Alterations in dopamine uptake sites and D1 and D2 receptors in cats symptomatic for and recovered from experimental parkinsonism

The administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to adult cats severely disrupts the dopaminergic innervation of the striatum. Animals display a parkinson-like syndrome, consisting of akinesia, bradykinesia, postural instability, and rigidity, which spontaneously recovers by 4–6 weeks after the last administration of MPTP. In this study we used quantitative receptor autoradiography to examine changes in DA uptake sites and DA receptors in the basal ganglia of normal, and symptomatic and recovered MPTP-treated cats. Consistent with the destruction of the nigrostriatal DA pathway, there was a severe loss of DA uptake sites, labeled with [³H]-mazindol, in the caudate nucleus (64–82%), nucleus accumbens (44%), putamen (63%), and substantia nigra pars compacta (SNc, 53%) of symptomatic cats. Following behavioral recovery, there were no significant changes in DA uptake site density. Significant increases of [³H]-SCH 23390 binding to D1 DA receptors were observed in the dorsal caudate (>24%; P < 0.05) of symptomatic cats and in all regions of the caudate-putamen (>30%; P < 0.05) of recovered animals. [³H]-SCH 23390 binding in tree substantia nigra pars reticulata was half of that in the striatum and showed no changes in symptomatic or recovered animals. No alterations in the binding of [¹²⁵1]-epidepride to D2 receptors was observed in any region of the striatum in either, symptomatic or recovered animals. [¹²⁵1]-Epidepride binding in the SNc was decreased by >36% (P < 0.05) following MPTP treatment. These data show that cats made parkinsonian by MPTP exposure have a significant decrease in the number of DA reuptake sites throughout the striatum and that recovery of sensorimotor function in these animals is not correlated with an increase in the number of striatal reuptake sites. Behavioral recovery, however, does seem to be correlated with a general elevation of Dl receptors throughout the striatal complex. The present data also show that direct correlations between changes in DA receptor regulation after a large DA depleting lesion and behavioral deficits or recovery from those deficits are difficult and that the relationships between DA receptors/transporters and behavior require further study. © 1995 Wiley-Liss, Inc.     

Autoradiographic localisation of [3H]-SCH23390 and [3H]-spiperone binding sites in honey bee brain

In the brain of the honey bee, dopamine receptors have been identified by using the vertebrate D1 dopamine antagonist [³H]-SCH23390 and the vertebrate D2 dopamine antagonist [³H]-spiperone. This study uses light microscope autoradiography to investigate the anatomical distributions of the binding sites labelled by [³H]-SCH23390 and [³H]-spiperone in tissue sections cut at three depths from the anterior surface of the brain. The binding of these radioligands differed significantly, in both density and distribution. Specific binding of [³H]-SCH23390, defined by using 5 × 10^-6M cis-(Z)-flupentixol, was densest in regions of somata, such as the deutocerebral somatal rind, the somatal layer beneath the calyces of the mushroom bodies and the ventral protocerebral somatal group. High levels of [³H]-SCH23390 binding were also measured in the lateral protocerebrum. [³H]-Spiperone binding site density estimates were consistently lower than those of [³H]-SCH23390. Specific binding of [³H]-spiperone, determined by subtracting binding in the presence of 10^-4M domperidone from the total binding, was highest in the alpha lobes, beta lobes, and calyces of the mushroom body neuropil. Relatively high binding was also measured in the central body and lateral protocerebral neuropil. These results suggest that the distribution of dopamine receptors in the brain of the bee is subtype specific, and they support the view that dopamine plays many roles in the insect central nervous system. J. Comp. Neurol. 394:29–37, 1998. © 1998 Wiley-Liss, Inc.     

In vivo labelling of rat brain dopamine D-2 receptors

Summary The stereospecific blockade by raclopride and FLB472 (the R enantiomer of raclopride) of the specific in vivo binding of [³H]-spiperone, [³H]-N,N-propylnorapomorphine (NPA) and [³H]-raclopride was studied in seven brain regions (e.g., caudate nucleus, olfactory tubercle, septum, hippocampus, frontal cortex, substantia nigra, pituitary gland) of the male albino rat. The binding of all three ligands was dose-dependently blocked by raclopride and FLB472. The blockade by FLB 472 occurred at doses 50–100 times higher than that obtained by raclopride. The maximal blockade by raclopride of [³H]-spiperone binding differed between brain areas. Thus, the largest blockade was obtained in the substantia nigra (95%), septum (90%), caudate nucleus (60%) and olfactory tubercle (60%), while the blockade of [³H]-spiperone binding in the frontal cortex and pituitary gland did not exceed 30% and 50%, respectively. In contrast to [³H]-spiperone, the in vivo binding of [³H]-NPA and [³H]-raclopride was prevented by 90–100% in all brain areas examined. Taken together, the present findings indicate that the in vivo binding of three radioactive ligands to a central dopamine D-2 receptor can be stereoselectively blocked by the enantiomers of raclopride. The findings suggest that, under in vivo conditions, [³H]-raclopride and [³H]-NPA may label a closely related receptor site. However only some of the [³H]-spiperone binding sites may be identical to the [³H]-raclopride binding sites. The findings indicate furthermore that the relative overlap of D-2 sites shared by [³H]-spiperone and [³H]-raclopride may vary between brain regions.Raclopride is identical to ()-(S)-3,5,dichloro-N-(1-ethyl-2-pyrrolidinyl)methyl-6-methoxysalicylamide tartrate [FLA870()]. The (R) enantiomer of this compound will in the present paper be referred to as FLB472.     

Nitric oxide synthase inhibition and MPTP-induced toxicity in the common marmoset

Nitric oxide, produced following activation of N-methyl-D-aspartate (NMDA) receptors, may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity since NMDA receptor antagonists have been shown to prevent MPTP induced nigral cell loss in primates. Common marmosets were treated with either saline or MPTP or L-N^Gnitro arginine methyl ester (L-NAME) or MPTP and L-NAME. MPTP-treated common marmosets showed motor deficits including bradykinesia, rigidity, and tremor accompanied by a marked loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta and of [³H]-mazindol binding in the caudate-putamen. MPTP treatment also caused an increase in glial fibrillary acidic protein (GFAP) staining in the substantia nigra compared to controls. However, MPTP treatment did not alter the number of constitutive nitric oxide synthase-immunoreactive neurones in the caudate-putamen. Furthermore, neurones or glial cells immunoreactive for inducible nitric oxide synthase were not observed in the substantia nigra pars compacta following MPTP treatment. L-NAME treatment alone did not produce any behavioural changes in marmosets and did not alter the number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta, the number of constitutive nitric oxide synthase-immunoreactive neurones or [³H]-mazindol binding in the caudate-putamen compared to saline-treated control animals. Furthermore, L-NAME did not affect the motor deficits, loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta, loss of [³H]-mazindol binding in the caudate-putamen, or the increase in GFAP staining in the substantia nigra induced by MPTP treatment of common marmosets. The failure of L-NAME to protect against MPTP-induced toxicity in the marmoset suggests that nitric oxide does not play a major role in such toxicity and casts doubt over the involvement of the NMDA:nitric oxide system in neurodegeneration in MPTP-treated primates. Synapse 26:301–316, 1997. © 1997 Wiley-Liss, Inc.     

Autoradiography of dopamine receptors and dopamine uptake sites in the spontaneously hypertensive rat

We examined the status of dopamine (DA) D1 and D2 receptors by using [3H]SCH 23390 and [3H]spiperone binding, respectively, and DA uptake sites by using [3H]mazindol binding in spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats. SHR showed significantly higher [3H]SCH 23390 and [3H]spiperone binding in the caudate-putamen (CPu), the nucleus accumbens (NAc) and the olfactory tubercle (OT) in comparison to the SD rats. There were no significant differences in [3H]mazindol-labeled DA uptake sites between the two strains. Unilateral 6-hydroxydopamine (6-OHDA) injection into the striatum resulted in more than 90% depletion of DA uptake sites in the CPu in both strains. 6-OHDA-induced DA depletion was associated with significant increases in striatal [3H]spiperone binding which were of similar magnitude in the SD rats (+64.1%) and SHR (+51.3%). There were only small decreases (-5.4%) in D1 receptor binding in the dorsolateral aspect of the CPu in the SHR, whereas there were no changes in striatal D1 receptors in the SD rats. These results indicate that, although the SHR have higher concentrations of both D1 and D2 receptors in the basal ganglia, these receptors are regulated in a fashion similar to DA receptors in SD rats after 6-OHDA-induced striatal DA depletion.     

Chronic high dose l-dopa treatment does not alter the levels of dopamine D-1, D-2 or D-3 receptor in the striatum of normal monkeys: an autoradiographic study

Summary. To assess the role of dopamine receptors in the genesis of dyskinesia, we have used quantitative autoradiography to determine the effect of chronic l-dopa administration on dopamine D-1 (using [³H]SCH 23390), D-2 (using [³H]spiperone) and D-3 (using [³H]7-OH-DPAT) receptor binding levels in the striatum of dyskinetic or non-dyskinetic monkeys. Total and subregional striatal analysis showed no difference in D-1, D-2 or D-3 receptor binding in the caudate and putamen between monkeys receiving high dose l-dopa treatment with marked dyskinesia and those without dyskinesia compared to untreated animals. It thus appears unlikely that changes in dopamine receptor expression are a primary cause of l-dopa induced dyskinesia. Rather, a functional dissociation of D-2 receptor coupling to co-expressed enkephalin/adenosine-2a receptor activity in the striato-GPe indirect pathway may be more important in the development or expression of l-dopa-induced involuntary movements. Received July 3, 2000; accepted March 13, 2001     

Hemiparkinsonism in a monkey after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is associated with regional ipsilateral changes in striatal dopamine D-2 receptor density

Infusion of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the right internal carotid artery of a cynomologus monkey (Macaca fascicularis) induced an almost complete loss of the dopaminergic innervation of the right caudate-putamen and hemiparkinsonism. Digital subtraction autoradiography revealed that at 8 weeks postinjection, a major increase in [3H]spiroperidol binding to D-2 sites in the lateral regions of the right caudate nucleus and putamen occurred, without a significant change in the medial caudate nucleus and putamen. The 92-96% decrease in specific [3H]mazindol binding observed in the right striatum extended into the medial caudate nucleus and putamen and confirmed the extensive loss of dopamine inputs to this structure. The region of the increase in D-2 receptor density is innervated by somatosensory, motor and parietal cortex. This indicates that the increase in D-2 receptor density in this region of the striatum may play a particularly important role in the L-dihydroxyphenylanine-induced motoric recovery observed in such animals.     

Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

Quantitative autoradiography using [3H]-SCH 23390, [3H]-sulpiride and [3H]-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both [3H]-SCH 23390 and of [3H]-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). [3H]-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral [3H]-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect [3H]-SCH 23390 or [3H]-forskolin binding, but largely removed [3H]-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce [3H]-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.     

PET demonstrates different behaviour of striatal dopamine D-1 and D-2 receptors in early Parkinson's disease

Striatal dopamine D-1 receptor binding was investigated in vivo with positron emission tomography (PET) in five patients with early Parkinson's disease using [11C]-SCH 23390. All patients had predominantly unilateral symptoms and showed a significant reduction in the accumulation of [18F]-6-F-DOPA in the striatum contralateral to the symptoms. None of the patients had received any antiparkinsonian medication. The striatal and cerebellar radioactivity was measured and corresponding striatum/cerebellum ratios were counted. The mean striatum/cerebellum ratio of [11C]-SCH 23390 binding was symmetric between the hemispheres. By contrast, the striatum/cerebellum ratio of [11C]raclopride binding, labelling dopamine D-2 receptors, was increased significantly in the hemisphere contralateral to the symptoms as compared with the opposite hemisphere. Thus, the present results show that the behaviour of striatal D-1 and D-2 receptors is different in early Parkinson's disease.     

D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis

The distribution of dopamine D-1 receptors has been determined in the rat brain by a quantitative in vitro light-microscopic autoradiographic method. The binding of [N-methyl-3H]-SCH 23390 to slide-mounted tissue sections takes place with characteristics expected of a substance that recognizes D-1 receptors. The binding is saturable, has high affinity, and exhibits an appropriate pharmacology and stereospecificity in several discrete microscopic brain regions as determined by quantitative autoradiography. The highest density of D-1 receptors occurs in the caudate-putamen, accumbens nucleus, olfactory tubercle, and the substantia nigra pars reticulata. High concentrations of D-1 receptors were associated with the intercalated and medial nuclei of the amygdala, entopeduncular nucleus, and major island of Calleja. Furthermore, moderate to low concentrations were observed in several other structures, such as the frontal cortex, subthalamic nucleus, and several thalamic, hypothalamic, and hippocampal areas. The distribution of D-1 receptors correlates very well with projection areas of dopaminergic pathways. This technique furnishes a powerful assay for the accumulation of detailed pharmacologic and anatomical data about D-1 receptors, and the results suggest possible CNS sites of action of D-1 dopamine receptor selective compounds.     

Localization of dopaminergic markers in the human subthalamic nucleus

The potential role for dopamine in the subthalamic nucleus was investigated in human postmortem tissue sections by examining; (1) immunostaining for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis; (2) binding of [(3)H]-SCH23390 (D1-like), [(3)H]-YM-09151-2 (D2-like), and [(3)H]-mazindol (dopamine uptake); and (3) expression of dopamine D1 and D2 receptor mRNAs. Immunostaining for tyrosine hydroxylase was visualized in Bouin's-fixed tissue by using a monoclonal antibody and the avidin-biotin-complex method. The cellular localization of the dopamine D1 and D2 receptor mRNAs was visualized by using a cocktail of human specific oligonucleotide probes radiolabeled with (35)S-dATP. Inspection of immunostained tissue revealed a fine network of tyrosine hydroxylase-immunostained fibers traversing the nucleus; no immunopositive cells were detected. Examination of emulsion-coated tissue sections processed for D1 and D2 receptor mRNA revealed, as expected, an abundance of D1 and D2 mRNA-positive cells in the caudate nucleus and putamen. However, no D1 or D2 receptor mRNA-expressing cells were detected in the subthalamic nucleus. Further, semiquantitative analysis of D1-like, D2-like and dopamine uptake ligand binding similarly revealed an enrichment of specific binding in the caudate nucleus and putamen but not within the subthalamic nucleus. However, a weak, albeit specific, signal for [(3)H]-SCH23390 and [(3)H]-mazindol was detected in the subthalamic nucleus, suggesting that the human subthalamic nucleus may receive a weak dopaminergic input. As weak D1-like binding is detected in the subthalamic nucleus, and subthalamic neurons do not express dopamine D1 or D2 receptor mRNAs, together these data suggest that the effects of dopaminergic agents on the activity of human subthalamic neurons may be indirect and mediated via interaction with dopamine D1-like receptors.     

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.     

Dopamine receptors status after unilateral nigral 6-OHDA lesion

The physiological effects of dopamine (DA) are mediated by several distinct receptor subtypes. The effects of unilateral nigral 6-hydroxydopamine (6-OHDA) lesions on DA receptors were investigated by receptor autoradiography using the D₁ selective ligand [³H]SCH 23390 as well as the D₂ ligand [³H]spiroperidol. mRNA distribution was studied byin situ hybridization. Lesioned rats were sacrificed at different time intervals. Receptor binding studies were performed on tissue sections using selective ligands. [³⁵S]UTP labeled RNA probes were prepared from the different cDNA (D₁, D₂, D₃) and used forin situ hybridization. A specific loss of receptor binding sites and mRNA hybridization was found in the lesioned substantia nigra pars compacta (SNc) at all times examined. Receptor binding studies revealed a different time-dependent increase in both D₁ and D₂ receptors.In situ hybridization showed that only D₂ receptor mRNA increased in the caudate-putamen (CPu) of the lesioned side 15 d after 6-OHDA. No changes were observed in D₁ and D₃ receptor mRNA during the entire time-course.     

Comparative distribution of dopamine D-1 and D-2 receptors in the basal ganglia of turtles, pigeons, rats, cats, and monkeys

The distribution and density of dopamine D-1 and D-2 receptors were studied in the basal ganglia of adult turtles, pigeons, rats, cats, and monkeys. Dopamine receptors were measured in vitro by quantitative autoradiography in alternate sections processed for D-1 and D-2 receptor subtypes and compared to adjacent sections stained for acetylcholinesterase (AChE) activity. [3H]-SCH 23390 and [3H]-spiroperidol were used to label the D-1 and D-2 dopamine receptor subtypes, respectively. The anatomic distribution of both D-1 and D-2 receptors in the basal ganglia was remarkably similar across all species examined. Whereas the absolute number of D-1 and D-2 receptors in the basal ganglia varied between species, the percentage of D-1 and D-2 receptors in a region was quite similar among species. The pattern of binding to the D-1 and D-2 receptor varied among the different species. The adult turtles, pigeons, and rats demonstrated nonpatchy D-1 and D-2 receptor binding in the striatum and pallidum. The adult cat and monkey caudate nucleus and putamen demonstrated mildly heterogeneous receptor binding in a pattern that differed from that seen with AChE staining, but did occasionally demonstrate similar patterns of the D-1 and D-2 receptor subtypes. The immature cat striatum was characterized by heterogeneous D-1 receptor binding that corresponded to heterogeneous AChE rich patches, whereas D-2 receptor binding was homogeneous. Heterogeneous binding was seen in other basal ganglia structures including the nucleus accumbens, olfactory tubercle, and substantia nigra pars compacta and reticulata. Complementary D-1 and D-2 receptor binding patterns were seen in the pallidum and substantia nigra of the mammals. The results of this study indicate that both D-1 and D-2 dopamine receptors are present in the basal ganglia of five different vertebrates. A common feature of dopamine receptors in the basal ganglia is their heterogeneity in distribution and density. The heterogeneity of dopamine receptors has similarities to and differences from the distribution of presynaptic dopamine and other neurotransmitter markers of the basal ganglia.     

In vivo labelling of pituitary dopamine D-2 receptors in the male rat using [3H]-raclopride

Summary The substituted benzamide drug [³H]-raclopride (Köhler et al., 1985) was used to label dopamine D-2 receptors within the individual lobes of the pituitary gland as well as in the brain of male rats in vivo. The in vivo [³H]-raclopride binding was found to be saturable, reversible and of high specificity. Between 5–30% of the binding was non-specific at saturating concentrations dependent upon the lobe of the pituitary gland as well as of the brain region (e.g., caudate nucleus and olfactory tubercle) studied. Saturation analyses revealed B_max-values of 12.9±1.6 and 2.2±0.9pmol·g^–1 wet weight in the intermediate and anterior lobes, respectively with respective K_D values of 6.5±4.6 and 7.3±2.4 nmol·kg^–1. Quantitative autoradiographic studies using a single concentration of [³H]-raclopride showed a similar relationship with regard to binding densities in the different lobes, and showed, in addition, that the posterior lobe contained the lowest number of specific [³H]-raclopride binding sites. The binding capacities and affinities of binding were 12.9±1.7 and 9.2±2.8 respectively in the caudate nucleus and 6.1±0.7 and 9.3±2.7 respectively in the olfactory tubercle.The pharmacological analysis revealed that (S)sulpiride, remoxipride and raclopride were 10 to 125 times more potent than their corresponding isomers [(R)sulpridie, FLA 731(–), and FLB 472, respectively] in blocking the in vivo [³H]raclopride binding in the pituitary gland as well as in brain. The in vivo potency of different D-2 antagonists in preventing the [³H]-raclopride binding in the anterior and intermediate lobes was: spiperone > domperidone > raclopride > (S)sulpiride > remoxipride. The D-1 selective antagonist SCH 23390 did not block the in vivo binding of [³H]-raclopride neither in the pituitary lobes nor in the brain. In agreement with these findings the D-2 agonists N,N-propylnorapomorphine and quinpirole (LY 171555) but not the D-1 agonist SKF 38393-A blocked the specific in vivo [³H]-raclopride binding in the pituitary gland as well as in the brain. Comparisons between the relative potencies of different drugs in blocking pituitary and brain D-2 receptors in vivo showed that some drugs, including sulpiride and domperidone, were more potent in the pituitary gland than in the brain, while remoxipride and raclopride were equipotent in the two areas. The D-2 agonists tested appeared to be slightly more potent in the brain than in the pituitary gland.     

Characterisation of dopamine receptors in insect (Apis mellifera) brain

In vitro binding experiments using the vertebrate D₁ dopamine receptor ligand [³H]SCH23390 and the vertebrate D₂ dopamine receptor ligand [³H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [³H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K_d of 6.3 ± 1.0 nM and B_max of 1.9 ± 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [³H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol ≥ chlorpromazine > fluphenazine> S(+)-butaclamol > spiperone. R(±)-SKF38393 and dopamine were the most effective agonists tested. [³H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D₁ dopamine receptor subtype. [³H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [³H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K_d of 0.11 ± 0.02 nM and B_max of 9.2 ± 0.5 fmol/mg protein. The calculated values for the low affinity site were a K_d of 19.9 nM and B_max of 862 fmol/mg protein. Kinetic analyses also indicated that [³H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [³H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [³H]spiperone binding was: spiperone > fluphenazine> S(+)-butaclamol > domperidone> R(+)-SCH23390 > S(−)-sulpiride.     

The effects of chronic continuous versus intermittent levodopa treatments on striatal and extrastriatal D1 and D2 dopamine receptors and dopamine uptake sites in the 6-hydroxydopamine lesioned rat — an autoradiographic study

The effects of chronic ‘continuous’ infusion and ‘intermittent’ modes of levodopa/carbidopa administration on apomorphine induced circling behaviour, DA uptake sites (labelled with [³H]mazindol) and D₁ and D₂ DA receptor binding (labelled with [³H]SCH 23390 and [³H]sulpiride, respectively) were investigated in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. The circling behaviour in response to apomorphine was greatly enhanced following chronic ‘intermittent’ but not ‘continuous’ levodopa treatments. Following the ‘intermittent’ regime, the lower dose of apomorphine induced a period of intense circling with delayed onset and rapid offset, than in rats given either ‘continuous’ infusion of levodopa or saline. The 6-OHDA lesion itself induced gross depletion of [³H]mazindol binding in all striatal subregions, NAc and OT, but not frontal cortex. [³H]Sulpiride binding in the ventrolateral striatal quadrant was increased on the denervated side and this correlated with the peak contralateral turns in response to 0.5 mg/kg apomorphine challenge. This asymmetry in striatal [³H]sulpiride binding was reduced in both groups of rats receiving levodopa. [³H]sulpiride binding in the NAc and OT and [³H]SCH 23390 binding in the striatum, NAc, OT and SNr were unaffected by DA denervation or either regime of levodopa treatments. ‘Continuous’ infusion and not ‘intermittent’ injections of levodopa reduced [³H]mazindol binding in the striatal subregions and the frontal cortex on both the denervated and intact sides. The potentiation of the behavioural response to apomorphine by chronic ‘intermittent’ levodopa treatment does not correspond with the levodopa induced alterations in striatal or extrastriatal DA receptors. In the same group of animals the narrowing of the duration of response to the lower dose of apomorphine may mimic the fluctuations in response to levodopa, seen clinically in long-term levodopa treated parkinsonian patients.     

Dopamine D-2 receptors in rat caudate-putamen: the lateral to medial gradient does not correspond to dopaminergic innervation

The topography of dopamine D-2 receptors within the rat caudate-putamen (CPU) was characterized by measuring [³H]spiroperidol binding to membranes from dissected regions of the CPU and to thin sections of rat forebrain using quantitative autoradiography. Specific binding, defined using 1 μM (+)-butaclamol, was considerably higher in the lateral CPU than the medial CPU, without any obvious gradient in the dorsoventral axis. The difference in B_max between the lateral and medial CPU was similar whether determined by digital subtraction autoradiography of coronal forebrain sections or by [³H]spiroperidol binding to membrane homogenates derived from CPU subregions. No regional differences were observed in the affinity of [³H]spiroperidol for D-2 sites. There were no lateral-to-medial differences in the dopaminergic innervation of the CPU, as determined by the measurement of the levels of dopamine, homovanillic acid and dihydroxyphenylacetic acid in subregions of the CPU using high pressure liquid chromatography. Thus, regional variations in the density of D-2 receptors within the CPU do not correspond to the degree of dopaminergic innervation. The functional importance of the organization of this dopamine receptor system is discussed in relation to the lateral-to-medial organization of the CPU.