Differential distributions and trafficking properties of dopamine D1 and D5 receptors in nerve cells

Objective

To explore the possible differential trafficking properties of the dopamine D1-like receptor subtypes, D1 receptor and D5 receptor.

Methods

To visualize distributions of dopamine D1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein (GFP) were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine (ACh). Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment.

Results

In resting conditions, D1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D1 receptors appeared in the cytosol, indicating that D1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties.

Conclusion

Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.     

Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A2A-dopamine D2 receptor heterodimers

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A_2A and dopamine D₂ receptors and the possible role of caveolin-1 in the cotrafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A_2A, D₁, and D₂ receptors obtained in two clones of Chinese hamster ovary cells—one transfected with A_2A and dopamine D₁ receptors and the other one with A_2A and D₂ receptors. Cells were treated for 3 h with 10 μM D₁ receptor agonist SKF 38393, 50 μM D₂-D₃ receptor agonist quinpirole, and 200 nM A_2A receptor agonist CGS 21680. In A_2A-D₁-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₁ receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D₁ receptors, with a preferential internalization of D₁ receptors colocalized with caveolin-1. In A_2A-D₂-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₂ receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A_2A and D₂ receptors, with a preferential internalization of A_2A and D₂ receptors colocalized with caveolin-1. The results suggest that A_2A and D₂ receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization. This paper is dedicated to Luciano Martini, Professor of Endocrinology in Milano and to Faustino Savoldi, Professor of Neurolgy in Pavia. Department of Human Anatomy and Physiology, Section of Antomy, University of Padova, 35121 Padova, Italy     

Adenosine A2A receptors in the nucleus accumbens bi-directionally alter cocaine seeking in rats

Repeated cocaine administration enhances dopamine D(2) receptor sensitivity in the mesolimbic dopamine system, which contributes to drug relapse. Adenosine A(2A) receptors are colocalized with D(2) receptors on nucleus accumbens (NAc) medium spiny neurons where they antagonize D(2) receptor activity. Thus, A(2A) receptors represent a target for reducing enhanced D(2) receptor sensitivity that contributes to cocaine relapse. The aim of these studies were to determine the effects of adenosine A(2A) receptor modulation in the NAc on cocaine seeking in rats that were trained to lever press for cocaine. Following at least 15 daily self-administration sessions and 1 week of abstinence, lever pressing was extinguished in daily extinction sessions. We subsequently assessed the effects of intra-NAc core microinjections of the A(2A) receptor agonist, CGS 21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride), and the A(2A) receptor antagonist, MSX-3 (3,7-dihydro-8-[(1E)-2-(3-methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)propyl-1-(2-propynyl)-1H-purine-2,6-dione disodium salt hydrate), in modulating cocaine- and quinpirole-induced reinstatement to cocaine seeking. Intra-NAc pretreatment of CGS 21680 reduced both cocaine- and quinpirole-induced reinstatement. These effects were specific to cocaine reinstatement as intra-NAc CGS 21680 had no effect on sucrose seeking in rats trained to self-administer sucrose pellets. Intra-NAc treatment with MSX-3 modestly reinstated cocaine seeking when given alone, and exacerbated both cocaine- and quinpirole-induced reinstatement. Interestingly, the exacerbation of cocaine seeking produced by MSX-3 was only observed at sub-threshold doses of cocaine and quinpirole, suggesting that removing tonic A(2A) receptor activity enables behaviors mediated by dopamine receptors. Taken together, these findings suggest that A(2A) receptor stimulation reduces, while A(2A) blockade amplifies, D(2) receptor signaling in the NAc that mediates cocaine relapse.     

Selective unilateral inactivation of striatal D1 and D2 dopamine receptor subtypes by EEDQ: turning behavior elicited by D2 dopamine receptor agonists

The unilateral intrastriatal injection of the irreversible dopamine (DA) receptor blocker N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induces a marked decrease in the density of D1 (-48%) and D2 (-51%) DA receptors available for binding to [3H]SCH 23390 and [3H]raclopride, respectively. A challenge dose of the D2 agonist LY 171555 (1 mg/kg, i.p., 24 h after EEDQ) causes intensive ipsiversive circling behavior, whereas the selective D1 agonist SKF 38393 (20 mg/kg, i.p., 24 h after EEDQ) is unable to induce rotations. The density of D1 and D2 DA receptors returns to basal levels by 7 days after the intrastriatal infusion of EEDQ. This biochemical recovery is associated with a progressive decrease in the number of rotations elicited by a challenge dose of LY 171555, suggesting that EEDQ does not cause any relevant neuronal damage. A selective inactivation of striatal D1 or D2 DA receptors can be obtained by injecting EEDQ 30 min after the administration of the D2 antagonist raclopride (20 mg/kg, i.p.) or of the D1 antagonist SCH 23390 (2 mg/kg, s.c.), respectively. The intensity of the circling behavior induced by LY 171555 24 h after EEDQ in animals with a selective inactivation of D2 DA receptors is similar to that found in rats in which both D1 and D2 DA receptors have been inactivated. In contrast, LY 171555 does not cause rotations when the density of D1 DA receptors is selectively decreased by EEDQ in rats pretreated with raclopride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine receptor-mediated regulation of RGS2 and RGS4 mRNA differentially depends on ascending dopamine projections and time

RGS2 and RGS4 mRNAs are regulated in the rat striatum by dopaminergic agents. The present study further characterizes this regulation in three experiments. First, dopamine type 1 (receptor) (D1)- and dopamine type 2 (receptor) (D2)-mediated regulator of G-protein signalling (RGS) gene regulation was investigated in animals with deleted ascending dopaminergic pathways. We showed that RGS2 expression is controlled by D1 receptors either by direct action on D1 receptors or indirectly by presynaptic D2 receptors. Conversely, RGS4 gene expression is independent of presynaptic D2 receptors. Second, the study of colocalization between RGS2 or RGS4 and D1 or D2 by double labelling in situ hybridization histochemistry revealed broad expression of RGS2 and RGS4 mRNA in striatal subpopulations with colocalization of RGS2 and RGS4 with both D1 and D2 receptors. Finally, to test how far their gene regulation is temporally concerted, changes in RGS2 and RGS4 mRNA levels were measured in parallel with receptor occupancy by specific dopaminergic drugs at different time-points. RGS2 was rapidly/transiently up-regulated by the D1 agonist SKF82958 and the D2 antagonist haloperidol (peak at 0.5 h) and down-regulated by the D1 antagonist SCH23390 and the D2 agonist quinpirole (trough at 1 and 2 h). RGS4 showed a delayed/transient up-regulation with SCH23390 and quinpirole (peak at 4 and 2 h) and down-regulation with haloperidol (trough at 8 h). Depending on the drug used, the degree of receptor occupancy did (D1 agonist and RGS2) or did not (D2 antagonist and RGS2) run parallel to RGS gene expression changes, indicating that certain drug effects are direct and others indirect. The precise control of RGS2 and RGS4 expression by dopamine receptors pleads in favour of their potential contribution to the fine-tuning of D1 and D2 receptor signalling cascades.     

Effects of dopamine D-1 and D-2 receptors on intraocular pressure in conscious rabbits

Summary This investigation was designed as a randomized, placebocontrolled, double-masked, crossover study in NZW rabbits with normal intraocular pressure (IOP) to investigate dopaminergic effects on IOP. SKF 38393, a selective D1-receptor agonist, increased, and SDZ PSD-958, a selective D1-receptor antagonist, decreased IOP, respectively. The selective D2-receptor agonist quinpirole decreased IOP, whereas the selective D2 receptor antagonist metoclopramide had no significant effect. Combinations of quinpirole with SDZ PSD-958 decreased IOP in an additive manner. SDZ GLC-756, a mixed D1-receptor antagonist/D2-receptor agonist, decreased IOP in a dose-dependent manner with a maximum effect greater than the maximum effects produced either by the D1-receptor antagonist SDZ PSD-958 and the D2-receptor agonist quinpirole. The effect of SDZ GLC-756 could only be partially blocked by the selective D2-receptor antagonist metoclopramide suggesting that both D1-receptor blockade and D2-receptor stimulation participate in its IOP-lowering effect. Tonography suggests that SDZ GLC-756 has no significant effect on outflow facility. Furthermore, the results suggest that both D1 and D2 receptors each play an independent role in the regulation of IOP in rabbits. Thus, simultaneous blockade of D1 receptors and stimulation of D2 receptors may provide a new pharmacological approach for the treatment of ocular hypertension frequently associated with glaucoma.     

Antagonism by olanzapine of dopamine D1, serotonin2, muscarinic, histamine H1 and alpha 1-adrenergic receptors in vitro

The atypical antipsychotic olanzapine has relatively high affinity for a number of neuronal receptors in radioreceptor binding assays. The ability of olanzapine to activate or antagonize a number of neuronal receptors was investigated in vitro, in cell lines transfected selectively with receptor subtypes and in receptor-selective isolated tissue studies. Olanzapine had no agonist activity at any of the receptors examined. However, olanzapine was a potent antagonist of 5-HT-stimulated increases in IP3 in cell lines transfected with 5-HT2A or 5-HT2B receptors with IC50 values of 30-40 nM. Olanzapine weakly blocked 5-HT-induced formation of IP3 in cell lines transfected with 5-HT2c receptors, but in this cell line potently inhibited 5-HT-stimulated [35S]GTP gamma S binding with a Ki value of 15 nM. Olanzapine blocked dopamine-stimulated adenylyl cyclase in rat retina with modest potency (Ki = 69 nM), consistent with its relatively low affinity for dopamine D1 receptors. Olanzapine blocked agonist-induced activities at the muscarinic receptor subtypes M1, M2, M3, and M5 with Ki values of 70, 622, 126, and 82 nM, respectively. In studies using cell lines transfected with muscarinic M4 receptors, olanzapine and the atypical antipsychotic clozapine did not have agonist activities as determined with cAMP inhibition and stimulation assays, arachidonic acid release and [35S]GTP gamma S binding assays. However, olanzapine antagonized agonist-induced effects in muscarinic M4 cells with a Ki value of 350 nM. In isolated tissue studies, olanzapine potently blocked agonist-induced effects at alpha 1-adrenergic and histamine H1 receptors (KB = 9 and 19 nM, respectively). Thus, olanzapine was an antagonist at all receptors investigated and was a particularly potent antagonist at 5-HT2A, 5-HT2B, 5-HT2C, alpha 1-adrenergic and histamine H1 receptors. Olanzapine was a weaker antagonist at muscarinic and dopamine D1 receptors.     

Facilitatory effect of the dopamine D4 receptor agonist PD168,077 on memory consolidation of an inhibitory avoidance learned response in C57BL/6J mice

The still unknown contribution of the D4 receptors to memory consolidation was studied examining the memory effects of the dopamine D4 agonist PD168,077, the putative dopamine D4 antagonist L745,870, their mutual combination, and the combination of the D4 agonist with representative compounds acting as agonist or antagonist on the D1, D2 and the D3 receptors. Memory consolidation was assessed in C57BL/6J mice using the one-trial step-through inhibitory avoidance task, the compounds being injected immediately after training (foot-shock) and performance measured 24h later. PD168,077 (0.5-10mg/kg) dose-dependently improved memory performance and L745,870 (0.05-5mg/kg) at doses lower than 1mg/kg increased and at doses higher than 1mg/kg impaired memory performance. PD168,077 did not affect the paradoxical promnesic effect of low doses (0.1-0.5mg/kg) of L745,870, but antagonised the memory-impairing effect induced by 5mg/kg L745,870. The D1 antagonist SCH23390 (0.025-0.05 mg/kg) and the D2 antagonist eticlopride (0.01-0.05 mg/kg) antagonised the promnesic effects of PD168,077, which attenuated the decreasing effect on memory consolidation of both D1 and D2 antagonists. Accordingly, the D1 agonist SKF38393 (5-20mg/kg) and the D2 agonist quinelorane (0.1-1 mg/kg) both synergistically magnified the memory-improving effects of the D4 agonist. The dopamine D3 antagonist U99194A (2.5-10mg/kg) did not affect the promnesic effects induced by the D4 agonist, which nevertheless abolished the U99194A-induced promnesic effects. Additionally, the amnesic effects produced by the D3 agonist 7-OH-DPAT (0.01-1 microg/kg) was attenuated by PD168,077. These results suggest a potential role of dopamine D4 receptors in memory consolidation, which would be similar to that of the D1 and D2 receptors and probably opposite to that of the D3 receptors.     

Roles of BCL-2 and caspase 3 in the adenosine A3 receptor-induced apoptosis

Selective A₃ adenosine receptor agonists have been shown to induce apoptosis in a variety of cell types. In this study we examined the effects of adenosine receptor agonists selective for A₁, A_2A, or A₃ receptors on the induction of apoptosis in primary cultures of rat astrocytes and in C6 glial cells. Treatment of the cells with the A₃ receptor agonist Cl-IB-MECA (10 μM) induced apoptosis in both cell types. The effects of Cl-IB-MECA were partially antagonized by the A₃ receptor-selective antagonist MRS 1191. In contrast, the A₁ and A_2A receptor agonists, CPA and CGS 21680, respectively, did not have significant effects on apoptosis in these cells. Cl-IB-MECA reduced the expression of endogenous Bcl-2, whereas it did not affect the expression of Bax. Overexpression of Bcl-2 in C6 cells abrogated the induction of apoptosis induced by the A₃ agonist. Cl-IB-MECA also induced an increase in caspase 3 activity and caspase inhibitors decreased the apoptosis induced by the A₃ agonist. These findings suggest that intense activation of the A₃ receptor is pro-apoptotic in glial cells via bcl2 and caspase-3 dependent pathways.     

Effects of mu-CPP and mesulergine on dietary choices in deprived rats: possible mechanisms of their action

Although it has been well established that compounds that stimulate 5-HT(2C) and/or 5-HT(1B) receptors induce hypophagia by promoting satiety process, the relative role of these receptor subtypes in dietary choices remains to be fully determined. m-CPP is considered a useful probe of 5-HT(2C) receptor function in vivo and its administration reduces food intake and appetite in humans and rats. Conversely, the non-selective 5-HT(2C) receptor antagonist mesulergine elicits feeding in rats. Food intake and dietary choices were measured in a food-deprivation experimental protocol employing male Wistar rats. Animals were given access for a 4-h period to a pair of isocaloric diets. These two diets were enriched in protein or carbohydrate proportions, respectively, but fat content was held constant. The mixed 5-HT(2C/1B) receptor agonist, m-CPP, led to a dose-dependent hypophagia, due to substantial reduction in carbohydrate consumption while protein intake was spared (0.62, 1.25 and 2.50 mg/kg i.p., respectively). The non-selective 5-HT(2C) receptor antagonist and also D2 agonist, mesulergine, on its own produced a significant dose-dependent increase in both protein and carbohydrate diets (1.0 and 3.0 mg/kg i.p., respectively). Combined treatment with m-CPP, at its maximum effective dose, and mesulergine dose-dependently reversed m-CPP-induced hypophagia, during the 4-h test period. In order to clarify the effects of mesulergine on dietary choices since it is simultaneously a dopamine agonist besides its antiserotonergic properties, the D2 agonist apomorphine was also used. Apomorphine caused a dose-dependent increase in protein intake while carbohydrate and total food intake remained nearly unchanged (0.5 and 1.0 mg/kg i.p., respectively). It is concluded that the mesulergine-induced hyperphagic response on both diets is the expression of a dual mode of action, due to its 5-HT(2C) antagonist activity together with D2 agonist properties. The results further indicate that the activation of hypothalamic 5-HT(2C) receptors may be involved in both protein sparing and carbohydrate suppressing effects of 5-HT (m-CPP-like effect), whereas an important role in increase of protein consumption seems to have the dopaminergic system probably through D2 receptors (apomorphine-like and mesulergine-like effects, respectively).     

An endogenous dopaminergic tone acting on dopamine D3 receptors may be involved in diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in estrogen-primed ovariectomized rats

The diurnal rhythm of tuberoinfundibular dopaminergic (TIDA) neuron activity, i.e., high in the morning and low in the afternoon, is prerequisite for the afternoon prolactin (PRL) surge in proestrous and estrogen-primed ovariectomized (OVX) female rats. Whether dopamine acts via D(3) receptors in regulating the rhythmic TIDA neuron activity and PRL secretion in estrogen-primed OVX (OVX+E(2)) rats is the focus of this study. Intracerebroventricular (icv) injection of a D(3) receptor agonist, PD128907 (0.1-10 μg/3 μl), in the morning significantly reduced the basal activity of TIDA neurons and increased plasma PRL level. The effects of PD128907 were reversed by co-administration of U99194A, a D(3) receptor antagonist, but not by raclopride, a D(2) receptor antagonist. To determine whether endogenous dopamine acts on D(3) receptors involved in the diurnal changes of the activities, we used both U99194A, a D(3) receptor antagonist, and an antisense oligodeoxynucleotide (ODN) against D(3) receptor mRNA in the study. U99194A (0.1 μg/3 μl, icv) given at 1200 h significantly reversed the lowered TIDA neuron activity and the afternoon PRL surge at 1500 h. Moreover, OVX+E(2) rats pretreated with the antisense ODN (10 μg/3 μl, icv) for 2 days had the same effects as the D(3) receptor antagonist on TIDA neuron activity and the PRL surge. The same treatment with sense ODN had no effect. In conclusion, an endogenous DA tone may act on D(3) receptors to inhibit TIDA neuron activity and in turn stimulate the PRL surge in the afternoon of OVX+E(2) rats.     

In vitro characterization of SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolone, monohydrochloride): a novel partial dopamine D2 and D3 receptor agonist and serotonin 5-HT1A receptor agonist

Present Parkinson's disease treatment strategies are far from ideal for a variety of reasons; it has therefore been suggested that partial dopamine receptor agonism might be a potential therapeutic approach with potentially fewer side effects. In the present study, we describe the in vitro characterization of the nonergot ligand SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolonemonohydrochloride). SLV308 binds to dopamine D(2), D(3), and D(4) receptors and 5-HT(1) (A) receptors and is a partial agonist at dopamine D(2) and D(3) receptors and a full agonist at serotonin 5-HT(1) (A) receptors. At cloned human dopamine D(2,L) receptors, SLV308 acted as a potent but partial D(2) receptor agonist (pEC(50) = 8.0 and pA(2) = 8.4) with an efficacy of 50% on forskolin stimulated cAMP accumulation. At human recombinant dopamine D(3) receptors, SLV308 acted as a partial agonist in the induction of [(35)S]GTPgammaS binding (intrinsic activity of 67%; pEC(50) = 9.2) and antagonized the dopamine induction of [(35)S]GTPgammaS binding (pA(2) = 9.0). SLV308 acted as a full 5-HT(1) (A) receptor agonist on forskolin induced cAMP accumulation at cloned human 5-HT(1) (A) receptors but with low potency (pEC(50) = 6.3). In rat striatal slices SLV308 concentration-dependently attenuated forskolin stimulated accumulation of cAMP, as expected for a dopamine D(2) and D(3) receptor agonist. SLV308 antagonized the inhibitory effect of quinpirole on K(+)-stimulated [(3)H]-dopamine release from rat striatal slices (pA(2) = 8.5). In the same paradigm, SLV308 had antagonist properties in the presence of quinpirole (pA(2) = 8.5), but the partial D(2) agonist terguride had much stronger antagonistic properties. In conclusion, SLV308 combines high potency partial agonism at dopamine D(2) and D(3) receptors with full efficacy low potency serotonin 5-HT(1) (A) receptor agonism and is worthy of profiling in in vivo models of Parkinson's disease.     

Multiple intramembrane receptor-receptor interactions in the regulation of striatal dopamine D2 receptors.

Adenosine A2A, group I mGlu and neurotensin receptors have been previously found to modulate the binding characteristics of dopamine D2 receptors in membrane preparations from rat striatum. In the present study it is shown that stimulation of different combinations of striatal A2A, group I mGlu and neurotensin receptors induce different effects on the modulation of D2 receptor binding to those obtained when they are separately stimulated using maximal effective concentrations. In competitive inhibition experiments of dopamine versus the D2 receptor antagonist [3H]raclopride the addition of the A2A receptor agonist CGS 21680, the group I mGlu receptor agonist DHPG or neurotensin induced a decrease in the affinity of the high affinity state of the dopamine D2 receptors for dopamine. When added together CGS 21680 plus neurotensin induced the same effect as when administered alone, CGS 21680 plus DHPG induced a synergistic effect and DHPG plus neurotensin lost their modulating effect on D2 receptor binding. These results demonstrate the existence of multiple intramembrane receptor-receptor interactions in the regulation of striatal D2 receptors.     

Combined treatment with citalopram and buspirone: effects on serotonin 5-HT2A and 5-HT2C receptors in the rat brain

INTRODUCTION: We wanted to elucidate whether the proposed advantages of citalopram-buspirone combination treatment are related to changes in 5-HT(2A/C) receptor-mediated neurotransmission. METHODS: The affinity of buspirone to 5-HT2A and 5-HT2C receptors was measured in vitro, and the influence of buspirone on 5-HT2C receptor-mediated phosphoinositide hydrolysis was estimated. Four groups of rats received citalopram (10 mg/kg), buspirone (6 mg/kg), citalopram-buspirone combination, or saline once a day s.c. for 14 days. Treatment effects on 5-HT2A and 5-HT2C receptors were investigated by receptor autoradiography with antagonist and agonist radioligands. RESULTS: Buspirone was found to be a weak 5-HT2C receptor antagonist, with a low affinity for 5-HT2A and 5-HT2C receptors. Repeated buspirone-citalopram combination treatment markedly decreased [3H]ketanserin and [125I]DOI binding to 5-HT2A receptors. Repeated administration of buspirone and buspirone-citalopram combination increased the affinity of [3H]mesulergine toward 5-HT2C receptors, and buspirone-citalopram combination also decreased [125I]DOI binding to 5-HT2C receptors. DISCUSSION: We suggest that downregulation of brain 5-HT2A receptors and possibly of 5-HT2C receptor agonist sites is involved in the beneficial clinical effects of buspirone-SSRI augmentation treatment. Furthermore, a conversion of brain 5-HT2C receptors from high- to low-affinity state may provide an additional mechanism for the anti-anxiety effects of buspirone.     

Dopaminergic regulation of dopamine D3 and D3nf receptor mRNA expression

Dopamine D3 receptors have the highest dopamine affinity of all dopamine receptors, and may thereby regulate dopamine signaling mediated by volume transmission. Changes in D3 receptor isoform expression may alter D3 receptor function, however, little is known regarding coordination of D3 isoform expression in response to perturbations in dopaminergic stimulation. To determine the effects of dopamine receptor stimulation and blockade on D3 receptor alternative splicing, we determined D3 and D3nf isoform mRNA expression following treatment with the D3 receptor antagonist NGB 2904, and the indirect dopamine agonist amphetamine. Expression of tyrosine hydroxylase (TH) mRNA, the rate‐limiting enzyme in dopamine synthesis, was also determined. The D3/D3nf mRNA expression ratio was increased in ventral striatum, prefrontal cortex, and hippocampus 6 h following D3 antagonist NGB 2904 treatment, and remained persistently elevated at 24 h in hippocampus and substantia nigra/ventral tegmentum. D3 mRNA decreased 65% and D3nf mRNA expression decreased 71% in prefrontal cortex 24 h following amphetamine treatment, however, these changes did not reach statistical significance. TH mRNA expression was unaffected by D3 antagonist NGB 2904, but was elevated by amphetamine in ventral striatum, hippocampus, and prefrontal cortex. These findings provide evidence for an adaptive response to altered D3 receptor stimulation involving changes in D3 receptor alternative splicing. Additionally, these data suggest D3 autoreceptor regulation of dopamine synthesis does not involve regulation of TH mRNA expression. Finally, the observation of regulated TH mRNA expression in dopamine terminal fields provides experimental support for the model of local control of mRNA expression in adaptation to synaptic activity. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Catalepsy induced by a blockade of dopamine D1 or D2 receptors was reversed by a concomitant blockade of adenosine A(2A) receptors in the caudate-putamen of rats

The present study sought to determine, in more detail, the effects of an unselective and a selective adenosine A(2A) receptor blockade on catalepsy induced by a blockade of dopamine D1 or D2 receptors in rats. The results demonstrated that systemic administration of the unselective A1/A2 receptor antagonist, theophylline and the selective A(2A) receptor antagonist, CSC potently reversed catalepsy induced by a systemic D2 receptor blockade with raclopride or by a bilateral blockade of D2 receptors in the caudate-putamen (CPu) with S(-)sulpiride. Likewise, systemic administration of theophylline and CSC reversed catalepsy induced by a systemic D1 receptor blockade with SCH23390; theophylline also counteracted catalepsy after an intra-CPu D1 receptor blockade with SCH23390. Intracerebral co-microinfusions of the selective A(2A) receptor antagonist, MSX-3 together with a D1 (SCH23390) or D2 receptor [S(-) sulpiride] antagonist revealed that catalepsy due to intra-CPu D1 or D2 receptor blockade can be potently reversed by an intra-CPu A2A receptor blockade. In conclusion, our results with systemic and intra-CPu drug administration demonstrate that D1 and D2 receptor-mediated catalepsy can both be reversed by a concomitant blockade of A(2A) receptors. Our results implicate that the CPu is a critical neural substrate for antagonistic interactions of a D1/D2 receptor blockade and an A(2A) receptor blockade in control of motor activity. The present results provide further support for the view that A(2A) receptor antagonists may be potential therapeutics for the treatment of Parkinson's disease.     

Adenosine A2A receptor knockout mice are partially protected against drug-induced catalepsy

Catalepsy assessed using the bar test was measured in both adenosine A2A receptor knockout (A2AR KO) and wild-type (A2AR WT) mice submitted to acute administration of the dopamine D2 receptor antagonist haloperidol (0.5, 2, 4, 6 mg/kg i.p.), the dopamine D1 antagonist SCH 23390 (0.3-3 mg/kg, s.c.), the vesicular monoamine transporter blocker reserpine (3-5 mg/kg, s.c.) or the acetylcholine muscarinic receptor agonist pilocarpine (25-50 mg/kg, i.p.). Except for reserpine, catalepsy scores were significantly lower in A2AR KO mice than in A2AR WT mice following low doses of these cataleptogenic agents. These results suggest that adenosine A2A receptors influence not only dopamine D2 and D1 receptor-mediated neurotransmission but also acetylcholine muscarinic receptor-mediated neurotransmission.     

D1 dopamine receptor--the search for a function: a critical evaluation of the D1/D2 dopamine receptor classification and its functional implications

The present review focuses on the hypothesized D1/D2 dopamine (DA) receptor classification, originally based on the form of receptor coupling to adenylate cyclase activity. The pharmacological effects of compounds exhibiting putative selective agonist or antagonist profiles at those DA receptors positively coupled to adenylate cyclase activity (D1 DA receptors) are extensively reviewed. Comparisons are made with the effects of putative selective D2 DA receptor agonists and antagonists, and on the basis of this work, the DA receptor classification is critically evaluated. A variety of biochemical, behavioral, and electrophysiological evidence is presented which supports the view that D1 and D2 DA receptors can interact in both an opposing and synergistic fashion. Particular attention is focused on the possibility that D1 receptor stimulation is required to enable the expression of certain D2 receptor-mediated effects, and the functional consequences of this form of interaction are considered. A hypothetical model is presented which considers how both the opposing and enabling forms of interaction between D1 and D2 DA receptors can control behavioral expression. Finally, the clinical relevance of this work is discussed and the potential use of selective D1 receptor agonists and antagonists in the treatment of psychotic states and Parkinson's disease is considered.     

Non-additivity of D2 receptor proliferation induced by dopamine denervation and chronic selective antagonist administration: evidence from quantitative autoradiography indicates a single mechanism of action

Experiments were conducted to investigate whether chronic dopamine (DA) D2 receptor blockade and DA denervation exert additive effects on striatal D2 receptor density. We employed for the first time chronic treatment with a pure D2 antagonist, metoclopramide, and measured regional striatal DA receptor binding with quantitative receptor autoradiography. Rats with extensive unilateral DA denervation induced by intracerebral 6-hydroxydopamine (6-OHDA) were injected daily for 21 days with either metoclopramide (30 mg/kg i.p.) or saline. Following a 72-h drug wash-out period, rats were sacrificed and brain sections through the caudate-putamen and nucleus accumbens were incubated with [3H]spiroperidol or [3H]SCH 23390 to assay D2 and D1 receptors, respectively. Autoradiographic analysis revealed that chronic metoclopramide treatment increased the density of D2 sites in the intact hemisphere for all regions examined without further augmenting the already increased density of D2 receptors seen in the 6-OHDA-treated hemisphere. In addition, chronic metoclopramide and 6-OHDA treatment by themselves exhibited remarkably parallel anterior-posterior gradients in their effects on D2 receptor density. D1 receptor density was not affected by metoclopramide treatment but was slightly reduced in the DA-denervated hemisphere. [3H]Mazindol labelling of high-affinity DA uptake sites indicated that the extent of DA denervation was greater than 98% in both saline- and metoclopramide-treated rats. These findings are consistent with the view that chronic D2 receptor blockade and DA denervation act via a single, common mechanism to increase D2 receptor density. Work from other laboratories, in which additive effects of denervation and chronic neuroleptic treatment have been purported, may have resulted from incomplete denervation. Experimental discrepancies may also be due to differing means by which the mesotelencephalic dopaminergic neurons are injured.