D2 dopamine receptor antagonists induce fos and related proteins in rat striatal neurons

Rats injected with haloperidol, which binds to both D2 dopamine and sigma receptors or the specific D2 dopamine receptor antagonist YM 09151-2, but not the specific D1 dopamine receptor antagonist SCH 23390, showed induction of c-fos protein and c-fos-related antigens in striatal neurons. This effect of haloperidol and YM 09151-2 was inhibited by the N-methyl-D-aspartate antagonist MK801 but was not affected by 1,3-di-O-tolylguanidine, a selective sigma receptor ligand. Two different antisera were used to detect c-fos protein: one was specific for c-fos protein itself while the other recognized c-fos protein as well as c-fos protein-related antigens. In time-course immunocytochemical studies, the c-fos protein was induced maximally by 1 h and had returned to baseline by 24 h. However, c-fos protein-related antigens were induced maximally after 2 h and remained elevated for at least three days after haloperidol injection. Furthermore, the c-fos protein-specific antiserum detected two to three times fewer immunopositive striatal cells than the antiserum which detected both c-fos protein-related antigens and c-fos protein in haloperidol-treated rats. This result suggests that some striatal neurons express c-fos protein-related antigens but not c-fos protein after haloperidol injection. In some striatal sections from haloperidol-injected rats immunostained with the antiserum which recognizes both c-fos protein and c-fos protein-related antigens, there were large areas of immunopositive neurons interspersed with "areas" of striatum devoid of immunostaining. The implications of these results for theories concerning the biochemical mechanism of action of haloperidol are discussed.     

Correlation between different memory systems and striatal dopamine D2/D3 receptor density: a single photon emission computed tomography study

OBJECTIVE: Although studies have indicated that the human basal ganglia have a specific role in different memory systems, the functional significance of the striatal dopamine activities for the basal ganglia remains less clear. This study assessed the relationship between measures of striatal dopamine activities and indices of different memory systems in healthy individuals. METHOD: Single photon emission computed tomography and [123I]IBZM (iodobenzamide) were used to assess the striatal dopamine D2/D3 receptor density in 62 healthy volunteers aged between 19 and 61 years. All subjects underwent a Wechsler Memory Scale-Revised test. RESULTS: Dopamine D2/D3 receptor densities in the striatum decline with age. Stepwise regression analysis showed that verbal delayed recall and working memory account for most of the variance in dopamine D2/D3 measurements. These relationships remain significantly after controlling for age effects. CONCLUSIONS: Brain striatal dopamine activities are also significantly associated with various memory systems, in addition to motor functions. This may explain why patients with neuropsychiatric disorders may have both memory and motor impairments.     

Enkephalin regulates acute D2 dopamine receptor antagonist-induced immediate-early gene expression in striatal neurons.

Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c-fos, zif 268) induced by the D2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.     

Relation between brain dopamine loss and D2 dopamine receptor density in MPTP monkeys

The relationship between dopamine (DA), dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA) concentrations and [3H]spiperone binding to D2 DA receptors in caudate nucleus, putamen and nucleus accumbens following DAergic lesion with MPTP in monkeys was investigated. The level of DA depletion varied from 37% to 100% while a mean elevation in [3H]spiperone binding density of 114.8 +/- 4.7% of control (P less than 0.01) was observed when DA depletion was at least 90% of control values. A logarithmic correlation (y = -7.19 In x +111.15; r = -0.54, P less than 0.01) between DA concentrations (x) and the density of [3H]spiperone binding sites (y) was observed in all brain regions. The correlation between DOPAC or HVA concentrations and [3H]spiperone binding was best represented by linear rather than logarithmic equations. These results indicate that supersensitivity of DA receptors develops after MPTP lesion in monkeys when the number of remaining nerve fibers becomes too few to compensate for the DA loss.     

Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.     

Chronic hypoxia increases beta-adrenergic receptor density in the lungs of young and old rats.

To test the hypothesis that the ability to regulate beta-adrenergic receptor (BAR) density in response to chronic hypoxic stress is impaired by aging, we measured BAR density in the lungs of young (age 3 months) and aged (age 20 months) rats exposed to hypobaric hypoxia (1/2 atm) for 3 weeks. BAR density increased by 63% in the lungs of both young and aged rats exposed to chronic hypoxia. Lung BAR density was unaffected by aging, independent of hypoxic conditions. We conclude that the ability to respond to chronic hypoxic stress with increased lung BAR density is unaffected by aging in rats.     

The correlation between striatal dopamine D2/D3 receptor availability and verbal intelligence quotient in healthy volunteers

BACKGROUND: Although a correlation between the central dopaminergic system and intelligence may exist, the results from imaging studies remain inconclusive. The aim of this study was to explore the relationship between striatal dopamine D2/D3 receptor availability and verbal intelligence quotient (VIQ) using single photon emission computed tomography (SPECT). METHOD: Striatal D2/D3 receptor availability of 64 healthy subjects was determined with the [123I]iodobenzamide ([123I]IBZM) ligand. Intelligence quotients (IQs) of the subjects were measured by the Wechsler Adult Intelligence Scale--Revised (WAIS-R). RESULTS: In addition to age, left striatal D2/D3 receptor availability correlated positively with VIQ. In females, left striatal D2/D3 receptor availability was the only variable that correlated significantly with the similarities subtest of VIQ. CONCLUSIONS: There is a relationship between left striatal D2/D3 receptor availability and verbal intelligence, which varies, predominantly in males.     

The D1 dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.     

Age-related changes in striatal dopamine D2 receptor binding in weaver mice and effects of ventral mesencephalic grafts

Summary Dopamine (DA) D2 receptor binding is increased in the striatum of 5–6 months old weaver mutant mice (Kaseda et al. 1987). This may occur in response to the loss of DA neurons in the midbrain and the decrease in DA content in the striatum of homozygous mutants. One purpose of the present study was to determine if the diminished DA innervation is associated with changes in D2 receptors at earlier ages and if the increase in DA D2 receptor binding seen at 5–6 months is a lasting phenomenon. Specific [³H]spiperone binding was measured in the dorsolateral (DL), dorsomedial (DM) and ventrolateral (VL) striatum and in the nucleus accumbens (AC) of homozygous weaver mutant mice (wv/wv), heterozygous littermates (wv/+) and wild-type controls (+/+). Mice were studied at 20 days and 1, 3, 6, 9 and 12 months of age. The difference in specific [³H]spiperone binding in DL striatum between wv/wv and +/+ mice was significantly greater at 6 months than the difference at 1 month and at 12 months of age. Foetal ventral mesencephalic grafts survive and establish functional innervation in the striatum of weaver mice as shown by the induction of a contralateral turning bias (Low et al. 1987). The second aim of the present studies was to determine if such grafts would also reverse the increase in DA D2 receptor binding in the striatum. Aspiration cavities were prepared in the cortex of weaver mice, and ventral mesencephalic tissue from E14–E15 +/+ foetuses was subsequently placed on the surface of the right dorsal striatum when the recipients were 3 months old. At 6 months of age, specifie [³H]spiperone binding was determined in DM and DL striatum and in AC. Compared with the non-grafted side, the decreases in [³H]spiperone binding on the right (grafted) side in DL and DM striatum of the transplanted group were significantly greater than the differences in the other two groups. Since the grafts were introduced at an age (3 months) at which no significant difference in binding is seen between wv/wv and + / +, it appears that the grafts may prevent the increase in DA D2 receptor binding which is seen in non-grafted weaver mice at 6 months of age.     

Adenosine A2a receptor/dopamine D2 receptor hetero-oligomerization: a hypothesis that may explain behavioral sensitization to psychostimulants and schizophrenia

The mechanisms underlying psychostimulant-induced behavioral sensitization and schizophrenia are yet to be fully elucidated. Evidence suggests that the dopamine D2 receptor (DRD2) as well as other neurotransmitter system receptors may be involved in the two conditions, and previous reports have hypothesized that oligomerization of dopamine receptors and analogs from other neurotransmitter systems may underlie the mechanism responsible. This paper describes findings which show co-localization of DRD2 and the adenosine A2a receptor (A2aAR) in the striatum, interaction between the two receptors and A2aAR/DRD2 hetero-oligomerization in the neuronal cell. The possibility that A2aAR/DRD2 hetero-oligomerization may be involved in the development of psychostimulant-induced behavioral sensitization or the pathogenesis of schizophrenia is explored. A2aAR/DRD2 hetero-oligomerization can enhance dopaminergic function, resulting in an increased behavioral response to psychostimulants or the development of psychotic symptoms. Receptor oligomerization can affect receptor degradation, which may affect the persistence of behavioral sensitization or psychotic symptoms. The proposed A2aAR/DRD2 hetero-oligomerization model may suggest new directions in the understanding and treatment of schizophrenia.     

Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish.

We mined the zebrafish genomic sequence database and identified contigs containing segments of several dopamine receptor genes. By using a polymerase chain reaction amplification strategy, we generated full-length cDNAs encoding a single dopamine D3 receptor and three distinct D2 receptor subtypes. Zebrafish dopamine receptor genes were mapped by using the T51 radiation hybrid panel. The D3 receptor gene (drd3) mapped to linkage group (LG) 24. The three D2 receptor genes were localized to LG 15 (drd2a), LG 16, (drd2b), and LG 5 (drd2c). With the exception of the drd2b gene, each of these map positions was syntenic with regions of human chromosomes containing orthologs of the zebrafish dopamine receptor genes. Whole-mount in situ hybridization was used to investigate expression of the D2 and D3 receptor genes. Expression of the drd3 gene was first detected at mid-somitogenesis and was particularly prominent in somites. Thereafter, the drd3 gene was expressed diffusely throughout the brain and spinal cord. The three D2 receptor genes were expressed throughout the central nervous system (CNS) in distinct but overlapping patterns. In early embryos, the drd2a gene was expressed exclusively in the epiphysis, whereas the drd2c gene was localized to the notochord. After 24 hpf, the drd2a, drd2b, and drd2c genes were differentially expressed throughout the CNS. The identification of dopamine receptor genes in zebrafish should allow us to use the power of zebrafish genetics to analyze the functional properties of this important class of neurotransmitter receptors.     

The effects of denervation and chronic haloperidol treatment on neostriatal dopamine receptor density are not additive in the rat

The effects of combined denervation and chronic haloperidol treatment on neostriatal D-2 receptor density were examined, and a lack of additivity was found. In the first experiment, haloperidol treatment for 14 days initiated on day 4 after the unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA) abolished the lesion-induced asymmetry of [3H]spiroperidol binding by elevating the density of sites in the intact striatum. These results were replicated in the second experiment, in which the drug treatment was begun 4-5 weeks after 6-OHDA. In both experiments the apomorphine-induced rotation did not differ significantly between lesioned rats treated with haloperidol or its vehicle.     

Functional recovery of the striatal cholinergic system in aged rats by adenoviral vector-mediated gene transfer of dopamine D2 receptor

We previously reported that the striatal dopamine-acetylcholine (ACh) interaction was affected by the aging process, possibly via a decrease of the striatal D(2)R expression. In the current study, the ACh responses to the infusion of 0.1microM of the D(2)R agonist quinpirole were measured with microdialysis techniques after adenoviral vector-mediated gene transfer of D(2)R into the striatum of 25-month-old rats. The D(2)R gene-transferred rats showed significantly stronger responses of the striatal cholinergic neurons to the infusion of the D(2)R agonist than did control vector-transferred rats. The current study suggests that age-associated functional decline with decreased gene expression of the receptor may be restored by intervention.     

Association of harm avoidance with dopamine D2/3 receptor availability in striatal subdivisions: a high resolution PET study

We examined the relationship between the personality trait of harm avoidance (HA) and the dopamine D_2/3 receptor availability in striatal subdivisions using high resolution positron emission tomography (PET) with [¹¹C]raclopride. Twenty-one healthy subjects completed 3 T magnetic resonance imaging and high resolution PET scans with [¹¹C]raclopride in order to measure D_2/3 receptor availability in subregions of the striatum. The D_2/3 receptor availability was obtained on the basis of the Logan graphical method. The Temperament and Character Inventory was used to measure the biogenetic temperament of HA. The analysis revealed that the HA score had significant negative correlations with D_2/3 receptor availability in the pre-commissural dorsal caudate and post-commissural putamen, suggesting that HA is associated with D_2/3 receptor availability in the associative and sensorimotor subdivisions of the striatum, which are mainly involved in cognition and locomotion. Further research is required to determine if pathological states have similar dopaminergic mechanisms in specific striatal subdivisions.     

Chronic ascorbate potentiates the effects of chronic haloperidol on behavioral supersensitivity but not D2 dopamine receptor binding.

Ample behavioral evidence suggests that ascorbate parallels the action of haloperidol, a widely used neuroleptic. To determine the extent to which this parallel extends to chronic treatment, 21 days of exposure to ascorbate (100 or 500 mg/kg) alone or combined with haloperidol (0.5 mg/kg) were assessed on stereotyped behavior and neostriatal D2 dopamine receptor binding in rats. Our results indicate that when challenged with the dopamine agonist, apomorphine (0.5 mg/kg), animals chronically treated with haloperidol or high-dose ascorbate alone display a supersensitive sniffing response relative to controls, while animals chronically treated with the combination of haloperidol and high-dose ascorbate display a further potentiation of sniffing relative to the haloperidol groups. In addition, [3H]spiperone saturation studies showed, as expected, an up-regulation of striatal D2 dopamine receptors in rats treated with haloperidol as reflected by a change in receptor density (Bmax) but not affinity (KD). Ascorbate treatment, however, had no effect on D2 receptor density or the distribution of [3H]apomorphine in whole brain. Even though chronic treatment with the haloperidol-high-dose-ascorbate combination produced an up-regulation of striatal D2 dopamine receptors, this treatment did not cause a further up-regulation relative to haloperidol alone nor did it have any effect on [3H]apomorphine distribution. Taken together, these findings indicate that although chronic ascorbate produces behavioral supersensitivity to apomorphine through central mechanisms, they appear to differ from those induced by chronic haloperidol.     

Reserpine-induced up-regulation of dopamine D2 receptors in the rat striatum is enhanced by denervation but not by chronic receptor blockade

Compensatory increases in the density of dopamine (DA) D2 receptors in the rat striatum occur following chronic interruption of dopaminergic neurotransmission. Substantia nigra lesions, DA depletion with reserpine and D2 receptor blockade by neuroleptics increase the number of striatal D2 receptors as identified with the D2 ligand, [3H]spiperone [( 3H]SPIP). Chronic administration of haloperidol to substantia nigra-lesioned rats causes an additive increase in binding over levels obtained with one treatment alone. In this study we have found a similar response when lesioned animals are treated with reserpine. However, compensatory increases in the number of [3H]SPIP binding sites found after combined administration of reserpine and haloperidol to intact rats do not exceed levels obtained following administration of either drug alone. The data suggest that up-regulation of striatal D2 binding sites occurring after substantia nigra lesions is unique relative to other forms of up-regulation and may involve the loss of a presynaptic regulatory factor other than DA.     

Pressure reversed extracellular striatal dopamine decrease produced by D1 receptor agonist SKF 38393, and D2 receptor agonist LY 171555, but failed to change the effect of the activation of both D1 and D2 receptors.

When human divers or experimental animals are exposed to high pressure, they develop the high-pressure neurological syndrome which is characterized by electroencephalographic changes, and behavioral disturbances. Recently, neurochemical disorders such as a pressure-induced increase in dopamine release have been demonstrated. In the present study, pharmacological experiments, using dopamine receptor agonists such as D1 receptor agonist SKF 38393, D2 receptor agonist LY 171555, and D1/D2 receptor agonist apomorphine, were performed to investigate dopamine receptor function at the neurochemical level. Only apomorphine and mixed SKF 38393 + LY 171555 prevented the pressure-induced increase in dopamine release while SKF 38393 or LY 171555 administered alone failed to do so. The results suggest that the D1-D2 link would be reduced under high pressure because of an abnormal function of D1 receptors which would allow high-affinity D2 states for dopamine. If so, such a preponderance of high-affinity states in D2 postsynaptic receptors could be associated with hyperbaric hyperlocomotor activity. Elsewhere, results also suggested that the pressure-induced disorders in dopamine receptor function could be involved in the pressure-induced elevation in dopamine release.     

Supportive evidence for contribution of the dopamine D2 receptor gene to heritability of stature: linkage and association studies

Increases in height were reported in children chronically exposed prenatally and postnatally to D2 receptor-blocking drugs. A possible haplotypic association between stature and the DRD2 gene was also reported. In this study, we examined linkage between stature and DRD2 by genotyping a dinucleotide repeat polymorphism in 79 sib-pairs aged 8–17 years. An association between stature and a putative functional polymorphism in the promoter region of the DRD2 gene was examined in the sib-pairs and in 125 unrelated male adults. All the subjects were Japanese. Linkage ( p = 0.004, SIBPAL) and an association ( p = 0.009, paired t -test, in the sib-pairs; p = 0.006, ANOVA, in the adults) with stature were suggested. These findings indicate that DRD2 is one of the genes that contribute to heritability of stature.     

Forskolin and dopamine D1 receptor activation increase huntingtin's association with endosomes in immortalized neuronal cells of striatal origin.

Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntington's disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.     

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen

Summary In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarily, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudateputamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.