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Journal of Neural TransmissionBasic Neurosciences, Genetics andImmunology, Movement disorders,Dementias, Biological Psychiatry,Biological Child and AdolescentPsychiatry ISSN 0300-9564Volume 118Number 12 J Neural Transm (2011) 118:1733-1742DOI 10.1007/s00702-010-0571-8

Role of dopamine D3 and serotonin 5-HT1A receptors in l-DOPA-induceddyskinesias and effects of sarizotan in the6-hydroxydopamine-lesioned rat model ofParkinson’s diseaseManfred Gerlach, Gerd D. Bartoszyk,Peter Riederer, Olivia Dean & Maartenvan den Buuse

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Role of dopamine D3 and serotonin 5-HT1A receptorsin L-DOPA-induced dyskinesias and effects of sarizotan in the6-hydroxydopamine-lesioned rat model of Parkinson’s disease

Manfred Gerlach • Gerd D. Bartoszyk •

Peter Riederer • Olivia Dean • Maarten van den Buuse

Received: 7 December 2010 / Accepted: 17 December 2010 / Published online: 21 January 2011

� Springer-Verlag 2011

Abstract Sarizotan, a 5-HT1A agonist with additional

affinity for D3 and D4 receptors, has been demonstrated to

have anti-dyskinetic effects. The mechanism by which these

effects occur is not clear. Using unilateral 6-hydroxydop-

amine-lesioned rats that received chronic intraperitoneal (ip)

administration of L-3,4-dihydroxyphenylalanine (L-DOPA)

we investigated the involvement of D3 and 5-HT1A receptors

in the effects of sarizotan on contraversive circling and

abnormal involuntary movements (AIMs). Before sensitiza-

tion by chronic L-DOPA treatment (12.5 with 3.25 mg/kg

benserazide ip, twice daily for 21 days), no effect of the

selective D3 agonist, PD128907 (1 or 3 mg/kg ip), or the

selective D3 antagonist, GR103691 (0.5 or 1.5 mg/kg ip), was

observed. Treatment with sarizotan (1 or 5 mg/kg ip) dose-

dependently inhibited the L-DOPA-induced contraversive

turning and AIMs. In co-treatment with the 5-HT1A antago-

nist, WAY100635 (1 mg/kg ip), sarizotan failed to affect this

behaviour, confirming the prominent 5-HT1A receptor-medi-

ated mechanism of action. In the presence of PD128907

(3 mg/kg ip), the effects of sarizotan on contraversive turning,

locomotive dyskinesia and axial dystonia, but not on orolin-

gual and forelimb dyskinesia, were blocked. On its own,

PD128907 had no effect on the behavioural effects of L-DOPA

except that it tended to reduce orolingual and forelimb dys-

kinesia. GR103691 had no effect on its own or in combination

with sarizotan. These data identify an involvement of D3

receptors in the action of sarizotan on some, but not all

L-DOPA-induced motor side effects. This selective involve-

ment is in contrast to the more general involvement of 5-HT1A

receptors in the anti-dyskinetic effects of sarizotan.

Keywords L-DOPA-induced dyskinesia � Abnormal

involuntary movements � Sarizotan � Parkinson therapy �Dopamine D3 receptors � Serotonin 5-HT1A receptors

Abbreviations

AIMs Abnormal involuntary movements

ip Intraperitoneally and introperitoneal, respectively

L-DOPA Levodopa, L-3,4-dihydroxyphenylalanine

LID L-DOPA-induced dyskinesia

PD Parkinson’s disease

5-HT Serotonin, 5-hydroxytryptamine

6-OHDA 6-Hydroxydopamine

Introduction

The mainstay of treatment in Parkinson’s disease (PD) has

been palliative treatment for dopamine deficiency with

M. Gerlach (&)

Department of Child and Adolescent Psychiatry,

Psychosomatics and Psychotherapy, Laboratory for Clinical

Neurobiology, University of Wurzburg, Fuchsleinstrasse 15,

97080 Wurzburg, Germany

e-mail: manfred.gerlach@uni-wuerzburg.de

G. D. Bartoszyk

Extended Profiling, Medical Science and Innovation,

Merck Serono Research and Development,

Frankfurter Strasse 250, 64293 Darmstadt, Germany

P. Riederer

Laboratory for Clinical Neurochemistry,

Department of Psychiatry, Psychosomatics and Psychotherapy,

and National Parkinson Foundation Centers of Excellence for

Neurodegenerative Diseases Research, University of Wurzburg,

Fuchsleinstrasse 15, 97080 Wurzburg, Germany

O. Dean � M. van den Buuse

Behavioural Neuroscience Laboratory, Mental Health Research

Institute, 155 Oak Street, Parkville, VIC 3052, Australia

123

J Neural Transm (2011) 118:1733–1742

DOI 10.1007/s00702-010-0571-8

Author's personal copy

L-3,4-dihydroxyphenylalanine (L-DOPA, levodopa) and a

peripherally active amino acid decarboxylase inhibitor

such as benserazide and carbidopa. This treatment usually

provides a sustained clinical benefit to patients. However,

after a few years of treatment, motor complications, par-

ticularly motor fluctuations such as wearing off, on–off

phenomena, and several different types of L-DOPA-

induced dyskinesia (LID) such as peak-dose, diphasic,

wearing-off, chorea, dystonia and athetosis, limit its use-

fulness (Jankovic 2005). Dyskinesia consists of two com-

ponents: the execution of involuntary movements in

response to drug administration, and the priming phe-

nomenon that underlies these movement’s establishment

and persistence (Jenner 2008; Nadjar et al. 2009). The

precise pathophysiology of these motor effects is not

defined, although many theories have been postulated.

Recent clinical and experimental findings indicate that

dyskinesia is associated with many functionally related

abnormalities in neurotransmitter systems which lead to

abnormalities in the rate, pattern and synchronisation of

neuronal activity within and outside the basal ganglia

(Brotchie 2005; Cenci 2007; Jenner 2008). Based on this

knowledge several approaches have been developed that

may prevent the development of LID in patients with PD or

that may reduce these involuntary movements without

antagonising the effects of the concomitant anti-Parkinso-

nian medication (Brotchie 2005; Fox et al. 2006; Muller

and Russ 2006). One of these approaches targets the

serotonin (5-hydroxytryptamine, 5-HT) system (for exam-

ple: Rylander et al. 2010; Zeng et al. 2010). Mounting

evidence (Arai et al. 1998; Muller and Russ 2006; Carta

et al. 2007; Eskow et al. 2009) suggests that the non-

physiological release of dopamine by 5-HT neurons leads

to the development of LID. Indeed, it was demonstrated in

experimental models of dyskinesia that lesions of rostral

raphe nuclei reduce the development of LID (Eskow et al.

2009) and administration of 5-HT1A agonists reduces LID

without impairing L-DOPA-improved motor performance

(Bibbiani et al. 2001; Srinivasan and Schmidt 2004; Dupre

et al. 2007; Eskow et al. 2009).

In rats, LID can be modelled via intracerebral injec-

tion of the dopaminergic neurotoxin 6-hydroxydopamine

(6-OHDA)—that damages the nigrostriatal pathway (Gerlach

and Riederer 1996)—followed by chronic administration of

low doses of L-DOPA. This treatment causes a sensitization

of the contraversive circling response (Henry et al. 1999)

and characteristic abnormal involuntary movements

(AIMs) with dyskinesia-associated cellular responses

(Lundblad et al. 2004). This model has been pharmaco-

logically validated and represents a cost-effective alterna-

tive to non-human primates for screening drugs with

potential anti-dyskinetic properties (Lundblad et al. 2002;

Dekundy et al. 2007).

Sarizotan is a novel compound with full 5-HT1A

receptor agonist properties and additional high affinity for

dopamine D3 and D4 receptors (Bartoszyk et al. 2004). In

rodent and primate models of PD, administration of sari-

zotan was shown to reduce LID without affecting the

efficacy of L-DOPA (Bibbiani et al. 2001; Gregoire et al.

2009, Marin et al. 2009). In PD patients with LID, sari-

zotan has been reported to attenuate these disabling motor

side effects at low doses (Olanow et al. 2004; Goetz et al.

2007) and prolong the efficacy of L-DOPA in patients with

advanced PD (Bara-Jimenez et al. 2005). The mechanism

of action of sarizotan has not been completely elucidated.

The present study was therefore focused on the role of

dopamine D3 receptors in LID and effects of sarizotan. The

effects of sarizotan on sensitization of the contraversive

circling response and AIMs were tested in combination

with PD128907 (a selective D3 agonist; Pugsley et al.

1995) or GR103691, a selective dopamine D3 antagonist

(Audinot et al. 1998). In addition, we confirmed a role of

the 5-HT1A receptor by testing the effect of WAY100635, a

selective HT1A antagonist (Ahlenius et al. 1999), in com-

bination with sarizotan.

Materials and methods

Materials

L-DOPA, benserazide and all other substances were of the

highest purity and were purchased from Sigma Chemical

Co. (St. Louis, MO, USA). Sarizotan HCl (EMD 128130;

batch EE79580) was a gift from Merck KGaA (Darmstadt,

Germany). L-DOPA was administered as the methyl ester

form, as it is more stable and soluble than non-esterified

DOPA. L-DOPA methyl ester is rapidly de-esterified in

vivo by non-specific esterases to form L-DOPA (Cooper

et al. 1984). In all experiments L-DOPA was administered

at the same time as benserazide. All drugs were dissolved

in sterile saline (except where indicated) and injected

intraperitoneally (ip) at a volume of 1 ml/kg of body

weight. Sarizotan and GR103691 were dissolved and son-

icated in DMSO which was then diluted with distilled

water to the required dose and volume to an end concen-

tration of 1–5% DMSO. PD128907 was dissolved in

straight 5% DMSO.

Animals

Animal experiments were carried out at the Behavioural

Neuroscience Laboratory, Mental Health Research Insti-

tute, Melbourne, Australia. All procedures complied with

the NIH Guide for the care and use of laboratory animals.

1734 M. Gerlach et al.

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The University of Melbourne Animal Experimentation

Ethics Committee approved all surgical techniques, treat-

ments and experimental protocols.

Male Sprague–Dawley rats (Animal Resource Centre,

Canning Vale, Western Australia) weighing 250–300 g

were housed in pairs on a 12-h light/dark cycle with an

ambient temperature of 21 ± 2�C. Rats had access to

standard rat chow and water ad libitum.

6-OHDA lesion surgeries

The rat hemiparkinsonian rat model with unilaterally

6-OHDA lesioning was generated as we described previ-

ously (Gerlach et al. 2004). In brief, the animals were

treated ip with pargyline (5 mg/kg) and desipramine

(20 mg/kg) to enhance the effect of 6-OHDA on dopamine

neurons and to block its effect on noradrenergic neurons,

respectively. The rats were then anesthetized with isoflu-

rane and mounted in a Kopf stereotaxic frame equipped

with a mouth mask to deliver gas anaesthetic. Unilateral

6-OHDA lesion of the median forebrain bundle was done

in the right side of the brain. Stereotaxic coordinates were

-2.8 mm from bregma, 2 mm lateral and 9 mm ventral to

the skull surface (Paxinos and Watson 1997). A volume of

2.5 ll of 6-OHDA HCl solution (5 mg/ml with 0.1%

ascorbic acid) was injected over 5 min. The rats were

allowed 3 weeks of post-operative recovery.

Treatment regimens

The entire experiment was done in one large cohort of

6-OHDA-lesioned rats that were selected on the basis of

preliminary experiments to assess spontaneous and apo-

morphine-induced turning responses (data not shown). On

day 0 (Table 1), animals were tested with sarizotan,

PD128907 or GR103691, to assess possible activity of the

various drugs to induce turning behaviour in the non-

sensitized state.

The next step was aimed at assessing the mechanisms

involved in the ability of sarizotan to inhibit L-DOPA-

induced turning and AIMs (Table 1). Therefore, all rats

were ip injected twice per day (around 9 a.m. and around

5 p.m.) with 12.5 mg/kg of L-DOPA methyl ester and

3.25 mg/kg of benserazide, except on experimental days

where only one injection of the combination was given.

The development of sensitization to this chronic treatment

was verified on day 7 and day 14 of treatment. The results

from these two measurement days were also used to select

the 36 best responders to L-DOPA before further tests with

sarizotan, PD128907, GR103691 and WAY100635 were

done. The studies on the mechanism of action of sarizotan

on L-DOPA responses were conducted on days 21, 24, 27

and 30 after the start of L-DOPA treatment.

Behavioural measurements

Rotational behaviour was measured by an automated

Rotacount system (Columbus Instruments, Columbus, OH,

USA) as described previously (Gerlach et al. 2004),

allowing unbiased recording of full and partial clockwise

and counter-clockwise turns. The number of full clockwise

(ipsiversive) and counter-clockwise (contraversive) turns

was recorded every 5 min during 2 h.

AIMs were assessed according to Cenci et al. (1998).

They were classified into four subtypes: locomotive dys-

kinesia, i.e. increased locomotion with contralateral side

bias; axial dystonia, i.e. contralateral twisted posturing of

the neck and upper body; orolingual dyskinesia; i.e. ste-

reotyped jaw movements and contralateral tongue protru-

sion; forelimb dyskinesia, i.e. repetitive rhythmic jerks of

dystonic posturing of the contralateral forelimb and/or

grabbing movements of the contralateral paw. For each of

these four subtypes, a score from 0 to 4 (0 = absence;

4 = severe) was given and a total score calculated. AIMs

were recorded by an observer every 5 min for about 10 s.

Data analyses

All rotation data and AIMs data were collected in an Excel

spreadsheet to calculate total summed scores and group

averages. From a small number of vehicle-treated controls

on every testing day, we compiled a combined vehicle

group to which the effects of all drug treatments were

compared. Data are presented as mean ± standard error of

the mean (SEM). Using the statistical software pack-

age Systat (version 9.0, SPSS Inc., USA), groups were

compared with analysis of variance (ANOVA). When

P \ 0.05, differences between groups were considered

statistically significant.

Results

Before the start of chronic L-DOPA treatment, no signifi-

cant effect was observed of any of the doses of sarizotan,

the selective D3 receptor agonist, PD128907, or the

selective D3 receptor antagonist, GR103691 (not shown).

Rats with a unilateral 6-OHDA lesion chronically treated

with L-DOPA showed increased total numbers of contra-

versive rotation on 7 and 14 days of treatment (data not

shown). The L-DOPA-treated rats also showed develop-

ment of AIMs on day 21 and later.

Effects of sarizotan on turning behaviour and AIMs

Injection of 12.5 mg/kg of L-DOPA with 3.25 mg/kg

of benserazide ip in chronically-treated rats induced

Role of dopamine D3 and serotonin 5-HT1A receptors in L-DOPA-induced dyskinesias 1735

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contraversive turning which was maximal at 15–75 min

after injection. Treatment with 1 and 5 mg/kg of sarizotan

caused a dose-dependent and significant reduction of this

turning behaviour (Fig. 1). Since the effect of L-DOPA and

of sarizotan was most clear at 15–75 min after injection,

the remaining analysis only includes data from this time

segment. The frequency of all AIMs, i.e. locomotive dys-

kinesia, axial dystonia, orolingual dyskinesia and forelimb

dyskinesia, between 15 and 75 min after L-DOPA injection

was dose-dependently and significantly reduced by pre-

treatment with sarizotan (Fig. 1).

Effects of PD128907 on turning behaviour and AIMs

Injection of 1 mg/kg ip of the selective dopamine D3

receptor agonist, PD128907, had no significant effect on

turning behaviour and AIMs (data not shown). Injection of

3 mg/kg of PD128907 significantly reduced forelimb dys-

kinesia and tended to reduce orolingual dyskinesia (Fig. 2).

In the presence of 3 mg/kg of PD128907, sarizotan failed

to significantly reduce contraversive turning, locomotive

dyskinesia and axial dystonia, but there was no change in

the effect of sarizotan on orolingual dyskinesia and fore-

limb dyskinesia (Fig. 2). In addition, axial dystonia scores

were significantly higher in the animals treated with both

PD128907 and sarizotan compared to sarizotan alone

(Fig. 2). Thus, stimulating the dopamine D3 receptor

appeared to influence only specific aspects of the AIMs.

Effects of GR103691 on turning behaviour and AIMs

Injection of 0.5 or 1.5 mg/kg ip of the selective dopamine

D3 receptor antagonist, GR103691, had no effect on turning

behaviour and AIMs (Table 2). In addition, co-treatment

with GR103691 (1.5 mg/kg) had no significant effect on

L-DOPA-induced turning behaviour following sarizotan

treatment. However, there was a trend that it enhanced some

effects of sarizotan, i.e. axial, orolingual and forelimb dys-

kinesia (Table 2).

Effects of WAY100635 on turning behaviour and AIMs

Injection of 1 mg/kg of the 5-HT1A receptor antagonist,

WAY100635, had no effect on the number of contralateral

turns or AIMs scores. In the presence of co-treatment with

WAY100635, 5 mg/kg of sarizotan had no effect on turn-

ing or AIMs (Table 2), confirming its prominent 5-HT1A

receptor-mediated mechanism of action.

Discussion

The major findings of the present study are: (1) sarizo-

tan and the dopamine D3 receptor agonist/antagonist,

PD128907/GR103691, have no anti-parkinsonian activity

in unilaterally 6-OHDA-lesioned rats; (2) acute treatment

with sarizotan (5 mg/kg ip) reduced sensitized contraver-

sive circling and attenuated AIMs induced by chronic

L-DOPA treatment in unilaterally 6-OHDA-lesioned rats

while eliciting no effect on the contraversive circling in

non-L-DOPA-primed animals; (3) co-treatment with the

higher dose of the dopamine D3 receptor agonist PD128907

(3 mg/kg ip) blocked at least some of the behavioural

effects of sarizotan, while co-treatment with the antagonist

GR103691 (0.5 or 1.5 mg/kg ip) had no effect; (4) co-

treatment with the serotonin 5-HT1A receptor antagonist

WAY100635 (1 mg/kg ip) blocked the action of sarizotan

on all AIM categories and on turning behaviour.

Table 1 Timetable for experiments

Day 21 Surgery of animals to induce unilateral 6-OHDA lesions of the median forebrain bundle

Day 0 Testing of ip administered drugs in non-sensitized rats: sarizotan 1 or 5 mg/kg, PD128907

1 or 3 mg/kg, GR103691 0.5 or 1.5 mg/kg, vehicle or (for comparison) apomorphine

Day 1 Start of chronic treatment with 12.5 mg/kg of L-DOPA methyl ester and 3.25 mg/kg of benserazide ip, twice daily

Day 7 Confirming L-DOPA-induced turning behaviour (no AIMs recorded)

Day 14 Confirming L-DOPA-induced turning behaviour (no AIMs recorded)

Day 21 L-DOPA sensitized rats were tested with sarizotan (1 or 5 mg/kg ip), PD128907

(1 or 3 mg/kg ip) or GR103691 (0.5 or 1.5 mg/kg ip), or vehicle

Day 24 L-DOPA sensitized rats were tested with sarizotan (1 or 5 mg/kg ip), PD128907 (1 or 3 mg/kg ip) or GR103691

(0.5 or 1.5 mg/kg ip), or vehicle

Day 27 L-DOPA sensitized rats were tested with vehicle, WAY100635 (1 mg/kg ip) or sarizotan 5 mg/kg ip plus WAY100635

Day 30 L-DOPA sensitized rats were tested with vehicle, PD128907 (3 mg/kg ip) plus sarizotan (5 mg/kg ip),

or GR103691 (1.5 mg/kg ip) plus sarizotan (5 mg/kg ip)

6-OHDA 6-hydroxydopamine, AIMs abnormal involuntary movements, ip, intraperitoneally, L-DOPA L-3,4-dihydroxyphenylalanine, levodopa,

GR103691 {49-acetyl-N-{4-[(2-methoxy-phenyl)-piperazin-1-yl]-butyl}-biphenyl-4-carboxamide (selective D3 receptor antagonist), PD128907{(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]-benzopyrano-[4,3-b]-1,4-oxazin-9-ol} (selective D3 receptor agonist), WAY100635N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane-carboxaminde (5-HT1A receptor selective antagonist)

1736 M. Gerlach et al.

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The unilateral 6-OHDA-lesioned rat is a widely used

animal model of PD which enables simple and automated

quantification of behavioural responses to drug treatments

(Ungerstedt and Arburthnott 1970; Gerlach and Riederer

1996). Classically, contraversive circling in rats with uni-

lateral 6-OHDA lesions is taken as an indication of the

potential of compounds to reverse the motor symptoms of

PD. L-DOPA and selective dopamine D2 receptor agonists

all evoke contraversive rotations in 6-OHDA-lesioned rats

as a result of post-synaptic dopamine stimulation and a

supersensitivity of dopamine receptors on the lesioned side.

In the first part of this study (before the start of the chronic

L-DOPA treatment), we were interested in determining the

potential anti-parkinsonian activity of sarizotan and the

dopamine D3 receptor agonist/antagonist, PD128907/

GR103691. No significant effect was observed of any of

these drugs. Recently, Lane et al. (2006) suggested that the

contraversive circling may not represent just anti-parkin-

sonian activity but may also indicate the ability to induce

or prime for dyskinesia. This is reinforced by the finding

that in contrast to L-DOPA and dopamine D2 receptor

agonists, BTS 74 398 (a monoamine uptake inhibitor)

neither sensitizes rotational behaviour in 6-OHDA-lesioned

rats nor induces dyskinesia in MPTP-treated primates

primed with L-DOPA (see Lane et al. 2006). Therefore, our

results suggest that sarizotan does have a low potential to

induce or prime for dyskinesia.

Chronic treatment of unilateral 6-OHDA-lesioned rats

with L-DOPA induced enhanced contraversive turning

response and was considered indicative of dyskinesiogenic

Fig. 1 The effect of

intraperitoneal treatment with

1 or 5 mg/kg of sarizotan (Sar1

and Sar5, respectively) on

contraversive turning and

abnormal involuntary

movements (AIMs) in rats

which had unilateral 6-OHDA-

induced lesions and were

chronically treated with

L-DOPA and benserazide.

Sarizotan pre-treatment dose-

dependently reduced L-DOPA-

induced turning and AIMs. Rats

were treated intraperitoneally

(ip) with L-DOPA methyl ester

(12.5 mg/kg) and benserazide

(3.25 mg/kg) twice daily for

20 days. On day 21, L-DOPA-

sensitized rats were treated ip

with sarizotan 30 min prior to

administration of L-DOPA

methyl ester and benserazide.

The number of animals per

group was n = 29 for the

combined vehicle treatment

group, n = 12 for the Sar1

group and n = 10 for the Sar5

group. *P \ 0.05 for

comparison with the vehicle

responses on all days combined

Role of dopamine D3 and serotonin 5-HT1A receptors in L-DOPA-induced dyskinesias 1737

123

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potential (Carey 1991). In recent years, however, the rel-

evance of rotational behaviour has been called in question

(Castaneda et al. 2005) and alternative methods that

resemble clinical manifestations of LID have been devel-

oped (Lundblad et al. 2002). The AIMs procedure used in

the present study employs discrete behavioural measures,

display face validity with known anti-dyskinetic com-

pounds, and demonstrate consistency throughout treatment

in L-DOPA-primed rats (Lundblad et al. 2002; Bishop et al.

2006).

Recent research suggests that the dopamine D3 receptor

(Bordet et al. 1997; Kostrzewa et al. 2004) and serotonin

5-HT1A receptor (Bibbiani et al. 2001; Iravani et al. 2003;

Bishop et al. 2006) may play a pivotal role in the sensiti-

zation of contraversive cycling and AIMs induced by

repeated L-DOPA administration in the 6-OHDA-lesioned

rat. We pharmacologically characterized the involvement

of the dopamine D3 receptor and the serotonin 5-HT1A

receptor in the anti-dyskinetic action of sarizotan. We

confirmed the potency of sarizotan to block not only

Fig. 2 The effect of intraperitoneal treatment with 3 mg/kg of

PD128907 on contraversive turning and abnormal involuntary

movements in rats which had unilateral 6-OHDA-induced lesions

and were chronically treated with L-DOPA and benserazide. The

effect of 5 mg/kg sarizotan to reduce L-DOPA-induced turning,

locomotive dyskinesia and axial dystonia was not observed after co-

treatment with 3 mg/kg of PD128907 (PD3). The effect of sarizotan

on orolingual dyskinesia and forelimb dyskineasia was not modified

by PD128907, which significantly reduced forelimb dyskinesia on its

own. Rats were treated intraperitoneally (ip) with L-DOPA methyl

ester (12.5 mg/kg) and benserazide (3.25 mg/kg) twice daily for

20 days. On day 21, L-DOPA-sensitized rats were treated 30 min

prior to administration of L-DOPA methyl ester and benserazide ip

with either sarizotan (5 mg/kg, Sar5), the selective D3 agonist

PD128907 (3 mg/kg, PD3), or the combination of these drugs. Data

for vehicle and sarizotan (Sar5) treatment are the same as in Fig. 1

and are included here for comparison. The number of animals per

group was n = 29 for the combined vehicle-treatment, n = 10 for the

PD3 group, n = 10 for the Sar5 group, and n = 9 for the PD3 ? Sar5

group. *P \ 0.05 for comparison with vehicle treatment on all days

combined. #P \ 0.05 for comparison with sarizotan treatment

1738 M. Gerlach et al.

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turning responses (Bibbiani et al. 2001), but also demon-

strated that sarizotan reduces L-DOPA-induced AIMs.

Co-treatment with the 5-HT1A receptor antagonist,

WAY100635, blocked the action of sarizotan, confirming

the importance of this receptor subtype in its mechanism of

action. Additionally, co-treatment with the higher dose of

PD128907 blocked at least some of the behavioural effects

of sarizotan, implying a major interaction with the dopa-

mine D3 receptor in the action of sarizotan as well. It

should be noted, however, that treatment with the dopa-

mine D3 antagonist, GR103691, had no effect on its own on

L-DOPA-induced turning behaviour or AIMs, nor did it add

to the effect of sarizotan, findings not directly supporting

an involvement of dopamine D3 receptor stimulation in

LID. There could be a number of reasons for this incon-

sistency. First, the effect of activation of dopamine D3

receptors on AIMs and turning behaviour could simply be

parallel and additive to the action of sarizotan, and not an

integral part of its in vivo pharmacology. Indeed, treatment

with PD128907 tended to induce reductions of some AIMs

on its own (Fig. 2). The second reason why GR103691 did

not have the same effect as sarizotan could be that its dose

was too low and that much higher doses of this antagonist

are needed to induce behavioural effects in combination

with sarizotan or the high dose of PD128907. Finally, the

agonist PD128907—as well as sarizotan—might act as a

competitive dopamine antagonist in the presence of

endogenous dopamine derived from L-DOPA.

There are some inconsistent findings concerning the role

of the dopamine D3 receptor in locomotor activity in nor-

mal rats and LID in 6-OHDA-lesioned rats (see Visanji

et al. 2006). In the intact rodent striatum, D3 receptor

expression is smaller than one percent of that of D1 and D2

receptors (Levesque et al. 1992). However, following

chronic L-DOPA treatment to 6-OHDA-lesioned rats, the

level of striatal D3 expression was demonstrated to rise

above that of vehicle-treated animals (Bordet et al. 1997).

Therefore, it was suggested that stimulation of striatal

dopamine D3 receptor expression may be involved in LID.

This suggestion leads to the idea that reduction of dopa-

mine D3 receptor-mediated neurotransmission may be a

useful strategy to attenuate LID without any effect on the

efficacy of L-DOPA treatment. However, this hypothesis is

inconsistent with findings showing anti-dyskinetic effects

of dopamine D3 receptor agonists (Hsu et al. 2004) and

with data from non-lesioned, normal animals in which D3

receptor stimulation has been shown to attenuate move-

ment (Millan et al. 2004). The first reports on the motor

effects of dopamine D3 receptor function in normal rats,

using the D3 receptor-preferring agonist 7-OH-DPAT,

demonstrated decreased locomotion at low, D3 receptor-

selective doses and enhanced locomotion at higher non-

selective doses (Daly and Waddington 1993; Svensson

et al. 1994). In addition, a D3-receptor-preferring antago-

nist, nafadotride, has been demonstrated to have the

opposite effect (Sautel et al. 1995). D3 knock-out mice

have also been reported to be hyperactive, providing more

support for an inhibitory role of D3 receptor stimulation on

locomotor activity (see for a review Holmes et al. 2004).

A surprising finding was that the interaction of

PD128907 and sarizotan was seen with locomotive dyski-

nesias, axial dystonia and turning behaviour, but not with

orolingual and forelimb dyskinesia (Fig. 2) as well as the

trend of GR103691 to enhance sarizotan’s effect on axial,

Table 2 The effect of treatment with 0.5 or 1.5 mg/kg of GR103691 on contraversive turning or abnormal involuntary movements (AIMs) in

rats which had unilateral 6-OHDA-induced lesions and were chronically treated with L-DOPA and benserazide

Treatment (dose mg/kg) Turns Locomotive dyskinesia Axial dystonia Orolingual dyskinesia Forelimb dyskinesia n

Vehicle 746 ± 107 17.4 ± 2.4 17.4 ± 2.7 14.6 ± 2.5 14.3 ± 2.5 29

Sarizotan (5) 242 ± 117* 5.3 ± 2.9* 3.5 ± 2.2* 2.6 ± 1.7* 3.0 ± 1.7* 10

GR103691 (0.5) 510 ± 185 13.6 ± 4.7 13.9 ± 5.0 11.4 ± 5.3 10.8 ± 5.2 10

GR103691 (1.5) 577 ± 150 15.8 ± 3.9 17.4 ± 4.5 14.8 ± 4.4 13.6 ± 4.3 11

GR103691 (1.5)

? sarizotan (5)

152 ± 64* 4.6 ± 2.1* 0.2 ± 0.1* 0.0 ± 0.0* 0.6 ± 0.4* 9

WAY100635 (1) 721 ± 163 18.6 ± 5.2 20.1 ± 5.9 19.0 ± 6.4 18.4 ± 6.4 7

WAY100635 (1)

? sarizotan (5)

647 ± 191 13.3 ± 5.2 14.6 ± 6.0 12.0 ± 5.9 12.0 ± 5.8 7

Treatment with 5 mg/kg sarizotan significantly reduced L-DOPA-induced turning and all AIMs categories on its own and in the presence of co-

treatment with GR103691, which had no effect on its own. In contrast, in the presence of WAY100635, sarizotan did not significantly alter

behaviour. Rats were treated intraperitoneally (ip) with L-DOPA methyl ester (12.5 mg/kg) and benserazide 3.25 mg/kg) twice daily for 20 days.

On day 21, L-DOPA-sensitized rats were treated 30 min prior to administration with L-DOPA methyl ester and benserazide ip with either

sarizotan, the selective D3 antagonist GR103691, the selective 5-HT1A receptor agonist WAY100635, or a combination of these drugs. Data for

vehicle and sarizotan treatment are the same as in Fig. 1 and are included here for comparison

Data are mean ± SEM and were analyzed with ANOVA. * P \ 0.05 for comparison with the vehicle responses on all days combined

Role of dopamine D3 and serotonin 5-HT1A receptors in L-DOPA-induced dyskinesias 1739

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orolingual and forelimb dyskinesia (Table 2). This sug-

gests that the mechanism responsible for AIMs is not the

same for all categories. This could indicate that different

brain systems are involved in the different AIMs, however,

at this point in time this is speculative.

Consistent with previous studies showing that the effects

of direct or indirect 5-HT1A receptor agonists with anti-

dyskinetic properties were blocked by administration of

selective 5-HT1A receptor antagonists (Bibbiani et al. 2001;

Iravani et al. 2003; Bishop et al. 2006), we found that co-

treatment with WAY100635 attenuated the action of sari-

zotan on all AIM categories and on turning behaviour. These

findings confirm that the anti-dyskinetic effects of sarizotan

involve the 5-HT1A receptor. An essential difference

between the interaction of WAY100635 and PD128907 with

sarizotan was that the former drug blocked all AIMs cate-

gories. This again supports that an involvement of 5-HT1A

receptors forms a fundamental component of the effect of

sarizotan; sarizotan reduced cortical and striatal glutamate

concentration in the corticostriatal glutamate pathway via

5-HT1A receptors in the cortex (Antonelli et al. 2005) and

L-DOPA-induced turning behaviour and AIMs in 6-OHDA-

lesioned rats when sarizotan was locally injected in the

subthalamic nucleus (Marin et al. 2009), a structure in basal

ganglia circuitry expressing 5-HT1A receptors. However, the

precise mechanism of this involvement remains further to be

elucidated. Ba et al. (2007) reported that 1 mg/kg 8-OH-

DPAT ip administered reduced L-DOPA-induced motor

complications. On the other hand, a lower dose (0.3 mg/kg)

induced ipsiversive circling in unilateral 8-OHDA-lesioned

rats (Mignon and Wolf 2007), whereas the higher doses

(1–3 mg/kg) gave rise to contraversive circling (Gerber et al.

1998; Matsubara et al. 2006).

In conclusion, chronic L-DOPA administration induced

behavioural responses in unilateral 6-OHDA-lesioned rats

similar to LID in PD patients that could be reduced by

sarizotan. The present experiments clearly identify an

involvement of the dopamine D3 receptor and the 5-HT1A

receptor in these behaviours, however, the results also

show the complexity of these interactions, highlighting

several details of the behavioural responses that can be

selectively and differentially modulated.

Acknowledgment This study was funded by Merck KGaA

(Darmstadt, Germany) without restriction.

Conflict of interest The authors declare that they have no conflict

of interest.

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