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Quinpirole attenuates the striatal immediate early gene expression, but not the hyperactivity, induced by the serotonin agonist RU-24969
Brain Research 852 Ž2000. 247–254 www.elsevier.comrlocaterbres
Research report
Quinpirole attenuates the striatal immediate early gene expression, but not the hyperactivity, induced by the serotonin agonist RU-24969 David F. Cook, David Wirtshafter
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The UniÕersity of Illinois at Chicago, Department of Psychology (M r C 285) 1009 BehaÕioral Sciences Building, 1007 West Harrison Street, Chicago, IL 60607-7137, USA Accepted 17 August 1999
Abstract Systemic administration of the mixed 5-HT1Ar1B agonist RU-24969 has been shown to produce a dramatic increase in locomotor activity and to induce robust c-Fos expression in the rat striatum. Previous studies have also shown that pretreatment with the D 2-like dopamine agonist quinpirole virtually abolishes RU-24969-induced striatal c-Fos expression. The present study was undertaken to determine whether the effects of RU-24969 on immediate early gene expression extend to the additional Fos family transcription factors FosB and Fra-2. Additionally, this study quantitatively examined the effect of quinpirole pretreatment on the ability of RU-24969 to induce both locomotor hyperactivity and striatal immediate early gene expression. RU-24969 alone produced elevations in locomotor activity and induced clear expression of c-Fos, FosB and Fra-2 throughout the entire striatal complex. Quinpirole pretreatment virtually abolished RU-24969-induced expression of all three transcription factors, but did not alter the elevated locomotor activity produced by RU-24969. These results demonstrate that the effects of RU-24969 on locomotor activity can be dissociated from its effects on immediate early gene expression within the striatum. q 2000 Elsevier Science B.V. All rights reserved. Keywords: c-Fos; FosB; Fra-2; Striatum; Locomotor activity; D 2 receptor; 5-HT1B receptor; Dopamine; Serotonin
1. Introduction Systemic administration of the mixed serotonin1Ar1B Ž5-HT1Ar1B . agonist RU-24969 dose dependently increases locomotor activity in both rats w6,19,28,43,46,53,66x and mice w8,14,18,19,45x. Recent data suggest that this locomotor response in the rat is mediated through the synergistic activation of both the 5-HT1A and 5-HT1B recognition sites w35,46x. The neural structures mediating the locomotor effects of RU-24969 have, however, not yet been identified. An interesting possibility is that some of the actions of RU-24969 may be exerted through the basal ganglia, a group of structures which are well known to play an important role in the control of behavioral activation and which contain high levels of 5HT 1B receptors w5,27,37,47,49x. Consistent with this notion, RU-24969 has been reported to increase dopamine release within the striatum w3,4x and dopamine antagonists have been found to inhibit RU-24969 induced hyperactivity w19,42,66x.
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Further evidence that RU-24969 influences basal ganglia functioning comes from studies which have shown that this drug induces pronounced expression of the immediate early gene ŽIEG. c-fos within the striatum of both the rat w10,11,72x and mouse w32x. It is striking that striatal Fos expression can also be promoted by a number of other drugs, such as the indirect dopamine agonists cocaine and amphetamine, which likewise induce increases in locomotor activity. Indeed, much more is known about the effects of these drugs on IEG expression than about the effects of serotonin agonists. For example, several studies have shown that the effects of dopamine agonists are not restricted to c-fos and that these drugs also induce the expression of a number of other immediate early genes including fosB, junB, zifr268 and several ‘‘Fos related antigens’’ w2,9,38,50x. Expression of multiple IEGs within the striatum has also been reported after blockade of dopamine receptors, although different patterns of gene expression may be produced by different drugs w24,33,34x. It is currently not known whether serotonin agonists also induce coordinated expression of multiple IEGs; accordingly, the first goal of the current experiments was to examine whether the effects of RU-24969 are restricted to
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c-Fos or whether this drug also induces expression of the additional Fos family transcription factors FosB and Fra-2. Although most workers have regarded immediate early genes primarily as markers of neuronal activity, it is possible that expression of these genes may actually be causally involved in the locomotor activation produced by dopamine agonists. For example, several authors have shown that intrastriatal injections of antisense oligonucleotides to c-fos are able to attenuate dopamine agonist induced Fos expression w26,62,63,67x and bilateral injections of antisense oligonucleotides into the ventral striatum have been shown to antagonize cocaine induced locomotion w22x. However, no data are available to indicate whether the ability of RU-24969 to induce locomotor activity is similarly related to its ability to induce immediate early gene expression in the striatum. In recent studies we have shown that pretreatment with the D 2-like dopamine receptor agonist quinpirole virtually abolishes the Fos response to injections of RU-24969 w11x. This observation provides a means of investigating the relationship between the effects of RU-24969 on locomotor activity and on striatal gene expression. If striatal Fos induction were causally involved in the production of hyperactivity, one would expect that quinpirole pretreatment would attenuate the effects of RU-24969 on both activity and on gene expression. If, in contrast, striatal Fos expression were not causally related to behavioral activation, quinpirole pretreatment would be expected to attenuate the effects of RU-24969 on gene expression, but not on locomotor activity. Accordingly, the second major goal of the current study was to compare the effects of quinpirole pretreatment on the locomotor and the IEG responses to RU-24969.
2. Materials and methods 2.1. Subjects Twenty-eight adult, male Sprague–Dawley rats weighing between 250 and 400 g were obtained from a colony maintained by the Psychology Department at the University of Illinois at Chicago. Rats were individually housed on a 12r12 h lightrdark cycle in standard suspended wire mesh cages and allowed free access to food and water except during behavioral testing. All experimental manipulations and behavioral observations were conducted during the light phase and were in compliance with protocols approved by the Animal Care Committee at the University of Illinois at Chicago. 2.2. Drugs Žy.-Quinpirole hydrochloride Žmol.wt.s 255.8. was obtained from Eli Lilly ŽIndianapolis, IN. and RU-24969 w5-methoxy-3-Ž1,2,3,6-tetrahydropyridin-4-yl.1 H-indole;
mol.wt.s 287.3x was obtained from the N.I.M.H. Chemical Synthesis Program administered through Research Biochemicals ŽNantick, MA.. Drug doses employed in the current experiments were selected on the basis of previous behavioral and Fos-like immunoreactivity studies w6,11,46,53,66x. All compounds were dissolved in distilled water and administered subcutaneously in a volume of 1 mlrkg body weight. 2.3. BehaÕioral procedures Locomotor activity was measured in infrared photocell activity boxes measuring 72.5 = 72.5 cm with 30.5 cm high walls. The insides of the boxes were painted flat black and lighting was provided by overhead fluorescent fixtures. Twenty-two animals were randomly assigned into four treatment conditions Žvehiclervehicle, vehiclerRU24969, quinpirolervehicle and quinpirolerRU-24969. which consisted of four to seven animals per group. Untreated animals were allowed to habituate to the activity boxes for 120 min periods on each of four consecutive days. On the day following the last habituation trial, rats were injected with either quinpirole Ž2.5 mgrkg. or its vehicle and returned to their home cages. Fifteen minutes later subjects were injected with either RU-24969 Ž5.0 mgrkg. or its vehicle and were then immediately placed into the activity boxes. Activity counts were collected for a two hour period after which subjects were anesthetized and perfused as described below. In order to confirm the behavioral results obtained in the experiment described above, the effects of quinpirole on RU-24969 induced hyperactivity were examined in six additional animals using a 3 = 2 Žquinpirole= RU-24969. factorial within-subjects design. Locomotor activity was measured as described above following injections of various combinations of quinpirole Ž0, 0.25 or 2.5 mgrkg. and RU-24969 Ž0 or 5.0 mgrkg.. Each animal was tested under each of the six treatment conditions in a counterbalanced order with at least three days separating sequential tests. 2.4. Perfusion and immunocytochemistry Animals were deeply anesthetized with sodium pentobarbital Ž50 mgrkg. and perfused transcardially at room temperature with 100 ml of normal saline followed by 900 ml of a 10% formalin solution prepared in phosphate buffer ŽpH 7.2.. The brains were then rapidly removed and placed in fresh fixative for 2 h at 48C after which they were transferred to a solution of 20% sucrose in phosphate buffered saline ŽPBS. and were stored overnight at 48C. The following day, the brains were rapidly frozen and 40 mm sections were taken through the region of the stratum immediately rostral to the decussation of the anterior commissure. Some free-floating sections were processed for c-Fos-like immunoreactivity immediately after sectioning while adjacent sections were stored for a maximum of two
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254
weeks in a cryoprotectant solution w70x at y208C before being processed for FosB-like or Fra-2-like immunoreactivity. c-Fos was detected using a rabbit anti-c-Fos serum ŽAb-5; Calbiochem, Cambridge, MA. prepared at a dilution of 50,000 = . FosB and Fra-2 were detected using rabbit antisera ŽSC-48 and SC-183; Santa Cruz Biotechnology, Santa Cruz, CA. at dilutions of 2000:1 and 1000:1, respectively. The FosB antibody we employed was directed against an epitope located near the N-terminal end of the molecule and thus would be expected to recognize both FosB proper and the truncated form of the molecule known as delta-FosB. All primary antibodies were prepared in Tris buffered saline ŽTBS. ŽpH 7.5. containing 0.2% Triton X-100, 0.05% sodium azide and 2% normal goat serum. Following incubation in the primary antibody for 72 h at 48C, sections were rinsed several times in PBS and then placed for 2 h at room temperature in biotinylated goat anti-rabbit serum ŽVector Laboratories, Burlingame,
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CA. diluted 200:1 in TBS containing 2.0% blocking serum. The sections were then rinsed several times in phosphate buffered saline and transferred to the Vectastain Elite ABC reagent ŽVector Laboratories. for a period of 90 min on a rotator at room temperature. Thereafter, they were rinsed thoroughly in PBS and developed using nickel intensified diaminobenzadine as the substrate. The reaction was terminated by transferring the sections into PBS after which the tissue was mounted, dried and cover-slipped using Eukitt mounting medium ŽCalibrated Instruments, Hawthorne, NY.. Control tissue was prepared by omitting the primary antibodies after which no specific staining could be seen. 2.5. Data analysis The number of striatal cells displaying either c-Fos-, FosB- or Fra-2-like immunoreactivity was quantified using a Leica Quantimet 500 image analysis system. Fields
Fig. 1. Photomicrographs of coronal sections taken through the medial striatum of rats sacrificed 2 h following RU-24969 Ž5.0 mgrkg. administration. The left hand column Žpanels A, B and C. displays fields from animals pretreated with vehicle and processed for c-Fos, Fra-2 and FosB, respectively; the right X X X hand column Žpanels A , B and C . displays fields from animals pretreated with quinpirole Ž2.5 mgrkg. and processed for c-Fos, Fra-2 and FosB, respectively. Scale bar represents 100 mm.
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measuring 1.0 = 1.0 mm located in the central-medial region of the striatum at a level immediately rostral to the decussation of the anterior commissure were digitized. Cells were automatically detected in the captured images based on their size, staining intensity relative to background, and aspect ratio; the detection parameters were determined following extensive preliminary study.
3. Results 3.1. Immediate early gene expression In animals treated with vehicle, virtually no expression of c-Fos or Fra-2 was seen in the striatum. In contrast, moderate numbers of cells displaying FosB-like immunoreactivity could be visualized in the striatum, especially in its ventromedial region, and in the adjacent nucleus accumbens. Basal staining was less pronounced in the more dorsal region examined here and many of the cells expressing Fos-B-like immunoreactivity in this area following saline treatment were too lightly labeled to be counted with the image analysis parameters employed.
Injections of RU-24969 resulted in a dramatic increase in staining for all three of the IEGs we examined and, for all three IEGs, pretreatment with quinpirole appeared to virtually eliminate the effects of RU-24969 throughout the entire striatal complex ŽFig. 1.. These impressions were supported by examination of the quantitative results displayed in Fig. 2a–c. For each of the three IEGs, analysis of the data by means of a 2 = 2 ŽRU-24969= quinpirole. factorial analysis of variance ŽANOVAs. indicated a significant effect of RU-24969 and a significant RU-24969= quinpirole interaction Ž p - 0.001 in each case., reflecting the fact that quinpirole attenuated the effects of RU-24969 while exerting little effect by itself. 3.2. Effects of RU-24969 on locomotor actiÕity Activity counts for the subjects examined by immunocytochemistry are shown in Fig. 2d where it can be seen that RU-24969 Ž5.0 mgrkg. induced a substantial increase in locomotor activity which was not attenuated by quinpirole pretreatment. Analysis of these data by means of a 2 = 2 Žquinpirole= RU-24969. ANOVA, indicated a significant effect of RU-24969, Ž F Ž1,18. s 17.1, P - 0.001., but not of quinpirole Ž p ) 0.2.. Although quinpirole actu-
Fig. 2. Effects of quinpirole and RU-24969 on striatal expression of c-Fos Žpanel A., Fos-B Žpanel B., Fra-2 Žpanel C. and locomotor activity ŽPanel D.. Error bars represent S.E.M.s.
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254
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Fig. 3. Effects of quinpirole and RU-24969 on locomotor activity examined using a within subject design in which each rat was tested under all six conditions. Error bars represent S.E.M.s.
ally tended to potentiate the response to RU-24969, the quinpirole= RU-24969 interaction did not approach significance Ž F - 1.. Fig. 3 shows the effects of quinpirole and RU-24969 on locomotor activity in the within-subjects experiment. A 3 = 2 Žquinpirole = RU-24969. repeated measures ANOVA indicated that RU-24969 again produced a significant increase in activity Ž F Ž1,5. s 55.9, P - 0.001.. Neither the effect of quinpirole Ž p ) 0.1., nor the quinpirole = RU-24969 interaction Ž F - 1. were significant.
4. Discussion The present results confirm our previous findings w10,11,72x that systemic administration of the mixed 5HT1Ar1B agonist RU-24969 induces intense c-Fos-like immunoreactivity ŽFLI. within the rat striatum. It is likely that this effect is due primarily to stimulation of the 5HT1B receptor since similar effects are also seen following injections of selective 5HT1B agonists, such as anpirtoline w72x and CP-94,253 Žin preparation., which lack affinity for the 5HT1A receptor. The current study also provides the first demonstration that RU-24969 induces expression of the additional Fos family transcription factors FosB and Fra-2. All of these proteins could be detected 2 h following drug treatment, but the possibility cannot currently be ruled out that peak expression of the various factors may occur at different times. Coordinated induction of c-Fos and FosB in the striatum has been previously shown to occur follow-
ing injections of a number of other drugs including amphetamine, neuroleptics and tetrahydrocannabinol ŽTHC., as well as in response to opiate withdrawal and cortical stimulation w1,24,40,50,52x. Substantially less is known about Fra-2 although this transcription factor has been reported to be expressed in the striatum following opiate withdrawal and in the nucleus accumbens following THC treatment w40,52x. It would appear that most pharmacological interventions tend to induce expression of multiple transcription factors within the striatum, but the extent to which different drugs are able to induce varied patterns of gene expression remains to be determined. It is also unclear at the current time whether all of these transcription factors are expressed in the same neurons, or whether some of them are specifically produced in discrete populations of cells. In several model systems, Fra-2 has been shown to regulate gene transcription in a direction opposite to that of c-Fos w58,65x and thus the effects of these two proteins might actually tend to oppose each other if they were co-expressed in individual neurons. In other brain structures, c-Fos and FosB have been found to be co-localized in individual cells w51x and it is likely that a similar situation occurs in the striatum, although no direct evidence is currently available. The precise mechanism by which RU-24969 activates striatal immediate early gene expression has not yet been identified. A number of studies have shown that injections of RU-24969 increase dopamine release or turnover within the striatum and other forebrain structures w3,4,6,39,41x. Since stimulation of dopamine receptors results in striatal FLI, these findings suggest that the induction of Fos expression by RU-24969 may occur as a result of stimu-
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lated dopamine release. This possibility is supported by findings that dopamine depleting lesions and pretreatment with the selective dopamine D 1 antagonist SCH-23390 markedly attenuate the c-Fos expression induced by RU24969 w10x. Alternatively, it is possible that RU-24969 produces its effects by a direct action on striatal neurons and that the role of dopamine is simply permissive. A number of workers have shown that the dopamine D 2-like agonist quinpirole is able to produce a regionally and compartmentally specific potentiation of the Fos response produced by administration of selective D 1 dopamine receptor agonists w16,29,48,71,73x. In contrast, the current results show that pretreatment with quinpirole virtually abolishes the striatal c-Fos, FosB and Fra-2 expression induced by RU-24969 alone. These findings are consistent with and extend previous studies in our laboratory which have shown that quinpirole is capable of attenuating the striatal c-Fos expression induced by the injection of several serotonergic, opiate and muscarinic receptor agonists w11x or by forcing animals to shuttle in order to escape electric shock w64x. Taken together, these findings suggest that quinpirole may attenuate the ability of a number of treatments to induce striatal FLI through a common mechanism, perhaps related to the capacity of this drug to suppress striatal unit activity w25x. It is possible, for example, that IEG expression in all of these situations is dependent on basal or drug-induced stimulation of D 1 dopamine receptors and that quinpirole acts as an ‘indirect D 1 receptor antagonist’ as a result of its ability to stimulate D 2-like autoreceptors. ŽIndeed, since quinpirole has been shown directly to decrease dopamine release w31,54,59,61x, an interesting question arises as to why this drug does not suppress locomotor activity when given by itself.. An alternative possibility is that quinpirole suppresses striatal FLI through a direct action on postsynaptic dopamine D 2 receptors located on striatal neurons. This interpretation is supported by the finding that while cAMP mediated protein phosphorylation has been implicated in the activation of the c-fos gene w17,56,68x, stimulation of D 2 receptors has been linked to an inhibition of cAMP formation w44,57,69x. Since D 2 receptors are heterogeneously distributed in different populations of striatal neurons w23,30x, this latter theory would require RU-24969 to induce IEG expression principally in cells containing D 2-like receptors. It should be noted in this context that dopamine agonists appear to induce Fos expression mainly in striatonigral neurons w7,55x which have been reported to express primarily D 1 receptors w15,21x. A third possibility is that the effects of quinpirole are mediated through a presynaptic suppression of corticostriatal transmission w36,60,74x. Future studies will be necessary to determine the precise mechanism through which quinpirole attenuates drug and behavioral induced FLI. Studies of antisense oligonucleotide injections have suggested that c-Fos may play an important role in mediating the motor responses produced by injections of dopamine
agonists w13,22,62,63,67x. It is not certain, however, whether the effects of these injections result from a blockade of drug induced Fos synthesis, or from a suppression of the constitutive expression of Fos which may occur at low levels w63x. The most striking result of the current experiments was the observation that injections of quinpirole, at doses which virtually abolished the induction of IEGs by RU-24969, failed entirely to attenuate the locomotor response to this serotonin agonist. These findings indicate that the induction of striatal IEGs cannot play a causal role in the production of hyperactivity by RU-24969, although it is still possible that constitutive expression of these transcription factors may by involved. This conclusion is analogous to that reached in previous studies in our laboratory w64x which found that quinpirole pretreatment almost abolished the expression of striatal FLI in rats shuttling to escape shock, but had no effect on escape latencies. Irrespective of its possible involvement in mediating effects on locomotor activity, striatal IEG expression would appear to provide a marker for the effects of RU-24969 in this region of the brain. Although RU-24969 has negligible affinity for catecholamine receptors, several reports have demonstrated that the hyperactivity induced by this drug is substantially attenuated in animals pretreated with reserpine or several dopamine receptor antagonists w19,42,66x. These findings and others w20x have suggested that RU24969 may induce hyperactivity as a result of its ability to release dopamine in the striatal complex. Several experimental results, however, are difficult to reconcile with this theory. For example, although the dopamine antagonist haloperidol blocks RU-24969 induced hyperactivity in intact animals, it fails to do so in subjects with electrolytic lesions of the globus pallidus w42x, which may attenuate the catalepsy normally produced by dopamine blockade w12x. Additionally, the nature of the behavior induced by RU24969 differs drastically from that seen after dopamine agonists in that it consists largely of bouts of explosive thigmotaxic locomotor activity and is associated with reductions in investigatory rearing and nose poking w19,46,53x. Both of these results suggest that the effects of RU-24969 on locomotor activity may result primarily from an action at extrastriatal sites. The current finding that quinpirole pretreatment blocks the effects of RU-24969 on striatal IEG expression without altering the effects of this drug on locomotor behavior provides novel and strong support for this viewpoint.
Acknowledgements The authors thank Eli Lilly for their generous gift of quinpirole. RU24969 was provided by Research Biochemcials International as a part of the N.I.M.H Chemical Synthesis Program, contract N01MH30003. These studies were supported by N.I.H. grant NS-33992.
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References w1x S. Baretta, H.B. Parthasarathy, A.M. Graybiel, Local release of GABAergic inhibition in the motor cortex induces immediate-early gene expression in indirect pathway neurons of the striatum, J. Neurosci. 17 Ž1997. 4752–4763. w2x S. Baretta, H.A. Robertson, A.M. Graybiel, Neurochemically specialized projection neurons of the striatum respond differently to psychomotor stimulants, in: G.W. Arbuthnott, P.C. Emson ŽEds.., Progress in Brain Research, Elsevier, New York, 1993, pp. 201–205. w3x S. Benloucif, M.P. Galloway, Facilitation of dopamine release in vivo by serotonin agonists: studies with microdialysis, Eur. J. Pharmacol. 200 Ž1991. 1–8. w4x S. Benloucif, J. Keegan, M.P. Galloway, Serotonin-facilitated dopamine release in vivo: pharmacological characterization, J. Pharmacol. Exp. Ther. 265 Ž1993. 373–377. w5x A.T. Bruninvels, B. Landwehrmeyer, E.L. Gustafson, M.M. Durkin, G. Mengod, T.A. Branchek, D. Hoyer, J.M. Palacios, Localization of 5-HT1B, 5-HT1Dalpha, 5-HT1E and 5-HT1F receptor messenger RNA in rodent and primate brain, Neuropharmacology 33 Ž1994. 367–386. w6x M.A. Carli, R. Invernizzi, L. Cervo, R. Samanin, Neurochemical and behavioral studies with RU-24969 in the rat, Psychopharmacology 94 Ž1988. 359–364. w7x M.A. Cenci, K. Campbell, K. Wictorin, A. Bjorklund, Striatal c-fos induction by cocaine or apomorphine occurs preferentially in output neurons projecting to the substantia nigra in the rat, Eur. J. Neurosci. 4 Ž1992. 376–380. w8x S.C. Cheetham, D.J. Heal, Evidence that RU-24969-induced locomotor activity in C57rB1r6 mice is specifically mediated by the 5-HT1B receptor, Br. J. Pharmacol. 110 Ž1993. 1621–1629. w9x A.J. Cole, R.V. Bhat, C. Patt, P.F. Worly, J.M. Baraban, D1 dopamine receptor activation of multiple transcription factor genes in rat striatum, J. Neurochem. 58 Ž1992. 1420–1426. w10x D.F. Cook, D. Wirtshafter, Serotonin agonist-induced c-fos expression in the rat striatum, Soc. Neurosci. Abst. 21 Ž1995. 1424. w11x D.F. Cook, D. Wirtshafter, Quinpirole attenuates striatal c-fos induction by 5-HT, opioid and muscarinic receptor agonists, Eur. J. Pharmacol. 349 Ž1998. 41–47. w12x B. Costall, J.E. Olley, Cholinergic and neuroleptic induced catalepsy: modification by lesions in the globus pallidus and substantia nigra, Neuropsychopharmacology 10 Ž1971. 581–594. w13x M. Dragunow, P. Lawlor, B. Chiasson, H. Robertson, c-fos antisense generates apomorphine and amphetamine-induced rotation, Neuroreport 5 Ž1993. 305–306. w14x C.R. Gardner, A.P. Guy, Behavioral effects of RU 24969, a 5-HT1 receptor agonist, in the mouse, Br. J. Pharmacol. 78 Ž1983. 96P. w15x C.R. Gerfen, The neostriatal mosaic: multiple levels of compartmental organization, J. Neur. Transm., Suppl. 36 Ž1992. 43–59. w16x C.R. Gerfen, K.A. Keefe, E.B. Gauda, D1 and D2 dopamine receptor function in the striatum: coactivation of D1- and D2-dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons, J. Neurosci. 15 Ž1995. 8167–8176. w17x A. Ghosh, D.D. Ginty, H. Bading, M.E. Greenberg, Calcium regulation of gene expression in neuronal cells, J. Neurobiol. 25 Ž1993. 294–303. w18x G.M. Goodwin, A.R. Green, A behavioral and biochemical study in mice and rats of putative selective agonists and antagonists for 5-HT1 and 5-HT2 receptors, Br. J. Pharmacol. 84 Ž1985. 743–753. w19x A.R. Green, A.P. Guy, C.R. Gardner, The behavioral effects of RU 24969, a suggested 5-HT1 receptor agonist in rodents and the effect on the behavior of treatment with antidepressants, Neuropharmacology 23 Ž1984. 655–661. w20x A.R. Green, D.J. Heal, The effects of drugs on serotonin-mediated behavioral models, in: R.A. Green ŽEd.., Neuropharmacology of Serotonin, Oxford, New York, 1985.
253
w21x M.B. Harrison, R.G. Wiley, G.F. Wooten, Selective localization of striatal D1 receptors to striatal neurons, Brain Res. 528 Ž1990. 317–322. w22x M. Heilig, J.A. Engel, B. Soderpalm, c-Fos antisense in the nucleus accumbens blocks the locomotor stimulant action of cocaine, Eur. J. Pharmacol. 236 Ž1993. 339–340. w23x S.M. Hersch, B.J. Cilax, C.A. Gutekunst, H.D. Rees, C.J. Heilman, K.K.L. Yung, J.P. Bolam, E. Ince, H. Yi, A.I. Levey, Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with corticostriatal afferents, J. Neurosci. 15 Ž1995. 5222–5237. w24x N. Hiroi, A.M. Graybiel, Atypical and typical neuroleptic treatments induce distinct programs of transcription factor expression in the striatum, J. Comp. Neurol. 374 Ž1996. 70–83. w25x K.C. Hooper, D.A. Banks, L.J. Stordahl, I.M. White, G.V. Rebec, Quinpirole inhibits striatal and excites pallidal neurons in freely moving rats, Neurosci. Lett. 237 Ž1997. 69–72. w26x M.L. Hooper, B.J. Chiasson, H.A. Robertson, Infusion into the brain of an antisense oligonucleotide to the immediate-early gene c-fos suppresses production of fos and produces a behavioral effect, Neuroscience 63 Ž1994. 917–924. w27x D. Hoyer, G. Engel, H.O. Kalkman, Characterization of the 5-HT1B recognition site in rat brain: binding studies with Žy. w125 Ixiodocyanopindolol, Eur. J. Pharmacol. 118 Ž1985. 1–12. w28x H.O. Kalkman, RU 24969-induced locomotion in rats is mediated by 5-HT1A receptors, Naunyn-Schmiedeberg’s Arch. Pharmacol. 352 Ž1995. 583–584. w29x G.J. LaHoste, J. Yu, J.F. Marshall, Striatal Fos expression is indicative of dopamine D1rD2 synergism and receptor supersensitivity, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 7451–7455. w30x C. LeMoine, B. Bloch, D1 and D2 dopamine receptor gene expression in the rat striatum: sensitive cRNA probes demonstrate prominent segregation of D1 and D2 mRNA’s in distinct neuronal populations of the dorsal and ventral striatum, J. Comp. Neurol. 355 Ž1995. 418–426. w31x A.C. Linthorst, H. De Lang, W. De Jong, D.H. Versteeg, Effect of the dopamine D2 receptor agonist quinpirole on the in vivo release of dopamine in the caudate nucleus of hypertensive rats, Eur. J. Pharmacol. 201 Ž1991. 125–133. w32x J.J. Lucas, L. Segu, R. Hen, 5-Hydroxytryptamine1B receptors modulate the effect of cocaine on c-fos expression: converging evidence using 5-hydroxytryptamine1B knockout mice and the 5-hydroxytryptamine1Br1D antagonist GR127935, Mol. Pharmacol. 51 Ž1997. 755–763. w33x G.A. MacGibbon, P.A. Lawlor, R. Bravo, M. Dragunow, Clozapine and haloperidol produce a differential pattern of immediate early gene expression in rat caudate–putamen, nucleus accumbens, lateral septum and island of Calleja, Mol. Brain Res. 23 Ž1994. 21–32. w34x G.A. MacGibbon, P.A. Lawlor, P. Huges, D. Young, M. Dragunow, Differential expression of inducible transcription factors in basal ganglia neurons, Mol. Brain Res. 34 Ž1995. 294–302. w35x D.L. Martinez, M.A. Geyer, A 5-HT1B antagonist blocks effects of RU 24969 on locomotor, but not investigatory, activity in rats, Soc. Neurosci. Abst. 23 Ž1997. 519. w36x G. Maura, A. Giardi, M. Raiteri, Release-regulating D2 dopamine receptors are located on striatal glutaminergic nerve terminals, J. Pharmacol. Exp. Ther. 247 Ž1998. 680–684. w37x D.N. Middlemiss, P.H. Hutson, The 5-HT1B receptors, Ann. N. Y. Acad. Sci. 600 Ž1990. 132–147. w38x R. Moratalla, E.A. Vickers, H.A. Robertson, B.H. Cochran, A.M. Graybiel, Coordinate expression of c-fos and junB is induced in the rat striatum by cocaine, J. Neurosci. 13 Ž1993. 423–433. w39x H. Nissbrandt, N. Waters, S. Hjorth, The influence of serotonergic drugs on dopaminergic neurotransmission in rat substantia nigra, striatum and limbic forebrain in vivo, Naunyn-Schmiedeberg’s Arch. Pharmacol. 346 Ž1992. 12–19. w40x H.E. Nye, E.J. Nestler, Induction of chronic Fos-related antigens in
254
w41x
w42x
w43x
w44x
w45x
w46x
w47x w48x
w49x
w50x
w51x
w52x
w53x
w54x
w55x
w56x
w57x
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254 rat brain by chronic morphine administration, Mol. Pharmacol. 49 Ž1996. 636–645. C. Oberlander, Effects of a potent 5-HT agonist, RU 24969, on the mesocorticolimbic and nigrostriatal dopamine systems, Br. J. Pharmacol. 80 Ž1983. 675P. C. Oberlander, B. Blaquiere, J.F. Pujol, Distinct functions for dopamine and serotonin in locomotor behavior: evidence using the 5-HT1 agonist RU 24969 in globus pallidus-lesioned rats, Neurosci. Lett. 67 Ž1986. 113–118. C. Oberlander, J.R. Boissier, Haloperidol blocks hyperlocomotion but not the circling behavior induced by the serotonin agonist RU 24969, Proc. 8th Int. Congr. Pharmacol. Abst., Tokyo, 1981, p. 839. P. Onali, M.C. Olianas, G.L. Gessa, Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum, Mol. Pharmacol. 28 Ž1985. 138–145. M.F. O’Neill, A.G. Fernandez, J.M. Palacios, GR 127935 blocks the locomotor and antidepressant-like effects of RU 24969 and the action of antidepressants in the mouse tail suspension tests, Pharmacol. Biochem. Behav. 53 Ž1996. 535–539. M.F. O’Neill, T. Parameswaran, RU-24969-induced behavioral syndrome requires activation of both 5-TH1A and 5-HT1B receptors, Psychopharmacology 132 Ž1997. 255–260. J.M. Palacios, C. Waeber, D. Hoyer, G. Mengod, Distribution of serotonin receptors, Ann. N. Y. Acad. Sci. 600 Ž1990. 36–52. M.L. Paul, A.M. Graybiel, J.C. David, H.A. Robertson, D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson’s disease, J. Neurosci. 12 Ž1992. 3729–3742. A. Pazos, J.M. Palacios, Quantitative autoradiographic mapping of serotonin receptors in the rat brain: I. serotonin-1 receptors, Brain Res. 346 Ž1985. 205–230. A.M. Persico, C.W. Schindler, B.F.O. O’Hara, M.T. Brannock, G. Uhl, Brain transcription factor expression: effects of acute and chronic amphetamine and injection stress, Mol. Brain Res. 20 Ž1993. 91–100. R.V. Peters, N. Aronin, W.J. Schwartz, c-Fos expression in the rat intergeniculate leaflet: photic regulation, co-localization with Fos-B and cellular identification, Brain Res. 728 Ž1996. 231–234. A. Porcella, G.L. Gessa, L. Pani, Delta9-tetrahydrocannabinol increases sequence-specific AP-1 DNA-binding activity and Fos-related antigens in the rat brain, Eur. J. Neurosci. 10 Ž1998. 1743–1751. N.L. Rempel, C.W. Callaway, M.A. Geyer, Serotonin1B receptor activation mimics behavioral effects of presynaptic serotonin release, Neuropsychopharmacology 8 Ž1993. 201–211. G.S. Robertson, C.S. Tham, C. Wilson, A. Jakubovic, H.C. Fibiger, In vivo comparisons of the effects of quinpirole and the putative presynaptic dopaminergic agonists B-HT 920 and SND 919 on striatal dopamine and acetylcholine release, J. Pharmacol. Exp. Ther. 264 Ž1993. 1344–1351. G.S. Robertson, S.R. Vincent, H.C. Fibiger, Striatonigral projection neurons contain D1 dopamine receptor activated c-fos, Brain Res. 523 Ž1990. 288–290. L.M. Robertson, T.K. Kerppola, M. Vendrell, D. Luk, R.J. Smeyne, C. Bocchaiaro, J.I. Morgan, T. Curran, Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements, Neuron 14 Ž1995. 241–252. S.W. Robinson, M.G. Caron, Selective inhibition of adenylyl cyclase type V by the dopamine D3 receptor, Mol. Pharmacol. 52 Ž1997. 508–514.
w58x S.E. Rutberg, E. Saez, S. Lo, S.I. Jang, N. Markova, B.M. Spiegelman, S.H. Yuspa, Opposing activities of c-Fos and Fra-2 on AP-1 regulated transcriptional activity in mouse keratinocytes induced to differentiate by calcium and phorbol esters, Oncogene 15 Ž1997. 1337–1346. w59x M. Santiago, B.H. Westerink, The regulation of dopamine release from nigrostriatal neurons in conscious rats: the role of somatodendritic autoreceptors, Eur. J. Pharmacol. 204 Ž1. Ž1991. 79–85. w60x R. Schwarcz, I. Creese, J.T. Coyle, S.H. Snyder, Dopamine receptors localized on cerebral cortical afferents to rat corpus striatum, Nature 271 Ž1978. 766–768. w61x R.E. See, B.A. Sorg, M.A. Chapman, P.W. Kalivas, In vivo assessment of release and metabolism of dopamine in the ventrolateral striatum of awake rats following administration of dopamine D1 and D2 receptor agonists and antagonists, Neuropharmacology 30 Ž1991. 1269–1274. w62x W. Sommer, B. Bjelke, D. Ganten, K. Fuxe, Antisense oligonucleotide to c-fos induces ipsilateral rotational behavior to d-amphetamine, Neuroreport 5 Ž1993. 277–280. w63x W. Sommer, K. Fuxe, On the role of c-fos expression in striatal transmission: the antisense oligonucleotide approach, Neurochem. Int. 31 Ž1997. 425–436. w64x W.M. Struthers, D. Wirtshafter, Quinpirole attenuates the striatal Fos expression induced by escape behavior, Brain Res. 785 Ž1998. 347–350. w65x T. Suzuki, H. Okuno, T. Yoshida, T. Endo, H. Nishina, H. Iba, Difference in transcriptional regulatory function between c-Fos and Fra-2, Nucleic Acids Res. 19 Ž1991. 5537–5542. w66x M.D. Tricklebank, D.N. Middlemiss, J. Neill, Pharmacological analysis of the behavioral and thermoregulatory effects of the putative 5-HT1 receptor agonist, RU 24969, in the rat, Neuropharmacology 25 Ž1986. 877–886. w67x T. Umekage, M. Namima, K. Fukushima, S. Sugita, T. Watanabe, c-fos antisense blocks methamphetamine-induced ambulatory activity reversibly, Neuroreport 8 Ž1997. 407–410. w68x F.M. Vaccarino, M.D. Hayward, H.N. Le, D.J. Hartigan, R.S. Duman, E.J. Nestler, Induction of immediate early genes by cyclic AMP in primary cultures of neurons from rat cerebral cortex, Brain Res., Mol. Brain Res. 19 Ž1993. 76–82. w69x L. Vallar, J. Meldolesi, Mechanisms of signal transduction at the dopamine D2 receptor, Trends Neurosci. 10 Ž1989. 74–77. w70x R.E. Watson, S.J. Wiegand, R.W. Clough, G.E. Hoffman, Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology, Peptides 7 Ž1986. 155–159. w71x D. Wirtshafter, K.E. Asin, Interactive effects of stimulation of D1 and D2 dopamine receptors on Fos-like immunoreactivity in the normosensitive rat striatum, Brain Res. Bull. 35 Ž1994. 85–91. w72x D. Wirtshafter, D.F. Cook, Serotonin-1B agonists induce compartmentally organized striatal Fos expression in rats, Neuroreport 9 Ž1998. 1217–1221. w73x D. Wirtshafter, G. Schardt, K.E. Asin, Compartmentally specific effects of quinpirole on the striatal Fos expression induced by stimulation of D1-dopamine receptors in intact rats, Brain Res. 771 Ž1997. 271–277. w74x B.K. Yamamoto, S. Davy, Dopaminergic modulation of glutamate release in striatum as measured by microdialysis, J. Neurochem. 58 Ž1992. 1742–1746.
Research report
Quinpirole attenuates the striatal immediate early gene expression, but not the hyperactivity, induced by the serotonin agonist RU-24969 David F. Cook, David Wirtshafter
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The UniÕersity of Illinois at Chicago, Department of Psychology (M r C 285) 1009 BehaÕioral Sciences Building, 1007 West Harrison Street, Chicago, IL 60607-7137, USA Accepted 17 August 1999
Abstract Systemic administration of the mixed 5-HT1Ar1B agonist RU-24969 has been shown to produce a dramatic increase in locomotor activity and to induce robust c-Fos expression in the rat striatum. Previous studies have also shown that pretreatment with the D 2-like dopamine agonist quinpirole virtually abolishes RU-24969-induced striatal c-Fos expression. The present study was undertaken to determine whether the effects of RU-24969 on immediate early gene expression extend to the additional Fos family transcription factors FosB and Fra-2. Additionally, this study quantitatively examined the effect of quinpirole pretreatment on the ability of RU-24969 to induce both locomotor hyperactivity and striatal immediate early gene expression. RU-24969 alone produced elevations in locomotor activity and induced clear expression of c-Fos, FosB and Fra-2 throughout the entire striatal complex. Quinpirole pretreatment virtually abolished RU-24969-induced expression of all three transcription factors, but did not alter the elevated locomotor activity produced by RU-24969. These results demonstrate that the effects of RU-24969 on locomotor activity can be dissociated from its effects on immediate early gene expression within the striatum. q 2000 Elsevier Science B.V. All rights reserved. Keywords: c-Fos; FosB; Fra-2; Striatum; Locomotor activity; D 2 receptor; 5-HT1B receptor; Dopamine; Serotonin
1. Introduction Systemic administration of the mixed serotonin1Ar1B Ž5-HT1Ar1B . agonist RU-24969 dose dependently increases locomotor activity in both rats w6,19,28,43,46,53,66x and mice w8,14,18,19,45x. Recent data suggest that this locomotor response in the rat is mediated through the synergistic activation of both the 5-HT1A and 5-HT1B recognition sites w35,46x. The neural structures mediating the locomotor effects of RU-24969 have, however, not yet been identified. An interesting possibility is that some of the actions of RU-24969 may be exerted through the basal ganglia, a group of structures which are well known to play an important role in the control of behavioral activation and which contain high levels of 5HT 1B receptors w5,27,37,47,49x. Consistent with this notion, RU-24969 has been reported to increase dopamine release within the striatum w3,4x and dopamine antagonists have been found to inhibit RU-24969 induced hyperactivity w19,42,66x.
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Further evidence that RU-24969 influences basal ganglia functioning comes from studies which have shown that this drug induces pronounced expression of the immediate early gene ŽIEG. c-fos within the striatum of both the rat w10,11,72x and mouse w32x. It is striking that striatal Fos expression can also be promoted by a number of other drugs, such as the indirect dopamine agonists cocaine and amphetamine, which likewise induce increases in locomotor activity. Indeed, much more is known about the effects of these drugs on IEG expression than about the effects of serotonin agonists. For example, several studies have shown that the effects of dopamine agonists are not restricted to c-fos and that these drugs also induce the expression of a number of other immediate early genes including fosB, junB, zifr268 and several ‘‘Fos related antigens’’ w2,9,38,50x. Expression of multiple IEGs within the striatum has also been reported after blockade of dopamine receptors, although different patterns of gene expression may be produced by different drugs w24,33,34x. It is currently not known whether serotonin agonists also induce coordinated expression of multiple IEGs; accordingly, the first goal of the current experiments was to examine whether the effects of RU-24969 are restricted to
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 9 3 - 9
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c-Fos or whether this drug also induces expression of the additional Fos family transcription factors FosB and Fra-2. Although most workers have regarded immediate early genes primarily as markers of neuronal activity, it is possible that expression of these genes may actually be causally involved in the locomotor activation produced by dopamine agonists. For example, several authors have shown that intrastriatal injections of antisense oligonucleotides to c-fos are able to attenuate dopamine agonist induced Fos expression w26,62,63,67x and bilateral injections of antisense oligonucleotides into the ventral striatum have been shown to antagonize cocaine induced locomotion w22x. However, no data are available to indicate whether the ability of RU-24969 to induce locomotor activity is similarly related to its ability to induce immediate early gene expression in the striatum. In recent studies we have shown that pretreatment with the D 2-like dopamine receptor agonist quinpirole virtually abolishes the Fos response to injections of RU-24969 w11x. This observation provides a means of investigating the relationship between the effects of RU-24969 on locomotor activity and on striatal gene expression. If striatal Fos induction were causally involved in the production of hyperactivity, one would expect that quinpirole pretreatment would attenuate the effects of RU-24969 on both activity and on gene expression. If, in contrast, striatal Fos expression were not causally related to behavioral activation, quinpirole pretreatment would be expected to attenuate the effects of RU-24969 on gene expression, but not on locomotor activity. Accordingly, the second major goal of the current study was to compare the effects of quinpirole pretreatment on the locomotor and the IEG responses to RU-24969.
2. Materials and methods 2.1. Subjects Twenty-eight adult, male Sprague–Dawley rats weighing between 250 and 400 g were obtained from a colony maintained by the Psychology Department at the University of Illinois at Chicago. Rats were individually housed on a 12r12 h lightrdark cycle in standard suspended wire mesh cages and allowed free access to food and water except during behavioral testing. All experimental manipulations and behavioral observations were conducted during the light phase and were in compliance with protocols approved by the Animal Care Committee at the University of Illinois at Chicago. 2.2. Drugs Žy.-Quinpirole hydrochloride Žmol.wt.s 255.8. was obtained from Eli Lilly ŽIndianapolis, IN. and RU-24969 w5-methoxy-3-Ž1,2,3,6-tetrahydropyridin-4-yl.1 H-indole;
mol.wt.s 287.3x was obtained from the N.I.M.H. Chemical Synthesis Program administered through Research Biochemicals ŽNantick, MA.. Drug doses employed in the current experiments were selected on the basis of previous behavioral and Fos-like immunoreactivity studies w6,11,46,53,66x. All compounds were dissolved in distilled water and administered subcutaneously in a volume of 1 mlrkg body weight. 2.3. BehaÕioral procedures Locomotor activity was measured in infrared photocell activity boxes measuring 72.5 = 72.5 cm with 30.5 cm high walls. The insides of the boxes were painted flat black and lighting was provided by overhead fluorescent fixtures. Twenty-two animals were randomly assigned into four treatment conditions Žvehiclervehicle, vehiclerRU24969, quinpirolervehicle and quinpirolerRU-24969. which consisted of four to seven animals per group. Untreated animals were allowed to habituate to the activity boxes for 120 min periods on each of four consecutive days. On the day following the last habituation trial, rats were injected with either quinpirole Ž2.5 mgrkg. or its vehicle and returned to their home cages. Fifteen minutes later subjects were injected with either RU-24969 Ž5.0 mgrkg. or its vehicle and were then immediately placed into the activity boxes. Activity counts were collected for a two hour period after which subjects were anesthetized and perfused as described below. In order to confirm the behavioral results obtained in the experiment described above, the effects of quinpirole on RU-24969 induced hyperactivity were examined in six additional animals using a 3 = 2 Žquinpirole= RU-24969. factorial within-subjects design. Locomotor activity was measured as described above following injections of various combinations of quinpirole Ž0, 0.25 or 2.5 mgrkg. and RU-24969 Ž0 or 5.0 mgrkg.. Each animal was tested under each of the six treatment conditions in a counterbalanced order with at least three days separating sequential tests. 2.4. Perfusion and immunocytochemistry Animals were deeply anesthetized with sodium pentobarbital Ž50 mgrkg. and perfused transcardially at room temperature with 100 ml of normal saline followed by 900 ml of a 10% formalin solution prepared in phosphate buffer ŽpH 7.2.. The brains were then rapidly removed and placed in fresh fixative for 2 h at 48C after which they were transferred to a solution of 20% sucrose in phosphate buffered saline ŽPBS. and were stored overnight at 48C. The following day, the brains were rapidly frozen and 40 mm sections were taken through the region of the stratum immediately rostral to the decussation of the anterior commissure. Some free-floating sections were processed for c-Fos-like immunoreactivity immediately after sectioning while adjacent sections were stored for a maximum of two
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254
weeks in a cryoprotectant solution w70x at y208C before being processed for FosB-like or Fra-2-like immunoreactivity. c-Fos was detected using a rabbit anti-c-Fos serum ŽAb-5; Calbiochem, Cambridge, MA. prepared at a dilution of 50,000 = . FosB and Fra-2 were detected using rabbit antisera ŽSC-48 and SC-183; Santa Cruz Biotechnology, Santa Cruz, CA. at dilutions of 2000:1 and 1000:1, respectively. The FosB antibody we employed was directed against an epitope located near the N-terminal end of the molecule and thus would be expected to recognize both FosB proper and the truncated form of the molecule known as delta-FosB. All primary antibodies were prepared in Tris buffered saline ŽTBS. ŽpH 7.5. containing 0.2% Triton X-100, 0.05% sodium azide and 2% normal goat serum. Following incubation in the primary antibody for 72 h at 48C, sections were rinsed several times in PBS and then placed for 2 h at room temperature in biotinylated goat anti-rabbit serum ŽVector Laboratories, Burlingame,
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CA. diluted 200:1 in TBS containing 2.0% blocking serum. The sections were then rinsed several times in phosphate buffered saline and transferred to the Vectastain Elite ABC reagent ŽVector Laboratories. for a period of 90 min on a rotator at room temperature. Thereafter, they were rinsed thoroughly in PBS and developed using nickel intensified diaminobenzadine as the substrate. The reaction was terminated by transferring the sections into PBS after which the tissue was mounted, dried and cover-slipped using Eukitt mounting medium ŽCalibrated Instruments, Hawthorne, NY.. Control tissue was prepared by omitting the primary antibodies after which no specific staining could be seen. 2.5. Data analysis The number of striatal cells displaying either c-Fos-, FosB- or Fra-2-like immunoreactivity was quantified using a Leica Quantimet 500 image analysis system. Fields
Fig. 1. Photomicrographs of coronal sections taken through the medial striatum of rats sacrificed 2 h following RU-24969 Ž5.0 mgrkg. administration. The left hand column Žpanels A, B and C. displays fields from animals pretreated with vehicle and processed for c-Fos, Fra-2 and FosB, respectively; the right X X X hand column Žpanels A , B and C . displays fields from animals pretreated with quinpirole Ž2.5 mgrkg. and processed for c-Fos, Fra-2 and FosB, respectively. Scale bar represents 100 mm.
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measuring 1.0 = 1.0 mm located in the central-medial region of the striatum at a level immediately rostral to the decussation of the anterior commissure were digitized. Cells were automatically detected in the captured images based on their size, staining intensity relative to background, and aspect ratio; the detection parameters were determined following extensive preliminary study.
3. Results 3.1. Immediate early gene expression In animals treated with vehicle, virtually no expression of c-Fos or Fra-2 was seen in the striatum. In contrast, moderate numbers of cells displaying FosB-like immunoreactivity could be visualized in the striatum, especially in its ventromedial region, and in the adjacent nucleus accumbens. Basal staining was less pronounced in the more dorsal region examined here and many of the cells expressing Fos-B-like immunoreactivity in this area following saline treatment were too lightly labeled to be counted with the image analysis parameters employed.
Injections of RU-24969 resulted in a dramatic increase in staining for all three of the IEGs we examined and, for all three IEGs, pretreatment with quinpirole appeared to virtually eliminate the effects of RU-24969 throughout the entire striatal complex ŽFig. 1.. These impressions were supported by examination of the quantitative results displayed in Fig. 2a–c. For each of the three IEGs, analysis of the data by means of a 2 = 2 ŽRU-24969= quinpirole. factorial analysis of variance ŽANOVAs. indicated a significant effect of RU-24969 and a significant RU-24969= quinpirole interaction Ž p - 0.001 in each case., reflecting the fact that quinpirole attenuated the effects of RU-24969 while exerting little effect by itself. 3.2. Effects of RU-24969 on locomotor actiÕity Activity counts for the subjects examined by immunocytochemistry are shown in Fig. 2d where it can be seen that RU-24969 Ž5.0 mgrkg. induced a substantial increase in locomotor activity which was not attenuated by quinpirole pretreatment. Analysis of these data by means of a 2 = 2 Žquinpirole= RU-24969. ANOVA, indicated a significant effect of RU-24969, Ž F Ž1,18. s 17.1, P - 0.001., but not of quinpirole Ž p ) 0.2.. Although quinpirole actu-
Fig. 2. Effects of quinpirole and RU-24969 on striatal expression of c-Fos Žpanel A., Fos-B Žpanel B., Fra-2 Žpanel C. and locomotor activity ŽPanel D.. Error bars represent S.E.M.s.
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254
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Fig. 3. Effects of quinpirole and RU-24969 on locomotor activity examined using a within subject design in which each rat was tested under all six conditions. Error bars represent S.E.M.s.
ally tended to potentiate the response to RU-24969, the quinpirole= RU-24969 interaction did not approach significance Ž F - 1.. Fig. 3 shows the effects of quinpirole and RU-24969 on locomotor activity in the within-subjects experiment. A 3 = 2 Žquinpirole = RU-24969. repeated measures ANOVA indicated that RU-24969 again produced a significant increase in activity Ž F Ž1,5. s 55.9, P - 0.001.. Neither the effect of quinpirole Ž p ) 0.1., nor the quinpirole = RU-24969 interaction Ž F - 1. were significant.
4. Discussion The present results confirm our previous findings w10,11,72x that systemic administration of the mixed 5HT1Ar1B agonist RU-24969 induces intense c-Fos-like immunoreactivity ŽFLI. within the rat striatum. It is likely that this effect is due primarily to stimulation of the 5HT1B receptor since similar effects are also seen following injections of selective 5HT1B agonists, such as anpirtoline w72x and CP-94,253 Žin preparation., which lack affinity for the 5HT1A receptor. The current study also provides the first demonstration that RU-24969 induces expression of the additional Fos family transcription factors FosB and Fra-2. All of these proteins could be detected 2 h following drug treatment, but the possibility cannot currently be ruled out that peak expression of the various factors may occur at different times. Coordinated induction of c-Fos and FosB in the striatum has been previously shown to occur follow-
ing injections of a number of other drugs including amphetamine, neuroleptics and tetrahydrocannabinol ŽTHC., as well as in response to opiate withdrawal and cortical stimulation w1,24,40,50,52x. Substantially less is known about Fra-2 although this transcription factor has been reported to be expressed in the striatum following opiate withdrawal and in the nucleus accumbens following THC treatment w40,52x. It would appear that most pharmacological interventions tend to induce expression of multiple transcription factors within the striatum, but the extent to which different drugs are able to induce varied patterns of gene expression remains to be determined. It is also unclear at the current time whether all of these transcription factors are expressed in the same neurons, or whether some of them are specifically produced in discrete populations of cells. In several model systems, Fra-2 has been shown to regulate gene transcription in a direction opposite to that of c-Fos w58,65x and thus the effects of these two proteins might actually tend to oppose each other if they were co-expressed in individual neurons. In other brain structures, c-Fos and FosB have been found to be co-localized in individual cells w51x and it is likely that a similar situation occurs in the striatum, although no direct evidence is currently available. The precise mechanism by which RU-24969 activates striatal immediate early gene expression has not yet been identified. A number of studies have shown that injections of RU-24969 increase dopamine release or turnover within the striatum and other forebrain structures w3,4,6,39,41x. Since stimulation of dopamine receptors results in striatal FLI, these findings suggest that the induction of Fos expression by RU-24969 may occur as a result of stimu-
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lated dopamine release. This possibility is supported by findings that dopamine depleting lesions and pretreatment with the selective dopamine D 1 antagonist SCH-23390 markedly attenuate the c-Fos expression induced by RU24969 w10x. Alternatively, it is possible that RU-24969 produces its effects by a direct action on striatal neurons and that the role of dopamine is simply permissive. A number of workers have shown that the dopamine D 2-like agonist quinpirole is able to produce a regionally and compartmentally specific potentiation of the Fos response produced by administration of selective D 1 dopamine receptor agonists w16,29,48,71,73x. In contrast, the current results show that pretreatment with quinpirole virtually abolishes the striatal c-Fos, FosB and Fra-2 expression induced by RU-24969 alone. These findings are consistent with and extend previous studies in our laboratory which have shown that quinpirole is capable of attenuating the striatal c-Fos expression induced by the injection of several serotonergic, opiate and muscarinic receptor agonists w11x or by forcing animals to shuttle in order to escape electric shock w64x. Taken together, these findings suggest that quinpirole may attenuate the ability of a number of treatments to induce striatal FLI through a common mechanism, perhaps related to the capacity of this drug to suppress striatal unit activity w25x. It is possible, for example, that IEG expression in all of these situations is dependent on basal or drug-induced stimulation of D 1 dopamine receptors and that quinpirole acts as an ‘indirect D 1 receptor antagonist’ as a result of its ability to stimulate D 2-like autoreceptors. ŽIndeed, since quinpirole has been shown directly to decrease dopamine release w31,54,59,61x, an interesting question arises as to why this drug does not suppress locomotor activity when given by itself.. An alternative possibility is that quinpirole suppresses striatal FLI through a direct action on postsynaptic dopamine D 2 receptors located on striatal neurons. This interpretation is supported by the finding that while cAMP mediated protein phosphorylation has been implicated in the activation of the c-fos gene w17,56,68x, stimulation of D 2 receptors has been linked to an inhibition of cAMP formation w44,57,69x. Since D 2 receptors are heterogeneously distributed in different populations of striatal neurons w23,30x, this latter theory would require RU-24969 to induce IEG expression principally in cells containing D 2-like receptors. It should be noted in this context that dopamine agonists appear to induce Fos expression mainly in striatonigral neurons w7,55x which have been reported to express primarily D 1 receptors w15,21x. A third possibility is that the effects of quinpirole are mediated through a presynaptic suppression of corticostriatal transmission w36,60,74x. Future studies will be necessary to determine the precise mechanism through which quinpirole attenuates drug and behavioral induced FLI. Studies of antisense oligonucleotide injections have suggested that c-Fos may play an important role in mediating the motor responses produced by injections of dopamine
agonists w13,22,62,63,67x. It is not certain, however, whether the effects of these injections result from a blockade of drug induced Fos synthesis, or from a suppression of the constitutive expression of Fos which may occur at low levels w63x. The most striking result of the current experiments was the observation that injections of quinpirole, at doses which virtually abolished the induction of IEGs by RU-24969, failed entirely to attenuate the locomotor response to this serotonin agonist. These findings indicate that the induction of striatal IEGs cannot play a causal role in the production of hyperactivity by RU-24969, although it is still possible that constitutive expression of these transcription factors may by involved. This conclusion is analogous to that reached in previous studies in our laboratory w64x which found that quinpirole pretreatment almost abolished the expression of striatal FLI in rats shuttling to escape shock, but had no effect on escape latencies. Irrespective of its possible involvement in mediating effects on locomotor activity, striatal IEG expression would appear to provide a marker for the effects of RU-24969 in this region of the brain. Although RU-24969 has negligible affinity for catecholamine receptors, several reports have demonstrated that the hyperactivity induced by this drug is substantially attenuated in animals pretreated with reserpine or several dopamine receptor antagonists w19,42,66x. These findings and others w20x have suggested that RU24969 may induce hyperactivity as a result of its ability to release dopamine in the striatal complex. Several experimental results, however, are difficult to reconcile with this theory. For example, although the dopamine antagonist haloperidol blocks RU-24969 induced hyperactivity in intact animals, it fails to do so in subjects with electrolytic lesions of the globus pallidus w42x, which may attenuate the catalepsy normally produced by dopamine blockade w12x. Additionally, the nature of the behavior induced by RU24969 differs drastically from that seen after dopamine agonists in that it consists largely of bouts of explosive thigmotaxic locomotor activity and is associated with reductions in investigatory rearing and nose poking w19,46,53x. Both of these results suggest that the effects of RU-24969 on locomotor activity may result primarily from an action at extrastriatal sites. The current finding that quinpirole pretreatment blocks the effects of RU-24969 on striatal IEG expression without altering the effects of this drug on locomotor behavior provides novel and strong support for this viewpoint.
Acknowledgements The authors thank Eli Lilly for their generous gift of quinpirole. RU24969 was provided by Research Biochemcials International as a part of the N.I.M.H Chemical Synthesis Program, contract N01MH30003. These studies were supported by N.I.H. grant NS-33992.
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References w1x S. Baretta, H.B. Parthasarathy, A.M. Graybiel, Local release of GABAergic inhibition in the motor cortex induces immediate-early gene expression in indirect pathway neurons of the striatum, J. Neurosci. 17 Ž1997. 4752–4763. w2x S. Baretta, H.A. Robertson, A.M. Graybiel, Neurochemically specialized projection neurons of the striatum respond differently to psychomotor stimulants, in: G.W. Arbuthnott, P.C. Emson ŽEds.., Progress in Brain Research, Elsevier, New York, 1993, pp. 201–205. w3x S. Benloucif, M.P. Galloway, Facilitation of dopamine release in vivo by serotonin agonists: studies with microdialysis, Eur. J. Pharmacol. 200 Ž1991. 1–8. w4x S. Benloucif, J. Keegan, M.P. Galloway, Serotonin-facilitated dopamine release in vivo: pharmacological characterization, J. Pharmacol. Exp. Ther. 265 Ž1993. 373–377. w5x A.T. Bruninvels, B. Landwehrmeyer, E.L. Gustafson, M.M. Durkin, G. Mengod, T.A. Branchek, D. Hoyer, J.M. Palacios, Localization of 5-HT1B, 5-HT1Dalpha, 5-HT1E and 5-HT1F receptor messenger RNA in rodent and primate brain, Neuropharmacology 33 Ž1994. 367–386. w6x M.A. Carli, R. Invernizzi, L. Cervo, R. Samanin, Neurochemical and behavioral studies with RU-24969 in the rat, Psychopharmacology 94 Ž1988. 359–364. w7x M.A. Cenci, K. Campbell, K. Wictorin, A. Bjorklund, Striatal c-fos induction by cocaine or apomorphine occurs preferentially in output neurons projecting to the substantia nigra in the rat, Eur. J. Neurosci. 4 Ž1992. 376–380. w8x S.C. Cheetham, D.J. Heal, Evidence that RU-24969-induced locomotor activity in C57rB1r6 mice is specifically mediated by the 5-HT1B receptor, Br. J. Pharmacol. 110 Ž1993. 1621–1629. w9x A.J. Cole, R.V. Bhat, C. Patt, P.F. Worly, J.M. Baraban, D1 dopamine receptor activation of multiple transcription factor genes in rat striatum, J. Neurochem. 58 Ž1992. 1420–1426. w10x D.F. Cook, D. Wirtshafter, Serotonin agonist-induced c-fos expression in the rat striatum, Soc. Neurosci. Abst. 21 Ž1995. 1424. w11x D.F. Cook, D. Wirtshafter, Quinpirole attenuates striatal c-fos induction by 5-HT, opioid and muscarinic receptor agonists, Eur. J. Pharmacol. 349 Ž1998. 41–47. w12x B. Costall, J.E. Olley, Cholinergic and neuroleptic induced catalepsy: modification by lesions in the globus pallidus and substantia nigra, Neuropsychopharmacology 10 Ž1971. 581–594. w13x M. Dragunow, P. Lawlor, B. Chiasson, H. Robertson, c-fos antisense generates apomorphine and amphetamine-induced rotation, Neuroreport 5 Ž1993. 305–306. w14x C.R. Gardner, A.P. Guy, Behavioral effects of RU 24969, a 5-HT1 receptor agonist, in the mouse, Br. J. Pharmacol. 78 Ž1983. 96P. w15x C.R. Gerfen, The neostriatal mosaic: multiple levels of compartmental organization, J. Neur. Transm., Suppl. 36 Ž1992. 43–59. w16x C.R. Gerfen, K.A. Keefe, E.B. Gauda, D1 and D2 dopamine receptor function in the striatum: coactivation of D1- and D2-dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons, J. Neurosci. 15 Ž1995. 8167–8176. w17x A. Ghosh, D.D. Ginty, H. Bading, M.E. Greenberg, Calcium regulation of gene expression in neuronal cells, J. Neurobiol. 25 Ž1993. 294–303. w18x G.M. Goodwin, A.R. Green, A behavioral and biochemical study in mice and rats of putative selective agonists and antagonists for 5-HT1 and 5-HT2 receptors, Br. J. Pharmacol. 84 Ž1985. 743–753. w19x A.R. Green, A.P. Guy, C.R. Gardner, The behavioral effects of RU 24969, a suggested 5-HT1 receptor agonist in rodents and the effect on the behavior of treatment with antidepressants, Neuropharmacology 23 Ž1984. 655–661. w20x A.R. Green, D.J. Heal, The effects of drugs on serotonin-mediated behavioral models, in: R.A. Green ŽEd.., Neuropharmacology of Serotonin, Oxford, New York, 1985.
253
w21x M.B. Harrison, R.G. Wiley, G.F. Wooten, Selective localization of striatal D1 receptors to striatal neurons, Brain Res. 528 Ž1990. 317–322. w22x M. Heilig, J.A. Engel, B. Soderpalm, c-Fos antisense in the nucleus accumbens blocks the locomotor stimulant action of cocaine, Eur. J. Pharmacol. 236 Ž1993. 339–340. w23x S.M. Hersch, B.J. Cilax, C.A. Gutekunst, H.D. Rees, C.J. Heilman, K.K.L. Yung, J.P. Bolam, E. Ince, H. Yi, A.I. Levey, Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with corticostriatal afferents, J. Neurosci. 15 Ž1995. 5222–5237. w24x N. Hiroi, A.M. Graybiel, Atypical and typical neuroleptic treatments induce distinct programs of transcription factor expression in the striatum, J. Comp. Neurol. 374 Ž1996. 70–83. w25x K.C. Hooper, D.A. Banks, L.J. Stordahl, I.M. White, G.V. Rebec, Quinpirole inhibits striatal and excites pallidal neurons in freely moving rats, Neurosci. Lett. 237 Ž1997. 69–72. w26x M.L. Hooper, B.J. Chiasson, H.A. Robertson, Infusion into the brain of an antisense oligonucleotide to the immediate-early gene c-fos suppresses production of fos and produces a behavioral effect, Neuroscience 63 Ž1994. 917–924. w27x D. Hoyer, G. Engel, H.O. Kalkman, Characterization of the 5-HT1B recognition site in rat brain: binding studies with Žy. w125 Ixiodocyanopindolol, Eur. J. Pharmacol. 118 Ž1985. 1–12. w28x H.O. Kalkman, RU 24969-induced locomotion in rats is mediated by 5-HT1A receptors, Naunyn-Schmiedeberg’s Arch. Pharmacol. 352 Ž1995. 583–584. w29x G.J. LaHoste, J. Yu, J.F. Marshall, Striatal Fos expression is indicative of dopamine D1rD2 synergism and receptor supersensitivity, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 7451–7455. w30x C. LeMoine, B. Bloch, D1 and D2 dopamine receptor gene expression in the rat striatum: sensitive cRNA probes demonstrate prominent segregation of D1 and D2 mRNA’s in distinct neuronal populations of the dorsal and ventral striatum, J. Comp. Neurol. 355 Ž1995. 418–426. w31x A.C. Linthorst, H. De Lang, W. De Jong, D.H. Versteeg, Effect of the dopamine D2 receptor agonist quinpirole on the in vivo release of dopamine in the caudate nucleus of hypertensive rats, Eur. J. Pharmacol. 201 Ž1991. 125–133. w32x J.J. Lucas, L. Segu, R. Hen, 5-Hydroxytryptamine1B receptors modulate the effect of cocaine on c-fos expression: converging evidence using 5-hydroxytryptamine1B knockout mice and the 5-hydroxytryptamine1Br1D antagonist GR127935, Mol. Pharmacol. 51 Ž1997. 755–763. w33x G.A. MacGibbon, P.A. Lawlor, R. Bravo, M. Dragunow, Clozapine and haloperidol produce a differential pattern of immediate early gene expression in rat caudate–putamen, nucleus accumbens, lateral septum and island of Calleja, Mol. Brain Res. 23 Ž1994. 21–32. w34x G.A. MacGibbon, P.A. Lawlor, P. Huges, D. Young, M. Dragunow, Differential expression of inducible transcription factors in basal ganglia neurons, Mol. Brain Res. 34 Ž1995. 294–302. w35x D.L. Martinez, M.A. Geyer, A 5-HT1B antagonist blocks effects of RU 24969 on locomotor, but not investigatory, activity in rats, Soc. Neurosci. Abst. 23 Ž1997. 519. w36x G. Maura, A. Giardi, M. Raiteri, Release-regulating D2 dopamine receptors are located on striatal glutaminergic nerve terminals, J. Pharmacol. Exp. Ther. 247 Ž1998. 680–684. w37x D.N. Middlemiss, P.H. Hutson, The 5-HT1B receptors, Ann. N. Y. Acad. Sci. 600 Ž1990. 132–147. w38x R. Moratalla, E.A. Vickers, H.A. Robertson, B.H. Cochran, A.M. Graybiel, Coordinate expression of c-fos and junB is induced in the rat striatum by cocaine, J. Neurosci. 13 Ž1993. 423–433. w39x H. Nissbrandt, N. Waters, S. Hjorth, The influence of serotonergic drugs on dopaminergic neurotransmission in rat substantia nigra, striatum and limbic forebrain in vivo, Naunyn-Schmiedeberg’s Arch. Pharmacol. 346 Ž1992. 12–19. w40x H.E. Nye, E.J. Nestler, Induction of chronic Fos-related antigens in
254
w41x
w42x
w43x
w44x
w45x
w46x
w47x w48x
w49x
w50x
w51x
w52x
w53x
w54x
w55x
w56x
w57x
D.F. Cook, D. Wirtshafterr Brain Research 852 (2000) 247–254 rat brain by chronic morphine administration, Mol. Pharmacol. 49 Ž1996. 636–645. C. Oberlander, Effects of a potent 5-HT agonist, RU 24969, on the mesocorticolimbic and nigrostriatal dopamine systems, Br. J. Pharmacol. 80 Ž1983. 675P. C. Oberlander, B. Blaquiere, J.F. Pujol, Distinct functions for dopamine and serotonin in locomotor behavior: evidence using the 5-HT1 agonist RU 24969 in globus pallidus-lesioned rats, Neurosci. Lett. 67 Ž1986. 113–118. C. Oberlander, J.R. Boissier, Haloperidol blocks hyperlocomotion but not the circling behavior induced by the serotonin agonist RU 24969, Proc. 8th Int. Congr. Pharmacol. Abst., Tokyo, 1981, p. 839. P. Onali, M.C. Olianas, G.L. Gessa, Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum, Mol. Pharmacol. 28 Ž1985. 138–145. M.F. O’Neill, A.G. Fernandez, J.M. Palacios, GR 127935 blocks the locomotor and antidepressant-like effects of RU 24969 and the action of antidepressants in the mouse tail suspension tests, Pharmacol. Biochem. Behav. 53 Ž1996. 535–539. M.F. O’Neill, T. Parameswaran, RU-24969-induced behavioral syndrome requires activation of both 5-TH1A and 5-HT1B receptors, Psychopharmacology 132 Ž1997. 255–260. J.M. Palacios, C. Waeber, D. Hoyer, G. Mengod, Distribution of serotonin receptors, Ann. N. Y. Acad. Sci. 600 Ž1990. 36–52. M.L. Paul, A.M. Graybiel, J.C. David, H.A. Robertson, D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson’s disease, J. Neurosci. 12 Ž1992. 3729–3742. A. Pazos, J.M. Palacios, Quantitative autoradiographic mapping of serotonin receptors in the rat brain: I. serotonin-1 receptors, Brain Res. 346 Ž1985. 205–230. A.M. Persico, C.W. Schindler, B.F.O. O’Hara, M.T. Brannock, G. Uhl, Brain transcription factor expression: effects of acute and chronic amphetamine and injection stress, Mol. Brain Res. 20 Ž1993. 91–100. R.V. Peters, N. Aronin, W.J. Schwartz, c-Fos expression in the rat intergeniculate leaflet: photic regulation, co-localization with Fos-B and cellular identification, Brain Res. 728 Ž1996. 231–234. A. Porcella, G.L. Gessa, L. Pani, Delta9-tetrahydrocannabinol increases sequence-specific AP-1 DNA-binding activity and Fos-related antigens in the rat brain, Eur. J. Neurosci. 10 Ž1998. 1743–1751. N.L. Rempel, C.W. Callaway, M.A. Geyer, Serotonin1B receptor activation mimics behavioral effects of presynaptic serotonin release, Neuropsychopharmacology 8 Ž1993. 201–211. G.S. Robertson, C.S. Tham, C. Wilson, A. Jakubovic, H.C. Fibiger, In vivo comparisons of the effects of quinpirole and the putative presynaptic dopaminergic agonists B-HT 920 and SND 919 on striatal dopamine and acetylcholine release, J. Pharmacol. Exp. Ther. 264 Ž1993. 1344–1351. G.S. Robertson, S.R. Vincent, H.C. Fibiger, Striatonigral projection neurons contain D1 dopamine receptor activated c-fos, Brain Res. 523 Ž1990. 288–290. L.M. Robertson, T.K. Kerppola, M. Vendrell, D. Luk, R.J. Smeyne, C. Bocchaiaro, J.I. Morgan, T. Curran, Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements, Neuron 14 Ž1995. 241–252. S.W. Robinson, M.G. Caron, Selective inhibition of adenylyl cyclase type V by the dopamine D3 receptor, Mol. Pharmacol. 52 Ž1997. 508–514.
w58x S.E. Rutberg, E. Saez, S. Lo, S.I. Jang, N. Markova, B.M. Spiegelman, S.H. Yuspa, Opposing activities of c-Fos and Fra-2 on AP-1 regulated transcriptional activity in mouse keratinocytes induced to differentiate by calcium and phorbol esters, Oncogene 15 Ž1997. 1337–1346. w59x M. Santiago, B.H. Westerink, The regulation of dopamine release from nigrostriatal neurons in conscious rats: the role of somatodendritic autoreceptors, Eur. J. Pharmacol. 204 Ž1. Ž1991. 79–85. w60x R. Schwarcz, I. Creese, J.T. Coyle, S.H. Snyder, Dopamine receptors localized on cerebral cortical afferents to rat corpus striatum, Nature 271 Ž1978. 766–768. w61x R.E. See, B.A. Sorg, M.A. Chapman, P.W. Kalivas, In vivo assessment of release and metabolism of dopamine in the ventrolateral striatum of awake rats following administration of dopamine D1 and D2 receptor agonists and antagonists, Neuropharmacology 30 Ž1991. 1269–1274. w62x W. Sommer, B. Bjelke, D. Ganten, K. Fuxe, Antisense oligonucleotide to c-fos induces ipsilateral rotational behavior to d-amphetamine, Neuroreport 5 Ž1993. 277–280. w63x W. Sommer, K. Fuxe, On the role of c-fos expression in striatal transmission: the antisense oligonucleotide approach, Neurochem. Int. 31 Ž1997. 425–436. w64x W.M. Struthers, D. Wirtshafter, Quinpirole attenuates the striatal Fos expression induced by escape behavior, Brain Res. 785 Ž1998. 347–350. w65x T. Suzuki, H. Okuno, T. Yoshida, T. Endo, H. Nishina, H. Iba, Difference in transcriptional regulatory function between c-Fos and Fra-2, Nucleic Acids Res. 19 Ž1991. 5537–5542. w66x M.D. Tricklebank, D.N. Middlemiss, J. Neill, Pharmacological analysis of the behavioral and thermoregulatory effects of the putative 5-HT1 receptor agonist, RU 24969, in the rat, Neuropharmacology 25 Ž1986. 877–886. w67x T. Umekage, M. Namima, K. Fukushima, S. Sugita, T. Watanabe, c-fos antisense blocks methamphetamine-induced ambulatory activity reversibly, Neuroreport 8 Ž1997. 407–410. w68x F.M. Vaccarino, M.D. Hayward, H.N. Le, D.J. Hartigan, R.S. Duman, E.J. Nestler, Induction of immediate early genes by cyclic AMP in primary cultures of neurons from rat cerebral cortex, Brain Res., Mol. Brain Res. 19 Ž1993. 76–82. w69x L. Vallar, J. Meldolesi, Mechanisms of signal transduction at the dopamine D2 receptor, Trends Neurosci. 10 Ž1989. 74–77. w70x R.E. Watson, S.J. Wiegand, R.W. Clough, G.E. Hoffman, Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology, Peptides 7 Ž1986. 155–159. w71x D. Wirtshafter, K.E. Asin, Interactive effects of stimulation of D1 and D2 dopamine receptors on Fos-like immunoreactivity in the normosensitive rat striatum, Brain Res. Bull. 35 Ž1994. 85–91. w72x D. Wirtshafter, D.F. Cook, Serotonin-1B agonists induce compartmentally organized striatal Fos expression in rats, Neuroreport 9 Ž1998. 1217–1221. w73x D. Wirtshafter, G. Schardt, K.E. Asin, Compartmentally specific effects of quinpirole on the striatal Fos expression induced by stimulation of D1-dopamine receptors in intact rats, Brain Res. 771 Ž1997. 271–277. w74x B.K. Yamamoto, S. Davy, Dopaminergic modulation of glutamate release in striatum as measured by microdialysis, J. Neurochem. 58 Ž1992. 1742–1746.