Role of Adrenoceptores in the Regulation of Dopamine-DARPP31 Signaling in Neostriatal Neurons

, ,

, ,

*Department of Anesthesiology, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Department of Pharmacology, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Department of Japan Science of Technology Agency, CREST, Kurume, Fukuoka, Japan

Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, USA

Noradrenaline has been shown to interact with the dopami-nergic system and regulate psychomotor functions. In animalmodels of Parkinsons disease, depletion of noradrenaline bydegeneration of noradrenergic neurons or genetic deletion ofdopamine b-hydroxylase potentiates the motor decits ofParkinsons disease (Srinivasan and Schmidt 2003; Romm-elfanger et al. 2007). These decits can be improved bynoradrenaline replacement (Rommelfanger et al. 2007),indicating a critical role for noradrenaline in the motordysfunction of Parkinsons disease. Adrenoceptors are sub-divided into three major classes by their differential couplingto G-proteins. a1-Adrenoceptors (a1A, a1B, a1D), coupled toGq, activate phospholipase C; a2-adrenoceptors (a2A2C),coupled to Gi, inhibit adenylyl cyclase (AC); and b-adrenoceptors (b13), coupled to Gs/olf, stimulate AC. It is

known that pre-synaptic a2-adrenoceptors negatively regu-late dopamine release from dopaminergic terminals (Tren-delenburg et al. 1994; Yavich et al. 1997; Gobert et al.2004). It is likely that noradrenaline also modulates

Received January 17, 2010; revised manuscript received February 24,2010; accepted February 24, 2010.Address correspondence and reprint requests to Akinori Nishi, M.D.,

Ph.D., Department of Pharmacology, Kurume University School ofMedicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.E-mail: [email protected]

Abbreviations used: AC, adenylyl cyclase; DARPP-32, dopamine- andcAMP-regulated phosphoprotein of Mr 32 kDa; DSP-4, N-(2-chloroeth-yl)-N-ethyl-2-bromobenzylamine hydrochloride; IP, immunoprecipita-tions; LC, locus coeruleus; PKA, protein kinase A; SDS, sodium dodecylsulfate.

Abstract

Studies in animal models of Parkinsons disease have re-

vealed that degeneration of noradrenaline neurons is involved

in the motor deficits. Several types of adrenoceptors are

highly expressed in neostriatal neurons. However, the selec-

tive actions of these receptors on striatal signaling pathways

have not been characterized. In this study, we investigated the

role of adrenoceptors in the regulation of dopamine/dopa-

mine- and cAMP-regulated phosphoprotein of Mr 32 kDa

(DARPP-32) signaling by analyzing DARPP-32 phosphoryla-

tion at Thr34 [protein kinase A (PKA)-site] in mouse neostri-

atal slices. Activation of b1-adrenoceptors induced a rapid and

transient increase in DARPP-32 phosphorylation. Activation of

a2-adrenoceptors also induced a rapid and transient increase

in DARPP-32 phosphorylation, which subsequently de-

creased below basal levels. In addition, activation of a2-

adrenoceptors attenuated, and blockade of a2-adrenoceptors

enhanced dopamine D1 and adenosine A2A receptor/DARPP-

32 signaling. Chemical lesioning of noradrenergic neurons

mimicked the effects of a2-adrenoceptor blockade. Under

conditions of a2-adrenoceptor blockade, the dopamine D2receptor-induced decrease in DARPP-32 phosphorylation

was attenuated. Our data demonstrate that b1- and a2-

adrenoceptors regulate DARPP-32 phosphorylation in neo-

striatal neurons. Gi activation by a2-adrenoceptors antago-

nizes Gs/PKA signaling mediated by D1 and A2A receptors in

striatonigral and striatopallidal neurons, respectively, and

thereby enhances D2 receptor/Gi signaling in striatopallidal

neurons. a2-Adrenoceptors may therefore be a therapeutic

target for the treatment of Parkinsons disease.

Keywords: D1 receptor, noradrenaline, phosphorylation,

striatum, a2-adrenoceptor.

J. Neurochem. (2010) 113, 10461059.

JOURNAL OF NEUROCHEMISTRY | 2010 | 113 | 10461059 doi: 10.1111/j.1471-4159.2010.06668.x

1046 Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 113, 10461059 2010 The Authors

dopamine receptor signaling post-synaptically in mediumspiny neurons. However, information on the interactionbetween noradrenaline and dopamine receptor signaling inmedium spiny neurons is limited.Despite the functional importance of noradrenaline in

dopaminergic neurotransmission, the striatum receives onlysparse noradrenergic innervation (Swanson and Hartman1975; Aston-Jones 2004). However, certain types of adreno-ceptors, such as b1-, a2A-, and a2C-adrenoceptors areexpressed in the striatum (Nicholas et al. 1996; MacDonaldet al. 1997). b1-Adrenoceptors were found to be expressed inmedium spiny neurons by radioligand binding (Nahorskiet al. 1979; Waeber et al. 1991) and immunohistochemistry(Pisani et al. 2003), and the loss of b1-adrenoceptors in thestriatum was reported in the late stages of Huntingtonschorea (Waeber et al. 1991). a2A-Adrenoceptors are widelydistributed in the brain including the striatum (MacDonaldet al. 1997), and mediate the inhibition of monoaminerelease and metabolism (Trendelenburg et al. 1994; Bucheleret al. 2002; Ihalainen and Tanila 2004). Generally, they areassociated with functions, such as sedation, analgesia, andhypotension (MacMillan et al. 1996; Lakhlani et al. 1997;Altman et al. 1999; Philipp et al. 2002). In contrast, a2C-adrenoceptors show a unique distribution pattern, and aremost abundantly expressed in the striatum, olfactory tuber-cle, hippocampus, and cerebral cortex (Nicholas et al. 1996;MacDonald et al. 1997; Winzer-Serhan et al. 1997; Holm-berg et al. 1999). a2C-Adrenoceptors are expressed inmedium spiny neurons in the striatum (Holmberg et al.1999), and are negatively coupled to AC via Gi (Lu andOrdway 1997; Zhang et al. 1999). a2C-Adrenoceptors arealso expressed in dopaminergic neurons in the substantianigra (Rosin et al. 1996; Lee et al. 1998) and possibly atdopaminergic terminals in the striatum. Together with a2A-adrenoceptors, they inhibit the release of dopamine (Bucheleret al. 2002). In mice lacking a2C-adrenoceptors, amphet-amine-induced locomotor activity, startle reactivity, andaggressive behavior were enhanced, whereas pre-pulseinhibition was attenuated (Sallinen et al. 1998a,b). Oppositechanges were reported in a2C-adrenoceptor over-expressingmice (Sallinen et al. 1998a,b). Thus, a2C-adrenoceptorslikely play an inhibitory role in the regulation of motor andemotional functions and a modulatory role in the processingof sensory information (Scheinin et al. 2001).Dopamine- and cAMP-regulated phosphoprotein of Mr

32 kDa (DARPP-32) is selectively enriched in medium spinyneurons in the striatum, and plays an essential role indopaminergic neurotransmission (Greengard et al. 1999;Svenningsson et al. 2004). Dopamine activates dopamineD1 receptors coupled to Gs/olf, leading to an activation ofcAMP/ protein kinase A (PKA) signaling and the phosphor-ylation of DARPP-32 at Thr34 (the PKA-site). WhenDARPP-32 is phosphorylated on Thr34, it is converted intoa potent inhibitor of protein phosphatase-1, and thereby

controls the phosphorylation state and activity of manydownstream physiological effectors, including various neu-rotransmitter receptors and voltage-gated ion channels(Svenningsson et al. 2004). The state of DARPP-32 phos-phorylation at Thr34 is also regulated by various othersignaling molecules, providing a mechanism for integratingdopamine and other neurotransmitter signals.Noradrenergic neurotransmission plays a critical role in

the regulation of motor function by interacting with dopa-mine signaling, and noradrenaline replacement therapy hasbeen proposed as a treatment for Parkinsons disease(Grimbergen et al. 2009). However, the molecular mecha-nisms by which adrenoceptors regulate dopamine signalingin the striatum have not been investigated. In this study, weinvestigated the regulation of DARPP-32 phosphorylation byb and a-adrenoceptors. We nd that activation of both typesof receptors affects the phosphorylation of DARPP-32 atThr34. Furthermore, activation of Gi by a2-adrenoceptorsand subsequent inhibition of adenosine A2A receptor/Gs/PKAsignaling are required for the actions of Gi-coupled D2receptors in striatopallidal neurons.

Materials and methods

Preparation, incubation, and processing of neostriatal slicesMale C57BL/6 mice at 68 weeks old were purchased from Japan

SLC (Shizuoka, Japan). All mice used in this study were handled in

accordance with the Guide for the Care and Use of Laboratory

Animals as adopted and promulgated by the U.S. National Institutes

of Health. The Institutional Animal Care and Use Committee of

Kurume University School of Medicine approved the specic

protocols. Male C57BL/6 mice were killed by decapitation. The

brains were rapidly removed and placed in ice-cold, oxygenated

Krebs-HCO3) buffer (124 mM NaCl, 4 mM KCl, 26 mM NaHCO3,

1.5 mM CaCl2, 1.25 mM KH2PO4, 1.5 mM MgSO4, and 10 mM D-

glucose, pH 7.4). Coronal slices (350 lm) were prepared using avibrating blade microtome, VT1000S (Leica Microsystems, Nuss-

loch, Germany). Striata were dissected from the slices in ice-cold

Krebs-HCO3) buffer. Each slice was placed in a polypropylene

incubation tube with 2 mL fresh Krebs-HCO3) buffer containing

adenosine deaminase (10 lg/mL). The slices were pre-incubated at30C under constant oxygenation with 95% O2/5% CO2 for 60 min.The buffer was replaced with fresh Krebs-HCO3

) buffer after

30 min of pre-incubation. Adenosine deaminase was included during

the rst 30 min of pre-incubation. Slices were treated with drugs as

specied in each experiment. Drugs were obtained from the following

sources: isoproterenol, propranolol, CGP20712A, ICI118551, ciraz-

oline, UK14304, yohimbine, nortriptyline, GR113808, SB258585,

SKF81297, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydro-chloride (DSP-4) from Sigma-Aldrich (St. Louis, MO, USA);

CGS21680 from Tocris Cookson (Bristol, UK). After drug treatment,

slices were transferred to Eppendorf tubes, frozen on dry ice, and

stored at )80C until assayed.Frozen tissue samples were sonicated in boiling 1% sodium

dodecyl sulfate (SDS) containing 50 mM sodium uoride and

boiled for an additional 10 min. Small aliquots of the homogenate

2010 The AuthorsJournal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 113, 10461059

Role of adrenoceptors in neostriatal neurons | 1047

were retained for protein determination by the bicinchoninic acid

protein assay method (Pierce, Rockford, IL, USA). Equal amounts

of protein (100 lg) were separated by SDS/polyacrylamide gelelectrophoresis (10% polyacrylamide gels), and transferred to

nitrocellulose membranes (0.2 lm; Schleicher and Schuell, Keene,NH, USA).

Immunoprecipitations of Flag- and Myc-tagged DARPP-32 inneostriatal slices from D1-DARPP-32-Flag/D2-DARPP-32-MycmiceD1-DARPP-32-Flag/D2-DARPP-32-Myc transgenic mice express

Flag- and Myc-tagged DARPP-32 under the control of dopamine D1and D2 receptor promoters, respectively (Bateup et al. 2008). In thestriatum, Flag-tagged DARPP-32 was shown to be expressed

selectively in D1 receptor-enriched striatonigral neurons, and Myc-

tagged DARPP-32 selectively in D2 receptor-enriched striatopallidal

neurons. Using antibodies against Flag and Myc tags, we selectively

immunoprecipitated DARPP-32 from D1 receptor- and D2 receptor-

expressing neurons and analyzed the phosphorylation state of

DARPP-32 in a neuronal subtype-specic manner. In each exper-

iment, six striatal slices were prepared from one mouse, and were

divided into three treatment conditions. In each treatment condition,

six slices, collected from three mice (two slices from each mouse),

were used for the analysis of DARPP-32 phosphorylation. Six

striatal slices were sonicated in 720 lL of immunoprecipitation (IP)lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA,

1% Triton X-100, 1% SDS, 100 nM okadaic acid, phosphatase

inhibitor cocktail (#P5726; Sigma-Aldrich), and protease inhibitor

cocktail (#11873580001; Roche, Basel, Switzerland)]. After deter-

mination of protein concentration, 15 lg of protein was saved forthe analysis of DARPP-32 phosphorylation in total striatal homog-

enate, and the residual homogenates were used for IP. In each IP

from striatal homogenate, 50 lL of washed EZView Red anti-FlagM2 afnity gel (Sigma-Aldrich) and 45 lL of anti-Myc antibody(Novus Biologicals, Littleton, CO, USA) coupled to magnetic beads

(3 lg of Myc antibody for every 5 lL of magnetic beads)(Dynabeads M-280 Tosylactivated; Invitrogen, Carlsbad, CA,

USA) were added. The homogenate/antibody mixture was gently

rotated overnight at 4C. Following the overnight incubation, theMyc magnetic beads were separated from the homogenate/antibody

mixture using a magnetic particle concentrator (Invitrogen), and

then the Flag afnity gels were separated by centrifugation. The

Myc magnetic beads and Flag afnity gels were washed with 1phosphate-buffered saline three times. After the nal wash, 30 lL ofsample buffer was added and the samples were boiled for 2 min.

Flag IP, Myc IP, and total striatal samples were loaded onto 4

12% polyacrylamide Bis-Tris gels (Bio-Rad, Hercules, CA, USA),

separated by electrophoresis, and transferred to nitrocellulose

membranes (0.2 lM; Schleicher and Schuell).

Lesioning of noradrenaline neuronsIn some experiments, N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine hydrochloride (DSP-4) was used to selectively lesion

noradrenergic neurons in the locus coeruleus (LC) (Gesi et al.2000; Rommelfanger et al. 2007). DSP-4 at a dose of 50 mg/kg orsaline (0.1 mL/10 g body weight) was injected intraperitoneally

(i.p.) to C57BL/6 mice. DSP-4 was dissolved in saline immediately

before injection and used within 2 min to avoid degradation. After

7 days of DSP-4 or saline injection, neostriatal slices were prepared

as described above and treated with SKF81297 or CGS21680.

ImmunoblottingThe membranes were immunoblotted using a phosphorylation state-

specic antibody raised against DARPP-32 phospho-peptides:

phospho-Thr34, the site phosphorylated by PKA (mAb-23,

1 : 750 dilution; CC500, 1 : 5004000; Snyder et al. 1992). Amonoclonal antibody (C24-5a, 1 : 7500 dilution) generated against

DARPP-32 (Hemmings and Greengard 1986), which is not

phosphorylation state-specic, was used to determine the total

amount of DARPP-32. None of the experimental manipulations

used in this study altered the total amount of DARPP-32.

The membrane was incubated with a goat anti-mouse or rabbit

Alexa 680-linked IgG (1 : 5000 dilution; Molecular Probes,

Eugene, OR, USA) or a goat anti-mouse or rabbit IRDyeTM800-

linked IgG (1 : 5000 dilution; ROCKLAND, Gilbertsville, PA,

USA). Fluorescence at infrared wavelengths was detected by the

Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA),

and quantied using Odyssey software. In an individual experiment,

samples from control- and drug-treated slices were analyzed on the

same immunoblot. For each experiment, values obtained for slices

were calculated relative to values for the control or drug-treated

slices, as described in gure legends. Normalized data from multiple

experiments were averaged and statistical analysis was carried out as

described in the gure legends.

ImmunohistochemistryUnder deep anesthesia induced with sodium pentobarbital, male

C57BL/6 mice at 68 weeks old were perfused rapidly through the

left ventricle with 50 mL of 4% paraformaldehyde in 0.1 M

phosphate buffer (pH 7.2) at 23C. Serial coronal sections 50 lmin thickness were cut with a vibrating microtome, VT1000S (Leica

Microsystems). Sections were processed for immunohistochemistry

with the use of the free-oating method as described (Nishi et al.2008). Sections were incubated with a rabbit anti-b1-adrenoceptorantibody (A-272; 1 : 500; Sigma), a rabbit anti-a2C-adrenoceptorantibody (RA19064; 1 : 200 dilution; Neuromics, Edina, MN,

USA), and a mouse anti-DARPP-32 antibody (C24-5a; 1 : 20 000

dilution) at 20C for 7 days. Antibody binding was visualized with auorescein isothiocyanate-conjugated donkey anti-mouse IgG

(1 : 100; Jackson ImmunoResearch, West Grove, PA) and a

rhodamine red-conjugated donkey anti-rabbit IgG (1 : 100; Jackson

ImmunoResearch). Sections were mounted in Vectashield (Vector

Laboratories, Burlingame, CA, USA) and examined with a confocal

laser-scanning microscope, LSM 5 PASCAL (Zeiss, Oberkochen,

Germany).

Results

Effects of b-adrenergic agonists on DARPP-32 Thr34phosphorylation in neostriatal slicesTreatment of neostriatal slices with a non-selective b-adrenergic agonist, isoproterenol (10 lM), rapidly andtransiently increased DARPP-32 Thr34 phosphorylation(Fig. 1a). Isoproterenol increased the level of phospho-Thr34 DARPP-32 by fourfold within 1 min of incubation,

Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 113, 10461059 2010 The Authors

1048 | M. Hara et al.

and the increased level of phospho-Thr34 DARPP-32subsequently returned to basal values at 10 min. Thestimulatory effect of isoproterenol on DARPP-32 Thr34

phosphorylation was attenuated by a non-selective b-adren-ergic antagonist, propranolol (10 lM) (Fig. 1b), althoughpropranolol itself did not affect the level of phospho-Thr34

(a)

(b)

(d)

(c)

Fig. 1 Effect of a b-adrenergic agonist on dopamine- and cAMP-

regulated phosphoprotein of Mr 32 kDa (DARPP-32) Thr34 phos-

phorylation in neostriatal slices. (a) Neostriatal slices were treated with

a non-selective b-adrenergic agonist, isoproterenol (10 lM), for the

indicated times. Typical immunoblots for detection of phospho-Thr34

DARPP-32 and total DARPP-32 in the same membrane are shown at

the left of panel. (b and c) Neostriatal slices were pre-treated for

10 min with (b) a non-selective b-adrenergic antagonist, propranolol

(10 lM), or a dopamine D1 receptor antagonist, SCH23390 (1 lM), or

(c) a b1-adrenergic antagonist, CGP20712A (10 lM), or a b2-adren-

ergic antagonist, ICI118551 (10 lM), followed by the addition of

isoproterenol (10 lM) for 1 min. (d) Neostriatal slices from D1-DARPP-

32-Flag/D2-DARPP-32-Myc mice were incubated with isoproterenol

(Iso; 10 lM) for 1 min. Flag-tagged DARPP-32, expressed in D1receptor-enriched striatonigral neurons, and Myc-tagged DARPP-32,

expressed in D2 receptor-enriched striatopallidal neurons, were im-

munoprecipitated. The panel shows data from total striatal homoge-

nate (Homog), Flag-tagged DARPP-32 in striatonigral neurons (D1-

Flag) and Myc-tagged DARPP-32 in striatopallidal neurons (D2-Myc).

Typical immunoblots for detection of phospho-Thr34 DARPP-32 and

total DARPP-32 in the same membrane are shown at the left of panel.

The levels of phospho-Thr34 DARPP-32 were quantified by the

Odyssey infrared imaging system, and the data were normalized to

values obtained with untreated slices. Data represent means SEM

for 412 experiments. **p < 0.01, ***p < 0.001 compared with un-

treated slices; p < 0.05, p < 0.001 compared with isoproterenol

alone; p < 0.001 compared with SCH23390 alone; p < 0.05

compared with ICI118551 alone; one-way ANOVA followed by New-

manKeuls test.



DARPP-32. To rule out the possibility that isoproterenolincreases DARPP-32 Thr34 phosphorylation by activatingdopamine D1 receptors (Vanderheyden et al. 1986), theeffect of isoproterenol was examined in the presence of adopamine D1 receptor antagonist, SCH23390 (1 lM). Pre-treatment with SCH23390 did not affect the stimulatoryeffect of isoproterenol on DARPP-32 Thr34 phosphorylation.To determine the subtype of b-adrenoceptors involved inDARPP-32 Thr34 phosphorylation, neostriatal slices werepre-treated with a b1-adrenergic antagonist, CGP20712A(10 lM), or a b2-adrenergic antagonist, ICI118551 (10 lM).Pre-treatment with CGP20712A, but not ICI118551, atten-uated the isoproterenol-induced increase in DARPP-32Thr34 phosphorylation (Fig. 1c), suggesting that activation

of b1-adrenoceptors induces the phosphorylation of DARPP-32 at Thr34 in neostriatal neurons.We next examined whether activation of b1-adreno-

ceptors increases DARPP-32 phosphorylation in D1 recep-tor-enriched striatonigral and/or D2 receptor-enrichedstriatopallidal neurons, using neostriatal slices from D1-DARPP-32-Flag/D2-DARPP-32-Myc transgenic mice (Ba-teup et al. 2008). Flag- and Myc-tagged DARPP-32 wereimmunoprecipitated from striatonigral and striatopallidalneurons, respectively, and the phosphorylation state ofDARPP-32 at Thr34 in the two types of neurons wasanalyzed. Treatment of neostriatal slices with isoproterenol(10 lM) for 1 min increased the level of phospho-Thr34DARPP-32 by approximately twofold in total striatal

(a) (b)

(c) (d)

Fig. 2 Effect of a1-adrenergic and a2-adrenergic agonists on dopa-

mine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32)

Thr34 phosphorylation in neostriatal slices. (a) Neostriatal slices were

treated with a selective a1-adrenergic agonist, cirazoline (1 lM), for

the indicated times. (b) Neostriatal slices were treated with a selective

a2-adrenergic agonist, UK14304 (1 lM), for the indicated times. (c)

Neostriatal slices were pre-treated with a selective a2-adrenergic

antagonist, yohimbine (1 lM), for 15 min, followed by the addition of

UK14304 (1 lM) for 1 min. (d) Neostriatal slices from D1-DARPP-32-

Flag/D2-DARPP-32-Myc mice were incubated with UK14304 (10 lM)

for 30 s. Flag-tagged DARPP-32 and Myc-tagged DARPP-32 were

immunoprecipitated. The panel shows data from total striatal homog-

enate (Homog), Flag-tagged DARPP-32 in striatonigral neurons (D1-

Flag) and Myc-tagged DARPP-32 in striatopallidal neurons (D2-Myc).




for four to nine experiments. *p < 0.05, **p < 0.01, ***p < 0.001

compared with untreated slices; p < 0.05 compared with UK14304

alone; one-way ANOVA followed by NewmanKeuls test.



homogenates (Fig. 1d). Isoproterenol increased the phos-phorylation of both Flag- and Myc-tagged DARPP-32 atThr34 by twofold, suggesting that isoproterenol activatesthe b1-adrenoceptor signaling cascades both in striatonigraland striatopallidal neurons. The results are consistent withimmunohistochemical data, demonstrating that b1-adreno-ceptors are expressed in all DARPP-32-positive striatalneurons (Fig. 3a).

Effects of a1- and a2-adrenergic agonists on DARPP-32Thr34 phosphorylation in neostriatal slicesThe role of a-adrenoceptors in the regulation of DARPP-32Thr34 phosphorylation was examined in neostriatal slices.Treatment with a selective a1-adrenergic agonist, cirazoline(1 lM), did not affect the level of phospho-Thr34 DARPP-32 by 5 min of incubation (Fig. 2a). However, treatmentwith a selective a2-adrenergic agonist, UK14304 (1 lM),increased DARPP-32 Thr34 phosphorylation by 2.5-foldwithin 1 min of incubation, and the increased level ofphospho-Thr34 DARPP-32 returned to basal values at5 min (Fig. 2b). After 20 min of incubation with UK14304,the level of phospho-Thr34 DARPP-32 decreased slightlybelow basal values (81.0 6.7% of control; p < 0.05 withStudents t-test, but not signicant with one-way ANOVAfollowed by NewmanKeuls test). The rapid and transientincrease in DARPP-32 Thr34 phosphorylation induced byUK14304 was antagonized by an a2-adrenergic antagonist,yohimbine (1 lM) (Fig. 2c). Immunohistochemical datashowed that a2C-adrenoceptors, the predominant subtype ofa2-adrenoceptors in the striatum, were expressed in allDARPP-32-positive neurons (Fig. 3b).We next examined whether UK14304 induces DARPP-32

Thr34 phosphorylation selectively in striatonigral or striato-pallidal neurons or both types of neurons. In neostriatal slices

from D1-DARPP-32-Flag/D2-DARPP-32-Myc mice, treat-ment with UK14304 signicantly increased the level ofphopho-Thr34 DARPP-32 in total striatal homogenate, butthe magnitude of the increase was less than that in C57BL/6mice possibly because of strain differences. The rapidincrease in DARPP-32 Thr34 phosphorylation induced byUK14304 was detected selectively in striatonigral but notstriatopallidal neurons (Fig. 2d). These results demonstrate acell-type specic effect of a2-adrenoceptors in the striatum,although a2C-adrenoceptors are present in all medium spinyneurons.

Interaction of a2-adrenoceptor signaling with dopamine D1and adenosine A2A receptor signalingWe next examined whether a2-adrenoceptors could modulateGolf/PKA/DARPP-32 signaling activated by dopamine D1and adenosine A2A receptors. Pre-treatment with the a2-adrenergic antagonist, yohimbine (10 lM), did not affect thebasal level of phospho-Thr34 DARPP-32. However, yohim-bine enhanced the increase in DARPP-32 Thr34 phosphor-ylation induced by SKF81297 (1 lM) (Fig. 4a) orCGS21680 (5 lM) (Fig. 4b), suggesting that tonic activityof a2-adrenoceptors inhibits dopamine D1 and adenosine A2Areceptor/PKA/DARPP-32 signaling in neostriatal neurons.To test this, we treated striatal slices with the a2-adrenergicagonist, UK14304 (1 lM for 20 min), which did notsignicantly affect the basal levels of phospho-Thr34DARPP-32 in this series of experiments. As expected fromantagonist experiments, UK14304 reduced the increase inDARPP-32 Thr34 phosphorylation induced by SKF81297(1 lM) (Fig. 4c) or CGS21680 (5 lM) (Fig. 4d). Theseresults suggest that a2-adrenoceptors negatively interact withdopamine D1 and adenosine A2A receptor/PKA/DARPP-32signaling.

(a)

(b)

Fig. 3 Expression of b1- and a2C-adreno-

ceptors in the striatum. Double immuno-

staining of striatal tissues with (a)

dopamine- and cAMP-regulated phospho-

protein of Mr 32 kDa (DARPP-32) and b1-

adrenoceptor antibodies and (b) DARPP-32

and a2C-adrenoceptor antibodies. The

expression of b1- and a2C-adrenoceptors

was detected in many DARPP-32-positive

neurons, suggesting their expression both

in striatonigral and striatopallidal neurons.

In agreement with Pisani et al. (2003), the

expression of b1-adrenoceptors was also

detected in DARPP-32-negative, large-

sized neurons (a; arrows), presumably

cholinergic interneurons. Scale bars,

10 lm.



We also examined the role of b-adrenoceptors in dopamineD1 and adenosine A2A receptor signaling. Pre-treatment withthe b-adrenergic antagonist, propranolol (10 lM), did notaffect the SKF81297 (1 lM)-induced or CGS21680 (5 lM)-induced increase in DARPP-32 Thr34 phosphorylation (datanot shown).

Interaction of a2-adrenoceptor signaling with dopamine D2receptor signalingWe next examined whether a2-adrenoceptors also play a rolein dopamine D2 receptor signaling. Treatment of slices with adopamine D2 receptor agonist, quinpirole (1 lM), decreasedthe level of phospho-Thr34 DARPP-32, as previouslyreported (Nishi et al. 1997). Pre-treatment with yohimbine(10 lM) attenuated the decrease in DARPP-32 Thr34phosphorylation induced by quinpirole (Fig. 5), suggestingthat activity of a2-adrenoceptors is required for the action ofdopamine D2 receptor to down-regulate DARPP-32 phos-phorylation in neostriatal neurons.

Effect of nortriptyline on DARPP-32 Thr34phosphorylation in neostriatal slicesThe expression of dopamine b-hydroxylase and noradrena-line transporters are reported to be low in the striatum(Swanson and Hartman 1975; Berridge et al. 1997; Mollet al. 2000; Moron et al. 2002; Arai et al. 2008), andtherefore the striatum is thought to receive a sparsenoradrenergic innervation. To determine whether the releaseand reuptake machinery of noradrenaline at noradrenergicterminals is functioning in the striatum, we examined theeffect of nortriptyline on DARPP-32 phosphorylation. Nor-triptyline inhibits the noradrenaline transporter with highpotency (Ki 3.4 nM) and the serotonin transporter with lowpotency (Ki 161 nM), but does not affect the dopaminetransporter (Ki 13 920 nM; Torres et al. 2003). Treatment ofneostriatal slices with nortriptyline (100 nM) increased thelevel of phospho-Thr34 DARPP-32 by vefold within 30 sof incubation, and the increased level of phospho-Thr34DARPP-32 returned to basal values at 2 min (Fig. 6a).

(a) (b)

(c) (d)

Fig. 4 Effect of a2-adrenoceptor blockade and activation on dopamine

D1 and adenosine A2A receptor/protein kinase A/dopamine- and

cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32) signaling

in neostriatal slices. (a and b) Neostriatal slices were pre-treated with

an a2-adrenergic antagonist, yohimbine (10 lM for 15 min), followed

by the addition of (a) a dopamine D1 receptor agonist, SKF81297

(1 lM for 5 min), or (b) an adenosine A2A receptor agonist, CGS21680

(5 lM for 2 min). (c and d) Neostriatal slices were pre-treated with an

a2-adrenergic agonist, UK14304 (1 lM for 20 min), followed by the

addition of (c) a dopamine D1 receptor agonist, SKF81297 (1 lM for

5 min), or (d) an adenosine A2A receptor agonist, CGS21680 (5 lM for

2 min). The levels of phospho-Thr34 DARPP-32 were quantified by

the Odyssey infrared imaging system, and the data were normalized to


for four experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared

with untreated slices; p < 0.05, p < 0.01 compared with SKF81297

or CGS21680 alone; one-way ANOVA followed by NewmanKeuls

test.



The nortriptyline-induced increase in DARPP-32 Thr34phosphorylation was partially but signicantly antagonizedby CGP20712A (10 lM) plus yohimbine (1 lM) (Fig. 6b);however, the antagonizing effect of either compound was notsignicant when slices were pre-incubated with CGP20712Aor yohimbine alone (data not shown). We also examined theeffect of serotonergic antagonists for 5-HT4 and 5-HT6receptors, which were reported to be the serotonin receptorsubtypes coupled to Gs/olf/cAMP/PKA signaling in thestriatum (Svenningsson et al. 2002). Treatment with a 5-

HT4 receptor antagonist, GR113808 (10 lM), plus a 5-HT6receptor antagonist, SB258585 (10 lM), also partiallyattenuated the nortriptyline-induced increase in DARPP-32Thr34 phosphorylation (Fig. 6c). Pre-treatment with a dopa-mine D1 receptor antagonist, SCH23390 (1 lM), did notattenuate the effect of nortriptyline (data not shown).Taken together, these data reveal a signicant contribution

of striatal noradrenaline release to DARPP-32 T34 phosphor-ylation in medium spiny neurons. Since we also observed anattenuation of the nortriptyline effect by blocking serotoninreceptors, it is likely that nortriptyline inhibited bothnoradrenaline and serotonin transporters in neostriatal slices,leading to increased extracellular noradrenaline andserotonin. Thus, nortriptyline increases phosphorylation ofDARPP-32 via activation of b1- and a2-adrenoceptors andserotonin 5-HT4 and 5-HT6 receptors.

Dopamine D1 and adenosine A2A receptor signaling inneostriatal slices from DSP-4-lesioned miceAfter determining that blockade of noradrenaline uptake instriatal slices affected DARPP-32 phosphorylation, weinvestigated whether depletion of noradrenaline in vivo hadan effect on D1 or A2A receptor-mediated phosphorylation ofDARPP-32. We injected mice with DSP-4 to selectivelylesion noradrenergic neurons of the LC. The expressionlevels of tyrosine hydroxylase in the LC, determined bywestern blotting, decreased to 45.7 2.9% of control (saline-treated mice; p < 0.05; Students t-test) after 7 days of DSP-4 administration, but the levels of tyrosine hydroxylase in thestriatum were not affected (103.5 5.2% of control),indicating that DSP-4 selectively lesions noradrenergicneurons in the LC without lesioning dopaminergic neuronsin the substantia nigra.

(a) (b) (c)

Fig. 6 Effect of nortriptyline on dopamine- and cAMP-regulated

phosphoprotein of Mr 32 kDa (DARPP-32) Thr34 phosphorylation in

neostriatal slices. (a) Neostriatal slices were treated with nortriptyline

(100 nM), an inhibitor of monoamine transporters relatively selective

for noradrenaline transporters, for the indicated times. (b and c)

Neostriatal slices were pre-incubated for 10 min with (b) CGP20712A

(10 lM) plus yohimbine (1 lM) or (c) a 5-HT4 receptor antagonist,

GR113808 (10 lM) plus a 5-HT6 receptor antagonist, SB258585

(10 lM), followed by the addition of nortriptyline (100 nM) for 30 s.




for four to six experiments. **p < 0.01, ***p < 0.001 compared with

untreated slices; p < 0.05 compared with nortriptyline alone;

p < 0.05 compared with CGP20712A plus yohimbine; one-way ANOVA

followed by NewmanKeuls test.

Fig. 5 Effect of a2-adrenoceptor blockade on dopamine D2 receptor/

protein kinase A/dopamine- and cAMP-regulated phosphoprotein of Mr

32 kDa (DARPP-32) signaling in neostriatal slices. Neostriatal slices

were pre-treated with an a2-adrenergic antagonist, yohimbine (10 lM

for 10 min), followed by the addition of a dopamine D2 receptor ago-

nist, quinpirole (1 lM for 10 min). The levels of phospho-Thr34

DARPP-32 were quantified by the Odyssey infrared imaging system,

and the data were normalized to values obtained with untreated slices.

Data represent means SEM for six to nine experiments.

***p < 0.001 compared with untreated slices; p < 0.001 compared

with quinpirole alone; one-way ANOVA followed by NewmanKeuls test.



We examined the effect of dopamine D1 and adenosineA2A receptor agonists on DARPP-32 Thr34 phosphorylationin neostriatal slices from saline- or DSP-4-treated mice.Treatment of slices with SKF81297 (1 lM) increasedDARPP-32 Thr34 phosphorylation threefold from 5 to15 min of incubation in saline-treated mice (p < 0.001compared with untreated slices; one-way ANOVA followedby NewmanKeuls test; Fig. 7a). In slices from DSP-4-treated mice, the SKF81297-induced increase in DARPP-32Thr34 phosphorylation was signicantly higher than that inslices from saline-treated mice at 10 and 15 min ofincubation (p < 0.05; two-way ANOVA followed by Bonfer-roni test).Treatment of slices with CGS21680 (1 lM) increased

DARPP-32 Thr34 phosphorylation threefold from 30 s to2 min of incubation in saline-treated mice (p < 0.01 com-pared with untreated slices; one-way ANOVA followed byNewmanKeuls test; Fig. 7b). In slices from DSP-4-treatedmice, the CGS21680-induced increase in DARPP-32 Thr34phosphorylation was signicantly greater than that in slicesfrom saline-treated mice at 1 min of incubation (p < 0.01;two-way ANOVA followed by Bonferroni test). We alsoexamined the effect of DSP-4-lesioning on dopamine D2receptor signaling. Treatment with quinpirole (10 nM1 lMfor 10 min) decreased DARPP-32 Thr34 phosphorylationsimilarly in slices from saline- and DSP-4-treated mice. Thendings demonstrate that the chemical lesioning of norad-renergic neurons by DSP-4 results in the enhancement of

dopamine D1 and adenosine A2A receptor/PKA/DARPP-32signaling in striatal neurons.

Discussion

We have demonstrated that b1- and a2-adrenoceptors regulatePKA/DARPP-32 signaling in the striatum (see Fig. 8).Dopamine D1 and adenosine A2A receptor signaling wasattenuated by prolonged activation of a2-adrenoceptors,whereas it was enhanced by pharmacological blockade ofa2-adrenoceptors and chemical lesioning of noradrenergicneurons in the LC. This indicates that a2-adrenoceptors,coupled to Gi, are tonically active and counteract Gs/olf-coupled dopamine D1 and adenosine A2A receptor signalingin striatonigral and striatopallidal neurons, respectively. Thetonic activity of Gi-coupled a2-adrenoceptors is also requiredfor the action of dopamine D2 receptors, another Gi-coupledreceptor expressed in striatopallidal neurons. Thus, a2-adrenoceptors induce two distinct effects on dopaminesignaling: inhibition of dopamine D1 receptor signaling instriatonigral neurons and enhancement of dopamine D2receptor signaling in striatopallidal neurons. Such functionalfeatures of a2-aderenoceptors suggest that a2-aderenoceptorscould be a target of therapeutic drugs for Parkinsons disease.

Noradrenergic innervation and the release of noradrenalinein the striatumDespite the functional importance of noradrenaline indopaminergic neurotransmission, striatum receives onlysparse noradrenergic innervation (Swanson and Hartman1975; Aston-Jones 2004). However, the presence of nor-adrenaline at the extracellular spaces in the striatum wasdemonstrated by microdialysis studies (Cenci et al. 1992; Liet al. 1998; Dawson et al. 2000; Gobert et al. 2004), and thesynthesis of noradrenaline from dopamine in striatal tissueswas detected in vitro (Udenfriend and Creveling 1959; Rossand Reis 1974) and in vivo (McGeer et al. 1963), suggestingthe presence of dopamine b-hydroxylase activity in thestriatum. Furthermore, the presence of noradrenaline trans-porter in the striatum was reported (Moll et al. 2000; Moronet al. 2002; Arai et al. 2008). Recently, Gobert et al. (2004)demonstrated that noradrenaline in the striatum was derivedfrom noradrenergic terminals and its release was subject toinhibitory control by a2-adrenoceptors. It is also possible thatnoradrenaline may be synthesized by dopamine b-hydroxy-lase expressed in striatal cells other than noradrenergicterminals. We found that the relatively selective noradrena-line transporter inhibitor, nortriptyline, increased DARPP-32Thr34 phosphorylation via activation of adrenergic as well asserotonergic receptors in neostriatal slices, indicating thepresence of functional noradrenergic terminals in the stria-tum. This nding is supported by the fact that the lesioningof noradrenergic neurons by DSP-4 enhanced striatal dopa-mine D1 and adenosine A2A receptor/PKA/DARPP-32

(a) (b)

Fig. 7 Dopamine D1 and adenosine A2A receptor/protein kinase A/

dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DAR-

PP-32) signaling in neostriatal slices from saline- and DSP-4-treated

mice. Mice were intraperitoneally injected with saline or DSP-4, and

neostriatal slices were prepared from the mice after 7 days of injec-

tion. Neostriatal slices from saline (closed circles)- or DSP-4 (open

circles)-treated mice were treated with (a) a dopamine D1 receptor

agonist, SKF81297 (1 lM), or (b) an adenosine A2A receptor agonist,

CGS21680 (1 lM), for the indicated times. The levels of phospho-

Thr34 DARPP-32 were quantified by the Odyssey infrared imaging

system, and the data were normalized to values obtained from un-

treated slices in saline-treated mice. Data represent means SEM for

six to eight experiments. *p < 0.05, **p < 0.01 compared with values in

saline-treated mice; two-way ANOVA followed by Bonferroni test.



signaling. Taken together, the presence of noradrenergicterminals in the striatum is supported by neuropharmacolog-ical and biochemical data, although contradictory data havealso been reported (Versteeg et al. 1976; Oke et al. 1978).

Functional roles of b1-adrenoceptors in the striatumb1-Adrenoceptors are highly expressed in striatal neurons,including cholinergic interneurons (Nahorski et al. 1979;Pazos et al. 1985; Pisani et al. 2003). Immunohistochemicalanalysis revealed the expression of b1-adrenoceptors both instriatonigral and striatopallidal neurons. Activation of b1-adrenoceptors, but not of b2-adrenoceptors, by isoproterenolinduced the up-regulation of cAMP/PKA signaling, leadingto the phosphorylation of DARPP-32 at Thr34 in the twotypes of striatal medium spiny neurons. The isoproterenol-induced phosphorylation of DARPP-32 at Thr34 was notbecause of the release of dopamine and subsequent activationof D1 receptors (Reisine et al. 1982) or the cross activation ofD1 receptors by isoproterenol (Vanderheyden et al. 1986),since the effect of isoproterenol was not antagonized by thedopamine D1 receptor antagonist. Under physiological

conditions in vivo, b1-adrenoceptors might be activated bynoradrenaline released from noradrenergic terminals and/orby physically released dopamine, resulting in increasedexcitability of both striatonigral and striatopallidal neuronsbecause of activation of PKA/DARPP-32 signaling. Suchchanges in the activity of the basal ganglia circuit may play arole in psychomotor functions. The b-adrenergic antagonist,propranolol, has been used for the treatment of essentialtremor and anxiety disorders (Emilien and Maloteaux 1998;Pahwa and Lyons 2003), although the mechanisms underly-ing its therapeutic effect are not understood. Whether theeffects of b1-adrenoceptors on DARPP-32 phosphorylationin the two types of striatal neurons presented here relates tothe pathophysiology of essential tremor or anxiety disordersremains to be determined.

Functional roles of a2-adrenoceptors in the striatumActivation of a2-adrenoceptors by UK14304 attenuated thedopamine D1 and adenosine A2A receptor-induced increasein DARPP-32 Thr34 phosphorylation in striatal neurons. Ourprevious studies using striatal slices from D1-DARPP-32-

cAMP

P-T34 DARPP-32

PP-1

DARPP-32PKA

AC

Gi

Dopaminergic terminal

DA release

A2AD2

Striatopallidal neuron (D2-type/indirect pathway)

D1

Striatonigral neuron (D1-type/direct pathway)

cAMP

P-T34 DARPP-32

PP-1

DARPP-32PKA

ACGi

Golf

Gi

Gi

NA

NANA

NA

NA

Golf

NA acting on 2A/CR: DA release

DA acting on D2R: motor function NA acting on 2A/CR: A2AR function

DA acting on D1R: motor function NA acting on 2A/CR: D1R function

Golf

Golf

NA NA

NA acting on 2A/CR: D2R function

Fig. 8 Interaction of b1- and a2A/C-adrenoceptor signaling with

dopamine D1 and D2 receptor signaling in the striatum. b1- and a2A/C-

adrenoceptors regulate protein kinase A (PKA)/dopamine- and cAMP-

regulated phosphoprotein of Mr 32 kDa (DARPP-32) signaling both in

striatopallidal and striatonigral neurons, in addition to the inhibitory

regulation of dopamine (DA) release by a2A/C-adrenoceptors. In stri-

atopallidal neurons, activation of a2A/C-adrenoceptors, coupled to Gi,

inhibits adenosine A2A receptor/Gs/olf/adenylyl cyclase (AC)/PKA/

DARPP-32 signaling, and therefore enhances dopamine D2 receptor/

Gi signaling. In striatonigral neurons, activation of a2A/C-adrenoceptors

inhibits dopamine D1 receptor/Gs/olf/AC/PKA/DARPP-32 signaling.

Thus, a2A/C-adrenoceptors differentially regulate dopamine signaling in

two pathways, and activation of a2A/C-adrenoceptors by noradrenaline

(NA) is required for dopamine to elicit its regulatory role in motor

function via D2 receptors.



Flag/D2-DARPP-32-Myc mice (Bateup et al. 2008; Kuroiwaet al. 2008) demonstrated that activation of dopamine D1 andadenosine A2A receptors selectively stimulates DARPP-32Thr34 phosphorylation in striatonigral and striatopallidalneurons, respectively. Taken together, these ndings indicatethat activation of a2-adrenoceptors inhibits PKA/DARPP-32signaling in both types of striatal neurons.Pharmacological blockade of a2-adrenoceptors and chem-

ical lesioning of noradrenergic neurons induced the enhance-ment of dopamine D1 and adenosine A2A receptor/PKA/DARPP-32 signaling, indicating that a2-adrenoceptors areactivated under basal conditions and tonically inhibit PKA/DARPP-32 signaling in both striatopallidal and striatonigralneurons. It has been demonstrated that a2-adrenoceptors,specically a2C-subtype adrenoceptors, are negatively cou-pled to AC in the striatum using mice with infusion ofantisense oligonucleotides against a2C-adrenoceptor mRNA(Lu and Ordway 1997) and with targeted inactivation of thea2C-adrenoceptor gene (Zhang et al. 1999). The possibilitythat dopamine is the endogenous activator of a2-adrenocep-tors in the striatum has been proposed (Zhang et al. 1999),because of the sparse noradrenergic innervation. However,the data presented here suggest that a2-adrenoceptors aretonically activated by noradrenaline, because lesioning ofnoradrenergic neurons with DSP-4 mimics the effect of a2-adrenoceptor antagonism.In behavioral studies, it has been shown that amphet-

amine-induced locomotor activity is enhanced in micelacking a2C-adrenoceptors, whereas the opposite change isobserved in mice over-expressing a2C-adrenoceptors (Salli-nen et al. 1998b). Thus, a2C-adrenoceptors likely play aninhibitory role in the regulation of motor function underconditions of high dopamine tone (Scheinin et al. 2001). Ourbiochemical data demonstrate that activation of a2-adreno-ceptors induces (i) down-regulation of dopamine D1 receptor/PKA signaling in striatonigral neurons, (ii) down-regulationof adenosine A2A receptor/PKA signaling in striatopallidalneurons, and (iii) up-regulation of dopamine D2 receptorsignaling in striatopallidal neurons (Fig. 8). The role of a2C-adrenoceptors in amphetamine-induced locomotor activitycan be explained by the modulation of dopamine D1receptor/PKA signaling in striatonigral neurons. Removalof the inhibitory effect of a2C-adrenoceptors on dopamine D1receptor/PKA signaling and the subsequent activation ofstriatonigral neurons presumably results in the enhancementof amphetamine-induced locomotor activity. If a2C-adreno-ceptors were predominantly to affect striatopallidal neurons,then removal of the modulatory effect of a2C-adrenoceptorson striatopallidal neurons would induce activation of aden-osine A2A receptor/PKA signaling and inhibition of dopa-mine D2 receptor signaling, resulting in the activation ofstriatopallidal neurons and an attenuation of amphetamine-induced locomotor activity. Thus, when dopamine tone ishigh, the inhibitory role of a2C-adrenoceptors might be

important to suppress the over-activation of dopamine D1receptor/PKA signaling in striatonigral neurons. However,the role of a2C-adrenoceptors observed under conditions ofamphetamine-induced high dopamine tone seems to bedifferent from that under conditions of low dopamine tonein Parkinsons disease, as described below.At early time points (30 s and 1 min), activation of a2-

adrenoceptors induced a transient increase in DARPP-32Thr34 phosphorylation selectively in striatonigral neurons.The effect was not mediated through activation of dopa-mine D1 receptors in striatonigral neurons by releaseddopamine, because a dopamine D1 receptor antagonist,SCH23390, failed to block the effect of a2-adrenoceptoractivation (data not shown) and a2-adrenoceptors are knownto pre-synaptically inhibit dopamine release in the striatum(Bucheler et al. 2002). Activation of a2-adrenoceptors hasbeen reported to induce the release of GABA (Zhang andOrdway 2003) and phospholipase C-mediated activation ofPKA (Karkoulias et al. 2007), both of which may result inthe phosphorylation of DARPP-32 at Thr34 (Snyder et al.1994). It is not clear why activation of a2-adrenoceptorsinduces DARPP-32 phosphorylation selectively in striato-nigral neurons, since a2C-adrenoceptors are expressed inboth striatonigral and striatopallidal neurons. It is possiblethat transient activation of dopamine D2 receptors counter-acts the a2C-adrenoceptor-induced phosphorylation of DAR-PP-32 in striatopallidal neurons, although this is justspeculation and requires further study. However, mecha-nisms for the activation of PKA/DARPP-32 signalingselectively in striatonigral neurons were not further inves-tigated, because the a2-adrenoceptor-induced phosphoryla-tion of DARPP-32 at early time points was relatively smalland transient, and the physiological signicance of thisphenomenon is not clear.

Role of a2-adrenoceptors in Parkinsons diseaseIn animal models of Parkinsons disease, the degeneration ofnoradrenaline neurons in addition to dopamine neurons isinvolved in motor decits of Parkinsons disease (Srinivasanand Schmidt 2003; Rommelfanger et al. 2007), and thesymptoms of Parkinsons disease are improved by restora-tion of noradrenaline (Rommelfanger et al. 2007). Inaddition, in reserpine-induced akinesia, administration ofL-DOPA improves akinesia, and the effect of L-DOPA isreported to be mediated in part by the synthesis ofnoradrenaline and activation of a2-adrenoceptors (Dolphinet al. 1976a,b). These results indicate that noradrenalineplays a critical role in the regulation of motor functionsby interacting with dopamine signaling pathways in thestriatum.a2-Adrenoceptors expressed in striatopallidal neurons

likely play a therapeutic role in Parkinsons disease. Here,we nd that activation of a2-adrenoceptors in striatopallidalneurons induces down-regulation of adenosine A2A receptor/



PKA signaling and up-regulation of dopamine D2 receptorsignaling (Fig. 8). These modulatory effects of a2-adreno-ceptors potentiate the action of dopamine through dopamineD2 receptors, which likely improves the symptoms ofParkinsons disease. Indeed, it has been shown that clonidine,an a2-adrenoceptor agonist, itself increases locomotor activ-ity (Hill and Brotchie 1999), and enhances locomotor activityinduced by dopamine receptor agonists (Pycock et al. 1977),muscarinic receptor antagonists (Carlsson et al. 1991), and aj-opioid receptor agonist (Hill and Brotchie 1999) inmonoamine-depleted animals.Interestingly, a2-adrenoceptor antagonists, such as idazo-

xan are also reported to have antiparkinsonian effects andattenuate L-DOPA-induced dyskinesia (Grondin et al.2000; Srinivasan and Schmidt 2004; Fornai et al. 2007).a2-Adrenoceptor antagonists have been demonstrated toenhance the release of noradrenaline by blocking pre-synaptic a2-adrenoceptors (Dennis et al. 1987). Theincrease in noradrenergic neurotransmission likely resultsin the potentiation of dopaminergic signaling (Dickinsonet al. 1988; Mavridis et al. 1991), thereby inducing anti-parkinsonian effects. Both the inhibition of pre-synaptica2-adrenoceptors, which activates noradrenergic neurotrans-mission, and the stimulation of post-synaptic a2-adreno-ceptors could be a useful therapeutic approaches for thetreatment of Parkinsons disease. The therapeutic potentialof either a2-adrenergic antagonists or agonists may bedetermined by the stage of Parkinsons disease. Whennoradrenergic innervation is conserved in early stages, thea2-adrenoceptor antagonist is expected to improve thesymptoms of Parkinsons disease, whereas the a2-adreno-ceptor agonist may be useful in late stages. Indirect anddirect activation of a2-adrenoceptors by a2-adrenoceptorantagonists and agonists, respectively, likely potentiatesdopamine D2 receptor signaling in striatopallidal neuronsand inhibits dopamine D1 receptor signaling in striatonigralneurons. The modulation of dopaminergic signaling by a2-adrenoceptor activation may improve the symptoms ofParkinsons disease and prevent the development of L-DOPA-induced dyskinesia (Santini et al. 2009). The long-term effects of a2-adrenoceptor antagonists and/or agonistsin various stages of Parkinsons disease need to beclaried.

Acknowledgements

This research was supported by a Grant-in-Aid for Scientic

Research from the Japan Society for the Promotion of Science

(18300128 to A.N.) and grants from the U.S.P.H.S. (MH074866

and DA10044 to PG), the Michael Stern Parkinsons Research

Foundation (to PG) and the Department of Defense (DOD/

USAMRAA W81XWH-09-1-402 to PG). The authors thank

Yukako Terasaki, Keiko Fujisaki and Michiko Koga for excellent

technical assistance.

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