Dop_JBC_Rev.doc July 31, 2004 Dopamine receptor-mediated ...
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Dop_JBC_Rev.doc
July 31, 2004
Dopamine receptor-mediated Ca2+ signaling in striatal medium spiny neurons
Tie-Shan Tang and Ilya Bezprozvanny*
Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390
Running title: Dopamine and Ca2+ signaling in MSN
Key words: calcium, inositol trisphosphate, dopamine, striatum, PKA, PP1, DARPP-32
*Corresponding author:
Dr. Ilya Bezprozvanny
Department of Physiology
UT Southwestern Medical Center at Dallas
Dallas, TX 75390-9040
tel: (214) 648-6737
fax: (214) 648-2974
E-mail: [email protected]
JBC Papers in Press. Published on August 2, 2004 as Manuscript M407389200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Abstract
Inositol (1,4,5)-trisphosphate (InsP3) and cyclic AMP (cAMP) are the two second
messengers that play an important role in neuronal signaling. Here we investigated the
interactions of InsP3 and cAMP-mediated signaling pathways activated by dopamine in
striatal medium spiny neurons (MSN). We found that in ~40% of MSN application of
dopamine elicited robust repetitive Ca2+ transients (oscillations). In pharmacological
experiments with specific agonists and antagonists we found that observed Ca2+
oscillations are triggered by activation of D1-class dopamine receptors. We further
demonstrated that activation of phospholipase C (PLC) is required for induction of
dopamine-induced Ca2+ oscillations, and that maintenance of dopamine-evoked Ca2+
oscillations requires both Ca2+ influx and Ca2+ mobilization from internal Ca2+ stores. In
“priming” experiments with a 5-HT2 receptor agonist we have shown a likely role for
calcyon in coupling D1-class dopamine receptors with Ca2+ oscillations in MSN. In
experiments with DAR-specific agonist SKF83959, we discovered that PLC activation
alone cannot account for dopamine-induced Ca2+ oscillations. We have further
demonstrated that direct activation of PKA by 8-Br-cAMP or inhibition of PP1 or
calcineurin (PP2B) phosphatases results in elevation of basal Ca2+ levels in MSN, but not
in Ca2+ oscillations. In experiments with competitive peptides we have shown an
importance of InsP3R1 association with PP1α and with AKAP9-PKA for dopamine-induced
Ca2+ oscillations. In experiments with MSN from DARPP-32 knockout mice we have
demonstrated a regulatory role of DARPP-32 in dopamine-induced Ca2+ oscillations. Our
results indicate that, following D1-class receptor activation, InsP3 and cAMP signaling
pathways converge on InsP3R1, resulting in Ca2+ oscillations in MSN.
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Dopamine is an important transmitter and neuromodulator in the brain. The cellular mechanisms
by which dopamine affects neuronal function are only beginning to be elucidated (1,2). Striatal
medium spiny neurons (MSN) express multiple subtypes of dopamine receptors (DARs) (3-5). On
the basis of their molecular structure and pharmacological properties, DARs are divided into D1-
class (D1R and D5R) and D2-class (D2R, D3R, and D4R) (6). D1-class DARs are coupled to
Gs/olf, activation of adenylyl cyclase and cAMP production (3). Activation of D2-class DARs has
dual effects of inhibiting cAMP production (3) and activating PLCβ (7). A putative D1-class DAR
subtype coupled to PLC activation and PIP2 hydrolysis, but not to cAMP production, has been
postulated (8-10), but has not yet been isolated or cloned. Recently, a specific agonist for this
receptor (SKF83959) was identified (11). A D1R-binding protein calcyon has been isolated by
yeast two-hybrid methods (12). Association of D1/D5 receptors with calcyon enables coupling of
D1/D5 receptors with Gq/11, resulting in PLC activation and InsP3 generation (12,13).
Cross talk between cAMP and Ca2+-signaling pathways plays an important role in
dopaminergic signaling in the neostriatum (1). Activation of D1-class DARs enhances L-type
Ca2+ channel activity (14-16) and currents via AMPA receptor (17) and NMDA receptor (18,19).
In contrast, activation of D2-class DARs reduces L-type Ca2+ currents (7) and NMDA receptor
activity (20). D1/5-mediated facilitation of L-type Ca2+ channels, AMPA and NMDA receptors
results from increased phosphorylation of these channels by protein kinase A (PKA) (16,21) and
decreased dephosphorylation of these channels by PP1 (17,22,23). Dopamine and cAMP-
regulated phosphoprotein of Mr 32,000 (DARPP-32) (24,25) is partly responsible for inhibition of
PP1 activity following activation of D1/5 receptors (26). DARPP-32 phosphorylated by PKA on a
single threonine residue (Thr-34) is transformed into a potent inhibitor of PP1, which in turn
regulates the phosphorylation state of many neurotransmitter receptors and voltage-gated ion
channels (1).
Type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) is a predominant InsP3R isoform in the
brain (27). The InsP3R1 plays an important role in neuronal Ca2+ signaling (28). Neuronal
InsP3R1 is one of the major substrates of protein kinase A (PKA) phosphorylation in the brain
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(29,30). PKA phosphorylates InsP3R1 at two sites, S1589 and S1755 (31-36). PKA
phosphorylation activates InsP3R1 by increasing the sensitivity of InsP3R1 to activation by InsP3
(35,37-39). In previous biochemical studies we discovered direct association of neuronal InsP3R1
with PP1α (39) and showed that InsP3R1-AKAP9-PKA complex is formed in the brain (40). In
experiments with striatal slices we demonstrated transient phosphorylation of striatal InsP3R1 by
PKA in response to dopamine and proposed that InsP3R1 may participate in a cross-talk between
cAMP and Ca2+ dopaminergic signaling pathways in the striatum (39). In the present study we
used Ca2+ imaging techniques to investigate dopamine-induced Ca2+ signals in primary cultures
of striatal MSN.
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EXPERIMENTAL PROCEDURES
DARPP32 knockout mice
Generation and breeding of DARPP32 knockout mice (C57BL/6) (kind gift of Dr Paul Greengard)
was previously described (26,41). The brains of E14.5-E15.5 embryos from wild type (D32(+/+))
and DARPP32 knockout (D32(-/-)) mice were collected, and the striata were dissected. The
striatal lysates of wild type and D32(-/-) embryos were analyzed by Western blotting with anti-
DARPP32 monoclonal antibodies (Cell Signaling Technologies).
Primary cultures of rat and mouse MSN
Primary cultures of rat MSN were established from E17-E18 rat embryos as previously described
(42). The mouse MSN cultures were established with some modifications of the protocol used for
rat MSN culture. Briefly, by using landmarks previously described (43), striata were dissected
from brains of D32 (+/+) and D32 (-/-) embryonic mice in ice-cold dissection solution (1× divalent-
free Hank's Balanced Salt Solution, 15 mM Hepes, 10 mM NaHCO3, and 100IU/ml
Penicillin/Streptomycin, pH 7.2). The striata from mice with identical genotypes were pooled and
treated with 0.25% trypsin for 7 minutes at 370C. After addition of 10% heat-inactivated fetal
bovine serum (Invitrogen) in DMEM (Invitrogen), the tissue was dissociated with trituration
solution (1× divalent-free Hank's Balanced Salt Solution, 1.0% DNase I, pH 7.2 (44)) by repetitive
pipetting. The cells were washed and plated at a density of 1 × 106 cells/ml on poly-D-lysine (MW
= 30,000–70,000 g/mol; 0.01% final concentration) pre-coated 12 mm round coverslips in plating
medium containing 60% DMEM, 30% Neurobasal media, 10% FBS, 100 units/ml penicillin-
streptomycin (Invitrogen) and maintained at 37°C, 5% CO2. 24 hr later, the cultures were
replaced by culture medium (containing 65% DMEM, 30% Neurobasal media, 1 × B27
(Invitrogen), 5% FBS, 100 units/ml penicillin-streptomycin (Invitrogen)). 4 µM of cytosine
arabinoside (AraC, Sigma) was added at 2–4 DIV to inhibit glial cell growth. The cultures were fed
with fresh culture medium every 7 days. The identity of established cultures was confirmed in
immunostaining experiments with GAD65 monoclonal antibodies (Chemicon Intl).
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Ca2+ imaging experiments
Ca2+ imaging experiments with 18-20 DIV rat and 16-18 DIV mouse MSN were performed as
previously described (42). Briefly, MSN neurons were loaded with 5 µM Fura 2-AM (Molecular
Probes) in artificial cerebrospinal fluid (ACSF) (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, 10 mM HEPES pH 7.3) for 45 min at 37°C. For imaging experiments the coverslips were
mounted onto a recording/perfusion chamber (RC-26G, Warner Instruments) maintained at 37°C
(PH1, Warner Instruments), positioned on the movable stage of an Olympus IX-70 inverted
microscope, and perfused with ACSF media by gravity flow. For some experiments (see text), the
culture was washed extensively with Ca2+-free ACSF (CaCl2 omitted from ACSF and
supplemented with 100 µM EGTA). Following Fura-2 loading, the MSN cells were intermittently
excited by 340 nm and 380 nm UV light (DeltaRAM illuminator, PTI) using a Fura-2 dichroic filter
cube (Chroma Technologies) and 60× UV-grade oil-immersed objective (Olympus). The emitted
light was collected by an IC-300 camera (PTI), and the images were digitized by ImageMaster
Pro software (PTI). Baseline (1-3 min) measurements were obtained prior to bath application of
dopamine dissolved in ACSF or Ca2+-free ACSF. Others drugs were applied to the recording
chamber as described in the text. The dopamine and drug solutions were prewarmed to 37°C
before application to MSN. Images at 340 and 380 nm excitation wavelengths were captured
every 5 s and 340/380 image ratio traces were recorded. Background fluorescence was
determined according to manufacturer's (PTI) recommendations and subtracted.
R9 peptide loading experiments
R9=RRRRRRRRR and R9-IC=RRRRRRRRRGHPPHMNVNPQQPA peptides were chemically
synthesized (UT Southwestern Protein Chemistry Technology Center), coupled to FITC at the
amino-terminus and dissolved in PBS. In loading experiments the R9-peptides were added to rat
MSN neurons for 10 min at 50 µM. Following R9-peptide loading, neurons were washed and
incubated in culture medium for ~2 hours prior to Ca2+ imaging experiments. As judged by FITC
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fluorescence, more that 90% of MSN were loaded with R9 and R9-IC peptides in these
experiments.
EGFP and EGFP-RT1-LIZ transfections
EGFP-RT1-LIZ construct was generated by subcloning PCR-amplified LIZ region of rat InsP3R1
(aa. 1251-1287) (40) into pEGFP-C3 expression vector (Clontech) and verified by sequencing.
pEGFP-C3 expression vector without an insert was used as a negative control (EGFP). The rat
MSN cultures at 19-20 DIV were transfected with EGFP plasmid or EGFP-RT1-LIZ plasmid by
the calcium-phosphate method as previously described (42,45) . 48 hr after transfection, the
MSN neurons were loaded with 5 µM Fura 2-AM and used in Ca2+ imaging experiments as
described above. Prior to Ca2+ imaging experiments EGFP and EGFP-RT1-LIZ transfected
MSN were identified by GFP imaging as previously described (42).
Drugs
SKF83959 was provided by the NIMH synthesis program (333 Ravenswood Ave., Menlo Park,
CA 94025, USA). Dopamine hydrobromide, Atropin sulfate, U73122, U73343, Thapsigargin, 8-Br-
cAMP, Calyculin A, Cyclosporin A, Okadaic acid were from Calbiochem (San Diego, California
92121, USA). (±)-SKF38393 hydrobromide [(±)-l-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-
7,8-diolhydrochloride], R(+)-SCH23390 hydrochloride, Spiperone hydrochloride, (+)-MK801
maleate, (-)-Quinpirole dihydrochloride, TTX (Tetrodotoxin), Ketanserin tartrate, Prazosin
hydrochloride, α-Methyl-5-hydroxytryptamine, CNQX, Nifedipine were purchased from Tocris
(Ellisville, MO 63021, USA).
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RESULTS
Dopamine induces Ca2+ oscillations in MSN
To test the effects of dopamine stimulation on Ca2+ signaling, we performed Ca2+ imaging
experiments with primary cultures of MSN established from E18 embryonic rats as previously
described (42,46). To monitor intracellular Ca2+ dynamics, MSN were loaded with the ratiometric
Ca2+ imaging dye Fura-2 and the intracellular free Ca2+ concentrations were estimated from the
ratio of Fura-2 emission at 340 nm and 380 nm excitation wavelengths (340/380 ratio). On
average, the basal levels of 340/380 ratios in unstimulated MSN were equal to 0.52 ± 0.05 (n =
29) (Table 1). Approximately 40% of cultured MSN (20 DIV) in our experiments displayed
repetitive Ca2+ transients (oscillations) in response to application of 400 µM dopamine (Figs 1A,
1B). On average (n = 29 MSN), these Ca2+ oscillations started 5.1 ± 2.2 min after dopamine
application, had an amplitude of 0.89 ± 0.1, occurred with the frequency of 12 ± 6 (spikes/20 min)
and lasted at least 20-30 min (Table 1).
D1-class receptors specifically mediate dopamine-evoked Ca2+ oscillations in MSN
Striatal medium spiny neurons abundantly express multiple subtypes of dopamine receptors
(DARs) (3-5,47). Based on pharmacological and molecular properties, DARs are divided into two
classes - D1-class and D2-class. Which DAR class mediates dopamine-induced Ca2+ oscillations
observed in our experiments (Fig 1)? To answer this question, we performed a series of
experiments with specific D1-class and D2-class antagonists and agonists. We found that the
dopamine-induced Ca2+ oscillations (Figures 1, 2A) in MSN were completely blocked in the
presence of 5 µM SCH23390 (D1-class DAR antagonist) and 5 µM Spiperone (D2-class DAR
antagonist) (Figure 2B). We further found that blockade of D2-class DARs by 5 µM Spiperone
had only a minor effect on dopamine-induced Ca2+ oscillations (Figure 2C), while blockade of D1-
class DARs by 5 µM SCH23390 resulted in almost complete suppression of dopamine-induced
Ca2+ oscillations (Figure 2D). In complementary experiments we found that the specific D1-class
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DAR agonist SKF38393 (50 µM) induced Ca2+ oscillations in MSN (Figure 2E), whereas the
specific D2-class DAR agonist Quinpirole (10 µM) was much less effective (Figure 2F).
Characteristically, application of Quinpirole resulted in an instant Ca2+ spike (Fig 2F), whereas
application of SKF38393 resulted in Ca2+ oscillations with a delay of several minutes (Fig 2E) as
observed after application of dopamine (Figs 1B, 2A). Taken together, these data suggested that
activation of either D1-class or D2-class DARs leads to Ca2+ responses in MSN, but that D1-class
DARs are coupled to Ca2+ oscillations more efficiently and via a different mechanism than the D2-
class DARs.
Classical D1-class DARs (D1R and D5R) are coupled to cAMP production and not to PLC
activation and InsP3-induced Ca2+ release. What is an explanation for Ca2+ signals observed in
our experiments (Figs 1 and 2)? One possibility is that 400 µM dopamine may facilitate action
potential firing in MSN cultures, which will lead to Ca2+ influx via voltage-gated L-type Ca2+
channels. To test this possibility, we performed experiments in the presence of 2 µM TTX, a
specific voltage-gated Na+ channel blocker which inhibits action potential firing in striatal neurons
(48,49). Pre-incubation of MSN with 2 µM TTX had no effect on dopamine-induced Ca2+
oscillations (Fig 3A), indicating that the observed responses are not due to action potential firing
in MSN cultures. Another possibility is that 400 µM dopamine can non-specifically activate other
PLC-linked neurotransmitter receptors expressed in MSN, such as 5-HT2, α1-Adrenoceptors and
muscarinic type Acetylcholine receptors (mAchR) (50-52). To determine if activation of any of
these receptors is involved in dopamine-induced Ca2+ oscillations, we repeated experiments in
the presence of 20 µM Ketanserin (5-HT2 receptor antagonist), 10 µM Prazosin (α1-
Adrenoceptors antagonist), and 5 µM Atropine (mAchR antagonist). To rule out activation of D2
receptors (also coupled to PLC), we also included 5 µM Spiperone (D2 dopamine receptor
antagonist). Preincubation of MSN with TTX-Spiperone-Ketanserin-Prazosin-Atropine mixture
had no significant effect on dopamine-induced Ca2+ oscillations (Fig 3B), confirming that
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dopamine-induced Ca2+ oscillations are mediated primarily by D1-class DARs and with a minor
contribution from D2-class DARs (Fig 2F).
In additional control experiments we studied Ca2+ signals in MSN induced by 5-HT2 receptor
activation. We found that application of 100 µM α-Methyl-5-hydroxytryptamine (5-HT2 receptor-
specific agonist) in the presence of 2 µM TTX induced one large Ca2+ spike, which was
occasionally followed by 1-2 spikes of much smaller amplitude (Fig 3C). These responses were
completely eliminated in the presence of 20 µM Ketanserin (5-HT2 receptor antagonist) (Fig 3D).
Thus, MSN Ca2+ signals mediated by 5-HT2 receptors differ from dopamine-induced responses,
because (1) Ca2+ transients in response to dopamine occurred with a lag time of 3-7 min, and the
first Ca2+ transient in response to α-Methyl-5-hydroxytryptamine occurred instantly (Fig 3C); (2)
multiple Ca2+ transients of equal amplitude (oscillations) were observed in response to dopamine,
but only one large transient and 1-2 much smaller transients were observed in response to α-
Methyl-5-hydroxytryptamine (Fig 3C); (3) dopamine-induced Ca2+ transients were not sensitive to
Ketanserin (Fig 3B), but α-Methyl-5-hydroxytryptamine-induced Ca2+ transients were completely
blocked by Ketanserin (Fig 3D). These results indicated that dopamine-induced Ca2+ oscillations
in our experiments are mediated predominantly by D1-class DARs but not by 5-HT2 receptors.
Ca2+ influx is required for maintenance of dopamine-induced Ca2+ oscillations in MSN
The dopamine-induced increase of intracellular free Ca2+ concentration observed in our
experiments (Figs 1-3) could result from Ca2+ release from internal Ca2+ stores and/or
extracellular Ca2+ influx. To test the role of extracellular Ca2+ influx in dopamine-evoked Ca2+
oscillations, we shifted the MSN from 2 mM Ca2+ in the extracellular medium to Ca2+-free
extracellular medium during dopamine-induced oscillations. We found that the dopamine-induced
Ca2+ oscillations quickly ceased in Ca2+-free medium, but restarted again following return to the
extracellular medium containing 2 mM Ca2+ (Fig 4A). These experiments demonstrated that
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extracellular Ca2+ influx is required for the maintenance of dopamine-induced Ca2+ oscillations in
MSN.
What was the source of extracellular Ca2+ influx in these experiments? Activation of D1-class
DARs enhances L-type Ca2+ currents (14-16), and activates AMPA receptors (17) and NMDA
receptors (16,18,19,53). To evaluate a possible role of these channels in dopamine-induced Ca2+
oscillations, we used a combination of specific blockers of L-type Ca2+ channels (10 µM
nifedipine), AMPA receptors (20 µM CNQX), and NMDA receptors (10 µM (+)-MK801). We found
that dopamine-induced Ca2+ oscillation quickly ceased after application of a (+)-
MK801/CNQX/Nifedipine mixture (Fig 4B). When MSN were preincubated with a (+)-
MK801/CNQX/Nifedipine mixture for 10 min, application of dopamine resulted in greatly
attenuated Ca2+ oscillations (Fig 4C). Pre-incubation with (+)-MK801/CNQX, CNQX/Nifedipine,
or (+)-MK801/Nifedipine mixtures had a partial inhibitory effect on dopamine-induced Ca2+
oscillations (Figs 4D-4F). From these experiments we concluded that L-type Ca2+ channels,
AMPA and NMDA receptors contribute jointly to maintenance of dopamine-induced Ca2+
oscillations in MSN, but are not required for initiation of these oscillations (Fig 4C).
PLC activation and intracellular Ca2+ mobilization are required for the initiation and maintenance
of dopamine-induced Ca2+ oscillations in MSN
The experiments described in the previous section (Fig 4) indicated that Ca2+ influx via L-type
Ca2+ channels, AMPA and NMDA receptors is necessary for maintenance of dopamine-induced
Ca2+ oscillations, but not required for their initiation. Thus, in the next series of experiments we
focused on mobilization of Ca2+ from intracellular Ca2+ stores. Activation of phospholipase C
(PLC) leads to hydrolysis of PIP2 and generation of DAG and InsP3. InsP3 activates InsP3R1 and
releases Ca2+ from intracellular Ca2+ stores. In MSN, activation of PLC-coupled class I mGluR
receptors efficiently evokes intracellular Ca2+ mobilization (42,54). To test if activation of PLC is
involved in dopamine-induced Ca2+ oscillations in MSN, we performed experiments with U73122,
a selective PLC inhibitor. We found that preincubation with 10 µM of U73122 for 10 min resulted
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in complete block of dopamine-induced Ca2+ oscillations (Fig 5A). In contrast, preincubation with
the same concentration of inactive analog U73343 had no effect on dopamine-induced Ca2+
oscillations (Fig 5B). These experiments demonstrated an essential role of PLC for initiation of
dopamine-induced Ca2+ oscillations in MSN. To test the importance of Ca2+ mobilization from
intracellular Ca2+ stores, we performed experiments with the specific SERCA Ca2+ pump inhibitor
thapsigargin. We found that acute application of 10 µM Thapsigargin quickly stopped dopamine-
induced Ca2+ oscillations (Fig 5C). Preincubation of MSN with 10 µM Thapsigargin for 10 min
resulted in almost complete block of dopamine-induced Ca2+ oscillations (Fig 5D). The
experiments described in this section (Fig 5) lead us to conclude that activation of PLC and
InsP3R1-mediated Ca2+ mobilization from intracellular Ca2+ stores play a critical role in initiation
and maintenance of dopamine-induced Ca2+ oscillations.
A role of the putative PLC-linked D1-class DAR in dopamine-induced Ca2+ oscillations
Classical D1-class DARs (D1/5) are coupled to cAMP production and not to PLC activation.
What is an explanation for a critical role of PLC (Fig 5) in D1-class mediated Ca2+ oscillations? In
control experiments we ruled out involvement of other PLC-coupled receptors expressed in MSN,
such as 5-HT2, α1-Adrenoceptors and mAchR receptors (Fig 3). Recent data suggested the
existence of a distinct D1-class DAR subtype that is coupled to PLC activation (8-10). This
receptor has not been purified or cloned, but recently a benzazepine compound SKF83959 has
been identified as a specific agonist for this putative PLC-linked D1-class DAR subtype (11). To
test a potential role of this putative D1-class subtype in observed Ca2+ signals, we performed
experiments with SKF83959. We found that application of 400 µM SKF83959 induced a single
instant Ca2+ transient in MSN (Fig 6A). A similar response to 400 µM SKF83959 was observed in
Ca2+-free media (Fig 6B). The response to 400 µM SKF83959 was eliminated in the presence of
the PLC inhibitor U73122 (Figure 6C). These results confirmed the existence of a PLC-coupled
DAR activated by SKF83959 in MSN. However, application of SKF83959 never resulted in Ca2+
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oscillations, indicating that activation of these receptors is not sufficient to support dopamine-
induced Ca2+ oscillations in MSN.
A role of calcyon in dopamine-induced Ca2+ oscillations
D1R and D5R are coupled to heterotrimeric G protein α subunit Gs/olf, and this activation
results in activation of adenylyl cyclase and cAMP production. Recently a D1R-binding protein
calcyon has been identified by yeast two hybrid screen (12,13). Calcyon is a single-pass
transmembrane protein of 24 kD that binds to the carboxy-terminal tail of D1R. Co-expression of
D1R or D5R with calcyon in a heterologous system enables coupling of these receptors to the
Gq/11 subunit, resulting in activation of PLCβ, generation of InsP3 and Ca2+ release (12). In
neurons, association of D1-class receptors with calcyon is promoted by “priming” resulting from
activation of other types of Gq/11-coupled receptors (12,55). To evaluate a possible role of
calcyon in the dopamine-induced Ca2+ transients observed in our experiments, we “primed” MSN
with 50 µM of α-Methyl-5-hydroxytryptamine (5-HT2 receptor-specific agonist) in the presence of
2 µM TTX. As described above (Fig 3C), application of α-Methyl-5-hydroxytryptamine to MSN
resulted in an instant single Ca2+ spike, consistent with transient activation of Gq/11 proteins and
PLC (Fig 7A). Application of 100 µM dopamine to “naïve” MSN was not sufficient to result in any
Ca2+ responses (Fig 7B). In contrast, application of the same concentration of dopamine to MSN
“primed” with 50 µM α-Methyl-5-hydroxytryptamine resulted in an instant single Ca2+ spike
followed by repetitive Ca2+ transients (oscillations) after 3-7 min delay (Fig 7C). Frequency and
amplitude of the observed Ca2+ oscillations were similar to those of oscillations induced by 400
µM of dopamine in experiments with naïve (non-primed) MSN (Fig 1). These experiments
indicated that “priming” of MSN with 50 µM α-Methyl-5-hydroxytryptamine increased the potency
of dopamine to induce Ca2+ oscillations, consistent with an involvement of calcyon (12,13).
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A role of PKA phosphorylation in dopamine-induced Ca2+ oscillations in MSN
Experiments described above (Figs 5 and 6) indicated that activation of PLC is necessary
but not sufficient to result in Ca2+ oscillations in MSN. To explain this result we reasoned that
increases in both cAMP and InsP3 might be required to support dopamine-induced Ca2+
oscillations. To test this hypothesis, we investigated the effects of a protein kinase A activator (8-
Br-cAMP) and of protein phosphatase inhibitors on Ca2+ signals in MSN. We found that
application of 500 µM 8-Br-cAMP to MSN resulted in elevation of basal Ca2+ levels in MSN (Fig
8A), most likely due to PKA-induced phosphorylation and activation of InsP3R1 (39). Similar
effects were observed in response to application of 10 nM Calyculin A (PP1/PP2A inhibitor) (Fig
8B) or 10 nM Cyclosporin A (PP2B inhibitor) (Fig 8C). In contrast, application of 1 nM Okadaic
acid, a specific PP2A inhibitor at this concentration, had less pronounced and more delayed
effect on basal Ca2+ levels in MSN (Fig 8D). The relative potencies of 8-Br-cAMP, Calyculin A,
Cyclosporin A, and Okadaic acid in these experiments are well correlated with the relative
potency of these drugs to shift striatal InsP3R1 into the PKA-phosphorylated state that was
compared in our previous experiments (39).
Persistent activation of PKA or block of PP1 or PP2B phosphatases results in Ca2+
elevation, most likely due to hyperphosphorylation and activation of InsP3R1 (39). Is it possible
to generate Ca2+ oscillations in MSN by activating both the PLC and PKA pathways? The answer
this question, we applied a mixture of 400 µM SKF83959 and 500 µM 8-Br-cAMP to MSN. We
found (Fig 8E) that application of SKF83959/8-Br-cAMP mixture resulted in an initial small Ca2+
transient (similar to SKF83959 application, Fig 6A), followed by persistent elevation in Ca2+ level
(similar to 8-Br-cAMP application, Fig 8A). Thus, SKF83959 and 8-Br-cAMP appear to affect
Ca2+ levels in an independent manner, and not lead to Ca2+ oscillations as observed in response
to dopamine. We further explored the connection between InsP3 and cAMP signaling pathways
by examining the effects of Calyculin A, Cyclosporin A and OA on dopamine-induced Ca2+
oscillations. We found that preincubation with Calyculin A suppressed dopamine-induced Ca2+
oscillations, presumably due to the rise in basal Ca2+ level (Fig 8F). Preincubation with
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Cyclosporin A initially increased frequency of Ca2+ oscillations and eventually suppressed
oscillations, presumably due to the rise in basal Ca2+ level (Fig 8G). In contrast to Calyculin A
and Cyclosporine A, OA did not have a significant effect on dopamine-induced Ca2+ oscillations
(Fig 8H). From these results we concluded that continuous hyper-phosphorylation of InsP3R1 by
PKA disrupts the in vivo regulatory mechanism required for dopamine-induced Ca2+ oscillations in
MSN.
A role of InsP3R1-PP1α association in dopamine-induced Ca2+ oscillations
In a previous paper we described a direct association between InsP3R1 carboxy-terminus and
PP1α (39). Is formation of an InsP3R1-PP1α complex physiologically relevant? To address this
question, we introduced the IC peptide (amino acids 2736-2749 of InsP3R1), which corresponds
to a minimal PP1α-binding site in the InsP3R1 sequence (39), into MSN. We reasoned that IC
peptide will displace PP1α from the complex with InsP3R1 and therefore may have a dominant
negative effect on the ability of PP1α to dephosphorylate InsP3R1. In order to introduce IC
peptide into MSN, we took advantage of recently developed protein delivery technology (PTD)
(56,57). In our experiments we used R9 signal (58) to deliver FITC-labeled R9-IC carboxy-
terminal peptide and R9 control peptide into cultured MSN (see Methods for details). Two hours
following FITC-R9 peptide loading, MSN neurons were washed, loaded with Fura-2 and used in
Ca2+ imaging experiments. Using FITC fluorescence, we estimated that >90% of MSN were
loaded with R9 and R9-IC peptides in our experiments.
Application of 400 µM dopamine induced Ca2+ oscillations in both R9 (Fig 9A) and R9-IC (Fig
9B) loaded MSN. However, Ca2+ oscillations were more frequent and had increased amplitude
in R9-IC loaded neurons (Fig 9B). Statistical analysis revealed that the average basal Ca2+
levels were similar (p < 0.05) in control, R9-loaded and R9-IC-loaded MSN (Table 1). The
average latency from dopamine application to the first Ca2+ spike was shorter in R9-IC loaded
neurons than in control or R9-loaded neurons (Table 1), but the difference did not reach statistical
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significance (p > 0.05, unpaired t-test). The average amplitude of Ca2+ transients was equal to
0.89 ± 0.1 (n = 29) in control MSN, 0.86 ± 0.09 (n = 18) in R9-loaded MSN, and 1.1 ± 0.15 (n =
29) in R9-IC-loaded MSN (Table 1). The average frequency of Ca2+ transients was equal to 12 ±
6 spikes/20 min (n = 29) in control MSN, 11 ± 6 spikes/20 min (n = 18) in R9-loaded MSN, and 20
± 8 spikes/20 min (n = 29) in R9-IC-loaded MSN (Table 1). From these results we determined
that loading of MSN with R9-IC peptide results in statistically significant (p <0.05) increases in
average spike amplitude and spike frequency (Table 1). These effects appear to be specific for
IC sequence, as loading with control R9 peptide had no significant effects (p < 0.05) on the main
properties of dopamine-induced Ca2+ oscillations in MSN (Table 1). Thus, we conclude that
disruption of InsP3R1-PP1α association by IC peptide has a significant potentiating effect on
amplitude and frequency of dopamine-induced Ca2+ oscillations in MSN.
A role of InsP3R1-AKAP9-PKA association in dopamine-induced Ca2+ oscillations
PKA phosphorylation increases InsP3R1 sensitivity to InsP3 (35,37-39). In recent biochemical
experiments we demonstrated a formation of InsP3R1-AKAP9-PKA ternary complex in the brain
(40 #3739). We found that InsP3R1-AKAP9 association is mediated via leucin/isoleucine zipper
(LIZ) motif in the InsP3R1 coupling domain and the 4th LIZ motif in AKAP9 (40 #3739). We
further showed that InsP3R1-AKAP9 association is disrupted in the presence of recombinant LIZ
fragment of InsP3R1 (RT1-LIZ) (40 #3739). To evaluate the functional consequences of InsP3R1-
AKAP9-PKA association for dopamine-induced Ca2+ signaling, we transiently expressed EGFP-
tagged RT1-LIZ construct (EGFP-RT1-LIZ) in MSN. As a negative control, MSN cultures were
transfected by EGFP plasmid. MSN transfected by EGFP or EGFP-RT1-LIZ constructs were
identified by GFP imaging as we previously described (42).
We found that 400 µM dopamine induced Ca2+ oscillations in MSN transfected with either
EGFP (Fig 9C) or EGFP-RT1-LIZ (Fig 9D) constructs. However, when compared to EGFP-
transfected MSN, Ca2+ oscillations in EGFP-RT1-LIZ-transfected MSN started after a longer
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delay and occurred with a reduced frequency (Figs 9C, 9D). Statistical analysis revealed that
the average basal Ca2+ levels were similar (p > 0.05) in control (untransfected), EGFP-transfected
and EGFP-RT1-LIZ-transfected MSN (Table 1). The average latency from dopamine application
to the first Ca2+ spike was equal to 5.1 ± 2.2 min (n = 29) in control (untransfected) MSN, 4 ± 2.8
min (n = 11) in EGFP-transfected MSN, and 16.2 ± 2.1 min (n = 9) in EGFP-RT1-LIZ-transfected
MSN (Table 1). The amplitude of Ca2+ transients was lower in EGFP-RT1-LIZ-transfected
neurons than in control (untransfected) or EGFP-transfected neurons, but the difference did not
reach a level of statistical significance (p > 0.05, unpaired t-test) (Table 1). The average
frequency of Ca2+ transients was equal to 12 ± 6 spikes/20 min (n = 29) in control (untransfected)
MSN, 15 ± 4 spikes/20 min (n = 11) in EGFP-transfected MSN, and 7 ± 3 spikes/20 min (n = 9) in
EGFP-RT1-LIZ-transfected MSN (Table 1). Thus, expression of EGFP-RT1-LIZ in MSN resulted
in significant (p <0.05) longer latency duration and reduced spiking frequency (Table 1).
Expression of EGFP alone had no significant effects (p < 0.05) on the main properties of
dopamine-induced Ca2+ oscillations in MSN when compared to control (untransfected) cells
(Table 1), indicating the observed effects are specific for RT1-LIZ sequence.
A role of DARPP-32 in dopamine-induced Ca2+ oscillations
DARPP-32 (D32) (59) is a regulatory phosphoprotein that has been suggested to play a key
role in dopaminergic signaling in the striatum by regulating PP1 activity (1). In contrast to other
components of the dopaminergic signaling pathway, no pharmacological blockers of D32 function
are currently available. To evaluate the importance of D32 in dopamine-induced Ca2+ signals, we
took advantage of D32 knockout mice (26,41). Western blotting experiments with anti-D32
monoclonal antibodies confirmed the presence of the DARRP-32 protein in striatal lysates
prepared from the wild-type (D32 +/+) mouse pups but not from the knockout (D32 -/-) mouse
pups (Figs 10A, 10B).
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We established primary MSN cultures from the wild type (D32 +/+) and knockout (D32 -/-)
embryonic brains and analyzed dopamine-induced Ca2+ signals in 16-18 DIV MSN by Ca2+
imaging with Fura-2 indicator. Similar to rat MSN (Fig 1), application of 400 µM dopamine
induced repetitive Ca2+ transients (oscillations) in wild type mouse MSN (Fig 10C) and in D32 -/-
MSN (Fig 10D). When compared to wild type (D32 +/+) mouse MSN, oscillations in D32 -/-
mouse MSN had similar amplitude (Figs 10C and 10D). However, when compared to the wild
type MSN, oscillations in D32 -/- mouse MSN started with a longer delay after dopamine
application and had reduced frequency of oscillations (Figs 10C and 10D). Statistical analysis
revealed that the main parameters of dopamine-induced Ca2+ spikes were not significantly
different (p > 0.05) between wild type mouse MSN and rat MSN (Table 1). Furthermore, the
average basal Ca2+ levels and the average amplitude of Ca2+ transients were similar (p > 0.05) in
D32 +/+ and D32 -/- mouse MSN (Table 1). In contrast, the average latency from dopamine
application to the first Ca2+ spike was equal to 4.5 ± 2.1 min (n = 38) in wild type MSN and 6.1 ±
2.5 min (n = 154) in D32 -/- mouse MSN (Table 1). Also, the average frequency of Ca2+
transients was equal to 13 ± 6 spikes/20 min (n = 38) in wild type MSN and 9 ± 4 spikes/20 min (n
= 154) in D32 -/- mouse MSN (Table 1). Thus, we conclude that genetic ablation of DARPP-32
leads to a statistically significant (p<0.05) increase in the lag time between dopamine application
and the first Ca2+ spike and reduction in the frequency of dopamine-induced Ca2+ spikes in MSN,
consistent with a regulatory role played by DARPP-32 in MSN.
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DISCUSSION
Cross-talk between InsP3, Ca2+ and cAMP dopaminergic signaling pathways in MSN
Cross-talk between cAMP and Ca2+-signaling pathways plays an important role in
dopaminergic signaling in the neostriatum (1). In the present study, we investigated dopamine-
evoked Ca2+ signals in cultured striatal medium spiny neurons (MSN). Our results have
elucidated a connection between class 1 DAR receptor activation and cAMP/Ca2+ signaling in
MSN and provided insights into cellular mechanisms of dopamine function in striatum. The
data obtained in our paper are consistent with the model shown on Fig 11. We propose that
dopamine acts on D1/5 DARs leading to activation of adenylyl cyclase, production of cAMP and
activation of PKA (3). PKA phosphorylates DARPP-32 and Inhibitor-1 proteins, converting them
into potent inhibitors of PP1 (1). Activation of PKA and inhibition of PP1 leads to increased
phosphorylation and activation of L-type Ca2+ channels, NMDA receptors and AMPA receptors
(16,17,21-23) and InsP3R1 (39). We recently discovered an association of neuronal InsP3R1 with
AKAP9-PKA, which is mediated by a Leucine/Isoleucine Zipper (LIZ) motif in the InsP3R1
sequence (40 #3739). EGFP-RT1-LIZ transfection experiments (Fig 9C-9D) demonstrated that
the PKA associated with InsP3R1-AKAP9 plays a major role in dopamine-induced InsP3R1
phosphorylation in MSN. PKA phosphorylation of Ca2+ influx channels and InsP3R1 is necessary,
but it is not sufficient to result in Ca2+ oscillations. As experiments with PLC antagonist U73122
demonstrated (Fig 5), activation of PLC is also required. Previous studies demonstrated coupling
of D2-class DARs to InsP3 production and Ca2+ release in striatal neurons (7). However,
dopamine-induced Ca2+ oscillations in our experiments were mediated by D1-class (cAMP-
coupled), not by D2-class (PLC-coupled), DARs (Figs 2-3).
We propose two potential solutions to this apparent contradiction (see Fig 11). One
possibility is that dopamine acts on a putative PLC-linked D1-class DAR (9,10,60). Indeed,
SKF83959, a specific agonist for this putative PLC-linked D1-class DAR (11) induced Ca2+
release from intracellular stores in MSN (Fig 6). Another possibility is that some fraction of D1/5
receptors in MSN is associated with calcyon, which enables coupling of these receptors to Gq/11
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and PLC (12,13). In support of the calcyon hypothesis, “priming” of MSN with a 5-HT2 agonist
facilitated dopamine-induced Ca2+ oscillations in our experiments (Fig 7C). It has been previously
reported that “priming” is not effective in striatal neurons (55). One potential explanation for this
discrepancy is different age of MSN cultures - 18-20 DIV in our experiments and 4-10 DIV in the
experiments of Lezcano and Bergson (55). Indeed, in control experiments we found that
responsiveness of MSN to dopamine increases with maturity of culture (data not shown). It
remains to be determined if D1/D5 DARs complexed with calcyon (12,13) and a putative PLC-
linked D1-class DAR (8-11) are the same or distinct (as shown on Fig 11) molecular entities.
Simultaneous activation of cAMP and InsP3 signaling pathways leads to activation of InsP3R1
(see Fig 11). Interestingly, dopamine-induced Ca2+ oscillations start with a delay of ~5 min (Fig
1). In contrast, activation of mGluR1/5 receptors (42), D2 DAR (Fig 2F), 5-HT2 receptors (Fig
3C), and putative PLC-coupled D1-class DAR (Fig 6A) leads to an instant Ca2+ transient in MSN.
The delay between application of dopamine and initiation of Ca2+ oscillations is similar to the
delay between application of 8-Br-cAMP (PKA activator) and Calyculin A (PP1 inhibitor) and
changes in basal Ca2+ levels in MSN (Figs 8A, 8B). To explain these results, we reasoned that
phosphorylation of InsP3R1 by PKA is necessary for initiation of Ca2+ oscillations. PKA
phosphorylation activates InsP3R1 by increasing the sensitivity of InsP3R1 to activation by InsP3
(35,37-39), and apparently InsP3 levels in dopamine-stimulated MSN are sufficient for activation of
phosphorylated InsP3R1, but not for activation of unphosphorylated InsP3R1.
Following InsP3R1 activation and release of Ca2+ from intracellular stores cytosolic Ca2+
concentration rises. InsP3R1 are under feedback regulation by cytosolic Ca2+ - low Ca2+
concentrations (< 300 nM) activate InsP3R1 and higher Ca2+ concentrations (>300 nM) inhibit
InsP3R1 (61-63). Thus, release of Ca2+ is expected to proceed in a highly cooperative manner and
terminate quickly due to Ca2+-inactivation of InsP3R1. Increase in cytosolic Ca2+ will also lead to
activation of the Ca2+-dependent phosphatase calcineurin (PP2B) (Fig 11). Activated calcineurin
dephosphorylates DARPP-32 and Inhibitor-1, resulting in disinhibition of PP1 (1), which in turn
dephosphorylates and “turns off” the InsP3R1 (39) and Ca2+ influx channels (16,17,21-23). Our
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model predicts that InsP3R1 must be phosphorylated by PKA again for the next Ca2+ spike to
occur. Thus, in contrast to most models of Ca2+ oscillations which are centered on biphasic
modulation of InsP3R1 by Ca2+ (64), dopamine-induced Ca2+ oscillations in MSN rely on an intricate
interplay between Ca2+ and cAMP signaling pathways leading to repetitive rounds of InsP3R1
phosphorylation by PKA and dephosphorylation by PP1.
This hypothesis is supported by the effects of cyclosporin A (calcineurin inhibitor) on the
phosphorylated state of striatal InsP3R1 (39) and dopamine-induced Ca2+ oscillations in MSN (Fig
8G). The proposed role of DARPP-32 is consistent with the analysis of dopamine-induced Ca2+
oscillations in MSN from DARPP-32 knockout mouse (Fig 10). Increase in lag time and reduction in
frequency of Ca2+ oscillations (Figs 10C, 10D, Table 1) are expected in the absence of DARPP-32,
a condition that should favor PP1-mediated dephosphorylation of target proteins, including InsP3R1.
Relatively mild effects observed in MSN from DARPP-32 knockout mice was result from the
redundant functions of DARPP-32 and Inhibitor-1, both of which are expressed in striatum (65,66).
The experiments with R9-IC competitive peptide and EGFP-RT1-LIZ indicate that InsP3R1-
associated PP1α (39) and InsP3R1-AKAP9-associated PKA (40 #3739) play a major role in control
of InsP3R1 phosphorylated state (Fig 9). Consistent with impaired PP1 function, MSN loaded with
R9-IC peptide display increased amplitude and frequency of dopamine-induced Ca2+ oscillations
(Figs 9A, 9B, Table 1). In contrast, disruption of InsP3R1-AKAP9-PKA complex by overexpressed
EGFP-RT1-LIZ construct resulted in delayed Ca2+ oscillations and reduced spike frequency (Figs
9C, 9D, Table 1).
Previous studies suggested an important role of L-type Ca2+ channels, NMDA receptors and
AMPA receptors in cross-talk between cAMP and Ca2+ signaling in striatal neurons (16,17,21-23).
We also observed that maintenance of dopamine-induced Ca2+ oscillations requires Ca2+ influx
from the extracellular space (Fig 4A), and established a role of L-type Ca2+ channels, NMDA
receptors and AMPA receptors in mediating Ca2+ influx (Figs 4C-4F). If Ca2+ influx is blocked,
Ca2+ oscillations can be initiated but can not be maintained (Fig 4B, 4C). These results indicate
that the most likely role of Ca2+ influx via L-type Ca2+ channels, NMDA receptors and AMPA
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receptors is to prevent depletion of intracellular Ca2+ stores. Indeed, application of the SERCA
pump inhibitor Thapsigargin resulted in rapid cessation of dopamine-induced Ca2+ oscillations
(Fig 5C, 5D), indicating that the Ca2+ stores in MSN can be easily depleted. Similar conclusions
have been reached earlier for Ca2+ oscillations induced by application of gonadotropin-releasing
hormone to cultured gonadotrophs (67). Thus, it appears that for both MSN and gonadotrophs
Ca2+ oscillations are driven by InsP3-mediated Ca2+ release from ER, but Ca2+ influx via plasma
membrane Ca2+ channels is necessary for ER refilling in order to mainitain the oscillations.
In conclusion, by using a Ca2+ imaging technique we described dopamine-induced Ca2+
oscillatory responses in MSN. The dopamine-induced Ca2+ oscillations are mediated primarily by
D1-class DARs and require an intricate interplay between cAMP, InsP3 and Ca2+ signaling
pathways which converge on InsP3R1 regulation (Fig 11). Direct association of InsP3R1 with
PP1α (39) and formation of InsP3R1-AKAP9-PKA complex (40) are important for fidelity of cAMP,
InsP3 and Ca2+ cross-talk. Future experiments will be needed to further test our model (Fig 11)
and to understand the possible physiological relevance of dopamine-induced Ca2+ oscillations for
striatal function.
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ACKNOWLEDGMENTS
We are grateful to Paul Greengard for providing DARPP-32 knockout mice, to Paul Greengard,
James Surmeier and Anita Aperia for helpful discussions, experimental advice, and comments on
the manuscript, to Zhengnan Wang, Zheng Yan and Phyllis Foley for expert technical and
administrative assistance. Supported by the Robert A. Welch Foundation, the Hereditary Disease
Foundation and NIH R01 NS38082 (I.B.).
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FIGURE LEGENDS
Figure 1. Dopamine-induced Ca2+ transients in rat MSN.
A, Dopamine induces Ca2+ transients in MSN. Representative images show Fura-2 340/380 ratios
in rat medium spiny neurons (MSN) before (-1 min) and after (0 min - 28 min) application of 400
µM dopamine. The images were collected in ACSF containing 2 µM of TTX. Dopamine was
applied to the bath at time 0 min. MSN 340/380 Fura-2 ratio images are shown in pseudocolor
for each minute of the experiment. The pseudocolor calibration scale for 340/380 ratios is shown
on the right.
B, A single MSN 340/380 ratio trace. A region of interest was chosen in the soma of a single
MSN (indicated by an arrow on panel A) and the 340/380 ratio in the selected region of interest
was plotted versus time in the experiment. The basal 340/380 ratio (before application of
dopamine) for the cell shown was 0.55. The time of 400 µM dopamine application (300 sec) is
indicated by the arrow. For the cell shown Ca2+ oscillations started 360 sec after dopamine
application (at time 660 sec), had an average amplitude of 0.9 and an frequency of 14 spikes/20
min. The data from the same experiment were used to generate panels A and B.
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Figure 2. D1-class DARs mediate dopamine-evoked Ca2+ transients in MSN.
The images were collected in ACSF and analyzed as described in Fig 1B legend.
A. Dopamine-induced Ca2+ transients in single MSN. The data are representative of n =48 MSN.
B. A combination of a D1-class DAR antagonist (5 µM SCH23390) and a D2-class DAR
antagonist (5 µM Spiperone) prevents dopamine-induced Ca2+ oscillations in MSN (n = 46).
C. A combination of a D2-class DAR antagonist (5 µM Spiperone) and an NMDA receptor
blocker (10 µM (+)MK801) does not prevent dopamine-induced Ca2+ oscillations in MSN (n = 27).
D. A combination of a D1-class DAR antagonist (5 µM SCH23390) and an NMDA receptor
blocker (10 µM (+)MK801) suppress dopamine-induced Ca2+ oscillations in MSN (n = 43).
E. The specific D1-class DAR agonist SKF38393 (50 µM) induces Ca2+ oscillations in MSN (n =
10).
F. The specific D2-class DAR agonist Quinpirole (10 µM) is less potent in inducing Ca2+
oscillations in MSN (n = 23).
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Figure 3. D2-class receptors, 5-HT2 receptors, mAch receptors and α1-Adrenoceptors are
not essential for dopamine-induced Ca2+ oscillations in MSN.
The images were collected in ACSF containing 2 µM TTX.
A. Dopamine-induced Ca2+ transients in single MSN. The data are representative of n = 31 MSN.
B, A combination of antagonists for 5-HT2 receptors (20 µM Ketanserin), α1-Adrenoceptors (10
µM Prazosin), mAchRs (5 µM Atropine), and D2-class DARs (5 µM Spiperone) does not prevent
dopamine-evoked Ca2+ oscillations in MSN (n=12).
C. Application of 5-HT2 receptor agonist (100 µM α-Methyl-5-hydroxytryptamine) to MSN
instantly induces a single instant Ca2+ spike followed by 1-2 much smaller spikes (n= 49).
D. 5-HT2 receptor antagonist (20 µM Ketanserin) blocks Ca2+ responses to 5-HT2 receptor
agonist (100 µM α-Methyl-5-hydroxytryptamine) in MSN (n=27).
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Figure 4. Ca2+ influx is required for maintenance but not for initiation of dopamine-induced
Ca2+ oscillations in MSN.
The images were collected in ACSF and analyzed as described in Fig 1B legend.
A, Dopamine-induced Ca2+ oscillations quickly ceased in Ca2+-free medium, and restarted after
re-addition of 2 mM Ca2+ to the extracellular medium (n=43). Changes in extracellular Ca2+
concentration are shown above the 340/380 ratio trace.
B, Addition of a mixture of a L-type Ca2+ channel blocker (10 µM nifedipine), AMPA receptor
blocker (20 µM CNQX) and NMDA receptor blocker (10 µM (+)-MK801) stopped dopamine-
induced Ca2+ oscillations in MSN (n = 44).
C, Preincubation of MSN with a mixture of an L-type Ca2+ channel blocker (10 µM nifedipine),
AMPA receptor blocker (20 µM CNQX) and NMDA receptor blocker (10 µM (+)-MK801) greatly
attenuate dopamine-induced Ca2+ oscillations in MSN (n = 36)
D - F, Preincubation of MSN with a mixture of an AMPA receptor blocker (20 µM CNQX) and an
NMDA receptor blocker (10 µM (+)-MK801) (D, n = 40), an L-type Ca2+ channel blocker (10 µM
nifedipine) and an AMPA receptor blocker (20 µM CNQX) (E, n = 28), or an L-type Ca2+ channel
blocker (10 µM nifedipine) and an NMDA receptor blocker (10 µM (+)-MK801) (F, n=20) had a
partial inhibitory effect on dopamine-induced Ca2+ oscillations in MSN.
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Figure 5. PLC activation and intracellular Ca2+ mobilization are required for the initiation
and maintenance of dopamine-induced Ca2+ oscillations in MSN
The images were collected in ACSF and analyzed as described in Fig 1B legend.
A, Preincubation with a PLC inhibitor (10 µM U73122) prevents dopamine-induced Ca2+
oscillations in MSN (n=40).
B, Preincubation with 10 µM of U73343 (inactive analog of U73122) has no effect on dopamine-
induced Ca2+ oscillations in MSN (n=41).
C, Application of SERCA Ca2+ pump inhibitor (10 µM Thapsigargin) stops dopamine-induced
Ca2+ oscillations in MSN (n= 37).
D, Preincubation of MSN with SERCA Ca2+ pump inhibitor (10 µM Thapsigargin for 10 min)
suppressed dopamine-induced Ca2+ oscillations in MSN (n=22).
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Figure 6. Role of the putative PLC-linked D1-class DAR in dopamine-induced Ca2+
oscillations
The images were collected in ACSF and analyzed as described in Fig 1B legend.
A. Application of 400 µM SKF83959 (specific agonist for the putative PLC-linked D1-class DAR
subtype (11)) induced an instant Ca2+ transient in MSN (n = 91).
B. Application of 400 µM SKF83959 induced a Ca2+ transient in MSN in Ca2+-free ACSF (n =
33).
C. Preincubation with PLC inhibitor U73122 prevented SKF83959-evoked Ca2+ response in MSN
(n=58).
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Figure 7. Dopamine-induced Ca2+ oscillations are “primed” by activation of 5-HT2
receptors.
The images were collected in ACSF containing 2 µM of TTX and analyzed as described in Fig 1B
legend.
A. Application of 50 µM α-Methyl-5-hydroxytryptamine (5-HT2 receptor-specific agonist) caused
an instant single Ca2+ spike in rat MSN (n=24).
B. 100 µM dopamine was not sufficient to induce any Ca2+ response in “naïve” MSN (n=78).
C. 100 µM dopamine induced Ca2+ oscillations in MSN “primed” with 50 µM α-Methyl-5-
hydroxytryptamine 5 min prior to dopamine application (n=38).
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Figure 8. Effects of PKA phosphorylation on Ca2+ signaling in MSN
The images were collected in ACSF and analyzed as described in Fig 1B legend.
A–D. Changes in basal Ca2+ levels in MSN induced by application of 500 µM 8-Br-cAMP
(membrane-permeable cAMP analog) (A, n = 64); 10 nM Calyculin A (PP1 inhibitor) (B, n = 34);
10 nM Cyclosporin A (PP2B inhibitor) (C, n = 33); 1 nM Okadaic acid (PP2A inhibitor) (D, n = 33).
E. Ca2+ responses in MSN induced by application of SKF83959/8-Br-cAMP mixture (n=39).
F– H. Effects of 10 nM Calyculin A (F, n=39), 10 nM Cyclosporin A (G, n=40) or 1 nM Okadaic
acid (H, n=23) on dopamine-induced Ca2+ oscillations in MSN.
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Figure 9. Effects of R9-IC and EGFP-RT1-LIZ on dopamine-induced Ca2+ oscillations in
MSN
The images were collected in ACSF containing 2 µM of TTX and analyzed as described in Fig 1B
legend.
A-B. Representative dopamine-induced Ca2+ oscillations in MSN loaded with R9 (panel A) or R9-
IC (panel B) peptides. The sequences of R9 and R9-IC peptides are shown above the traces.
C-D. Representative dopamine-induced Ca2+ oscillations in MSN transfected with EGFP (panel
C) or EGFP-RT1-LIZ (panel D) constructs.
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Figure 10. A role of DARPP-32 in dopamine-induced Ca2+ oscillations in MSN.
The images were collected in ACSF containing 2 µM of TTX and analyzed as described in Fig 1B
legend.
A-B. Western blotting of striatal lysates from the wild type (D32(+/+), panel A) and DARPP-32
knockout (D32(-/-), panel B) pup brain lysates with anti-DARPP32 monoclonal antibodies. The
lysate from each pup (1-6 for D32(+/+), 1-7 for D32(-/-)) was loaded on a separate lane. The
expected position of DARPP-32 (D32) on the gel is indicated.
C-D. Representative dopamine-induced Ca2+ oscillations in wild type (D32(+/+)) mouse MSN
(panel C) and in DARPP32 knockout (D32(-/-)) mouse MSN (panel D).
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Fig 11. Model of D1-class DAR mediated cAMP, InsP3 and Ca2+ signaling in MSN.
Application of dopamine activates D1/5 DARs leading to activation of adenylyl cyclase, production
of cAMP and activation of PKA. Actions of dopamine can be mimicked by D1-specific agonist
SKF38393 and prevented by D1-specific antagonist SCH23390, but not by D2-specific antagonist
Spiperone. Activated PKA phosphorylates DARPP-32 and Inhibitor-1, converting them into potent
inhibitors of PP1 (1). Activation of PKA and inhibition of PP1 leads to increased phosphorylation
and activation of Ca2+ influx channels (L-type Ca2+ channels, NMDA receptors and AMPA
receptors) and InsP3R1. InsP3R1 is associated with AKAP9-PKA via LIZ motif (40 #3739) and
with PP1α via carboxy-terminal IC region (39). Dopamine also acts on a putative PLC-linked D1-
class DAR (activated by SKF83959 (11)) and/or PLC-coupled D1/5-calcyon complex (12).
Formation of D1/5-calcyon complex is promoted by “priming” of MSN with 5-HT2 receptor agonist
(not shown). It remains to be determined if D1/D5 DARs complexed with calcyon (12,13) and a
putative PLC-linked D1-class DAR (8-11) are the same or distinct (as shown) molecular entities.
Simultaneous activation of cAMP and InsP3 signaling pathways leads to activation of InsP3R1
(39), which themselves are under biphasic regulation by Ca2+ (61-63). Increase in cytosolic Ca2+
activates calcineurin (PP2B), which dephosphorylates DARPP-32 and Inhibitor-1, resulting in
disinhibition of PP1 (1), which in turn dephosphorylates InsP3R1 and Ca2+ influx channels. Ca2+
influx via L-type Ca2+ channels, NMDA receptors, AMPA receptors and SERCA pump activity are
necessary to prevent depletion of intracellular Ca2+ stores during oscillations. Proposed model
indicates that dopamine-induced Ca2+ oscillations in MSN rely on an intricate interplay between
Ca2+ and cAMP signaling pathways leading to repetitive rounds of InsP3R1 phosphorylation by PKA
and dephosphorylation by PP1. The model is supported by pharmacological experiments (U73122
– PLC blocker, CalA – PP1 blocker, CsA – PP2B blocker, Thapsigargin – SERCA blocker,
(+)MK801 – NMDA receptor blocker, CNQX – AMPA receptor blocker, Nifedipine – L-type Ca2+
channel blocker), effects of R9-IC and EGFP-RT1-LIZ competitive peptides and analysis of
DARPP-32 knockout (KO).
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Table 1 Statistical analysis of dopamine-induced Ca2+ oscillations in MSN.
The quantitative parameters of Ca2+ oscillations are shown as mean ± SD. A number of cells
used in the analysis is shown in the first column (n). Significant (p < 0.05, unpaired t test)
differences from control MSN are shown as (***).
MSN n Basal Ca2+
R340/380
lag time (min)
Amplitude R340/380
Frequency (/20 min)
Control 29 0.52 ± 0.05 5.1 ± 2.2 0.89 ± 0.1 12 ± 6
R9 18 0.51 ± 0.03 4.7 ± 2.1 0.86 ± 0.09 11 ± 6
R9-IC 29 0.49 ± 0.04 4.3 ± 2.2 1.1± 0.15 (***) 20 ± 8 (***)
EGFP 17 0.57 ± 0.03 3.9 ± 2.8 0.82 ± 0.1 15 ± 4
EGFP- RT1-LIZ 15 0.57 ± 0.05 16.2 ± 6.3 (***) 0.76 ± 0.04 7 ± 3 (***)
Mouse (D32+/+) 38 0.51 ± 0.03 4.5 ± 2.1 0.87 ± 0.08 13 ± 6
Mouse (D32-/-) 154 0.53 ± 0.04 6.1 ± 2.5 (***) 0.87 ± 0.11 9 ± 4 (***)
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REFERENCES
1. Greengard, P., Allen, P. B., and Nairn, A. C. (1999) Neuron 23, 435-447.
2. Nicola, S. M., Surmeier, J., and Malenka, R. C. (2000) Annu Rev Neurosci 23, 185-215
3. Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., and Caron, M. G. (1998) Physiol
Rev 78, 189-225.
4. Vallone, D., Picetti, R., and Borrelli, E. (2000) Neurosci Biobehav Rev 24, 125-132.
5. Surmeier, D. J., Song, W. J., and Yan, Z. (1996) J Neurosci 16, 6579-6591.
6. Jackson, D. M., and Westlind-Danielsson, A. (1994) Pharmacol Ther 64, 291-370
7. Hernandez-Lopez, S., Tkatch, T., Perez-Garci, E., Galarraga, E., Bargas, J., Hamm, H.,
and Surmeier, D. J. (2000) J Neurosci 20, 8987-8995.
8. Undie, A. S., and Friedman, E. (1990) J Pharmacol Exp Ther 253, 987-992.
9. Friedman, E., Jin, L. Q., Cai, G. P., Hollon, T. R., Drago, J., Sibley, D. R., and Wang, H. Y.
(1997) Mol Pharmacol 51, 6-11.
10. Panchalingam, S., and Undie, A. S. (2001) Neuropharmacology 40, 826-837.
11. Jin, L. Q., Goswami, S., Cai, G., Zhen, X., and Friedman, E. (2003) J Neurochem 85, 378-
386.
12. Lezcano, N., Mrzljak, L., Eubanks, S., Levenson, R., Goldman-Rakic, P., and Bergson, C.
(2000) Science 287, 1660-1664
13. Bergson, C., Levenson, R., Goldman-Rakic, P. S., and Lidow, M. S. (2003) Trends
Pharmacol Sci 24, 486-492
14. Hernandez-Lopez, S., Bargas, J., Surmeier, D. J., Reyes, A., and Galarraga, E. (1997) J
Neurosci 17, 3334-3342
15. Surmeier, D. J., Bargas, J., Hemmings, H. C., Jr., Nairn, A. C., and Greengard, P. (1995)
Neuron 14, 385-397
16. Cepeda, C., Colwell, C. S., Itri, J. N., Chandler, S. H., and Levine, M. S. (1998) J
Neurophysiol 79, 82-94.
by guest on February 15, 2018http://w
ww
.jbc.org/D
ownloaded from
37
17. Yan, Z., Hsieh-Wilson, L., Feng, J., Tomizawa, K., Allen, P. B., Fienberg, A. A., Nairn, A.
C., and Greengard, P. (1999) Nat Neurosci 2, 13-17
18. Levine, M. S., Altemus, K. L., Cepeda, C., Cromwell, H. C., Crawford, C., Ariano, M. A.,
Drago, J., Sibley, D. R., and Westphal, H. (1996) J Neurosci 16, 5870-5882
19. Flores-Hernandez, J., Cepeda, C., Hernandez-Echeagaray, E., Calvert, C. R., Jokel, E.
S., Fienberg, A. A., Greengard, P., and Levine, M. S. (2002) J Neurophysiol 88, 3010-
3020
20. Kotecha, S. A., Oak, J. N., Jackson, M. F., Perez, Y., Orser, B. A., Van Tol, H. H., and
MacDonald, J. F. (2002) Neuron 35, 1111-1122
21. Blank, T., Nijholt, I., Teichert, U., Kugler, H., Behrsing, H., Fienberg, A., Greengard, P.,
and Spiess, J. (1997) Proc Natl Acad Sci U S A 94, 14859-14864
22. Rajadhyaksha, A., Leveque, J., Macias, W., Barczak, A., and Konradi, C. (1998) Dev
Neurosci 20, 204-215
23. Snyder, G. L., Fienberg, A. A., Huganir, R. L., and Greengard, P. (1998) J Neurosci 18,
10297-10303.
24. Ouimet, C. C., Miller, P. E., Hemmings, H. C., Jr., Walaas, S. I., and Greengard, P. (1984)
J Neurosci 4, 111-124
25. Walaas, S. I., and Greengard, P. (1984) J Neurosci 4, 84-98
26. Fienberg, A. A., Hiroi, N., Mermelstein, P. G., Song, W., Snyder, G. L., Nishi, A.,
Cheramy, A., O'Callaghan, J. P., Miller, D. B., Cole, D. G., Corbett, R., Haile, C. N.,
Cooper, D. C., Onn, S. P., Grace, A. A., Ouimet, C. C., White, F. J., Hyman, S. E.,
Surmeier, D. J., Girault, J., Nestler, E. J., and Greengard, P. (1998) Science 281, 838-842
27. Furuichi, T., Kohda, K., Miyawaki, A., and Mikoshiba, K. (1994) Current Opinion Neurobiol
4, 294-303
28. Berridge, M. J. (1998) Neuron 21, 13-26.
29. Walaas, S. I., Nairn, A. C., and Greengard, P. (1986) J Neurosci 6, 954-961.
by guest on February 15, 2018http://w
ww
.jbc.org/D
ownloaded from
38
30. Supattapone, S., Danoff, S. K., Theibert, A., Joseph, S. K., Steiner, J., and Snyder, S. H.
(1988) Proc. Natl. Acad. Sci. USA 85, 8747-8750
31. Danoff, S. K., Ferris, C. D., Donath, C., Fischer, G. A., Munemitsu, S., Ullrich, A., Snyder,
S. H., and Ross, C. A. (1991) Proc. Natl. Acad. Sci. USA 88, 2951-2955
32. Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. S. (1991)
Biochem. and Biophys. Res. Comm. 175, 192-198
33. Haug, L. S., Jensen, V., Hvalby, O., Walaas, S. I., and Ostvold, A. C. (1999) J Biol Chem
274, 7467-7473.
34. Pieper, A. A., Brat, D. J., O'Hearn, E., Krug, D. K., Kaplin, A. I., Takahashi, K., Greenberg,
J. H., Ginty, D., Molliver, M. E., and Snyder, S. H. (2001) Neuroscience 102, 433-444
35. Wagner, L. E., 2nd, Li, W. H., and Yule, D. I. (2003) J Biol Chem 278, 45811-45817
36. Soulsby, M. D., Alzayady, K., Xu, Q., and Wojcikiewicz, R. J. (2004) FEBS Lett 557, 181-
184
37. Nakade, S., Rhee, S. K., Hamanaka, H., and Mikoshiba, K. (1994) J. Biol. Chem. 269,
6735-6742
38. Wojcikiewicz, R. J., and Luo, S. G. (1998) J Biol Chem 273, 5670-5677
39. Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J Neurosci 23, 403-415.
40. Tu, H., Tang, T. S., Wang, Z., and Bezprozvanny, I. (2004) J Biol Chem 279, 19375-
19382
41. Fienberg, A. A., and Greengard, P. (2000) Brain Res Brain Res Rev 31, 313-319.
42. Tang, T.-S., Tu, H., Chan, E. Y., Maximov, A., Wang, Z., Wellington, C. L., Hayden, M. R.,
and Bezprozvanny, I. (2003) Neuron 39, 227-239
43. Howe, A. R., and Surmeier, D. J. (1995) J Neurosci 15, 458-469
44. Goslin, K., Asmussen, H., and Banker, G. (1998) in Culturing Nerve Cells (Banker, G.,
and Goslin, K., eds), pp. 339-370, MIT Press, Cambridge, MA
45. Maximov, A., and Bezprozvanny, I. (2002) J Neurosci 22, 6939-6952
46. Mao, L., and Wang, J. Q. (2001) Brain Res Mol Brain Res 86, 125-137.
by guest on February 15, 2018http://w
ww
.jbc.org/D
ownloaded from
39
47. Gerfen, C. R. (1992) Trends Neurosci 15, 133-139.
48. Nisenbaum, E. S., and Wilson, C. J. (1995) J Neurosci 15, 4449-4463
49. Wilson, C. J., and Kawaguchi, Y. (1996) J Neurosci 16, 2397-2410
50. Hoyer, D., Hannon, J. P., and Martin, G. R. (2002) Pharmacol Biochem Behav 71, 533-
554
51. Harrison, J. K., Pearson, W. R., and Lynch, K. R. (1991) Trends Pharmacol Sci 12, 62-67
52. Caulfield, M. P. (1993) Pharmacol Ther 58, 319-379
53. Cepeda, C., and Levine, M. S. (1998) Dev Neurosci 20, 1-18
54. Morikawa, H., Khodakhah, K., and Williams, J. T. (2003) J Neurosci 23, 149-157
55. Lezcano, N., and Bergson, C. (2002) J Neurophysiol 87, 2167-2175
56. Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Trends Cell Biol 10, 290-295
57. Becker-Hapak, M., McAllister, S. S., and Dowdy, S. F. (2001) Methods 24, 247-256
58. Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L., and
Rothbard, J. B. (2000) Proc Natl Acad Sci U S A 97, 13003-13008
59. Walaas, S. I., Aswad, D. W., and Greengard, P. (1983) Nature 301, 69-71.
60. Pacheco, M. A., and Jope, R. S. (1997) J Neurochem 69, 639-644
61. Iino, M. (1990) J. Gen. Physiol. 95, 1103-1122
62. Bezprozvanny, I., Watras, J., and Ehrlich, B. E. (1991) Nature 351, 751-754
63. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252, 443-446
64. Keizer, J., and Deyoung, G. W. (1992) Biophys. J. 61, 649-660
65. Gustafson, E. L., and Greengard, P. (1990) Exp Brain Res 79, 447-458
66. Gustafson, E. L., Girault, J. A., Hemmings, H. C., Jr., Nairn, A. C., and Greengard, P.
(1991) J Comp Neurol 310, 170-188
67. Stojilkovic, S. S., Kukuljan, M., Iida, T., Rojas, E., and Catt, K. J. (1992) Proc Natl Acad
Sci U S A 89, 4081-4085
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A 400 M dopamine
-1 min 0 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min 8 min
9 min 10 min 11 min 12 min 13 min 14 min 15 min 16 min 17 min 18 min
19 min 20 min 21 min 22 min 23 min 24 min 25 min 26 min 27 min 28 min
400 M dopamine
400 M dopamine
B
340/
380
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500
340/
380
400 M dopamine
time (sec)
Fig 1
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0.30.50.70.91.11.31.5
0 500 1000 1500 2000time (sec)
340/
380 5 M Spiperone + 10 M (+)MK801
400 M Dopamine
0.30.50.70.91.11.31.5
0 500 1000 1500 2000time (sec)
340/
380 5 M SCH23390 + 5 M Spiperone
400 M Dopamine
0.30.50.70.91.11.31.5
0 500 1000 1500 2000time (sec)
340/
380
400 M DopamineA
340/
380
D
0.30.50.70.91.11.31.5
0 500 1000 1500 2000
5 M SCH23390 + 10 M (+)MK801
400 M Dopamine
time (sec)
B
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500
time (sec)
340/
380 50 M SFK 38393
E
F
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500
time (sec)
340/
380
10 M QuinpiroleC
Fig 2
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CA
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500time (sec)
340/
380
400 M Dopamine
2 M TTX
Fig 3
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500time (sec)
2 M TTX+5 M Spiperone+20 M Ketanserin+5 M Atropine+10 M Prazosin
400 M DopamineB D
0.30.50.70.91.11.31.5
0 500 1000 1500 200 2500time (sec)
340/
380
2 M TTX
100 M -Methyl-5-hydroxytryptamine
340/
380
0.30.50.70.91.11.31.5
0 500 1000 1500 2000 2500
2 M TTX + 20 M Ketanserin
100 M -Methyl-5-hydroxytryptamine
time (sec)
340/
380
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0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000 3600time (sec)
340/
380
400 M Dopamine
2 mM Ca2+
A
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
10 M (+)MK801 + 20 M CNQX + 10 M Nifedipine
400 M DopamineC
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
E
2 mM Ca2+
20 M CNQX + 10 M Nifedipine
400 M Dopamine
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
10 M (+)MK801 + 20 M CNQX+ 10 M Nifedipine
400 M DopamineB
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
D
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
10 M (+)MK801+20 M CNQX
400 M Dopamine
10 M (+)MK801 + 10 M Nifedipine
400 M DopamineF
Fig 4
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Fig 5
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000time (sec)
340/
380 10 M U73343
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000time (sec)
340/
380
400 M Dopamine
10 M U73122
A
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000time (sec)
340/
380
10 M Thapsigargin
C 400 M Dopamine
B400 M Dopamine
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
time (sec)
340/
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10 M Thapsigargin
D400 M Dopamine by guest on February 15, 2018
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A
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 600 1200 1800 2400 3000
400 M SKF8395934
0/38
0
time (sec)
B
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 600 1200 1800 2400 3000
400 M SKF83959
Ca2+ free ACSF
340/
380
time (sec)
C
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500
time (sec)
340/
380
400 M SKF83959
10 M U73122
Fig 6
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A
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
50 M -Methyl-5-hydroxytryptamine34
0/38
0
B time (sec)
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
100 M Dopamine
340/
380
time (sec)
C
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 500 1000 1500 2000 2500time (sec)
340/
380
100 M Dopamine
50 M -Methyl-5-hydroxytryptamine
Fig 7
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0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000time (sec)
340/
380
10 nM Cyclosporin A
0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000time (sec)
340/
380
10 nM Calyculin A
0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000time (sec)
340/
380
1.0 nM OA
0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000
time (sec)
340/
380
500 M 8-Br-cAMPA
B
C
D
0.3
0.50.7
0.91.1
1.31.5
0 600 1200 1800 2400 3000time (sec)
340/
380 10 nM Calyculin
400 M Dopamine
0.30.50.70.91.11.31.5
500 1100 1700 2300 2900 3500time (sec)
340/
380 10 nM Cyclosporin A
400 M Dopamine
0.30.50.70.91.11.31.5
0 600 1200 1800 2400 3000time (sec)
340/
380 1.0 nM OA
400 M Dopamine
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 600 1200 1800 2400 3000time (sec)
340/
380
400 M SKF83959 +500 M 8-Br-cAMPE
F
G
H
Fig 8
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0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
time (sec)
340/
380
400 M Dopamine
A C
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
time (sec)
340/
380
400 M Dopamine
EGFPR9
R9=RRRRRRRRR
B D
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
time (sec)
340/
380
400 M Dopamine
EGFP-RT1-LIZ
R9-IC =RRRRRRRRRGHPPHMNVNPQQPA
400 M Dopamine
R9-IC
0 400 800 1200 1600 2000
time (sec)
0.3
0.5
0.7
0.9
1.1
1.3
1.5
340/
380
Fig 9
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A B
33 kDDARPP-32
#1 #2 #3
D32 +/+
#4 #5 #6
MW, kDa
#1 #2 #3
D32 -/-
#4 #5 #6 #7
MW, kDa
33 kD DARPP-32
DC D32 +/+ D32 -/-
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 1600 2000
time (sec)
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400 M Dopamine
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0 400 800 1200 2000 2600
time (sec)
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400 M Dopamine
Fig 10
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NMDARAMPARL-type CaCh
Ca2+
D1/5
cAMP
PKADARPP-32
pDARPP-32
InsP3R1 PP1
PP2B
D1/5
PLC
InsP3
CsA
CalA
calc
yon
priming
SKF38393Dopamine
SKF83959
DAR
SCH23390 (+)MK801CNQX
Nifedipine
U73122
Ca2+
R9-IC
KO
P
SERCA
AKAP9 PKA
I-1
pI-1
Thapsigargin
D2
Spiperone
EGFP-RT1-LIZ
Fig 11
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Tie-Shan Tang and Ilya BezprozvannyDopamine receptor-mediated Ca2+ signaling in striatal medium spiny neurons
published online August 2, 2004J. Biol. Chem.
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