduced when compared with that of
AC5+/+ (p < 0.05). ** indicates a difference between two groups
(p < 0.01). Base, Baseline;
FSK, forskolin.
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General motor behaviors of naïve mutant animals
We examined whether the genetic ablation of the AC5
gene with the concomitant abolishment of the AC5 activity in the brain could produce any abnormality in the expression of motor behaviors. Regarding spontaneous motor activities including walking, running, climbing, grasping, writhing, and motor skills related to eating, AC5/ mice were
indistinguishable from their wild-type littermates. AC5/ animals displayed no
overt sign of ataxia or tremor, despite the fact that the
forskolin-stimulated AC activity in the cerebellum was reduced to 60%
of that in AC5+/+ (Fig. 2C).
Thus, spontaneous general motor activities of
AC5/ appeared to be normal.
Coupling of D1 dopamine receptors and AC5
The genetic disruption of the AC5 and the presence of
the forskolin-activated residual AC activity in the striatum of
AC5/ prompted us to
examine the receptor-effector interactions in the context of AC5. To
delineate the interactions, we relied on biochemical and
pharmaco-behavioral assay methods. First of all, we explored whether
AC5 is the essential component downstream of D1
dopamine receptor activation. In the striatum of
AC5+/+, the D1
agonist SKF38393 (100 µM)-stimulated AC
activity was increased markedly (29.04 ± 2.04 
53.16 ± 4.24 pmol of cAMP per milligram per minute; p < 0.01;
183% of the baseline). Similarly, the AC activity stimulated by
dihydrexidine (DHX; 100 µM), a full agonist for
D1, was enhanced more than twofold in
AC5+/+ (29.04 ± 2.04 60.84 ± 5.69 pmol of cAMP per milligram per minute; p < 0.01; 210% of the baseline). In the striatum of
AC5/, the SKF38393 (100 µM)-stimulated AC activity was increased at a
low level, but it was consistently higher than the baseline control in
repeated experiments (5.05 ± 0.16 5.73 ± 0.15 pmol of
cAMP per milligram per minute; p < 0.01; 113% of the
baseline). Consistently, the treatment with DHX (100 µM) produced a comparable level of increase in
AC5/ (5.05 ± 0.16 5.80 ± 0.21 pmol of cAMP per milligram per minute; p < 0.01; 115% of the baseline), implying that a
functional
D1-Gs-AC system was present in the striatum of
AC5/ (Fig.
3A). Overall, however, the
increase of SKF38393- or DHX-stimulated AC activity in
AC5/ was reduced to
10-11% of that in AC5+/+. We assessed
separately the DHX-stimulated AC activity in the dorsal (caudate and
putamen) and ventral (nucleus accumbens and its surrounding areas)
striatum and found no difference in their activation folds in these
subdivisions of striatum (data not shown). Together, these results
suggest that D1 receptors use AC5 as their primary AC and other ACs as their alternative pathway. In the frontal
cortex of cerebrum in AC5+/+, where
D1 receptor density is not as high as in
striatum, we observed that the DHX (100 µM)-stimulated AC activity was relatively low
but substantially increased when compared with the baseline control
(10.75 ± 0.26 14.27 ± 0.73 pmol of cAMP per milligram per minute; p < 0.01; 133% of the baseline). In
AC5/, the DHX-stimulated
AC activity was also enhanced in a similar fold (7.91 ± 0.24 10.87 ± 0.29 pmol of cAMP per milligram per minute;
p < 0.01; 137% of the baseline), despite the fact
that overall the DHX-stimulated AC activity in
AC5/ was decreased to 73%
of that in AC5+/+. Thus, other ACs
available in the frontal cortex appear to interact effectively with
D1.
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Figure 3.
D1 and AC coupling. A,
AC assays with D1 agonists, SKF38393 and DHX. The fold
increment of AC activity by SKF38393 (100 µM) or DHX (100 µM) in the striatum (A) of
AC5/ was 11 and 10%,
respectively, of that in AC5+/+
(p < 0.01 for both genotypes). GTP addition
itself increases the baseline AC activity in
AC5+/+ as well as in
AC5/.
B, E, The D1 dopamine
agonist, SKF38393 (50 mg/kg, i.p.), increased the locomotor activity in
both AC5+/+ and
AC5/
(B). The locomotor activity of
AC5/ was dramatically
increased 20 min after administration of SKF38393
(E); however, the mechanism underlying the
behavioral response remains unknown. D, The
D1-specific dopamine agonist, DHX (30 mg/kg, i.p.),
increased the locomotor activity in both
AC5+/+ and
AC5/. Note that
D1 agonist-induced locomotion of
AC5/
was markedly increased, despite the fact that D1
agonist-induced AC activation was severely impaired. C,
F, The D1 receptor antagonist, SCH23390 (0.3 mg/kg,
i.p.), produced the suppression of locomotion in both genotypes
(C). The time-dependent change of the locomotion
induced by SCH23390 is shown in F. * and ** indicate a
difference between two groups at p < 0.05 and
p < 0.01, respectively. Base,
Baseline; Veh, vehicle.
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We questioned whether the severe but incomplete loss of the
D1 agonist-stimulated AC activity in the striatum
of AC5/ would result in
any impaired pharmaco-behaviors in response to D1
agonists or antagonists. To address this, we relied on a behavioral assay paradigm using the open field test. The open field test has been
used widely for the measurement of neuronal outputs of the basal
ganglia system responding to agonists and antagonists of dopamine
receptors, psychomotor stimulants, or antipsychotic drugs (Xu et al.,
1994; Dulawa et al., 1999). Administration of the
D1 agonist, SKF38393 (50 mg/kg, i.p.), induced an
increase of the horizontal locomotor activity in wild-type littermates as reported previously (Gomeza et al., 1999). In
AC5/, the same dose of
SKF38393 produced a robust enhancement of the locomotion.
Interestingly, the increase in the locomotor effects of the D1 agonist
in
AC5/
animals was higher than that observed in
AC5+/+ (Fig.
3B,E). Similar results were
obtained by the administration of the D1-specific
agonist, DHX (30 mg/kg, i.p.) (Fig. 3D). In addition, the
locomotor activity in the open field test after administration of the
D1 antagonist, SCH23390 (0.3 mg/kg, i.p.), was
fully suppressed in AC5/
as seen in AC5+/+ (Fig.
3C,F). Therefore, the
D1 system in
AC5/ appeared to be
functional, at least in part, at the behavioral level, despite the fact
that the AC activity induced by D1 stimulation was diminished severely. The identity of the non-AC5 effector(s) responsible for the observed D1-dependent
pharmaco-behavioral responses was not known in the present study.
Coupling of A2A adenosine receptor and AC5
Similar to those of D1 dopamine receptors,
A2A receptors are preferentially expressed in the
striatum and positively coupled to AC (Ferre et al., 1997; Moreau and
Huber, 1999). We tested whether the anatomical and biochemical analogy
between A2A and D1
receptors can be extended to the receptor-effector coupling mode.
Biochemical assessment indicated that the A2A
agonist CGS21680 (10 µM)-stimulated AC activity in the
striatum of AC5+/+ was highly increased
(26.14 ± 2.51 46.51 ± 6.36 pmol of cAMP per milligram
per minute; p < 0.05; 178% of the baseline). In the
striatum of AC5/, the AC
activity stimulated by CGS21680 was significantly increased (4.94 ± 0.17 7.34 ± 0.52 pmol of cAMP per milligram per minute; p < 0.01; 149% of the baseline), suggesting the
existence of a functional A2A/AC system in the
striatum of AC5/ (Fig.
4A). However, the AC
activity stimulated by CGS21680 (10 µM) in
AC5/ was reduced to 16%
of that in AC5+/+, indicating that AC5 was
the major AC for A2A receptors.
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Figure 4.
A2A and AC5 coupling.
A, The A2A adenosine receptor agonist,
CGS21680 (10 µM), induced an increase of AC activity in
the striatum of AC5+/+ and
AC5/. The
CGS21680-induced increment of AC activity in
AC5/ was 16% of that
in wild-type littermates. B, C, The
A2A agonist CGS21680 (0.5 mg/kg, i.p.) produced the
suppression of locomotion in both
AC5+/+ and
AC5/
(B). The time-dependent change of locomotion
induced by CSG21680 is shown in C. D,
Administration of caffeine (15 mg/kg, i.p.) increased the
locomotion of both AC5+/+ and
AC5/ mice. * and **
indicate a difference between two groups at p < 0.05 and
p < 0.01, respectively. Base, Baseline;
CGS, CGS21680; Veh, vehicle;
Caff, caffeine.
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We examined whether the severe loss of the A2A
agonist-stimulated AC activity in the striatum of
AC5/ could result in
impaired behavior in response to A2A agonist treatment. Administration of the A2A agonist
CGS21680 (0.5 mg/kg, i.p.) induced akinesis broadly in
AC5+/+. Similarly, administration of the
same dose of CGS21680 produced a strong suppression of motor behaviors
in
AC5/
(Fig. 4B,C). The locomotor activity
in the open field after administration of the nonselective adenosine
receptor antagonist, caffeine (15 mg/kg, i.p.), was increased in
AC5/ as seen in
AC5+/+ (Fig. 4D). These results indicate
that the typical A2A receptor-specific pharmacology was retained, at least in part, at behavioral levels in
AC5/.
Coupling of D2 dopamine receptors and AC5
A possibility of coupling between AC5 and D2
was investigated. Because D2 dopamine receptors
are negatively coupled to AC via
Gi protein, the effect
of D2 activation on AC activity can be measured
clearly when D2 is coactivated together with
D1 or other
Gs-coupled
receptors. Indeed, in the striatum of
AC5+/+, D2
activation by quinpirole, a D2 agonist,
suppressed the D1 agonist DHX (0.5 µM)-stimulated AC activity in a dose-dependent manner, as was reported previously (Mottola et al., 1992). However, D2 activation by quinpirole even at high doses
failed to inhibit the DHX-stimulated AC activity in
AC5/ (Fig.
5A). Similarly, the D2 agonist
quinpirole did not produce any inhibitory effects on the AC activation
induced by the neuropeptide VIP (1 µM) or the
A2AR agonist, CGS21680 (0.1 µM) in
AC5/ (Fig.
5B,C). More decisively, the
inhibitory effect of D2 activation on the
forskolin (0.1 µM)-stimulated AC activity,
unlike that in AC5+/+, was totally
abolished in AC5/ (Fig.
5D). Together, these data consistently indicate that the D2-Gi-AC
system is completely impaired in the striatum of
AC5/.
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Figure 5.
D2 and AC5 coupling. A,
The D1 agonist DHX (0.5 µM)-induced AC
activation in AC5+/+ was suppressed in
a dose-dependent manner by quinpirole, but not in
AC5/.
Q(1) and Q(10) denote, respectively, 1 and 10 µM of quinpirole. Base,
Baseline. B-D, The AC activity stimulated by
A2A agonist CGS21680 (0.1 µM)
(B), neuropeptide VIP (1 µM)
(C), or forskolin (0.1 µM)
(D) was suppressed in a dose-dependent manner by
quinpirole in AC5+/+ but not in
AC5/. The
concentrations of DHX (0.5 µM), CGS21680 (0.1 µM), VIP (1 µM), and forskolin
(0.1 µM) for B-D were
chosen on the basis of preliminary experiments, because they produced
~110-130% of AC activation of the baseline control, which was also
applied for the assay of muscarinic acetylcholine receptor activation
in Figure 6. * and ** indicate a difference between two groups at
p < 0.05 and p < 0.01, respectively.
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Coupling of other Gi-coupled receptors
and AC5
We explored whether other
Gi-coupled receptors
also strictly require AC5. The receptors that are expressed abundantly
in the striatum and negatively coupled to AC through Gi protein include
muscarinic acetylcholine receptors M4 and
M2 (Weiner et al., 1990; Gomeza et al., 1999). Biochemical assessment indicated that in the striatum of
AC5+/+, the forskolin-stimulated AC
activity was inhibited in a dose-dependent manner by oxotremorine, an
agonist for muscarinic acetylcholine receptors. The forskolin-induced
AC activity in the striatum of AC5/ was notably
suppressed by oxotremorine, although the oxotremorine-induced inhibition appeared to be partially defective (Fig.
6A). In the frontal cortex of cerebrum in
AC5/, the
forskolin-induced AC activation was also suppressed by oxotremorine (Fig. 6B). Although this study did not aim at
differentiating the identity of the oxotremorine-activated receptors,
the oxotremorine-responsive receptors in the striatum might be
M4 or M2 (Gomeza et al.,
1999). Therefore, despite the fact that >80% of forskolin-stimulated AC activity was abolished and the
D2-Gi-AC system was completely defective in the striatum of
AC5/, at least a part of
the machinery of the
M4-Gi-AC and
M2-Gi-AC
system in the striatum of AC5/ appeared to be
functional.
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Figure 6.
A functional muscarinic acetylcholine receptor
system is present in the striatum of
AC5/.
A, B, Oxotremorine (Oxo),
an agonist for muscarinic acetylcholine receptors, suppressed the
forskolin (0.1 µM)-stimulated AC activity, in a
dose-dependent manner, in the striatum (A) and
frontal cortex (B) of both
AC5+/+ and
AC5/. The muscarinic
acetylcholine receptors that are negatively coupled to AC and expressed
abundantly in the striatum include M4 and M2 receptors. The amount of
AC activity suppressed strongly in
AC5+/+ by a high dose of oxo (1,000 µM) appears to include the contribution of the
GTP-stimulated AC activity as well as the forskolin-stimulated AC
activity. * and ** indicate a difference between two groups at
p < 0.05 and p < 0.01, respectively.
Base, Baseline; FSK, forskolin.
Oxo 10, Oxo 100, and Oxo
1000 were, respectively, 10, 100, and 1,000 µM of oxotremorine.
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D1 and D2 receptor densities and other
gene expression
To test the possibility that the biochemical deficit of the
D2-Gi-AC
system is caused indirectly by the severe reduction of
D2 expression in
AC5/, we measured the
dopamine receptor density in the striatum. The receptor binding study
with D2 antagonist
3H-spiperone revealed no significant
difference in D2 density between AC5/ and
AC5+/+
(Bmax was 1.47 ± 0.10 (+/+) and
1.25 ± 0.09 (/) pmol/mg protein; p > 0.05).
Consistently, the receptor binding study to brain tissue sections with
another D2 ligand,
125I-sulpiride, indicated that the
D2 receptor density in the brain of
AC5/ was comparable to
that in wild-type littermates (data not shown). We observed that
D1 density also was unchanged (Table
1). In addition, the expressions of
Gs,
Gi, Go, and
Gz proteins and of the
PKA catalytic subunit and PKA regulatory
subunits, RI and RII, in the striatum of
AC5/ were unchanged (data
not shown). Hence, the impairment of the D2-Gi-AC
system in AC5/ may be
explained not by the reduced expression of D2 or
Gi and
Go or the increased
expression of D1, but by the total deficit of the
D2-Gi-AC5 signaling in the striatal neurons.
We examined potential compensatory changes in the expression of genes
for substance P, dynorphin, neuropeptide Y, enkephalin, tyrosine
hydroxylase, GAD, and parvalbumin. Light microscopic examination of
brain sections stained with antibody for each of those showed no
obvious change in the expression of these genes in the brain of
AC5/ (data not shown). The
expression of striatal peptides, for example, substance P, dynorphin,
neuropeptide Y, and enkephalin, was known to be influenced by the
activity of D1 and D2
receptors. In particular, the expression of dynorphin was greatly
reduced in
D1/, and
the expression of GAD and enkephalin was significantly increased in D2/
(Xu et al., 1994; Baik et al., 1995). However, the expression of
dynorphin and enkephalin in the brain of
AC5/ mice was not altered.
These results may suggest that AC5 is the principal AC but not the sole
signaling effector for D1 and D2 receptors.
Responses to the antipsychotic drugs sulpiride, haloperidol,
and clozapine
We investigated whether the biochemical deficit of the
D2-Gi-AC
system could be manifested with altered responses to
D2 antagonists at behavioral levels.
Administration of the D2 antagonist, sulpiride
(50 mg/kg, i.p.), produced severe immobilization of wild-type
littermates over the course of the monitoring period. In contrast, the
same dose of the drug did not elicit such suppression in
AC5/ animals (Fig.
7A,B).
These data are consistent with the notion that AC5 is a crucial
component for the D2 receptor signaling in
striatal neurons.
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Figure 7.
Loss of neuroleptic responsiveness to
antipsychotic drugs in
AC5/ mice.
A, The D2 antagonist, sulpiride (50 mg/kg,
i.p.), induced a decrease of motor activity in
AC5+/+ but produced a marked
enhancement of locomotion in
AC5/
(p < 0.01 for +/+, and
p < 0.05 for /). B, The
locomotion of AC5/ was
gradually accelerated after administration of sulpiride.
C, Haloperidol induced akinesis broadly in wild-type
littermates but did not elicit such behavioral suppression in mutant
animals. Note the dramatic enhancement of locomotion in
AC5/ at lower doses of
haloperidol. D, Time-dependent changes of locomotion
induced by 1 mg/kg of haloperidol are presented. E, As
opposed to that in AC5+/+, cataleptic
response was induced by haloperidol (3 mg/kg, i.p.) in
AC5/. For convenience,
180 sec of cutoff time was set for all cases that lasted >180 sec.
F, Clozapine produced a substantial decrease of
locomotion in both mutant and wild-type littermates in a dose-dependent
manner, but its depression effect on the motor activity was markedly
diminished in AC5/.
Note the sharply increased locomotion of
AC5/ by a low dose
(0.6 mg/kg) of clozapine. * and ** indicate a difference between two
groups at p < 0.05 and p < 0.01, respectively.
Veh, Vehicle; Sulp, sulpiride;
HAL, haloperidol; CZP, clozapine.
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We further examined the effect of haloperidol, a typical
antipsychotic drug the primary target of which is
D2 (Seeman and Van Tol, 1994; Blin, 1999).
Haloperidol administration (0.3-3 mg/kg, i.p.) quickly led to the
immobilization of wild-type littermates for hours of observation. On
the contrary, AC5/ animals
that received the same doses of haloperidol displayed no sign of
behavioral suppression but instead showed hyperactive locomotion (Fig.
7C,D). Consistently, as opposed to that in
AC5+/+, administration of haloperidol (3 mg/kg, i.p.) did not lead
AC5/ animals to catalepsy
(Fig. 7E). Thus, despite the presence of other ACs in the
striatum, and potential non-AC effectors such as ion channels (Cai et
al., 2000; Sidhu and Niznik, 2000), the neuroleptic effects of
haloperidol were totally eliminated in AC5/. These results
indicate that AC5 acts as an indispensable signaling route for the
typical neuroleptic effects of haloperidol.
Similar to that for haloperidol,
AC5/ mice were also
hyperactive in response to clozapine, an atypical antipsychotic drug
(Fig. 7F). After administration of clozapine, the
locomotor activity of wild-type littermates was suppressed in a
dose-dependent manner, whereas the locomotion of
AC5/ marked consistently
high scores in the same doses tested (Fig. 7F). These
data suggest that AC5 is an important component for the neuroleptic
action of clozapine, regardless of the fact that the behavioral
response to clozapine in the open field test was slightly different
from that of haloperidol.
The increased locomotion by haloperidol or clozapine was
antagonized by D1 antagonist
Because a list of reports indicated that haloperidol has a
property to induce dopamine release (Di Chiara et al., 1977; Pehek, 1999; Westerink et al., 2001), we tested the possibility that the
paradoxical haloperidol-induced increase of locomotion in AC5/ was related to the
increased activation of dopamine receptors. First, we determined that
the dose of 0.03 or 0.3 mg/kg of D1 antagonist
SCH23390 (i.p.) produced, respectively, intermediate or almost complete
suppression of locomotion of normal mice in the open field test (data
not shown). In AC5/, the
enhancement of locomotion by haloperidol (0.3 mg/kg, i.p.) was
repressed intermediately by 0.03 mg/kg (i.p.) of SCH23390 and
completely by 0.3 mg/kg (i.p.) of SCH23390. The total distance traveled
for the given period reached the level of the SCH23390 treatment alone
(Fig. 8A). Furthermore,
the enhanced locomotion by clozapine (0.6 mg/kg, i.p.) was also
antagonized intermediately by 0.03 mg/kg (i.p.) of SCH23390 and
completely by 0.3 mg/kg (i.p.) of SCH23390 (Fig. 8B).
These results suggest that the enhanced locomotion of
AC5/ in response to
haloperidol and clozapine resulted from the increased activation of
SCH23390-sensitive D1 class dopamine
receptors.
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Figure 8.
The increased locomotion by haloperidol or
clozapine was antagonized by D1 antagonist.
A, The enhancement of locomotion by haloperidol (0.3 mg/kg, i.p.) was repressed in a dose-dependent manner by D1
antagonist SCH23390. B, The enhanced locomotion by
clozapine (0.6 mg/kg, i.p.) was also antagonized in a dose-dependent
manner by SCH23390. ** indicates a difference between two groups at
p < 0.01. HAL, Haloperidol;
SCH, SCH23390; SCH 0.03, 0.03 mg/kg SCH;
SCH 0.3, 0.3 mg/kg SCH.
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DISCUSSION |
Roles of AC5 in D2 receptor function
Targeted disruption of the AC5 gene produced the
complete elimination of AC5 expression in the brain, which
made it possible to delineate the indispensable role of AC5 downstream
D2 receptors. The typical inhibitory effect of
D2 activation on D1
agonist-, A2A agonist-, and forskolin-stimulated
AC activity was completely abolished in
AC5/ (Fig. 5). It appeared
that other types of ACs present in the striatum were not responsive to
D2 receptor activation. In accordance with this
notion, administration of the D2 antagonists,
haloperidol and sulpiride, did not produce the typical neuroleptic
effects in AC5/ animals,
unlike those in AC5+/+ (Fig.
7A-D). Together, these results are consistent
with the conclusion that AC5 is the physiologically relevant AC for
D2 receptors and that AC5 is a necessary route
for the neuroleptic action of these antipsychotic drugs.
It is unlikely, however, that the absence of the neuroleptic effects
and the total impairment of the
D2-Gi-AC system in AC5/ were
produced indirectly by the reduced expression of the
Gi system or the
D2 receptor itself. First, it was observed that the forskolin-induced AC activation in
AC5/, although it was low,
was suppressed by oxotremorine, an agonist for muscarinic acetylcholine
receptors (Fig. 6). This result indicates the presence of the
functional Gi-coupled receptor system in AC5/.
Furthermore, the expression level of
Gi in the striatum of
AC5/ was comparable to
that of AC5+/+ (data not shown).
Therefore, the complete absence of D2-mediated AC
inhibition in
AC5/
cannot be attributed to the general property of receptors that are
negatively coupled to AC or to the low levels of forskolin-stimulated AC activity in the striatum of
AC5/ (Fig.
2A). Second, receptor-binding studies with
3H-spiperone revealed that the
D2 receptor density was slightly reduced in
AC5/, which was
insignificant (Table 1). Given that heterozygous mice deficient for
dopamine receptor D2
(D2+/) that
carry ~50% of D2 receptors were notably
suppressed by D2 antagonist in the open field
test (Kelly et al., 1998), the lack of neuroleptic effects in
AC5/ cannot be explained
by the loss of D2 receptors in the striatum. It
remains unknown at present whether the total impairment of the
D2-AC system in the striatum of
AC5/, even in the presence
of other ACs and the Gi protein system, was caused by the failure of colocalization of D2 receptor with other ACs in the same neurons or
the inability of D2 to pair with other ACs.
Interestingly, the neuroleptic drugs, sulpiride, haloperidol, and
clozapine, produced a markedly enhanced locomotion in
AC5/, especially at low
doses (Fig. 7). Although no direct evidence is available at present,
these results are tempting us to speculate that inhibition by these
drugs of presynaptic D2, or
D2S as implied by Usiello et al. (2000), might
result in an increase of dopamine release in the striatum, which in
turn produces the enhanced locomotion in
AC5/. This hypothesis is
consistent with our finding that the paradoxical enhancement of
locomotion by haloperidol (0.3 mg/kg, i.p.) or clozapine (0.6 mg/kg,
i.p.) was antagonized by SCH23390 (0.3 mg/kg, i.p.) as shown in Figure
8, but not by the A2A agonist, CGS23390, at the
dose that produced the complete immobilization (K.-W. Lee and P.-L.
Han, unpublished observations). So, D1 receptor
activity in postsynaptic neurons seems to be important for the
D2 antagonist-induced increase of locomotion in
AC5/. This speculation
agreed with the fact that D1-dependent
pharmaco-behaviors were retained in
AC5/ (Fig. 3). However,
given that dopamine receptors are expressed at low levels in many brain
regions, including frontal cortex, we do not exclude the possibility
that AC5-uncoupled D2 receptors in striatal or
extrastriatal regions contribute the neuroleptic-induced enhanced
locomotion in AC5/.
Nonetheless, the remarkable increase of locomotion by haloperidol or
clozapine in AC5/ is
consistent with the notion that both haloperidol and clozapine had a
property of increasing locomotion in the open field test that was
produced in part by a common mechanism that involved the increased
release of dopamine, as was suggested previously (Di Chiara et al.,
1977; Pehek, 1999; Westerink et al., 2001).
Issues of D2-AC5 coupling
Colocalization of AC5 and D2 receptors in
striatal neurons (Mons and Cooper, 1994) and the coupling between
D2 and AC5 as demonstrated in this study raised
the question of whether
AC5/ mice were the
phenocopy of
D2/
(Baik et al., 1995; Jung et al., 1999; Wang et al., 2000). The phenotype of AC5/ mice has
a similarity to that of
D2/. For
example,
AC5/
mice were not driven into catalepsy by haloperidol (Fig.
7E), as were
D2/
and
D2L/
(Kelly et al., 1998; Usiello et al., 2000).
It was somewhat unique to
AC5/, however, that the
locomotion was highly increased by haloperidol (Fig. 7). In addition,
despite the complete deficit of
D2-Gi-AC
signaling in the striatum (Fig. 6),
AC5/ mice did not show
Parkinsonian-like phenotypes or the reduced spontaneous general motor
activities displayed by
D2/
TOP
ABSTRACT
INTRODUCTION
