Widespread distribution of brain dopamine receptors evidenced with [125I]iodosulpride, a highly selective ligand.

The new benzamide derivative [125I]iodosulpride is a highly sensitive and selective ligand for D-2 dopamine receptors and displays a very low nonspecific binding to membrane or autoradiographic sections. On autoradiographic images, D-2 receptors are present not only in well-established dopaminergic areas but also, in a discrete manner, in a number of catecholaminergic regions in which the dopaminergic innervation is still unknown, imprecise, or controversial, as in the sensorimotor cerebral cortex or cerebellum. This widespread distribution suggests larger physiological and pathophysiological roles for cerebral dopamine receptors than was previously thought.

Dopamine D3 receptors expressed by all mesencephalic dopamine neurons.

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D(3) receptor (D(3)R). Using transfected cells expressing recombinant D(3)R, but not D(2) receptor, this antibody labeled 45-80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [(125)I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D(3)R binding, revealed using [(125)I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin ([(125)I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D(3)R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D(3)R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D(3)R binding in both AccSh and SN/VTA. D(3)R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D(3) autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D(3)R-deficient mice.

Novel dopamine receptors half a decade later.

Only half a decade ago, the effects of dopamine were all attributed to activation of two receptor subtypes, the D1 and D2, with opposing effects on adenylate cyclase, and for which apparently selective ligands were available. From the end of 1988, however, the application of homology cloning techniques starting from sequences of the seven transmembrane domain catecholamine receptors, particularly that of the D2 receptor, led to the identification of 'novel', previously uncharacterized dopamine receptors. In this article, Pierre Sokoloff and Jean-Charles Schwartz discuss the functional significance of such diversity, as well as the new therapeutic perspectives it offers.

Dopamine-related genes and their relationships to monoamine metabolites in CSF.

Monoamine metabolite (MM) levels in lumbar cerebrospinal fluid (CSF) are extensively used as indirect estimates of monoamine turnover in the brain. In this study we investigated genotypes for DNA polymorphisms in the D2 (DRD2), D3 (DRD3), and D4 (DRD4) dopamine receptor and tyrosine hydroxylase (TH) genes and their relationships to CSF MM in healthy volunteers (n = 66). Concentrations of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were corrected for back length, a confounding variable. Corrected MM levels were not related to age, gender, height, weight heredity, season or atmospheric pressure at sampling. Individuals with specific DRD2 and TH allele and genotype configurations significantly differed in HVA and MHPG concentrations. DRD3 homo- and heterozygotic genotypes had significantly different CSF 5-HIAA levels. DRD4 genotypes were not related to MM concentrations. The results suggest that specific DRD2, DRD3, and TH genotypes participate in the regulation of monoamine turnover in the central nervous system. Accordingly monoamine receptors and synthesizing enzyme genotypes appear to be variance factors influencing MM concentrations in CSF. The relationships found in this study support MM concentrations as markers for monoamine transmission in the human brain.

Failure to find evidence for linkage or association between the dopamine D3 receptor gene and schizophrenia.

OBJECTIVE: This study was performed to assess the possible involvement of the dopamine D3 receptor gene (DRD3) in the etiology of schizophrenia. The authors' approach included a population study and a family study using both parametric (lod score) and nonparametric (affected pedigree member) methods of linkage analysis. METHOD: Two different DNA markers were studied at the DRD3 locus. The family study included 35 multiplex families of schizophrenic subjects for the linkage analyses. The population study involved 50 unrelated schizophrenic subjects and 50 normal comparison subjects from the same ethnic and geographic origin. RESULTS: Whichever clinical classification was used to define the pathological phenotype (schizophrenia or schizophrenia spectrum), the results of the lod score and affected pedigree member studies did not provide any evidence of linkage of the DRD3 gene to the illness. The negative results of the association study reinforce these results. CONCLUSIONS: The hypothesis that the DRD3 gene has a predisposing role in schizophrenia was not supported by these population and family studies. However, the possibility that this gene has a role in the etiology of the disease cannot be definitely excluded because of the intrinsic limitations of the methods of analysis and the number of subjects studied.

Activation of dopamine D4 receptor inhibits an L-type calcium current in cerebellar granule cells.

The functions of the D4 receptor, a newly cloned D2-like receptor, as well as the identity of cells expressing it, are still poorly defined. Using quantitative polymerase chain reaction we detected the messenger RNA of the D4, but not other D2-like receptor, in cultured granule cells from neonatal rat cerebellum. In these neurons, dopamine reduced high-voltage-activated calcium current, with a pharmacology corresponding to that of the D4 receptor. The response declined from one to three days, when calcium currents were mostly sensitive to nifedipine, to 15 days, when nifedipine-insensitive calcium currents were also present and D4 receptor messenger RNA had declined. The dopamine response was abolished after pretreatment of the cells by pertussis toxin, was potentiated and made irreversible by infusion of guanosine 5'-O-(3-thiotriphosphate) but persisted in the presence of cyclic AMP and isobutylmethylxanthine. These results indicate the presence in the neonatal cerebellum of a functional D4 receptor inhibiting an L-type calcium current, an action involving a Gi/Go protein but independent from adenylate cyclase inhibition.

Phenotypical characterization of neurons expressing the dopamine D3 receptor in the rat brain.

We have established the cellular distribution of the dopamine D3 receptor using tritiated 7-hydroxy-N-N-di-n-propyl-2-aminotetralin and a complementary RNA probe to visualize autoradiographically the protein in binding studies and the gene transcripts by in situ hybridization, respectively. Studies with these two markers confirm the restricted expression of the D3 receptor in few brain areas, i.e. mainly the ventral striatal complex, the substantia nigra-ventral tegmental area and the cerebellum. In nucleus accumbens, the D3 receptor was mainly expressed in medium-sized neurons of the rostral pole and ventromedial shell subdivisions, but not of the core or septal pole, i.e. accumbal subdivisions expressing the D2 receptor. In the ventromedial shell, about 60% of the D3 receptor-expressing neurons were neurotensin neurons, presumably projecting to the ventral pallidum. In the islands of Calleja, both D3 receptor binding and messenger RNA were abundant in the entire population of granule cells. These cells are known to make sparse contacts with dopaminergic axons and also to express the D1 receptor. In the mesencephalon, low levels of D3 messenger RNA were detected in few dopamine neurons of substantia nigra pars lateralis and ventral tegmental area. In addition, some D3 receptor binding but not messenger RNA was detected in medial substantia nigra and lateral ventral tegmental area, where the receptor is presumably located presynaptically on afferents. In the archicerebellum, Purkinje cell perikarya in lobules 9 and 10 expressed the D3 receptor messenger RNA, whereas binding sites were found in the molecular layer, where corresponding dendrites but no known dopaminergic projection from mesencephalon are found. The occurrence of D3 receptor gene expression in some brain areas receiving low dopamine innervation supports the hypothesis that this receptor may mediate non-synaptic actions of dopamine.

Identification of the dopamine autoreceptor in the guinea-pig retina as D(2) receptor using novel subtype-selective antagonists.

1. Dopamine release in the retina is subject to modulation via autoreceptors, which belong to the D(2) receptor family (encompassing the D(2), D(3) and D(4) receptors). The aim of the present study was to determine the receptor subtype (D(2) vs D(3)) involved in the inhibition of dopamine release in guinea-pig retinal discs, using established (haloperidol, (S)-nafadotride) and novel dopamine receptor antagonists (ST-148, ST-198). 2. hD(2L) and hD(3) receptors were expressed in CHO cells and the pK(i) values determined in binding studies with [(125)I]-iodosulpride were: haloperidol 9.22 vs 8.54; ST-148 7.85 vs 6.60; (S)-nafadotride 8.52 vs 9.51; ST-198 6.14 vs 7.92. 3. The electrically evoked tritium overflow from retinal discs preincubated with [(3)H]-noradrenaline (which represents quasi-physiological dopamine release) was inhibited by the dopamine receptor agonists B-HT 920 (talipexole) and quinpirole (maximally by 82 and 71%; pEC(50) 5.80 and 5.83). The concentration-response curves of these agonists were shifted to the right by haloperidol (apparent pA(2) 8.69 and 8.23) and ST-148 (7.52 and 7.66). (S)-Nafadotride 0.01 microM and ST-198 0.32 microM did not affect the concentration-response curve of B-HT 920. 4. The dopamine autoreceptor in the guinea-pig retina can be classified as a D(2) receptor. ST-148 and ST-198 show an improved selectivity for D(2) and D(3) receptors when compared to haloperidol and (S)-nafadotride, respectively.

Multiple dopamine receptors: the D3 receptor and actions of substances of abuse.

Our knowledge of dopamine receptor diversity has markedly increased during the past few years as a result of discovery of five distinct genes, splice variants and polymorphic receptors. The genes can be classified in two subfamilies: the intronless genes that encode the D1 and D5 receptors positively linked to adenylyl cyclase and genes with introns that encode the two isoforms of the D2 receptor and the D3 and D4 receptors. The various dopamine receptor subtypes can be distinguished by their sequence, intracellular signalling systems, pharmacology and localisation. The localisation of the D3 receptor in the shell of nucleus accumbens suggests its participation in brain reward circuits and actions of substances of abuse.

Meta-analysis of DRD3 gene and schizophrenia: ethnic heterogeneity and significant association in Caucasians.

The involvement of dopamine in the etiology of schizophrenia is suggested by a number of neurobiological and pharmacological data, the dopamine D3 receptor (DRD3) being selectively expressed in brain regions which may be specifically involved in the risk for schizophrenia. The gene coding for DRD3 has thus been extensively analyzed. Since the initial report providing substantial evidence for an association of homozygosity of either allele of the gene coding for DRD3 (BalI polymorphisms) with schizophrenia, a flurry of replicating studies has appeared, which have been split into confirmations and nonreplications in North European Caucasian, Mediterranean, Asian, American, and African populations. The involvement of DRD3 polymorphisms thus remains questionable, particularly as no linkage studies have favored a nonrandom segregation of DRD3 alleles and schizophrenia. We performed a metaanalysis from 29 independent samples, from 24 different association studies so far published, allowing the examination of 2,619 schizophrenic patients and 2,517 controls. No significant differences of genotype counts were noted between patients and controls for the whole sample, considering frequency of any genotype. Starting from the high variability of the genotypes in different geographical areas, the impact of ethnic heterogeneity was taken into account. When the studies were reorganized in five groups according to geographical origin of samples, both homozygosity and 1-1 genotype revealed significant heterogeneity (P < 0.05). We specifically found an excess of homozygosity and 1-1 genotype in schizophrenic patients only in the African and Caucasian groups (P < 0.05). The present analysis suggests a small but significant effect of DRD3 in the susceptibility to schizophrenia, at least in Caucasians.

No association between schizophrenia and homozygosity at the D3 dopamine receptor gene.

The D3 dopamine receptor gene is an important candidate gene for schizophrenia, since (because of its almost exclusive expression in the limbic system) it combines the dopamine receptor hypothesis with the limbic system hypothesis of schizophrenia. A BalI restriction fragment length polymorphism of the D3 dopamine receptor gene has been typed in 107 schizophrenic patients and 98 normal controls from Sichuan (China). With regard to alleles or genotypes, no significant differences were obtained between controls from Europe and China, between patients and controls, and between patient subgroups and controls. These results indicate a lack of association between schizophrenia and the D3 dopamine receptor gene in our sample. Our findings are at variance with reports of a significant excess of homozygosity at the D3 dopamine receptor gene in schizophrenic patients from Wales (United Kingdom) and Alsace (France). In conclusion, further studies will be needed with larger samples of patients from Wales and Alsace as well as with samples of different racial groups to prove or disprove the initial positive association between schizophrenia and genotypes of the D3 dopamine receptor gene.

Lack of association between alcohol-dependence and D3 dopamine receptor gene in three independent samples.

Numerous studies on the involvement of dopamine receptors in the genetics of alcoholism focused on associations between a polymorphism of the D2 dopamine receptor (DRD2) gene and alcohol dependence. However, the results of these studies are conflicting. Another receptor, the D3 dopamine receptor (DRD3), may be of additional interest since it is specifically located in the limbic area, and in particular in the nucleus accumbens which plays a significant role in the reward process of addiction behavior. We thus tested the association in three independent samples of alcoholic patients, with different origins and various inclusion criteria. No difference in the DRD3 gene polymorphism emerged between controls and alcoholic patients, regardless of their origin, inclusion criteria, or presence or absence of the DRD2 TaqI A1-allele. Despite the fact that more information could have been considered and that association studies provide limited information, there is good evidence that this DRD3 polymorphism does not play a major role in the genetic component of alcoholism.

Dopamine D3 receptor gene: organization, transcript variants, and polymorphism associated with schizophrenia.

DNA fragments from a genomic library were used to establish the partial structure of the human dopamine D3 receptor gene (DRD3). Its coding sequence contains 6 exons and stretches over 40,000 base pairs. The complete DRD3 transcript and three shorter variants, in which the second and/or third exon are deleted, were detected in similar proportions in brains from four controls and three psychiatric patients. The Msp I polymorphism was localized in the fifth intron of the gene, 40,000 base pairs downstream the Bal I polymorphism and a PCR-based method was developed for genotyping this polymorphism. The distribution of the Msp I and Bal I genotypes were not independent in 297 individuals (chi 2 = 10.5, df = 4, P = 0.03), but only a weak association was found between allele 1 of the Bal I polymorphism and allele 2 of the Msp I polymorphism (chi 2 = 3.99, df = 1, P = 0.04). The previously reported association between homozygosity at both alleles of the Bal I polymorphism and schizophrenia was presently maintained in an extended sample, comprising 119 DSM-III-R chronic schizophrenics and 85 controls (chi 2 = 5.3, df = 1, P = 0.02) and found more important in mal than in females. The presence of the Bal I allele 2 is associated with an early age at onset, particularly in males (df = 35, t value = 2.6, P = 0.014). In the same sample, allelic frequencies, genotype counts, and proportion of homozygotes for the Msp I polymorphism did not differ between schizophrenics and controls (chi 2 = 0.06, df = 1, P = 0.80, chi 2 = 0.22, df = 1, P = 0.90 and chi 2 = 0.16, df = 1, P = 0.69, respectively). The large distance of the Msp I polymorphism from the Bal I polymorphism and its localization in the 3' part of the gene may explain the discrepant results obtained with the two polymorphisms.

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907.

The selective D3-dopamine receptor agonist 4aR, 10bR-(+)-trans-3,4,4a, 10b-tetrahydro-4-[N-propyl-2,3-3H]-2H,5H-[1] benzopyrano[4,3-b]-1,4-oxazin-9-ol ([3H]PD 128907) was used to visualise D3-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [3H]PD 128907 has an 18- to 40-fold selectivity for D3- over D2-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [3H]7-OH-DPAT. [3H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [3H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D2/D3-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D3-dopamine receptors in the human brain. The binding obtained with [3H]PD 128907 was qualitatively similar to that using [3H]7-OH-DPAT in the presence of GTP. However, [3H]7-OH-DPAT labelled, in contrast to [3H]PD 128907, also D3-dopamine receptors in neocortex. The new compound [3H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D3-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype.

Role of dopamine D3 receptors in thermoregulation: a reappraisal.

Dopamine agonist-induced hypothermia has been proposed to be mediated by the D3 receptor (D3R), as it is elicited by (+)7-OH-DPAT and antagonized by S 14297, two putative D3R-preferential ligands. Here we show, however, that S 14297 is a full and partial agonist at D3R and D2R, respectively. Hypothermia was induced in rats by agonists with potencies correlated with their D3R and D2R functional potencies, and was reversed by antagonists, with a rank order of potency typical of the D2R. Moreover, BP 897, a highly potent and selective but partial D3R agonist was inactive in producing hypothermia or reversing (+)7-OH-DPAT-induced hypothermia. (+)7-OH-DPAT was as potent and efficient in inducing hypothermia in wild-type as in D3R-deficient mice. Hence, our results suggest that hypothermia does not result from a selective stimulation of the D3R.

A functional test identifies dopamine agonists selective for D3 versus D2 receptors.

The functional potency of a series of dopamine agonists for increasing mitogenesis, measured by incorporation of [3H]thymidine, was established in transfected cell lines expressing human D2 or D3 receptors. The functional selectivity of agonists markedly differs from their binding selectivity. (+)7-OH-DPAT, pramipexole, quinerolane and PD 128,907, the most D3 receptor-selective compounds in binding studies, were 7, 15, 21 and 54 times more potent, respectively, at the D3 than at the D2 receptor in the functional test. Bromocriptine displayed a 10-fold functional selectivity toward the D2 receptor. The known behavioural actions of D3 selective agonists support a role for the D3 receptor in motor inhibitions, which should be taken into account for the treatment of motor dysfunctions by dopamine agonists.

D3 receptor test in vitro predicts decreased cocaine self-administration in rats.

The three dopamine agonists with highest reported D3 receptor selectivity in vitro, pramipexole, quinelorane and PD128,907, decreased self-administration of a high dose of cocaine in rats as a result of a leftward shift in the cocaine dose-effect function. In contrast the D3 preferring antagonist nafadotride increased cocaine self-administration. Moreover the relative potencies of these and other D2-like dopamine agonists (lisuride, 7-OH-DPAT, quinpirole, apomorphine, bromocriptine) to modulate cocaine self-administration were highly correlated with their relative potencies for increasing mitogenesis in vitro in cell lines expressing D3 but not D2 receptors. These results support the hypothesis that the D3 receptor may be an important target for pharmacotherapies for cocaine abuse and dependence.

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA.

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

D3 dopamine receptor mRNA is widely expressed in the human brain.

Considerable attention has been given to the association of the D3 dopamine receptor subtype and limbic function based on the abundant localization of D3 receptor sites and mRNA expression in the islands of Calleja and nucleus accumbens in experimental animals. Though most human anatomical studies have focused on the role of D3 receptors in limited brain structures, detailed information about the overall anatomical organization of the D3 receptor in the human brain is still, however, not available. In the current study, we examined the anatomical distribution of D3 receptor mRNA expression at different levels of the human brain in whole hemisphere horizontal cryosections using in situ hybridization. This approach made it possible to establish for the first time the wide and heterogenous expression of the D3 receptor gene throughout the human brain. As expected, the most abundant D3 mRNA expression levels were found in the islands of Calleja and discrete cell cluster populations within the ventral striatum/nucleus accumbens region. High levels were also evident within the dentate gyrus and striate cortex. Low to moderate D3 mRNA expression levels were apparent in most brain areas including all other cortical regions (highest in the anterior cingulate/subcallosal gyrus), caudate nucleus, putamen, anterior and medial thalamic nucleus, mammillary body, amygdala, hippocampal CA region, lateral geniculate body, substantia nigra pars compacta, locus coeruleus, and raphe nuclei. While the current anatomical map of D3 receptor mRNA expression in the human brain does confirm previous reports that D3 receptors may play important roles in limbic-related functions such as emotion and cognition, the findings also suggest other non-limbic functions for D3 mRNA-expressing cell populations such as processing of motor and sensory information.

Functional potencies of new antiparkinsonian drugs at recombinant human dopamine D1, D2 and D3 receptors.

We measured the affinities of bromocriptine, pramipexole, pergolide and ropinirole at human recombinant dopamine D1, D2 and D3 receptors in binding and functional tests. All four compounds bound with high affinity at the dopamine D3 receptor; bromocriptine and pergolide also had high affinity for the dopamine D2 receptor, while only pergolide had significant, although moderate, affinity for the dopamine D1 receptor. Only pergolide had high potency and intrinsic activity at the dopamine D1 receptor for stimulating cyclic AMP accumulation. In addition, the potencies and efficacies of pergolide and bromocriptine, as well as that of dopamine, at the dopamine D1 receptor were increased in the presence of forskolin, an adenylate cyclase activator. All four compounds were highly potent agonists at dopamine D2 and D3 receptors, as measured in a mitogenesis assay. Bromocriptine was ten times more potent and pramipexole and ropinirole ten times less potent at the dopamine D2 than at the dopamine D3 receptor, whereas pergolide was equipotent at the two receptors. These results suggest that the activity of recently developed antiparkinsonian drugs at either the dopamine D1 or the dopamine D3 and not only the dopamine D2 receptors should be taken into account in analyses of their mechanisms of action in therapeutics.