Paradoxical Striatal Cellular Signaling Responses to Psychostimulants in Hyperactive Mice
2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês
10.1074/jbc.m606062200
ISSN1083-351X
AutoresJean‐Martin Beaulieu, Tatyana D. Sotnikova, Raul R. Gainetdinov, Marc G. Caron,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoRecent investigations have shown that three major striatal-signaling pathways (protein kinase A/DARPP-32, Akt/glycogen synthase kinase 3, and ERK) are involved in the regulation of locomotor activity by the monoaminergic neurotransmitter dopamine. Here we used dopamine transporter knock-out mice to examine which particular changes in the regulation of these cell signaling mechanisms are associated with distinct behavioral responses to psychostimulants. In normal animals, amphetamine and methylphenidate increase extracellular levels of dopamine, leading to an enhancement of locomotor activity. However, in dopamine transporter knock-out mice that display a hyperactivity phenotype resulting from a persistent hyperdopaminergic state, these drugs antagonize hyperactivity. Under basal conditions, dopamine transporter knock-out mice show enhanced striatal DARPP-32 phosphorylation, activation of ERK, and inactivation of Akt as compared with wild-type littermates. However, administration of amphetamine or methylphenidate to these mice reveals that inhibition of ERK signaling is a common determinant for the ability of these drugs to antagonize hyperactivity. In contrast, psychostimulants activate ERK and induce hyperactivity in normal animals. In hyperactive mice psychostimulant-mediated behavioral inhibition and ERK regulation are also mimicked by the serotonergic drugs fluoxetine and 5-carboxamidotryptamine, thereby revealing the involvement of serotonin-dependent inhibition of striatal ERK signaling. Furthermore, direct inhibition of the ERK signaling cascade in vivo using the MEK inhibitor SL327 recapitulates the actions of psychostimulants in hyperactive mice and prevents the locomotor-enhancing effects of amphetamine in normal animals. These data suggest that the inhibitory action of psychostimulants on dopamine-dependent hyperactivity results from altered regulation of striatal ERK signaling. In addition, these results illustrate how altered homeostatic state of neurotransmission can influence in vivo signaling responses and biological actions of pharmacological agents used to manage psychiatric conditions such as Attention Deficit Hyperactivity Disorder (ADHD). Recent investigations have shown that three major striatal-signaling pathways (protein kinase A/DARPP-32, Akt/glycogen synthase kinase 3, and ERK) are involved in the regulation of locomotor activity by the monoaminergic neurotransmitter dopamine. Here we used dopamine transporter knock-out mice to examine which particular changes in the regulation of these cell signaling mechanisms are associated with distinct behavioral responses to psychostimulants. In normal animals, amphetamine and methylphenidate increase extracellular levels of dopamine, leading to an enhancement of locomotor activity. However, in dopamine transporter knock-out mice that display a hyperactivity phenotype resulting from a persistent hyperdopaminergic state, these drugs antagonize hyperactivity. Under basal conditions, dopamine transporter knock-out mice show enhanced striatal DARPP-32 phosphorylation, activation of ERK, and inactivation of Akt as compared with wild-type littermates. However, administration of amphetamine or methylphenidate to these mice reveals that inhibition of ERK signaling is a common determinant for the ability of these drugs to antagonize hyperactivity. In contrast, psychostimulants activate ERK and induce hyperactivity in normal animals. In hyperactive mice psychostimulant-mediated behavioral inhibition and ERK regulation are also mimicked by the serotonergic drugs fluoxetine and 5-carboxamidotryptamine, thereby revealing the involvement of serotonin-dependent inhibition of striatal ERK signaling. Furthermore, direct inhibition of the ERK signaling cascade in vivo using the MEK inhibitor SL327 recapitulates the actions of psychostimulants in hyperactive mice and prevents the locomotor-enhancing effects of amphetamine in normal animals. These data suggest that the inhibitory action of psychostimulants on dopamine-dependent hyperactivity results from altered regulation of striatal ERK signaling. In addition, these results illustrate how altered homeostatic state of neurotransmission can influence in vivo signaling responses and biological actions of pharmacological agents used to manage psychiatric conditions such as Attention Deficit Hyperactivity Disorder (ADHD). Dopaminergic neurotransmission mediates a series of physiological functions ranging from the control of locomotion and cognition to attention, emotion, and reward (1Carlsson A. Annu. Rev. Neurosci. 1987; 10: 19-40Crossref PubMed Scopus (122) Google Scholar, 2Gainetdinov R.R. Caron M.G. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 261-284Crossref PubMed Scopus (296) Google Scholar, 3Zhou Q.Y. Palmiter R.D. Cell. 1995; 83: 1197-1209Abstract Full Text PDF PubMed Scopus (590) Google Scholar). This neurotransmitter system is also the main target for the actions of psychostimulants. By interfering with dopamine transporter (DAT) 3The abbreviations used are: DAT, dopamine transporter; KO, knock-out; WT, wild type; 5CT, 5-carboxamidotryptamine; ADHD, attention deficit hyperactivity disorder. 3The abbreviations used are: DAT, dopamine transporter; KO, knock-out; WT, wild type; 5CT, 5-carboxamidotryptamine; ADHD, attention deficit hyperactivity disorder. functions, compounds like methylphenidate and amphetamine normally increase extracellular levels of dopamine, leading to their well known locomotor-promoting/stimulant effect. However, under certain conditions psychostimulants can antagonize hyperactivity through a mechanism of action that is not well understood. Indeed, psychostimulants are used therapeutically to manage the symptoms of hyperactivity, impulsivity, and inattention associated with Attention Deficit Hyperactivity Disorder (ADHD), a common psychiatric condition affecting a significant portion of pediatric and adult populations (4Horrigan J.P. Expert. Opin. Pharmacother. 2001; 2: 573-586Crossref PubMed Scopus (7) Google Scholar, 5Greenhill L.L. Psychiatr. Clin. North. Am. 1992; 15: 1-27Abstract Full Text PDF PubMed Google Scholar, 6Barkley R.A. Attention Deficit Hyperactivity Disorder: a Handbook for Diagnosis and Treatment. Guiford, New York1990Google Scholar). The therapeutic efficacy of psychostimulants that typically enhance monoaminergic neurotransmission has led to the postulate that imbalance in dopamine (DA), norepinephrine, and/or serotonin (5HT) synaptic transmission may contribute to the etiology of ADHD (7Biederman J. Biol. 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Toxicol. 2003; 43: 261-284Crossref PubMed Scopus (296) Google Scholar, 10Gainetdinov R.R. Wetsel W.C. Jones S.R. Levin E.D. Jaber M. Caron M.G. Science. 1999; 283: 397-401Crossref PubMed Scopus (705) Google Scholar, 11Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2034) Google Scholar, 12Jones S.R. Gainetdinov R.R. Jaber M. Giros B. Wightman R.M. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4029-4034Crossref PubMed Scopus (555) Google Scholar). Moreover, the same psychostimulants that induce locomotor hyperactivity in normal mice exert a paradoxical antihyperkinetic effect in DAT-KO mice (10Gainetdinov R.R. Wetsel W.C. Jones S.R. Levin E.D. Jaber M. Caron M.G. Science. 1999; 283: 397-401Crossref PubMed Scopus (705) Google Scholar), thus providing a model system to identify potential mechanisms by which psychostimulants, instead of exerting their normal stimulatory action, can counteract these behavioral manifestations.Recent investigations on the mechanisms of action of psychostimulants and other psychoactive compounds have shown that many of these drugs can act simultaneously on multiple neurotransmitter systems (13Roth B.L. Sheffler D.J. Kroeze W.K. Nat. Rev. Drug. Discov. 2004; 3: 353-359Crossref PubMed Scopus (961) Google Scholar), thus suggesting that distinct cellular signaling mechanisms may determine their pharmacological actions (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 15Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. 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Three major signaling pathways have been shown to be associated with striatal DA neurotransmission and concomitant locomotor responses to psychostimulants. First, activation or inhibition of the cAMP pathway through D1 and D2 dopamine receptors leads to regulation of protein kinase A and modulation of the dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32), an inhibitor of protein phosphatase-1 (21Greengard P. Allen P.B. Nairn A.C. Neuron. 1999; 23: 435-447Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). Phosphorylation of DARPP-32 on Thr-34 has been associated with locomotor activation and biochemical responses to multiple psychotropic drugs, including psychostimulants (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 16Fienberg A.A. Hiroi N. Mermelstein P.G. Song W. Snyder G.L. Nishi A. Cheramy A. O'Callaghan J.P. Miller D.B. Cole D.G. Corbett R. Haile C.N. Cooper D.C. Onn S.P. Grace A.A. Ouimet C.C. White F.J. Hyman S.E. Surmeier D.J. Girault J. Nestler E.J. Greengard P. Science. 1998; 281: 838-842Crossref PubMed Scopus (389) Google Scholar, 17Fukui R. Svenningsson P. Matsuishi T. Higashi H. Nairn A.C. Greengard P. Nishi A. J. Neurochem. 2003; 87: 1391-1401Crossref PubMed Scopus (51) Google Scholar). Second, a distinct signaling pathway involves the serine/threonine kinase Akt that is negatively regulated by D2-class receptors through a signaling complex involving protein phosphatase-2A and β-arrestin 2 (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Stimulation of dopamine D2-class receptors leads to a dephosphorylation of Akt on its regulatory Thr-308 residue (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 15Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5099-5104Crossref PubMed Scopus (672) Google Scholar). Inactivation of Akt by DA results in activation of glycogen synthase kinase 3, which in turn contributes to the development of locomotor hyperactivity (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 15Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5099-5104Crossref PubMed Scopus (672) Google Scholar). In addition, DA also positively regulates ERK by acting through multiple signaling mechanisms that involve D1 and/or D2-class dopamine receptors (18Zhang L. Lou D. Jiao H. Zhang D. Wang X. Xia Y. Zhang J. Xu M. J. Neurosci. 2004; 24: 3344-3354Crossref PubMed Scopus (189) Google Scholar, 19Valjent E. Corvol J.C. Pages C. Besson M.J. Maldonado R. Caboche J. J. Neurosci. 2000; 20: 8701-8709Crossref PubMed Google Scholar, 20Pozzi L. Hakansson K. Usiello A. Borgkvist A. Lindskog M. Greengard P. Fisone G. J. Neurochem. 2003; 86: 451-459Crossref PubMed Scopus (113) Google Scholar, 22Chen J. Rusnak M. Luedtke R.R. Sidhu A. J. Biol. Chem. 2004; Google Scholar, 23Wang C. Buck D.C. Yang R. Macey T.A. Neve K.A. J. Neurochem. 2005; 93: 899-909Crossref PubMed Scopus (56) Google Scholar). Although ERK has been shown to participate in acute responses to cocaine (19Valjent E. Corvol J.C. Pages C. Besson M.J. Maldonado R. Caboche J. J. Neurosci. 2000; 20: 8701-8709Crossref PubMed Google Scholar), it appears to be mostly involved in the development of long-term changes of gene expression, synaptic plasticity, and locomotor responses following repeated exposure to this drug (19Valjent E. Corvol J.C. Pages C. Besson M.J. Maldonado R. Caboche J. J. Neurosci. 2000; 20: 8701-8709Crossref PubMed Google Scholar, 24Valjent E. Pages C. Herve D. Girault J.A. Caboche J. Eur. J. Neurosci. 2004; 19: 1826-1836Crossref PubMed Scopus (356) Google Scholar, 25Berhow M.T. Hiroi N. Nestler E.J. J. Neurosci. 1996; 16: 4707-4715Crossref PubMed Google Scholar).Here we have used DAT-KO mice to investigate the involvement of striatal signaling events in the inhibitory action of psychostimulants on hyperactivity. We demonstrate that in the presence of an overactive brain dopaminergic system created by the absence of DAT, inhibition of ERK signaling is a common property that accounts for the action of psychostimulants to diminish the hyperactivity of DAT-KO mice. These findings may have implications for the mechanisms by which psychostimulants achieve their pharmacological effects in ADHD.MATERIALS AND METHODSExperimental Animals—C57BL/129SvJ DAT-KO and WT littermates were described previously (11Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2034) Google Scholar, 26Cyr M. Beaulieu J.M. Laakso A. Sotnikova T.D. Yao W.D. Bohn L.M. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11035-11040Crossref PubMed Scopus (123) Google Scholar). These mice have been inbred for more than 30 generations to generate mice sharing a similar genetic background between KO and WT control animals. For all experiments mice of 3 to 4 months of age were used. Before experiments, animals were housed four or five to a cage at 23 °C on a 12-h light/12-h dark cycle with ad libitum access to food and water. Animal care was approved by the Institutional Animal Care and Use Committee and followed National Institutes of Health guidelines.Antibodies—The anti-phospho-Akt (Thr-308), anti-total-Akt, antiphospho-ERK1/2 (Thr-202/Tyr-204), anti-ERK were purchased from Cell Signaling Technology (Beverly, MA). The anti-phospho-DARPP-32 Thr-34 was from Phosphosolutions (Aurora, CO). The anti-DARPP-32 was obtained from BD Transduction Laboratories (Lexington, KY).Drug Administration—Amphetamine (Sigma), methylphenidate (Sigma), and 5-carboxamidotryptamine (5CT; Sigma) were dissolved in saline and injected intraperitoneally. Fluoxetine (Tocris Cookson Inc., Ellisville, MO) was dissolved in water and injected subcutaneously. SL327 (Tocris Cookson Inc.) was injected intraperitoneally after suspension in a minimal amount of Tween and made up to volume with distilled water. Corresponding vehicle solutions were administered to control animals.Western Blot Analyses—Western blot analyses were performed as described in Beaulieu et al. (15Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5099-5104Crossref PubMed Scopus (672) Google Scholar). For quantitative analysis, total proteins were used as loading controls for phosphoprotein signals.Measurement of Locomotor Activity—Locomotion was evaluated under illuminated conditions in an automated Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH). Locomotor activity was measured in terms of the total distance covered (horizontal activity), and the stereotypy time refers to the total time that stereotypic behaviors (repetitive beam breaks of a given beam or beams with intervals <1 s) were observed (10Gainetdinov R.R. Wetsel W.C. Jones S.R. Levin E.D. Jaber M. Caron M.G. Science. 1999; 283: 397-401Crossref PubMed Scopus (705) Google Scholar).Statistical Analyses—Data were analyzed by two-tailed t test or one-way analysis of variance. Values in graphs were expressed as mean ± S.E. n represents the number of animals used for each experiment.RESULTSTo examine the activity of DA-associated signaling molecules in hyperactive DAT-KO mice under basal conditions, we performed Western blot analysis on striatal extracts prepared from DAT-KO mice and control wild-type (WT) littermates. This analysis revealed an elevation of Thr-34-DARPP-32 phosphorylation in DAT-KO mice (Fig. 1A). As previously reported (14Beaulieu J.M. Sotnikova T.D. Marion S. Lefkowitz R.J. Gainetdinov R.R. Caron M.G. Cell. 2005; 122: 261-273Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 15Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5099-5104Crossref PubMed Scopus (672) Google Scholar), DAT-KO mice also exhibited reduced phosphorylation of Akt on its regulatory Thr-308 residue (Fig. 1B). Finally, enhanced phosphorylation/activation of ERK2 was detected using an anti-phospho-ERK1/2 antibody (Fig. 1C), thus indicating that all three signaling pathways were responding to persistently enhanced DA neurotransmission in the striatum of DAT-KO mice.Administration of the psychostimulants methylphenidate and amphetamine to DAT-KO mice resulted in a marked reduction of locomotor activity (Fig. 2, A and C). To identify common signaling events underlying this behavioral effect, we examined the impact of psychostimulants on phospho-DARPP-32, phospho-Akt, and phospho-ERK2 levels in hyperactive DAT-KO mice. Relative phosphoprotein levels were measured by Western blots in striatal extracts obtained from DAT-KO mice treated either with methylphenidate (30 mg/kg) or amphetamine (2 mg/kg) under conditions where a maximal effect on locomotion was observed. As shown in Fig. 2B, administration of methylphenidate to DAT-KOs resulted in a modest dephosphorylation of Thr-308-Akt and in a more pronounced inhibition of ERK2 while leaving DARPP-32 essentially unaffected. In comparison, treatment of DAT-KO mice with amphetamine led to a slight increase in Akt phosphorylation and a reduction of ERK2 phosphorylation (Fig. 2D), thus indicating that inactivation of ERK is a common effect of psychostimulants in DAT-KO mice.FIGURE 2Psychostimulants antagonize ERK activity and locomotion in hyperactive mice. Locomotor activity and phosphoprotein levels following administration of methylphenidate 30 mg/kg intraperitoneal (A, B) or amphetamine 2 mg/kg intraperitoneal (C, D) to DAT-KO mice. For locomotor activity measurements, mice were placed in an activity monitor, habituated for 30 min, and monitored for 90 min following drug administration. A and C, locomotor activity was continuously recorded as total distance traveled in blocks of 5 min. Arrows indicate drug administration; n indicates the number of animals used per experiment. Data are average ± S.E. B and D, Western blot analyses of pDARPP32, pAkt, and pERK2 were carried out from striata collected at time points for which drugs showed significant behavioral action: methylphenidate 90 min post-injection, amphetamine 60 min post-injection. V, vehicle; M, methylphenidate; A, amphetamine. B and D, n = 5 mice/group. Data are average ± S.E. *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The paradoxical behavioral action of psychostimulants in hyperactive DAT-KO mice has been related to changes in 5HT neurotransmission (10Gainetdinov R.R. Wetsel W.C. Jones S.R. Levin E.D. Jaber M. Caron M.G. Science. 1999; 283: 397-401Crossref PubMed Scopus (705) Google Scholar, 27Gainetdinov R.R. Mohn A.R. Bohn L.M. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11047-11054Crossref PubMed Scopus (143) Google Scholar). We therefore evaluated the effect of serotonergic drugs on behavior and signaling protein phosphorylation. The selective serotonin reuptake inhibitor fluoxetine exerts its pharmacological action by blocking 5HT transporter-mediated reuptake, thus enhancing extracellular 5HT levels (28Fuller R.W. Wong D.T. Robertson D.W. Med. Res. Rev. 1991; 11: 17-34Crossref PubMed Scopus (172) Google Scholar). As shown previously (10Gainetdinov R.R. Wetsel W.C. Jones S.R. Levin E.D. Jaber M. Caron M.G. Science. 1999; 283: 397-401Crossref PubMed Scopus (705) Google Scholar), fluoxetine (20 mg/kg) had a powerful inhibitory effect on hyperactivity in DAT-KO mice (Fig. 3A). Western blots prepared from fluoxetine or vehicle-treated DAT-KO mice showed that this specific serotonin reuptake inhibitor produced a potent inhibition of ERK2 under conditions leading to suppression of locomotor hyperactivity (Fig. 3B). Notably, fluoxetine had no significant effect on Akt or DARPP-32 phosphorylation (Fig. 3B). To further support these observations we then used 5CT a non-selective 5HT receptor agonist (29Lanfumey L. Hamon M. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 1-10Crossref PubMed Scopus (135) Google Scholar, 30Nelson D.L. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 53-58Crossref PubMed Scopus (83) Google Scholar, 31Molewijk H.E. Hartog K. van der Poel A.M. Mos J. Olivier B. Psychopharmacology (Berl.). 1996; 128: 31-38Crossref PubMed Scopus (51) Google Scholar). Administration of 5CT (0.1 mg/kg) produced marked and prolonged suppression of hyperactivity in DAT-KO mice (Fig. 3C). Remarkably, this behavioral effect of 5CT was correlated with reduced ERK phosphorylation without any effect on Akt or DARPP-32 (Fig. 3D).FIGURE 3Serotonergic drugs reproduce behavioral and biochemical actions of psychostimulants in hyperactive mice. Locomotor activity and phosphoprotein levels following administration of fluoxetine 20 mg/kg subcutaneously (A, B) or 5-carboxamidotryptamine (5CT) 0.1 mg/kg intraperitoneally (C, D) to DAT-KO mice. For locomotor activity measurements, mice were placed in an activity monitor, habituated for 30 min, and monitored for 90 min following drug administration. A and C, locomotor activity was continuously recorded as total distance traveled in blocks of 5 min. Arrows indicate drug administration; n indicates the number of animals used/experiment. Data are average ± S.E. B and D, Western blot analyses of pDARPP32, pAkt, and pERK2 were conducted from striatal extracts collected at the time of maximal behavioral action: fluoxetine 20 min post-injection, 5CT 30 min post-injection. V, vehicle; F, fluoxetine; 5CT, 5-carboxamidotryptamine. B and D, n = 5 mice/group. Data are average ± S.E. *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the dynamic of ERK regulation and its correlation with the behavioral action of psychostimulants, phospho-ERK levels were measured at different time points following administration of amphetamine (2 mg/kg) or methylphenidate (30 mg/kg). Amphetamine caused a sustained dephosphorylation of ERK that reached its maximum at 60 min and lasted over a period of 120 min post-injection (Fig. 4A), thus correlating with the long lasting action of this drug on hyperactivity in DAT-KO mice. In contrast, the action of methylphenidate on ERK phosphorylation was progressive and reached its maximum at 90 min post-injection (Fig. 4B). Taken together, these results indicate that inhibition of ERK represents a common biochemical outcome of psychostimulants and 5HT drugs that suppress locomotor hyperactivity in DAT-KO mice.FIGURE 4Time course of striatal ERK2 regulation by psychostimulants and fluoxetine. ERK2 phosphorylation was measured by Western blot analysis in the striatum of DAT-KO mice after injection of amphetamine (2 mg/kg, intraperitoneal) (A) or methylphenidate (30 mg/kg, intraperitoneal) (B). n = 4–10 mice/group. Data are average ± S.E. *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Psychostimulants are potent inducers of locomotor hyperactivity in normal rodents. Furthermore, administration of the psychostimulant cocaine is known to activate ERK in the striatum (18Zhang L. Lou D. Jiao H. Zhang D. Wang X. Xia Y. Zhang J. Xu M. J. Neurosci. 2004; 24: 3344-3354Crossref PubMed Scopus (189) Google Scholar, 19Valjent E. Corvol J.C. Pages C. Besson M.J. Maldonado R. Caboche J. J. Neurosci. 2000; 20: 8701-8709Crossref PubMed Google Scholar), thus suggesting that changes in ERK signaling induced by psychostimulants in hyperactive mice may differ from their action in normal animals. To test this possibility, we proceeded to evaluate the action of psychostimulants and 5HT drugs on locomotion and ERK signaling in WT animals. As expected, administration of methylphenidate or amphetamine to WT mice resulted in a marked increase of locomotor activity (Fig. 5, A and C). Moreover, instead of inhibiting ERK activity like in DAT-KO mice, these two psychostimulants enhanced ERK2 phosphorylation in the striatum of WT littermates (Fig. 5, B and D). In contrast, administration of the 5HT drugs fluoxetine and 5CT to WT mice resulted in noticeable reduction in basal locomotor activity and in a significant reduction of ERK2 phosphorylation at least in the case of 5CT (Fig. 6, A–D). These results indicate that psychostimulants not only induce opposite behavioral locomotor effects in WT and DAT-KO mice but that these effects are paralleled by paradoxical changes in ERK-mediated signaling. Moreover, both the behavioral and signaling effects of psychostimulants in hyperactive mice are similar to those triggered by 5HT drugs.FIGURE 5Behavioral and biochemical actions of psychostimulants in normal mice. Locomotor activity and phospho-ERK levels following administration of methylphenidate 30 mg/kg intraperitoneal (A, B), amphetamine 2 mg/kg intraperitoneal (C, D) to WT mice. For locomotor activity measurements, mice were placed in an activity monitor, habituated for 30 min, and monitored for 90 min following drug administration. A and C, locomotor activity was continuously recorded as total distance traveled in blocks of 5 min. Arrows indicate drug administration; n indicates the number of animals used/experiment. Data are average ± S.E. Western blot analyses of pERK2 levels from striatal extracts were collected under conditions used in previous experiments: methylphenidate 90 min post-injection, amphetamine 60 min post-injection. B and D, V, vehicle; M, methylphenidate; A, amphetamine; n = 5 mice/group. Data are average ± S.E. *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Behavioral and biochemical actions of serotonergic drugs in normal mice. Locomotor activity and phospho-ERK levels following administration of fluoxetine 20 mg/kg subcutaneously (A, B) or 5-carboxamidotryptamine (5CT) 0.1 mg/kg intraperitoneally (C, D) to WT mice. For locomotor activity measurements, mice were placed in an activity monitor, habituated for 30 min, and monitored for 90 min following drug administration. A and C, locomotor activity was continuously recorded as total distance traveled in blocks of 5 min. Arrows indicate drug administration; n indicates the number of animals used/experiment. Data are average ± S.E. Western blot analyses of pERK2 levels from striatal extracts were collected under conditions used in previous experiments: fluoxetine 20 min post-injection, 5CT 30 min post-injection. B and D, V, vehicle; F, fluoxetine; 5CT, 5-carboxamidotryptamine; n = 5 mice/group. Data are average ± S.E. *, p ≤ 0.05.View Large Image
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