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The Journal of Neuroscience, June 15, 2000, 20(12):4480-4488
Regulation of Phosphorylation of the GluR1 AMPA Receptor in the
Neostriatum by Dopamine and Psychostimulants In Vivo
Gretchen L.
Snyder1,
Patrick B.
Allen1,
Allen
A.
Fienberg1,
Carmina G.
Valle1,
Richard L.
Huganir2,
Angus C.
Nairn1, and
Paul
Greengard1
1 Laboratory of Molecular and Cellular Neuroscience,
The Rockefeller University, New York, New York 10021, and
2 Department of Neuroscience, Howard Hughes Medical
Institute, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205
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ABSTRACT |
The activation of cAMP-dependent protein kinase regulates the
physiological activity of AMPA-type glutamate receptors. In this study,
phosphorylation of the AMPA receptor subunit GluR1 at
Ser845 was increased in neostriatal slices by
activation of D1-type dopamine receptors and by inhibitors of protein
phosphatase 1/protein phosphatase 2A. In contrast,
Ser831, a residue which, when phosphorylated by
protein kinase C or calcium/calmodulin-dependent kinase II, increases
AMPA receptor channel conductance, was unaffected by either D1 or D2
receptor agonists in neostriatal slices. The phosphorylation of
Ser845, but not Ser831, was
strongly increased in neostriatum in vivo in
response to the psychostimulants cocaine and methamphetamine. The
effects of dopamine and psychostimulants on the phosphorylation of
GluR1 were attenuated in dopamine and cAMP-regulated phosphoprotein Mr 32 kDa (DARPP-32) knock-out mice. These
results identify DARPP-32 and AMPA-type glutamate receptors as likely
essential cellular effectors for psychostimulant actions.
Key words:
dopamine; DARPP-32; methamphetamine; cocaine; protein
phosphatase 1; D1 receptor; protein kinase A
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INTRODUCTION |
Ionotropic glutamate receptors
function as ligand-gated ion channels and are believed to play a role
in certain forms of learning and memory (Bliss and Collingridge, 1993 ;
Linden, 1994), in the development of psychomotor stimulant
sensitization (Wolf, 1998) and in various forms of neurodegenerative
disease (Lipton and Rosenberg, 1994). They are classified by their
physiological and pharmacological characteristics as members of AMPA,
kainate, or NMDA receptor subclasses (Monaghan et al., 1989).
AMPA-type glutamate receptors are abundantly expressed on dendritic
processes of medium spiny neostriatal neurons (Petralia and Wenthold,
1992) where they mediate a large proportion of the glutamate-induced
excitation (Cepeda et al., 1993).
Considerable evidence indicates that AMPA receptors are regulated by
protein phosphorylation. For example, activation of cAMP-dependent protein kinase (PKA) in hippocampal (Greengard et al., 1991; Wang et
al., 1991; Rosenmund et al., 1994; Kameyama et al., 1998) or neostriatal (Yan et al., 1999) neurons increases AMPA currents recorded from these cells. The notion that these effects occur as the
result of phosphorylation of the receptor is supported by the
observation that the GluR1 subunit is phosphorylated in response to PKA
activation in human embryonic kidney-293 cells (Blackstone et al.,
1994; Tan et al., 1994) (but see McGlade-McCulloh et al., 1993).
Moreover, a PKA-dependent residue, Ser845,
is required for PKA-mediated potentiation of peak current carried by
homomeric GluR1 channels (Roche et al., 1996). AMPA receptors also can
be regulated by phosphorylation of Ser831
after activation of calcium/calmodulin-dependent kinase II (CaMKII) (Barria et al., 1997) or protein kinase C (PKC) leading to potentiation of AMPA currents in hippocampal neurons (Barria et al., 1997; Mammen et
al., 1997). Whereas PKA-mediated phosphorylation at Ser845 enhances AMPA currents by
increasing channel open time probability (Roche et al., 1996),
phosphorylation at Ser831 increases
single-channel conductance of AMPA receptors (Derkach et al., 1999).
Thus, PKA and CaMKII/PKC signaling cascades control distinct properties
of the AMPA receptor by regulating unique GluR1 residues.
Dopamine and cAMP-regulated phosphoprotein
Mr 32 kDa (DARPP-32); is a
neostriatum-enriched substrate for PKA (Walaas et al., 1983; Ouimet et
al., 1984). Phosphorylation at Thr34 by
PKA converts DARPP-32 into an efficient inhibitor of protein phosphatase 1 (PP1) (Hemmings et al., 1984), a serine/threonine phosphatase enriched in the dendritic spines of neostriatal neurons (Ouimet et al., 1995). Studies using mice lacking the gene for DARPP-32
indicate that DARPP-32 mediates many physiological and behavioral
effects of dopamine (Fienberg et al., 1998). A recent study (Yan et
al., 1999) showed that whole-cell AMPA currents are enhanced by D1
dopamine receptors via a pathway involving DARPP-32, PP1, and the
PP1-targeting protein spinophilin (Allen et al., 1997). A principal
goal of the present study was to determine whether regulation of
neostriatal GluR1 subunit phosphorylation by this pathway is a
plausible mechanism for the physiological control of AMPA receptors by dopamine.
Psychostimulants increase the synaptic availability of dopamine in the
brain (Hyman, 1996; Koob and LeMoal, 1997), leading to both acute and
long-term plastic changes in dopaminoceptive neurons (Nestler and
Aghajanian, 1997). Some of these effects appear to require the
activation of glutamate receptors, although the precise role that
glutamate plays in addiction is unclear (Wolf, 1998). Thus, a second
objective of this study was to investigate the possibility that
regulation of AMPA receptor phosphorylation in vivo by
psychostimulants might contribute to these effects.
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MATERIALS AND METHODS |
Preparation and treatment of neostriatal slices. Male
C57BL/6 mice (8-12 weeks in age) were killed by decapitation. The
brain was rapidly transferred to an ice-cold surface where it was
blocked and fixed to the cutting surface of a Vibratome (Ted Pella)
maintained at 4°C. The brain was placed in cold, oxygenated (95%
O2 and 5% CO2) Krebs'
bicarbonate buffer of the following composition (in mM):
125 NaCl, 5 KCl, 26 NaHCO3, 1.5 CaCl2, 1.5 MgSO4, and 10 glucose, pH 7.4. Coronal slices of mouse brain (400 µm in thickness) were cut and pooled in 10 ml of cold buffer. Neostriatal slices were
cut from the coronal sections under a dissecting microscope. The slices
were pooled, then transferred individually to 4 ml polypropylene tubes
containing 2 ml of fresh, cold, oxygenated buffer. The tissue was
preincubated for 15 min at 30°C, the buffer was replaced, and tissue
sections were preincubated for an additional 30 min. At the end of this
second preincubation period, the buffer was replaced with Krebs'
buffer or buffer containing the indicated test substances for 30 sec to
60 min. After treatment, the slices were immediately frozen in liquid
nitrogen and stored at  80°C until assayed.
In some experiments, neostriatal slices were prepared from C57BL/6 mice
(8-10 weeks of age) lacking the gene for DARPP-32 (Fienberg et al.,
1998). DARPP-32 knock-out mice and wild-type controls were generated
from the offspring of heterozygous mating pairs. All mice were
age-matched, and only males were used.
Preparation of GluR1 fusion protein for stoichiometric analysis
of Ser845 and
Ser831 phosphorylation. A bacterial
protein comprising the last putative intracellular domain of GluR1
(residues 809-889) fused to glutathione-S-transferase (GST)
was prepared as follows: rat brain cDNA was used as a template for PCR
amplification of the GluR1 sequence using the primers TGC GTC GAC CGA
GTT CTG CTA CAA ATC CC; TTC GCG GCC GCT CCA GTT ACA ATC CTG TG
(Operon). The resulting 272 bp fragment was digested with
SalI-NotI and ligated in frame with the GST
coding sequence contained in a bacterial expression plasmid pGex-4T-2
(Pharmacia, Piscataway, NJ). This plasmid was sequenced to ensure
fidelity of PCR amplification and transformed into Escherichia
coli strain BL21 DE3. Bacteria were grown at 37°C to an
OD600 of 0.5. The temperature was reduced to
30°C, and expression was induced over 2 hr by the addition of
isopropylthio- -galactoside to a final concentration of 0.1 mM. Cells were harvested by centrifugation and
lysed in a French press. After clarification of the lysate by
centrifugation at 30,000 × g for 20 min, the fusion
protein was purified by affinity chromatography on glutathione-agarose beads (Pharmacia), using 5 mM glutathione and 50 mM Tris, pH 8.0, for elution.
GST-GluR1 (35 µM) was preparatively phosphorylated under
the following conditions: 10 µg/ml purified PKA in 50 mM
Tris, pH 8.0, 10 mM magnesium chloride, 0.4 mM
EGTA, 50 µM [ 32P] ATP
or 1 µg/ml purified CaMKII in 50 mM Tris, pH 8.0, 10 mM magnesium chloride, 0.4 mM EGTA, 2 mM DTT, 1 µM calmodulin, 1.5 mM
calcium chloride, and 50 µM
[32P]ATP. Reactions were stopped by
the addition of Laemmli buffer. Stoichiometry of the phosphorylated
fusion proteins was calculated based on PhosphorImager (Molecular
Dynamics, Eugene, OR) analysis of phosphate incorporation. Known
quantities of these proteins were compared with tissue samples, the
levels of phospho-GluR1 and GluR1 were detected by ECL, and the
resulting values used to calculate stoichiometries of phosphorylation
for the Ser845 and
Ser831 sites in neostriatal tissue.
Drug treatment and microwave irradiation of mice. Male
C57BL/6 mice (8-12 weeks in age) were injected with vehicle (0.9%
NaCl in water) or vehicle containing various concentrations of either methamphetamine HCl (20 or 30 mg/kg, s.c.) or cocaine HCl (10 or 20 mg/kg, i.p.). To insure preservation of phosphoproteins in their
in vivo phosphorylation state, the animals were
killed at various times (15-60 min) after injection by focused
microwave irradiation (4.5-5 kW for 1.4 sec) using a small-animal
microwave (Murimachi Kikai, Tokyo, Japan). The brains were rapidly
removed, and the neostriatum was dissected and stored at 80°C until
assayed for phosphoprotein levels.
Immunoblotting for phospho-GluR1. Frozen tissue samples were
sonicated in 1% SDS. Small aliquots of the homogenate were retained for protein determination by the BCA protein assay method (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts
of protein (50 µg for slice experiments; 250 µg for microwave experiments) were loaded onto 7.5% acrylamide gels. The proteins were
separated by SDS-PAGE (Laemmli, 1970), and transferred to nitrocellulose membranes (0.2 µm) (Schleicher and Schuell) by the
method of Towbin et al. (1979). Membranes were blocked for 30-60 min
in PBS (in mM: 124 NaCl, 4 KCl, 10 Na2HPO4, and 10 KH2PO4, pH 7.2) containing
5% nonfat dry milk and 0.2% Tween 20 (Blotto). The membranes were
immunoblotted using antisera that selectively detect either the
Ser845-phosphorylated or the
Ser831-phosphorylated form of GluR1 (1:200
dilutions for each antibody) (Kameyama et al., 1998), or an antiserum
(PharMingen, San Diego, CA; 1:10,000 dilution) that detects the
C-terminal region of GluR1, irrespective of phosphorylation state. In
some experiments these samples were also immunoblotted with monoclonal
antibody 23 (1:750 dilution) (Snyder et al., 1992), a phosphorylation
state-specific monoclonal antibody raised against a DARPP-32 peptide
containing phospho-Thr34, the site
phosphorylated by PKA.
Antibody binding was revealed by incubation with either a goat
anti-rabbit horseradish peroxidase-linked IgG or a goat anti-mouse horseradish peroxidase-linked IgG (each at a 1:6000-8000 dilution) (Pierce) and the ECL immunoblotting detection system (Amersham, Arlington Heights, IL). Chemiluminescence was detected by
autoradiography using DuPont NEN (Boston, MA) autoradiography film, and
bands were quantified by analysis of scanned images by NIH Image 1.52 software. Because the linear range for quantitation of ECL signals by
densitometry is limited, several film exposures were obtained for each
set of samples to insure that signals were within a density range that
allowed accurate quantitation.
In all of the experiments for this study, nitrocellulose membranes were
sequentially analyzed for phospho-Ser845
GluR1 or for phospho-Ser831 GluR1, and
then for total levels of C-terminal GluR1. After probing a membrane for
phospho-GluR1, the filter was washed three times for 5 min each in PBS
to remove any remaining chemiluminescent reagent. The membrane was then
stripped of antibody by incubation at 60°C for 60 min in a buffer
containing 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris/HCl, pH 6.7. The filter was then washed several
times in large volumes of PBS and blocked in Blotto for 30-60 min
before immunoblotting with the C-terminal GluR1 antibody.
Data were analyzed by Student's t-test or Mann-Whitney
U test, as indicated, with significance defined as
p < 0.05.
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RESULTS |
Differential phosphorylation of GluR1 in neostriatum by protein
kinase activators
GluR1 is phosphorylated in vitro at
Ser845 by PKA (Blackstone et al., 1994)
and at Ser831 by PKC (Mammen et al., 1997)
or CaMKII (McGlade-McCulloh et al., 1993). Phosphorylation of GluR1 at
Ser845, in response to activation of PKA,
potentiates AMPA currents by increasing open time probability of the
receptor channel (Roche et al., 1996). The phosphorylation of GluR1 at
Ser831 in response to activation of CaMKII
or PKC increases AMPA receptor function by enhancing channel
conductance (Derkach et al., 1999). Treatment of neostriatal slices
with forskolin (50 µM), an adenylyl cyclase
activator, increased phosphorylation of
Ser845 approximately eightfold (776 ± 20% of control in three experiments) (Fig.
1A). The PKC activator
phorbol 12,13-dibutyrate (PDBu) (5 µM) (Fig.
1B) increased the phosphorylation of GluR1 at
Ser831 by an average of 10-fold (1005 ± 28% of control in three experiments) in neostriatal
slices.
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Figure 1.
Effects of protein kinase activators on GluR1
phosphorylation in neostriatal slices. Neostriatal slices were prepared
from normal C57BL/6 mice and incubated for 5 min in the absence
(Control) or presence of the adenylyl cyclase
activator forskolin (A) or the PKC activator PDBu
(B). Levels of phospho-Ser845
(A) or phospho-Ser831
(B) were determined by SDS-PAGE and
immunoblotting. Phospho-GluR1 levels were detected, quantitated by
densitometry, and expressed as percentage of the level detected in
control slices. Arrows (top panels)
indicate the position of the phospho-GluR1 bands in representative
autoradiograms. Data (bottom panels) are expressed as
means ± SEM for three experiments
(*p < 0.05 compared with control, Mann-Whitney
U test).
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Phosphorylation state-specific antibodies were used to compare the
levels of phospho-Ser845 and
phospho-Ser831 GluR1 in neostriatal
slices with known quantities of GluR1 fusion proteins phosphorylated by
PKA or CaMKII, respectively. A low, basal stoichiometry of
phosphorylation at Ser845 was measured
(~0.05 mol/mol) in slices, which was increased to ~0.33 mol/mol
after 5 min of forskolin treatment (50 µM).
Ser831 was also phosphorylated at a low
level in untreated slices (a stoichiometry of 0.06 mol/mol). The
stoichiometry of phosphorylation at Ser831
was increased to ~0.26 mol/mol after 5 min of PDBu treatment (5 µM). These data indicate that GluR1 is phosphorylated in
intact neostriatal cells at Ser845 or
Ser831, after activation of either PKA or
PKC/CaMKII, respectively.
Phosphorylation of GluR1 in response to dopamine
receptor stimulation
Dopamine receptors are subdivided into a D1 class (i.e., D1, D5
subtype receptors) and a D2 class (i.e., D2, D3, and D4 subtype receptors) with the D1 and D2 subtypes being the most highly enriched in mammalian neostriatum (Sibley and Monsma, 1992). D1 class receptors stimulate adenylyl cyclase and increase cAMP formation (Stoof and
Kebabian, 1981). D2 class receptors, via interactions with specific G-proteins, can be coupled to multiple effector systems, including adenylyl cyclase, Ca2+ and
K+ channels, and phospholipase C (Huff,
1996). To evaluate the potential contribution of D1-subtype and
D2-subtype dopamine receptors to the regulation of GluR1
phosphorylation, we measured the effects of dopamine and selective D1
and D2 agonists and antagonists on phosphorylation at
Ser845 and
Ser831.
Dopamine increased the phosphorylation of GluR1 at
Ser845 (428 ± 68% compared with
control) (Fig. 2A).
Levels of phosphorylation were maximal at between 2.5 and 5 min, and
declined to basal values by 10 min of incubation with dopamine (data
not shown). The effect of dopamine was blocked by pretreatment of
slices with a D1-type receptor antagonist, SCH23390 (1 µM) (127 ± 21% compared with control). In contrast, the D2-type receptor antagonist sulpiride (1 µM) had no effect on dopamine-stimulated
phosphorylation of GluR1 (565 ± 81% compared with control)
or on D1 agonist-induced phosphorylation of GluR1 (data not shown).
Neither the D1 nor the D2 antagonist alone had a significant effect on
the basal level of GluR1 phosphorylation (data not shown) indicating
that any endogenously released dopamine was not exerting a significant regulation of this site. The phosphorylation of
Ser845 was increased by treatment of
slices with SKF81297, a D1 agonist (337 ± 82% of control;
Fig. 2A), whereas quinpirole, a D2 agonist, had no
effect on basal levels (91 ± 12% of control; Fig.
2A). Quinpirole did not reduce phosphorylation of
Ser845 induced by a D1 agonist (data not
shown). These data indicate that dopamine-induced phosphorylation of
GluR1 is most likely mediated by activation of a
D1-receptor/PKA-dependent signaling pathway.
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Figure 2.
Effect of activation of D1-type or D2-type
dopamine receptors on neostriatal GluR1 phosphorylation. Neostriatal
slices were prepared from normal C57BL/6 mice. A, At
time 0 slices were incubated in the absence or presence of the D1
receptor antagonist SCH23390 (1 µM; D1 Antag) or the D2
receptor antagonist sulpiride (1 µM; D2 Antag), as
indicated. At 10 min slices were incubated in normal buffer alone
(Control), with dopamine (DA) (100 µM) plus the dopamine uptake inhibitor nomifensine (10 µM), SKF81297 (1 µM) (D1) or quinpirole (1 µM) (D2), with or without SCH23390 or sulpiride for an
additional 5 min. B, Slices were incubated with either
normal buffer (Control) or with SKF81297 (1 µM) or quinpirole (1 µM) for 5 min. The
solid arrows indicate the position of
phospho-Ser845 GluR1 (A) or
phospho-Ser831 GluR1 (B). The
open arrows indicate cross-reactive bands detected by
the phosphorylation state-specific antibody. The intensity of these
bands did not change as a function of dopamine agonist or antagonist
treatment. Phospho-Ser845 GluR1
(A) or phospho-Ser831 GluR1
(B) was detected in the samples, quantitated by
densitometry, and expressed as percentage of the level in control
slices. Data are presented as means ± SEM for three
experiments (*p < 0.05 compared with control,
Mann-Whitney U test).
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D2 agonists have recently been shown to increase intracellular
Ca2+ levels in dissociated neostriatal
cells (Hernandez et al., 1999), an effect that could result in
activation of Ca2+-dependent kinases such
as CaMKII. Thus, we evaluated the effects of D1 and D2 receptor
activation on Ser831 phosphorylation. In
contrast to the robust increase in phosphorylation of
Ser845 observed in response to dopamine or
a D1-selective agonist (Fig. 2A), neither dopamine,
SKF81297 (106 ± 11% of control), quinpirole (82 ± 12% of control), nor SKF81297 plus quinpirole (107 ± 15% of control) had a significant effect on
Ser831 phosphorylation (Fig.
2B).
Regulation of Ser845 phosphorylation:
involvement of a DARPP-32/PP1 cascade
The ability of serine/threonine protein phosphatases to regulate
the basal phosphorylation of GluR1 in neostriatal cells was examined.
Neostriatal slices were incubated with either calyculin A (500 nM) or okadaic acid (1 µM), inhibitors of
both PP1 and protein phosphatase 2A (PP2A). Calyculin A increased
phospho-Ser845 GluR1 to a level 600 ± 129% of control after 60 min of incubation (Fig.
3A). Similarly, okadaic acid
increased phospho-Ser845 GluR1 to a
maximal level of 783 ± 102% of control after 60 min. By
comparison, cyclosporin A, an inhibitor of calcineurin (protein phosphatase 2B, PP2B), had no significant effect on the basal level of
phosphorylated Ser845 GluR1 after a 60 min
incubation period (104 ± 4% compared with control; Fig.
3A) or on the state of phosphorylation of DARPP-32 at
Thr34 (data not shown). Phosphorylation of GluR1 at
Ser831 was slightly increased by calyculin
A (256 ± 78% of control) in contrast to the absence of
effect of okadaic acid (137 ± 36% of control) or
cyclosporin A (104 ± 25% of control) (Fig. 3B). However, the effect of calyculin A was not statistically significant (p > 0.05; Mann-Whitney U test, for
three experiments). These data indicate that
Ser845 is a better substrate than
Ser831 for regulation by PP1 and/or PP2A
in neostriatal neurons.
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Figure 3.
Effects of protein phosphatase inhibitors on the
phosphorylation state of neostriatal GluR1 at Ser845
and Ser831. Mouse neostriatal slices were incubated
for 60 min in the absence (Control) or presence
of either okadaic acid (1 µM) (OKA) or
calyculin A (0.5 µM) (Cal A), inhibitors
of PP1/PP2A, or cyclosporin A (5 µM) (Cyc
A), an inhibitor of PP2B. Ser845
phospho-GluR1 (A) and Ser831
phospho-GluR1 (B) were detected, quantitated by
densitometry, and expressed as percentage of the level in control
slices. Data are expressed as means ± SEM for three
experiments (*p < 0.05 compared with control,
Mann-Whitney U test).
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To investigate the specific contribution of PP1 to dopamine-induced
phosphorylation of GluR1, we compared
Ser845 phosphorylation in wild-type mice
and in mice bearing a targeted deletion of the gene for the selective
PP1 inhibitor DARPP-32 (Fienberg et al., 1998). These experiments
revealed that there was no significant difference in the basal
phosphorylation of Ser845 as the result of
the DARPP-32 knock-out. Dopamine (100 µM) induced a
threefold to fourfold increase in phosphorylation of GluR1 at Ser845 in neostriatal slices from
wild-type mice (324 ± 85% of control at 2.5 min and 383 ± 26% of control at 5 min), but not from DARPP-32 knock-out mice (94 ± 41% of control at 2.5 min and 174 ± 72% of control at 5 min; Fig.
4A). A similar
attenuation of dopamine-induced phosphorylation was observed in nucleus
accumbens slices (data not shown). These data indicate that inhibition
of PP1 is required for dopamine-induced increases in GluR1
phosphorylation. In support of this conclusion, we found that direct,
pharmacological inhibition of PP1/PP2A affected GluR1 phosphorylation
comparably in wild-type and knock-out slices. In this series of
experiments okadaic acid increased Ser845
phosphorylation in slices from both wild-type and DARPP-32 knock-out mice (878 ± 210% of control in wild-type mice and 1008 ± 50% of control in knock-out mice) after 60 min of
incubation (Fig. 4B).
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Figure 4.
Role of DARPP-32 in the dopamine-induced
phosphorylation of neostriatal GluR1 on Ser845.
A, Mouse neostriatal slices prepared from wild-type mice
(left) or DARPP-32 knock-out mice (right)
were incubated in the absence or presence of dopamine (100 µM) plus nomifensine (10 µM) for the
indicated times. Samples were immunoblotted for
Ser845-phosphorylated GluR1. The level of
phospho-GluR1 was quantitated by densitometry, and the data were
expressed as percentage of either the wild-type or knock-out control
level. The results are expressed as means ± SEM for
five experiments (*p < 0.05 compared with 0 time,
Mann-Whitney U test). B, Slices from
wild-type or knock-out mice were incubated with a maximally effective
concentration of the PP1/2A inhibitor, okadaic acid
(OKA; 1 µM) for 60 min. The level of
phospho-Ser845 GluR1 was quantitated by
densitometry, and the data were expressed as percentage of the
wild-type control level. Results are expressed as means
± SEM for three experiments (*p < 0.05 compared with control, Mann-Whitney U test).
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Regulation of GluR1 phosphorylation by cocaine
in vivo
To evaluate the possible involvement of psychostimulants in the
control of AMPA receptor phosphorylation in vivo, intact
mice were treated with drugs that are known to enhance dopamine
availability in the neostriatum, and the effect on the phosphorylation
of GluR1 was examined. We first measured the effect of cocaine (20 mg/kg, i.p.), a drug that increases dopamine availability through
blockade of the dopamine transporter, on the phosphorylation of GluR1
at Ser845. Cocaine administration produced
a behavioral activation in mice characterized by increased locomotor
behavior that was evident within 15 min of injection. Cocaine also
caused a large increase in the phosphorylation state of GluR1 at
Ser845 in neostriatum, as observed 15 min
(saline, 100 ± 18%; cocaine, 612 ± 198%) and
30 min (saline, 100 ± 22%; cocaine, 604 ± 170%) after injection (Fig. 5). The
effect of cocaine treatment on GluR1 phosphorylation rapidly
dissipated, as Ser845 phosphorylation
returned to the basal level within 60 min (saline, 100 ± 4%; cocaine, 89 ± 25%) of drug administration (Fig. 5). In contrast, cocaine treatment did not significantly affect the level
of phosphorylation of GluR1 at Ser831 in
neostriatum compared with time-matched saline-injected controls at any
time point examined (89 ± 30% of control at 15 min; 80 ± 18% of control at 30 min; and 98 ± 8% of
control at 60 min) (Fig. 5). These data indicate that acute cocaine
injection selectively regulates phosphorylation of GluR1 at
Ser845.
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Figure 5.
Effect of acute cocaine on neostriatal GluR1
phosphorylation at Ser845 and
Ser831 in vivo. Normal C57Bl/6 mice
were injected (intraperitoneally) with vehicle alone
(Saline) or with vehicle containing cocaine (20 mg/kg)
and killed by focused microwave irradiation at 15, 30, or 60 min after
injection. Neostriatum was dissected from each brain and analyzed for
phospho-Ser845 and
phospho-Ser831 GluR1. The arrows
indicate the position of GluR1, as verified by immunoblotting with an
antibody against a C-terminal sequence of GluR1. A,
Representative experiment showing immunoblots of
phospho-Ser845 GluR1 from three individual animals
for each treatment condition. B, Quantitation of
phospho-Ser845 and phospho-Ser831
in three experiments, each analyzed in triplicate
(*p < 0.05, compared with saline; Mann-Whitney
U test).
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Role of DARPP-32 in cocaine-mediated regulation of
Ser845 phosphorylation in vivo
It seemed possible that cocaine-stimulated dopamine release could
increase GluR1 phosphorylation by at least two mechanisms. First,
dopamine and D1-receptor-mediated activation of PKA would be expected
to lead directly to phosphorylation of Ser845.
Second, PKA-mediated phosphorylation of DARPP-32 at
Thr34 would be expected to inhibit PP1
activity, leading to an increase in phosphorylation state of PP1
substrates, including GluR1. Consistent with this idea, cocaine did
induce a severalfold increase in
phospho-Thr34 DARPP-32 (605 ± 205% of control) (Fig.
6A), concurrent with
the increase in Ser845 phosphorylation in
wild-type mice. A possible role for DARPP-32 in the in vivo
phosphorylation of GluR1 was further evaluated by measuring
cocaine-induced GluR1 phosphorylation in DARPP-32 knock-out mice. The
level of GluR1 expressed in neostriatum was unaffected by the genetic
deletion of DARPP-32 (data not shown). However, the increase in GluR1
phosphorylation at Ser845 in neostriatum
observed after treatment with a low dose of cocaine (10 mg/kg, i.p.)
(671 ± 75% of control in this series of experiments) was
greatly attenuated in mice lacking DARPP-32 (138 ± 18% of control; Fig. 6A).
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Figure 6.
Effect of acute cocaine on in vivo
phosphorylation of GluR1 at Ser845: comparison of
wild-type and DARPP-32 knock-out mice. Wild-type or DARPP-32 knock-out
mice were injected (intraperitoneally) with saline vehicle
(Sal) or with (10 mg/kg)
(A) or (20 mg/kg) (B)
cocaine (Coc) and killed by focused microwave
irradiation 30 min later. The levels of
phospho-Ser845 GluR1 and
phospho-Thr34 DARPP-32 were detected in neostriatum
by immunoblotting, and the data were quantitated by densitometry.
Arrows indicate the position of phospho-GluR1 and
phospho-DARPP-32 bands. A, Top, An autoradiogram of a
representative experiment shows the level of phospho-GluR1 in
saline-injected or cocaine-injected wild-type and DARPP-32 knock-out
mice. The bar graph summarizes phospho-Ser845 GluR1
levels in the neostriatum of the wild-type and knock-out mice. Data are
expressed as means ± SEM for eight mice per group (*p < 0.01 compared with saline-injected mice; Student's t test).
A, Bottom, A typical autoradiogram shows the level of
phospho-DARPP-32 in a saline-injected and a cocaine-injected wild-type
mouse neostriatum. The bar graph shows phospho-Thr34
DARPP-32 levels in the neostriata of mice. Data are expressed as means
± SEM for four mice per group (*p < 0.05 compared with saline-injected mice; Mann-Whitney
U test). B, Data are expressed as means
± SEM for 3-5 mice per group (*p < 0.05 compared with respective saline-injected controls;
Mann-Whitney U test).
|
|
A previous report from this laboratory (Fienberg et al., 1998) showed
that the effect of DARPP-32 knock-out on cocaine-induced locomotor
behavior was dose-dependent. Thus, the increase in locomotor behavior
elicited by a low (10 mg/kg), but not by a high (20 mg/kg) dose of
cocaine was blocked in DARPP-32 knock-out mice. We examined the dose
dependency of the effects of cocaine on GluR1 phosphorylation. A 20 mg/kg concentration of cocaine induced an approximately sevenfold increase in Ser845 phosphorylation (709 ± 198% of control; Fig. 6B) in wild-type mice. This increase was not significantly reduced in DARPP-32 knock-out
mice (479 ± 165% of control; Fig.
6B).
Regulation of GluR1 phosphorylation by methamphetamine
in vivo
Methamphetamine-HCl, a substituted analog of
D-amphetamine with profound abuse potential, also increases
dopamine availability in the neostriatum. The effect of systemic
injection of methamphetamine on the in vivo phosphorylation
of GluR1 in mouse neostriatum was examined. Injection of mice with
methamphetamine (i.e., 20 or 30 mg/kg, s.c.) induced behavioral
activation, eliciting intense grooming behavior and hypersensitivity to
touch. Treatment of mice with methamphetamine (20 mg/kg, s.c.)
increased phospho-Ser845 levels by twofold
to threefold in neostriatum 30 min after drug injection (254 ± 32% of saline-injected wild-type) (Fig.
7B). In contrast,
methamphetamine treatment had no effect on the level of
phospho-Ser831 (93 ± 12% of
saline-injected control) (Fig. 7D). This dose of methamphetamine also increased the phosphorylation of DARPP-32 on
Thr34 by more than twofold in the
neostriatum of mice (270 ± 82% of control) (Fig.
7E). The increase in
phospho-Ser845 induced by this
concentration of methamphetamine was fully blocked in DARPP-32
knock-out mice (120 ± 28% of saline-injected wild type)
(Fig. 7B). A higher concentration of methamphetamine (30 mg/kg, s.c.) induced a fourfold to fivefold increase in
Ser845 phosphorylation in the neostriatum
of wild-type mice compared to vehicle-treated controls (486 ± 80% of saline-injected wild types) (Fig. 7C).
The higher concentration of methamphetamine induced a significant
increase in Ser845 phosphorylation even in
the DARPP-32 knock-out mice (325 ± 40% of saline-injected
knock-out mice) (Fig. 7C). The level of
Ser845 phosphorylation that was observed
in the knock-out mice, however, was significantly reduced compared to
wild-type mice (Fig. 7C).
View larger version (32K):
[in this window]
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|
Figure 7.
Effect of acute methamphetamine administration on
in vivo phosphorylation of GluR1 at
Ser845 and Ser831 in wild-type
and DARPP-32 knock-out mice. Wild-type (A-E) or
DARPP-32 knock-out (B, C) mice were injected with
vehicle or methamphetamine (Meth) 20 mg/kg
(subcutaneously) (B, D, E)
or 30 mg/kg (A, C) and killed by focused microwave
irradiation 30 min later. Neostriatal phospho-Ser845
GluR1 (A-C) and
phospho-Ser831 GluR1 (A, D) and
phospho-Thr34 DARPP-32 (E)
were detected by immunoblotting and quantitated by densitometry. Data
are expressed as percentage of values for saline-injected wild-type
mice and represent means ± SEM for nine (B,
C), three (D), or eight animals
(E) per group (*p < 0.05 compared with saline-injected mice;  p < 0.05, compared with wild-type Meth alone; Students' t
test).
|
|
| |
DISCUSSION |
Using neostriatal slices, we have demonstrated that dopamine
stimulates phosphorylation of GluR1 at
Ser845 through activation of D1-type
dopamine receptors and stimulation of adenylyl cyclase and PKA. We have
also demonstrated that psychostimulants rapidly and reversibly increase
the phosphorylation of GluR1 at Ser845 in
the neostriatum in vivo. Estimates of the stoichiometry of GluR1 phosphorylation indicate that a substantial proportion of the
GluR1 is phosphorylated at Ser845 in
response either to dopamine in slices or to cocaine and methamphetamine in vivo. These data indicate that regulation of the state of
phosphorylation of GluR1 represents a probable mechanism for control of
AMPA receptor currents by dopamine and psychostimulants. Results from a
previous study involving a Ser845-Ala
mutation of GluR1 indicated that phosphorylation of GluR1 at
Ser845 was required to potentiate peak
currents carried by homomeric GluR1 receptors (Roche et al., 1996). In
addition, PKA agonists reversed synaptic depression of AMPA currents in
hippocampal slices by increasing phosphorylation at
Ser845 (Kameyama et al., 1998; Lee et al.,
1998). Taken together, these data suggest that dopamine-induced,
PKA-mediated, phosphorylation of Ser845 in
slices and in vivo is likely to enhance glutamatergic
transmission through AMPA receptors.
AMPA receptor currents are also modulated by phosphorylation of GluR1
at Ser831 (Barria et al., 1997; Derkach et
al., 1999). This residue, which is phosphorylated in vitro
by PKC or CaMKII (McGlade-McCulloh et al., 1993; Blackstone et al.,
1994), is also phosphorylated to a low but measurable stoichiometry in
untreated neostriatum. However, the phosphorylation of this site was
not regulated in response to dopaminergic activity in slices or
in vivo with either wild-type mice (Fig.
2B) or in DARPP-32 knock-out mice (data not shown).
The data further suggest that dopamine and D1 agonists regulate AMPA
receptor currents through the selective phosphorylation of
Ser845. The proportion of GluR1 subunits
phosphorylated at Ser831 is greatly
increased by activation of PKC (Fig. 1B) or CaMKII signaling pathways (our unpublished observations), suggesting that both Ser845 and
Ser831 can serve to mediate
neurotransmitter-specific effects on AMPA receptors.
Dopamine-induced phosphorylation at Ser845
in slices and psychostimulant-induced phosphorylation of
Ser845 in vivo were reduced in the
neostriatum of DARPP-32 knock-out mice. These data are consistent with
the loss of D1-mediated enhancement of whole-cell AMPA currents in
neostriatal neurons from DARPP-32 knock-out mice (Yan et al., 1999).
Taken together, the results indicate that a pathway involving PKA and
DARPP-32/PP1 functionally regulates AMPA receptors in basal ganglia
neurons. It is unlikely that the deficits in psychostimulant action
seen in the absence of DARPP-32 are attributable to a reduced ability
to release dopamine. This conclusion is supported by a recent study in
which dopamine overflow in neostriatum was measured by amperometry in
response to stimulation of the medial forebrain bundle.
Stimulation-evoked dopamine release in vivo was found to be
equivalent in wild-type and DARPP-32 knock-out mice (F. Gonon, A. Fienberg, and P. Greengard, unpublished observations). In addition,
deletion of the DARPP-32 gene does not significantly affect either the
density of D1 receptors or their affinity for dopamine ligands (P. Svenningsson, B. Fredholm, A. Fienberg, and P. Greengard, unpublished
observations). Moreover, Ser845
phosphorylation is increased comparably in slices from wild-type and
DARPP-32 knock-out mice in response either to forskolin, which induces
PKA activity (G. Snyder, unpublished observations), or to drugs that
inhibit PP1/PP2A activity (Fig. 4B). Together, the results indicate that the loss of GluR1 regulation is caused by a loss
of ability to regulate PP1 activity.
The phosphorylation of Ser845 elicited by
either cocaine or methamphetamine is blocked in DARPP-32 knock-out mice
at low but not at high concentrations of these drugs. These data are
consistent with previous reports (Fienberg et al., 1998, Snyder et al.,
1998) showing that DARPP-32 knock-out blocks responses to low
(physiological) levels of activation of transmission, but not to high
(often supraphysiological) levels of activation. For example,
cocaine-induced locomotor activation in mice induced by a 10 mg/kg dose
of drug is fully blocked in DARPP-32 knock-out mice, whereas activation
induced by a 20 mg/kg cocaine injection is unaffected by DARPP-32
knock-out (Fienberg et al., 1998).
Taken together, we propose the following interpretation of these data:
that PKA increases phosphorylation of
Ser845 by at least two mechanisms: (1) by
directly phosphorylating Ser845, and (2)
by increasing phosphorylation of DARPP-32 at
Thr34, leading to inhibition of PP1
activity. Thus, our data suggest that high concentrations of
psychostimulants (i.e., 30 mg/kg methamphetamine or 20 mg/kg cocaine)
provide sufficient activation of PKA to sustain PKA-mediated
phosphorylation of GluR1 in the absence of PP1 inhibition. The results
obtained at the lower concentrations of psychostimulants support a role
for PKA-dependent phosphorylation of DARPP-32 at Thr34 as an obligatory component of
pathways that mediate a variety of biochemical, physiological, and
behavioral effects of dopamine in neostriatal neurons (Fienberg et al.,
1998; Greengard et al., 1999). However, a recent study (Bibb et al.,
1999) has demonstrated that DARPP-32 is phosphorylated at
Thr75 by a cyclin-dependent kinase family
member, cdk5, converting the protein into a PKA inhibitor. It will be
important in future studies to evaluate the possible contribution of
the regulation of Thr75 of DARPP-32
to the control of AMPA channels by dopamine and psychostimulants.
Psychostimulants are known to regulate various biochemical indices of
glutamate function including glutamate release, metabolism, and
receptor expression (Wolf, 1998). Most of these changes do not occur in
response to acute drug use, but appear only after repeated drug
presentation, indicating that they represent indirect, long-term
adaptive responses. The present results show that the regulation of
AMPA receptor current through phosphorylation is likely to be a rapid,
acute, and dramatic response to neostriatal dopamine release by
psychostimulant drugs. The enhancement of AMPA receptor currents by
these drugs represents an attractive mechanism by which glutamate
receptors and their signaling pathways might be recruited to mediate a
variety of effects, such as increased immediate early gene (IEG)
expression (Konradi et al., 1996), which may eventually lead to
sensitization. In support of this idea, AMPA or NMDA receptor
antagonists block amphetamine-induced IEG expression (Konradi et al.,
1996) and prevent the development of behavioral sensitization to
amphetamine (Karler et al., 1989; Wolf, 1998).
| |
FOOTNOTES |
Received Dec. 14, 1999; revised March 1, 2000; accepted April 7, 2000.
This work was supported by United States Public Health Service Grants
DA10044 and MH40899 (P.G.). We thank Peter Ingrassia and Stacey Galdi
for excellent technical assistance.
Correspondence should be addressed to Dr. Gretchen L. Snyder,
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, Box 296, 1230 York Avenue, New York, NY 10021. E-mail: snyderg{at}rockvax.rockefeller.edu.
| |
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[Abstract]
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C. L. Palmer, L. Cotton, and J. M. Henley
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E. C. Muly, P. Allen, M. Mazloom, Z. Aranbayeva, A. T. Greenfield, and P. Greengard
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T. Hills, P. J. Brockie, and A. V. Maricq
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H. K. Manji, I. I. Gottesman, and T. D. Gould
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L. Ding, D. J. Perkel, and M. A. Farries
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A. M. Vanhoose and D. G. Winder
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F. Miskevich, W. Lu, S.-Y. Lin, and M. Constantine-Paton
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R. R. Gainetdinov, A. R. Mohn, L. M. Bohn, and M. G. Caron
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D. C. Goff and J. T. Coyle
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J. Wang and P. O'Donnell
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TOP
ABSTRACT
INTRODUCTION
