[3H]GBR 12935 Binding to Dopamine Uptake Sites in the Human Brain

Binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine ([3H]GBR 12935) was studied in membrane preparations of several human brain regions. In putamen, the substituted piperazine derivates cis- and trans-flupenthixol displaced 90% of the total [3H]GBR 12935 binding. Computer-assisted analysis of the competition curves revealed a high-affinity site (30%; KiH = 54 nM) and a low-affinity site (60%; KiL = 4.5 microM). The dopamine uptake blockers mazindol and nomifensine only displaced 30% of the total [3H]GBR 12935 binding in a monophasic way. Binding of [3H]GBR 12935 to the dopamine uptake sites, i.e., that displaced by dopamine uptake blockers, corresponded to part of the binding having low affinity for flupenthixol and was only detected in putamen, nucleus caudatus, nucleus accumbens, and substantia nigra. Even after masking the high-affinity binding site for flupenthixol by including 1 microM cis-flupenthixol in the binding assays, no dopamine uptake sites could be detected in globus pallidus, amygdala, thalamus, hippocampus, and cerebral cortex. Binding of [3H]GBR 12935 to dopamine uptake sites was lost in the nucleus caudatus ipsilateral to ventral midbrain infarctions, confirming their location on nigrostriatal nerve endings. Gross unilateral lesions of the striato- and pallidonigral pathways did not affect the number of dopamine uptake sites in the ipsilateral substantia nigra, suggesting that they may reside on the soma or dendrites of nigral neurons.     

Imaging the Dopamine Uptake Site with Ex Vivo [18F]GBR 13119 Binding Autoradiography in Rat Brain

We studied the binding of [18F]GBR 13119 (1-[[(4-[18F]fluorophenyl) (phenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine) to rat brain with autoradiography after intravenous injection. The rank order of binding was dorsal striatum greater than nucleus accumbens = olfactory tubercle greater than substantia nigra = ventral tegmental area greater than other areas. Binding was blocked by prior injection of dopamine uptake blockers but not by injection of dopamine receptor antagonists or drugs that bind to the dialkylpiperazine site. Unilateral 6-hydroxy-dopamine lesions of dopamine neurons caused a marked decrease in striatal and nigral binding on the side of the lesion. We conclude that intravenous injection of [18F]GBR 13119 provides a useful marker of presynaptic dopamine uptake sites.     

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Active uptake of 3,4-dihydroxyphenylethylamine (dopamine) is sodium- and temperature-dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+-dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low- and high-affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.     

Differences in Dopamine Clearance and Diffusion in Rat Striatum and Nucleus Accumbens Following Systemic Cocaine Administration

Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCl (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopamine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine.     

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Abstract: Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17β-estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17β-estradiol (10 μg/0.2 ml), progesterone (0.72 mg/0.2 ml). 17β-estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [³H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 ± 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16-23%. Chronic treatment of ovariectomized rats with 17β-estradiol progesterone, or their combination increased to the same extent and uniformly [³H]-GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17β-estradiol and/ or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.     

[3H] GBR-12935 Binding to Human Cerebral Cortex Is Not to Dopamine Uptake Sites

Abstract: The binding of the dopamine uptake inhibitor [³H] GBR-12935 to 16 regions of the human brain was investigated in competition experiments with increasing concentrations of GBR-12909, mazindol, and dopamine. The methodology used included a relatively high tissue concentration (8 mg/ml) and addition of 5 m M KCI in the assay buffer. GBR-12909 inhibited 80–90% of the binding in most regions, whereas dopamine only inhibited the binding in the striatum. Mazindol inhibited only part of the cortical binding at concentrations of >1 μ M , whereas the inhibition in the caudate and the putamen also contained a high-affinity component representing the dopamine uptake site. It is concluded that the [³H] GBR-12935 binding sensitive to GBR-12909 cannot be regarded as specific binding to the dopamine uptake site because the displaceable binding most likely is not related to the dopamine uptake site.     

Correlation between (3H)dopamine specific uptake and (3H)GBR 12783 specific binding during the maturation of rat striatum

The development of the specific uptake of dopamine in the rat striatum during the early postnatal period is compared with the ontogenetic changes of the specific binding of (3H)GBR 12783 to the site of uptake inhibition. During maturation, the increase in the specific binding of (3H)GBR 12783 parallels the increase in the specific uptake of dopamine. (3H)GBR 12783 specific binding sites increase in number from day 1 postpartum until 40 days, when they reach the adult level. In 40 day-old rats, the weight of the striatum represents 80% of adult values. The affinity of (3H)GBR 12783 for the inhibition site is similar in membrane preparations obtained from 6 day-old pups and adults; this results in a same ability of the inhibitor to block the specific uptake of dopamine into synaptosomes obtained from pups or adult rats. These data support the hypothesis of the existence of a single molecular entity including both the inhibition site and the carrier itself.     

Autoradiographic localization of a non-reducible somatostatin analog (125I-CGP 23996) binding sites in the rat brain: comparison with membrane binding

The regional distribution of somatostatin binding sites in the rat brain was determined by quantitative autoradiography, using 125I-CGP 23996, a non-reducible somatostatin analog. In preliminary experiments, kinetic properties of 125I-CGP 23996 binding to rat brain membranes and slide mounted frozen brain sections were compared and found similar. In addition, distribution of 125I-CGP 23996 and 125I-N-Tyr-SRIF14 binding sites on membrane prepared from 10 different rat brain structures were closely correlated (r = 0.91, 2 p less than 0.01), indicating that the non-reducible analog recognizes the same binding site as the Tyr-extended native peptide. Highest levels of 125I-CGP 23996 binding sites were found in anterior temporal, frontal and cingular cortex as well as hippocampus. Moderate levels were found in the remaining part of the limbic system including amygdala, olfactory tubercles and bed nucleus of the stria terminalis. In the brain stem, nuclei involved in the auditory system such as the ventral cochlear nucleus and the superior olive nucleus, contained high levels of 125I-CGP 23996 binding sites. The distribution of 125I-CGP 23996 binding sites roughly correlated with that of the endogenous peptide in most structures, except in the mediobasal hypothalamus.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Ligand binding to dopamine-receptors: Analysis & interpretation

Dopamine receptor interaction was studied by concentration dependent inhibition of ³H-Spiperone binding to calf caudate nucleus homogenates with (+)-butaclamol, haloperidol, ergometrine, apomorphine and dopamine. The results were analyzed in terms of a one and a two site receptor model using computerized curve fitting procedures. Evidence is given in favour of a two site receptor model on basis of statistical criteria. The presence of two independent non-interconverting receptor sites for dopamine in calf caudate nucleus is consistent with binding data from others and with in vivo experiments.     

[3H]2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN)

The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [³H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursorl-3,4-dihydroxyphenylalanine (l-dopa). Chronic administration of haloperidol orl-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [³H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P₃. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.     

Differential Interaction of Phencyclidine-Like Drugs with the Dopamine Uptake Complex In Vivo

The phencyclidine (PCP) derivative, [3H]N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine ([3H]BTCP), was used to label in vivo the dopamine uptake complex in mouse brain. The striatum accumulated the highest level of total and specific binding. Drugs which bind to the dopamine uptake site inhibited [3H]BTCP binding on an order similar to their in vitro affinities for the high-affinity [3H]BTCP site. Drugs which label selectively other monoamine uptake complexes. PCP, or sigma recognition sites were ineffective at doses up to 40 mg/kg. PCP bound to and dissociated from the dopamine uptake complex very rapidly. N-[1-(2-Thienyl)cyclohexyl]pideridine (TCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) had no effect at any time or at any dose. These results imply that the pharmacological effects of PCP are due to its simultaneous interaction with the dopamine uptake complex and the PCP receptor. Conversely, TCP and MK-801, which have the same behavioral properties as PCP, exert their action only through the interaction with the PCP receptor.     

Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging

Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p = 0.0001) in the Bmax of mazindol binding and a 23% decrease (p = 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p = 0.0156) but not young rats (p = 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.     

Syntheses of novel diphenyl piperazine derivatives and their activities as inhibitors of dopamine uptake in the central nervous system

A new series of diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, which were modified at sites between the diphenyl and piperazine moieties, was prepared and evaluated for dopamine transporter binding affinity with [(3)H]GBR12935 in rat striatal membranes. These synthesized compounds showed apparent dopamine transporter binding affinities (IC(50)<30 nM) and some of them were approximately equivalent in activity to GBR12909 known as a potent dopamine uptake inhibitor, showing the activities with IC(50) values of nanomolar range. Among them, 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 2 was evaluated for extracellular dopamine levels in rat striatum using in vivo brain microdialysis. The intraperitoneal administration of 2 (0.01, 0.03, or 0.1 mmol/kg) induced dose-dependent increases of dopamine levels in rat striatal dialysates. The maximum increases in dopamine levels induced by 2 were greater than those by GBR12909. The pharmacological data of these novel diphenyl piperazine derivatives show that the compounds have potent dopamine uptake inhibitory activities in the central nervous system.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

The regional distribution of dopamine and serotonin uptake and transmitter concentrations in the human brain

The regional distribution of the dopamine and serotonin uptake sites in human brain have been assessed and compared with the distribution of the transmitters and their metabolites measured in the same brains and also with a limited regional distribution of the uptake sites in rat and sheep brain. The affinity of the uptake sites for both transmitters was determined and found to be c. 0.2 μ M in all 3 species. Most dopamine uptake in all species was in caudate and putamen samples. Many regions of the human brain showed no dopamine uptake and little dopamine uptake was seen in sheep cortex or nigral preparations. Dopamine and metabolite concentrations were highest in the caudate, putamen and substantia nigra. Most serotonin uptake was seen in the hypothalamus in all 3 species; less was observed in the striatal regions; the cortical and nigral preparations of sheep brain showed little serotonin uptake though cortical preparations of rat brain had high levels of uptake. In the human brain, other regions did not show serotonin uptake. Highest concentrations of serotonin were found in the substantia nigra and medulla, intermediate concentrations in the putamen, globus pallidus, hypothalamus, olfactory tubercle and thalamus; very low concentrations of serotonin were found in other regions. The use of the human uptake site for pharmacological studies and as a marker for monoaminergic afferents in human health and disease is discussed.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).     

D(3) receptor ligands modulate extracellular dopamine clearance in the nucleus accumbens

An involvement of the D(3) dopamine receptor in the regulation of extracellular dopamine has been suggested. However, the mechanisms mediating this effect are unclear. We have used the technique of no net flux microdialysis under transient conditions to examine the influence of the D(3) -preferring agonist (+)-PD128907 upon extracellular dopamine levels in the nucleus accumbens of the mouse. (+)-PD 128907 (0.1 mg/kg intraperitoneally) significantly decreased extracellular dopamine. This decrease was associated with a marked increase in the extraction fraction, which suggests an increase in dopamine clearance. The ability of D(3) -preferring compounds to modulate dopamine uptake was investigated in vitro using rotating disk electrode voltammetry. (+)-PD 128907 (10 nm) significantly increased the initial clearance rate of 3 microm dopamine in rat nucleus accumbens tissue suspensions. Kinetic analysis revealed no change in the apparent K (m) of uptake but it showed a 33% increase in V (max). In contrast, the D(3) antagonist GR 103691 (10 nm) significantly decreased dopamine uptake. Consistent with the low levels of D(3) receptors in the dorsal striatum, neither compound affected uptake in tissue suspensions from this brain region. These data indicate that D(3) receptor activation increases dopamine uptake in the nucleus accumbens and suggest that this receptor subtype can regulate extracellular dopamine by modulating the DA transporter activity.