DARPP-32 mediates serotonergic 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
Serotonin is implicated in the regulation of complex sensory, motor,
affective, and cognitive functions. However, the biochemical mech-
anisms whereby this neurotransmitter exerts its actions remain
largely unknown. DARPP-32 (dopamine- and cAMP-regulated phos-
phoprotein of molecular weight 32,000) is a phosphoprotein that has
primarily been characterized in relation to dopaminergic neurotrans-
mission. Here we report that serotonin regulates DARPP-32 phos-
phorylation both in vitro and in vivo. Stimulation of 5-hydroxy-
tryptamine (5-HT4 and 5-HT6) receptors causes an increased
phosphorylation state at Thr34–DARPP-32, the protein kinase A site,
and a decreased phosphorylation state at Thr75–DARPP-32, the cyclin-
dependent kinase 5 site. Furthermore, stimulation of 5-HT2 receptors
increases the phosphorylation state of Ser137–DARPP-32, the casein
kinase-1 site. Behavioral and gene transcriptional effects induced
by compounds that selectively release serotonin were greatly re-
duced in DARPP-32 knockout mice. Our data indicate that DARPP-32
is essential not only for dopaminergic but also for serotonergic
neurotransmission.
T
he serotonergic and dopaminergic neurotransmitter systems
regulate emotion, mood, reward, and cognition. Pertubations of
these neurotransmitter systems are thought to contribute to the
etiology of several common neuropsychiatric disorders, such as
schizophrenia, bipolar disorder, depression, and drug addiction.
Indeed, the serotonergic and the dopaminergic systems appear to
be the primar y targets for most of the current medications used for
the treatment of psychiatric disorders. To better understand the
etiology of these disorders and to improve therapeutic options,
there is a considerable interest in elucidating the biochemical
mechanisms that underlie the behavioral effects of serotonin and
dopamine, in particular in prefrontal cortex and striatum, two
forebrain areas that receive converging serotonergic and dopami-
nergic inner vation (1, 2). Most reports favor the notion that
serotonin and dopamine interact in a cooperative manner to exert
biochemical (3, 4) and electrophysiological (5) actions, but there are
also numerous publications reporting opposing actions of these
neurotransmitter systems (for a recent review see ref. 6).
One well-characterized mediator of the biochemical, electro-
physiological, transcriptional, and behavioral effects of dopamine is
DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of
molecular weight 32,000) (7–9). DARPP-32 is highly enriched in
prefrontal cortex and striatum. Activation of dopamine D1 recep-
tors, by means of stimulation of protein kinase A (PKA), phos-
phor ylates DARPP-32 at Thr34 and thereby converts DARPP-32
into a potent inhibitor of protein phosphatase-1 (PP-1) (10). This
effect is counteracted by activation of D2 receptors, by means of
inhibition of PKA (11) and stimulation of the Ca2protein phos-
phatase-2B (PP-2B) signaling cascade, which dephosphor ylates
Thr34–DARPP-32 (12). Activation of D1 receptors also decreases
the phosphor ylation state of DARPP-32 at Thr75 (13). This site is
phosphor ylated by cyclin-dependent kinase 5 (Cdk5) (14) and
dephosphor ylated by protein phosphatase 2A (PP-2A) (13). The
dopaminergic regulation of the phosphor ylation state at Thr75–
DARPP-32 is mediated by means of a PKAPP-2A signaling
cascade (13). Thus, enhanced dopaminergic transmission by means
of D1 receptors leads to a decreased phosphor ylation of Thr75–
DARPP-32, which reduces inhibition of PKA (14), and thereby
facilitates transmission by means of the PKAThr34–DARPP-32
PP-1 signaling cascade. The efficacy of this signaling cascade also is
regulated by the phosphor ylation state of DARPP-32 at Ser137,
because an increased phosphor ylation at Ser137–DARPP-32, by
casein kinase-1 (CK-1), prevents the dephosphor ylation at Thr34–
DARPP-32 by protein PP-2B and thereby potentiates the PKA
Thr34–DARPP-32PP-1 cascade (15).
In view of the critical role for serotonin in modulating mental
functions, we have conducted a detailed characterization of the
regulation of DARPP-32 phosphor ylation by serotonin in vitro and
in vivo. By the use of DARPP-32 knockout (KO) mice, we also have
evaluated the possible involvement of DARPP-32 in mediating
biochemical and behavioral effects of compounds that selectively
release serotonin.
Materials and Methods
Preparation and Treatment of Brain Slices. Slices (300 m) from the
neostriatum (dorsal striatum), nucleus accumbens (ventral stria-
tum), and prefrontal cortex were prepared from adult male C57
Bl6 mice as described (12). The slices were preincubated in Krebs
buffer (118 mM NaCl4.7 mM KCl1.5 mM Mg2SO41.2 mM
KH2PO425 mM NaHCO311.7 mM glucose1.3 mM CaCl2) at
30°C under constant oxygenation (95% O25% CO2) for 60 min,
with a change of buffer after 30 min. To prevent reuptake of
serotonin into ner ve terminals, slices were pretreated with the
serotonin reuptake inhibitor, f luoxetine (10 M), 2 min before the
application of serotonin. The slices were thereafter treated with
serotonin and other drugs as specified in each experiment. After
drug treatment, the buffer was removed and the slices were rapidly
frozen on dr y ice and stored at 80°C until immunoblotted.
Whole Animal Studies. In experiments examining the effects of
para-chloroamphetamine (PCA) and 5-hydroxytr yptophan (5-
HTP) on DARPP-32 phosphor ylation, adult male C57Bl6 mice
were injected i.p. with saline, PCA (4 mgkg), or 5-HTP (50 mgkg)
and killed 15 min after the injection by focused microwave irradi-
ation (4.5–5 kW for 1.4 s), using a small animal microwave
(Muromachi Kikai, Tokyo). In these and all other experiments with
5-HTP, carbidopa (50 mgkg) was administered 30 min before
5-HTP. The brains were rapidly removed, and striata and frontal
cortices were dissected out and stored at 80°C until assayed.
Immunoblotting. Immunoblot procedures were as described (16). In
experiments using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-pretreated mice, immunoblotting was also carried out
Abbreviations: Cdk5, cyclin-dependent kinase 5; CK-1, casein kinase 1; DARPP-32,
dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000; EMDT,
2- ethyl - 5- methoxy- N, N- di methyl tryptami ne; 5- HT, 5- hydroxytryptami ne; 5- HTP, 5-
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,
2-[1-(4-piperonyl)piperazinyl]benzothiazole; TTX, tetrodotoxin; WT, wild type.
‡
To whom reprint requests should be addressed. E-mail: greengd@mail.rockefeller.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
3188 –3193 PNAS March 5, 2002 vol. 99 no. 5 www.pnas.orgcgidoi10.1073pnas.052712699
with an antibody against tyrosine hydroxylase (Chemicon). Data on
protein phosphor ylation 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
mgkg), or 5-HTP (50 mgkg) 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 44
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 mgkg), or 5-HTP (50 mgkg; 30 min after 50 mgkg
carbidopa i.p.) was injected i.p., and locomotor activity was mea-
sured for 50 min. In some experiments, adult male C57Bl6 mice
were pretreated with clozapine (0.3 mgkg) 10 min before the
administration of saline, PCA (4 mgkg), or 5-HTP (50 mgkg).
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 phosphor ylation 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-
phor ylation at all three phosphor ylation 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
DARPP-32 phosphor ylation at Thr75 was followed by a pronounced
decrease in the state of phosphor ylation at this site. No changes in
the total levels of DARPP-32 were found at any time point. Based
on these data, slices were incubated for 2 min for studies of
phospho-Thr34–DARPP-32 and phospho-Ser137–DARPP-32, and
for 10 min for studies of phospho-Thr75–DARPP-32 in all subse-
quent experiments. A lower concentration of serotonin was re-
quired to regulate DARPP-32 phosphor ylation at Thr34 than at
Thr75 and Ser137 (Fig. 1 d–f ). The EC50 values for the effects of
serotonin on DARPP-32 phosphor ylation at Thr34, Thr75, and
Ser137 were 13.5, 29.6, and 37.6 M, respectively.
Using slices prepared from nucleus accumbens, it was found that
2 min of incubation with serotonin (100 M) increased phosphor-
ylation at Thr34 (307 25%, n 12) and Ser137 (143 7%, n
12), and that 10 min of incubation with serotonin decreased
phosphor ylation at Thr75 (75 6%, n 12). Experiments carried
out in slices from the prefrontal cortex showed that 2 min of
incubation with serotonin increased phospho-Thr34–DARPP-32
(185 15%, n 6) and that 10 min of incubation with serotonin
decreased phospho-Thr75–DARPP-32 (85 3%, n 6). Because
of a low signal in prefrontal cortex, the level of phospho-Ser137–
DARPP-32 could not be accurately assayed in this area. Thus, the
regulation of phosphor ylation of DARPP-32 by serotonin was
similar in all three brain regions. Because more slices can be
collected from neostriatum than from nucleus accumbens, slices
made from neostriatum were used in most of the subsequent
experiments.
Effects of 5-Hydroxytryptamine (5-HT) Receptor Agonists on DARPP-32
Phosphorylation in Neostriatal and Cortical Slices. Several serotonin
receptors, most notably 5-HT1B, 5-HT2AC, 5-HT3, 5-HT4, and
5-HT6, are expressed throughout the striatum. The identity of the
serotonin receptors involved in the serotonin-mediated regulation
of DARPP-32 phosphor ylation was determined by the use of
selective agonists and antagonists. Results obtained with various
serotonin agonists in neostriatal slices are shown in Table 1.
2-[1-(4-Piperonyl)piperazinyl]benzothiazole (PPB, a 5-HT4 recep-
tor agonist) and 2-ethyl-5-methox y-N,N-dimethyltr ypt amine
(EMDT, a 5-HT6 receptor agonist), alone or together, qualitatively
mimicked the action of serotonin at phospho-Thr34–DARPP-32
and phospho-Thr75–DARPP-32, but not at phospho-Ser137–
DARPP-32. The EC50 values for the actions of PPB and EMDT at
phospho-Thr34–DARPP-32 were 27.3 and 20.5 M, and at phos-
pho-Thr75–DARPP-32 were 45.5 and 27.4 M, respectively. In
addition, 5-methoxytr yptamine (100 M), which has a high affinity
as an agonist at 5-HT4 and 5-HT6 receptors, increased phospho-
Thr34 –DARPP-32 (261 31%, n 12) and decreased phospho-
Thr75–DARPP-32 (76 7%, n 12). Conversely, DOI [()-2,5-
Fig. 1. Regulation by serotonin of DARPP-32 phosphorylation in slices of
neostriatum. (a–c) Time course and (d–f ) dose–response studies of regulation by
serotonin of phosphorylation of DARPP-32 at (a and d) Thr34, (b and e) Thr75, and
(c and f ) Ser137 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-
Ser137–DARPP-32 and for 10 min for studies of phospho-Thr75–DARPP-32. Data
represent means ( SE) (n 6 –10). *, P 0 05; **, P 0.01; ***, P 0.001
compared with control; one-way ANOVA followed by Dunnett’s test.
Svenningsson et al. PNAS March 5, 2002 vol. 99 no. 5 3189
N
EU
RO
B
IO
LO
G
Y
di met hox y- 4- i odoamphet ami ne hydr ochl or i de; ( ) - 1- ( 2, 5
dimethoxy-4-iodophenyl)-2-aminopropane], a 5-HT2A/C agonist,
mimicked the action of serotonin at phospho-Ser137–DARPP-32,
but not at phospho-Thr34–DARPP-32 and phospho-Thr75–
DARPP-32. The EC50 value for DOI at phospho-Ser137–
DARPP-32 was 18.2 M. The 5-HT1B receptor agonist, anpirtoline
(presumably by decreasing cAMP), and the 5-HT3 agonist,
SR57227A (presumably by increasing Ca2), decreased phospho-
Thr34–DARPP-32, but were without significant effects on phospho-
Thr75–DARPP-32 and phospho-Ser137–DARPP-32.
Using slices prepared from the prefrontal cortex, it was found
that 2 min of incubation with the 5-HT4 receptor agonist, PPB, or
the 5-HT6 receptor agonist, EMDT, increased phospho-Thr34–
DARPP-32 (203 15%, n 6, and 185 19%, n 6, respectively),
whereas 10 min of incubation with PPB or EMDT decreased
phospho-Thr75–DARPP-32 (84 4%, n 6, and 80 7%, n 6,
respectively).
Effects of 5-HT Receptor Antagonists on Serotonin-Mediated
DARPP-32 Phosphorylation in Neostriatal Slices. The results obtained
with selective serotonin receptor antagonists (Table 1) were con-
sistent with the results obtained with agonists. Thus, SDZ 205,557,
a 5-HT4 receptor antagonist, and Ro 04 – 6790, a 5-HT6 receptor
antagonist, alone or together, reduced the effects of serotonin on
phospho-Thr34–DARPP-32 and phospho-Thr75–DARPP-32, but
not on phospho-Ser137–DARPP-32. Conversely, ketanserin, a
5-HT2A/C receptor antagonist, reduced the effects of serotonin on
phospho-Ser137–DARPP-32 phosphor ylation, but not on phospho-
Thr34–DARPP-32 or phospho-Thr75–DARPP-32. The multirecep-
tor antagonist, clozapine, which has a particularly high affinity for
5-HT2 and 5-HT6 receptors (17), significantly counteracted sero-
tonin-mediated phosphor ylation of DARPP-32 at all sites exam-
ined. Taken together, the results obtained with serotonin receptor
agonists and antagonists indicate that the effects of serotonin on
DARPP-32 phosphor ylation can be accounted for primarily by
activation of 5-HT2, 5-HT4, and 5-HT6 receptors.
Effects of Signal Transduction Modulators on Serotonin-Mediated
DARPP-32 Phosphorylation in Neostriatal Slices. To investigate
whether the effects of serotonin on DARPP-32 phosphor ylation
were mediated through a direct action on DARPP-32-containing
neurons, slices were pretreated with tetrodotoxin (TTX), which
inhibits action potential-dependent synaptic transmission by block-
ing voltage-gated sodium channels. The actions of serotonin on
DARPP-32 phosphor ylation were unaffected by TTX, indicating a
direct action (Table 2). The principal signal transduction pathways
used by 5-HT2 and 5-HT45-HT6 receptors involve activation of
phospholipase C and PKA, respectively. To investigate the contri-
butions of these signaling pathways to the action of serotonin,
striatal slices were pretreated with either U-73122, a selective
phospholipase C inhibitor, or Rp-cAMPS, a selective PKA inhib-
itor, before the application of serotonin. As shown in Table 2,
U-73122 blocked the effect of serotonin on phospho-Ser137–
DARPP-32, but not on phospho-Thr34–DARPP-32 or phospho-
Thr75–DARPP-32. Conversely, Rp-cAMPS inhibited the action of
serotonin on phospho-Thr34–DARPP-32 and phospho-Thr75–
DARPP-32, but not on phospho-Ser137–DARPP-32.
Regulation of DARPP-32 Phosphorylation in Vivo by PCA and 5-HTP.
Mice were injected i.p. with the serotonin releaser, PCA (4 mgkg),
or the serotonin precursor, 5-HTP (50 mgkg). The results shown
in Fig. 2 demonstrate that in striatum PCA and 5-HTP cause a
common pattern of alteration of DARPP-32 phosphor ylation, i.e.,
an increase at phospho-Thr-34 –DARPP-32 and phospho-Ser137–
DARPP-32 and a decrease at phospho-Thr75–DARPP-32. Further-
more, in prefrontal cortex PCA and 5-HTP increased phospho-
Thr34–DARPP-32 (147 4.8%, n 5 mice, and 144 8.3%, n
5 mice, respectively), but decreased phospho-Thr75–DARPP-32
(79 3.8%, n 5 mice, and 74 8.0%, n 5, respectively). Thus,
serotonin regulates DARPP-32 phosphor ylation in a qualitatively
similar fashion both in vitro and in vivo.
Involvement of DARPP-32 in the Regulation of c-fos mRNA Expression
by PCA and 5-HTP. To evaluate the relevance of DARPP-32 in
mediating actions of PCA and 5-HTP on gene transcription, WT
and DARPP-32 KO mice were injected with PCA or 5-HTP, and
the effects on c-fos mRNA expression were investigated. As illus-
trated in Fig. 3a, both PCA and 5-HTP induced a strong expression
of c-fos mRNA throughout striatum and cerebral cortex of WT
mice. This effect, which was quantitated in the periventricular
region of striatum (Fig. 3b) and in the cingulate cortex (Fig. 3c), was
strongly attenuated in DARPP-32 KO mice.
Table 1. Regulation of DARPP-32 phosphorylation in vitro by
5-HT receptor agonists and antagonists
Thr34 Thr75 Ser137
Control 100 (17.0) 100 (5.0) 100 (3.9)
Serotonin 342 (26.7)*** 76 (7.5)** 141 (6.0)**
5-HT receptor agonists
PPB (5HT4 agonist) 218 (36.1)* 79 (8.8)* 107 (2.0)
EMDT (5HT6 agonist) 301 (39.5)** 76 (7.6)** 103 (5.5)
PPB EMDT 397 (41.0)*** 70 (3.8)** 105 (7.9)
DOI (5HT2 agonist) 144 (10.7) 105 (4.5) 158 (8.3)**
Anpirtoline (5HT1B agonist) 86 (3.7)* 102 (22.2) 93 (10.0)
SR 57227 (5HT3 agonist) 78 (3.5)* 88 (6.8) 105 (9.6)
5-HT receptor antagonists
SDZ 205,557 (5HT4 antagonist) 94 (16.7) 103 (17.5) 102 (8.8)
SDZ serotonin 257 (34.4)**† 95 (11.5)† 129 (5.0)*
Ro 04-6790 (5HT6 antagonist) 90 (8.7) 101 (12.5) 105 (7.3)
Ro serotonin 225 (28.5)*† 96 (8.4)† 131 (7.2)*
Ro SDZ 84 (6.5) 110 (11.5) 107 (6.2)
SDZ Ro serotonin 198 (44.5)*‡ 98 (6.1)‡ 134 (18.1)*
Ketanserin (5HT2 antagonist) 94 (2.9) 105 (8.9) 97 (4.8)
Ketanserin serotonin 322 (28.4)*** 78 (12.1)* 101 (9.1)†
Clozapine (5HT2/6 antagonist) 124 (19.8) 101 (13.4) 105 (5.5)
Clozapine serotonin 156 (15.4)*‡ 98 (5.2)‡ 110 (4.9)†
Effects of serotonin (100 M), PPB (100 M), a 5-HT4 receptor agonist,
EMDT (100 M), a 5-HT6 receptor agonist, DOI (100 M), a 5-HT2 receptor
agonist, anpirtoline, (100 M), a 5-HT1B receptor agonist, and SR 57227 (100
M), a 5-HT3 receptor agonist, on Thr34, Thr75, and Ser137. In antagonist
experiments, ketanserin (10 M), a 5-HT2 receptor antagonist, SDZ 205,557 (10
M), a 5-HT4 receptor antagonist, Ro 04-6790 (10 M), a 5-HT6 receptor
antagonist, or clozapine (10 M), a multireceptor antagonist with high affin-
ity for 5-HT2 and 5-HT6 receptors, was applied to striatal slices 10 min before
serotonin (100 M). Data represent means ( SEM) (n 12–30). *, P 0.05; **,
P 0.01; ***, P 0.001 compared with control; †, P 0.05; ‡, P 0.01
compared with serotonin alone; one-way ANOVA followed by Dunnett’s test.
Table 2. Regulation of serotonin-mediated DARPP-32
phosphorylation in vitro by signal transduction modulators
Thr34 Thr75 Ser137
Control 100 (17.0) 100 (5.0) 100 (3.9)
Serotonin 342 (26.7)*** 76 (7.5)** 141 (6.0)**
TTX 92 (5.8) 103 (3.5) 98 (12.5)
TTX serotonin 348 (63.2)*** 83 (8.1)* 144 (3.3)**
U-73122 (PLC inhibitor) 117 (10.9) 90 (6.6) 86 (6.2)
U-73122 serotonin 297 (34.0)** 70 (6.1)** 89 (3.7)‡
RpcAMPs (PKA inhibitor) 88 (13.1) 105 (7.2) 108 (10.0)
RpcAMPs serotonin 150 (44.7)‡ 102 (9.5)† 147 (4.0)**
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
applied to striatal slices 10 min before serotonin (100 M). Data represent
means ( SEM) (n 10 –30). *, P 0.05; **, P 0.01; ***, P 0.001 compared
with no serotonin; †, P 0.05; ‡, P 0.01 compared with serotonin alone;
one-way ANOVA followed by Dunnett’s test.
3190 www.pnas.orgcgidoi10.1073pnas.052712699 Svenningsson et al.
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 (6,133 293 cm20 min) and DARPP-32 KO (6,268 418
cm20 min) mice.
Blockade by Clozapine of Behavioral Effects of PCA and 5-HTP. As
demonstrated above, the atypical neuroleptic agent, clozapine,
which has a ver y high affinity at 5-HT2 and 5-HT6 receptors,
strongly counteracted the effects of serotonin on DARPP-32
phosphor ylation. We therefore evaluated the effects of pretreat-
ment with a relatively low dose of clozapine (0.3 mgkg) on PCA-
and 5-HTP-induced behaviors. The results shown in Fig. 5 dem-
onstrate that clozapine abolished the hyperactivity induced by PCA
or 5-HTP. It should be noted that clozapine also has a moderate
affinity for several dopamine receptors (17). However, previous
work has shown that at least 10 times higher concentrations (2–10
mgkg) of clozapine are necessar y to inhibit dopamine-dependent
hyperactivity (17).
Characterization of Serotonin-Dopamine Interactions in the Regula-
tion of DARPP-32 Phosphorylation. To determine whether the effects
of serotonin on DARPP-32 phosphor ylation were mediated
through modulation of dopaminergic signaling pathways, several
types of experiments were carried out (Table 3). Serotonin and SKF
82958, a D1 receptor-selective agonist, had additive effects in
increasing phospho-Thr34–DARPP-32 and decreasing phospho-
Thr75–DARPP-32. Serotonin and quinpirole, a D2 receptor-
selective agonist, had antagonistic effects on phospho-Thr34–
DARPP-32 and phospho-Thr75–DARPP-32 and additive effects in
increasing phosphor ylation at phospho-Ser137–DARPP-32. The
effects of serotonin on DARPP-32 phosphor ylation were not
significantly altered in slices from D1 receptor KO mice (unpub-
lished results) or from mice pretreated with the dopaminergic
neurotoxin, MPTP (Table 3), indicating that the effects of seroto-
nin on DARPP-32 phosphor ylation were not mediated through
regulation of dopamine release.
Discussion
The present studies, and those described in ref. 16, have demon-
strated that behavioral as well as biochemical effects induced by
enhanced serotonergic neurotransmission are attenuated in
DARPP-32 KO mice. The results show an important role for
DARPP-32 in mediating the actions of serotonin in vivo. We have
investigated the underlying molecular mechanisms.
Neostriatum, nucleus accumbens, and prefrontal cortex receive
a moderate to high serotonergic inner vation (1), which is further
increased when the levels of dopamine in this region are decreased
(18). Several serotonin receptors, most notably 5-HT1B, 5-HT1E,
Fig. 2. Regulation of DARPP-32 phosphorylation in vivo by PCA and 5-HTP. Mice
were injected i.p. with saline, PCA (4 mgkg), or 5-HTP (50 mgkg). Fifteen
minutes later mice were killed by focused microwave irradiation. Data represent
means SE for 5–10 mice per group. *, P 0.05; **, P 0.01; ***, P 0.001
compared with saline-treated mice; one-way ANOVA followed by Dunnett’s test.
Fig. 3. Regulation of c-fos mRNA expression by PCA and 5-HTP in WT and
DARPP-32 KO mice. (a) Dark-field autoradiograms showing the expression of
c-fos mRNA 20 min after treatment with saline, PCA (4 mgkg), or 5-HTP (50
mgkg) in WT and DARPP-32 KO mice. (Magnification: 5.) (b and c) Histograms
show quantification of the expression of c-fos mRNA in (b) periventricular area of
striatum and (c) cingulate cortex after each treatment. WT, filled bars; DARPP-32
KO, open bars. Data represent means SE for 5– 8 mice per group. **, P 0.01;
***, P 0.001 compared with saline-treated mice; #, P 0.05; ##, P 0.01
compared with WT mice given the same treatment; one-way ANOVA followed by
Dunnett’s test.
Svenningsson et al. PNAS March 5, 2002 vol. 99 no. 5 3191
N
EU
RO
B
IO
LO
G
Y
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-
erol formation; 5-HT3 receptors increase Na and Ca2 inf lux; and
5-HT4 and 5-HT6 receptors increase cAMP formation.
Serotonin caused an increased phosphor ylation of DARPP-32 at
Thr34 (the PKA site) and Ser137 (the CK-1 site) and a decreased
phosphor ylation at Thr75 (the Cdk5 site), in brain slices. The use of
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
pathways. The actions of serotonin in regulating DARPP-32 phos-
phor ylation at Thr34 and Thr75 were mediated primarily by means
Fig. 4. Behavioral responses to treatment with PCA and 5-HTP in WT and
DARPP-32 KO mice. Effects of (Left) PCA (4 mgkg) and (Right) 5-HTP (50 mgkg)
on total locomotion (Upper) and stereotypic movements (Lower) in WT and
DARPP-32 KO mice. Data represent means SE for 6 –10 mice per group. *, P
0.05; **, P 0.01; ***, P 0.001 compared with saline-treated mice; ###, P
0.001; ##, P 0.01; #, P 0.05 compared with WT mice given the same treatment;
two-way ANOVA followed by Duncan’s test.
Fig. 5. Effect of clozapine on behavioral responses to treatment with PCA and
5-HTP in WT mice. Effect of pretreatment with clozapine (0.3 mgkg) on loco-
motion induced by (Left) PCA (4 mgkg) or (Right) 5-HTP (50 mgkg). Data
represent means SE for 6 –10 mice per group. **, P 0.01 compared with
saline-treated mice; ##, P 0.01 compared with PCA-treated or 5-HTP-treated
mice; two-way ANOVA followed by Duncan’s test.
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-
ylation of Thr34. 5-HT2 receptors activate PLC, increasing casein kinase-1 (CK1)
activity (27), phosphorylation of Ser137, and inhibition of dephosphorylation by
protein phosphatase-2B (PP2B) of Thr34 (15). These various actions of serotonin on
DARPP-32 phosphorylation are synergistic, each leading to an increased state of
phosphorylation of Thr34 and an increased inhibition of PP-1 (10). Solid lines
indicate activation; dashed lines indicate inhibition.
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 serotonin 717 (42.4)***#§ 56 (1.1)***#§ 121 (14.4)
Quinpirole (D2 agonist) 65 (4.7)** 112 (8.5) 135 (5.1)*
Quinpirole serotonin 125 (12.7)###† 106 (13.2)## 186 (11.1)***#†
MPTP treatment 93 (8.7) 118 (9.3) 104 (7.9)
MPTP-treatment
serotonin 307 (44.1)*** 80 (9.2)* 154 (21.1)**
SKF 82958 (10 M), a D1 receptor agonist, or quinpirole (10 M), a D2
receptor agonist, was applied 10 min before serotonin (100 M). MPTP (50
mgkg, 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 ( SEM) (n
16 –30). *, P 0.05; **, P 0.01; ***, P 0.001 compared with control; #, P
0.05; ##, P 0.01; ###, P 0.001 compared with serotonin alone; §, P 0.05
compared with SKF 82958 alone; †, P 0.05 compared with quinpirole alone;
one-way ANOVA followed by Dunnett’s test.
3192 www.pnas.orgcgidoi10.1073pnas.052712699 Svenningsson et al.
of activation of 5-HT4 and 5-HT6 receptors, whereas the regulation
at Ser137 was mediated primarily by means of 5-HT2 receptors. The
three pathways appear to inhibit PP-1 through synergistic mecha-
nisms (Fig. 6) and provide a model by which serotonin as well as
compounds which increase serotonin transmission may produce
their effects. These latter compounds include, in addition to PCA
and 5-HTP, the selective serotonin reuptake inhibitor, f luoxetine,
a widely used antidepressant agent.
The pattern of DARPP-32 phosphor ylation induced by elevated
serotonergic neurotransmission is ver y similar to that induced by
elevated dopaminergic neurotransmission (13). However, the se-
rotonin-mediated regulation of DARPP-32 phosphor ylation is
largely independent of altered dopaminergic neurotransmission
(Table 3). Numerous psychoactive compounds act directly on both
the serotonergic and dopaminergic systems. For example, cocaine
inhibits the reuptake of both dopamine and serotonin from the
synapse. It has recently been shown that the reinforcing properties
of cocaine in the place preference test remain intact in dopamine
transporter KO mice or serotonin transporter KO mice, but are
severely impaired in mice that lack both transporters (19). These
data imply that certain biochemical effects of cocaine can be
mediated either by dopamine or serotonin. Our data indicate that
signaling through the DARPP-32 pathway may be responsible for
this redundancy. In support of this suggestion, the diminished
responsiveness to the reinforcing properties of cocaine in the place
preference test of the double transporter KO mice is mimicked by
the DARPP-32 KO mice (20).
Evidence that the effects of serotonin on DARPP-32 phosphor-
ylation obser ved in vitro are of physiological relevance came from
studies in which we injected mice with PCA or 5-HTP and examined
the effects on DARPP-32 phosphor ylation in the striatum and
prefrontal cortex. Both treatments led to a pattern of DARPP-32
phosphor ylation that closely resembled that obser ved in slices in
response to serotonin, namely increases at phospho-Thr34–
DARPP-32 and phospho-Ser137–DARPP-32 and a decrease at
phospho-Thr75–DARPP-32. It seemed possible that the effects of
5-HTP on DARPP-32 phosphor ylation were attributable mainly to
ectopically released serotonin produced in dopaminergic ner ve
terminals. However, the fact that PCA, which acts selectively on
serotonergic ner ve terminals, at the dose used, also had pronounced
effects on DARPP-32 phosphor ylation in the striatum and the
prefrontal cortex, shows that an enhanced release of serotonin in
these areas regulates DARPP-32 phosphor ylation. The conclusion
that DARPP-32 mediates the behavioral effects of serotonin is
supported by experiments showing that 0.3 mg/kg of clozapine, a
dose that antagonize responses by means of 5-HT2 and 5-HT6
receptors, counteracts the locomotor effects of PCA and 5-HTP.
Strong evidence for an involvement of DARPP-32 in the bio-
chemical and behavioral actions of serotonin under in vivo condi-
tions came from studies in which we examined effects of PCA and
5-HTP in WT and DARPP-32 KO mice. An induction of c-fos
mRNA was found throughout striatum and cerebral cortex in WT
mice treated with PCA or 5-HTP. The ability of both PCA and
5-HTP to induce c-fos mRNA expression was strongly reduced in
DARPP-32 KO mice. It is well known that the transcription of c-fos
mRNA is negatively regulated by PP-1 (21, 22). The critical
involvement of DARPP-32 in PCA- and 5-HTP-mediated c-fos
induction is likely explained, at least in part, by its ability to inhibit
PP-1.
Previous work implicated serotonin in the regulation of locomo-
tion and explorator y behavior (23). Depletion of serotonin caused
a long-term reduction in exploration (24), whereas local application
of serotonin into nucleus accumbens caused an increase in loco-
motion (25) in rats. It is generally agreed that agents that increase
the release of serotonin, such as PCA, 5-HT P, and 3,4-
methylenedioxymethamphetamine (‘‘Ecstasy’’) increase locomotor
activity (23, 26). To further investigate the functional relevance of
DARPP-32 phosphor ylation in the actions of PCA and 5-HTP, the
effects of these drugs on locomotor activity and stereotypic behav-
iors were examined in WT and DARPP-32 KO mice. As expected,
both PCA and 5-HTP increased locomotor activity and stereotypic
behaviors in WT mice. Both of these responses were greatly
attenuated in DARPP-32 KO mice.
In conclusion, the studies described here, together with those
described in ref. 16, indicate that DARPP-32 plays a central role in
serotonergic signaling. Moreover, the data provide the outline of a
molecular mechanism for the behavioral actions of those psycho-
stimulants and antidepressant agents that achieve their effects
through pertubation of serotonergic transmission.
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
antibodies against phospho-Thr34 DARPP-32, phospho-Thr75 DARPP-32,
and total DARPP-32 and S. Col, S. Galdi, and L. Lannebo for technical
assistance. This work was supported by National Institute of Mental Health
Grants MH-40899 and DA10044 (to P.G.). P.S. is supported by a postdoc-
toral fellowship from Stiftelsen fo¨r Internationalisering av Ho¨gre utbildning
och forskning. 2-Ethyl-5-methoxy-N,N-dimethyltr yptamine was synthesized
by Eli Lilly & Company.
1. Steinbusch, H. W. (1981) Neuroscience 6, 557– 618.
2. Lindvall, O. & Bjo¨rklund, A. (1984) in Handbook of Chemical Neuroanatomy,
eds. Bjo¨rklund, A. & Ho¨kfelt, T. (Elsev ier, Amsterdam), pp. 55–122.
3. Bhat, R. V. & Baraban, J. M. (1993) J. Phar macol . Exp. Ther. 267,
496 –505.
4. Genova, L. M. & Hyman, S. E. (1998) Synapse 30, 71–78.
5. White, F. J., Hu, X. & Henr y, D. J. (1993) J. Phar macol . Exp. Ther. 266,
1075–1084.
6. Barnes, N. M. & Sharp, T. (1999) Neurophar macolog y 38, 1083–1152.
7. Fienberg, A. A., Hiroi, N., Mer melstein, P. G., Song, W. J., Snyder, G. L.,
Nishi, A., Cheramy, A., OCallaghan, J. P., Miller, D. B., Cole, D. G., et al.
(1998) Science 281, 838 – 842.
8. Greengard, P., Allen, P. B. & Nairn, A. C. (1999) Neuron 23, 435– 447.
9. Greengard, P. (2001) Science 294, 1024 –1030.
10. Hemmings, H. C., Jr., Greengard, P., Tung, H. Y. L. & Cohen, P. (1984) Nature
(London) 310, 503–505.
11. Lindskog, M., Svenningsson, P., Fredholm, B. B., Greengard, P. & Fisone, G.
(1998) Neuroscience 88, 1005–1008.
12. Nishi, A., Snyder, G. L. & Greengard, P. (1997) J. Neurosci . 17, 8147– 8155.
13. Nishi, A., Bibb, J. A., Snyder, G. L., Higashi, H., Nairn, A. C. & Greengard,
P. (2000) Proc. Natl . Acad. Sci . USA 97, 12840 –12845.
14. Bibb, J. A., Snyder, G. L., Nishi, A., Yan, Z., Meijer, L., Fienberg, A. A., Tsai,
L. H., Kwon, Y. T., Girault, J. A., Czernik, A. J., et al. (1999) Nature (London)
402, 669 – 671.
15. Desdouits, F. Siciliano, J. C. Greengard, P. & Girault, J.-A. (1995) Proc. Natl .
Acad. Sci . USA 92, 2682–2685.
16. Svenningsson, P., Tzavara, E. T., Witkin, J. M., Fienberg, A. A., Nomikos, G. G.
& Greengard P. (2002) Proc. Natl . Acad. Sci . USA 99, 3182–3187.
17. Leysen, J. E. (2000) in At ypical Antipsychotics, eds. Ellenbroek, B. A. & Cools,
A. R. (Birkha
¨user, Basel), pp. 57– 81.
18. Rozas, G., Liste, I., Guerra, M. J. & Labandeira-Garcia, J. L. (1998) Neurosci .
Lett . 245, 151–154.
19. Sora, I., Hall. S., Andrews, A. M., Itokawa, M., Li, X.-F., Wei, H.-B., Wichems,
C., Lesch, K.-P., Murphy, D. L. & Uhl, G. R. (2001) Proc. Natl . Acad. Sci . USA
98, 5300 –5305.
20. Zachariou, V., Benoit-Marandl, M., Allen, P. B., Ingrassia, P., Fienberg, A. A.,
Gonon, F., Greengard, P. & Picciotto, M. (2002) Biol . Psychiatr y, in press.
21. Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T.,
Karin, M., Shenolikar, S. & Montminy, M. (1992) Cell 70, 105–113.
22. Liu, F.-C. & Graybiel, A. M. (1996) Neuron 17, 1133–1144.
23. Geyer, M. A. (1996) Behav. Brain Res. 73, 31–36.
24. Lipska, B. K., Jaskiw, G. E., Ar ya, A. & Weinberger, D. R. (1992) Phar macol .
Biochem. Behav. 43, 1247–1252.
25. Sasaki-Adams, D. M. & Kelley, A. E. (2001) Neuropsychophar macolog y 25,
440 – 452.
26. Modigh, K. (1972) Psychophar macolog y 23, 48 –54.
27. Liu, F., Ma, X. H., Ule, J., Bibb, J. A., Nishi, A., DeMaggio, A. J., Yan, Z., Nairn,
A. C. & Greengard, P. (2001) Proc. Natl. Acad. Sci. USA 98, 11062–11068.
Svenningsson et al. PNAS March 5, 2002 vol. 99 no. 5 3193
N
EU
RO
B
IO
LO
G
Y
No comments:
Post a Comment