DARPP-32 mediates neurotransmission in the forebrain

Per Svenningsson*, Eleni T. Tzavara†, Feng Liu*, Allen A. Fienberg*, George G. Nomikos†, and Paul Greengard*‡

*Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021; and †Eli Lilly and Company, Lilly Corporate Center, Neuroscience Discovery Research, Indianapolis, IN 46285

Contributed by Paul Greengard, December 31, 2001 is implicated in the regulation of complex sensory, motor, DARPP-32, which reduces inhibition of PKA (14), and thereby affective, and cognitive functions. However, the biochemical mech- facilitates transmission by means of the PKA͞Thr34–DARPP-32͞ anisms whereby this neurotransmitter exerts its actions remain PP-1 signaling cascade. The efficacy of this signaling cascade also is largely unknown. DARPP-32 (- and cAMP-regulated phos- regulated by the phosphorylation state of DARPP-32 at Ser137, phoprotein of molecular weight 32,000) is a phosphoprotein that has because an increased phosphorylation at Ser137–DARPP-32, by primarily been characterized in relation to dopaminergic neurotrans- casein kinase-1 (CK-1), prevents the dephosphorylation at Thr34– mission. Here we report that serotonin regulates DARPP-32 phos- DARPP-32 by protein PP-2B and thereby potentiates the PKA͞ phorylation both in vitro and in vivo. Stimulation of 5-hydroxy- Thr34–DARPP-32͞PP-1 cascade (15). (5-HT4 and 5-HT6) receptors causes an increased In view of the critical role for serotonin in modulating mental phosphorylation state at Thr34–DARPP-32, the protein kinase A site, functions, we have conducted a detailed characterization of the and a decreased phosphorylation state at Thr75–DARPP-32, the cyclin- regulation of DARPP-32 phosphorylation by serotonin in vitro and dependent kinase 5 site. Furthermore, stimulation of 5-HT2 receptors in vivo. By the use of DARPP-32 knockout (KO) mice, we also have increases the phosphorylation state of Ser137–DARPP-32, the casein evaluated the possible involvement of DARPP-32 in mediating kinase-1 site. Behavioral and gene transcriptional effects induced biochemical and behavioral effects of compounds that selectively by compounds that selectively release serotonin were greatly re- release serotonin. duced in DARPP-32 knockout mice. Our data indicate that DARPP-32 is essential not only for dopaminergic but also for serotonergic Materials and Methods neurotransmission. Preparation and Treatment of Brain Slices. Slices (300 ␮m) from the neostriatum (dorsal striatum), nucleus accumbens (ventral stria- ͞ he serotonergic and dopaminergic neurotransmitter systems tum), and prefrontal cortex were prepared from adult male C57 Bl6 mice as described (12). The slices were preincubated in Krebs Tregulate emotion, mood, reward, and cognition. Pertubations of ͞ ͞ ͞ buffer (118 mM NaCl 4.7 mM KCl 1.5 mM Mg2SO4 1.2 mM these neurotransmitter systems are thought to contribute to the ͞ ͞ ͞ KH2PO4 25 mM NaHCO3 11.7 mM glucose 1.3 mM CaCl2)at etiology of several common neuropsychiatric disorders, such as ͞ schizophrenia, bipolar disorder, depression, and drug addiction. 30°C under constant oxygenation (95% O2 5% CO2) for 60 min, Indeed, the serotonergic and the dopaminergic systems appear to with a change of buffer after 30 min. To prevent reuptake of serotonin into nerve terminals, slices were pretreated with the be the primary targets for most of the current medications used for ␮ the treatment of psychiatric disorders. To better understand the serotonin reuptake inhibitor, (10 M), 2 min before the etiology of these disorders and to improve therapeutic options, application of serotonin. The slices were thereafter treated with there is a considerable interest in elucidating the biochemical serotonin and other drugs as specified in each experiment. After drug treatment, the buffer was removed and the slices were rapidly mechanisms that underlie the behavioral effects of serotonin and Ϫ dopamine, in particular in prefrontal cortex and striatum, two frozen on dry ice and stored at 80°C until immunoblotted. forebrain areas that receive converging serotonergic and dopami- nergic innervation (1, 2). Most reports favor the notion that Whole Animal Studies. In experiments examining the effects of serotonin and dopamine interact in a cooperative manner to exert para-chloroamphetamine (PCA) and 5-hydroxytryptophan (5- HTP) on DARPP-32 phosphorylation, adult male C57͞Bl6 mice biochemical (3, 4) and electrophysiological (5) actions, but there are ͞ ͞ also numerous publications reporting opposing actions of these were injected i.p. with saline, PCA (4 mg kg), or 5-HTP (50 mg kg) neurotransmitter systems (for a recent review see ref. 6). and killed 15 min after the injection by focused microwave irradi- One well-characterized mediator of the biochemical, electro- ation (4.5–5 kW for 1.4 s), using a small animal microwave (Muromachi Kikai, Tokyo). In these and all other experiments with physiological, transcriptional, and behavioral effects of dopamine is ͞ DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 5-HTP, carbidopa (50 mg kg) was administered 30 min before molecular weight 32,000) (7–9). DARPP-32 is highly enriched in 5-HTP. The brains were rapidly removed, and striata and frontal cortices were dissected out and stored at Ϫ80°C until assayed. prefrontal cortex and striatum. Activation of dopamine D1 recep- tors, by means of stimulation of protein kinase A (PKA), phos- Immunoblotting. Immunoblot procedures were as described (16). In phorylates DARPP-32 at Thr34 and thereby converts DARPP-32 experiments using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into a potent inhibitor of protein phosphatase-1 (PP-1) (10). This (MPTP)-pretreated mice, immunoblotting was also carried out effect is counteracted by activation of D2 receptors, by means of inhibition of PKA (11) and stimulation of the Ca2ϩ͞protein phos- phatase-2B (PP-2B) signaling cascade, which dephosphorylates Abbreviations: Cdk5, cyclin-dependent kinase 5; CK-1, casein kinase 1; DARPP-32, 34 Thr –DARPP-32 (12). Activation of D1 receptors also decreases dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000; EMDT, the phosphorylation state of DARPP-32 at Thr75 (13). This site is 2-ethyl-5-methoxy-N,N-dimethyltryptamine; 5-HT, 5-hydroxytryptamine; 5-HTP, 5- phosphorylated by cyclin-dependent kinase 5 (Cdk5) (14) and hydroxytryptophan; KO, knockout; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PCA, para-chloroamphetamine; PKA, protein kinase A; PP-1, protein phosphatase-1; PPB, dephosphorylated by protein phosphatase 2A (PP-2A) (13). The 2-[1-(4-piperonyl)piperazinyl]benzothiazole; TTX, tetrodotoxin; WT, wild type. 75 dopaminergic regulation of the phosphorylation state at Thr – ‡ ͞ To whom reprint requests should be addressed. E-mail: [email protected]. DARPP-32 is mediated by means of a PKA PP-2A signaling The publication costs of this article were defrayed in part by page charge payment. This cascade (13). Thus, enhanced dopaminergic transmission by means article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 75 of D1 receptors leads to a decreased phosphorylation of Thr – §1734 solely to indicate this fact.

3188–3193 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052712699 Downloaded by guest on September 30, 2021 with an antibody against tyrosine hydroxylase (Chemicon). Data on protein phosphorylation are expressed as percentage of control. In situ hybridization wild type (WT) mice and DARPP-32 KO mice (7) were given an i.p. injection with either saline, PCA (4 mg͞kg), or 5-HTP (50 mg͞kg) and killed 20 min postinjection by decapitation. In situ hybridization experiments against c-fos mRNA were carried out as described (16).

Locomotor Activity Measurements. Locomotor activity of WT and DARPP-32 KO mice was measured in activity monitors (43 ϫ 44 ϫ 45 cm) equipped with both horizontal and vertical sensors (Opto- Varimex, Columbus Instruments, Columbus, OH). Mice were acclimated to the boxes for 1 h, 2–4 days before the day of the experiment. The day of the experiment, mice were placed in the boxes for 20 min (habituation period) before drug administration. Saline, PCA (4 mg͞kg), or 5-HTP (50 mg͞kg; 30 min after 50 mg͞kg carbidopa i.p.) was injected i.p., and locomotor activity was mea- sured for 50 min. In some experiments, adult male C57͞Bl6 mice were pretreated with (0.3 mg͞kg) 10 min before the administration of saline, PCA (4 mg͞kg), or 5-HTP (50 mg͞kg). Locomotor activity parameters (distance traveled and stereotypic movements) were quantified by using the AutoTrack System (Columbus Instruments). Results Regulation by Serotonin of DARPP-32 Phosphorylation in Brain Slices. It was shown that serotonin regulates the phosphorylation of DARPP-32 at Thr34 (the PKA site), Thr75 (the Cdk5 site), and Ser137 (the CK-1 site) (16). Here we have studied this phenomenon in greater detail, using slices prepared from neostriatum (dorsal striatum), nucleus accumbens (ventral striatum), and prefrontal cortex. Time-course experiments in neostriatal slices showed that serotonin (100 ␮M) caused a rapid increase in DARPP-32 phos- phorylation at all three phosphorylation sites (Fig. 1 a–c), with the effect at Thr34 being the most pronounced. The state of phosphor- ylation of DARPP-32 at Thr34 and Ser137 returned to basal levels within 10 min (Fig. 1 a–c) and remained there at 30 min (not shown). In contrast, the small, but significant, increase in Fig. 1. Regulation by serotonin of DARPP-32 phosphorylation in slices of DARPP-32 phosphorylation at Thr75 was followed by a pronounced neostriatum. (a–c) Time course and (d–f) dose–response studies of regulation by 34 75 decrease in the state of phosphorylation at this site. No changes in serotonin of phosphorylation of DARPP-32 at (a and d) Thr ,(b and e) Thr , and 137 ␮ the total levels of DARPP-32 were found at any time point. Based (c and f) Ser in neostriatum. (a–c) Serotonin was used at 100 M. (d–f) Slices were incubated for 2 min for studies of phospho-Thr34–DARPP-32 and phospho- on these data, slices were incubated for 2 min for studies of Ser137–DARPP-32 and for 10 min for studies of phospho-Thr75–DARPP-32. Data 34 137 phospho-Thr –DARPP-32 and phospho-Ser –DARPP-32, and represent means (Ϯ SE) (n ϭ 6–10). *, P Ͻ 0 05; **,PϽ0.01; ***, P Ͻ 0.001 for 10 min for studies of phospho-Thr75–DARPP-32 in all subse- compared with control; one-way ANOVA followed by Dunnett’s test. quent experiments. A lower concentration of serotonin was re- quired to regulate DARPP-32 phosphorylation at Thr34 than at 75 137 Effects of 5-Hydroxytryptamine (5-HT) Receptor on DARPP-32 Thr and Ser (Fig. 1 d–f). The EC50 values for the effects of serotonin on DARPP-32 phosphorylation at Thr34, Thr75, and Phosphorylation in Neostriatal and Cortical Slices. Several serotonin Ser137 were 13.5, 29.6, and 37.6 ␮M, respectively. receptors, most notably 5-HT1B, 5-HT2A͞C, 5-HT3, 5-HT4, and Using slices prepared from nucleus accumbens, it was found that 5-HT6, are expressed throughout the striatum. The identity of the serotonin receptors involved in the serotonin-mediated regulation 2 min of incubation with serotonin (100 ␮M) increased phosphor- of DARPP-32 phosphorylation was determined by the use of ylation at Thr34 (307 Ϯ 25%, n ϭ 12) and Ser137 (143 Ϯ 7%, n ϭ selective agonists and antagonists. Results obtained with various 12), and that 10 min of incubation with serotonin decreased serotonin agonists in neostriatal slices are shown in Table 1. phosphorylation at Thr75 (75 Ϯ 6%, n ϭ 12). Experiments carried 2-[1-(4-Piperonyl)piperazinyl]benzothiazole (PPB, a 5-HT4 recep- out in slices from the prefrontal cortex showed that 2 min of tor ) and 2-ethyl-5-methoxy-N,N-dimethyltryptamine incubation with serotonin increased phospho-Thr34–DARPP-32 Ϯ ϭ (EMDT, a 5-HT6 receptor agonist), alone or together, qualitatively NEUROBIOLOGY (185 15%, n 6) and that 10 min of incubation with serotonin mimicked the action of serotonin at phospho-Thr34–DARPP-32 75 Ϯ ϭ decreased phospho-Thr –DARPP-32 (85 3%, n 6). Because and phospho-Thr75–DARPP-32, but not at phospho-Ser137– 137 of a low signal in prefrontal cortex, the level of phospho-Ser – DARPP-32. The EC50 values for the actions of PPB and EMDT at DARPP-32 could not be accurately assayed in this area. Thus, the phospho-Thr34–DARPP-32 were 27.3 and 20.5 ␮M, and at phos- regulation of phosphorylation of DARPP-32 by serotonin was pho-Thr75–DARPP-32 were 45.5 and 27.4 ␮M, respectively. In similar in all three brain regions. Because more slices can be addition, 5-methoxytryptamine (100 ␮M), which has a high affinity collected from neostriatum than from nucleus accumbens, slices as an agonist at 5-HT4 and 5-HT6 receptors, increased phospho- made from neostriatum were used in most of the subsequent Thr34–DARPP-32 (261 Ϯ 31%, n ϭ 12) and decreased phospho- experiments. Thr75–DARPP-32 (76 Ϯ 7%, n ϭ 12). Conversely, DOI [(Ϯ)-2,5-

Svenningsson et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 3189 Downloaded by guest on September 30, 2021 Table 1. Regulation of DARPP-32 phosphorylation in vitro by Table 2. Regulation of serotonin-mediated DARPP-32 5-HT receptor agonists and antagonists phosphorylation in vitro by signal transduction modulators Thr34 Thr75 Ser137 Thr34 Thr75 Ser137

Control 100 (17.0) 100 (5.0) 100 (3.9) Control 100 (17.0) 100 (5.0) 100 (3.9) Serotonin 342 (26.7)*** 76 (7.5)** 141 (6.0)** Serotonin 342 (26.7)*** 76 (7.5)** 141 (6.0)** 5-HT receptor agonists TTX 92 (5.8) 103 (3.5) 98 (12.5)

PPB (5HT4 agonist) 218 (36.1)* 79 (8.8)* 107 (2.0) TTX ϩ serotonin 348 (63.2)*** 83 (8.1)* 144 (3.3)** EMDT (5HT6 agonist) 301 (39.5)** 76 (7.6)** 103 (5.5) U-73122 (PLC inhibitor) 117 (10.9) 90 (6.6) 86 (6.2) PPB ϩ EMDT 397 (41.0)*** 70 (3.8)** 105 (7.9) U-73122 ϩ serotonin 297 (34.0)** 70 (6.1)** 89 (3.7)‡

DOI (5HT2 agonist) 144 (10.7) 105 (4.5) 158 (8.3)** RpcAMPs (PKA inhibitor) 88 (13.1) 105 (7.2) 108 (10.0) ‡ † Anpirtoline (5HT1B agonist) 86 (3.7)* 102 (22.2) 93 (10.0) RpcAMPs ϩ serotonin 150 (44.7) 102 (9.5) 147 (4.0)** SR 57227 (5HT3 agonist) 78 (3.5)* 88 (6.8) 105 (9.6) ␮ 5-HT receptor antagonists TTX (2 M), an inhibitor of voltage-gated sodium channels, U-73122 (10 ␮M), a phospholipase C inhibitor, or Rp-cAMPS (300 ␮M), a PKA inhibitor, were SDZ 205,557 (5HT4 antagonist) 94 (16.7) 103 (17.5) 102 (8.8) ␮ SDZ ϩ serotonin 257 (34.4)**† 95 (11.5)† 129 (5.0)* applied to striatal slices 10 min before serotonin (100 M). Data represent means (Ϯ SEM) (n ϭ 10–30). , P Ͻ 0.05; , P Ͻ 0.01; , P Ͻ 0.001 compared Ro 04-6790 (5HT6 antagonist) 90 (8.7) 101 (12.5) 105 (7.3) * ** *** Ͻ Ͻ Ro ϩ serotonin 225 (28.5)*† 96 (8.4)† 131 (7.2)* with no serotonin; †, P 0.05; ‡, P 0.01 compared with serotonin alone; Ro ϩ SDZ 84 (6.5) 110 (11.5) 107 (6.2) one-way ANOVA followed by Dunnett’s test. SDZ ϩ Ro ϩ serotonin 198 (44.5)*‡ 98 (6.1)‡ 134 (18.1)*

Ketanserin (5HT2 antagonist) 94 (2.9) 105 (8.9) 97 (4.8) ϩ serotonin 322 (28.4)*** 78 (12.1)* 101 (9.1)† agonists and antagonists indicate that the effects of serotonin on Clozapine (5HT2/6 antagonist) 124 (19.8) 101 (13.4) 105 (5.5) DARPP-32 phosphorylation can be accounted for primarily by ϩ ‡ ‡ † Clozapine serotonin 156 (15.4)* 98 (5.2) 110 (4.9) activation of 5-HT2,5-HT4, and 5-HT6 receptors. Effects of serotonin (100 ␮M), PPB (100 ␮M), a 5-HT receptor agonist, 4 Effects of Signal Transduction Modulators on Serotonin-Mediated EMDT (100 ␮M), a 5-HT6 receptor agonist, DOI (100 ␮M), a 5-HT2 receptor DARPP-32 Phosphorylation in Neostriatal Slices. agonist, anpirtoline, (100 ␮M), a 5-HT1B receptor agonist, and SR 57227 (100 To investigate 34 75 137 ␮M), a 5-HT3 receptor agonist, on Thr , Thr , and Ser . In antagonist whether the effects of serotonin on DARPP-32 phosphorylation experiments, ketanserin (10 ␮M), a 5-HT2 receptor antagonist, SDZ 205,557 (10 were mediated through a direct action on DARPP-32-containing ␮M), a 5-HT4 receptor antagonist, Ro 04-6790 (10 ␮M), a 5-HT6 receptor neurons, slices were pretreated with tetrodotoxin (TTX), which antagonist, or clozapine (10 ␮M), a multireceptor antagonist with high affin- inhibits action potential-dependent synaptic transmission by block- ity for 5-HT2 and 5-HT6 receptors, was applied to striatal slices 10 min before ing voltage-gated sodium channels. The actions of serotonin on serotonin (100 ␮M). Data represent means (Ϯ SEM) (n ϭ 12–30). *, P Ͻ 0.05; **, Ͻ Ͻ Ͻ Ͻ DARPP-32 phosphorylation were unaffected by TTX, indicating a P 0.01; ***, P 0.001 compared with control; †, P 0.05; ‡, P 0.01 direct action (Table 2). The principal signal transduction pathways compared with serotonin alone; one-way ANOVA followed by Dunnett’s test. ͞ used by 5-HT2 and 5-HT4 5-HT6 receptors involve activation of phospholipase C and PKA, respectively. To investigate the contri- dimethoxy-4-iodoamphetamine hydrochloride; (Ϯ)-1-(2,5 butions of these signaling pathways to the action of serotonin, striatal slices were pretreated with either U-73122, a selective dimethoxy-4-iodophenyl)-2-aminopropane], a 5-HT2A/C agonist, mimicked the action of serotonin at phospho-Ser137–DARPP-32, phospholipase C inhibitor, or Rp-cAMPS, a selective PKA inhib- 34 75 itor, before the application of serotonin. As shown in Table 2, but not at phospho-Thr –DARPP-32 and phospho-Thr – 137 137 U-73122 blocked the effect of serotonin on phospho-Ser – DARPP-32. The EC50 value for DOI at phospho-Ser – 34 DARPP-32 was 18.2 ␮M. The 5-HT receptor agonist, anpirtoline DARPP-32, but not on phospho-Thr –DARPP-32 or phospho- 1B Thr75–DARPP-32. Conversely, Rp-cAMPS inhibited the action of (presumably by decreasing cAMP), and the 5-HT3 agonist, ϩ serotonin on phospho-Thr34–DARPP-32 and phospho-Thr75– SR57227A (presumably by increasing Ca2 ), decreased phospho- DARPP-32, but not on phospho-Ser137–DARPP-32. Thr34–DARPP-32, but were without significant effects on phospho- Thr75–DARPP-32 and phospho-Ser137–DARPP-32. Regulation of DARPP-32 Phosphorylation in Vivo by PCA and 5-HTP. Using slices prepared from the prefrontal cortex, it was found Mice were injected i.p. with the serotonin releaser, PCA (4 mg͞kg), that 2 min of incubation with the 5-HT4 receptor agonist, PPB, or ͞ 34 or the serotonin precursor, 5-HTP (50 mg kg). The results shown the 5-HT6 receptor agonist, EMDT, increased phospho-Thr – Ϯ ϭ Ϯ ϭ in Fig. 2 demonstrate that in striatum PCA and 5-HTP cause a DARPP-32 (203 15%, n 6, and 185 19%, n 6, respectively), common pattern of alteration of DARPP-32 phosphorylation, i.e., whereas 10 min of incubation with PPB or EMDT decreased an increase at phospho-Thr-34–DARPP-32 and phospho-Ser137– 75 Ϯ ϭ Ϯ ϭ phospho-Thr –DARPP-32 (84 4%, n 6, and 80 7%, n 6, DARPP-32 and a decrease at phospho-Thr75–DARPP-32. Further- respectively). more, in prefrontal cortex PCA and 5-HTP increased phospho- Thr34–DARPP-32 (147 Ϯ 4.8%, n ϭ 5 mice, and 144 Ϯ 8.3%, n ϭ Effects of 5-HT Receptor Antagonists on Serotonin-Mediated 5 mice, respectively), but decreased phospho-Thr75–DARPP-32 DARPP-32 Phosphorylation in Neostriatal Slices. The results obtained (79 Ϯ 3.8%, n ϭ 5 mice, and 74 Ϯ 8.0%, n ϭ 5, respectively). Thus, with selective serotonin receptor antagonists (Table 1) were con- serotonin regulates DARPP-32 phosphorylation in a qualitatively sistent with the results obtained with agonists. Thus, SDZ 205,557, similar fashion both in vitro and in vivo. a5-HT4 receptor antagonist, and Ro 04–6790, a 5-HT6 receptor antagonist, alone or together, reduced the effects of serotonin on Involvement of DARPP-32 in the Regulation of c-fos mRNA Expression phospho-Thr34–DARPP-32 and phospho-Thr75–DARPP-32, but by PCA and 5-HTP. To evaluate the relevance of DARPP-32 in not on phospho-Ser137–DARPP-32. Conversely, ketanserin, a mediating actions of PCA and 5-HTP on gene transcription, WT 5-HT2A/C receptor antagonist, reduced the effects of serotonin on and DARPP-32 KO mice were injected with PCA or 5-HTP, and phospho-Ser137–DARPP-32 phosphorylation, but not on phospho- the effects on c-fos mRNA expression were investigated. As illus- Thr34–DARPP-32 or phospho-Thr75–DARPP-32. The multirecep- trated in Fig. 3a, both PCA and 5-HTP induced a strong expression tor antagonist, clozapine, which has a particularly high affinity for of c-fos mRNA throughout striatum and cerebral cortex of WT 5-HT2 and 5-HT6 receptors (17), significantly counteracted sero- mice. This effect, which was quantitated in the periventricular tonin-mediated phosphorylation of DARPP-32 at all sites exam- region of striatum (Fig. 3b) and in the cingulate cortex (Fig. 3c), was ined. Taken together, the results obtained with serotonin receptor strongly attenuated in DARPP-32 KO mice.

3190 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052712699 Svenningsson et al. Downloaded by guest on September 30, 2021 Fig. 2. Regulation of DARPP-32 phosphorylation in vivo by PCA and 5-HTP. Mice were injected i.p. with saline, PCA (4 mg͞kg), or 5-HTP (50 mg͞kg). Fifteen minutes later mice were killed by focused microwave irradiation. Data represent means Ϯ SE for 5–10 mice per group. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001 compared with saline-treated mice; one-way ANOVA followed by Dunnett’s test.

Involvement of DARPP-32 in the Behavioral Effects of PCA and 5-HTP. To investigate the role of DARPP-32 in mediating behavioral actions exerted by an enhanced release of serotonin, the effects of PCA and 5-HTP on locomotor activity and stereotypic behaviors were examined in WT and DARPP-32 KO mice. PCA as well as 5-HTP caused a robust increase both in locomotor activity and stereotypic behavior in WT mice (Fig. 4). The actions on both of these behavioral parameters were abolished in DARPP-32 KO mice. The attenuated responsiveness of DARPP-32 KO mice to PCA and 5-HTP cannot be explained by a loss of ability of these mice to be hyperactive. In experiments in which drug-naive animals were exposed to the novel environment represented by the test cages, we found no difference in locomotor activity scores between WT (6,133 Ϯ 293 cm͞20 min) and DARPP-32 KO (6,268 Ϯ 418 cm͞20 min) mice.

Blockade by Clozapine of Behavioral Effects of PCA and 5-HTP. As Fig. 3. Regulation of c-fos mRNA expression by PCA and 5-HTP in WT and demonstrated above, the atypical neuroleptic agent, clozapine, DARPP-32 KO mice. (a) Dark-field autoradiograms showing the expression of which has a very high affinity at 5-HT2 and 5-HT6 receptors, c-fos mRNA 20 min after treatment with saline, PCA (4 mg͞kg), or 5-HTP (50 strongly counteracted the effects of serotonin on DARPP-32 mg͞kg) in WT and DARPP-32 KO mice. (Magnification: ϫ5.) (b and c) Histograms phosphorylation. We therefore evaluated the effects of pretreat- show quantification of the expression of c-fos mRNA in (b) periventricular area of ment with a relatively low dose of clozapine (0.3 mg͞kg) on PCA- striatum and (c) cingulate cortex after each treatment. WT, filled bars; DARPP-32 and 5-HTP-induced behaviors. The results shown in Fig. 5 dem- KO, open bars. Data represent means Ϯ SE for 5–8 mice per group. **, P Ͻ 0.01; onstrate that clozapine abolished the hyperactivity induced by PCA ***, P Ͻ 0.001 compared with saline-treated mice; #, P Ͻ 0.05; ##, P Ͻ 0.01 or 5-HTP. It should be noted that clozapine also has a moderate compared with WT mice given the same treatment; one-way ANOVA followed by Dunnett’s test. affinity for several dopamine receptors (17). However, previous work has shown that at least 10 times higher concentrations (2–10 ͞ mg kg) of clozapine are necessary to inhibit dopamine-dependent lished results) or from mice pretreated with the dopaminergic hyperactivity (17). neurotoxin, MPTP (Table 3), indicating that the effects of seroto- nin on DARPP-32 phosphorylation were not mediated through Characterization of Serotonin-Dopamine Interactions in the Regula- regulation of dopamine release. tion of DARPP-32 Phosphorylation. To determine whether the effects of serotonin on DARPP-32 phosphorylation were mediated Discussion through modulation of dopaminergic signaling pathways, several The present studies, and those described in ref. 16, have demon- NEUROBIOLOGY types of experiments were carried out (Table 3). Serotonin and SKF strated that behavioral as well as biochemical effects induced by 82958, a D1 receptor-selective agonist, had additive effects in enhanced serotonergic neurotransmission are attenuated in increasing phospho-Thr34–DARPP-32 and decreasing phospho- DARPP-32 KO mice. The results show an important role for 75 Thr –DARPP-32. Serotonin and quinpirole, a D2 receptor- DARPP-32 in mediating the actions of serotonin in vivo. We have selective agonist, had antagonistic effects on phospho-Thr34– investigated the underlying molecular mechanisms. DARPP-32 and phospho-Thr75–DARPP-32 and additive effects in Neostriatum, nucleus accumbens, and prefrontal cortex receive increasing phosphorylation at phospho-Ser137–DARPP-32. The a moderate to high serotonergic innervation (1), which is further effects of serotonin on DARPP-32 phosphorylation were not increased when the levels of dopamine in this region are decreased significantly altered in slices from D1 receptor KO mice (unpub- (18). Several serotonin receptors, most notably 5-HT1B, 5-HT1E,

Svenningsson et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 3191 Downloaded by guest on September 30, 2021 Table 3. Interactions of serotonin and dopamine signaling pathways on DARPP-32 phosphorylation in slices Thr34 Thr75 Ser137

Control 100 (17.0) 100 (5.0) 100 (3.9) Serotonin 342 (26.7)*** 76 (7.5)** 141 (6.0)**

SKF 82958 (D1 agonist) 552 (59.1)*** 63 (1.7)*** 105 (14.7) SKF 82958 ϩ serotonin 717 (42.4)***#§ 56 (1.1)***#§ 121 (14.4)

Quinpirole (D2 agonist) 65 (4.7)** 112 (8.5) 135 (5.1)* Quinpirole ϩ serotonin 125 (12.7)###† 106 (13.2)## 186 (11.1)***#† MPTP treatment 93 (8.7) 118 (9.3) 104 (7.9) MPTP-treatment ϩ serotonin 307 (44.1)*** 80 (9.2)* 154 (21.1)**

SKF 82958 (10 ␮M),aD1 receptor agonist, or quinpirole (10 ␮M),aD2 receptor agonist, was applied 10 min before serotonin (100 ␮M). MPTP (50 mg͞kg, i.p.) was administered i.p. four times at 12-h intervals, 4 weeks before the slice experiment. This pretreatment caused a 85–93% reduction in the levels of tyrosine hydroxylase in striatum. Data represent means (Ϯ SEM) (n ϭ 16–30). *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001 compared with control; #, P Ͻ 0.05; ##, P Ͻ 0.01; ###, P Ͻ 0.001 compared with serotonin alone; §, P Ͻ 0.05 compared with SKF 82958 alone; †, P Ͻ 0.05 compared with quinpirole alone; one-way ANOVA followed by Dunnett’s test.

selective serotonin receptor agonists and antagonists, and other pharmacological reagents, including TTX, U-73122, and Rp- cAMPs, has enabled us to elucidate the underlying signaling Fig. 4. Behavioral responses to treatment with PCA and 5-HTP in WT and ͞ ͞ pathways. The actions of serotonin in regulating DARPP-32 phos- DARPP-32 KO mice. Effects of (Left) PCA (4 mg kg) and (Right) 5-HTP (50 mg kg) 34 75 on total locomotion (Upper) and stereotypic movements (Lower)inWTand phorylation at Thr and Thr were mediated primarily by means DARPP-32 KO mice. Data represent means Ϯ SE for 6–10 mice per group. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001 compared with saline-treated mice; ###, P Ͻ 0.001; ##, P Ͻ 0.01; #, P Ͻ 0.05 compared with WT mice given the same treatment; two-way ANOVA followed by Duncan’s test.

5-HT2A,5-HT2C,5-HT3, 5-HT4, and 5-HT6, are expressed in striatum (6). All of them are metabotropic receptors, with the exception of 5-HT3 receptors, which are ionotropic. These seroto- nin receptors act primarily by means of the following second messenger systems: 5-HT1B/E receptors decrease cAMP formation; 5-HT2A/C receptors increase inositol triphosphate and diacylglyc- ϩ 2ϩ erol formation; 5-HT3 receptors increase Na and Ca influx; and 5-HT4 and 5-HT6 receptors increase cAMP formation. Serotonin caused an increased phosphorylation of DARPP-32 at Thr34 (the PKA site) and Ser137 (the CK-1 site) and a decreased phosphorylation at Thr75 (the Cdk5 site), in brain slices. The use of

Fig. 6. Model summarizing pathways by which enhanced serotonergic trans- mission regulates DARPP-32 phosphorylation at Thr34, Thr75, and Ser137. Activa- tion of 5-HT4 or 5-HT6 receptors sequentially increases cAMP levels, activity of PKA, and phosphorylation of Thr34. Activated PKA also phosphorylates and activates protein phosphatase 2A (PP-2A) (13), causing dephosphorylation of the Cdk5 site, Thr75 (14), removing inhibition of PKA (14) and increasing phosphor- 34 Fig. 5. Effect of clozapine on behavioral responses to treatment with PCA and ylation of Thr . 5-HT2 receptors activate PLC, increasing casein kinase-1 (CK1) 5-HTP in WT mice. Effect of pretreatment with clozapine (0.3 mg͞kg) on loco- activity (27), phosphorylation of Ser137, and inhibition of dephosphorylation by motion induced by (Left) PCA (4 mg͞kg) or (Right) 5-HTP (50 mg͞kg). Data protein phosphatase-2B (PP2B) of Thr34 (15). These various actions of serotonin on represent means Ϯ SE for 6–10 mice per group. **, P Ͻ 0.01 compared with DARPP-32 phosphorylation are synergistic, each leading to an increased state of saline-treated mice; ##, P Ͻ 0.01 compared with PCA-treated or 5-HTP-treated phosphorylation of Thr34 and an increased inhibition of PP-1 (10). Solid lines mice; two-way ANOVA followed by Duncan’s test. indicate activation; dashed lines indicate inhibition.

3192 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052712699 Svenningsson et al. Downloaded by guest on September 30, 2021 of activation of 5-HT4 and 5-HT6 receptors, whereas the regulation supported by experiments showing that 0.3 mg/kg of clozapine, a 137 at Ser was mediated primarily by means of 5-HT2 receptors. The dose that antagonize responses by means of 5-HT2 and 5-HT6 three pathways appear to inhibit PP-1 through synergistic mecha- receptors, counteracts the locomotor effects of PCA and 5-HTP. nisms (Fig. 6) and provide a model by which serotonin as well as Strong evidence for an involvement of DARPP-32 in the bio- compounds which increase serotonin transmission may produce chemical and behavioral actions of serotonin under in vivo condi- their effects. These latter compounds include, in addition to PCA tions came from studies in which we examined effects of PCA and and 5-HTP, the selective serotonin reuptake inhibitor, fluoxetine, 5-HTP in WT and DARPP-32 KO mice. An induction of c-fos a widely used agent. mRNA was found throughout striatum and cerebral cortex in WT The pattern of DARPP-32 phosphorylation induced by elevated mice treated with PCA or 5-HTP. The ability of both PCA and serotonergic neurotransmission is very similar to that induced by 5-HTP to induce c-fos mRNA expression was strongly reduced in elevated dopaminergic neurotransmission (13). However, the se- DARPP-32 KO mice. It is well known that the transcription of c-fos rotonin-mediated regulation of DARPP-32 phosphorylation is mRNA is negatively regulated by PP-1 (21, 22). The critical largely independent of altered dopaminergic neurotransmission involvement of DARPP-32 in PCA- and 5-HTP-mediated c-fos (Table 3). Numerous psychoactive compounds act directly on both induction is likely explained, at least in part, by its ability to inhibit the serotonergic and dopaminergic systems. For example, cocaine PP-1. inhibits the reuptake of both dopamine and serotonin from the Previous work implicated serotonin in the regulation of locomo- synapse. It has recently been shown that the reinforcing properties tion and exploratory behavior (23). Depletion of serotonin caused of cocaine in the place preference test remain intact in dopamine a long-term reduction in exploration (24), whereas local application transporter KO mice or serotonin transporter KO mice, but are of serotonin into nucleus accumbens caused an increase in loco- severely impaired in mice that lack both transporters (19). These motion (25) in rats. It is generally agreed that agents that increase data imply that certain biochemical effects of cocaine can be the release of serotonin, such as PCA, 5-HTP, and 3,4- mediated either by dopamine or serotonin. Our data indicate that methylenedioxymethamphetamine (‘‘Ecstasy’’) increase locomotor signaling through the DARPP-32 pathway may be responsible for activity (23, 26). To further investigate the functional relevance of this redundancy. In support of this suggestion, the diminished DARPP-32 phosphorylation in the actions of PCA and 5-HTP, the responsiveness to the reinforcing properties of cocaine in the place effects of these drugs on locomotor activity and stereotypic behav- preference test of the double transporter KO mice is mimicked by iors were examined in WT and DARPP-32 KO mice. As expected, the DARPP-32 KO mice (20). both PCA and 5-HTP increased locomotor activity and stereotypic Evidence that the effects of serotonin on DARPP-32 phosphor- behaviors in WT mice. Both of these responses were greatly ylation observed in vitro are of physiological relevance came from attenuated in DARPP-32 KO mice. studies in which we injected mice with PCA or 5-HTP and examined In conclusion, the studies described here, together with those the effects on DARPP-32 phosphorylation in the striatum and described in ref. 16, indicate that DARPP-32 plays a central role in prefrontal cortex. Both treatments led to a pattern of DARPP-32 serotonergic signaling. Moreover, the data provide the outline of a phosphorylation that closely resembled that observed in slices in molecular mechanism for the behavioral actions of those psycho- response to serotonin, namely increases at phospho-Thr34– stimulants and antidepressant agents that achieve their effects DARPP-32 and phospho-Ser137–DARPP-32 and a decrease at through pertubation of serotonergic transmission. phospho-Thr75–DARPP-32. It seemed possible that the effects of 5-HTP on DARPP-32 phosphorylation were attributable mainly to We thank D. Nelson and A. Nishi for their critical reading of this manuscript. We thank G. L. Snyder, J. A. Bibb, and H. C. Hemmings Jr. for ectopically released serotonin produced in dopaminergic nerve 34 75 terminals. However, the fact that PCA, which acts selectively on antibodies against phospho-Thr DARPP-32, phospho-Thr DARPP-32, and total DARPP-32 and S. Col, S. Galdi, and L. Lannebo for technical serotonergic nerve terminals, at the dose used, also had pronounced assistance. This work was supported by National Institute of Mental Health effects on DARPP-32 phosphorylation in the striatum and the Grants MH-40899 and DA10044 (to P.G.). P.S. is supported by a postdoc- prefrontal cortex, shows that an enhanced release of serotonin in toral fellowship from Stiftelsen fo¨r Internationalisering av Ho¨gre utbildning these areas regulates DARPP-32 phosphorylation. The conclusion och forskning. 2-Ethyl-5-methoxy-N,N-dimethyltryptamine was synthesized that DARPP-32 mediates the behavioral effects of serotonin is by Eli Lilly & Company.

1. Steinbusch, H. W. (1981) Neuroscience 6, 557–618. 15. Desdouits, F. Siciliano, J. C. Greengard, P. & Girault, J.-A. (1995) Proc. Natl. 2. Lindvall, O. & Bjo¨rklund, A. (1984) in Handbook of Chemical Neuroanatomy, Acad. Sci. USA 92, 2682–2685. eds. Bjo¨rklund, A. & Ho¨kfelt, T. (Elsevier, Amsterdam), pp. 55–122. 16. Svenningsson, P., Tzavara, E. T., Witkin, J. M., Fienberg, A. A., Nomikos, G. G. 3. Bhat, R. V. & Baraban, J. M. (1993) J. Pharmacol. Exp. Ther. 267, & Greengard P. (2002) Proc. Natl. Acad. Sci. USA 99, 3182–3187. 496–505. 17. Leysen, J. E. (2000) in Atypical Antipsychotics, eds. Ellenbroek, B. A. & Cools, 4. Genova, L. M. & Hyman, S. E. (1998) Synapse 30, 71–78. A. R. (Birkha¨user, Basel), pp. 57–81. 5. White, F. J., Hu, X. & Henry, D. J. (1993) J. Pharmacol. Exp. Ther. 266, 18. Rozas, G., Liste, I., Guerra, M. J. & Labandeira-Garcia, J. L. (1998) Neurosci. 1075–1084. Lett. 245, 151–154. 6. Barnes, N. M. & Sharp, T. (1999) Neuropharmacology 38, 1083–1152. 19. Sora, I., Hall. S., Andrews, A. M., Itokawa, M., Li, X.-F., Wei, H.-B., Wichems, 7. Fienberg, A. A., Hiroi, N., Mermelstein, P. G., Song, W. J., Snyder, G. L., C., Lesch, K.-P., Murphy, D. L. & Uhl, G. R. (2001) Proc. Natl. Acad. Sci. USA Nishi, A., Cheramy, A., OCallaghan, J. P., Miller, D. B., Cole, D. G., et al. 98, 5300–5305. (1998) Science 281, 838–842. 20. Zachariou, V., Benoit-Marandl, M., Allen, P. B., Ingrassia, P., Fienberg, A. A., 8. Greengard, P., Allen, P. B. & Nairn, A. C. (1999) Neuron 23, 435–447. Gonon, F., Greengard, P. & Picciotto, M. (2002) Biol. Psychiatry, in press. 9. Greengard, P. (2001) Science 294, 1024–1030. 21. Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T., 10. Hemmings, H. C., Jr., Greengard, P., Tung, H. Y. L. & Cohen, P. (1984) Nature Karin, M., Shenolikar, S. & Montminy, M. (1992) Cell 70, 105–113.

(London) 310, 503–505. 22. Liu, F.-C. & Graybiel, A. M. (1996) Neuron 17, 1133–1144. NEUROBIOLOGY 11. Lindskog, M., Svenningsson, P., Fredholm, B. B., Greengard, P. & Fisone, G. 23. Geyer, M. A. (1996) Behav. Brain Res. 73, 31–36. (1998) Neuroscience 88, 1005–1008. 24. Lipska, B. K., Jaskiw, G. E., Arya, A. & Weinberger, D. R. (1992) Pharmacol. 12. Nishi, A., Snyder, G. L. & Greengard, P. (1997) J. Neurosci. 17, 8147–8155. Biochem. Behav. 43, 1247–1252. 13. Nishi, A., Bibb, J. A., Snyder, G. L., Higashi, H., Nairn, A. C. & Greengard, 25. Sasaki-Adams, D. M. & Kelley, A. E. (2001) Neuropsychopharmacology 25, P. (2000) Proc. Natl. Acad. Sci. USA 97, 12840–12845. 440–452. 14. Bibb, J. A., Snyder, G. L., Nishi, A., Yan, Z., Meijer, L., Fienberg, A. A., Tsai, 26. Modigh, K. (1972) Psychopharmacology 23, 48–54. L. H., Kwon, Y. T., Girault, J. A., Czernik, A. J., et al. (1999) Nature (London) 27. Liu, F., Ma, X. H., Ule, J., Bibb, J. A., Nishi, A., DeMaggio, A. J., Yan, Z., Nairn, 402, 669–671. A. C. & Greengard, P. (2001) Proc. Natl. Acad. Sci. USA 98, 11062–11068.

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