Dopamine d3 Schizophrenia

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CNS & Neurological Disorders - Drug Targets, 2006,5, 25-43 25

1871-5273/06 $50. 00+. 00 2006 Bentham Science Publishers Ltd.

The Dopamine D3 Receptor: A Therapeutic Target for the Treatment ofNeuropsychiatric Disorders

P. Sokoloff*,1, J. Diaz2, B. Le Foll1, O. Guillin1, L.Leriche1, E. Bezard3 and C. Gross3

1

INSERM, Unit de Neurobiologie et Pharmacologie, Molculaire (U573), Centre Paul Broca, 2ter rue d'Alsia, 75014Paris, France2

Universit Ren Descartes, Laboratoire de Physiologie, Facult de Pharmacie, Avenue de l'Observatoire, 75005Paris, France3CNRS UMR 5543, Basal Gang,

Laboratoire de Neurophysiologie, Universit Victor Segalen, 33076 Bordeaux, France

Abstract: The role of the D3 receptor has remained largely elusive before the development of selective research

tools, such as selective radioligands, antibodies, various highly specific pharmacological agents and knock-out

mice. The data collected so far with these tools have removed some of the uncertainties regarding the functions

mediated by the D3 receptor. The D3 receptor is an autoreceptor that controls the phasic, but not tonic activity of

dopamine neurons. The D3 receptor, via regulation of its expression by the brain-derived neurotrophic factor

(BDNF), mediates sensitization to dopamine indirect agonists. This process seems responsible for side-effects of

levodopa (dyskinesia) in the treatment of Parkinsons disease (PD), as well as for some aspects of conditioning to

drugs of abuse. The D3 receptor mediates behavioral abnormalities elicited by glutamate/NMDA receptor blockade,which suggests D3 receptor-selective antagonists as novel antipsychotic drugs. These data allow us to propose

novel treatment options in PD, schizophrenia and drug addiction, which are awaiting evaluation in clinical trials.

Keywords: Brain-derived neurotrophic factor, Autoreceptor, Parkinson's disease, Schizophrenia, Drug addiction, Depression.

1. INTRODUCTION

The pleiotropic actions of dopamine have long beenassumed to result from the interaction with two types ofreceptors, termed D1 and D2 receptors. In spite of previoussuggestions of additional dopamine receptors, the discoveryof the D3 receptor [1] was rather unexpected, as was that ofD4 and D5 receptors that followed [2, 3]. From the

beginning, attention has been attracted to the restricteddistribution of the D3 receptor in the brain, seemingly relatedto functions of dopamine associated with the limbic brain.Hence, the hypothesis has been put forward that the D3receptor could be involved in the pathophysiology of severalpsychiatric disorders, such as schizophrenia and drugaddiction, which result from dysfunction of dopamineneurotransmission. The involvement of D3 receptors inpathological conditions has received some support frompost-mortem clinical studies. Nevertheless, the initial lack ofselective pharmacological tools, which delayed assessment ofthe role of D3 receptor in vivo, raised questions about thephysiological significance of the D3 receptor.

The D3 receptor belongs to the family of G protein-

coupled receptors, whose topography is characterized by theoccurrence of seven transmembrane domains. Its primarysequence is close to that of the D2 receptor, and to a lesserextent, to the D4 receptor. Information on the structure of theD3 receptor and its intracellular signaling has been publishedelsewhere [4-6]. The present chapter aims at measuring the

*Address correspondence to this author at the Institut de Recherche PierreFabre, Exploratory R&D Centre, Neurology-Psychiatry, 17 avenue Jean-Moulin, 81106 Castres cedex, France; Tel: (33 ) 5 63 71 42 65; Fax: (33 1)5 63 37 09 62; E-mail: [email protected]

progress accomplished in the field fifteen years after thidentification of the D3 receptor and reviews the anatomicapharmacological, genetic and clinical data presentlavailable. These data, still incomplete to some extent, nowreveal functions mediated by the D3 receptor and itinvolvement in the pathophysiology of several neuropsychiatric disorders. D3 receptor-selective pharmacologicaagents should also be considered as novel treatment option

in these disorders.

2. PRE- AND POST-SYNAPTIC LOCALIZATIONOF THE D3 RECEPTOR IN THE BRAIN

In rat brain, in which the phenotypes of neuronexpressing the D3 receptor have been characterized, the largesreceptor densities occur in granule cells of the islands oCalleja and in medium-sized spiny neurons of the rostral anventromedial shell of nucleus accumbens, which co-expresthe D1 receptor, substance P, dynorphin and/or neurotensi[7-9]. These output neurons from the nucleus accumbenreceive their dopaminergic innervation from the ventrategmental area and reach the entorhinal and prefrontal cortexafter relays in the ventral pallidum and mediodorsa

thalamus. In turn, the shell of nucleus accumbens receiveprojections from the cerebral cortex (infralimbic, ventraagranular, insular and piriform areas), hippocampus anamygdala and also projects to the ventral tegmental area fromwhich dopaminergic afferents originate [10, 11]. Thesvarious specific connections of the shell of nucleuaccumbens, a part of the "extended amygdala" [12], suggesthat this area is involved in a series of feedback or feedforward loops, involving notably the prefrontal cortex anventral tegmental area and subserving control of emotionsmotivation and reward.


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26 CNS & Neurological Disorders - Drug Targets, 2006, Vol. 5, No. 1 Sokoloff et al

In the human and non-human primate brains, thephenotype of neurons expressing the D3 receptor are not yetidentified, but several studies show their distribution to berather similar to that in the rat (highest levels in islands ofCalleja and nucleus accumbens, see Fig. 1) with, however,higher densities and larger distribution in the ventral part ofthe caudate putamen and the cerebral cortex [13-19].

One aspect of the localization and function of the D3receptor, which has remained highly debated is its occurrenceas an autoreceptor, regulating the activity of dopamineneurons. The existence of D3 autoreceptors was originallyproposed on the basis of the expression of D3 receptormRNA in substantia nigra and the ventral tegmental area,which strongly decreases after lesion of dopamine neurons[1]. This lesion, however, also downregulates postsynapticD3 receptor in nucleus accumbens [20], by deprivation ofbrain-derived neurotrophic factor (BDNF), an anterogradefactor of dopamine neurons (see below). Hence, the lesion-induced decrease in areas of dopamine cell bodies couldreflect a similar process occurring in non-dopaminergicneurons. Dopamine release [21] and synthesis [22] areinhibited by stimulation of the D3 receptor expressed in atransfected mesencephalic cell line and various agonists, withlimited preference for the D3 receptor [23], inhibit dopaminerelease, synthesis and neuron electrical activity (see [6] for areview), giving support to the existence of D3 autoreceptors.However, the selectivity of these agonists towards the D3receptor in vivo has been strongly questioned, because theyelicit similar inhibition of dopamine neuron activities inwild-type and D3 receptor-deficient mice [24]. In addition,dopamine autoreceptor functions are suppressed in D2receptor-deficient mice [25, 26]. Nevertheless, dopamineextracellular levels in the nucleus accumbens [24] and

Fig. (1). D3 receptor distribution in the human brain. A, D3receptor binding, revealed with [3H]7-OHDPAT, a D3 receptor-

selective radioligand. Nuc. acc., nucleus accumbens; Par. Cx,

parietal cortex. B, In situ hybridization signals with a D3receptor-specific probe. C, In situ hybridization signals were

quantified from 3 individuals and expressed as percentage of

the level in nucleus accumbens (Nuc. acc.). Caudate, caudate

nucleus; Cing. Cx, cingulate cortex; Dent Gyrus, dentate gyrus;

Frontal Cx, frontal cortex; Isl Calleja, islands of Calleja; Par. Cx,

parietal cortex; Sub. Nigra, substantia nigra pars compacta;

Subth. nuc., subthalamic nucleus. Data from [17] and [18].

striatum [27] are twice as high in D3 receptor-deficient as iwild-type mice, suggesting a control of dopamine neuronactivity by the D3 receptor.

A selective anti-D3 receptor antibody has been developedthe immuno-reactivity of which perfectly matches D3 receptobinding (Fig. 2). This antibody allowed us to confirm thpresence of D3 autoreceptors at the somato-dendritic level oall dopaminergic neurons in substantia nigra and ventrategmental area [28]. D3 autoreceptors, together with Dautoreceptors [25], may thus control the electrical activity odopamine neurons, which would explain the elevateextracellular dopamine levels in projections areas of thesneurons in D3 receptor-deficient mice. This control coulhave been masked in experiments using compoundinadequately selective of the D3 receptor [24], since thcompounds used also activate the D2 receptor. The existencof a control of dopamine release by the D3 receptor harecently received support from the use of selective Dreceptor antagonists (see section 6). Alternatively, Dautoreceptors could not be operant in anesthetized animals oin vitro in brain slices used in electrophysiological studie[24, 25], whereas dopamine extracellular levels wermeasured in freely moving animals.

Fig. (2). Immunohistochemical localization of the D3 receptor i

rat brain. A, B Superimposable distr ibutions of binding o

[125I]trans-7-OH-PIPAT, a D3 receptor-selective ligand (A) D

receptor immunoreactivity (B), with highest levels in th

islands of Calleja (IcjM and IC) and moderate levels in the shel

of nucleus accumbens (AccSh) (ac, anterior commissura. C, D

expression of D3 receptor immunoreactivity alone (in red in C

and in combination with tyrosine hydroxylas

immunoreactivity (in green in B). All tyrosine hydroxylase

positive neurons in the mesencephalon express the D3 recepto

Data from [28].

The use of knockout mice has brought somclarifications, and also raised new questions. The decrease iextracellular levels of dopamine, induced by low doses oPD 128,907, a D3 receptor-preferring agonist, measured b


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The Dopamine D3 Receptor CNS & Neurological Disorders - Drug Targets, 2006, Vol. 5, No. 1 27

brain microdialysis, is attenuated in D3 receptor-deficientmice [29], suggesting that D3 receptors control dopaminerelease. However, the decrease in dialysate dopamine levelsis abolished in D2 receptor-deficient mice [30]. Recentstudies in transfected cells have shown that co-expression ofD2 and D3 receptors enhances the potency of D2-like agonistsfor the inhibition of adenylate cyclase [31]. If there were asimilar functional D2/D3 receptor interaction in dopamine

neurons where the two receptors co-exist [28, 32], this wouldexplain why D3 receptors could modulate extracellulardopamine levels by increasing the potency of dopamine at D2receptor [30]. The hypothesis of a control exerted by D3autoreceptors is also supported by the observations that D3receptor-deficient mice display signs reminiscent ofhyperdopaminergia, presumably resulting from the lack ofautoreceptors controlling dopamine neuron activity. Thus,they have decreased expression of tyrosine hydroxylase andincreased expression and function of the dopaminetransporter; these alterations may represent adaptive changesto increased dopamine tone [33]. Another study found thatsubchronic treatment with pramipexole, D3-preferringagonist, lowered the Vmax of [

3H]dopamine uptake, which is

in line with the previous study, but that D3 receptor-deficientmice had lower Vmax of [

3H]dopamine uptake than wild-type

mice [34]. Finally, D3 autoreceptors may mediate yetunrecognized control by dopamine of other activities ofdopamine neurons, such as synthesis or release ofneuropeptides co-expressed with dopamine in these neurons,e.g. neurotensin, cholecystokinin or neurotrophins.

3. BRAIN-DERIVED NEUROTROPHIC FACTOR

CONTROLS THE EXPRESSION OF THE D3

RECEPTOR AND TRIGGERS BEHAVIORAL

SENSITIZATION

The expression of the D3 receptor in medium-sized

neurons of the nucleus accumbens is highly dependent upondopaminergic innervation : in a rat model of PD, obtainedby unilateral and extensive destruction of dopamine neuronsby local infusion of 6-hydroxydopamine (6-OHDA), D3receptor expression is decreased in the shell of the nucleusaccumbens of the denervated side [20]. D3 receptor density isalso decreased in PD patients [35] and in a non-humanprimate model of PD, i.e. in MPTP-treated monkeys[19]This paradoxical change (the D2 receptor is upregulatedunder these circumstances) was shown to depend on thedeprivation of an anterogradely-transported factor fromdopaminergic neurons, distinct from dopamine itself and itsknown peptide co-transmitters [20]. In rats, D3 receptorexpression appears in the shell of the nucleus accumbens

during the first postnatal week, coincident with itsinnervation by dopamine neurons, suggesting that the factormaintaining D3 receptor expression in adulthood also triggersthis expression during development [36]. Brain-derivedneurotrophic factor (BDNF) was a candidate factor for theregulation of D3 receptor expression.

BDNF, like other neurotrophins, had initially beenregarded as responsible for neuron proliferation,differentiation and survival, after its neuronal uptake andretrograde transport to the soma [37]. A more diverse role forBDNF as an extracellular transmitter has, nevertheless, beeninferred from observations that it is anterogradely transported[38, 39], released upon neuron depolarization and triggers

rapid intracellular signals [37, 40] and action potentials incentral neurons [41] via intracellular transduction of its highaffinity membrane receptor TrkB. TrkB (tropomyosireceptor kinase B) is a high affinity receptor for BDNFwhich activates intracellular signaling viautophosphorylation of tyrosine residues. BDNF can altefast synaptic transmission by speeding up the developmenof excitatory and inhibitory synapses [42], and b

modulating synaptic efficacy [43]. In particular, BDNF inecessary for induction and maintenance of hippocampalong-term potentiation [43-45]. Although some observationsuggest a role of BDNF in nociception [46] and learning [4748], little is known, about the consequences of BDNFinduced synaptic plasticity on physiological functions opathophysiological conditions.

The first insight for a role of BDNF in the regulation oD3 receptor expression came from the observation that Trk Band D3 receptor mRNAs co-localized in a high proportion oneurons in the shell of nucleus accumbens [49]. Moreover, iwild-typeBDNF

+/+mice, D3 receptor binding and mRNA i

the shell of the nucleus accumbens increased sharply frompostnatal days 9-14 (P9-P14) to P17-P23, whereas ihomozygousBDNF-/- mice, D3 receptor binding and mRNAwere low at P9-P14, and did not increase at later stages (Fig3A), showing that BDNF is required for normadevelopment of D3 receptor expression in the shell of thnucleus accumbens [49]. However, in the islands of Callejaanother brain region which expresses high levels of Dreceptor, the expression level was high at early stages odevelopment and not decreased in D3 receptor-deficient mic[49, 50]. This suggests that there was both BDNF-dependen(in the nucleus accumbens) and BDNFindependent (in thislands of Calleja) population of D3 receptor-expressinneurons. This is consistent with the observation that thislands of Calleja do not express TrkB [49]. The BDNF gen

mutation does not impair the early development of dopaminneurons [51], nor their later development, since tyrosinhydroxylase, a marker of these neurons, was not significantlaffected by the lack of BDNF [49]. This suggests that BDNFacts directly on dopamine D3 receptor-expressing neuronrather than indirectly via an effect on the development odopamine neurons. Note, however, that other studies suggesthat BDNF can act directly on dopamine neurodevelopment [52, 53]. It is also worth noting that BDNFdeprivation selectively reduces the expression of the Dreceptor, and not that of the homologous D1 and D2 receptor[49], which are not, or only marginally, down-regulated b6-OHDA lesions [54].

In unilaterally 6-OHDA-lesioned rats, repeate

administration of levodopa, leading to extraneuronadopamine formation, triggers D3 receptor overexpression noonly in the shell of the nucleus accumbens, but also in thdenervated striatum, a brain structure in which D3 receptoexpression is hardly detectable [54] (Fig. 3B). Durinlevodopa treatment of 6-OHDA-lesioned rats, infusion intthe denervated striatum of a selective BDNF antagonisformed by fusion between the Fc-tail of humaimmunoglobin G (IgG) and a part of TrkB (IgG-TrkB) [55]impairs induction of both D3 receptor mRNA and proteiexpression (Fig. 3B). This indicates that BDNF is necessarfor this process [49].


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D3 receptor overexpression induced by levodopa has beenshown to be responsible for the development of behavioralsensitization to this drug, i.e. a progressive enhancement ofresponsiveness, which appears as an increased number oflevodopa-induced rotations: the development and extinctionof behavioral sensitization parallel D3 receptor expression inthe striatum during the treatment with levodopa and after itscessation (Fig. 3C). Moreover, the increase in the number of

rotations is blocked by a preferential D3 receptor antagonis[54] and induced by a selective partial D3 receptor agonis[56]. Infusion of IgG-TrkB dose-dependently inhibitbehavioral sensitization (Fig. 3D), indicating that behaviorasensitization is triggered by BDNF.

Striatal BDNF in fact originates mainly from corticaneurons [39]. In agreement, cortical ablation partially impairthe induction of D3 receptor overexpression in the striatum

Fig. (3). BDNF controls D3 receptor expression during development and in adults. A, ( top) Autoradiographic pictures of D3 recepto

binding, obtained with [125I]7-OH-PIPAT, in wild-type BDNF+/+ or BDNF-/- mice at post-natal day 23 (P23). (bottom) D3 recepto

binding (Ci/g) with animals at P9-P10 or P14-P23 were analysed. There was a signif icant effect of genotype (P < 0.0001), age (P =

0.0023) and genotype X age interaction (P = 0.019). * P < 0.05 vs.BDNF+/+ littermates. B, 7-day striatal infusion of IgG-TrkB, a BDNF

antagonist, blocks the induction of D3 receptor binding by L-DOPA. (top ): In situ hybridisation signals of D3 receptor mRNA in

animals receiving continuous infusions into the striatum of IgG (control animal, left) or IgG-TrkB (right) for 7 days and levodopa fo

5 days.(bottom): Quantitative analysis showing a significant effect of treatment (P = 0.006). * P < 0.05 and ** P


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and behavioral sensitization, indicating that both processesrequire the participation of corticostriatal neurons [49].Levodopa induces BDNF mRNA in the frontal cortex in the6-hydroydopamine-lesioned side, mainly in cortical layer Vcontaining pyramidal cell bodies and in layer VI, whichsends projections to various subcortical areas, notably striataland accumbal areas [57]. This effect critically depends uponactivation of a D1 or D5 receptor [49] and is consistent with

the presence of D1 receptors on cortical pyramidal cells [58]and with the observation that stimulation of a D1 or D5receptor under similar circumstances phosphorylates cAMPresponse element-binding protein [59], a factor that activatesBDNF gene transcription [60, 61]. Hence, induction of D3receptor expression in striatum is triggered by a D1/D5receptor stimulation-dependent elevation of BDNF incortico-striatal neurons, a process that is more pronounced inthe 6-OHDA-lesioned side as compared to the control side,which accounts for the induction of D3 receptor expressionrestricted to the lesioned side. Moreover, BDNF-induced D3receptor expression might cause a more pronounceddisequilibrium in responsiveness to dopamine between thetwo sides, which might lead to enhanced rotational behavior.

4. D3 RECEPTOR-SELECTIVE PHARMACOLOGI-CAL AGENTS

Initial pharmacological studies with recombinant D3receptor showed that dopamine, as well as some of itsagonists, display higher affinity at D3 receptor than at D2receptor [1, 62]. Among antagonists, antipsychotics displayvery similar affinities at D2 and D3 receptors (Table 1), but(+)AJ-76 and (+)UH 232, two aminotetralin derivativesacting as preferential dopamine autoreceptor antagonists [63],show a little preference for the D3 receptor, as compared toD2 receptor [1]. In fact, (+) UH 232 has been shown toexhibit partial agonistic properties at the D3 receptor [64].

An important step towards identifying selective D3receptor ligands was the discovery that the dopamine agonist[3H](+)7-OH-DPAT selectively binds in vitro to the natural

D3 receptor [65], permitting visualization of this receptor inrat [65] and human [66] brain slices and confirmation of itspharmacological properties. While an often used tool, thereare some questions about selectivity in vivo (discussed in[67]), and a retention of activity in mice deficient for the D3receptor [68, 69]. More recent compounds have beendeveloped with a higher degree of selectivity both in vitroand in vivo. For instance, three putative D3 receptorantagonists, nafadotride [70], PNU-99194A [71] and S14297[72] , display 7-20 times higher affinity for the D3 than theD2 receptor (Table 1). Nafadotride and PNU-99194A are

devoid of agonistic activity, and they increase locomotoractivity at low doses without affecting dopamine synthesisor release in rats [70, 71, 73], suggesting an inhibitory roleof post-synaptic D3 receptors. This hypothesis is consistentwith the observation that D3 receptor mutant mice displayhyperactivity in a novel environment [74]. However, theselectivity of nafadotride and PNU-99194A have beenquestioned, since their stimulant effects on locomotoractivity persist in D3 receptor mutant mice [68]. S14297,previously assumed as an antagonist [72], actually acts as afull agonist on D3 receptor-mediated mitogenesis andinhibition of cyclic AMP accumulation in recombinant cells[69], which questions the nature of the effects shown with

this compound. It follows that highly selective ligands werneeded for assigning functional role(s) for the D3 receptor.

Table 1. Dopamine Receptor Affinity for Antagonists

Affinity (Ki, nM)a

Drug Receptor subtype

D1 D2 D3 D4 D5

Non selective:

Amisulpride >1,000 2.8 3.2 >1,000 >1,000

(+) Butaclamol 5 1 4.5 45 6.1

Chlorpromazine 35 5 4 16 33

Clozapine 35 145 238 29 343

Eticlopride >10,000 0.1 0.25 25 >10,000

Flupentixol, cis 4 1.5 2.5 - 12

Fluphenazine 6 0.6 0.8 30 8

Haloperidol 150 2 5 6.5 170

Olanzapine 48 30 41 36 74

Pimozide >10,000 3 4 30 -

Quetapine 390 380 260 1,050 -

Raclopride >50,000 1 1.2 2,100 -

Remoxiprideb

>10,000 588 1,600 3,200 -

Risperidone 560 6 11 16 560

Spiperone 380 0.08 0.4 0.1 2,400

Sulpiride >10,000 38 60 280 >10,000

Thioridazine 34 7 8 10 300

YM-09151-2 2,600 0.05 0.09 0.13 -

D3- selective:

BP 897(partial agonist)

3,000 61 0.9 300 -

GR 103,691 - 24 0.4 81 -

GR 218,231 >1,000 63 1 10,000 -

Nafadotride 890 3 0.3 1,780 -

NGD 2904 >10,000 217 1.4 >5,000 >10,000

S 33084 500 32 0.3 2,000 1,300

SB-277011A >1,000 1,030 11 >1,000 >1,000

ST 198 25,000 780 12 5,000 -

PNU-99194A - 2,280 223 >10,000 -

aData reviewed in [233] and data from [234], [56] and [81].bMetabolites of this compound with higher affinities for D2 and D3 receptors hav

been identified.

Screening of a series of newly designed molecules [56led to the identification of BP 897 (see the chemicastructure in Fig. 8). This compound displays a high affinitat the D3 receptor (Ki = 0.92 nM), a 70 times lower affinityat the D2 receptor (Ki = 61 nM) and much lower affinity aD1 and D4 receptors (Table 1), as well at a variety of nondopamine receptors. In NG 108-15 cells expressing thhuman D3 receptor, BP 897 potently inhibits forskolininduced cyclic AMP accumulation and produces mitogenesi


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(EC50 of ~1 nM), however to a maximal extent ( ~55 %) ofthat maximally elicited by dopamine or the full agonistquinpirole. In contrast, in cells expressing the D2 receptor,BP 897 fails to either inhibit cyclic AMP accumulation ortrigger mitogenesis; it reversibly antagonizes quinpirole-induced mitogenesis, however, only at concentrations largelyexceeding those required to stimulate the D3 receptor. Hence,BP 897 appears as the first selective, potent, but partial

(intrinsic activity ~ 0.6) D3 receptor agonist, and a weak D2receptor antagonist in vitro. Other studies using [

35S]GTPS

binding or microphysiometry [75, 76] failed to sort out theagonistic activity of BP 897; however, in these studies, theagonist 7-OH-DPAT, which has high intrinsic activity in themitogenesis assay [23], displays very low intrinsic activitysuggesting that coupling was not optimal in these studies.

In vivo, BP 897 at high dosage (10-20 mg/kg) occupiesbrain dopamine D2, receptors and, as a result of blockade ofthese receptors, displays typical neuroleptic-like properties(induction of catalepsy, antagonism of apomorphine-inducedstereotypies). This indicates that selective occupancy of D3receptors should be obtained at doses not exceeding 1mg/kg, taking into ,account the in vitro selectivity of thecompound (Table 1). At these low dosages, BP 897 does notaffect spontaneous locomotor activity and body temperature.BP 897 agonist/antagonist potency in vivo was assessed ontwo presumably D3 receptor-mediated responses. Inhemiparkinsonian rats repeatedly pretreated with levodopa,BP 897 potentiates rotations elicited by a D1 receptor-selective agonist; this potentiation does not occur beforelevodopa treatment, i.e. before induction of D3 receptorexpression and is abolished by co-treatment with nafadotride,indicating that BP 897 acts as an agonist for this response[56]. In the islands of Calleja of rats, BP 897 enhanced c-

fos mRNA, an effect similar in direction and amplitude tothat produced by nafadotride [77]; in addition, in contrast to

D2/D3 receptor agonists, BP 897 potentiates the response to aD1 receptor agonist [56]. This effect is abolished in D3receptor mutant mice. Thus, in vivo, BP 897 increases D1receptor-mediated responses by acting as either an agonist oran antagonist, depending upon the response considered,consistent with its partial agonist properties in vitro. BP 897acts as a receptor agonist on rotations elicited in dopamine-depleted brain and as a receptor antagonist on c-fosexpression maintained by a dopaminergic tone.

More recently, two highly selective D3 receptorantagonists have been identified. S33084 has a Ki value of0.25 nM at the D3 receptor and a 120 times lower affinity atthe D2 receptor [78]. SB-277011A has a Ki value of 11 nMat the D3 receptor and a 100 times lower affinity at the D2

receptor [79]. Both compounds have much lower affinity atdopamine D1 and D4 receptors (Table 1), as well as variousother non-dopaminergic receptors. In several functional invitro assays, they display no agonistic activity andcompetitively antagonize dopamine or dopamine agonist-induced responses. Remarkably, both compounds have noeffect on spontaneous locomotor activity or dopamine efflux,measured by brain microdialysis, but antagonize preferentialD3 dopamine agonist-induced inhibition of dopamine efflux.This indicates that D3 receptors, presumably D3autoreceptors, exert a phasic, but not tonic control of theactivity of dopamine neurons. In agreement, the two D3receptor antagonists do not modify spontaneous or

psychostimulant-induced locomotion. Finally, another Dreceptor-selective antagonist, ST 198 has been described [8081], which has Ki values of 12 nM and 780 nM foinhibiting binding to D3 and D2 receptors, respectivel(Table 1). Interestingly, the selectivity of this compound, awell as that of BP 897 and nafadotride, for the D3 receptor ivivo has been assessed by using D3 receptor-deficient micethe inhibition of dizocilpine (MK-801)-induced locomoto

hyperactivity by the three compounds is abolished in Dreceptor mutant mice [81, 82].

5. THE D3 RECEPTOR AND PARKINSON'

DISEASE

PD is associated with several symptoms such as akinesiarigidity and tremor, which result from the lack of the braineurotransmitter dopamine [83]. Substitution treatment foPD, e.g. by levodopa, initially reduces motor symptomsbut eventually induces, in most of patients, debilitating anpharmacoresistant involuntary movements, i.e. dyskinesiapresumably resulting from an excessive response tdopamine [84]. This excessive response to dopamine, forme

from levodopa, results in behavioral sensitization to thdrug. Enhanced responses to levodopa in 6-OHDA-lesionerats could reflect either the progressive motor recoveroccurring at treatment initiation or the development olevodopa-induced dyskinesia (LID) as is seen in long-termtreated PD patients [85]. This could not be assessed in PDlike rats, which do not develop typical LID. For this reasonmonkeys treated with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), which destroys dopaminneuron terminals and cell bodies and produces a variety oPD-like symptoms, including akinesia and rigidity [86]were used. Long-term treatment of these monkeys witlevodopa elicits dyskinesia, the repertoire and the severity owhich are not distinguishable from LID occurring in PD

patients [84].MPTP alone produces a severe loss of D3 recepto

binding in the caudate nucleus [81], a brain structurinvolved in associative locomotion (goal-orientelocomotion), an effect which is compensated by treatmenwith levodopa in MPTP-intoxicated animals without LID(Fig. 4). However, in MPTP-intoxicated monkeys with LIDD3 receptor binding is higher than in non-dyskinetimonkeys in the putamen and internal part of the globupallidus and even higher than in normal monkeys [81] (Fig4). Moreover, D3 receptor binding levels in the putamecorrelate with the occurrence and severity of LID. Thesresults show that PD-like symptoms and LID araccompanied by down- and up-regulation of D3 recepto

expression, respectively, while such a correlation does noexist for either D1 or D2 receptors under comparablexperimental conditions [81, 84]. In addition, the occurrencof dyskinesia does not correlate with the severity of thlesion [81], which indicates that the level of D3 receptoexpression level in the putamen and globus palliducorrelates well with dyskinesia .

The changes in D3 receptor expression are likely to reflecfluctuations in D3 receptor function, as it is the case in PDlike rats, in which such changes are responsible foalterations of motor responses [49, 54]. To test thihypothesis, BP 897 was administered to dyskinetic MPTP


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intoxicated monkeys in combination with levodopa, andevaluated for LID and PD-like symptoms by experimentersunaware of the treatment received [81]. BP 897 attenuatesLID by 66% (Fig. 5A), but has almost no influence on thetherapeutic effect of levodopa, i.e. it does not reverse theimprovement of PD-like symptoms (Fig. 5B). Bothhyperkinesia (that is not true dyskinesia and thus not rated asone) and choreic/athetoid movements are improved. The

kinetics of activity counts (Fig. 5C) highlighted thischaracteristic pharmacological behavior by showing theability of BP 897 to undermine the levodopa-inducedactivity around the dyskinesia threshold, defined from acorrelation with clinically assessed LID. The dissociationbetween the effects of BP 897 on LID and the therapeuticeffects of levodopa seems to be related to the mixed agonist-antagonist property of this compound, because nafadotrideand ST 198, two D3 receptor-selective antagonists devoid ofagonistic effects, elicit a reduction of LID similar to thatobtained with BP 897 (Fig. 5A), which is, however,accompanied by a reappearance of PD-like symptoms (Fig.5B-D).

Fig. (4). Dyskinesia is accompanied by D3 receptor

overexpression. A, typical receptor autoradiograms obtained

with [125I]7-OH-PIPAT, a selective D3 receptor radioligand, in the

brain from a control monkey, untreated MPTP-intoxicated

monkeys (MPTP), and non-dyskinetic and dyskinetic MPTP-

intoxicated monkeys treated by levodopa (LD). Cd, caudate

nucleus; GPe, external part of globus pallidus; GPi, internal part

of globus pallidus; Pu, putamen. b, quantitative analysis of

autoradiograms obtained as in A. Results are mean + S.E.M. ofvalues in Ci/mg. Treatments had significant effects in Cd (P =

0.007 by analysis of variance), Pu (P = 0.026) and GPi (P =

0.028), but not in GPe (P = 0.22). *, P < 0.05 vs. control; , P


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ultimate question relates to how useful BP 897 will be intreating LIDs in PD patients. The current status of D3dopamine receptors in the basal ganglia of patients with PDis uncertain. D3 receptor levels have been reported eitherdecreased in the striatum of pathologically-defined PD casestreated with levodopa [35] or increased, however only inpatients with a robust response to levodopa therapy [90].These results confirm that D3 receptors might be a factor

contributing to dopaminergic drug responsiveness in PD, butclinical trials with D3 receptor-selective agent are necessaryfor assessing their relevance to LID.

Activation of D3 receptor has been suggested to protectdopaminergic neurons from degeneration in Parkinson'sdisease. [34, 91]. Clinical studies with pramipexole andropinirole, two D3 receptor-preferring agonists, showed lessdecline in markers of dopamine neuron integrity, comparedto levodopa [92, 93]. Nevertheless, the interpretations ofthese data is equivocal, insofar as chronic treatment withpramipexole lowers the Vmax of the dopamine transporter[34], which is related to dopamine neuron degeneration inone study [92]. In addition, although the data suggest abeneficial effect of dopamine agonist treatment, a detrimentaleffect of levodopa on progressive dopamine neurondegeneration cannot be completely ruled out [94, 95].

6. THE D3 RECEPTOR AND SCHIZOPHRENIA

Before the discovery of the novel dopamine receptorsubtype, it was assumed that the therapeutic action ofantipsychotic drugs could be attributed to blockade ofdopamine D2 rather than D1 receptors [96]. However, thecloning of several dopamine D2-like receptors, i.e. D2, D3,and D4 receptors, has raised the question of the role of thesethree proteins as targets for antipsychotics. Initially, thepositive correlation between drug affinity at D2-like receptorbinding site and drug plasma level identified these receptors

as common targets for all antipsychotics [97, 98]. This wasapparently confirmed when D2-like receptor occupancy instriatum was determined by PET and found to be 70-80 %in most cases [99, 100]. It remains to be established whethersuccessful treatment of schizophrenia is also accompanied bysignificant occupancy of D3 or D4 receptor, a difficult task inview of their relative low abundance, which could not beundertaken so far because of the lack of a suitable PET scanprobe. Nevertheless, acomparison of the affinities ofantipsychotic drugs at recombinant D2 and D3 receptor (Table1) indicates that these compounds generally show some, butvery limited preference for the D2 receptor. Hence, it can beassumed that significant D3 receptor occupancy occurs duringantipsychotic treatments resulting in 70-80 % D2 receptor

occupancy (D2 receptor occupancy up to 80 % resulting inextrapyramidal side effect [101]). In contrast with D2 or D3,D4 receptor occupancy cannot account for the antipsychoticactivity of all drugs, since effective compounds, such asthioproperazine or the benzamides show low affinity at theD4 receptor (Table 1). In addition, clinical trials with D4selective antagonists have shown a lack of antipsychoticactivity [16]. Remoxipride apparently shows markedpreference for the D2 over D3 receptor, but the compoundmay act indirectly, by generating active metabolites, whichdisplay an inverse pharmacological profile [101, 102]. In thecase of clozapine, only limited D3 receptor occupancy can beexpected in view of the rather low affinity for this receptor.

Several lines of evidence suggest that the efficacy oclozapine might rely on D3 receptor antagonism. Clozapinplasma levels of successfully treated patients [103] exceedby one order of magnitude the affinity constant of clozapinat D3 receptors, suggesting that the D3 receptor may bblocked by clozapine. Moreover, a higher affinity oclozapine for the D3 over the D2L isoform, which acts mainlat postsynaptic sites [104], was described [105]

Furthermore, the atypical response to clozapine on _FosBlike immunoreactivity in rats is abolished in D3 receptordeficient mice [106]. Finally, clozapine inhibits MK-801induced locomotor activity, a test predictive to thantipsychotic activity, with the same pattern as D3 receptorselective ligands, contrary to haloperidol (see below) [82].

Tolerance to antipsychotic drug-induced extrapyramidaside-effect develops in treated psychotic patients, whpresent a progressive decrease in extrapyramidal side-effectoccurring during the first weeks of treatment and thappearance of tardive dyskinesia after several years. Bycontrast, no tolerance seems to occur at the level of (thedopamine receptor subtype(s) involved in the antipsychotiactivity, since this activity does not diminish upon lonterm treatment. It is, therefore, noteworthy that repeateadministration of haloperidol for two weeks to rats failed ttrigger any significant upregulation in D3 receptor mRNA obinding in various brain areas, whereas in the same treatmenupregulated D2 receptor mRNA [20]. The absence oupregulation of D3 receptor mRNA and binding followinrepeated antipsychotic treatment is in agreement with othestudies [107-109]. In addition, the reduction in neurotensigene transcript in the ventromedial shell of nucleuaccumbens, a typical response to D3 receptor blockade [7]does not show tolerance following repeated administration ohaloperidol, whereas the reverse response in the core onucleus accumbens, mediated by the D2 receptor, diminishe

upon repeated haloperidol administration [7]. Thesobservations indirectly support the idea that thantipsychotic activity of neuroleptic drugs is related to Drather than D2 receptor blockade

A link between the D3 receptor and schizophrenia waalso suggested from the main expression of this receptor inforebrain limbic area that participates in the control of thactivity of the prefrontal cortex (see above). Indeed, manfunctional imaging and neuropsychological studies havimplicated the prefrontal cortex in integrative function(memory, speech, focused attention) and in the disorder othese functions observed in schizophrenia [110]. MoreoverD3 receptor levels have been found elevated in the brain odrug-free schizophrenic patients, but not in patients unde

medication with antipsychotics at the time of death [108]This suggests that the increase in D3 receptor expression is hallmark of the disease, and not of its treatment, and thaantipsychotic medications normalize this receptor expression

D3 receptor overexpression in the etiology oschizophrenia raises the question of mechanisms governinthis receptor expression during development. D3 receptoexpression during embryonic and early postnatadevelopment is characterized by an appearance of transcriptat early stages in neuroblasts or migrating neurons in the ra[36] or human brain [101], in which it was already detecteat week 6. However, the cortical neuroepithelium giving ris


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to the cerebral neocortex was heavily labeled in the human,but not in the rat embryo [101]. Tentatively, the D3 receptorexpressed in neuroepithelial cells lining the cerebralventricles might regulate their mitotic activity since,activation of the recombinant receptors enhances mitogenesis[111]. Putative dopaminergic neurons were seen in thehuman brain, adjacent to the ventricle, presumably in thegerminal zone at 16 days of gestation [112]. Being the only

dopamine receptor subtype expressed in these dividingneuroepithelial cells and owing to its high sensitivity todopamine (allowing dopamine to act at a certain distancefrom its release), the D3 receptor could have a role in thecontrol of the proliferation of these cells elicited bydopamine and, therefore, in the number of neurons of theirprogeny [113, 114]. Note, however, that a role of D3 receptorin neurogenesis in the adult subventricular zone isdocumented only in rats [115], and that D3 receptor-knockoutmice have apparently normal development [76]. The latterobservations suggest that the role of D3 receptor in neuronproliferation and differentiation may not be critical.

Among the cerebral morphometric abnormalities detectedin either in vivo or post-mortem studies, ventricularenlargement, reduction in neocortical and hippocampalneuronal density could result from an abnormal control oftheir progenitors in the neuroepithelium during earlydevelopment. Moreover, a role for BDNF can behypothesized in this process, as well as in the etiology ofschizophrenia, since BDNF controls D3 receptor expressionduring development and in adults (see above). However, therole of BDNF is difficult to establish in schizophrenia,because both an increase [116, 117] and a decrease [118, 119]in BDNF levels or BDNF mRNA have been found incortical areas of antipsychotic-treated schizophrenic patients.

Genetic factors are possible other factors controlling D3receptor in schizophrenia, which is an inheritable disorder. A

very large series of studies (nearly seventy) has beenpublished so far aiming at assessing the implication of theD3 receptor gene in schizophrenia, responsiveness to itstreatments and the side-effects of these treatments. Thesestudies have started after the identification of polymorphismsof this gene, notably a mutation substituting a Gly

9for a

Ser9

in the N-terminus of the receptor and creating a Bal Irestriction site [120]. Crocq and coworkers were the first todetect, in French and Welsh populations, evidence for anassociation of homozygosity of either allele 1 (Ser-Ser) orallele 2 (Gly-Gly) with schizophrenia [121]. A flurry of otherassociation studies has followed, the majority of which didnot confirm the initial studies. Nevertheless, twoindependent meta-analyses of a large number of association

studies were performed in an attempt to minimize the lack ofstatistical power of individual studies and to control forpopulation heterogeneity in a total population of over 2,500patients and 2,500 controls and convergent conclusions wereobtained [122, 123]. Significant, although limited excess ofhomozygotes for both alleles were found, clearly suggestingthat having the 1-1 allele (or the 2-2 allele) of the D3 receptorgene slightly enhances susceptibility to the disease overhaving the 1-2 allele. However, a recent meta-analysis [124]including more studies (comprising a total of 8,761 subjects)indicates that the association, while persisting, is very looseand that, at most, the D3 receptor gene contributes verymodestly to the susceptibility to schizophrenia. Convergent

genetic studies also suggest a role of the D3 receptor gene itardive dyskinesia, a frequent motor side-effect oantipsychotic drugs [125].

Glutamate has also been suggested to be involved inschizophrenia from the following clinical observationsPhencyclidine (PCP), a non-competitive antagonist at the Nmethyl-D-aspartate (NMDA) subtype of glutamate receptoris an anesthetic agent that has psychotomimetic properties iman [126]. PCP produces schizophrenic-like symptoms ihealthy volunteers or abusers [127, 128] and precipitatepsychosis in schizophrenic patients [129]. By contrast, drugfacilitating glutamate neurotransmission by acting at thglycine accessory site of the NMDA receptor, such as Dcycloserine, improve schizophrenia and enhance the efficacyof antipsychotic drugs, notably against the negativsymptoms of the disease [130]. Moreover, direct evidence foaltered NMDA receptor function in schizophrenia has beerecently reported [131] and genetic linkage studies inschizophrenia now show that the most plausiblsusceptibility genes (neuregulin 1, dysbindin, prolindehydrogenase and others) functionally interact witglutamate pathways [132]. These observations led to thproposal that schizophrenia may result from glutamatdeficiency.

In animals, PCP or dizocilpine (MK-801), another nocompetitive NMDA receptor antagonist [133], elicitbehavioral abnormalities, including hyperactivity, disruptioof sensorimotor gating and social deficit that arcircumvented by antipsychotic drugs, particularly of thatypical type [128]. The ability of PCP to simulatschizophrenia has led to the proposal that the behavioraabnormalities evident in schizophrenic patients and PCPexposed humans are caused by dysfunction of commonneural substrates. The effect of PCP or its cognate moleculeon brain dopamine systems has received particular attentio

[128, 134], since alteration in dopaminergic systems habeen hypothesized in schizophrenia [135]. Systemiadministration of phencyclidine or MK-801 increasedopamine cell firing rate in the ventral tegmental area (VTAdopamine metabolism [136-138] and extracellular dopaminconcentrations in the nucleus accumbens [139]Hyperlocomotion induced in rodents by systemicalladministered NMDA receptor antagonists is blocked by 6hydroxydopamine lesions of the VTA [137] or depletion odopamine from neuronal stores [140].Hence, convergent datstrongly suggest that locomotor effects of NMDA receptoantagonists at low doses are mediated via increasedopamine function, but the dopamine receptors involvehave not been fully characterized.

To investigate the role of the D3 receptor in thschizophrenia-like behavioral abnormalities produced bNMDA receptor blockade, the effects of D3 receptor-selectivagents have been studied in wild-type and D3 receptorknockout mice administered with MK-801 [82]Hyperactivity produced by low doses of MK-801 wapotently and completely inhibited by haloperidol, clozapinenafadotride, or BP 897 with ED50 values ranging from 0.0to 0.2 mg/kg (Fig. 6). Unlike the other agents, haloperidoalso inhibited spontaneous locomotor activity at the sam


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Fig. (6). Effect of antipsychotic drugs and D3 receptor

antagonists on MK-801-induced locomotor hyperactivity.

Saline (Sal), BP 897, nafadotride, clozapine, or haloperidol were

administered at the indicated dosage and the spontaneous

activity (empty columns) and activity after MK-801 (0.12 mg/kg,

filled columns) recorded. * P < 0.05, ** P < 0.01, *** P < 0.005

vs. respective Sal. Data from [82].

doses. That the D3 receptor mediated the effects of MK-801,was confirmed by the dramatic decrease in MK-801-inducedhyperactivity in D3 receptor knockout mice (Fig. 7). Theinhibitory effects nafadotride and BP 897 were completely

suppressed, whereas that of clozapine was partiallysuppressed (Fig. 7B). These results indicate that the MK-801-induced hyperactivity involves stimulation of the D3receptor. Nevertheless, the hyperactivity remaining in D3receptor-knockout mice depends on other receptors.

These results show that activation of the D3 receptor is amajor mechanism underlying locomotor effects of NMDAreceptor antagonists at low doses. Blockade of the D3receptor produces an effect very similar to that produced byantipsychotics on MK-801-induced hyperactivity, which,therefore, supports the growing evidence suggesting that D3receptor blockers might have antipsychotic properties. Theyalso suggest that the D3 receptor may be an intermediary

linking glutamate and dopamine dysfunctions inschizophrenia. This hypothesis has received recent supportby the preliminary results of the first double-blind placebo-controlled study of BP 897 in schizophrenia, which showedexposure-dependent antipsychotic effects [141]. Takentogether, these recent results add to the previously gainedevidence indicating that the D3 receptor plays an importantrole in schizophrenia and its treatment.

7. THE D3 RECEPTOR AND DRUG ADDICTION

Converging evidence supports the idea that themesocorticolimbic system, which projects from the ventral

tegmental area to the nucleus accumbens, frontal cortexolfactory tubercle, amygdala and septal area, is an importansubstrate for the rewarding/reinforcing effects of abusedrugs. One of the major projection areas of mesolimbidopamine neurons, the shell of nucleus accumbens wherboth D3 receptor protein and mRNA are highly expressedprojects to the ventral tegmental area and receives projectionfrom the prefrontal cortex, hippocampus and amygdala [10

11]. These various specific connections within the shell onucleus accumbens are part of the "extended amygdala[142], making it a critical structure in the control omotivation and the effects of drug-associated conditionestimuli. The density of D3 receptor is elevated in long-termcocaine abusers [143, 144], and in animals chronicallytreated with cocaine [145] or nicotine [146], suggesting thaD3 receptor over-expression is due to drug exposure. Theseffects appear selective for the D3 receptor, since no changein D1 or D2 expression have been found in the brains of thesanimals [145]. Although the functional consequences of sucan upregulation are not fully understood, preclinical evidencsuggests that this upregulation is involved in phenomensuch as behavioral sensitization that are thought to play an

important role in drug dependence [147].

Fig. (7). Effect of D3 receptor gene deletion on MK-801-induce

hyperactivity and its inhibition by clozapine and D3 recepto

antagonists. A, Following a 30-min period of habituation, salin

(Sal) or MK-801 (MK, 0.12 mg/kg) was injected (arrow) to wild

type (D3R+/+) or D3 receptor-deficient mice (D3R

-/-) and locomoto

activity was recorded during 60 min. * P < 0.05, ** P < 0.01, vs

respective saline-treated mice, # P < 0.05, ## P < 0.01 vs. MK

801-treated D3R-/- mice. B, Effect of saline (SAL), BP 897 (

mg/kg), nafadotride (NAF, 1 mg/kg) or clozapine (CLOZ,

mg/kg) on MK-801-induced hyperactivity on wild-type (D3R+/+

or D3 receptor-deficient (D3R-/-) mice. The clozapine effects in D

/- mice indicate that the MK-801-induced hyperactivity in thes

mice is independent of the D3 receptor, and may involve othe

clozapines targets (noradrenalin or serotonin receptors). * P

0.001 vs. saline-treated D3R+/+ mice, # P < 0.05 vs. saline-treate

D3R-/- mice. Data from [82].

Repeated intermittent administration of drugs of abusinduces a sensitization strikingly similar to that produced bylevodopa in hemiparkinsonian rats, which is characterized benhanced behavioral responses (see [148] for a review)Interestingly, it appears that D3 receptor overexpression ifacilitated by the interaction of the effects of drugs withdrug-associated environmental stimuli [145]. Moreover, Dreceptor overexpression that is induced by repeated nicotin


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administration is more pronounced in rats repeatedlyreceiving nicotine injections in a distinctive environmentdesigned to facilitate drug-induced conditioning, than in ratsreceiving nicotine in their home-cages [149].

Interestingly, BDNF, which controls D3 receptorexpression, has also been directly implicated in drugaddiction processes. BDNF is synthesized in thehippocampus, amygdala, dopamine neurons and prefrontalcortex [150] that project to the nucleus accumbens. BDNFinfusion in the nucleus accumbens enhances, whereas areduction in BDNF levels reduces cocaine-conditioning [151,152]. Furthermore, BDNF expression increases followingexposure to cocaine-associated stimuli and after prolongedcocaine withdrawal [145, 153], factors that may promotelong-lasting changes facilitating drug-seeking behavior [154]or cocaine craving [155]. Both BDNF [145, 151] and D3receptors (see below) have been implicated in paradigms thatinvolve classically conditioned effects of drugs of abuse.Drug conditioning necessitates repeated associations ofdistinctive environmental stimuli with drug effects.Nevertheless, it has been shown that a single stimulusassociated with a single cocaine experience can acquiremotivational value and elicit long-lasting cocaine-seeking inrats [156]. A single administration of various drugs of abuse,including cocaine, methamphetamine, morphine and, to alesser extent, nicotine, induces a transient increase in BDNFexpression in the prefrontal cortex, associated with a long-lasting increase in D3 receptor binding and mRNA levels inthe shell of the nucleus accumbens [157]. Thus, the lack ofpermanent increase of BDNF expression after cocaineadministration, allows phasic changes of BDNF release to beassociated with each presentation of environmental stimuli.On the contrary, the much longer time scale for increase inD3 receptor levels permits additive effects of repeatedpresentations. Therefore, these results suggest that the

BDNF/D3 receptor pathway, activated by drugs as soon as attheir first administration, is involved in the initiation andmaintenance of drug conditioning.

Pharmacological data, obtained with non selectivedopamine receptor ligands, suggested the involvement of theD3 receptor in the reinforcing effects of cocaine (see [158] fora review). Pretreatment with the D3 receptor-preferringagonists, pramipexole, quinelorane and PD 128,907, dosedependently decreases cocaine self-administration [159, 160]in a manner suggesting that these agonists potentiate, ratherthan reduce the reinforcing effects of cocaine. In addition, therelative potencies of these different agonists in decreasingcocaine self-administration are highly correlated with theirfunctional potency at the D3, but not the D2 receptor [161].

Finally, preferential agonists at D3 receptor substitute forcocaine in rhesus monkeys trained to discriminate cocainefrom saline [162], and there is a correlation between thepotencies of several preferential agonists at D3 receptor inreproducing the discriminative stimulus effects of cocaine insquirrel monkeys and their in vitro potencies at the D3receptor, but not D2 receptor [163]. All of these observationssuggest that stimulation of D3 receptor can enhance thereinforcing effects of cocaine.

However, several recent studies using more selectiveligands and D3 receptor-deficient mice now indicate that theability of D3 receptor blockade to decrease the motivation to

take drugs appears to depend on the price the animal has topay (in terms of efforts) to get the drug. In other words, thmore the number of lever pressures is high to get the drugthe more self-administration is inhibited by D3 receptoblockade. Thus, under small fixed-ratio schedules, where onor two responses are required to produce each injection, BP897 or SB-277011A does not alter cocaine selfadministration in rats [56, 164]. Recent findings obtaine

using transgenic mice confirm the lack of involvement of Dreceptor in the reinforcing effects of cocaine, since deletion othe D3 receptor in transgenic mice does not produce anchange in cocaine self-administration [165]. Similar findinghave been described with nicotine [166] and ethanol [167168].

Nevertheless, the D3 receptor controls the motivation tself-administered drugs under FR (fixed ratio) schedules oreinforcement with high response requirements oprogressive-ratio schedules. Thus, SB-277011A reducecocaine self-administration under both a progressive-ratischedule and a fixed-ratio schedule with a relatively higresponse requirement [169]. The fact that blockade of the Dreceptor produces different effects under different schedules oreinforcement is consistent with a behavioral economianalysis [170]: the ability of D3 receptor blockade to decreasthe motivation to take drugs depends on the price

of drug

drugs being self-administered under high price conditionappear to be more sensitive to the effects of D3 receptoblockade.

Drug-associated environmental stimuli are major factorthat can cause relapse to drug use in abstinent drug addictsThis process is critical for psychostimulants, but also fonicotine and heroin addiction [171-174]. Moreover, ianimals, such stimuli can induce and maintain drug-seekinbehavior in the absence of drug and can also reinstatextinguished drug-seeking behavior [175-180]. In a second

order schedule of reinforcement in which a drug-associatestimulus progressively gains motivational salience and, as conditioned reinforcer, maintains and controls drug-seekinbehavior, BP 897 [56] (see Fig. 8) and SB-277011A [164dose-dependently reduce cocaine-seeking behavior. Theseffects occur in the absence of any observable non-specifieffects (e.g. motor disturbances), effects that might havdisrupted performance. Thus, these ligands have ndemonstrated reinforcing effects;for instance BP 897 is noself-administered in rats [56] and monkeys [181], but arable to reduce cocaine-seeking behavior maintained bpresentation of cocaine-associated stimuli.

Priming injections of drug and environmental stressor

have also been identified as factors that can trigger relapse todrug-seeking and drug-taking behavior in humans anexperimental animals [182, 183]. SB-277011A administration inhibits stress-induced reinstatement of cocaine-seekinbehavior [184] and also reinstatement of nicotine-seekinbehavior produced by a priming injection of nicotine in rat[166]. Thus, it appears that D3 blockade can decrease thinfluence of various factors producing relapse in animals anhumans.

Since D3 receptor-selective ligands are able to disrupdrug-seeking behavior induced by the presentation ococaine-associated stimuli, it has been suggested that thesligands may be useful tools for suppressing reactivity to


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drug-associated environmental stimuli [56, 149, 185]. Thesestimuli are thought to gain motivational value throughPavlovian conditioning processes. A variety of procedureshave been used to assess the influence of D3 receptor onPavlovian-conditioned behavioral responses [145, 149].Thus, an animal model frequently used to explore the controlover behavior that can be exerted by drugs of abuse is theconditioned place preference (CPP) procedure. CPP occurswhen repeated administration of a drug followed byplacement in a distinctive environment (e.g. one compart-ment of a two- or three-compartment apparatus) results in theability of that environment to elicit approach behavior andincreased time contact (conditioned place preference) in theabsence of the previously administered drug. It has been

argued that CPP, like drug self-administration and a numberof related phenomena, can be considered a measure of drug-seeking behavior [186, 187]. Thus, CPP procedures, likesecond-order schedules of drug injection, provide a means ofmeasuring the influence of drug-related environmentalstimuli on behavior. D3 receptor-selective ligands blockCPP, as well as various conditioned responses to differentdrugs of abuse (Table 2). Remarkably, these ligands have noeffect on responses associated with natural reward, such asfood.

With CPP procedures, it is possible to assess the effectsof D3 receptor-selective ligands on the acquisition of drug-

induced place preferences, as well as on the expression oCPP in the absence of the drug. Although acquisition odrug-induced CPP has often been considered a reliablmeasure of the rewarding effects of drugs, in fact, thiprocedure measures the progressive acquisition by initiallneutral stimuli, of conditioned motivational effects resultingfrom their association with the interoceptive effects producedby drug administration. SB-277011A prevents thdevelopment of cocaine- [188] and heroin-[189] induceCPP. Similar findings have been described with BP 897[190]. In contrast, the development of morphine-induceCPP was not significantly altered by BP 897 in rats [190]BP 897, however, facilitates the acquisition of morphineinduced CPP in mice [191]. Effects of D3 receptor-selectiv

ligands during the acquisition of CPP processes may reflecblockade of the direct rewarding effects of the drug, but mayalso reflect blockade of the ability to form conditionedassociations, which subsequently allow the stimuli tinfluence the behavior. However, BP 897 has no effect othe recall of environmental stimuli or on recognition oenvironmental stimuli, not associated with drug effects. Foinstance, BP 897 does not alter habituation to an open fielduring consecutive sessions and has no effect on passivavoidance of a stressful stimulus, a mild electric shoc[145].

Fig. (8). The D3 receptor-selective partial agonist BP 897 inhibits cue-controlled cocaine-seeking but has no reinforcing effects. A

during repeated sessions, rats are trained to self administer i.v. cocaine by a lever pressing. BP 897, tested when a stable pattern of

self-adminis tration is acquired, has no effect on cocaine self-administrat ion session, i.e. no reinforcing effect. B, Progressively, a

light stimulus is associated with cocaine self-administration, which gains reinforcing properties and finally maintains drug-seeking

behavior, even without drug delivery (C). During this last phase, the behavior of the animal, which reflects the motivation to take

drug induced by the conditioned stimulus is dose-dependently reduced by BP 897. (*P


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The mechanisms underlying the effects of D3 receptor-selective ligands in animal models of drug dependence arebeginning to be revealed. There has been considerableattention to D3 receptor in the nucleus accumbens, but D3receptors are also expressed in the ventral tegmental area andthe amygdala, brain regions implicated in the effects of drugsof abuse and in reactivity to drug associated cues. Since D3receptors are overexpressed in the brain of cocaine addicts[143] and of cocaine and nicotine-treated animals [145, 149],D3 receptor-selective ligands may act by attenuatingexacerbated dopamine transmission in the nucleusaccumbens. In agreement, Xi et al. recently reported thatlocal administration of SB-277011A, into the nucleus

accumbens block the reinstatement of cocaine-seekingbehavior induced by stress in rats [184].

Nevertheless, there is evidence that BP 897 may also actthrough D3 autoreceptors. Thus, stimulation of D3autoreceptors by BP 897, acting as an agonist, would reducedopaminergic transmission and, therefore, drug-seekingbehavior [192]. This hypothesis has been put forward basedon the effects of BP 897 on c-fos gene expression in variousbrain regions of cocaine-conditioned mice. Exposure tococaine-associated stimuli increases in c-fos gene expressionin various subcortical limbic areas, including the nucleusaccumbens, caudate-putamen, ventral tegmental area andamygdala [145]. Surprisingly, although BP 897 reduces

hyperactivity induced by presentation of cocaine-associatedstimuli, there is no effect on c-fos gene expression in thenucleus accumbens, supporting the hypothesis that BP 897does not act directly at postsynaptic D3 receptors.Nevertheless, this hypothesis is not consistent with theobservation that infusion of BP 897 into the nucleusaccumbens blocks the expression of amphetamine-conditioned locomotor hyperactivity [193]. In contrast, BP897 decreased c-fos gene expression in the ventral tegmentalarea, probably reflecting inhibition of activity ofdopaminergic neurons, the major neuronal population in thisarea [194]. This hypothesis is consistent with the activationof dopaminergic neurons induced by reward-predicting

stimuli [195] and by increases of dopaminergineurotransmission often associated with presentation of drugassociated stimuli [196, 197]. These putative and distincmechanisms of action may explain why D3 receptor partiaagonists and antagonists, behave slightly differentially idifferent models of addiction [56, 164].

D3 receptor ligands may also act through D3 receptors ithe amygdala [65]. This brain area in particular thbasolateral nucleus of amygdala, is involved in drugaddiction [198, 199] and in associative functions underlyindrug-conditioned behavior,[179, 200-202]. Moreover, drugassociated stimuli have been reported to increase extracellula

levels of dopamine in the amygdala [197] and to increase cfos gene expression [145]. Interestingly, BP 89administration increases c-fos gene expression in thamygdala of cocaine-conditioned mice, an effect that may bdue to a direct effect of BP 897 on D3 receptors in thamygdala, or to an indirect effect through regulation odopaminergic neuron activity [145]. In agreement with thihypothesis. BP 897 blocks the expression of amphetamineconditioned locomotor hyperactivity when infused into thbasolateral amygdala [193]. Another important brain aremay be the somatosensory cortex, where environmentastimuli are integrated. BP 897 inhibits c-fos expression ithe somatosensory cortex, which is induced by thpresentation of cocaine-associated stimuli in rodents [145]

In view of the low D3 receptor expression level in thsomatosensory cortex, the effect of BP 897 may be indirecin this region, via decreased activity of VTA neurons ansubsequent changes in the amygdala and thalamo-corticapathways. These effects have been confirmed by c-foanalysis in the brain of mice after development of morphineinduced CPP; BP 897 decreased c-fos expression in thsomatosensory cortex of wild-type animals, and these effectwere abolished in D3 receptor-deficient mice [203] (Fig. 9).

Thus, D3 receptor-selective ligands appear to modulatthe motivation to self-administer drugs under situationwhere the drugs have a high price. Numerous experimentalso indicate that the D3 receptor is involved in reactivity t

Table 2. Effects of D3 Receptor Ligands on Various Conditioned Responses

Reinforcer Drug cue-controlled response Effects of D3 receptor ligands Reference

Cocaine Drug-seeking behavior BP 897a; SB-277011A [235]

Cocaine Conditioned place preference BP 897 [190]

Cocaine Conditioned locomotor activity BP 897; SB-277011A [145]

Amphetamine Conditioned locomotor activity BP 897 [236]

Heroin Drug-seeking behavior BP 897 is inactive [235]

Heroin Conditioned place preference SB-277011A [189]

Morphine Conditioned place preference (mice) BP 897 [203]

Morphine Conditioned place preference (rats) BP 897 is inactive [190]

Nicotine Conditioned place preference BP 897; ST 198 [237]

Nicotine Conditioned locomotor activity BP 897; SB-277011A [149]

Ethanol Seeking behavior SB-277011A [238]

Food Seeking behavior BP 897 and SB-277011A are inactive [235]aattenuate.


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environmental stimuli associated with the effects of manyabused drugs. These stimuli, especially those associated withpsychostimulants such as cocaine and nicotine, can elicitreports of craving and precipitate relapse in abstinent humansubstance abusers [171-173, 204]. D3 receptor-selectiveligands may therefore provide an effective means ofpreventing relapse to drug use. Moreover, a therapeuticintervention with D3 receptor-selective ligands would notinterfere with normal activities, since these ligands do not

alter conditioned responses to aversive stimuli or to naturareinforcers, such as food.

8. THE D3 RECEPTOR AND DEPRESSION

BDNF is an important factor for the plasticity of thdopamine mesolimbic system, of which the role inappetitive motivation and positive reinforcement has beeemphasized above. Experimental evidence also supports thview that secondary adaptive changes in the function o

Fig. (9). Effect of BP 897 on morphine-induced CPP (A) and on c-fos expression (B) in wild-type (D3R+/+) and D3 receptor-deficien

(D3R-/-)mice. A, Time spent on the least preferred floor measured before and after pairing with saline or morphine at the dose of 32

mg/kg and changes is expressed as the time difference. Saline or BP 897 (1 mg/kg), was injected 30 min before the test session. *P

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