D1 Dopamine Receptor Binding and mRNA Levels Are Not Altered After Neonatal 6-Hydroxydopamine Treatment: Evidence Against Dopamine-Mediated Induction of D1 Dopamine Receptors During Postnatal Development

Abstract: The role of dopaminergic innervation on the postnatal developmental expression of D₁ dopamine receptors was investigated. Bilateral destruction of dopa-mine-containing neurons was achieved by treating rats intracisternally with 6-hydroxydopamine (6-OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D₁ receptor activation by residual dopamine, a group of 6-OHDA-lesioned rats was given twice daily injections of the D₁ receptor antagonist SCH-23390, from day 4 to 20. D₁ dopamine receptor binding was assessed in the caudate—putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [³H]SCH-23390 binding. In addition, the relative amount of D_1A receptor mRNA was assessed by in situ hybridization of a ³⁵S-labeled riboprobe. In the developing rats, neither the amount of [³H]SCH-23390 binding nor the amount of D_1A receptor mRNA was altered by 6-OHDA lesioning followed by chronic treatment with SCH-23390. Thus, bilateral destruction of dopamine-containing neurons and treatment with SCH-23390 in neonatal rats did not interfere with the developmental expression of D₁ receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH-23390 from postnatal day 4 to 20 also did not alter [³H]SCH-23390 binding or levels of D₁ receptor mRNA. However, adult rats treated chronically with SCH-23390 exhibited increased [³H]SCH-23390 binding but did not show a significant change in D₁ receptor mRNA levels.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Repeated Reserpine Increases Striatal Dopamine Receptor and Guanine Nucleotide Binding Protein RNA

Abstract: In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty-four hours after seven consecutive daily injections of reserpine—a treatment that is known to produce functional sensitization of D₁ and D₂ dopamine receptors—the level of striatal D₁ dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein G_sα was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D₁ DA receptor and G_sα mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D₂ dopamine receptor and of G proteins G_i2α and G_oα by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D₂ dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins G_i2α and G_oα were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal G_sα mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D₁ dopamine receptor system following long-term dopamine depletion.     

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.     

Neurochemical Evidence that Postsynaptic Nucleus Accumbens D3 Receptor Stimulation Enhances Cocaine Reinforcement

Abstract: The mechanism by which two D₃ receptor-preferring agonists, 7-hydroxydipropylaminotetralin (7-OH-DPAT) and quinelorane, modulate cocaine reinforcement was examined by monitoring nucleus accumbens dopamine levels with in vivo microdialysis while rats intravenously self-administered the following four different drug solutions consecutively: (1) cocaine; (2) a combination of cocaine plus a low dose of either agonist; (3) either agonist alone; and finally, (4) a physiological saline solution. Both 7-OH-DPAT (4 µg/infusion) and quinelorane (0.25 µg/infusion) decreased cocaine (0.25 mg/infusion) intake in a manner indicating an enhancement of cocaine reinforcement and simultaneously decreased the cocaine-induced elevations in nucleus accumbens dopamine levels by >50%. Subsequent self-administration of either 7-OH-DPAT (4 µg/infusion) or quinelorane (0.25 µg/infusion) alone resulted in significant, but stable, increases in drug intake, with a concurrent decrease in nucleus accumbens dopamine levels to ∼50% below nondrug baseline levels. These findings indicate that postsynaptic D₃ receptor stimulation in the nucleus accumbens enhances the reinforcing properties of cocaine. In a second experiment, local application of 7-OH-DPAT via reverse dialysis (30 and 100 n M perfusate concentrations) dose-dependently decreased nucleus accumbens dopamine efflux to 76 ± 3.9 and 61 ± 6.3% of baseline, respectively, whereas there was no effect of this agonist on dopamine efflux in the ipsilateral striatum of these same animals. Coperfusion with the D₃ receptor-preferring antagonist nafadotride dose-dependently blocked the effect of 7-OH-DPAT on nucleus accumbens dopamine efflux. These results suggest that, at low concentrations, 7-OH-DPAT selectively activates D₃ receptors in vivo.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Activation of post-synaptic dopamine D₁ receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D₁ receptor (D₁R) agonist SKF38393, but not D₂ receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D₁R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D₁Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D₂ receptors are also involved in the release of tPA induced by morphine and methamphetamine.     

Dopaminergic Regulation of Septohippocampal Cholinergic Neurons

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D₁ agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D₂ receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d -Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D₁ receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D₂ antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D₁ receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.     

Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment

Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson's disease pathology and l -DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. l -DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D₂/D₃ agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by l -DOPA but not by DA depletion alone. Similarly, l -DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by l -DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson's disease. Thus, these proteins may be targets for antiparkinsonian therapy.     

Postmortem Stability of Monoamines, Their Metabolites, and Receptor Binding in Rat Brain Regibns

Abstract: The effects of postmortem delay, time of storage, and freezing, thawing, and refreezing tissue samples were studied in postmortem rat brain using conditions that reflect the handling of postmortem human brain before neurochemical analysis. The levels of monoamines and metabolites in the striatum and cingulate and occipital cortex were measured using alumina extraction and HPLC methods. Binding of raclopride to dopamine D₂, SCH- 23390 to dopamine D₁, ketanserin to serotonin 5-HT₂, 8- hydroxy-2-(di- n -propylamino)tetralin to serotonin 5-HT_1A, and cholecystokinin (CCK)-8 to CCK-B sites was measured in tissue homogenates from the striatum or frontoparietal cortex. An 18-h postmortem delay before dissection and storage resulted in region-specific changes in monoamine and metabolite levels. Binding to striatal D₁and frontoparietal cortex CCK-B sites was reduced over the course of a 27-h postmortem delay. Binding to D₂ and 5-HT sites was relatively stable. Storage of tissue for up to 8 months also resulted in region-specific changes in monoamine and metabolite levels. No changes in receptor binding were seen after long-term storage. Freezing, thawing, and refreezing tissue samples resulted in increased levels of striatal 3, 4-dihydroxyphenylacetic acid and decreased binding to striatal D₂ sites. These results demonstrate time-, temperature-, and storage-dependent regional differences in.the stability of monoamines and their metabolites and in binding to various receptor sites. These differences in stability and binding should be accounted for to interpret accurately the effects of neurological disorders on neurotransmitter dynamics in postmortem human brain tissue.     

Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu₅ receptors (mGlu₅R) and dopamine D₂ receptors (D₂R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu₅R, D₂R and adenosine A_2A receptor (A_2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu₅R, D₂R and A_2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu₅R/D₂R/A_2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.     

Zinc Allosterically Modulates Antagonist Binding to Cloned D1 and D2 Dopamine Receptors

Abstract: Cations of various size and charge were used as atomic scale probes of D₁ and D₂ dopamine receptors. Those cations that perturbed the binding of D₁- and D₂-selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [³H]SCH-23390 and [³H]methylspiperone to cloned D_1A and D_2L dopamine receptors in either the presence or absence of Zn²⁺. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K _D) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D₁- and D₂-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Effects of Cerebral Ischemia on Regional Dopamine Release and D1 and D2 Receptors

Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D₁ and D₂) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D₁ and/or D₂ receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (¹²⁵I-SCH23982 and [³H]YM-09151-2, respectively). The ischemic release of DA from the striatum was greater (400-fold over preischemic level) than that from the cortex (12-fold over preischemic content). The affinity for the D₁-receptor ligand was lower ( K _D= 1.248 ± 0.047 n M ) after ischemia than that for sham controls ( K _D= 0.928 ± 0.032 n M, p < 0.001). The number of binding sites for D₂ receptors decreased in striatum ( B _max= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls ( B _max= 510 ± 25.2 fmol/mg of protein, p < 0.05). D₁ or D₂ binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D₁- and D₂-mediated DA neurotransmission.     

In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

Tonic autoinhibition contributes to the heterogeneity of evoked dopamine release in the rat striatum

Electrically evoked dopamine release as measured by voltammetry in the rat striatum is heterogeneous in both amplitude and temporal profile. Previous studies have attributed this heterogeneity to variations in the density of dopamine (DA) terminals at the recording site. We reach the alternate conclusion that the heterogeneity of evoked DA release derives from variations in the extent to which DA terminals are autoinhibited. We demonstrate that low-amplitude, slow evoked DA responses occur even though recording electrodes are close to DA terminals. Moreover, the D₂ agonist and antagonist, quinpirole and raclopride, respectively, affect the slow responses in a manner consistent with the known functions of pre-synaptic D₂ autoreceptors. Recording sites that exhibit autoinhibited responses are prevalent in the dorsal striatum. Autoinhibition preceded electrical stimulation, which is consistent with our prior reports that the striatum contains a tonic pool of extracellular DA at basal concentrations that exceed the affinity of D₂ receptors. We conclude that the striatum contains DA terminals operating on multiple time courses, determined at least in part by the local variation in autoinhibition. Thus, we provide direct, real-time observations of the functional consequence of tonic and phasic DAergic signaling in vivo .