Dopamine D2 receptors exert tonic regulation over discrete neurotensin systems of the rat brain

Blockade of dopamine D₂ receptors with either the selective antagonist, sulpiride, or the non-selective antagonist, haloperidol, induces 2- to 3-fold increases in the content of neurotensin-like immunoreactivity in the striatum and the nucleus accumbens of the rat brain. Quantitatively similar increases were also observed (a) in the striatum following selective degeneration of more than 85% of the nigrostriatal dopamine pathway with 6-hydroxydopamine and (b) in both the striatum and the nucleus accumbens after non-selective depletion of brain dopamine using reserpine plus α-methyl-p-tyrosine. Interestingly, treatment of animals with sulpiride or haloperidol, following the depletion of dopamine by either 6-hydroxydopamine or reserpine plus α-methyl-p-tyrosine, did not add to the elevation in neurotensin content of either structure caused by the dopamine depletion alone. These data suggest that an intact dopamine system is required for the neuroleptics to exert effects on individual neurotensin systems. In addition, the same mechanism appears to underlie the responses of the neurotensin pathways to treatments with the neuroleptics or dopamine-depleting drugs. A likely explanation for the effects of neuroleptics and dopamine-depleting drugs is that they eliminate tonic activity on D₂ receptors by basally released dopamine in the striatum and the nucleus accumbens. Supportive evidence for this hypothesis is that concurrent administration of the D₂ receptor agonist, LY 171555, with reserpine, completely blocked the effects of reserpine-induced dopamine depletion on neurotensin systems of the striatum and the nucleus accumbens.     

Autoradiographic analysis of D1 and D2 dopaminergic receptors in rat brain after paradoxical sleep deprivation

Previous work had shown that paradoxical sleep deprivation (PSD) results in potentiation of several apomorphine-induced behaviors, leading to the suggestion that PSD induces an upregulation of brain dopamine receptors. In this study, quantitative receptor autoradiography was used to verify whether PSD does, in fact, induce alterations in D₁ or D₂ receptor binding, and to investigate the regional brain specificity of such effects. After 96 h of PSD, [³H]SCH-23390 binding to D₁ receptors was examined in 30 different brain areas of 10 experimental and 10 cage control rats. [³H]Spiperone was used to label D₂ sites in adjacent tissue sections. Results revealed a 39% increase in [³H]SCH 23390 binding in the entorhinal cortex of PSD rats (p < 0.05), but no other changes in any of the remaining 29 brain areas examined. In contrast, [³H]spiperone binding was significantly elevated in the n. accumbens (+45%) and in all subrogions of the caudate-putamen (range: +13% to +23%). These results, thus, provide evidence that PSD increases D₂ but not D₁ receptor binding in brain. The present results also suggest that upregulated D₂ receptors can account for the previously reported changes in apomorphine-induced behaviors after PSD.     

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [I]epidepride

The regional distribution of striatal and extrastriatal dopamine D₂ receptors in human brain was studied in vitro with(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide, [¹²⁵I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D₂ receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D₂ receptors (0.17–0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31–0.46 pmol/g tissue). Inhibition of [¹²⁵I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [¹²⁵I]epidepride binding was potently inhibited only by dopamine D₂ ligands. The present study demonstrates that dopamine D₂ receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D₂ receptors is comparable to many limbic regions and is considerably higher than in cortex. Very few frontal lobe D₂ receptors are present in man.     

Autoradiographic localization of D1 dopamine receptors in the rat brain with [H]SCH 23390

The regional distribution of D₁ dopamine (DA) receptors in the rat brain has been studied by quantitative autoradiography using the specific D₁ antagonist [³H]SCH 23390 as a ligand. The binding of [³H]SCH 23390 to striatal sections was saturable, stereospecific, reversible and of high affinity (K_d = 2.05nM); it occurred at single population of sites and possessed the pharmacological features of the D₁ DA receptor. The highest densities of [³H]SCH 23390 binding sites were found in the caudate-putamen, olfactory tubercle, nucleus accumbens and substantia nigra (especially in the pars compacta). High densities were also observed in the nucleus interstitialis striae terminalis, the anterior olfactory nucleus, the entopeduncular nucleus, the subthalamic nucleus, the claustrum and the amygdalohippocampal area. An intermediate labelling was found in the anteromedial and suprarhinal DA terminal fields of the cerebral cortex, the basolateral, medial and lateral amygdaloid nuclei, the endopiriform nucleus, the primary olfactory cortex, the globus pallidus, the superior colliculus (especially the superficial layer), the nucleus amygdaloideus corticalis and the dorsal hippocampus (molecular layer of the CA₁ and dentate gyrus). In the anteromedial and suprarhinal cortices, [³H]SCH 23390 binding was more concentrated in layers V and VI. Moderate levels of [³H]SCH 23390 were found in the thalamus, hypothalamus, the habenula, the ventral tegmental area, the posterior cingulate and entorhinal cortices, the supragenual dopamine terminal system and the cerebellum (molecular layer). This regional distribution of [³H]SCH 23390 closely correlated (except for the cerebellum) with the reported distribution of dopaminergic terminals. The topographical distribution of [³H]SCH 23390 has also been studied in detail in striatal subregions. The density of D₁ receptors was much greater in the ventrolateral sector and medial margin of the striatum than in the ventromedial and dorsolateral sectors. A rostrocaudal decrease in the densities of D₁ sites was also found along the rostrocaudal axis of the caudate-putamen. These lateral to medial and anteroposterior gradients overlapped with the density of the dopaminergic afferents.     

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 blockerN-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 the 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. These results indicate that the imbalance in striatal D₂ receptors, but not in D₁ receptors, is a critical factor for the expression of the motor effects elicited by LY 171555 in EEDQ-treated rats.     

[H]Spiperone binds selectively to rat striatal D2 dopamine receptors in vivo: a kinetic and pharmacological analysis

We have determined the kinetic, equilibrium saturation, and pharmacological characteristics of [³H]spiperone ([³H]SPIP) binding to rat brain regional particulate fractions following i.v. injections of [³H]SPIP and compared these parameters to those determined in vitro with traditional ligand-homogenate binding assays. [³H]SPIP binding to rat striatum in vivo and in vitro occurs to a single class of non-interacting binding sites which possess the pharmacological properties of a D₂ dopamine (DA) receptor. The potencies of neuroleptic drugs in inhibiting DA receptor-mediated behaviors correlate with their potencies at displacing striatal [³H]SPIP binding in vivo. While striatum possesses a similar density of [³H]SPIP binding sites in vivo (34 pmol/g) and in vitro (31 pmol/g), binding affinity in vivo is about 200 times lower than in vitro. This difference in binding affinities appears to arise from alterations of [³H]SPIP association and dissociation rate constants brought about by tissue homogenization. The implications of our findings for external imaging of DA receptors and studies of DA receptor function in human brain homogenates are discussed.     

Specific bindings of prostaglandin D2, E2 and F2α in postmortem human brain

Binding activities specific for each of [³H]prostaglandin (PG) D₂, E₂ and F_2α were detected in the P₂ fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity;K_dvalues were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [³H]PG-D₂, [³H]PG-E₂ and [³H]PG-F_2α in the P₂ fractions from 17 brain regions. The PG-D₂ binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E₂ binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D₂ binding, no significant binding of [³H]PG-3₂ was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F_2α binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D₂, PG-E₂ and PG-F_2α, in the human brain.     

Dopamine D3 receptors in the basal ganglia of the common marmoset and following MPTP and l-DOPA treatment

The distribution of the dopamine D3 receptor was studied by receptor autoradiography using [³H]7-OH-DPAT in striatal and extrastriatal brain regions of the common marmoset (Callithrix jacchus). Saturation studies demonstrated that [³H]7-OH-DPAT bound with similar affinity to different regions of marmoset brain. In normal marmosets, specific [³H]7-OH-DPAT binding was found in both striatal and extrastriatal regions. Very high levels of specific [³H]7-OH-DPAT binding were detected in the islands of Calleja and nucleus accumbens but in addition high levels of binding were detected in rostral caudate nucleus and putamen. In common marmosets treated with the selective nigral neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the levels of specific [³H]7-OH-DPAT binding in striatal and extrastriatal regions were not different to those in normal animals. Chronic treatment of MPTP-treated marmosets with l-DOPA/carbidopa did not alter the levels of specific [³H]7-OH-DPAT binding in any brain region. These results demonstrate that in common marmosets D₃ receptors are located in both striatal and limbic regions. The receptor density is not altered by dopaminergic denervation or by chronic l-DOPA administration. The D₃ receptor may, therefore, be important in both the therapeutic and adverse effects of drugs used to treat Parkinson's disease.     

The role of dopamine in the maintenance and breakdown of D1/D2 synergism

The neurochemical factors involved in the maintenance and breakdown of dopamine D₁/D₂ receptor synergism were investigated by giving rats various pharmacological treatments that diminish the ability of dopamine to interact with its D₁ and/or D₂ receptors. Following these treatments, rats were observed for the expression of stereotyped motor behavior in response to independent stimulation of D₁ or D₂ receptors. Independent D₂-mediated responses were observed: (a) 2 h after the last of three daily reserpine (1 mg/kg) injections, (b) 48 h after bilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathways, (c) 24 h after a concentrated 48-h regimen (one injection/6 h) of eticlopride (0.5 mg/kg) or eticlopride + SCH 23390 (0.5 mg each), and (d) 2 h after a concentrated 48-h regimen (one injection/6 h) of α-methyl-p-tyrosine (αMPT; 100 mg/kg), but not after control treatments or a concentrated regimen of SCH 23390 alone. By contrast, independent D₁-mediated responses were observed only after three daily reserpine injections or 48 h after bilateral 6-OHDA lesions. Independent D₁-mediated stereotypy was not observed under control conditions or following a concentrated 48-h regimen of (a) SCH 23390 or eticlopride (0.5 mg/kg each) alone or in combination, (b) a high dose of SCH 23390 (1.0 mg/kg), (c) αMPT (100 mg/kg), or (d) αMPT (100 mg/kg)+SCH 23390 (1.0 mg/kg). Reserpine, bilateral 6-OHDA, and αMPT treatments produced striatal dopamine depletions of 96%, 92%, and 71%, respectively. These data indicate that the breakdown in D₁/D₂ synergism consists of two components: (a) D₁ independence from the controlling influence of D₂ receptors, and (b) D₂ independence from the controlling influence of D₁ receptors. The interaction of synaptic DA with its D₂ receptors plays a major role in determining whether these receptors can function independently of D₁ receptors, whereas reduced DA-D₁ activity alone appears insufficient to elicit D₁ independence.     

Age-related changes in the turnover rates of D1-dopamine receptors in the retina and in distinct areas of the rat brain

The steady-state density and the turnover rates of D₁-dopamine receptors were investigated in the striatum, nucleus accumbens, substantia nigra, and retina of adult (3-month-old) and aged (23-month-old) rats. The turnover rates were measured by monitoring the repopulation kinetics of D₁-dopamine receptors labeled with [³H]-SCH 23390 after the irreversible inactivation induced by a single dose of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg, s.c.). In all the neural tissues examined, the repopulation of D₁ dopamine receptors could be adequately described by a theoretical model that assumes a constant rate of receptor production (i.e. zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e. first order). The results obtained indicate that the reduction in the density of D₁-dopamine receptors in the striatum, nucleus accumbens and substantia nigra of aged rats is the result of a larger decrease in the receptor production rate (−44 to −60%) than in the receptor degradation rate (−21 to −46%). By contrast, the production rate of D₁-dopamine receptors in the retina of aged rats remains unchanged, whilst the degradation rate is reduced by 25%. This results in an age-related increase in the density of D₁-dopamine receptors in the rat retina.     

A single (−)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra

The in vitro and in vivo effects of (−)-nicotine on dopamine D₂ receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (−)-nicotine resulted in a reduction of theK_D value of the D₂ antagonist [³H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in theB_max value. This action of (−)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, theK_D and theB_max values of the D₂ agonist [³H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5–48 h after a single i.p. injection with 1 mg/kg of (−)-nicotine. No significant change in neostriatal D₂ receptor mRNA levels was observed at any time interval after the (−)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4–24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 μM) concentrations of (−)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [³H]raclopride or [³H]NPA binding. These results indicate that a single (−)-nicotine injection can produce a selective and delayed increase in the affinity of D₂ receptors for the antagonist, but not for the agonist without modifying the levels of D₂ receptor mRNA, probably via the activation of central nicotinic receptors.     

Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathways in the rat

The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from rats which had received unilateral 6-hydroxydopamine (6-OHDA) injections destroying ascending nigrostriatal neurones. The binding of both ligands to striatal sections was first shown to be saturable, reversible and of high affinity and specificity [( 3H]SCH 23390: Bmax 2.16 pmol/mg protein, Kd 1.4 nM; [3H]sulpiride; Bmax 0.67 pmol/mg protein, Kd 10.7 nM). After unilateral stereotaxic 6-OHDA injections, rats rotated contralaterally when challenged with apomorphine (0.5 mg/kg), or specific D1 or D2 agonists, SKF 38393 (1.0-5.0 mg/kg) and LY 171555 (0.05-0.5 mg/kg), respectively. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A loss of approximately 90-95% of uptake sites was reproducibly accompanied by an enhanced density of binding ipsilaterally for the D2 ligand, [3H]sulpiride, in all areas of the striatum, but most markedly in the lateral areas. An increase in the D2 binding site density was also seen in the ipsilateral nucleus accumbens and the olfactory tubercle. In contrast, in the same animals, the striatal D1 receptors were far less affected by dopaminergic denervation, with no consistent changes seen in the binding of [3H]SCH 23390. These results suggest that dopamine D2 receptors are more susceptible than D1 receptors to changes after dopaminergic denervation, which is expressed as an increase in the density of binding sites revealed here with [3H]sulpiride.     

D1 and D2 receptor modulation in rat striatum and nucleus accumbens after subchronic and chronic haloperidol treatment

The antipsychotic effects of neuroleptic drugs are believed to be achieved by chronic blockade of dopaminergic transmission in the limbic system. Nevertheless, the effects of chronic (3-12 months) haloperidol administration on the dopaminergic transmission in the nucleus accumbens of rodents remains poorly understood. Studies of spontaneous locomotor activity (SLA), a behavioral measure related to limbic dopamine transmission, and of dopamine D2 receptor density in the nucleus accumbens after chronic oral haloperidol treatment have yielded conflicting results. We evaluated these indices after 8 months of parenteral administration of haloperidol decanoate. We report here that, after 8 months of parenteral treatment, SLA stays significantly decreased and D2 receptors in the nucleus accumbens exhibit the same up-regulation as in the striatum (about 50%). These results fail to support the notion of a different pattern of D2 receptor adaptation to neuroleptic treatment between the nucleus accumbens and the striatum. In contrast, dopamine D1 receptors were found to be unaffected in the nucleus accumbens but decreased in the striatum by 22% after 8 months of treatment. This observation could be relevant to the pathogenesis of tardive dyskinesia.     

Blockade of in vivo binding of I-labeled 3-iodobenzamide (IBZM) to dopamine receptors by D2 antagonist and agonist

The in vivo binding of [¹²⁵I]3-iodobenzamide (IBZM), a substituted benzamide, to DA receptor binding sites in the caudate nucleus, nucleus accumbens, and olfactory tubercle was investigated by using ex vivo autoradiography. The in vivo binding of IBZM seems to be selective to D₂ dopamine receptors, since the binding was blocked by pretreatment of animals with D₂ agonist LY-171555 or antagonist YM-09151-2. Furthermore, in vitro binding assays in striatal membranes confirmed that IBZM binding was highly selective to D₂ sites. Thus, IBZM, when labeled with ¹²³I (T1/2: 13 h; 159 kev), could be a potential ligand for imaging D₂ dopamine receptors by single photon emission computerized tomography procedures.     

Infusion of the dopamine D1 receptor antagonist SCH 23390 into the amygdala blocks fear expression in a potentiated startle paradigm

Dopamine (DA) D₁ receptors are distributed in the nucleus accumbens and the amygdala, two regions of the mesocorticolimbic DA system known to be activated by aversive environmental stimuli. The objective of the present study was to determine the contribution of D₁ receptors in these brain regions to the expression of a fear-motivated behavior, notably, potentiated startle in rats. Injection of the DA D₁ receptor antagonist SCH 23390 into the amygdala blocked the ability of a conditioned light stimulus previously paired with footshock to enhance acoustic startle amplitudes. Bilateral intracerebral administration of SCH 23390 into the nucleus accumbens had no effect on fear-potentiated startle. The observed opposing effects of amygdaloid DA D₁ receptor antagonism on fear expression, along with earlier research demonstrating the involvement of ventral tegmental area (VTA) DA neurons on fear-potentiated startle, suggest a role for mesoamygdaloid activity in conditioned excitatory fear reactions.     

Local injection of alkylating and nonalkylating dopamine receptor antagonists into rat basal forebrain: autoradiographic assessment of D2-like and D3 sites

N-chloroethyl derivatives of 7-hydroxy-1,2,3,4-tetrahydronaphthalene (7-OH-DPAT), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), or fluphenazine were microinjected into rat nucleus accumbens (Acc), and receptor binding quantified autoradiographically after 24 h. EEDQ reduced

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nemonapride (D₂-like receptors) binding in Acc (by 84%) and islands of Calleja (IC; 44%), without affecting

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(+)-7-OH-DPAT (D₃); N-chloroethyl-7-OH-DPATs blocked both radioligands in Acc and IC (30%–70%); fluphenazine had no effect.     

Dopamine denervation leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

We have previously found, in striatal membrane preparations from young (2 months old) rats, that stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) increases the dissociation constants of high- (K_h) and low-affinity (K_l) dopamine D₂ binding sites (labelled with the selective dopamine D₂ antagonist [³H]raclopride) without changing the proportion of high affinity binding sites (R_h). In the present study in striatal preparations from adult (6 months old) rats, it was found that in addition to the increase in both K_h and K_l values, stimulation of adenosine A₂ receptors is associated with an increase in R_h. These result suggest that, in the adult rat, adenosine A₂ stimulation may inhibit the behavioural effects induced by dopamine D₂ stimulation both by decreasing the affinity and the transduction of dopamine D₂ receptors. We have also studied the intramembrane A₂-D₂ receptor interaction in an experimental model of Parkinson's disease, namely in rats with a unilateral 6-OH-dopamine-induced lesion of the nigro-striatal dopamine pathway. It was found that a unilateral dopamine denervation is associated with a higher density of striatal dopamine D₂ receptors in the order of 20%, without any change in their affinity compared with the unlesioned neostriatum. Furthermore, the density (B_max values) of dopamine D₂ receptors in the contralateral neostriatum was significantly higher (about 20%) than in the striatum from native animals. This finding suggests that an unilateral dopamine denervation also induces compensatory long-lasting changes of dopamine D₂ receptors in the contralateral neostriatum. In addition to the hightened sensitivity to dopamine agonists, it is known that the dopamine denervated striatum is more sensitive to adenosine antagonists like methylxanthines. If the adenosine A₂-dopamine D₂ interaction is the main mechanism of action mediating the central effects of methylxanthines, the dopamine denervation might also potentiate this interaction, i.e., dopamine D₂ receptors could be not only more sensitive to dopamine but also to adenosine A₂ receptor activation. Our results support this hypothesis, since membrane preparations from the denervated neostriatum are more sensitive to the effect of CGS 21680 on dopamine D₂ receptors. Thus a low dose of CGS 21680 (3 nM), which is not effective in membrane preparations from the neostriatum of naive animals, is still effective in membranes from the denervated neostriatum. These results underline the potential antiparkinsonian activity of adenosine A₂ antagonists.     

Involvement of dopamine D1 and D2 receptors in the ethanol-associated place preference in rats exposed to conditioned fear stress

The present study was designed to investigate: (1) the involvement of dopamine D₁ and D₂ receptors, and (2) the roles of these receptors and endogenous opioid systems (endorphinergic and enkephalinergic systems) in the ethanol-induced place preference in rats exposed to conditioned fear stress using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference. The selective D₁ receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H3-benzazepine)hydrochloride (SCH23390; 0.01 and 0.03 mg/kg, s.c.) and the selective D₂ receptor antagonist S(−)-5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride; 20 and 40 mg/kg, s.c.) significantly attenuated the ethanol-induced place preference. The administration of ethanol (75 mg/kg, i.p.) tended to produce a place preference, but this effect was not significant. SCH23390 (0.03 mg/kg, s.c.) and sulpiride (40 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.). In addition, SCH23390 (0.03 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3,-g]isoquinoline (TAN-67; 20 mg/kg, s.c.). On the other hand, sulpiride (40 mg/kg) had no significant effect on the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by TAN-67. These results suggest that D₁ and D₂ receptors may be involved in the rewarding mechanism of ethanol under psychological stress. In addition, D₁ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ- and δ-opioid receptors, whereas D₂ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ-opioid receptors, but not in that modulated by the activation of δ-opioid receptors.     

D3 dopamine receptor mRNA is widely expressed in the human brain

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

Evidence for the behavioral supersensitivity of dopamine D2 receptors without receptor up-regulation in morphine-abstinent rats

The effect of morphine tolerance-dependence and abstinence on the characteristics of dopamine D₂ receptors in brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with 6 morphine pellets for a 7-day period, each containing 75 mg of morphine free base. Rats implanted with placebo served as controls. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to a challenge dose of morphine. Similarly, the development of physical dependence was evidenced by a decreased in body weight and colonic temperature after morphine pellet removal (withdrawal). The binding characteristics (B_max andK_d values) of [³H]spiroperidol to dopamine D₂ receptors were determined in the tissues of morphine-tolerant and morphine-abstinent rats. In the tolerant rats, the pellets were left intact at the time of sacrificing, whereas, in the abstinent rats the pellets were removed 18 h prior to sacrificing. The binding of [³H]spiroperidol was determined in membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups. [³H]Spiroperidol bound to brain regions and spinal cord at a single high affinity site. TheB_max or theK_d values in brain regions and spinal cord of morphine-tolerant and -abstinent rats did not differ from their respective placebo controls. The behavioral responses to a selective dopamine D₂ receptor agonist, 2-bromo-α-ergocryptine were also determined in the morphine-abstinent rats. In morphine-abstinent rats, increased behavioral activity, such as total distance travelled, number of movements, and the number of stereotypic movements was seen as compared to placebo controls. The dose of 2-bromo-α-ergocryptine which by itself had no effect on any type of behavioral activity in placebo-treated rats, increased the total distance travelled, horizontal activity, number of movements, and movement time in morphine-abstinent rats. Although in morphine-tolerant or morphine-abstinent rats, the characteristics of [³H]spiroperidol binding to dopamine D₂ receptors in brain regions and spinal cord were unchanged, the supersensitivity was observed to behavioral responses of 2-bromo-α-ergocryptine, a selective dopamine D₂ receptor agonist. These results provide an evidence for behavioral responses of 2-bromo-α-ergocryptine, a selective up-regulation in morphine abstinent rats. Previously, we have show that dopamine D₁ receptors are unaffected in morphine tolerant rats but are modified in morphine-abstinent rats. Thus, in the morphine abstinent process a significant difference was noted in the biochemical characteristics of dopamine D₁ and D₂ receptors.