Effects of NaCl and sultopride on striatal [H]spiperone binding in neonatal, adult and senescent rats

Effects of NaCl, (+)-and (−)-sultopride on striatal [³H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [³H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [³H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [³H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (−)-sultopride for [³H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (−)-sultopride at every stage. It is suggested that stabilizing effect of Na⁺ on dopamine (DA) receptor complexes and increasing effect of Na⁺ on binding affinity of benzamide to DA₂ receptors keep functions through development and aging.     

Dopamine receptors in the anterior lobe of the human pituitary gland: autoradiographic localization

We examined the distribution of dopamine and serotonin receptors in human pituitary gland by light microscopic autoradiography using [³H]spiperone as a binding ligand. Clusters of specific binding sites for [³H]spiperone were observed in the anterior pituitary. This clustering is similar to the clustering of lactotrophs observed in immunohistochemical studies. No specific binding sites for [³H]spiperone were present in the posterior lobe of the pituitary gland. We found no evidence for the presence of serotonin-2 receptors (i.e. cinanserin-displaceable [³H]spiperone binding) in human pituitary.     

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.     

Brain dopamine transporter: gender differences and effect of chronic haloperidol

Gender differences and the effect of chronic haloperidol on the rat brain dopamine transporter is reported. The density of striatal dopamine transporter sites labelled with [³H]GBR 12935, and of substantia nigra dopamine transporter mRNA measured by in situ hybridization were higher in female compared to male rats whereas striatal D2 specific binding labelled with [³H]spiperone was not significantly higher. Daily haloperidol treatment (1 mg/kg, i.p.) for 21 days increased striatal [³H]spiperone specific binding but left unchanged striatal [³H]GBR 12935 binding density and affinity as well as substantia nigra dopamine transporter mRNA levels. A reduce clearance rate of dopamine in the striatum after acute and chronic haloperidol was previously reported; the present results indicate that this may occur without changes in the sites of dopamine transport or in gene expression of this transporter.     

Effects of NaCl and sultopride on striatal [3H]spiperone binding in neonatal,adult and senescent rats

Effects of NaCl, (+)-and (?)-sultopride on striatal [³H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [³H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [³H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [³H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (?)-sultopride for [³H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (?)-sultopride at every stage. It is suggested that stabilizing effect of Na⁺ on dopamine (DA) receptor complexes and increasing effect of Na⁺ on binding affinity of benzamide to DA₂ receptors keep functions through development and aging.     

Autoradiographic analysis of regional alterations in brain receptors following chronic administration and withdrawal of typical and atypical neuroleptics in rats

Summary Rats were administered haloperidol, clozapine, raclopride, or no drug for 28 days or 8 months. Following a 3 week withdrawal period, in vitro autoradiography was utilized to examine receptor binding for dopamine D2 ([³H]spiperone and [³H]raclopride), dopamine D1 ([³H]SCH23390), GABA_A ([³H]muscimol), benzodiazepine ([³H]RO15-1788), and muscarinic ACh receptors ([³H]QNB). [³H]spiperone was elevated in striatal subregions only in haloperidol-treated rats, with the largest increases seen in the 8 month duration animals. Striatal [³H]raclopride binding was increased after both short- and long-term treatment in both haloperidol and raclopride, but not clozapinetreated animals. Clozapine-treated rats showed significant increases in [³H]SCH23390 in the nucleus accumbens after 28-day administration; otherwise no changes were seen for this ligand in any other groups. Increases in [³H]muscimol binding in the substantia nigra reticulata were seen in haloperidol-treated rats after 8 month treatment. Binding of [³H]QNB and [³H]RO15-1788 were not significantly different from control for any of the drug-treated groups. These data suggest that persisting alterations in receptor binding are primarily seen in dopamine D2 and GABA receptors after withdrawal from chronic administration of haloperidol but not the atypical neuroleptics, clozapine and raclopride.     

Localization of [H]N-propylnorapomorphine binding in mouse striatum

The anatomic localization of specific striatal [³H]N-propylnorapomorphine ([³H]PNA) binding was determined in male C57BL/6J mice. Striatal [³H]PNA binding was of high affinity and sensitive to guanine nucleotides. Frontal cortical ablation did not alter striatal [³H]PNA binding, but reduced [³H]spiperone binding by 36%. Kainic acid reduced and 6-hydroxydopamine elevated [³H]PNA binding. A combined frontal cortical ablation and striatal kainic acid lesion was similar to that of kainate alone. These data are consistent with a localization of [³H]PNA binding sites on neurons intrinsic to the mouse striatum.     

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.     

Long-term treatment with haloperidol or clozapine does not affect dopamine D4 receptors in rat frontal cortex

Summary We examined the effects of long-term treatment with haloperidol and clozapine on dopamine D₄ receptors in rat frontal cortex. Dopamine D₄ receptor binding sites were indirectly determined from the displacement experiments of [³H]clozapine binding using nemonapride. Three-weeks administration of haloperidol (0.5mg/kg) or clozapine (10mg/kg) did not significantly affect the D₄ receptors in the frontal cortex. The density of D₂ receptors, determined by [³H]spiperone binding to striatum, was increased by long-term treatment with haloperidol, but it was not significantly changed by that with clozapine.     

Age-Related Changes in the N-Methyl- -aspartate Receptor Binding Sites within the Human Basal Ganglia

The present study examined the regional differences in dopamine transporter binding sites and NMDA receptor complex binding based on autoradiographic images obtained in postmortem sections of human normal brain tissues. In middle-aged control tissues, high and comparable levels of [³H]CFT binding were observed in the caudate nucleus, putamen, and accumbens nucleus without significant alteration along the rostrocaudal axis and ventral and dorsal parts of these nuclei. In aging normal brain tissues, dopamine binding sites for [³H]CFT were significantly reduced in the caudate nucleus, putamen, and accumbens nucleus. -[³H]Glutamate, [³H]MK-801, and [³H]glycine binding to the NMDA receptor complex was lower in aging brain tissues than in middle-aged controls. Significant correlation did occur between age and [³H]CFT binding and between age and -[³H]glutamate, [³H]MK-801, and [³H]glycine binding sites. These results demonstrate that the basal ganglia have age-associated reductions in dopamine transporter uptake and NMDA receptors. These data support hypoactive activity of the NMDA receptor complex system with advancing age. The dopamine transporter uptake and NMDA receptors appear to be vulnerable to the aging process in the basal ganglia.     

Dopamine receptor changes following destruction of the nigrostriatal pathway: Lack of a relationship to rotational behavior

Phenomena consistent with postsynaptic supersensitivity developed in the rat neostriatum following the destruction of dopaminergic afferent neurons. A gradual in-increase in the densoty of binding sites for [³H]spiperone occurred over a 2–3 week period. This increase was apparent only after the almost complete loss of dopamine-containing nerve terminals as measured by the depletion of endogenous dopamine. The properties of the receptor labeled by [³H]spiperone were not altered by denervation. Elimination of dopamine-containing nerve endings in the neostriatum was accompanied by the gradual development of an increase in dopamine-sensitive adenylate cyclase activity in homogenates of the caudate ipsilateral to the lesion as compared to the contralateral side. The administration of apomorphine led to pronounced circling behavior. This effect occurred rapidly and was maximal within 3 days following destruction of dopaminergic neurons. The increase in the density of dopamine receptors and in a receptor-mediated function may partially account for the development of enhanced electrophysiological responses to dopamine agonists in the neostriatum. However, the results do not explain the drug-induced rotational behavior which develops after destruction of the dopamine-containing nigrostriatal pathway. This behavioral phenomenon clearly preceded the appearance of receptor alterations in the corpus striatum.     

Light-dependent regulation of dopamine receptors in mammalian retina

The specific binding of [3H]spiperone, a D-2 dopamine receptor ligand, in in retinas from rabbits kept one week in constant light was significantly lower than in retinas from rabbits exposed to constant dark. Constant light did not alter the binding of [3H]spiperone in the striatum, where melatonin does not inhibit dopamine release. The decrease in [3H]spiperone binding induced by constant light in retina appears to be associated with the activation of inhibitory melatonin receptors on dopaminergic neurons. In support of this hypothesis, treatments that elevate melatonin concentrations, such as dark or melatonin administration, reversed the light-induced down-regulation of D-2 dopamine binding sites in retina. It is concluded that the decrease in melatonin levels in constant light disinhibits the dopamine-containing retinal neurons in vivo leading to elevated dopamine release and subsequent D-2 dopamine receptor down-regulation.     

[H]Nemonapride Binding in Human Caudate and Putamen

The binding of [³H]nemonapride to human postmortem caudate and putamen tissue was autoradiographically investigated using several antipsychotic drugs. Saturation experiments revealed a single population of binding sites (dissociation constant (K_D) 0.38 ± 0.01 nM, and total binding capacity (B_MAX) 55 fmol/TE). Prototypic dopamine (DA) receptors antagonists displaced [³H]nemonapride in a monophasic manner. The order of displacement potency was expected for DA D₂-like receptors: spiperone > (+)butaclamol ≥ chlorpromazine > (−)sulpiride > ketanserin. Displacement with serotonergic antagonists suggests that in human caudate and putamen tissue [³H]nemonapride may have a very low affinity serotonergic component. However, [³H]nemonapride displays a high affinity and selectivity for DA D₂-like receptors and should make it a preferred compound for tritium-based autoradiography.     

Two distinct serotonin receptors: regional variations in receptor binding in mammalian brain

Two distinct serotonin receptors in mammalian brain are labeled respectively with [³H]serotonin (5-HT₁) and [³H]spiperone (5-HT₂). In general, agonists display highest affinities for 5-HT₁ while antagonists prefer 5-HT₂ sites. To conduct regional studies of 5-HT receptors, we estimated 5-HT₂ sites with [³H]spiperone, using the 5-HT₂ specific antagonist cinanserin to displace binding to 5-HT₂ but not dopamine receptors and sulpiride to displace [³H]spiperone from dopamine but not 5-HT receptors. About 15% of cerebral cortical [³H]spiperone binding appears to involve dopamine sites and the remainder involves 5-HT₂ receptors. In the corpus striatum about 80% of [³H]spiperone binding labels dopamine receptors and the rest involves 5-HT₂ sites. [³H]mianserin binds about equally to 5-HT₂ sites are studies selectively by displacing histamine H₁-receptor binding with the H₁-antihistamine triprolidine. [³H]LSD labels both 5-HT₁ and 5-HT₂ receptors. Its binding to 5-HT₁ sites is displaced selectively with 5-HT, while its binding to 5-HT₂ receptors is displaced with cinanserin. [³H]5-HT labels only 5-HT₁ receptors. The regional distribution of the two 5-HT receptors is similar in rat, guinea pig and bovine brain. However, the regional patterns of 5-HT₁ and 5-HT₂ receptors differ considerably in all 3 species. The hippocampus is quite high in 5-HT₁ receptors but low in 5-HT₂ sites. The cerebellum contains the lowest levels of both 5-HT₁ and 5-HT₂ receptors. In bovine brain, most areas contain similar numbers of 5-HT₁ and 5-HT₂ receptors. However, the substantia nigra, the richest 5-HT₁ area inbovine brain, possesses 10 times more 5-HT₁ than 5-HT₂ sites.     

GABAergic and dopaminergic transmission in the brain of Roman high-avoidance and Roman low-avoidance rats

The GABAergic and dopaminergic pathways in the central nervous system (CNS) play a pivotal role in the control of emotions and in the adaptive responses to stressful stimuli. The present study was aimed at characterizing a range of biochemical markers of GABA- and dopamine-mediated neurotransmission in the CNS of Roman high-avoidance (RHA/Verh) and Roman low-avoidance (RLA/Verh) rats, two psychogenetically selected lines that differ in their level of emotionality. The stimulatory effect of GABA on ³⁶Cl⁻ uptake was less pronounced in the cerebral cortex of RLA/Verh rats as compared to RHA/Verh rats, whereas no line-related changes were detected in [³H]GABA and [³H]flunitrazepam binding. On the other hand, the density of D₁ dopamine receptors labeled with [³H]SCH 23390 was lower in the nucleus accumbens of RLA/Verh rats as compared to their RHA/Verh counterparts, whilst no line-dependent changes were observed in the binding parameters of D₁ dopamine receptors in the striatum, amygdala, and prefrontal cortex. These biochemical differences may contribute to the distinct emotionality and responsiveness to the effects of psychoactive drugs of RHA/Verh and RLA/Verh rats.     

Characterization of specific binding sites labeled by [H]LSD in coronal sections of paraformaldehyde-fixed rat brain

Specific, high-affinity binding of [³H]lysergic acid diethylamide (LSD) in coronal sections of paraformaldehyde-fixed rat brain is described. Intracardiac perfusion of paraformaldehyde selectively altered 40% of the total binding sites normally labeled by [³H]LSD in unfixed sections. Competition by unlabeled LSD and serotonin (5-HT) was not altered by the fixation procedure. Competition by spiperone, however, revealed that the fixation procedure preferentially altered sites for which spiperone has high affinity. This technique should facilitate the combination of neuroanatomical techniques such as radioautography and immunocytochemistry.     

Evidence for loss of brain [H]spiroperidol and [H]ADTN binding sites in rabbit brain with aging

[³H]Spiroperidol and [³H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [³H]spiroperidol binding sites and a 140% higher number of [³H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [³H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [³H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [³H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.     

The effect of chronic treatment with a novel aryl-piperazine antipsychotic on monoamine receptors in rat brain

The effects of chronic treatment of rats with RWJ 3776, a novel aryl-piperazine containing antipsychotic drug, on brain monoamine receptors were studied. Rats were treated daily with RWJ 37796 (1.3 mg/kg), the typical antipsychotic haloperidol (1 mg/kg) or vehicle (control) for 21 days, and were sacrificed 3 days after the last injection. Binding of [³H]Sch-23390 and [³H]spiperone to D₁ and D₂ dopamine receptors, respectively, and [³H]8-hydroxy-2-(di-n-propylamino)-tetralin ([³H]8OH-DPAT) to 5-HT_1A receptors were measured in various brain regions using quantitative autoradiography. Binding to D₂ dopamine receptors was significantly elevated in the caudate-putamen of rats treated with haloperidol or RWJ 37796 as compared to controls. However, the magnitude of the increase in D₂ binding was significantly greater in haloperidol-treated (+ 38%) compared to RWJ 37796-treated (+ 21%) rats. Haloperidol treatment also increased binding (+ 35%) to D₂ dopamine receptors in the nucleus accumbens, where RWJ 37796 treatment had a considerably smaller effect (+ 12). No changes in D₁ dopamine or 5-HT_1A receptor binding were detected following either antipsychotic treatment in any brain regions studied. Thus, at comparable doses, the novel antipsychotic RWJ 37796 produces less up-regulation of D₂ dopamine receptor binding in the striatum than does the typical antipsychotic haloperidol.     

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.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [³H]N-n-propylnorapomorphine ([³H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [³H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (K_d = 0.27–0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [³H]NPA binding sites possess the pharmacological features of the dopamine D₂ receptor. The highest levels of [³H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the ³H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anteior frontal cortex. Thorough examination of the subregional distribution of [³H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogenous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [³H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [³H]NPA binding were observed in the putamen. The present data indicate that [³H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D₂ receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.