Age-related reductions in [3H]WIN 35,428 binding to the dopamine transporter in nigrostriatal and mesolimbic brain regions of the fischer 344 rat.
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In the present study, we used the potent cocaine analog [3H]WIN 35, 428 to map and quantify binding to the dopamine transporter (DAT) within the dorsal striatum, nucleus accumbens, substantia nigra, and ventral tegmental area in young (6-month-old), middle-aged (12-month-old), and aged (18- and 24-month-old) Fischer 344 rats. Quantitative autoradiographic analysis of indirect [3H]WIN 35,428 saturation curves revealed two-site binding for all four brain regions in every age group. The percentage of binding to the high- or low-affinity sites did not differ with age or region and was approximately 50%. However, significant age-related decreases in the overall density (Bmax) of [3H]WIN 35,428-binding sites were observed in the striatum, nucleus accumbens, substantia nigra, and ventral tegmental area. The Bmax within all brain regions declined by more than 15% every 6 months, with the Bmax in the aged (24-month-old) group being approximately half that measured in the young adult (6-month-old) group. Competition experiments indicated that nomifensine also exhibited two-site binding to the DAT in Fischer 344 rats. No consistent age-related differences in binding affinities were noted with either [3H]WIN 35,428 or nomifensine. Taken together, these results support the hypothesis that functional DATs within the nigrostriatal and mesolimbic systems are down-regulated with age, without changing their affinity for ligands. (+info)
Behavioral and neurochemical effects of the dopamine transporter ligand 4-chlorobenztropine alone and in combination with cocaine in vivo.
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The current studies evaluated the novel diphenylmethoxytropane analog 4-chlorobenztropine (4-Cl-BZT), cocaine, and combinations of the two drugs for their abilities to stimulate locomotor activity, produce cocaine-like discriminative stimulus effects, and elevate extracellular dopamine (DA) in the nucleus accumbens (NAc) as measured by in vivo microdialysis. Peripherally administered cocaine was approximately twice as efficacious as 4-Cl-BZT as a locomotor stimulant and was behaviorally active at a lower dose than was 4-Cl-BZT. Cocaine also was more efficacious than 4-Cl-BZT in producing discriminative-stimulus effects in rats trained to discriminate i.p. injections of 10 mg/kg cocaine from saline. The time course of behavioral activation differed markedly between the two drugs, with much shorter onset and duration of locomotor stimulant effects for cocaine relative to 4-Cl-BZT. Similarly, i.p. cocaine (10 and 40 mg/kg) induced a pronounced, rapid, and short-lived increase in DA in the NAc, whereas i.p. 4-Cl-BZT was effective only at the higher dose and produced a more gradual, modest, and sustained (>/=2 h) elevation in accumbens DA. In contrast to i.p. administration, local infusion of 4-Cl-BZT (1-100 microM) into the NAc through the microdialysis probe elevated extracellular DA to a much greater extent than did local cocaine (nearly 2000% of baseline maximally for 4-Cl-BZT versus 400% of baseline for cocaine) and displayed a much longer duration of action than cocaine. However, when microinjected bilaterally into the NAc at 30 or 300 nmol/side, cocaine remained a more efficacious locomotor stimulant than 4-Cl-BZT. Finally, pretreatment with i.p. 4-Cl-BZT dose dependently enhanced the locomotor stimulant, discriminative stimulus effects, and NAc DA response to a subsequent low-dose i.p. cocaine challenge. The diphenylmethoxytropane analog also facilitated the emergence of stereotyped behavior and convulsions induced by high-dose cocaine. The current results demonstrate that DA transporter ligands that do not share the neurochemical and behavioral profiles of cocaine nevertheless may enhance the effects of cocaine in vivo. (+info)
In vivo dopamine clearance rate in rat striatum: regulation by extracellular dopamine concentration and dopamine transporter inhibitors.
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Dopamine transporter (DAT) inhibitors are expected to decrease dopamine (DA) clearance from the extracellular space of the brain. However, mazindol and cocaine have been reported to "anomalously" increase DA clearance rate. To better understand in vivo DAT activity both in the absence and presence of DAT inhibitors, clearance of exogenously applied DA was measured in dorsal striata of urethane-anesthetized rats using high-speed chronoamperometry. As higher amounts of DA were ejected, DA signal amplitudes, but not time courses, increased. Clearance rates increased until near maximal rates of 0.3 to 0.5 microM/s were attained. Provided baseline clearance rates were relatively low (< 0.1 microM/s), local application of either nomifensine or cocaine markedly increased exogenous DA signal amplitudes and time courses. Relative to the low baseline group, locally applied nomifensine decreased clearance rate when baseline clearance was high ( approximately 0.4 microM/s). However, even when baseline clearance rates were high, systemic injection of nomifensine, mazindol, GBR 12909, or benztropine increased DA signal amplitudes to a greater extent than time courses, consistent with the observed increases in clearance rates. In contrast, despite low baseline clearance rates, systemic injection of cocaine, WIN 35,428, or d-amphetamine preferentially increased DA signal time course, consistent with the observed decreases in clearance rates. Our results emphasize that as extracellular DA concentrations increase, DAT velocity increases to a maximum, partially explaining the ability of DAT inhibitors to increase DA clearance rates. However, by itself, kinetic activation is not sufficient to explain the ability of certain systemically administered DAT inhibitors to anomalously increase DA clearance. (+info)
Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo.
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1. Chronic treatment with low doses of the selective monoamine oxidase (MAO) type B inhibitors selegiline [(-)-deprenyl] and rasagiline, causes elevation in extracellular level of 3,4-dihydroxyphenylethylamine (dopamine) in the rat striatum in vivo (Lamensdorf et al., 1996). The present study was carried out to determine whether this effect of selegiline could be the result of an inhibition of the high-affinity dopamine neuronal transport process. 2. Changes in activity of the dopamine transporter (DAT) in vivo following selegiline treatment were evaluated indirectly by microdialysis technique in the rat, from the change in striatal dopamine extracellular concentration following systemic amphetamine administration (4 mg kg(-1), i.p.). Striatal levels of the DAT molecule were determined by immunoblotting. Uptake of [3H]-dopamine was determined in synaptosomes from selegiline-treated animals. 3. Amphetamine-induced increase in striatal extracellular dopamine level was attenuated by one day and by chronic (21 days) treatment with selegiline (0.25 mg kg(-1), s.c.). 4. Striatal levels of DAT were elevated after 1 and 21 days treatment with selegiline, but were not affected by clorgyline, rasagiline, nomifensine or amphetamine. 5. The increase in DAT expression, and attenuation of amphetamine-induced dopamine release, were not accompanied by a change in [3H]-dopamine uptake in synaptosomes of selegiline-treated animals. 6. The results suggest that a reversible inhibition of dopamine uptake occurs following chronic low dose selegiline treatment in vivo which may be mediated by an increase in endogenous MAO-B substrates such as 2-phenylethylamine, rather than by the inhibitor molecule or its metabolites. Increased DAT expression appears to be a special property of the selegiline molecule, since it occurs after one low dose of selegiline, and is not seen with other inhibitors of MAO-A or MAO-B. The new DAT molecules formed following selegiline treatment appear not to be functionally active. (+info)
Functional morphometry of the striatum in Parkinson's disease on three-dimensional surface display of 123I-beta-CIT SPECT data.
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The purpose of this study was to evaluate whether striatal morphology on a three-dimensional surface display of 123I-2beta-carbomethoxy-3beta-(4-iodophenyl)tropane (123I-beta-CIT) SPECT data can be used as a diagnostic index for Parkinson's disease. METHODS: We studied 11 patients with mild Parkinson's disease and 21 age-matched controls. Triple-head SPECT scans were acquired for 30 min at 20 h after injection of 123I-beta-CIT. We measured the vertical height of the caudate head (H) and the length of the long axis of the striatum (L) on the three-dimensional surface display generated from SPECT data. The morphometric index of the striatum was defined as L/H. The power of L/H to discriminate Parkinson's disease and control groups was evaluated by discriminant function analysis and was compared with that of region of interest (ROI)-based 123I-beta-CIT binding measurements (V"3) and their ratios. RESULTS: The mean L/H ratios (ipsilateral/contralateral) to the most affected limbs were (33%/45%) lower in the Parkinson's disease group compared with the control group, respectively. All other ROI-based measures confirmed that dopamine transporter reductions were most severe in the contralateral posterior putamen (a 68% reduction in V"3). In 1 patient with a subsequent clinical diagnosis of drug-induced parkinsonism, all SPECT measures were normal. The contralateral putamen contributed most to the discriminatory power, and the contralateral L/H showed the best discriminatory power of all SPECT measures. CONCLUSION: These results suggest that striatal morphology on a three-dimensional display of 123I-beta-CIT SPECT data provides information of diagnostic significance for Parkinson's disease. This morphometry can be done without requiring technically demanding ROI analysis, and thus this technique may be suitable for routine clinical use. (+info)
Simultaneous SPECT studies of pre- and postsynaptic dopamine binding sites in baboons.
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The central nervous system dopamine transporters (DATs) and dopamine D2/D3 receptors are implicated in a variety of neurological disorders. Both sites are also targets for drug treatment. With the successful development of [99mTc]TRODAT-1, single-isotope imaging studies using this ligand for DAT imaging can be complemented by additional use of 123I-labeled D2/D3 receptor ligand co-injected to assess both pre- and postsynaptic sites of the dopaminergic system simultaneously. METHODS: Twelve SPECT scans of the brain were obtained in two baboons after intravenous administration of 740 MBq (20 mCi) [99mTc]-TRODAT-1 (technetium, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3,2,1]oct-2-yl]methyl ](2-mercaptoethyl) amino]ethyl]-amino]ethanethiolato (3-)]- oxo-[1R-(exo-exo)]) and 185 MBq (5 mCi) [123I]iodobenzamide or [123I]iodobenzofuran. SPECT data were acquired by a triple-head gamma camera equipped with ultra-high-resolution fanbeam collimators (scan duration = 210 min). Two sets of SPECT data were obtained using energy windows of 15% centered on 140 keV for 99mTc and 10% asymmetric with a lower bound at 159 keV for 123I. After coregistration with MRI, region-of-interest analysis was performed using predefined templates from coregistered MRI. In blocking studies, baboons were pretreated with N-methyl-2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT, 14 mg) or raclopride (14 mg) to block DAT or D2/D3 binding site, respectively. RESULTS: Image quality of dual-isotope studies was similar to that obtained from single-isotope studies. When one site was blocked with CFT or raclopride, the binding of the respective ligand to the other site was not affected. CONCLUSION: This is the first example that clearly demonstrates the feasibility of simultaneous imaging of both pre- and postsynaptic sites of the dopaminergic system in baboons with dual-isotope SPECT studies. With or without corrections for cross-contamination of 123I into the 99mTc window, striatum-to-cerebellum ratios (target-to-nontarget) of dual-isotope experiments did not differ significantly from single-isotope experiments. This method may be a valuable and cost-effective tool for gaining comprehensive information about the dopaminergic system in one SPECT imaging session. (+info)
Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type.
(7/1177)
We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd = 1. 83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k-1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists. (+info)
Sodium depletion and aldosterone decrease dopamine transporter activity in nucleus accumbens but not striatum.
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Motivated behaviors, including sodium (Na) appetite, are correlated with increased dopamine (DA) transmission in the nucleus accumbens (NAc). DA transporter (DAT) modulation affects DA transmission and may play a role in motivated behaviors. In vivo Na depletion, which reliably induces Na appetite, was correlated with robust decreases in DA uptake via the DAT in the rat NAc with rotating disk electrode voltammetry [1,277 +/- 162 vs. 575 +/- 89 pmol. s-1. g-1; Vmax of transport for control vs. Na-depleted tissue]. Plasma aldosterone (Aldo) levels increase after in vivo Na depletion and contribute to Na appetite. Decreased DAT activity in the NAc was observed after in vitro Aldo treatment (428 +/- 28 vs. 300 +/- 25 pmol. s-1. g-1). Neither treatment affected DAT activity in the striatum. These results suggest that a direct action of Aldo is one possible mechanism by which Na depletion induces a reduction in DAT activity in the NAc. Reduced DAT activity may play a role in generating increased NAc DA transmission during Na appetite, which may underlie the motivating properties of Na for the Na-depleted rat. (+info)