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J Biol Chem, Vol. 274, Issue 41, 29406-29412, October 8, 1999
From the The acute administration of dopamine
D1 receptor agonists induces the expression of the
immediate early gene c-fos. In wild type mice, this
induction is completely abolished by pretreatment with the
D1-selective antagonist SCH23390, and pretreatment with the
D2-like receptor antagonist eticlopride reduces the levels of c-fos expressed in response to D1 receptor
stimulation. Mice deficient for the dopamine D3 receptor
express levels of D1 agonist-stimulated c-fos
immunoreactivity that are lower than c-fos levels of their wild type littermates. Moreover, the acute blockade of D2
receptors in D3 mutant mice further reduces
c-fos expression levels. These data indicate that the basal
activity of both D2 and D3 receptors contributes to D1 agonist-stimulated c-fos
responses. The findings therefore indicate that not only D2
but also D3 receptors play a role in dopamine-regulated
gene expression.
An increase in neuronal activity triggers the transcription of
immediate early genes, including the proto-oncogene c-fos, which, in turn, stimulates the transcription of
AP-1-promotor-containing late response genes that are responsible for
adaptive changes in mature neurons (1, 2). It is well established that
the concentration of second messengers and differences in the
activation threshold for calcium-dependent signaling are
important factors controlling cellular responses to stimulatory
signals, including the transcriptional activation of Fos genes (3, 4).
Two key regulators of c-fos transcription are the
phosphorylated forms of mitogen-activated protein kinase
(MAPK)1 and CREB (4). CREB is
a Ca2+/cAMP-responsive transcription factor that in its
phosphorylated form (pCREB; phosphorylated at Ser133)
stimulates transcription of the c-fos gene by interacting
with the CRE transcriptional regulatory element (for review see Ref. 2). The MAPK cascade is thought to participate in the activation of
c-fos transcription either by phosphorylating CREB at
Ser133 or by phosphorylating ternary complex factors that,
together with serum response factor, bind to the SRE element of the
c-fos promotor (2). Activation of the MAPK cascade occurs in
response to serum or growth factor stimulation (5) as well as in
response to increased calcium levels via a Ras-dependent
pathway (6).
Induction of c-fos expression is an important mechanism in
the control of neurotransmitter-regulated gene expression. One extensively studied example is the dopamine-regulated expression of Fos
(7). In the striatum, for example, induction of c-fos expression has been shown to occur in response to the acute
administration of cocaine and amphetamine (8-13). These drugs
indirectly activate dopamine receptors, and the Fos responses elicited
by them have been linked to the activation of dopamine
D1-like receptors, which couple to stimulatory subsets of
heterotrimeric proteins to stimulate cytosolic second messengers
(14).
Moreover, blockade of dopamine D2-like receptors (which
couple to inhibitory G proteins) by neuroleptic drugs results in
increased c-fos expression levels. Anatomically, a close
topographic relationship exists between c-fos effects and
the expression of receptors upon which various typical and atypical
neuroleptics act (15-18). Furthermore, if mesotelencephalic
dopaminergic neurons are destroyed by 6-hydroxydopamine, neuroleptic
drugs no longer increase c-fos expression levels, indicating
that dopamine exerts a tonic inhibitory effect on basal c-fos levels via its action on D2-like receptors
(15).
Pharmacological studies further revealed synergistic c-fos
responses to combined D1- and D2-like receptor
stimulation in the striatum of normal and dopamine-depleted rats
(19-21). Whereas the topographic overlap between increase in
c-fos expression and expression of receptors blocked by
various neuroleptic drugs suggest a cellular mechanism that regulates
basal c-fos-expression levels, synergistic c-fos
responses to combined D1- and D2-like receptor stimulation are more likely to be regulated at the level of neuronal circuitry.
The studies that found synergistic effects of D1- and
D2-like receptor activation on c-fos responses
(20, 21) used D2-like agonists that do not discriminate
between the D2 and D3 receptor subtypes (for
review see Ref. 22). Thus, it remains unresolved whether D2
and D3 receptors contribute similarly or differently to the
modulation of c-fos responses. However, the current lack of
antagonists specific for each member of the D2 class of
dopamine receptors complicates attempts to elucidate the role of both
receptor subtypes in the modulation of c-fos expression
levels. Therefore, the present study used mutant mice generated by gene
targeting via homologous recombination that lack D2,
D3, and D2/D3 receptors (23) to
test whether the absence of D2 and D3 receptors
alters dorsal striatal and extrastriatal c-fos responses to
D1 receptor stimulation.
Animals--
The generation of D2, D3,
and D2/D3 mutant mice is described elsewhere
(23). For studies that compared c-fos responses to application of the D1 agonist SKF82958 across different
mutants, homozygous mutants and wild type littermates were derived from crosses of heterozygous D2 and D3 single
mutants with a hybrid 129/Sv × C57Bl/6 genetic background.
Heterozygous D2/D3 double mutants were
generated via cross-breeding of homozygous D2 males with
homozygous D3 females and were subsequently cross-bred to generate wild type and the various genetic combinations of
D2/D3 double mutants. For studies that compared
c-fos responses to D1 agonist stimulation
between wild type and D3 single mutants, congenic (8th generation of back-crossing) C57Bl/6 D3
mutants and their wild type littermates were used.
Male animals at postnatal ages P15, P30, P60, and P70 were used in this
study. Their genotypes were verified by Southern blotting as described
in Jung et al. (23). Animals were housed in a 12-h light/dark cycle colony room at 22 °C with free access to food and
water. The following drugs were administered: 1 mg/kg SKF82958, 1 mg/kg
SCH23390, and 0.5 mg/kg eticlopride. All drugs were purchased from
Research Biochemicals, Inc. (Natick, MA). Drugs were dissolved in
saline and administered intraperitoneally between 2 and 5 p.m. Animals were killed at defined time points after systemic drug administration, and their brains were quickly removed. The dorsal striatum (caudoputamen) was immediately dissected from all brains. This
dissection did not include the ventral part of the striatum containing
the nucleus accumbens. The dorsal striatal tissue and the remaining
brain tissue were collected in separate tubes and stored at Expression of c-fos, pMAPK, and pCREB
Immunoreactivities--
For immunoblot analyses (c-fos,
pMAPK, and pCREB), proteins were extracted in a buffer containing 1×
phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate,
and 0.1% SDS (RIPA buffer) supplemented with 2 mM
Na3VO4, 20 mM NaF, 1 mM
EGTA, 1 mM dithiothreitol, 1 µM microcystin,
and protease inhibitors. The protein concentration in each lysate was
determined using the BCA protein assay kit (Pierce).
c-fos immunoreactivity was detected with two different
rabbit polyclonal anti-c-fos antibodies. One antibody was
purchased from Santa Cruz (Santa Cruz, CA; lot number H067) and used at a dilution of 1:1000. The other antibody (dilution, 1:2,500 to 1:5000)
was obtained from Oncogene Research (San Diego, CA; lot number D08330).
A sheep polyclonal anti-phospho MAPK antibody (Upstate Biotechnology,
Inc., Lake Placid, NY) was used at a dilution of 1:1000, and a rabbit
polyclonal anti-pCREB antibody (Upstate Biotechnology) was used at a
dilution of 1:2,500. A mouse monoclonal anti-TH antibody (Incstar,
Stillwater, MN; dilution, 1:10,000) was used to reprobe all
immunoblots. Bound antigen was visualized using the appropriate
peroxidase-conjugated secondary antibodies (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) in conjunction with ECL (Pierce). The
ECL signals on autoradiograms were assessed densitometrically using the
National Institutes of Health Image Analysis Software. Optical density
measurements of standards on the film were made to construct a standard
correlation curve. Relative optical densities were determined for
optical densities of signals located in equal size sample areas.
The Role of D2 and D3 Receptors in
Modulating c-fos Responses to D1 Agonist
Stimulation--
To test whether mice lacking dopamine D2,
D3, and D2/D3 receptors maintain
the wild type specificity between stimulation of D1
receptors and transcription of the c-fos gene, mice were
treated with the full D1 agonist SKF82958 (1 mg/kg
intraperitoneal), a ligand known to stimulate c-fos
expression in rat striatum (21, 24). A first series of experiments
performed on wild type animals determined the time course of
c-fos induction and tested whether D1- and
D2-like receptor antagonists block or reduce this induction.
As shown in Fig. 1A, the
earliest time point at which a robust induction of expression of
c-fos can be detected on Western blots is 60 min after the
systemic application of the drug. In general, however, the onset of
detectable c-fos expression varies between 60 and 90 min, a
variability that is most likely accounted for by the route of drug
administration (i.e. intraperitoneal). c-fos
immunoreactivity remains detectable up to 120 min after drug
administration and then returns to base-line levels (not shown). In
addition to the induction of c-fos expression, SKF82958 administration also leads to phosphorylation of the MAPK. The kinetics
of MAPK phosphorylation is more rapid and increased levels of pMAPK
immunoreactivity are detected 20 min after drug administration (Fig.
1A). The expression levels of pMAPK return to base line 30-45 min after drug administration (not shown). Interestingly, although the anti-pMAPK antibody recognizes the phosphorylated pMAPK
isoforms ERK 1 and ERK 2 (see "Experimental Procedures"), only the
42-kDa protein pERK 2 was detected following SKF treatment.
SKF-induced c-fos responses are completely abolished when
mice are pretreated with the D1-selective antagonist
SCH23390 (1 mg/kg intraperitoneal). The results of duplicate
experiments with dorsal striatal and extrastriatal tissues of mice are
shown in Fig. 1B. Furthermore, as shown in Fig.
1C, pretreatment of mice with the D2-like
antagonist eticlopride (0.5 mg/kg intraperitoneal) reduces
c-fos responses to SKF stimulation, an effect that is most
apparent in the dorsal striatum and that is further examined below.
The next series of experiments analyzed the expression of
c-fos immunoreactivity 60 and 90 min after SKF-82958 (1 mg/kg) application in the dorsal striatum and brain without dorsal
striatum tissues of mutant mice lacking D2, D3,
and D2/D3 receptors. These experiments were
performed on mice with a hybrid C57Bl/6 × 129Sv genetic
background (see "Experimental Procedures"), and the results are
shown in Fig. 2. In both striatal and
extrastriatal tissues of mice at P30, the most striking difference in
the levels of c-fos immunoreactivity is seen in
D3 single mutants, which express lower levels compared with
wild type, D2 single mutants, and
D2/D3 double mutants. In Fig. 2A,
other differences include an earlier induction of c-fos expression in D2 single mutants and a slightly stronger
induction in D2/D3 double mutants, but these
differences are no longer apparent in mice at P60. A representative
example of c-fos levels expressed in extrastriatal tissues
of P60 mice is shown in Fig. 2B. Similar to results obtained
with P30 mice, c-fos levels of D3 single mutants are decreased when compared with wild type, D2 single
mutants, and D2/D3 mutants. The optical density
of signals measured 60 and 90 min following drug administration is 5.17 for wild type and 1.98 for D3 single mutants and indicate a
62% decrease in c-fos responses in the mutant animals.
Optical density measurements of corresponding signals obtained from
D2 single mutants (optical density, 3.51) and
D2/D3 double mutants (optical density, 3.01) also indicate a reduction of c-fos expression. However, this
reduction (32% in D2 single mutants and 42% in the double
mutants) is substantially less than the reduction seen in
D3 single mutants. Furthermore, c-fos levels
expressed in the dorsal striatum of both D2 and
D2/D3 mutants are reduced by only 20% (not
shown).
When SKF82958 was administered to mice at P15, only a marginal (and
barely detectable) c-fos response was detected in all animals, regardless of their genotype. A representative example of
c-fos responses in the striatum of these mice is shown in
Fig. 2C. Thus, a robust c-fos expression in
response to D1 agonist stimulation occurs only at most
advanced postnatal ages. In fact, the differences in c-fos
responses of P30 and P60 mice suggest that the maturity of
c-fos responses correlates with the maturity of the animal.
Despite of the differences in c-fos responses seen in
D2 and D2/D3 mutants at P30 and
P60, c-fos levels of D3 single mutants are
consistently lower at both postnatal ages. To test whether possible
differences in the genetic background could have accounted for the
lower c-fos responses to SKF82958 in brains of
D3 single mutants, additional experiments were performed
with congenic (C57Bl/6) D3 mutants and their wild type
littermates. As shown in Fig. 3, also
congenic D3 mutants showed a substantial reduction in the levels of c-fos expressed following SKF treatment. Fig.
3A shows a representative example of a Western blot of
proteins extracted from brain (without striatum) tissue that was probed
with the same antibody used to probe the blots shown in Fig. 2 (a
rabbit polyclonal anti-c-fos antibody; Santa Cruz).
Identical results were obtained when the blot was probed with another
polyclonal anti-c-fos antibody (Oncogene Research) (Fig.
3B). This antibody, like the first antibody, recognizes an
epitope located within peptide sequences that constitute the amino
terminus of the c-fos protein. Unlike the first antibody,
however, the second antibody does not recognize other nonspecific
protein bands (like the one migrating above the c-fos band
in Fig. 2). In fact, it recognizes exclusively one major
c-fos protein of 55 kDa and a second protein of 57 kDa that
is known to result from posttranslational modifications of the 55-kDa
c-fos protein (25). A densitometric analysis of signals
shown in Fig. 3B revealed an optical density of 2.95 for the
entire field containing c-fos signals of wild type animals 45, 60, 90, and 120 min after SKF treatment. The optical density for an
equal size field containing corresponding signals of D3 mutants is only 1.68, indicating a 57% reduction in c-fos
expression levels. Thus, the results obtained with congenic
D3 mutants are very similar to the results obtained with
hybrid (129Sv × C57Bl/6) D3 mutant mice (Fig.
2B). Moreover, Fig. 3C illustrates that, similar
to the results shown in Fig. 2A, congenic D3
mutants also express lower levels of dorsal striatal c-fos
immunoreactivity in response to D1 agonist stimulation.
Optical density measurements of these signals (wild type, 4.84;
D3 mutants, 1.42) revealed a 70% reduction of
c-fos levels in D3 mutants.
The next experiments tested whether the D2-like antagonist
eticlopride would further affect the D1 agonist-stimulated
c-fos response of D3 mutants. The results are
summarized in Fig. 4. As expected, in
both extrastriatal and striatal tissue, administration of eticlopride
(0.5 mg/kg) alone does not lead to detectable c-fos expression levels. It should be noted that in the experiment shown in
Fig. 4, the c-fos responses of mice treated with SKF alone are low at 60 min and robust at 90 min after drug administration, whereas c-fos responses of mice treated with eticlopride
and SKF are clearly detectable at both time points. As outlined above, the onset of detectable c-fos expression following the
systemic administration of SKF varies between 60 and 90 min. This
variability in the kinetics of c-fos induction necessitates
that c-fos levels are measured at 60 and 90 min after drug
administration so that the sums of optical densities of both time
points can be compared. In Fig. 4A, results of duplicate
experiments on extrastriatal tissues of mice treated with SKF and
eticlopride and mice treated with SKF alone are compared. The optical
density of the 60- and 90-min signals obtained from SKF-treated animals
is 7.74. By comparison, the mean optical density of the 60- and 90-min
c-fos signals of animals treated with SKF and eticlopride is
4.43. Thus, a 43% reduction of c-fos expression levels is
found in mice pretreated with eticlopride. Similar results are obtained
for the dorsal striatum (Fig. 4B). The optical density of
c-fos signals following SKF treatment alone (2.51) is 20%
higher than the corresponding optical density of c-fos
signals following SKF and eticlopride treatment (2.01). It is further
noted that in contrast to mice treated with SKF alone, striatal tissues
of mice treated with SKF and eticlopride express similar levels of the
55- and 57-kDa c-fos proteins (see Figs. 1C and
4B). The reason for this expression pattern is presently
unclear. Finally, as shown in Fig. 4B, the D1-selective antagonist SCH23390 completely abolishes the
c-fos response to SKF82958 administration in D3
single mutants (see also Fig. 1B).
In summary, the results shown above indicate that D3 single
mutants express lower levels of c-fos in response to SKF
treatment alone and that the D2-like antagonist eticlopride
further reduces this response. These results are consistent with the
results obtained with wild type mice (Fig. 1C), and they
suggest that the basal activity of both D2 and
D3 receptors is required for the expression of maximum
levels of c-fos responses to D1 agonist
stimulation. It is therefore very surprising that compared with
D3 single mutants, the c-fos responses of
D2/D3 double mutants are reduced to a
substantially lesser extent. In fact, given that c-fos
responses are also reduced in D2 single mutants (albeit to
a much lesser extent compared with D3 single mutants), one
would expect that D2/D3 double mutants express
the lowest levels of c-fos in response to D1
agonist stimulation. Results of two additional experiments, however,
suggest that mice lacking D2 and
D2/D3 receptors develop compensatory mechanisms that enable them to express higher levels of c-fos in
response to D1 receptor stimulation. First, as shown in
Fig. 5A, it is obvious at a
glance that double-mutant mice that are homozygous for the
D3 mutation and heterozygous for the D2
mutation (D3 Expression of pCREB and pMAPK in Response to D1 Agonist
Stimulation--
It has previously been shown that differences in MAPK
activity and CREB phosphorylation are critical in regulating
c-fos expression (4). The following experiments therefore
tested whether the expression of these two key transcriptional
regulators is affected by the lack of D3 and/or
D2 receptors.
Immunoblotting experiments were performed with pCREB- and
pMAPK-specific antibodies to test whether the levels of pCREB and pMAPK
differ between wild type and the various mutants that received SKF82958
treatment (1 mg/kg). These experiments were done with C57Bl/6 × 129Sv hybrid mice. As shown in Fig. 6,
SKF administration led to the expression of similar levels of pCREB and
pMAPK in wild type, D2 and D3 single mutants,
and D2/D3 double mutants. The slightly lower
level of pCREB immunoreactivity shown in Fig. 6 for D3
mutants could not be verified with additional pCREB immunoprecipitation experiments (not shown). Thus, the results indicate that differences in
the expression levels of pCREB and pMAPK do not account for the
differences seen in the c-fos responses described above.
D1 Agonist-stimulated c-fos Responses in Wild Type Mice
and D3 Mutant Mice--
The present study revealed a
blunted c-fos response to D1 agonist stimulation
in mice lacking the dopamine D3 receptor. In both wild
type and D3 mutants, c-fos responses to
stimulation with the full D1 agonist SKF82958 could be
completely abolished by pretreatment with the D1-selective
antagonist SCH23390. Furthermore, in both wild type and D3
mutant mice, pretreatment with the D2-like antagonist
eticlopride reduced the magnitude of c-fos responses to
D1 agonist stimulation. These results indicate that
although the induction of c-fos responses is dependent on
D1 receptor stimulation (26), maximum c-fos
responses require a steady-state activity of both
D2 and D3 receptors. The reduced
c-fos response to D1 agonist stimulation in
D3 mutants and the further reduction of c-fos
expression levels following blockade of D2 receptors in these mutants indicate cooperativity between D1,
D2, and D3 receptors in the modulation of
c-fos responses to D1 receptor stimulation.
Differences in c-fos responses between wild type and
D3 mutants were found in mice with a hybrid C57Bl/6 × 129Sv genetic background as well as in congenic C57Bl/6 mice. Thus, it
is unlikely that differences in the genetic background contributed to
the reduced c-fos expression found in SKF-treated
D3 mutants. Furthermore, the observed differences in
c-fos responses to D1 agonist stimulation cannot
be explained by differences in D1 receptor expression
levels in D3 mutants. Previous studies (27, 28), as well as
our own unpublished results, have shown that the expression of
D1 receptor ligand-binding sites is unaltered in these mutants.
It is also unlikely that the D1-selective agonist used in
the present study exerts its effects on c-fos induction not
only via D1 but also via D2 receptors. SKF82958
has recently been reported to act as a D2-like autoreceptor
agonist to inhibit the basal firing rate of midbrain dopaminergic units
(29). However, as shown in the present study, c-fos
responses elicited by SKF82598 are completely abolished by pretreatment
with the D1-selective antagonist SCH23390. Moreover, if
c-fos responses were modulated by an agonist action of this
drug at D2-like autoreceptors, the autoreceptor effect
would be inhibitory and, in contrast to our results, pretreatment with
eticlopride should have increased c-fos responses. In
addition, studies on mutant mice have now shown clearly that
autoreceptor functions are mediated by D2 but not D3 receptors (30, 31). c-fos responses of
D3 single mutants (in which D2 receptors remain
expressed), however, are also reduced by eticlopride. Altogether, these
results are inconsistent with a possible agonist action of SKF82958 at
D2 autoreceptors to modulate c-fos responses.
The present study demonstrates a similar role for D2 and
D3 receptors in the potentiation of c-fos
responses to D1 agonist stimulation. Our results resemble
results of previous pharmacological studies that found synergistic
effects of concurrent D1- and D2-like receptor
stimulation on c-fos responses in normal and
6-hydroxydopamine-lesioned animals (20, 21). The D2-like
agonists quinpirole and quinelorane used in these studies, however,
have high affinities for both D2 and D3
receptors (22). Thus, it remained unclear whether both D2
and D3 receptors participate in this synergism. The present study, however, employed D3 mutant mice that were treated
either with a D1 agonist alone or a combination of the
D1 agonist and a D2-like receptor antagonist.
The results revealed not only a cooperative involvement of both
D2 and D3 receptors in the modulation of
D1 agonist-stimulated c-fos responses, they also
illustrate that the basal activity of D2 and D3
receptors (i.e. activity in the absence of exogenous
agonist) is sufficient to mediate this synergistic effect.
D1 Agonist-stimulated c-fos Responses in D2
and D2/D3 Mutants--
Studies on
D3 mutant mice revealed a role for both D2 and
D3 receptors in enhancing c-fos responses to
D1 receptor stimulation. However, the minimal reduction of
c-fos expression levels observed in
D2/D3 mutants does not reflect the magnitude of
the combined contribution of D2 and D3
receptors to the modulation of D1 agonist-stimulated c-fos responses. In fact, c-fos levels of double
mutants are substantially higher than those seen in D3
single mutants. It is therefore possible that
D2/D3 double mutants have developed
compensatory mechanisms that enable them to maintain relatively high
(but still subnormal) c-fos responses to D1
agonist stimulation, processes that, at least to some extent, are
also likely to operate in brains of D2 single mutants.
Three observations support this possibility: First,
D2+/
Adaptive changes are known to occur during the development of mice
generated with constitutive knockout techniques, and they can be a
serious complication for the interpretation of mutant phenotypes. It is
perhaps not surprising that D2/D3 and possibly also D2 mutants (but not D3 mutants) develop
adaptive mechanisms to compensate for the loss of the receptor(s).
D2 receptors are far more abundant than D3
receptors (14), and their absence is likely to have more serious
consequences for the normal functioning of the brain. Indeed, a
comparison of the motor phenotypes of all three mutants revealed that
mice lacking D2 receptors are more severely impaired than
mice lacking D3 receptors and that D2/D3 double mutants are most severely impaired
(23). Furthermore, other adaptations have already been described for
D2 single mutants that include, for example, alterations in
the expression of glutamic acid decarboxylase mRNA, whose encoded
protein is involved in the synthesis of the neurotransmitter What Are the Mechanisms Mediating the Cooperative Interactions
between D1 and D2-like Receptors in the
Modulation of c-fos Responses?--
Synergistic or cooperative effects
of D1- and D2-like receptor stimulation have
not only been observed for the induction of c-fos
expression, they have also been observed in electrophysiological and
behavioral experiments. For example, the co-administration of
D1- and D2-like agonists results in a
synergistic inhibition of spontaneous and glutamate-evoked firing (33).
Furthermore, in dopamine-depleted animals, unconditional behavioral
responses to psychostimulants (increased locomotor activity, sniffing,
licking, and biting) have been shown to require stimulation of both
D1 and D2 receptors (34). However, it is not
yet established whether the apparent cooperative interaction between
D1, D2, and D3 receptors result
from interactions at the cellular level or whether they involve
intercellular pathways. For the striatum, for example, anatomic studies
suggest a predominant expression of D1 receptors in neurons
of the striatonigral pathway, whereas D2 receptors are
expressed in neurons of the striatopallidal pathway (35). This
different cellular distribution would imply that an
intercellular interaction between different pathways
underlies the D1/D2 receptor synergism,
pathways that may modulate the tonic inhibitory influence of enkephalin
and/or
Despite the present uncertainty regarding the contribution of cellular
or intercellular mechanisms, determining the point at which
D1, D2, and D3 receptor-mediated
signals converge to regulate the transcriptional activity of the
c-fos gene will be an important next step toward
understanding the mechanism of dopaminergic regulation of
c-fos expression. Our finding that the expression of two key
regulators of c-fos transcription, pCREB and pMAPK, is not
significantly altered in D1 agonist-treated mice that lack either D2 or D3 receptors suggests that the
activity of other components of the signaling/transcriptional activator
cascade is modulated by D2 and D3 receptors and
that these components are most likely operating downstream of CREB and
MAPK activation.
Implications of the Findings--
Although D3
receptors were identified by molecular cloning almost a decade ago, it
has proven difficult to elucidate the function of these receptors with
conventional pharmacological studies (22). Recently, however, a
behavioral study on D3 mutant mice showed that one role of
D3 receptors is to diminish the normal cooperative effects
of D1 and D2 receptor stimulation in the
regulation of motor activity and responses to the rewarding properties
of psychostimulants (28). The present study identified another role,
namely a cooperative participation of D3 receptors in the
regulation of c-fos responses to D1 agonist
stimulation. The finding that D3 receptors play a role in
dopamine-regulated gene expression adds an important new aspect to our
understanding of D3 receptor expression and function.
*
This work was supported by National Science Foundation Grant
IBN-9808567 (to C. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Columbia
University, Depts. of Psychiatry and Neuroscience, 1051 Riverside Dr., Unit 42, New York, NY 10032. Tel.: 212-543-6505; Fax: 212-543-6017; E-mail: schmauss@neuron.cpmc.columbia.edu.
2
E. A. Nimchinsky, P. R. Hof, W. G. M. Janssen, C. Schmauss, and J. H. Morrison, unpublished observation.
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
Pn, postnatal day
n.
Decreased c-fos Responses to Dopamine D1
Receptor Agonist Stimulation in Mice Deficient for D3
Receptors*
§ and¶
Department of Psychiatry/Neuroscience,
Columbia University, New York, New York 10032 and the
§ Graduate Program in Neurobiology, Mount Sinai School of
Medicine, New York, New York 10029
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C
until use. All animal procedures were approved by the Institutional
Animal Care and Use Committee.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of c-fos and pMAPK
expression following SKF82958 (1 mg/kg) treatment of wild type
mice. A, left, expression of extrastriatal
c-fos immunoreactivity 30-90 min after drug administration.
The migration of the c-fos immunoreactive protein band,
detected with a rabbit polyclonal antibody (Santa Cruz), is indicated
by an arrowhead. Right, pMAPK expression in brain
and dorsal striatum at 10-20 min time points after drug
administration. Time points are indicated on top of each
lane. The anti-pMAPK antibody detects only one
phosphorylated MAPK of 42 kDa (ERK 2). Lanes s, saline.
B, c-fos induction in brain (without striatum)
and striatum 90 min following SKF82958 (SKF; 1 mg/kg)
administration to mice pretreated with either SCH23390 (SCH;
1 mg/kg) or saline. Results of duplicate experiments are shown.
C, SKF82958-induced expression of c-fos
immunoreactivity 60 min (lanes a) and 90 min (lanes
b) with or without eticlopride pretreatment (Et; 0.5 mg/kg). Total striatal and extrastriatal (brain( st))
protein (100 µg) extracted from the combined tissues of two animals
was loaded onto each lane. The blots shown in B and
C were reprobed with an antibody directed against tyrosine
hydroxylase (TH) to demonstrate that equal amount of protein
was loaded onto each lane.
View larger version (19K):
[in a new window]
Fig. 2.
Expression of striatal and extrastriatal
c-fos immunoreactivity following SKF82958 (1 mg/kg)
treatment of wild type (wt) and mutant mice.
Shown is the expression of c-fos immunoreactivity 60 and 90 min after drug administration to P30 (A), P60
(B), and P15 mice (C). c-fos
immunoreactivity (arrowhead) was detected with a rabbit
polyclonal antibody (Santa Cruz). DM, homozygous
D2/D3 double mutants. Lanes s,
saline; DM*, homozygous D2/D3 double
mutants at P30. 100 µg of total protein extracted from combined brain
tissues of two animals/genetic group was loaded onto each lane. Each
blot was reprobed with an anti-TH antibody.
View larger version (28K):
[in a new window]
Fig. 3.
Expression of striatal and extrastriatal
c-fos immunoreactivity following SKF82958 treatment in
congenic wild type and D3 mutants. A,
extrastriatal expression of c-fos immunoreactivity 45, 60, 90, and 120 min following SKF82598 treatment of P30 mice.
c-fos immunoreactivity (arrowhead) was detected
with a rabbit polyclonal antibody (Santa Cruz). B, protein
samples loaded onto the gel shown in A were probed with a
rabbit polyclonal anti-c-fos antibody that exclusively
recognizes c-fos immunoreactivities of 55 and 57 kDa
(Oncogene Research). C, striatal expression of
c-fos immunoreactivity 60, 90, and 120 min following
SKF82598 treatment of P30 mice. On all gels, 100 µg of protein
extracted from brain tissue of one animal/time point was loaded.
Lanes S, saline. The blots shown in B and
C were reprobed with an anti-TH antibody.
View larger version (43K):
[in a new window]
Fig. 4.
Expression of c-fos immunoreactivity following treatment of congenic D3
mutants with eticlopride and SKF82958. Shown is expression of
extrastriatal (brain (without striatum)) (A) and striatal
c-fos immunoreactivity (B) 60 and 90 min after
SKF administration to mice that were pretreated either with eticlopride
(Et; 0.5 mg/kg) or saline. The last lane on the blot shown in
B demonstrates that SCH23390 (SCH; 1 mg/kg)
pretreatment completely abolishes the SKF-induced c-fos
induction. A rabbit polyclonal anti-c-fos antibody (Oncogene
Research) was used to probe both blots. Total protein (100 µg)
extracted from brain tissue of one animal/time point was loaded onto
each lane.
/;D2+/; labeled
D2+/ in Fig. 5A) express drastically reduced
levels of c-fos compared with mice that are either
heterozygous for the D3 mutation and homozygous for the
D2 mutation (D3+/;D2/;
labeled D3+/ in Fig. 5A) or homozygous for both
mutations (D3/;D2/; labeled
MD/ in Fig. 5A). These results suggest that
on a D3 mutant genetic background, c-fos levels
are reduced substantially when only one intact D2-encoded
allele is lacking. Second, as shown in Fig. 5B,
c-fos responses of homozygous D2 single mutants are unaffected by eticlopride, an antagonist with high affinity for
both D2 and D3 receptors (22). This result
suggests that adaptive mechanisms involving systems other then the
dopaminergic system also operate in brains of D2 single
mutants.
View larger version (25K):
[in a new window]
Fig. 5.
SKF82958-induced expression of extrastriatal
c-fos immunoreactivity in D2 and
D2/D3 mutants. A,
c-fos expression in three different genetic combinations of
D2/D3 double mutants. D3+/ ,
D3+/;D2/; D2+/,
D3/;D2+/; DM/,
D3/;D2/. Total protein (100 µg)
extracted from brain tissues of two P70 animals/genetic group was
loaded onto each lane. c-fos immunoreactivity
(arrowhead) was detected with a rabbit polyclonal
anti-c-fos antibody (Santa Cruz), and the blot was reprobed
with an anti-TH antibody. B, SKF-induced expression of
c-fos immunoreactivity in D2 single mutants
pretreated with eticlopride (Et; 0.5 mg/kg) or saline. A
rabbit polyclonal anti-c-fos antibody (Oncogene Research)
was used to probe the blot which contains 100 µg of total
protein/lane. Time points after drug administration (60 and 90 min) are
indicated on top of each lane.
View larger version (7K):
[in a new window]
Fig. 6.
Expression of pCREB and pMAPK
immunoreactivities in striatal and extrastriatal brain tissue of wild
type (wt) and mutant mice treated with SKF82958.
Left, striatal pCREB levels (PCREB) were
determined 5 min following drug administration. 60 µg of total
protein extracted from brains of three animals/genetic group was loaded
onto each lane. The blot was probed with an anti-pCREB antibody. pCREB
levels remained unchanged up to 2 h after drug administration (not
shown). Right, the expression of pMAPK (ERK 2; 42 kDa) was
determined 20 min after drug administration using an anti-pMAPK
antibody. D2, homozygous D2 mutants;
D3, homozygous D3 mutants; DM,
homozygous D2/D3 double mutants.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
;D3/ double mutants express
substantially lower levels of c-fos in response to
D1 agonist stimulation compared with homozygous double
mutants (D2/;D3/). This result is
consistent with the cooperative role of D2 receptors in the
induction of c-fos responses (i.e. the reduced
expression of D2 receptors leads to reduced cooperativity)
and suggests further that the much lesser reduction of c-fos
responses of homozygous D2 single mutants results from
compensatory adaptations that developed in mice with a homozygous and
not a heterozygous D2 mutant genotype. Second, in contrast to wild type and D3 single mutants, c-fos
responses to SKF treatment of homozygous D2 mutants are not
affected by pretreatment with the D2-like antagonist
eticlopride. Because eticlopride has a high affinity for both
D2 and D3 receptors (22), it should have blocked the effect mediated by D3 receptors. The inability
of eticlopride to alter c-fos responses of D2
mutants suggests that compensatory mechanisms involve systems other
than the dopaminergic receptor system. Such systems could, for example,
involve the adenosine A2A receptor, which has been shown to
modulate c-fos responses to D1 receptor
stimulation (21). Third, as shown in Fig. 2A,
c-fos responses of D2/D3 double
mutants at P30 but not at P60 are higher than corresponding wild type
responses. This may indicate that, at P30, the "fine tuning" of
developing compensatory mechanisms is not yet completed.
-amino
butyric acid (32). Regardless of the nature of adaptations that result
in relatively high c-fos responses in mice lacking
D2 receptors, our results suggest that these adaptations
obscure the interpretation of the magnitude of effect mediated by
D2 receptors in the complex interaction between
D1, D2, and D3 receptors to
modulate c-fos responses.
-amino butyric acid released from local axon collaterals of
D1-containing striatonigral neurons and/or alter the
activity of striatal interneurons. However, recent single-cell reverse
transcription polymerase chain reaction experiments detected an
extensive co-localization of D1- and D2-like
receptor-encoded mRNA in medium spiny neurons of the striatonigral
pathway (36). Furthermore, in contrast to previous reports (37, 38),
results of our studies on the expression of D3 receptor
protein (detected with D3-specific monoclonal antibodies)
and D3-encoded mRNA revealed a significant amount of
D3 receptor expression in the dorsal striatum (23, 39). In
addition, imunocytochemical studies on rodent brain tissues identified
a high number of D3-immunoreactive neurons that possibly
represent medium spiny cells and many (but not all) large
interneurons.2 Results of
single-cell polymerase chain reaction studies further revealed that
~50% of the substance P/D1 receptor-expressing neurons also expressed D3 and D4 receptor mRNA, and
results of electrophysiological studies suggest that ~50% of all
medium-spiny projection neurons co-express functional D1-
and D2-like receptors (36). These findings give weight to
the possibility that a cellular mechanism underlies the modulatory
effects of D2 and D3 receptors. However, it
should be noted that the effects of D2 and D3
receptor expression on c-fos responses to D1
agonist stimulation are not restricted to the dorsal striatum as
previously suggested (20). We found that it also operates in
extrastriatal brain regions. Because the extrastriatal tissues that we
analyzed comprised the entire brain (without dorsal striatum), we can
only suggest that the c-fos immunoreactivity detected
therein is derived mainly from anatomic regions representing the
mesolimbic/mesocortical dopaminergic projection areas that express
D1, D2, and D3 receptors. For these anatomic structures, however, evidence for co-localization of all three
receptors is still lacking.
FOOTNOTES
ABBREVIATIONS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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