Dopamine Receptor mRNA Research Articles

Dopamine depolarizes podocytes via a D1-like receptor.

Dopamine influences glomerular haemodynamics and dopamine receptors have been demonstrated in the glomerulus, but little is known about the cellular effects of dopamine in glomerular cells. The aim of this study was to investigate the influence of dopamine on the cellular functions of podocytes. The effect of dopamine on membrane voltage was investigated in differentiated mouse podocytes. The membrane voltage was measured using the patch clamp technique. Reverse transcribed-polymerase chain reaction (RT-PCR) studies were performed to investigate the expression of dopamine receptor mRNA in mouse glomeruli and podocytes. The addition of dopamine (100 nM-1000 microM) caused a concentration-dependent depolarization of podocytes (EC50 is approximate to 10 microM). Like dopamine, the selective agonist of the D1-like receptor, SKF 82958, depolarized podocytes in a concentration-dependent manner. (EC50 is approximate to 50 microM). SKF 82958 stimulated a time-and concentration-dependent accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in podocytes (EC50 is approximate to microM). RT-PCR studies with primers derived from mouse sequences amplified mouse mRNA for the D1-like and the D2-like receptor in glomeruli, which were obtained by the sieve technique, whereas only mRNA for the D1-like receptor was detected in cultured mouse podocytes. The data indicate that dopamine induces a cAMP-dependent depolarization via a D1-like receptor in podocytes.

Chronic morphine exposure and spontaneous withdrawal are associated with modifications of dopamine receptor and neuropeptide gene expression in the rat striatum.

The influence of chronic morphine and spontaneous withdrawal on the expression of dopamine receptors and neuropeptide genes in the rat striatum was investigated. Morphine dependence was induced by subcutaneous implantation of two morphine pellets for 6 days. Rats were made abstinent by removal of the pellets 1, 2 or 3 days before they were killed. The mRNA levels coding for D1- and D2-dopamine receptors, dynorphin, preproenkephalin A and substance P were determined by quantitative in situ hybridization. The caudate putamen and the nucleus accumbens showed equivalent modifications in dopamine receptor and neuropeptide gene expression. After 6 days of morphine, a decrease in D2-dopamine receptor and neuropeptide mRNA levels was observed (-30%), but there was no change in D1-dopamine receptor mRNA. In abstinent rats, both D1- and D2-dopamine receptor mRNA levels were decreased 1 day after withdrawal (-30% compared with chronic morphine). In contrast, neuropeptide mRNA levels were unaffected when compared with those observed after 6 days of morphine. During the second and third day of withdrawal, there was a gradual return to the levels seen in the placebo-treated group, for both dopamine receptor and neuropeptide mRNAs. Phenotypical characterization of striatal neurons expressing mu and kappa opioid receptor mRNAs showed that, in striatonigral neurons, both mRNAs were colocalized with D1-receptor and Dyn mRNAs. Our results suggest that during morphine dependence, dopamine and morphine exert opposite effects on striatonigral neurons, and that effects occurring on striatopallidal neurons are under dopaminergic control. We also show that withdrawal is associated with a down regulation of the postsynaptic D1 and D2 receptors.

Targeted Expression of a Toxin Gene to D1 Dopamine Receptor Neurons byCre-Mediated Site-Specific Recombination

Idiopathic Parkinson's disease involves the loss of midbrain dopaminergic neurons, resulting in the presynaptic breakdown of dopaminergic transmission in the striatum. Huntington's disease and some neurodegenerative diseases with Parkinsonian features have postsynaptic defects caused by striatal cell death. Mice were generated in which an attenuated form of the diphtheria toxin gene (tox-176) was expressed exclusively in D1 dopamine receptor (D1R)-positive cells with the aim of determining the effect of this mutation on development of the basal ganglia and on the locomotor phenotype. Transgenic mice expressing Cre, a site-specific DNA recombinase, were crossed with a second line in which a transcriptionally silenced tox-176 gene was inserted into the D1R gene locus by homologous recombination. Young doubly transgenic mutant mice expressing the tox-176 gene displayed bradykinesia, dystonia, and had falls caused by myoclonic jerks. The mutant brain had evidence of apoptosis and reactive gliosis and, consistent with the D1R expression pattern, the striatum was reduced in volume, and the Islands of Calleja were absent. In contrast, the cortex was of normal thickness. D1Rs were not detectable in mutants by in situ hybridization or ligand autoradiography, whereas D2 dopamine receptor (D2R) mRNA and protein was present in the striatum. In addition, substance P and dynorphin, neuropeptides known to be expressed in D1R-positive striatonigral projection neurons were not detectable. Enkephalin, a marker found in D2-positive striatopallidal projection neurons was expressed in the mutant brain. The mutant represents a novel neurodegenerative disease model with a dramatic extrapyramidal phenotype.

Changes in the Midbrain-Rostral Forebrain Dopamine Circuitry in the Cocaine-Exposed Primate Fetal Braina.

To ascertain cocaine's effects in the fetus, we developed a nonhuman primate model in which pregnant monkeys were administered cocaine (3 mg/kg im) or saline four times a day from day 20 through days 40-70 of a 165-day gestation. At the time of cesarean section, plasma levels of cocaine in fetal blood were 231 ± 70 ng/ml. Fetal brains were examined using immunocytochemistry, in situ hybridization, receptor autoradiography, and nuclease protection assay analysis. No differences were found in the expression of tyrosine hydroxylase and dopamine receptor mRNAs by days 40-45 of gestation. However, by day 60 the midbrain of monkeys exposed to cocaine had significantly reduced expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Moreover, dopamine D1 and D2 receptor mRNAs were significantly elevated in the rostral forebrain as were D1 and D2 receptor binding sites in days 60-70 cocaine-exposed fetuses. Cocaine treatment from day 20 to days 60 and 70 of gestation also significantly increased the mRNA concentrations of dynorphin and enkephalin in the rostral forebrain. These findings suggest that in utero cocaine exposure has profound effects on the developing dopamine neurocircuitry.

Dopamine receptor mRNAs in the rat lymphocytes

It has been suggested that dopamine might play a role in the regulation of the immune system. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNA for the different subtypes of dopamine receptors in the rat lymphocytes. D1, D3 and D5 receptor mRNAs were identified. These results provide further evidence for the interaction of dopamine systems and the immune system, and suggest to further investigate whether the immunosuppressive actions of dopamine and dopaminergic drugs might depend on a direct interaction with dopamine receptors on the lymphocyte membrane. Moreover, they suggest the suitability of this animal species to further investigate the correlation between changes in the expression of central and peripheral dopamine receptors produced by manipulations of the dopamine systems.

Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization.

In order to improve the identification and characterization of dopaminoceptive neurons, the rat brain was mapped for D1 dopamine receptor mRNA by non-radioactive in situ hybridization (ISH) with a 45mer digoxigenin-labeled oligonucleotide probe. The specificity of the results was controlled with the help of a 396-bp D1 receptor riboprobe. Labeled hybrids were visualized with an alkaline phosphatase-coupled anti-digoxigenin antibody. The high resolution obtained permitted individual labeled cells to be identified and to distinction between cell bodies and processes. D1 mRNA was largely confined to neurons. With the exception of ependymal cells, glial cells were not distinctly labeled. Subcellularly, D1 mRNA was localized to perikarya but not to dendrites or axons. D1 receptor-expressing neurons were present in all of the known terminal fields of mesencephalic or diencephalic dopaminergic neurons. However, D1 message was also detected in brain areas which are not known to contain D1 ligand binding sites or in which the presence or the cellular source of this receptor subtype had previously not been unequivocally established, such as the hippocampus or cerebellar cortex. Moreover, labeled neurons were present in regions not known to receive dopaminergic projections, such as the thalamic and some brainstem nuclei. We conclude that this ISH technique provides a considerable gain in sensitivity and resolution with regard to neurotransmitter receptor mapping.

Elevation of D 4 dopamine receptor mRNA in postmortem schizophrenic brain

The D 4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D 4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D 4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D 4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase ( G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D 4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level ( P<0.011) of D 4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D 4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

Layer V neurons bear the majority of mRNAs encoding the five distinct dopamine receptor subtypes in the primate prefrontal cortex.

In situ hybridization histochemistry was used to determine the laminar distribution of D1, D2, D3, D4, and D5 dopamine receptor mRNAs in the primate prefrontal cortex and to compare striatal and cortical levels of these messages within the same tissue sections. All five subtypes of dopamine receptor mRNA are present in both the monkey striatum and the cerebral cortex but in different proportions within each structure. Thus, levels of D1 and D2 mRNAs are noticeably stronger in the striatum than in the cortex, whereas D4 and D5 expression is clearly higher in the cortex. The D3 transcripts appear nearly equivalent in the striatum and the cortex. A major finding is that, within the prefrontal cortex, mRNAs encoding all dopamine receptor subtypes are expressed most strongly in layer V. This laminar pattern of mRNA distribution does not hold in all cortical regions. The relatively high levels of mRNAs encoding known dopamine receptor subtypes in the primate cerebral cortex, including the D4 receptor, underscore the importance of this structure as a target for therapeutic actions of antipsychotic drugs. Further, their prominence in layer V of the prefrontal cortex, which contains the corticostriatal and corticotectal projection neurons, provides a neural basis for dopaminergic regulation of the descending control systems.

D 3 dopamine receptor mRNA is widely expressed in the human brain

Considerable attention has been given to the association of the D 3 dopamine receptor subtype and limbic function based on the abundant localization of D 3 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 D 3 receptors in limited brain structures, detailed information about the overall anatomical organization of the D 3 receptor in the human brain is still, however, not available. In the current study, we examined the anatomical distribution of D 3 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 D 3 receptor gene throughout the human brain. As expected, the most abundant D 3 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 D 3 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 D 3 receptor mRNA expression in the human brain does confirm previous reports that D 3 receptors may play important roles in limbic-related functions such as emotion and cognition, the findings also suggest other non-limbic functions for D 3 mRNA-expressing cell populations such as processing of motor and sensory information.

Changes in dopamine receptor mRNA expression following chronic mild stress and chronic antidepressant treatment

This study used in-situ hybridization to examine D1 and D2 receptor mRNA expression in the brains of rats subjected to chronic mild stress and/or chronic treatment (5 weeks) with the antidepressant drugs imipramine and fluoxetine. As in previous studies using the chronic mild stress procedure, stress decreased the consumption of a palatable sucrose solution, and both antidepressants reversed this effect. In-situ hybridization to D1 and D2 receptor probes was studied in midbrain and forebrain sections. In the midbrain sections, stress decreased the D2 receptor message in the substantia nigra and in the lateral part of the ventral tegmental area (VTA), but not in the medial part of the VTA. Imipramine, but not fluoxetine, increased the D2 receptor message in the VTA, but only in the non-stressed group. In the basal ganglia, stress decreased the D2 receptor message in the nucleus accumbens (core and shell) and in the lateral, but not the medial, part of the caudate nucleus, in both control and stressed groups. Both imipramine and fluoxetine increased the D2 receptor message in the lateral, but not the medial, part of the caudate nucleus and in the shell, but not the core, of the nucleus accumbens. There were no significant changes in D1 receptor message. The results, which are consistent in some respects, and inconsistent in others, with predictions from earlier work, support the hypothesis that forebrain dopamine systems are involved in mediating the behavioural effects of chronic mild stress and their reversal by antidepressants.

Ontogeny of the striatal neurons expressing the D2 dopamine receptor in humans: an in situ hybridization and receptor-binding study.

D2 dopamine receptor (D2R) gene expression was analyzed by in situ hybridization and D2R ligand autoradiography in the human striatum during ontogeny. D2R mRNA and ([3H]YM-09151-2)-binding sites were detected in the striatum from week 12 of fetal life. At this time, D2R mRNA and binding sites were predominant in the putamen and occurred in a pattern of clusters. D2R-binding sites displayed a similar pattern. The signal in the caudate nucleus was weak from weeks 12 to 16. From week 20 of fetal life, D2R mRNA and D2R-binding sites signals became intense in the ventral striatum. At birth, D2R mRNA became homogeneously distributed while D2R-binding sites kept an heterogeneously distribution. Comparative topological and temporal analysis of the D2R, enkephalin and D1 dopamine receptor (D1R) mRNAs showed a distinct developmental pattern for each mRNA. Before birth, the neurons expressing enkephalin and D1R mRNAs were preferentially distributed in the matrix and in the striosomes, respectively, while the neurons expressing D2R mRNA did not display a preferential localization. At birth, high levels of enkephalin mRNA were restricted to the matrix; D1R mRNA level was homogeneous throughout the striatum. D2R mRNA was heterogeneously distributed in the whole striatum with high signals located both in the striosomes and the matrix. These results demonstrate that functional D2R are expressed as early as week 12 in the striatum with a heterogeneous distribution. Our findings also demonstrate that, in contrast to what was expected from similar studies in rodents, D2R mRNA and enkephalin mRNA do not display identical, overlapping expression patterns in striatal neurons during human ontogeny.

Opioidergic and dopaminergic gene expression in the caudate-putamen and accumbens of the mutant mouse, tottering (tg/tg).

Expression of preproenkephalin, dynorphin and D2 dopamine receptor mRNAs was examined in selected regions of the forebrain of homozygous and heterozygous tottering mice, using in situ hybridization histochemistry. Homozygous tottering mice carry an autosomal recessive mutation causing them to exhibit petit mal-like epilepsy. Preproenkephalin mRNA levels were significantly higher in the lateral caudate and the core of the nucleus accumbens of homozygous tottering mice compared to wild-type controls. No differences were observed in the expression of dynorphin and D2 receptor mRNA distribution in brain regions examined in the mutant mice as compared to wild-type controls.

Functional D1 and D5 dopamine receptors are expressed in the suprachiasmatic, supraoptic, and paraventricular nuclei of primates.

In rodents, D1 dopamine receptors are expressed in the suprachiasmatic nucleus and are believed to play important roles in regulating circadian rhythms. It is not currently known if the primate circadian system can be influenced by dopaminergic agents, which have broad clinical use. To determine if dopamine receptors can potentially influence primate circadian function, we examined the expression of D1 dopamine receptors in the anterior hypothalamus of ring-tailed macaques (Macaca nemestrema), baboons (Papto sp.), and humans. Because D5 dopamine receptors also stimulate adenylyl cyclase activity, D5 dopamine receptor expression was studied as well. We used [125I]SCH 23982, which binds to D1 and D5 dopamine receptors, and labeling of the suprachiasmatic (SCN), supraoptic (SON), and paraventricular (PVN) nuclei was detectable in each species. In situ hybridization studies revealed differential expression of D1 and D5 dopamine receptor mRNA in the hypothalamus. D1 dopamine receptor mRNA was expressed in the SCN, SON, and PVN. By contrast, D5 dopamine receptor mRNA was expressed only in the SON and PVN of baboons and humans. Injection of the D1/D5 dopamine receptor agonist SKF 38393 at night increased the uptake of 2-deoxy-D-[14C]glucose in the SCN, SON, and PVN of newborn baboons. By contrast, c-fos mRNA expression was induced in the SON and PVN, but not in the SCN. These data show that D1 and D5 dopamine receptors are present in the hypothalamus of primates and show that activation of these receptors acutely influences SCN, SON, and PVN activity.

Up-regulation of D3 dopamine receptor mRNA in the nucleus accumbens of human cocaine fatalities.

The effects of chronic cocaine use on D3 receptor mRNA expression in the human nucleus accumbens was assessed by reverse transcription-polymerase chain reaction. D3 receptor/cyclophilin mRNA ratios in the nucleus accumbens were increased 6-fold in cocaine overdose victims as compared to age-matched and drug-free control subjects. This finding demonstrates that chronic cocaine exposure leads to adaptive increases in the expression of D3 receptor mRNA in a critical reward center in brain.

D1 dopamine receptors are not expressed in human melanoma.

D1 dopamine receptor mRNA has been demonstrated in mouse melanoma cells, and the expression of these G-protein-coupled receptors in human melanoma was therefore presumed when dopamine receptor binding radiopharmaceuticals were found to be useful for the detection of metastases in whole-body scintigraphy. The aim of this study was thus to investigate if D1 dopamine receptor mRNA or protein could be directly demonstrated in melanoma cells. The presence of D1 dopamine receptor mRNA was investigated in six human melanoma cell lines from metastases using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, in vitro binding assays with the D1 dopamine receptor agonist 125I-Sch 23982 were performed in 19 melanoma metastases. No D1 dopamine receptor mRNA could be detected by RT-PCR. All melanotic metastases were found to accumulate 125I-Sch 23982, with the presence of binding sites and intensity of 125I-Sch 23982 labelling correlating to the amount of melanin present in the metastases. Two amelanotic melanomas did not accumulate 125I-Sch 23982. D1 dopamine receptors could not be detected by means of RT-PCR or in vitro binding assays in human melanomas. Detection of antagonists is best explained by non-specific binding to melanin.

Molecular anatomy of the development of the human substantia nigra.

A series of 15 fetal and perinatal human brains (from week 12 of fetal life to day 2 after birth) was studied in order to describe the anatomical and molecular correlates of the substantia nigra ontogeny. In situ hybridization, immunohistochemistry and binding studies were used to detect D2 dopamine receptor (D2R) mRNA, D2R binding sites, dopamine membrane transporter (DAT) mRNA, tyrosine hydroxylase (TH) protein D1 dopamine receptor (D1R) protein and D1R binding sites. Dopaminergic (DA) neurons of the substantia nigra were detected through TH immunoreactivity from week 12. At week 16, the substantia nigra was clearly delineated as a compact group of intermingled neurons and fibers. From week 19, groups of DA neurons were segregated from the pars reticulata. These groups have been divided into the substantia nigra pars compacta, the ventral tegmental area and the retrorubral area. The DA neurons exhibited a gradual increase in size and branching development until birth. From week 12 onward they expressed several other markers of dopamine transmission, i.e., D2R mRNA, D2R binding sites and DAT mRNA. The ventral tegmental area expressed lower levels of mRNA for DAT and D2R than the pars compacta. From week 12, D1R immunoreactivity and D1R binding sites were also present in the substantia nigra pars reticulata. This suggests that projecting striatonigral neurons, known to express the D1R gene, have developed pathways connecting with the substantia nigra by week 12. Our results demonstrate that the developing substantia nigra in human displays early transcriptional and translational activity for the main constituents of dopaminergic transmission from week 12 and receives at this time dopaminoceptive inputs bearing D1 receptors from the striatum.

D2 dopamine receptors reduce N-type Ca2+ currents in rat neostriatal cholinergic interneurons through a membrane-delimited, protein-kinase-C-insensitive pathway.

Dopamine has long been known to regulate the activity of striatal cholinergic interneurons and the release of acetylcholine. Yet, the cellular mechanisms by which this regulation occurs have not been elucidated. One way in which dopamine might act is by modulating voltage-dependent Ca2+ channels. To test this hypothesis, the impact of dopaminergic agonists on Ca2+ channels in neostriatal cholinergic interneurons was studied by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reactions. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly, all interneurons tested (90%, n = 17) coexpressed D2 (short and long isoforms) and D1b (D5) dopamine receptor mRNAs. D1a receptor mRNA was found in only a small subset (20%) of the sample and D3 and D4 receptor mRNAs were undetectable. D2 receptor agonists rapidly and reversibly reduced N-type Ca2+ currents. D1b/D1a receptor activation had little or no effect on Ca2+ currents. The D2 receptor antagonist sulpiride blocked the effect of D2 agonists. Dialysis with guanosine-5'-O-(2-thiodiphosphate) or brief exposure to the G protein (Gi/o) alkylating agent N-ethylmaleimide also blocked the D2 modulation. The reduction in N-type currents was neither accompanied by kinetic slowing nor significantly reversed by depolarizing prepulses. The D2 receptor effects were mediated by a membrane-delimited pathway, because the modulation was not seen in cell-attached patches when agonist was applied to the bath and was not disrupted by perturbations in cytosolic signaling pathways known to be linked to D2 receptors. Activation of M2 muscarinic receptors occluded the D2 modulation, suggesting a shared signaling element. However, activation of protein kinase C attenuated the M2 modulation without significantly affecting the D2 modulation. Taken together, our results suggest that activation of D2 dopamine receptors in cholinergic interneurons reduces N-type Ca2+ currents via a membrane-delimited, Gi/o class G protein pathway that is not regulated by protein kinase C. This signaling pathway may underlie the ability of D2 receptors to reduce striatal acetylcholine release.

Interrelationships between tyrosine hydroxylase-immunoreactive dopaminergic afferents and somatostatinergic neurons in the rat central amygdaloid nucleus.

Interrelationships between dopaminergic afferents and somatostatinergic neurons of the rat central amygdaloid nucleus were studied using tyrosine hydroxylase/somatostatin double immunolabeling for light and electron microscopy. Additionally, morphological features of somatostatin neurons in different subnuclei of the central nucleus were studied, and the results were complemented by codistribution studies of somatostatin and D1 and D2 dopamine receptor mRNA expression. Dense axonal immunolabeling for tyrosine hydroxylase was colocalized with somatostatin-immunoreactive or somatostatin mRNA-reactive neurons in the medial and the central lateral part of the central nucleus. The number of somatostatinergic neurons detected was higher using in situ hybridization than using immunolabeling. Somatostatin-immunoreactive neurons of the medial central nucleus possessed deeply indented nuclei, and immunoreaction product was confined to the Golgi apparatus and its vicinity. On the other hand, those in the central lateral subnucleus possessed nuclei without indentations and showed diffuse staining of the cytoplasm and/or in large vesicles. Double labeling showed that in the central lateral central nucleus, somatostatin-immunoreactive neurons were contacted by tyrosine hydroxylase-immunoreactive terminals, and on the electron microscopic level synaptic contacts between differently labeled structures were observed. D1 and D2 receptor mRNA-reactive neurons were differentially distributed in central nucleus subnuclei. D1 receptor mRNA-expressing neurons were found only in the medial subnucleus, while D2 receptor mRNA was expressed by a number of neurons in the lateral central and a few in the medial one. Thus, the study proves that somatostatin-immunoreactive neurons of the central lateral central nucleus are directly innervated by dopaminergic afferents and may express the D2 dopamine receptor.

Antisense strategies in dopamine receptor pharmacology.

Recent advances in molecular biology have provided pharmacologists the opportunity of developing an entirely new type of agent for studying and treating a variety of biological disorders. These agents, termed antisense oligodeoxynucleotides, have as their target the messenger RNAs encoding specific proteins. They act by binding to selected portions of these mRNAs through complimentary interactions and thereby prevent the synthesis of these proteins. These novel pharmacological tools have the promise of being easier to design and being more selective and predictable in their actions. In addition, insofar as agents targeted to receptors for neurotransmitters are concerned, unlike the classical pharmacological agents, these new compounds may not lead to the upregulation of the very receptors the drugs are designed to inhibit. The present review summarizes briefly studies on the effect of oligodeoxynucleotides antisense to the mRNAs encoding the various subtypes of the dopamine receptor. The studies show that oligodeoxynucleotides antisense to the D2 dopamine receptor when intracerebroventricularly into brains of rodents are rapidly taken up into the brain tissue, distributed to brain cells, and produce effects characteristic of highly selective D2 dopamine antagonists. The compounds also produced specific reductions in the levels of D2 dopamine receptor mRNA and D2 dopamine receptors. Similarly, injecting an antisense oligodeoxynucleotide targeted to the D1 dopamine receptor mRNA produces effects characteristic of D1 dopamine receptor antagonists. Other studies using these agents has produced evidence that there is a small pool of receptors that turn over very rapidly and which constitute the functional pool of these receptors. The evidence suggests further that antisense oligodeoxynucleotides inhibit the synthesis of this small functional pool of dopamine receptors, thereby providing an explanation of why there is often a discordance between changes in dopaminergic function and changes in the levels of dopamine receptors. Studies of antisense oligodeoxynucleotides targeted to the other subtypes of dopamine receptor may help reveal the biological roles that these and other newly discovered subtypes of neurotransmitter receptors have. They may also provide an entirely new and potentially more selective therapeutic regimen for altering the functions of these receptors.

Cellular distribution of the rat D 1B receptor in central nervous system using anti-receptor antisera

Polyclonal antisera have been generated against two unique polypeptide fragments in the rat D 1B dopamine (DA) receptor, as deduced from the cDNA sequence. Antisera titers were monitored using solid-phase ELISA. Once the titers were established, antisera specificity was determined using Chinese Hamster ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D 1B DA receptor. Immunoreactivity following staining with either anti-D 1B DA receptor antisera was equivalent, selective for the D 1B DA receptor-transfected CHO cells, and expressed at their membrane and within the cell cytoplasm. Minimal immunofluorescent staining for D 1B DA receptor proteins was detected in untransfected CHO cells, or in D 1A DA receptor-transfected CHO cells. The regional and cellular distribution patterns for the D 1B DA receptor subtype were examined in various brain areas and illustrated significant protein levels within the frontal and parietal cortices and in the hippocampus and dentate gyrus. Lesser amounts of receptor protein staining were seen in the dorsal striatum, olfactory tubercle, and cerebellar vermis. D 1B DA receptor protein staining was correlated with the cellular expression of D 1B DA receptor mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D 1B DA receptor transcripts and encoded proteins in identified neurons of the frontal cortex and striatum showed variations in receptor expression in these identified basal ganglia pathways.