Dopamine Transporter Radioligand Research Articles

Differential effects of scopolamine on in vivo binding of dopamine transporter and vesicular monoamine transporter radioligands in rat brain

The in vivo equilibrium specific binding of d-threo-[ 3H]methylphenidate, a radioligand for the dopamine transporter (DAT), and (+)-α-[ 3H]dihydrotetrabenazine, a radioligand for the vesicular monoamine transporter (VMAT2), were examined in rat brain with and without prior administration of 5 mg/kg scopolamine. Drug-treated animals exhibited a 30% increase in d-threo-[ 3H]methylphenidate binding to the DAT in the striatum relative to controls. No changes in specific binding of (+)-α-[ 3H]dihydrotetrabenazine were observed in any brain region following scopolamine pretreatment. Cholinergic drugs thus differentially affect in vivo specific binding of DAT and VMAT2 radioligands, suggesting this should be a consideration in selection of in vivo markers for imaging studies of dopaminergic terminals in the brain of animals and humans.

Phosphatidylinositol 3-Kinase, Protein Kinase C, and MEK1/2 Kinase Regulation of Dopamine Transporters (DAT) Require N-terminal DAT Phosphoacceptor Sites

The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca2+/calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake Vmax. PKC principally modulates Vmax. Neither p38 nor Ca2+/calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.

Cocaine induction of dopamine transporter trafficking to the plasma membrane.

Several previous human postmortem experiments have detected an increase in striatal [(3)H]WIN 35428 binding to the dopamine transporter (DAT) in chronic cocaine users. However, animal experiments have found considerable variability in DAT radioligand binding levels in brain after cocaine administration, perhaps caused by length and dose of treatment and type of radioligand used. The present experiments tested the hypothesis that [(3)H]WIN 35428 binding and [(3)H]dopamine uptake would be increased by exposure to cocaine through alterations in DAT cellular trafficking, rather than increased protein synthesis. Experiments were conducted in stably hDAT-transfected N2A cells and assessed the dose response and time course of cocaine effects on [(3)H]WIN 35428 binding to the DAT, [(3)H]dopamine uptake, measures of DAT protein and mRNA, as well as DAT subcellular location. Cocaine doses of 10(-6) M caused statistically significant increases in [(3)H]WIN 35428 binding and [(3)H]dopamine uptake after 12 and 3 h, respectively. Despite these increases in DAT function, there was no change in DAT total protein or mRNA. Immunofluorescence and biotinylation experiments indicated that cocaine treatment induced increases in plasma membrane DAT immunoreactivity and intracellular decreases. The present model system may further our understanding of regulatory alterations in DAT radioligand binding and function caused by cocaine exposure.

Loss of Dopamine Transporters in Methamphetamine Abusers Recovers with Protracted Abstinence

Methamphetamine is a popular drug of abuse that is neurotoxic to dopamine (DA) terminals when administered to laboratory animals. Studies in methamphetamine abusers have also documented significant loss of DA transporters (used as markers of the DA terminal) that are associated with slower motor function and decreased memory. The extent to which the loss of DA transporters predisposes methamphetamine abusers to neurodegenerative disorders such as Parkinsonism is unclear and may depend in part on the degree of recovery. Here we assessed the effects of protracted abstinence on the loss of DA transporters in striatum, in methamphetamine abusers using positron emission tomography and [(11)C]d-threo-methylphenidate (DA transporter radioligand). Brain DA transporters in five methamphetamine abusers evaluated during short abstinence (<6 months) and then retested during protracted abstinence (12-17 months) showed significant increases with protracted abstinence (caudate, +19%; putamen, +16%). Although performance in some of the tests for which we observed an association with DA transporters showed some improvement, this effect was not significant. The DA transporter increases with abstinence could indicate that methamphetamine-induced DA transporter loss reflects temporary adaptive changes (i.e., downregulation), that the loss reflects DA terminal damage but that terminals can recover, or that remaining viable terminals increase synaptic arborization. Because neuropsychological tests did not improve to the same extent, this suggests that the increase of the DA transporters was not sufficient for complete function recovery. These findings have treatment implications because they suggest that protracted abstinence may reverse some of methamphetamine-induced alterations in brain DA terminals.

3H]MFZ 2-12: A Novel Radioligand for the Dopamine Transporter

In an effort to develop a tritiated dopamine transporter radioligand with higher affinity than the widely used [ 3H]WIN 35,428, we have synthesized [ 3H]2β-carbomethoxy-3β-(3′,4′-dichlorophenyl)tropane ([ 3H]MFZ 2-12). Unlabeled MFZ 2-12 and the N-demethylated intermediate (MFZ 2-13) inhibited dopamine uptake by the human dopamine transporter with IC 50's of 1.1 and 1.4 nM, respectively. The N-nor-intermediate (MFZ 2-13) was treated with CT 3I resulting in [ 3H]MFZ 2-12; S.A.=80 Ci/mmol). [ 3H]MFZ 2-12 reversibly bound with a K D of 2.8 nM to human dopamine transporter expressed heterologously in EM4 cells.

Changes in dopamine availability in the nigrostriatal and mesocortical dopaminergic systems by gait in Parkinson's disease

The basal ganglia play a role in controlling movement during gait. The aim of the present study was to investigate changes in dopamine transporter (DAT) availability in the striatum and extrastriatal region in association with walking exercise in six normal subjects and seven age-matched unmedicated patients with Parkinson's disease. This was done by comparing DAT radioligand uptake in the dopaminergic projection areas after gait with that under the resting condition using a DAT probe, 11C-labelled 2-beta-carbomethoxy-3beta-(4-fluorophenyl) tropane ([11C]CFT) and PET. Physiological parameters were stable during and after gait in both groups. The regions of interest method for measuring differences in [11C]CFT uptake level and voxel-based statistical parametric mapping (SPM96) showed that [11C]CFT uptake in the striatum (specifically the putamen) was decreased by gait to a greater extent in normal subjects, whereas a significant reduction in [11C]CFT uptake was not found in the putamen but in the caudate and orbitofrontal cortex in Parkinson's disease patients. These results are the first in vivo evidence that DAT availability is reduced in the nigrostriatal projection area by basic human behaviour, i.e. gait. Alterations in this availability in Parkinson's disease suggested that shifted activation in the medial striatum and the mesocortical dopaminergic system might reflect the pathophysiology of parkinsonian gait.

Synthesis and preliminary characterization of a high‐affinity novel radioligand for the dopamine transporter

Synthesis and preliminary characterization of a high‐affinity novel radioligand for the dopamine transporter

Pallidal border cells: an anatomical and electrophysiological study in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkey.

A dopamine transporter-radioligand binding study demonstrated a dopaminergic innervation around the pallidal complex in the normal monkey (n=5), i.e. where a subpopulation of pallidal neurons known as "border cells" is classically identified. Surprisingly, this peripallidal binding persists in monkeys rendered parkinsonian (n=5) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The border cell electrophysiological activity was then analysed in normal and parkinsonian monkeys (n=2), either in the untreated state or following administration of levodopa. Pallidal border cell firing frequency was significantly decreased after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (8.9+/-0.7 vs 31.4+/-1.6Hz, P<0.05). This decrease was partly corrected by levodopa administration (19.2+/-1.0Hz, P<0.05 vs both normal and parkinsonian situations). The peripallidal dopaminergic innervation suggests that pallidal border cells are under a direct dopaminergic control, arising from the ventral tegmental area and/or the basal forebrain magnocellular complex, the role of which remains unknown. Moreover, the relative sparing of these dopaminergic fibers in parkinsonian monkeys suggests that they would exhibit specific adaptive properties totally different from those described in the nigrostriatal pathway.

Effects of route of administration on cocaine induced dopamine transporter blockade in the human brain

The route of administration influences the reinforcing effects of cocaine. Here we assessed whether there were differences in the efficacy of cocaine to block the dopamine transporters (major target for cocaine's reinforcing effects), as a function of route of administration. Positron emission tomography and [ 11C]cocaine, a dopamine transporter radioligand, were used to compare the levels of dopamine transporter blockade induced by intravenous, smoked and intranasal cocaine in 32 current cocaine abusers. In parallel, the temporal course for the self-reports of “high” were obtained. Cocaine significantly blocked dopamine transporters. The levels of blockade were comparable across all routes of administration and a dose effect was observed for intravenous and intranasal cocaine but not for smoked cocaine. For equivalent levels of cocaine in plasma and DAT blockade, smoked cocaine induced significantly greater self reports of “high” than intranasal cocaine and showed a trend for a greater effect than intravenous cocaine. The time to reach peak subjective was significantly faster for smoked (1.4 ± 0.5 min) than for intravenous cocaine (3.1 ± 0.9 min), which was faster than intranasal cocaine (14.6 ± 8 min). Differences in the reinforcing effects of cocaine as a function of the route of administration are not due to differences in the efficacy of cocaine to block the dopamine transporters. The faster time course for the subjective effects for smoked than intravenous and for intravenous than for intranasal cocaine highlights the importance of the speed of cocaine's delivery into the brain on its reinforcing effects.

Regulation of Rat Dopamine Transporter mRNA and Protein by Chronic Cocaine Administration

This study describes a direct comparison of dopamine transporter (DAT) mRNA and protein, as well as its binding sites, in tissue from the same animals after chronic cocaine administration. Rats were treated twice daily with 25 mg/kg cocaine or with saline. After 8 days of cocaine administration, changes in DAT mRNA levels in the substantia nigra pars compacta and ventral tegmental area were measured by in situ hybridization, and DAT protein in the striatum was quantified by immunoblotting. Whereas chronic cocaine treatment significantly reduced levels of DAT mRNA in the substantia nigra pars compacta and ventral tegmental area as compared with vehicle-treated controls, cocaine treatment did not alter DAT protein levels in the striatum. Furthermore, the density of DAT binding sites was also measured in the striatum by quantitative autoradiography using two DAT radioligands, 33-(4-[125I]iodophenyl)tropane-2-carboxylic acid methyl ester ([125I]RTI-55) and [3H]propanoyl-3beta-(4-tolyl)tropane ([3H]PTT). Similar to the results of immunoblotting of DAT protein, [1251]RTI-55 and [3H]PTT binding site levels also remained unaltered. These results indicate a dissociation in the regulation of DAT mRNA and its protein levels as a result of cocaine administration in rats. This study also indicates that the DAT ligands [3H]PTT and [125I]RTI-55 provide an accurate assessment of DAT protein levels.

Improved syntheses of the PET radioligands, [11C]FLB 457, [11C]MDL 100907 and [11C]β-CIT-FE, by the use of [11C]methyl triflate

The highly reactive labelling agent, [11C]methyl triflate, was used to synthesise three recently developed PET radioligands. The labelling reactions were 11C-methylations of phenols for the preparations of the D2 receptor radioligand, [11C]FLB 457, and the 5-HT2A receptor radioligand, [11C]MDL 100907, and 11C-methylation of a carboxylic acid for the preparation of the dopamine transporter radioligand, [11C]β-CIT-FE. The synthesis of the 5-HT1A receptor radioligand, [O-methyl-11C]WAY-100635, was used to establish general reaction conditions for the methylation of phenols with [11C]methyl triflate. Compared to the previous use of [11C]methyl iodide in these radiosyntheses, [11C]methyl triflate demanded less precursor, allowed faster reactions and improved radiochemical yields. Normal-phase HPLC was used to speed up product purifications (except for [11C]β-CIT-FE). Hence, preparation times were shorter, resulting in radioligands with higher specific radioactivity. © 1998 John Wiley & Sons, Ltd.

Improved syntheses of the PET radioligands, [11C]FLB 457, [11C]MDL 100907 and [11C]β-CIT-FE, by the use of [11C]methyl triflate

The highly reactive labelling agent, [11C]methyl triflate, was used to synthesise three recently developed PET radioligands. The labelling reactions were 11C-methylations of phenols for the preparations of the D2 receptor radioligand, [11C]FLB 457, and the 5-HT2A receptor radioligand, [11C]MDL 100907, and 11C-methylation of a carboxylic acid for the preparation of the dopamine transporter radioligand, [11C]β-CIT-FE. The synthesis of the 5-HT1A receptor radioligand, [O-methyl-11C]WAY-100635, was used to establish general reaction conditions for the methylation of phenols with [11C]methyl triflate. Compared to the previous use of [11C]methyl iodide in these radiosyntheses, [11C]methyl triflate demanded less precursor, allowed faster reactions and improved radiochemical yields. Normal-phase HPLC was used to speed up product purifications (except for [11C]β-CIT-FE). Hence, preparation times were shorter, resulting in radioligands with higher specific radioactivity. © 1998 John Wiley & Sons, Ltd.

18F]CFT [(18F)WIN 35,428], a radioligand to study the dopamine transporter with PET: characterization in human subjects.

We have characterized the usage of [18F]CFT (also known as [18F]WIN 35,428) as a radioligand for in vivo studies of human dopamine transporter by PET. CFT was labeled with 18F to a high specific activity, and dynamic PET scans were conducted in healthy volunteers at various time points up to 5 h from [18F]CFT injection. The regional distribution of [18F]CFT uptake correlated well with the known distribution of dopaminergic nerve terminals in the human brain and also with that of other dopamine transporter radioligands. Striatal binding peaked at 225 min after injection and declined thereafter, demonstrating the reversible nature of the binding to the dopamine transporter. Therefore, due to the relatively long half-life of 18F (109.8 min), PET scans with [18F]CFT could easily be conducted during the binding equilibrium, allowing estimation of Bmax/Kd values (i.e., binding potential). Binding potentials for putamen and caudate measured at equilibrium were 4.79+/-0.11 and 4.50+/-0.23, respectively. We were able to also visualize midbrain dopaminergic neurons (substantia nigra) with [18F]CFT in some subjects. In conclusion, the labeling of CFT with 18F allows PET scans to be conducted at binding equilibrium, and therefore a high signal-to-noise ratio and reliable quantification of binding potential can be achieved. With a high resolution 3D PET scanner, the quantification of extrastriatal dopamine transporters should become possible.

14] Specificity and ion dependence of binding of GBR analogs

This chapter describes the methodological aspects, which play an important role in the binding of GBR derivatives to dopamine transporter (DAT), starting from a description of standard experimental procedures used for a GBR binding study. It also explains the binding specificity and changes in Na + dependence resulting from the modifications of the composition of incubation medium. GBR compounds have played an important role in the characterization of structural and functional properties of DAT. It is suggested that a better definition of the nature of the nonspecific sites and the use of rational experimental conditions of binding would allow the study of DAT with radiolabeled GBR compounds to be expanded for a better comparison of the properties of their binding with those of other DAT radioligands. Binding of GBR derivatives can be obtained in preparations from rat or mouse brain area containing dopaminergic cells and projections, such as striatum and olfactory tubercules.

125I]β-CIT-FE and [ 125I]β-CIT-FP are superior to [ 125I]β-CIT for dopamine transporter visualization: Autoradiographic evaluation in the human brain

The binding of the three dopamine transporter radioligands ([ 125I]β-CIT, [ 125I]β-CIT-FE, and [ 125I]β-CIT-FP) was studied using whole-hemisphere autoradiography on postmortem human brains. The autoradiograms revealed an intense and homogeneous labeling of the nucleus caudatus and putamen but also to varying extent to serotonergic and noradrenergic transporters of neocortex and thalamus. The order of specificity estimated (striatum over neocortex ratios) was β-CIT-FP > β-CIT-FE ⪢ β-CIT, suggesting that β-CIT-FE and β-CIT-FP should be preferred for in vivo studies of the dopamine transporter in the human brain.

Positron Emission Tomography Radioligands for Dopamine Transporters and Studies in Human and Nonhuman Primates

Several radioligands have been examined for their suitability as positron emission tomography (PET) probes of the nerve terminal Dopamine transporter (DAT). These include [ 11 C]nomifensine, [ 11 C]cocaine, [ 11 C]RTI-55, [ 11 C]WIN 35 428, [ 11 C]methylphenidate, and [ 11 C] d-threo -methylphenidate. F-18 GBR compounds have also been prepared as DAT radioligands. These PET radioligands differ with respect to their affinities for DAT, their specific-to-nonspecific binding ratios, and their specificity for the DAT as well as their kinetics. These radioligands also have been used to measure DAT occupancies by drugs that inhibit the DAT and to assess if there is a relation between DAT blockade and the “high.”For example, PET and [ 11 Clcocaine have been used to evaluate the relation between cocaine doses and DAT occupancies and to evaluate the time course of the effects of RTI-55 on cocaine binding in the baboon brain. These radioligands also have been used to assess if there is a relation between DAT blockade by psychostimulant drugs and their behavioral effects in human subjects. This is illustrated by a study that evaluated the relation between methylphenidate-induced “high” and DAT inhibition. PET and [ 11 C] d-threo -methylphenidate were used to estimate DAT occupancies at different times after methylphenidate.

Dopamine transporter binding in rat striatum and nucleus accumbens is unaltered following chronic changes in dopamine levels

The effects of chronic treatment with the tyrosine hydroxylase inhibitor α-methyltyrosine or the dopamine precursor l -3,4-dihydroxyphenylalanine on radioligand binding to the dopamine transporter in homogenates of rat striatum and nucleus accumbens were assessed. Chronic α-methyltyrosine administration decreased dopamine levels in both the striatum and nucleus accumbens without a resulting change in [ 3 H]mazindol binding to the dopamine transporter in either brain region. In a second experiment, chronic administration of α-methyltyrosine or l -3,4-dihydroxyphenylalanine produced significant decreases and increases, respectively, in striatal dopamine levels without altering the binding of [ 3 H]WIN-35,428, a chemically distinct dopamine transporter radioligand. These data suggest that even dramatic fluctuations in brain dopamine levels do not modulate the abundance of the dopamine transporter.

Dopamine transporter binding in rat striatum and nucleus accumbens is unaltered following chronic changes in dopamine levels

The effects of chronic treatment with the tyrosine hydroxylase inhibitor α-methyltyrosine or the dopamine precursor l-3,4-dihydroxyphenylalanine on radioligand binding to the dopamine transporter in homogenates of rat striatum and nucleus accumbens were assessed. Chronic α-methyltyrosine administration decreased dopamine levels in both the striatum and nucleus accumbens without a resulting change in [ 3H]mazindol binding to the dopamine transporter in either brain region. In a second experiment, chronic administration of α-methyltyrosine or l-3,4-dihydroxyphenylalanine produced significant decreases and increases, respectively, in striatal dopamine levels without altering the binding of [ 3H]WIN-35,428, a chemically distinct dopamine transporter radioligand. These data suggest that even dramatic fluctuations in brain dopamine levels do not modulate the abundance of the dopamine transporter.