Dopamine Transporter Binding Sites Research Articles

Altered Dopamine Synaptic Markers in Postmortem Brain of Obese Subjects.

Dopaminergic signaling in the reward pathway in the brain has been shown to play an important role in food intake and the development of obesity. Obese rats release less dopamine (DA) in the nucleus accumbens (NAc) after food intake, and amphetamine stimulated striatal DA release is reduced in vivo in obese subjects. These studies suggest that DA hypofunction associated with hedonic dysregulation is involved in the pathophysiology of obesity. To identify brain changes in obesity, quantitative measures of DA synaptic markers were compared in postmortem brain tissues of normal weight and obese subjects over a range of increasing body mass indices (BMI). DA transporter (DAT) numbers in the striatum were compared to the relative expression of DAT, tyrosine hydroxylase (TH) and D2 dopamine receptors (DRD2) in midbrain DA neurons. Radioligand binding assays of [3H]WIN35,428 demonstrated that the number of striatal DAT binding sites was inversely correlated with increasing BMI (r = −0.47; p < 0.01). DAT and TH gene expression were significantly decreased in the somatodendritic compartment of obese subjects (p < 0.001), with no significant change in DRD2 compared to normal weight subjects. The reduced density of striatal DAT with corresponding reductions in DAT and TH gene expression in substantia nigra (SN) suggests, that obesity is associated with hypodopaminergic function. A DA reward deficiency syndrome has been suggested to underlie abnormal eating behavior that leads to obesity. Neurobiological changes in presynaptic DA markers demonstrated postmortem in human brain support a link between hedonic DA dysregulation and obesity.

Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

Characterization of [123I]FP-CIT binding to the dopamine transporter in the striatum of tree shrews by quantitative in vitro autoradiography.

Aim of this study was to quantify the binding of [(123) I]FP-CIT in striatum of healthy tree shrews. [(123) I]FP-CIT is widely used in clinical SPECT imaging to reveal nigrostriatal degeneration in aid of the diagnosis of clinically uncertain parkinsonian syndromes. Despite its wide clinical use, the saturation binding parameters of [(123) I]FP-CIT for the dopamine transporter (DAT) have not yet been determined in any mammalian brain. Tree shrews are genetically and neuroanatomically more similar to humans than are rodents and might therefore be a valuable animal model for research of neurological disorders involving brain dopamine. Quantitative in vitro autoradiography with [(123) I]FP-CIT was performed with brains of healthy tree shrews and, for comparison, brains of healthy rats. Dopamine D2/3 receptor autoradiography with [(3) H]raclopride was also performed. Saturation analysis revealed high specificity of [(123) I]FP-CIT for DAT in the striatum with considerably higher affinity in tree shrews than in rats (KD = 10.3 versus 36.4 nM). The density of DAT binding sites also was higher in tree shrews than in rats (Bmax = 2499 versus 1495 pmol/g wet weight (ww)). [(3) H]raclopride revealed D2/3 receptors in the tree shrew striatum with about the same density as in rats (Bmax = 78.4 versus 84.1 pmol/g ww), but with slightly lower affinity in tree shrews (KD = 1.27 versus 0.59 nM). The higher affinity in combination with the higher abundance of DAT binding sites compared to rat striatum predicts substantially higher binding of [(123) I]FP-CIT in SPECT studies of living tree shrews.

Increased Sensitivity to Cocaine Self-Administration in HIV-1 Transgenic Rats is Associated with Changes in Striatal Dopamine Transporter Binding.

Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [(125)I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125)I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the ascending limb of the cocaine dose-response curve observed in HIV-1 Tg vs. F344 rats, and has major implications for the function of cocaine binding to DAT in HIV patients. The absence of HIV-related changes in heroin intake are likely due to less dopaminergic involvement in the mediation of heroin reward, further emphasizing the preferential influence of HIV on dopamine-related behaviors.

Mice expressing markedly reduced striatal dopamine transporters exhibit increased locomotor activity, dopamine uptake turnover rate, and cocaine responsiveness

Variations in the expression levels of the dopamine transporter (DAT) can influence responsiveness to psychostimulant drugs like cocaine. To better understand this relationship, we studied a new DAT-low expresser (DAT-LE) mouse model and performed behavioral and biochemical studies with it. Immunoblotting and [(3) H]WIN 35,428 binding analyses revealed that these mice express ∼35% of wildtype (WT) mouse striatal DAT levels. Compared to WT mice, DAT-LE mice were hyperactive in a novel open-field environment. Despite their higher basal locomotor activity, cocaine (10 or 20 mg/kg, i.p.) induced greater locomotor activation in DAT-LE mice than in WT mice. The maximal velocity (Vmax ) of DAT-mediated [(3) H]DA uptake into striatal synaptosomes was reduced by 46% in DAT-LE mice, as compared to WT. Overall, considering the reduced number of DAT binding sites (Bmax ) along with the reduced Vmax in DAT-LE mice, a 2-fold increase in DA uptake turnover rate (Vmax /Bmax ) was found, relative to WT mice. This suggests that neuroadaptive changes have occurred in the DAT-LE mice that would help to compensate for their low DAT numbers. Interestingly, these changes do not include a reduction in tyrosine hydroxylase levels, as was previously reported in DAT knockout homozygous and heterozygous animals. Further, these changes are not sufficient to prevent elevated novelty- and cocaine-induced locomotor activity. Hence, these mice represent a unique model for studying changes of in vivo DAT function and regulation that result from markedly reduced levels of DAT expression.

Amphetamine and methamphetamine differentially regulate biophysical properties of dopamine transporter

Dopamine transporter (DAT) substrates, amphetamine (AMPH) &amp; methamphetamine (METH), compete with dopamine (DA) uptake &amp; induce DA reverse transport via DAT albeit by poorly understood mechanism. Whole‐cell recordings of cells overexpressing DAT indicate that small differences in chemical structure or chirality of substrates influence the nature of DAT‐mediated currents. D‐AMPH elicits a DAT‐mediated persistent depolarizing current larger than that of the racemic mixture of METH (S(+), R(‐) isomers) or R(‐)‐METH alone. Extracellular S(+)‐METH elicits larger DAT currents than R(‐)‐METH or the racemic mixture. Thus, R(‐)‐METH may be a DAT partial agonist which decrease DAT current elicited by S(+)‐METH, the more effective stereoisomer (or full agonist). Ongoing experiments examine whether drug access to the internal DAT binding site is required to elicit a response or whether simultaneous or stepwise drug access to the internally accessible binding site, in addition to an externally accessible DAT binding site, is required for maximum response. Via recording pipette, mouse DA neurons will be directly dialyzed with these compounds or their stereoisomers while measuring DAT current &amp; firing activity. Funding was provided by DA026947/DA/NIDA and NS071122/NS/NINDS

Ectopic expression of TrKA in the adult rat basal ganglia induces both nerve growth factor‐dependent and ‐independent neuronal responses

Ectopic expression of tropomyosin-related kinase A (TrkA), the high-affinity receptor of nerve growth factor (NGF), has been widely used in cell culture systems to uncover its role in cell survival or death events. In contrast, little is known about the consequences of its expression in vivo. To address this question, adeno-associated virus (AAV) vectors were used to express TrkA in the substantia nigra (SN) and striatum of adult rats. Nine weeks after transfer, tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNAs were slightly decreased in the ipsilateral SN. This decrease was no longer significant when NGF was delivered into the striatum. There was no change of DAT binding sites or D1 or D2 receptor mRNAs and binding sites in the striatum, suggesting that ectopic TrkA exerts a limited effect on the pool of TH and DAT transcripts, without affecting overall dopamine signaling. When transferred into the striatum, TrkA transgene had no effect on the size of the cholinergic interneurons, but it exerted typical neurotrophic effects, as shown by an enlargement of the projection neurons and nitric oxide synthase (nNOS)-expressing interneurons. This trophic action was amplified by a delivery of NGF. No toxic effect of the transgene was noted. These data indicate that ectopic expression of TrkA may result in the promotion of neurotrophic effects or can influence neuronal plasticity in the absence of exogenous NGF in neuronal populations that naturally fail to respond to this factor.

No Differential Regulation of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter 2 (VMAT2) Binding in a Primate Model of Parkinson Disease

Radioligands for DAT and VMAT2 are widely used presynaptic markers for assessing dopamine (DA) nerve terminals in Parkinson disease (PD). Previous in vivo imaging and postmortem studies suggest that these transporter sites may be regulated as the numbers of nigrostriatal neurons change in pathologic conditions. To investigate this issue, we used in vitro quantitative autoradioradiography to measure striatal DAT and VMAT2 specific binding in postmortem brain from 14 monkeys after unilateral internal carotid artery infusion of 1-Methyl-4-Phenyl-1,2,3,6-tetrahydropyridine (MPTP) with doses varying from 0 to 0.31 mg/kg. Quantitative estimates of the number of tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in substantia nigra (SN) were determined with unbiased stereology, and quantitative autoradiography was used to measure DAT and VMAT2 striatal specific binding. Striatal VMAT2 and DAT binding correlated with striatal DA (rs = 0.83, rs = 0.80, respectively, both with n = 14, p<0.001) but only with nigra TH-ir cells when nigral cell loss was 50% or less (r = 0.93, n = 8, p = 0.001 and r = 0.91, n = 8, p = 0.002 respectively). Reduction of VMAT2 and DAT striatal specific binding sites strongly correlated with each other (r = 0.93, n = 14, p<0.0005). These similar changes in DAT and VMAT2 binding sites in the striatal terminal fields of the surviving nigrostriatal neurons demonstrate that there is no differential regulation of these two sites at 2 months after MPTP infusion.

Synthesis and evaluation of novel N-fluoropyridyl derivatives of tropane as potential PET imaging agents for the dopamine transporter

A series of novel N-fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT (K(i)=4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [(18)F]6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [(18)F]6d may be useful as a potential radioligand for imaging DATs with PET.

Restorative Effects of Platelet Derived Growth Factor-BB in Rodent Models of Parkinson's Disease

Parkinson's disease is characterized by motor deficits caused by loss of midbrain dopaminergic neurons. Neurotrophic factors and cell transplantation have partially restored function in models of Parkinson's disease, but have had limited effects in humans. Here we show that intracerebroventricular administration of platelet-derived growth factor-BB can offer an alternative strategy to restore function in Parkinson's disease; In animal models of nigrostriatal injury, a two weeks treatment with platelet-derived growth factor-BB resulted in long-lasting restoration of striatal dopamine transporter binding sites and expression of nigral tyrosine hydroxylase. It also normalized amphetamine-induced rotational behavior in 6-hydroxydopamine lesioned rats. Platelet-derived growth factor-BB promoted proliferation of neural progenitor cells in the subventricular zone. The effects on dopaminergic neurons and functional recovery could be blocked by co-infusion with a proliferation inhibitor, indicating a link between the proliferative and anti-parkinsonian effects. Based on the current data, we consider platelet-derived growth factor-BB a clinical candidate drug for treatment of Parkinson's disease.

ChemInform Abstract: Synthesis, Dopamine and Serotonin Transporter Binding Affinities of Novel Analogues of Meperidine.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Cocaine Seeking Habits Depend upon Dopamine-Dependent Serial Connectivity Linking the Ventral with the Dorsal Striatum

A neuroanatomical principle of striatal organization has been established through which ventral domains, including the nucleus accumbens, exert control over dorsal striatal processes mediated by so-called "spiraling," striato-nigro-striatal, circuitry. We have investigated the functional significance of this circuitry in the control over a cocaine-seeking habit by using an intrastriatal disconnection procedure that combined a selective, unilateral lesion of the nucleus accumbens core and infusion of a dopamine receptor antagonist into the contralateral dorsolateral striatum, thereby disrupting striato-midbrain-striatal serial connectivity bilaterally. We show that this disconnection selectively decreased drug-seeking behavior in rats extensively trained under a second-order schedule of cocaine reinforcement. These data thereby define the importance of interactions between ventral and dorsal domains of the striatum, mediated by dopaminergic transmission, in the neural mechanisms underlying the development and performance of cocaine-seeking habits that are a key characteristic of drug addiction.

99mTc]TRODAT-1/[ 123I]IBZM SPECT studies of the dopaminergic system in Tourette syndrome

The good clinical effectiveness of dopamine depleter and receptor antagonists on tics suggests dopaminergic hyperactivity in Tourette syndrome (TS). In this case-control study of 10 TS patients and 15 age-matched healthy controls, we evaluated (i) presynaptic and postsynaptic striatal dopaminergic function using [ 99mTc]TRODAT-1/[ 123I]IBZM single photon emission computed tomography (SPECT) and (ii) correlations between dopamine transporter (DAT)/D2 receptor binding sites and tic severity scores. Patients 1–5 were pretreated with haloperidol and were drug free for at least 3 months before SPECT imaging. Patients 6–10 were drug-naïve. We found no significant difference in DAT and D2 receptor binding sites between TS patients and healthy controls nor any association between striatal DAT or D2 receptor binding sites and tic severity assessed using the Modified Rush Videotape Rating Scale. Our findings provided no direct evidence of abnormally available striatal DAT or dopamine D2 receptors in TS. However, functional abnormalities of the dopaminergic system, e.g., alterations in the synaptic release of endogenous dopamine, cannot be completely ruled out.

MDMA modifies active avoidance learning and recall in mice

Several studies have suggested the existence of cognitive deficits after repeated or high doses of 3,4-methylenedioxymethamphetamine (MDMA) in humans and experimental animals. However, the extent of the impairments observed in learning or memory tasks remains unclear. The objective of this study was to evaluate the effects of different dosing regimens of MDMA on the ability of mice to learn and recall an active avoidance task. Animals were treated with MDMA (0, 1, 3, 10 and 30 mg/kg) under four different experimental conditions, and active avoidance acquisition and recall were evaluated. In experiments 1 and 2, MDMA was administered 1 h before different active avoidance training sessions. In experiments 3 and 4, mice received a repeated treatment with MDMA before or after active avoidance training, respectively. Changes in presynaptic striatal dopamine transporter (DAT) binding sites were evaluated at two different time points in animals receiving a high dose of MDMA (30 mg/kg) or saline twice a day over 4 days. MDMA administered before the active avoidance sessions interfered with the acquisition and the execution of a previously learned task. A repeated treatment with high doses of MDMA administered before training reduced acquisition of active avoidance in mice, while pre-treatment with both high and low doses of MDMA impaired recall of this task. A reduction in DAT binding was observed 4 days but not 23 days after the last MDMA administration. Acute MDMA modifies the acquisition and execution of active avoidance in mice, while repeated pre-treatment with MDMA impairs acquisition and recall of this task.

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuTAa leucine transporter as a template

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.

In-vivo quantification of dose-dependent dopamine transporter blockade in the rat striatum with small animal SPECT

This study investigated dopamine transporter blockade in the rat striatum after treatment with various doses of methylphenidate using a high-resolution small animal SPECT ('TierSPECT') and I-FP-CIT. I-FP-CIT was administered intravenously 1 h after intraperitoneal injection of methylphenidate (3 mg.kg, 10 mg.kg) or vehicle. Rats underwent scanning 2 h after radioligand application. From the spatial resolution of the imaging system and the size of the rat striatum followed that 'true' radioactivity concentrations were underestimated by approximately 50%. From cerebellar and partial volume corrected striatal radioactivity concentrations, striatal equilibrium ratios (V3'') were computed as estimations of the binding potential. Vehicle-treated animals yielded striatal V3'' values of 3.5+/-0.9 (mean+/-SD). After pre-treatment with 3 mg.kg and 10 mg.kg methylphenidate, striatal V3'' values were reduced to 2.4+/-0.8 (independent t-test, two-tailed, P=0.026) and 1.7+/-0.6 (P<0.001), respectively. This first in-vivo study of rat dopamine transporter binding after pre-treatment with various doses of methylphenidate showed a dose-dependent reduction of striatal dopamine transporter binding. Results indicate that in-vivo quantification of dopamine transporter binding is feasible with I-FP-CIT and the TierSPECT method. This may be of future relevance for investigating in-vivo binding properties as well as pharmacological profiles of novel agents acting at the dopamine transporter binding site. Moreover, alterations of striatal transporter densities may be investigated in animal models of neurological and psychiatric diseases such as attention-deficit/hyperactivity disorder and Parkinson's disease.

Methamphetamine‐Induced Selective Dopaminergic Neurotoxicity Is Accompanied by an Increase in Striatal Nitrate in the Mouse

Exposure to high doses of methamphetamine (METH), a major drug of abuse, may cause neuronal damage. Previous studies have implicated the role of peroxynitrite, produced by nitric oxide (NO) and reactive oxygen species, in dopaminergic neurotoxicity produced by METH in mice. The present article was undertaken to investigate if a neurotoxic regimen of METH is associated with changes in tissue levels of nitrate and nitrite, which are the stable products of NO. Administration of METH (5 mg/kg x 3) to Swiss Webster mice resulted in marked depletion of dopamine (DA) and DA transporter (DAT) binding sites but no change in 5-hydroxytryptamine (5-HT) and 5-HT transporter (5-HTT) binding sites in the striatum, amygdala, frontal cortex, and hippocampus, suggesting that METH causes selective neurotoxicity to DA nerve terminals. The concentration of nitrate in the striatum was increased by about two-fold after METH administration; however, no changes in nitrate concentration were detected in other brain regions that endured dopaminergic neurotoxicity. These findings suggest that (a) a neurotoxic regimen of METH produces selective increase in NO in the striatum, which may generate toxic species such as peroxynitrite, and (b) toxins other than NO-related derivatives may mediate dopaminergic neurotoxicity in the amygdala and frontal cortex.

Postnatal manganese exposure attenuates cocaine-induced locomotor activity and reduces dopamine transporters in adult male rats

In the present study, we examined whether exposing rats to manganese (Mn) during the preweanling period would affect basal or cocaine-induced locomotor activity in adulthood and reduce the number of striatal dopamine transporter binding sites. On postnatal day (PD) 1–21, rats were given oral supplements of vehicle or Mn chloride (250 or 750 μg/day). Striatal Mn and iron (Fe) accumulation as well as serum Fe levels were measured on PD 14, PD 21, and PD 90. Throughout the dosing period, rats were evaluated on standard measures of sensory and motor development. During adulthood, the basal and cocaine-induced locomotor activity of vehicle- and Mn-exposed rats was assessed using automated testing chambers. After completion of behavioral testing, striatal dopamine transporter binding sites were measured using [ 3H]GBR 12935. Results showed that early Mn exposure enhanced striatal Mn accumulation on PD 14 and PD 21, while depressing serum Fe levels on PD 21. Exposure to Mn on PD 1–21 did not affect striatal or serum Mn or Fe levels on PD 90. During the second postnatal week, Mn-exposed rat pups performed more poorly than controls on a negative geotaxis task, however basal motor activity of preweanling rat pups was not affected by Mn treatment. When tested in adulthood, basal locomotor activity of vehicle- and Mn-exposed rats also did not differ. In contrast, adult rats previously exposed to 750 μg/day Mn showed an enhanced locomotor response when challenged with 10 mg/kg cocaine. A different pattern of results occurred after treatment with a higher dose of the psychostimulant, because Mn-exposed rats showed an attenuated locomotor response when given 20 mg/kg cocaine. Importantly, Mn-exposed rats exhibited long-term reductions in striatal dopamine transporter binding sites. Considered together, these results indicate that postnatal Mn exposure has long-term behavioral and neurochemical effects that can persist into adulthood.

A reliable model of intravenous MDMA self-administration in naïve mice

MDMA is one of the most widely consumed recreational drugs in Europe. However, the mechanisms involved in the reinforcing properties of MDMA are still unclear. In this sense, the establishment of a reliable model of MDMA self-administration in mice could represent an important approach to study the neuronal substrates associated with MDMA reward by using genetically modified mice. To develop a reliable model of operant intravenous MDMA self-administration in drug-naïve mice. Mice were trained to acquire intravenous self-administration of MDMA at different doses (0, 0.06, 0.125, 0.25, 0.5 and 1.0 mg/kg/infusion) on a FR1 schedule of reinforcement for 15 consecutive days. The motivational value of different doses of MDMA (0.125, 0.25 and 0.5 mg/kg/infusion) was then tested using a progressive ratio paradigm. Finally, [3H]-mazindol autoradiographic studies were carried out in order to quantitatively assess presynaptic dopamine transporter (DAT) binding sites in the striatum of mice trained to self-administer MDMA (0 and 1.0 mg/kg/infusion) during 15 days. The latency for discrimination between the active and inactive holes, as well as the number of animals acquiring stability criteria, varied as a function of the dose of MDMA. The mice responding for intermediate doses (0.125, 0.25 and 0.5 mg/kg/infusion) discriminated earlier than those responding for low (0.06 mg/kg/infusion) or high (1.0 mg/kg/infusion) doses. The percentage of animals achieving stability criteria increased with days of testing and was inversely proportional to the dose of MDMA. The breaking points achieved for doses of 0.125 and 0.25 mg/kg/infusion were significantly higher than for a dose of 0.5 mg/kg/infusion. No significant DAT neurotoxicity was observed in the striatum of animals self-administering MDMA at a dose of 1 mg/kg/infusion. The present results show that MDMA can be reliably self-administered by drug-naïve mice.

Acute manganese administration alters dopamine transporter levels in the non-human primate striatum

We used positron emission tomography (PET) to measure non-invasively the effect of acute systemic administration to manganese sulfate (MnSO 4) on dopamine transporter (DAT) levels in the living non-human primate brain. Baboons received [ 11C]-WIN 35,428 PET scans to measure DAT levels before and after acute MnSO 4 administration. In one animal, we observed a 46% increase in DAT binding potential (BP), a measure of DAT binding site availability, 1 week after Mn administration. DAT levels returned to baseline values at 4 months and remained constant at 10 months after treatment. A subsequent single MnSO 4 injection to the same animal also resulted in a 57% increase in DAT-BP, 2 days after administration. In a second animal, a 76% increase in DAT-BP relative to baseline was observed at 3 days after Mn injection. In this animal, the DAT-BP returned to baseline levels after 1 month. Using in vitro receptor binding assays, we found that Mn inhibits [ 3H]-WIN 35,428 binding to rat striatal DAT with an inhibitory constant ( K i) of 2.0 ± 0.3 mM ( n = 4). Saturation isotherms and Scatchard analysis of [ 3H]-WIN 35,428 binding to rat striatal DAT showed a significant decrease (30%, p < 0.001) in the maximal number of binding sites ( B max) in the presence of 2 mM MnSO 4. No significant effect of Mn was found on binding affinity ( K d). We also found that Mn inhibits [ 3H]-dopamine uptake with an IC 50 of 11.4 ± 1.5 mM ( n = 4). Kinetic studies and Lineweaver–Burk analysis showed a significant decrease (40%, p < 0.001) in the maximal velocity of uptake ( V max) with 5 mM MnSO 4. No significant effect of Mn was found on Michaelis–Menton constant ( K m). These in vitro findings suggest that the increase in DAT levels in vivo following acute Mn administration may be a compensatory response to its inhibitory action on DAT. These findings provide helpful insights on potential mechanisms of Mn-induced neurotoxicity and indicate that the DAT in the striatum is a target for Mn in the brain.