dop_jbc_rev.doc july 31, 2004 dopamine receptor-mediated

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.


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


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


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


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


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.


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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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


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

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0.7

0.9

1.1

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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


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)

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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

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0.9

1.1

1.3

1.5

340/

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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

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0.7

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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

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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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