Association between equine temperament and polymorphisms in dopamine D4 receptor gene

The variable number of tandem repeats (VNTR) polymorphism of the dopamine D4 receptor (DRD4) gene has been reported to be associated with the personality trait of novelty-seeking in humans. In the genus Equus, this region includes an 18-bp repeat unit and there are inter- and intraspecies differences in the number of repetitions. Because horses are unique among livestock species in that their temperament is considered important, we investigated the possible role of this region on equine temperament in thoroughbred horses. We simultaneously determined the sequences of this polymorphic region and administered a questionnaire survey to horse caretakers with questions about 20 different traits of their horses’ temperament. Although there was no difference in the number of repeats among the 136 thoroughbred horses studied, two single nucleotide polymorphisms (SNPs), one of which might cause an amino acid change (A-G substitution), existed. By analyzing the association between these SNPs and temperament scores, a significant association was revealed between two temperament traits (Curiosity and Vigilance) and the A-G substitution. Horses without the A allele had significantly higher Curiosity and lower Vigilance scores than those with the A allele at the A-G substitution. In addition, similar associations between both temperament scores and each genotype of the A-G substitution were observed in two subgroups divided according to the time of their introduction to the farm. These results suggested that the SNP in the VNTR region of the equine DRD4 gene might be related to individual differences in equine temperament.     

Association of seven polymorphisms of the D2 dopamine receptor gene with brain receptor-binding characteristics

Association of alleles at the Taql A, Taql B, intron 6, Taql D, exon 7, exon 8, and promoter-141C sites of the D2 dopamine receptor gene with D2 dopamine receptor binding characteristics in the caudate nucleus of Caucasian alcoholic and nonalcoholic subjects was determined. For the Taql D, exon 7, exon 8, and promoter-141C sites there were no significant allelic differences in Bmax (number of binding sites) or Kd (binding affinity) of the D2 dopamine receptors. However, subjects having the minor alleles at the Taql A, Taql B, and intron 6 sites had significantly lower Bmax than subjects not having them. None of these three polymorphisms had any significant effect on Kd. Highly significant linkage disequilibria were observed among the Taql A, Taql B, and intron 6 polymorphic sites, but linkage disequilibria between these three sites and each of the Taql D, exon 7, exon 8, and promoter-141C sites were of lesser or of no significance. Taken together, these findings suggest that the Taql A, Taql B, and intron 6 polymorphisms, but not the Taql D, exon 7, exon 8, and promoter-141C polymorphisms, are in linkage disequilibrium with a functional allelic variant that affects D2 dopamine receptor expression.     

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene ( DRD4 ) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a ) were detected in this breed, and genotype frequencies were in Hardy–Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele ( P  = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.     

A new polymorphism in the gene for the dopamine D2 receptor

A new polymorphism has been identified in the gene for the dopamine D₂ receptor. The polymorphism was found by single stranded conformational polymorphism (SSCP) analysis. Sequencing revealed an insertion at a BsoF1 restriction site. This polymorphism in the 3- untranslated region was found at a frequency of 0.07 in Centre d'Etude du Polymorphisme Humain (CEPH) parents.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Purification of brain D2 dopamine receptor.

D2 dopamine receptors have been extracted from bovine brain using the detergent cholate and purified approximately 20,000-fold by affinity chromatography on haloperidol-sepharose and wheat germ agglutinin-agarose columns. The purified preparation contains D2 dopamine receptors as judged by the pharmacological specificity of [3H]spiperone binding to the purified material. The sp. act. of [3H]spiperone binding in the purified preparation is 2.5 nmol/mg protein. The purified preparation shows a major diffuse band at Mr 95,000 upon SDS-polyacrylamide gel electrophoresis and there is evidence for microheterogeneity either at the protein or glycosylation level. Photoaffinity labelling of D2 dopamine receptors also shows a species of Mr 95,000. The D2 dopamine receptor therefore is a glycoprotein of Mr 95,000.     

The dopamine D2 receptor gene in lamprey, its expression in the striatum and cellular effects of D2 receptor activation

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates.     

Direct sequencing of the dopamine D2 receptor (DRD2) in schizophrenics reveals three polymorphisms but no structural change in the receptor.

The dopamine D2 receptor gene (gene symbol DRD2) is a candidate gene for schizophrenia because the potency of certain neuroleptics correlates with their affinity for this receptor. Seven regions of likely functional significance including the coding sequences and the splice junctions were fully sequenced in the dopamine D2 receptor of 14 schizophrenics (and partially in several others) meeting DSM-III-R diagnostic criteria and in four unaffected non-Caucasians (97 kb of total sequence). No structural changes were found, suggesting that alteration in the structure of the dopamine D2 receptor is not commonly involved in the etiology of schizophrenia. However, two common and one uncommon intragenic polymorphisms were found. At least one of the polymorphisms was informative for linkage in 70% of Caucasians and 78% of Koreans.     

Polymorphism of the dopamine D2 receptor gene in populations from the Volga-Ural region

The PCR technique was used to analyze the TaqIA- and NcoI-polymorphisms at the dopamine D2 receptor gene (DRD2) in eight populations of the Volga-Ural region belonging to Turkic (Bashkirs, Tatars, and Chuvashes), Finno-Ugric (Maris, Komis, Mordovians, and Udmurts), and Eastern-Slavic (Russians) ethnic groups. Population-specific patterns of the main TaqIA- and NcoI-polymorphisms distribution were established. Specific trends in changes of genotype and allele frequency of the dopamine D2 receptor gene depending on the ethnicity of the population were revealed.     

Repeat sequences in the gene encoding the human D4 dopamine receptor.

The gene encoding the human D4 dopamine receptor has evolved by gaining at least five internal repeats which are located within exons 3 and 4, and in the intervening intron 3 sequence. The amino acid sequence in the cytoplasmic loop of the receptor, involved in G protein coupling, has been altered by these gene changes.     

An antibody to dopamine D2 receptor inhibits dopamine antagonist and agonist binding to dopamine D2 receptor cDNA transfected mouse fibroblast cells.

A polyclonal antibody to dopamine D2 receptor (D2-receptor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [3H]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM09151-2 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 +/- 5.26 and 76.20 +/- 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltk- and Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB cells with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor.     

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

D2 dopamine receptor involvement in spinal dopamine-produced antinociception.

Experiments were performed on 79 lightly pentobarbital-anesthetized rats. Rats displayed a dose-dependent increase in tail-flick latencies following the injection of dopamine (DA) into the lumbar subarachnoid space through an intrathecal tube. Sulpiride, a D2-subtype receptor antagonist, antagonized the DA-induced analgesia (antinociceptive) effect; while SCH-23390, a D1-subtype receptor antagonist, had no effect even in a higher dose. To further investigate whether the well-known spinal serotonergic, noradrenergic and opioidergic receptor systems were involved in DA-induced antinociception, their antagonists, methysergide, phentolamine, and naloxone were tested respectively. The results showed that phentolamine, but not methysergide or naloxone, could block the DA-induced antinociception. The present data provide evidence that DA exerts antinociceptive effects through D2-subtype dopamine receptor(s) at the spinal level, and that spinal alpha-adrenergic receptors may mediate this effect.     

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.     

A rare FokI RFLP in the human dopamine D2 receptor gene (DRD2)

Summary A new biallelic polymorphism for FokI restriction enzyme due to CT transition in the fourth intron of human DRD2 is described. It must be a usefull marker of this candidate gene for several mental disorders.