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• W2058163535 abstract "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-fosimmunoreactivity that are lower than c-fos levels of their wild type littermates. Moreover, the acute blockade of D2receptors 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-fosresponses. The findings therefore indicate that not only D2but also D3 receptors play a role in dopamine-regulated gene expression. 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-fosimmunoreactivity that are lower than c-fos levels of their wild type littermates. Moreover, the acute blockade of D2receptors 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-fosresponses. The findings therefore indicate that not only D2but 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 (1Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1981) Google Scholar, 2Ginty D.D. Neuron. 1997; 18: 183-186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 (3Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1246) Google Scholar, 4Fields R.D. Eshete F. Stevens B. Itoh K. J. Neurosci. 1997; 17: 7252-7266Crossref PubMed Google Scholar). Two key regulators of c-fos transcription are the phosphorylated forms of mitogen-activated protein kinase (MAPK) 1The abbreviations used are:MAPKmitogen-activated protein kinasePnpostnatal dayn and CREB (4Fields R.D. Eshete F. Stevens B. Itoh K. J. Neurosci. 1997; 17: 7252-7266Crossref PubMed Google Scholar). 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.2Ginty D.D. Neuron. 1997; 18: 183-186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 (2Ginty D.D. Neuron. 1997; 18: 183-186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Activation of the MAPK cascade occurs in response to serum or growth factor stimulation (5York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar) as well as in response to increased calcium levels via a Ras-dependent pathway (6Finkbeiner S. Greenberg M.E. Neuron. 1996; 16: 233-236Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). mitogen-activated protein kinase postnatal dayn 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 (7Hughes P. Dragunow M. Pharmacol. Rev. 1995; 47: 133-178PubMed Google Scholar). In the striatum, for example, induction of c-fosexpression has been shown to occur in response to the acute administration of cocaine and amphetamine (8Graybiel A.M. Moratalla R. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6912-6916Crossref PubMed Scopus (877) Google Scholar, 9Young S.T. Rorrino L.J. Iadarola M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1291-1295Crossref PubMed Scopus (458) Google Scholar, 10Moratalla R. Robertson H.A. Graybiel A.M. J. Neurosci. 1992; 12: 2609-2622Crossref PubMed Google Scholar, 11Nguyen T.V. Kosofsky B. Birnbaum R. Cohen B.M. Hyman S.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4270-4274Crossref PubMed Scopus (329) Google Scholar, 12Konradi C. Cole R.L. Keckers S. Hyman S.E. J. Neurosci. 1994; 14: 5623-5634Crossref PubMed Google Scholar, 13Cole R.L. Konradi C. Douglass J. Hyman S.E. Neuron. 1995; 14: 813-823Abstract Full Text PDF PubMed Scopus (323) Google Scholar). 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 (14Missale C. Nash S.R. Robinson S.W. Jaber M Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2749) Google Scholar). 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 (15Robertson G.S. Fibiger H.C. Neuroscience. 1992; 46: 315-328Crossref PubMed Scopus (445) Google Scholar, 16Merchant K.M. Dorsa D.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3447-3451Crossref PubMed Scopus (168) Google Scholar, 17Robertson G.S. Matsumura H. Fibiger H.C. J. Pharmacol. Exp. Ther. 1994; 271: 1058-1066PubMed Google Scholar, 18Merchant K.M. Dobie D.J. Filloux F.M. Totzke M. Aravagiri M. Dorsa D.M. J. Pharmacol. Exp. Ther. 1994; 271: 460-471PubMed Google Scholar). 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 (15Robertson G.S. Fibiger H.C. Neuroscience. 1992; 46: 315-328Crossref PubMed Scopus (445) Google Scholar). Pharmacological studies further revealed synergistic c-fosresponses to combined D1- and D2-like receptor stimulation in the striatum of normal and dopamine-depleted rats (19Paul M.L. Graybiel A.M. David J.-C. Robertson H.A. J. Neurosci. 1992; 12: 3729-3742Crossref PubMed Google Scholar, 20Keefe K.A. Gerfen C.R. Neuroscience. 1995; 66: 903-913Crossref PubMed Scopus (114) Google Scholar, 21Le Moine C. Svenningsson P. Fredholm B.B. Bloch B. J. Neurosci. 1997; 17: 8038-8048Crossref PubMed Google Scholar). 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-fosresponses 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 (20Keefe K.A. Gerfen C.R. Neuroscience. 1995; 66: 903-913Crossref PubMed Scopus (114) Google Scholar, 21Le Moine C. Svenningsson P. Fredholm B.B. Bloch B. J. Neurosci. 1997; 17: 8038-8048Crossref PubMed Google Scholar) used D2-like agonists that do not discriminate between the D2 and D3 receptor subtypes (for review see Ref. 22Levant B. Pharmacol. Rev. 1997; 49: 231-252PubMed Google Scholar). Thus, it remains unresolved whether D2and 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 (23Jung M.-Y. Skryabin B.V. Arai M. Abbondanzo S. Fu D. Brosius J. Robakis N.K. Polites H.G. Pintar J.E. Schmauss C. Neuroscience. 1999; 91: 911-924Crossref PubMed Scopus (117) Google Scholar) to test whether the absence of D2 and D3 receptors alters dorsal striatal and extrastriatal c-fos responses to D1 receptor stimulation. The generation of D2, D3, and D2/D3 mutant mice is described elsewhere (23Jung M.-Y. Skryabin B.V. Arai M. Abbondanzo S. Fu D. Brosius J. Robakis N.K. Polites H.G. Pintar J.E. Schmauss C. Neuroscience. 1999; 91: 911-924Crossref PubMed Scopus (117) Google Scholar). 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 D3mutants 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. (23Jung M.-Y. Skryabin B.V. Arai M. Abbondanzo S. Fu D. Brosius J. Robakis N.K. Polites H.G. Pintar J.E. Schmauss C. Neuroscience. 1999; 91: 911-924Crossref PubMed Scopus (117) Google Scholar). 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 −80 °C until use. All animal procedures were approved by the Institutional Animal Care and Use Committee. 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 mmNa3VO4, 20 mm NaF, 1 mmEGTA, 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. To test whether mice lacking dopamine D2, D3, and D2/D3 receptors maintain the wild type specificity between stimulation of D1receptors 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-fosexpression in rat striatum (21Le Moine C. Svenningsson P. Fredholm B.B. Bloch B. J. Neurosci. 1997; 17: 8038-8048Crossref PubMed Google Scholar, 24Wang J.Q. McGinty J.F. Neuroscience. 1996; 72: 601-616Crossref PubMed Scopus (69) Google Scholar). 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. 1 A, 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-fosimmunoreactivity 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.1 A). 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. 1 B. Furthermore, as shown in Fig.1 C, 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. 2 A, other differences include an earlier induction of c-fosexpression 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. 2 B. 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. 2 C. 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-fosresponses 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.3 A 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.3 B). 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 (25Curran T. Bravo R. Muller R. Cancer Surveys. 1985; 4: 655-681PubMed Google Scholar). A densitometric analysis of signals shown in Fig. 3 B 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 D3mutants is only 1.68, indicating a 57% reduction in c-fosexpression 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.2 B). Moreover, Fig. 3 C illustrates that, similar to the results shown in Fig. 2 A, congenic D3mutants also express lower levels of dorsal striatal c-fosimmunoreactivity 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-fosexpression 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. 4 A, 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. 4 B). The optical density of c-fos signals following SKF treatment alone (2.51) is 20% higher than the corresponding optical density of c-fossignals 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. 1 C and4 B). The reason for this expression pattern is presently unclear. Finally, as shown in Fig. 4 B, the D1-selective antagonist SCH23390 completely abolishes the c-fos response to SKF82958 administration in D3single mutants (see also Fig. 1 B). 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. 1 C), 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-fosresponses 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 D1agonist 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. 5 A, it is obvious at a glance that double-mutant mice that are homozygous for the D3 mutation and heterozygous for the D2mutation (D3−/−;D2+/−; labeledD2+/− in Fig. 5 A) 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. 5 A) or homozygous for both mutations (D3−/−;D2−/−; labeledMD−/− in Fig. 5 A). 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. 5 B, c-fos responses of homozygous D2 single mutants are unaffected by eticlopride, an antagonist with high affinity for both D2 and D3 receptors (22Levant B. Pharmacol. Rev. 1997; 49: 231-252PubMed Google Scholar). This result suggests that adaptive mechanisms involving systems other then the dopaminergic system also operate in brains of D2 single mutants. It has previously been shown that differences in MAPK activity and CREB phosphorylation are critical in regulating c-fos expression (4Fields R.D. Eshete F. Stevens B. Itoh K. J. Neurosci. 1997; 17: 7252-7266Crossref PubMed Google Scholar). 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 D3mutants 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. 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 D3mutant 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 (26Moratalla R. Xu M. Tonegawa S. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14928-14933Crossref PubMed Scopus (168) Google Scholar), maximum c-fosresponses require a steady-state activity of bothD2 and D3 receptors. The reduced c-fos response to D1 agonist stimulation in D3 mutants and the further reduction of c-fosexpression 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 (27Accili D. Fishburn C.S. Drago J. Steiner H. Lachowicz J.E. Park B.-H. Gauda E.B. Lee E.J. Cool M.H. Sibley D.R. Gerfen C.R. Westphal H. Fuchs S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1945-1949Crossref PubMed Scopus (437) Google Scholar, 28Xu M. Koeltzow T.E. Santiago G.T. Moratalla R. Cooper D.C. Hu X.-T. White N.M. Graybiel A.M. White F.J. Tonegawa S. Neuron. 1997; 19: 837-848Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), 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 (29Ruskin D.N. Rawji S.S. Walters J.R. J. Pharmacol. Exp. Ther. 1998; 286: 272-281PubMed Google Scholar). However, as shown in the present study, c-fosresponses 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 (30Mercuri N.B. Saiardi A. Bonci A. Picetti R. Calabresi P. Bernadi G. Borelli E. Neuroscience. 1997; 79: 323-327Crossref PubMed Scopus (175) Google Scholar, 31Koeltzow T.E. Xu M. Cooper D.C. Hu X.-T. Tonegawa S. Wolf M.E. White F.J. J. Neurosci. 1998; 18: 2231-2238Crossref PubMed Google Scholar). 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-fosresponses. The present study demonstrates a similar role for D2 and D3 receptors in the potentiation of c-fosresponses 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 (20Keefe K.A. Gerfen C.R. Neuroscience. 1995; 66: 903-913Crossref PubMed Scopus (114) Google Scholar, 21Le Moine C. Svenningsson P. Fredholm B.B. Bloch B. J. Neurosci. 1997; 17: 8038-8048Crossref PubMed Google Scholar). The D2-like agonists quinpirole and quinelorane used in these studies, however, have high affinities for both D2 and D3receptors (22Levant B. Pharmacol. Rev. 1997; 49: 231-252PubMed Google Scholar). Thus, it remained unclear whether both D2and 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 D3receptors (i.e. activity in the absence of exogenous agonist) is sufficient to mediate this synergistic effect. 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 D3receptors 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 D3single 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 D1agonist 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+/−;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-fosresponses 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-fosresponses 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 (22Levant B. Pharmacol. Rev. 1997; 49: 231-252PubMed Google Scholar), it should have blocked the effect mediated by D3 receptors. The inability of eticlopride to alter c-fos responses of D2mutants 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 (21Le Moine C. Svenningsson P. Fredholm B.B. Bloch B. J. Neurosci. 1997; 17: 8038-8048Crossref PubMed Google Scholar). Third, as shown in Fig. 2 A, 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. 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 D3receptors (14Missale C. Nash S.R. Robinson S.W. Jaber M Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2749) Google Scholar), 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 (23Jung M.-Y. Skryabin B.V. Arai M. Abbondanzo S. Fu D. Brosius J. Robakis N.K. Polites H.G. Pintar J.E. Schmauss C. Neuroscience. 1999; 91: 911-924Crossref PubMed Scopus (117) Google Scholar). 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 γ-amino butyric acid (32Baik J.-H. Picetti R. Dalardi A. Thirlet G. Dierich A. Depaulis A. Le Meur M. Borelli E. Nature. 1995; 377: 424-428Crossref PubMed Scopus (589) Google Scholar). 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. Synergistic or cooperative effects of D1- and D2-like receptor stimulation have not only been observed for the induction of c-fosexpression, 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 (33White F.J. Hu X.-T. Waddington J.L. Dopamine Receptor Interactions. Academic Press, San Diego1993: 79-114Google Scholar). 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 (34Waddington J.L. Daly S.A. Waddington J.L. Dopamine Receptor Interactions. Academic Press, San Diego1993: 51-78Google Scholar). 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 (35Gerfen C. Trends Neurosci. 1992; 15: 133-139Abstract Full Text PDF PubMed Scopus (961) Google Scholar). This different cellular distribution would imply that anintercellular interaction between different pathways underlies the D1/D2 receptor synergism, pathways that may modulate the tonic inhibitory influence of enkephalin and/or γ-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 (36Surmeier D.J. Song W.-J. Yan Z. J. Neurosci. 1996; 16: 6579-6591Crossref PubMed Google Scholar). Furthermore, in contrast to previous reports (37Sokoloff P. Giros B. Martres M.-P. Bouthenet M.-L. Schwartz J.-C. Nature. 1990; 347: 146-151Crossref PubMed Scopus (2420) Google Scholar, 38Bouthenet M.-L. Souil E. Martres M.-P. Sokoloff P. Giros B. Schwartz J.-C. Brain Res. 1991; 564: 203-219Crossref PubMed Scopus (788) Google Scholar), 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 (23Jung M.-Y. Skryabin B.V. Arai M. Abbondanzo S. Fu D. Brosius J. Robakis N.K. Polites H.G. Pintar J.E. Schmauss C. Neuroscience. 1999; 91: 911-924Crossref PubMed Scopus (117) Google Scholar, 39Nimchinsky E.A. Hof P.R. Janssen W.G.M. Morrison J.H. Schmauss C. J. Biol. Chem. 1997; 272: 29229-29237Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). 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. 2E. A. Nimchinsky, P. R. Hof, W. G. M. Janssen, C. Schmauss, and J. H. Morrison, unpublished observation. 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 (36Surmeier D.J. Song W.-J. Yan Z. J. Neurosci. 1996; 16: 6579-6591Crossref PubMed Google Scholar). 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 D3receptor expression on c-fos responses to D1agonist stimulation are not restricted to the dorsal striatum as previously suggested (20Keefe K.A. Gerfen C.R. Neuroscience. 1995; 66: 903-913Crossref PubMed Scopus (114) Google Scholar). 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. 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. Although D3receptors were identified by molecular cloning almost a decade ago, it has proven difficult to elucidate the function of these receptors with conventional pharmacological studies (22Levant B. Pharmacol. Rev. 1997; 49: 231-252PubMed Google Scholar). 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 (28Xu M. Koeltzow T.E. Santiago G.T. Moratalla R. Cooper D.C. Hu X.-T. White N.M. Graybiel A.M. White F.J. Tonegawa S. Neuron. 1997; 19: 837-848Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). 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." @default.
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