Address correspondence and reprint requests to Gilberto Fisone, Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 171 77 Stockholm, Sweden. E-mail: email@example.com
In the striatum, stimulation of dopamine D2 receptors results in attenuation of glutamate responses. This effect is exerted in large part via negative regulation of AMPA glutamate receptors. Phosphorylation of the GluR1 subunit of the AMPA receptor has been proposed to play a critical role in the modulation of glutamate transmission, in striatal medium spiny neurons. Here, we have examined the effects of blockade of dopamine D2-like receptors on the phosphorylation of GluR1 at the cAMP-dependent protein kinase (PKA) site, Ser845, and at the protein kinase C and calcium/calmodulin-dependent protein kinase II site, Ser831. Administration of haloperidol, an antipsychotic drug with dopamine D2 receptor antagonistic properties, increases the phosphorylation of GluR1 at Ser845, without affecting phosphorylation at Ser831. The same effect is observed using eticlopride, a selective dopamine D2 receptor antagonist. In contrast, administration of the dopamine D2-like agonist, quinpirole, decreases GluR1 phosphorylation at Ser845. The increase in Ser845 phosphorylation produced by haloperidol is abolished in dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) knockout mice, or in mice in which the PKA phosphorylation site on DARPP-32 (i.e. Thr34) has been mutated (Thr34 → Ala mutant mice), and requires tonic activation of adenosine A2A receptors. These results demonstrate that dopamine D2 antagonists increase GluR1 phosphorylation at Ser845 by removing the inhibitory tone exerted by dopamine D2 receptors on the PKA/DARPP-32 cascade.
dopamine- and cAMP-regulated phosphoprotein of 32 kDa
cAMP-dependent protein kinase, PKA
In the striatum, dopamine exerts a complex modulation of glutamatergic transmission by regulating different subtypes of glutamate ionotropic receptors. Previous studies have shown that, in striatal medium spiny neurons, activation of dopamine D1 receptors enhances responses mediated by N-methyl-d-aspartate (NMDA) (Cepeda et al. 1993; Colwell and Levine 1995; Cepeda et al. 1998; Flores-Hernández et al. 2002) and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) (Yan et al. 1999; Lin et al. 2003) glutamate receptors. Dopamine D2 receptors are highly expressed by a large population of striatal medium spiny neurons, where they modulate glutamate function by inhibiting responses elicited via activation of non-NMDA receptors. Thus, AMPA current amplitude is reduced by quinpirole, a dopamine D2-like receptor agonist (Hernández-Echeagaray et al. 2004), and glutamatergic transmission is potentiated in dopamine D2 receptor knockout mice (Cepeda et al. 2001).
Phosphorylation of the GluR1 subunit plays an essential role in the regulation of AMPA receptors. Protein kinase C and calcium/calmodulin-dependent protein kinase II catalyse GluR1 phosphorylation at Ser831 (Barria et al. 1997; Mammen et al. 1997), thereby increasing AMPA channel conductance (Derkach et al. 1999). A similar effect also occurs following phosphorylation of GluR1 catalysed by cAMP-dependent protein kinase (PKA) at Ser845 (Roche et al. 1996; Banke et al. 2000). The involvement of the cAMP pathway in the regulation of AMPA receptor function is clearly demonstrated by the observation that D1 receptors prevent current rundown at AMPA receptors via activation of a PKA/DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) cascade (Yan et al. 1999), which promotes Ser845 phosphorylation (Snyder et al. 2000). In addition, it has been reported that PKA-dependent phosphorylation of GluR1 is involved in dopamine D1 receptor-mediated insertion of AMPA receptors onto the surface of striatal medium spiny neurons (Mangiavacchi and Wolf 2004).
Administration of dopamine D2 receptor antagonists, including antipsychotic drugs (e.g. haloperidol) used in the treatment of schizophrenia, results in the activation of the cAMP/PKA pathway via blockade of postsynaptic dopamine D2 receptors located on medium spiny neurons and coupled to inhibition of cAMP production. This effect is accompanied by increased phosphorylation of DARPP-32 at the PKA site, Thr34 (Svenningsson et al. 2000; Pozzi et al. 2003) and may be involved in the dopamine D2 receptor-mediated regulation of AMPA receptor function. Virtually nothing, however, is known about the involvement of dopamine D2 receptors in the control of the state of phosphorylation of the AMPA receptor. The present study was designed to determine the effect of blockade of dopamine D2 receptors on the state of phosphorylation of GluR1, and to examine the involvement of DARPP-32 in dopamine D2 receptor-dependent regulation of GluR1 phosphorylation.
Materials and methods
Male C57BL/6 mice (20–30 g) were obtained from B&K (Stockholm, Sweden) or from Charles River (Kingston, NY, USA). Wild-type and DARPP-32 knockout mice (Fienberg et al. 1998) were generated from the offspring of DARPP-32+/+ × DARPP-32+/+ and DARPP-32–/– × DARPP-32–/– mating pairs. These mating pairs were obtained from heterozygous mice, which were backcrossed for at least 20 generations on a C57BL/6 background. DARPP-32+/+ × DARPP-32+/+ and DARPP-32–/– × DARPP-32–/– mating was carried out separately for no more than two generations. Mice bearing a mutation in which Thr34 was replaced by a non-phosphorylatable Ala (Thr34 → Ala) (Svenningsson et al. 2003) were obtained from heterozygous animals generated from C57BL/6 × 129SV hybrids bred for one generation on a C57BL/6 background. All experiments were approved by the Swedish Animal Welfare Agency and the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medical College.
Haloperidol, eticlopride, quinpirole and SCH23390 were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). KW6002 was a gift from Hoffman-LaRoche (Basel, Switzerland). Haloperidol was dissolved in a solution of 10% acetic acid in saline and the pH was brought to 6.0 with 1.0 m NaOH. Eticlopride, quinpirole and SCH23390 were dissolved in 0.9% saline. KW6002 was suspended by sonication in a solution of 8% Tween-80 in saline.
Determination of phosphorylated GluR1 and DARPP-32
Mice were injected i.p. with vehicle or drugs and killed by decapitation at specific time points (15–60 min). The heads of the animals were immediately immersed in liquid nitrogen for 6 s. The brains were then removed, and the striata rapidly (20 s) dissected out on an ice-cold surface. Each striatum was sonicated in 750 μL of 1% sodium dodecylsulfate and boiled for 10 min. Aliquots of the homogenate were used for protein determination. Equal amounts of protein (30 μg; containing equal amounts of GluR1) from each sample were loaded onto 7.5 or 10% polyacrylamide gels. The proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) (Towbin et al. 1979). PhosphoSer831- and phosphoSer845-GluR1 were detected using commercial polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY, USA). The state of phosphorylation of DARPP-32 was determined using a monoclonal antibody against phosphoThr34-DARPP-32 (Snyder et al. 1992). Antibodies generated against GluR1 and DARPP-32 (Cell Signaling Technology, Beverly, MA, USA) that are not phosphorylation state specific, were used to estimate the total amount of GluR1 and DARPP-32. Antibody binding was revealed using HRP-linked IgGs as a secondary antibodies (diluted 1 : 10 000) and enhanced chemiluminescence immunoblotting detection. Quantification of the bands corresponding to phosphorylated or total proteins was done by densitometry, using NIH Image (version 1.63) software.
Blockade of dopamine D2 receptors increases GluR1 phosphorylation at Ser845
In a first series of experiments we examined the ability of haloperidol, a typical antipsychotic drug with high affinity for dopamine D2-like receptors, and eticlopride, a selective dopamine D2-like receptor antagonist, to regulate phosphorylation of GluR1 at Ser845 and Ser831. Haloperidol increased Ser845 phosphorylation at the doses of 0.25 and 0.5 mg/kg (Fig. 1a). The levels of phosphoSer845-GluR1 were maximally enhanced 15 min after injection, still elevated at 30 min and returned to basal levels after 60 min (Fig. 1b). Eticlopride (0.2 mg/kg) produced a comparable increase (172% of control) in Ser845 phosphorylation, as determined 15 min after administration (Fig. 1c). Haloperidol (Fig. 2a) and eticlopride (Fig. 2b) did not affect the state of phosphorylation of GluR1 at Ser831.
Phosphorylation of DARPP-32 at Thr34 is involved in the increase in GluR1 phosphorylation produced by haloperidol
Blockade of dopamine D2 receptors following administration of haloperidol or eticlopride results in increased phosphorylation of DARPP-32 at Thr34 (Svenningsson et al. 2000; Pozzi et al. 2003). Phosphorylation at Thr34 converts DARPP-32 into an inhibitor of protein phosphatase-1 (Hemmings et al. 1984), thereby amplifying PKA-mediated responses by reducing dephosphorylation of downstream target proteins (Greengard 2001). In this study, we confirmed the ability of haloperidol and eticlopride to increase the state of phosphorylation of DARPP-32 at Thr34 (Fig. 3c). We therefore examined the possible involvement of Thr34 phosphorylation of DARPP-32 in the haloperidol-induced increase in GluR1 phosphorylation. We found that administration of 0.5 mg/kg of haloperidol to DARPP-32 knockout mice (Fig. 3a) or to Thr34 → Ala mice (Fig. 3b) did not produce any effect on Ser845 phosphorylation.
Quinpirole reduces phosphorylation of GluR1 and DARPP-32
In support of the idea that dopamine D2-like receptors are involved in the regulation of the state of phosphorylartion of GluR1, we found that quinpirole, a dopamine D2-like receptor agonist, reduced the basal levels of phosphoSer845-GluR1 by 23%(Fig. 4a). A similar effect was exerted on the phosphorylation of DARPP-32 at Thr34, which was reduced by 21% (Fig. 4b).
Blockade of adenosine A2A, but not of dopamine D1, receptors prevents the ability of haloperidol to increase GluR1 phosphorylation
The results described above indicated that haloperidol increases the state of phosphorylation of GluR1 in striatal neurons by removing the inhibitory tone exerted by dopamine D2 receptors on the cAMP/PKA pathway. Adenosine A2A and dopamine D1 receptors are selectively expressed in striatopallidal and striatonigral projection neurons, respectively (Gerfen et al. 1990; Fink et al. 1992; Schiffmann and Vanderhaeghen 1993), where they stimulate the cAMP/PKA pathway via Golf-mediated activation of adenylyl cyclase (Corvol et al. 2001). We examined the effects of blockade of adenosine A2A and dopamine D1 receptors on haloperidol-induced phosphorylation of GluR1. We found that the selective adenosine A2A receptor antagonist, KW-6002 (3 mg/kg), abolished the increase in Ser845 phosphorylation produced by haloperidol (Fig. 5a). In contrast, blockade of dopamine D1 receptors, achieved with SCH23390 (0.15 mg/kg), did not modify the effect of haloperidol (Fig. 5b). These results indicate that the increase produced by haloperidol on the state of phosphorylation of GluR1 occurs specifically at the level of striatopallidal neurons, as it requires A2A receptor-mediated, but not D1 receptor-mediated, activation of the cAMP/PKA pathway.
Total levels of GluR1 and DARPP-32
Total levels of GluR1 and DARPP-32 were measured in the same tissue extracts used for the determination of phosphorylated proteins and were not modified by any of the treatments described in the study (Figs 1, 3, 4 and 5, upper panels). The striata of wild-type mice, DARPP-32 knockout mice, and Thr34 → Ala mutant mice contained similar levels of GluR1 (Figs 3a and b, upper panels).
In this study, we report for the first time the involvement of dopamine D2-like receptors in the regulation of the state of phosphorylation of the AMPA receptor. Our data show that administration of the dopamine D2 receptor antagonists, haloperidol or eticlopride, results in a large increase in the state of phosphorylation of the PKA site, Ser845, of the GluR1 subunit. This finding indicates the existence, under normal conditions, of a negative control exerted by dopamine, via activation of D2 receptors, on AMPA receptor phosphorylation. Several lines of evidence indicate that dopamine D2 receptors inhibit AMPA receptor transmission in striatal medium spiny neurons (Cepeda et al. 2001; Hernández-Echeagaray et al. 2004). The present results suggest that a decrease in phosphorylation of GluR1 at Ser845 is involved in such regulation.
Dopamine D2 receptors are coupled to Gi/o mediated reduction of adenylyl cyclase activity and inhibition of the cAMP/PKA pathway. Therefore, we propose that haloperidol and eticlopride increase Ser845 phosphorylation by removing the inhibitory tone normally exerted by D2 receptors on PKA activity. Blockade of dopamine D2 receptors and disinhibition of PKA also results in phosphorylation of DARPP-32 at Thr34 (Svenningsson et al. 2000; Pozzi et al. 2003). Our data show that phosphoThr34-DARPP-32 is crucially involved in the effect of haloperidol on AMPA receptors. Therefore, it appears that haloperidol increases GluR1 phosphorylation at Ser845 via activation of PKA and concomitant inhibition of protein phosphatase-1 mediated via phosphorylation of DARPP-32 at Thr34.
Adenosine A2A receptors and dopamine D2 receptors are preferentially expressed in striatopallidal neurons (Gerfen et al. 1990; Fink et al. 1992; Schiffmann and Vanderhaeghen 1993), where they stimulate and inhibit, respectively, the cAMP/PKA/DARPP-32 pathway. In this study, we show that, in intact mice, the positive modulation exerted by haloperidol on GluR1 phosphorylation at Ser845 is prevented by blockade of the cAMP/PKA/DARPP-32 cascade, achieved with the A2A receptor antagonist KW-6002. These data further support the idea of a close functional relationship between adenosine A2A and dopamine D2 receptors and indicate that, in striatopallidal neurons, the state of phosphorylation of GluR1 is in part determined by the opposite actions of adenosine, which stimulates GluR1 phosphorylation via A2A receptors, and dopamine, which inhibits GluR1 phosphorylation via D2 receptors.
The ability of haloperidol to increase the state of phosphorylation of GluR1, thereby promoting glutamate transmission at the level of the striatopallidal pathway, may have important physiological consequences. It is generally believed that activation of striatopallidal neurons leads to inhibition of thalamocortical projection neurons and reduction of motor activity (Gerfen 1992). Administration of haloperidol produces a marked depression of locomotion, characterized by rigid immobility (catalepsy), which is thought to occur via blockade of the inhibitory action exerted by dopamine D2 receptors on striatopallidal neurons. It is conceivable that such an inhibitory action involves suppression of excitatory glutamatergic transmission. Therefore, the reduction of locomotor activity produced by haloperidol may involve activation of the cAMP/PKA/DARPP-32 cascade, increased phosphorylation of GluR1 and facilitation of glutamate AMPA receptor transmission in striatopallidal neurons. This hypothesis is supported by the observation that catalepsy produced by raclopride, a dopamine D2 receptor antagonist, is attenuated in DARPP-32-null mice (Fienberg et al. 1998).
This work was supported by Swedish Research Council grant 13482 (GF), The Peter Jay Sharp Foundation, NIH grants MH40899 and DA10044 (PG), and DAMD17-03-1-0396 (ITI). The excellent technical assistance of Alberta Alickaj, Mario Clementi, Christopher Joynes, Jonathan Kukol, Minal Rana and Ayesha Sattar is gratefully acknowledged.