Genotype influences in vivo dopamine transporter availability in human striatum.

In vivo availability of striatal dopamine transporter (DAT) protein has been reported to be reduced among alcoholics, and allelic variation of the DAT gene (SLC6A3) has been associated with severity of alcohol withdrawal. We examined the VNTR polymorphism of the 3' untranslated region of SLC6A3 and DAT protein availability in 14 abstinent alcoholics and 11 control subjects. Single photon emission computed tomography (SPECT) and plasma levels of the radioligand [I-123]beta-CIT were used to quantify DAT protein availability. Individuals with the 9-repeat/10-repeat genotype had a mean 22% reduction of DAT protein availability in putamen compared with 10-repeat homozygous individuals (t = 2.14, df = 23, p < .05). Consistent with earlier studies, alcoholism, per se, was not significantly associated with either DAT availability or DAT genotype. These findings suggest that the VNTR polymorphism of the DAT gene effects translation of the DAT protein. This effect may explain a variety of clinical associations that have been reported with this polymorphism.     

Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter

Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca(2+) in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na(+) and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca(2+) by inclusion of 50 microM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 microM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[(3)H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca(2+) that was blocked by prior treatment with 5 microM thapsigargin or 10 microM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca(2+) and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca(2+) levels through its interaction with DAT.     

Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo

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.     

Striatal dopamine transporter density in major depression

Rationale: There are no previous data available regarding [¹²³I]β-CIT binding to the dopamine transporter sites in the basal ganglia in depressed patients. Objective: The present study tested the hypothesis that the brain DAT density in depressed patients is lower than that in matched healthy controls. Methods: Fifteen drug-naive outpatients with major depression and 18 healthy controls were investigated using single photon emission computerized tomography (SPECT) with a high-affinity dopamine transporter specific radioligand, ¹²³I-labeled β-CIT (2β-carbomethoxy-3β-(4-iodophenyl)-tropane). Results: We found a significantly higher [¹²³I]β-CIT uptake in both sides of the basal ganglia in patients with major depression than in the controls (Mann-Whitney U-test, P = 0.002 on the right and P = 0.003 on the left). Conclusions: The radioligand uptake reflecting the DAT density was significantly higher among the patients than in the controls. This finding is unexpected, since it is generally believed that monoaminergic neurotransmission is lower in depression, and therefore it could be assumed that a reduction in dopamine transmission would lead to secondary down-regulation of DAT density. However, it is possible that up-regulation of the DAT may be the primary alteration, which leads to lower intrasynaptic dopamine concentration and to lower dopamine neural transmission. Received: 20 October 1998/Final version: 25 January 1999     

Antipsychotic-induced extrapyramidal symptoms in patients with schizophrenia: associations with dopamine and serotonin receptor and transporter polymorphisms

Background Little is known about the influence of polymorphisms of the dopamine and serotonin system on the risk for extrapyramidal symptoms (EPS) during treatment with antipsychotic drugs. Methods Of 119 subjects with schizophrenia treated with antipsychotics, 63 had current or previous EPS (acute dystonia, parkinsonism, tardive dyskinesia), and 56 had no such symptoms. All subjects were genotyped for a total of eight dopamine and serotonin receptor and transporter polymorphisms: the Taq1A polymorphism of the dopamine D₂ receptor (DRD2) gene, the Msc1 polymorphism of the dopamine D₃ receptor (DRD3) gene, the variable number of tandem repeat (VNTR) polymorphism of the dopamine transporter (DAT1) gene, four polymorphisms (102T/C, His452Tyr, 516 C/T, and Thr25Asn) of the serotonin 5-HT_2A receptor (5HTR2A) gene, and the 5HTTLPR polymorphism of the serotonin transporter (5HTT) gene. Results The frequency of the A1 allele of the DRD2 Taq1A polymorphism was significantly higher in the EPS group than in the control group [16% vs. 7%, P = 0.040; odds ratio (OR) 2.4; 95% confidence interval (CI) 1.1–5.7]. Also, the 9 repeat allele of the DAT1 VNTR polymorphism was significantly more common in the EPS group (42% vs. 28%, P = 0.030; OR 1.9; 95% CI 1.1–3.3). Being a carrier of both DRD2 Taq1A A1 and DAT1 VNTR 9 repeat alleles was also significantly associated with the occurrence of EPS (19% vs. 6%, P = 0.040; OR 4.0; 95% CI 1.05–15.2) No significant differences in allele frequencies were found for the other polymorphisms. Conclusion Presence of the Taq1A A1 allele of the DRD2 and the 9 repeat allele of the DAT1 VNTR polymorphisms might be risk factors for EPS caused by antipsychotic drugs.     

Displacement of serotonin and dopamine transporters by venlafaxine extended release capsule at steady state: a [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)-tropane single photon emission computed tomography imaging study

Both positron emission tomography and single photon emission computed tomography (SPECT) studies suggest that saturation of serotonin transporters (SERT) is present during treatment with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). Selective serotonin reuptake inhibitors also appear to increase the availability of dopamine transporters (DAT). The current study measured SERT occupancy and modulation of DAT by the serotonin/norepinephrine reuptake inhibitor (SNRI) venlafaxine using [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)-tropane SPECT. Eight healthy subjects were administered open-label venlafaxine extended release capsules (75 mg/d for 4 days followed by 150 mg/d for 5 days). Venlafaxine significantly inhibited [123I]beta-CIT binding to SERT in the brainstem (55.4%) and the diencephalon (54.1%). In contrast, venlafaxine increased [123I]beta-CIT binding to DAT in the striatum (10.1%) after 5 days of administration of 150 mg/d. The displacement of [123I]beta-CIT from brain SERT and the increase in striatal [123I]beta-CIT binding to DAT appear similar to previous work with the SSRI citalopram (40 mg/d). A literature review of SERT occupancy by marketed SSRIs and the SNRI venlafaxine using SPECT ([123I]beta-CIT) or positron emission tomography ([11C](N, N-Dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine) imaging suggests that therapeutic doses of SNRI are associated with virtual saturation of the serotonin transporter.     

Serotonin and dopamine transporter availabilities correlate with the loudness dependence of auditory evoked potentials in patients with obsessive-compulsive disorder.

Brain monoaminergic function is involved in the pathophysiology of psychiatric disorders. The loudness dependence (LD) of the N1/P2 component of auditory evoked potentials has been proposed as a noninvasive indicator of central serotonergic function, whereas single photon emission computed tomography (SPECT) and [123I]beta-CIT can be used to visualize both serotonin (SERT) and dopamine transporters (DAT). The aim of the study was to correlate LD and SPECT measures in patients with obsessive-compulsive disorder, a condition with evidence for a serotonergic dysfunction. A total of 10 subjects received both neurophysiological and imaging investigations. Evoked potentials were recorded following the application of acoustic stimuli with increasing intensities. The LD of the relevant subcomponents (tangential dipoles) was investigated using dipole source analysis. SPECT was performed 20-24 h after injection of a mean 140 MBq [123I]beta-CIT. As a measure of brain SERT and DAT availabilities, a ratio of specific to nonspecific [123I]beta-CIT binding for the midbrain . pons region (SERT) and the striatum (DAT) was used. The LD of the right tangential dipole correlated significantly with both SERT and DAT availabilities (Pearson's correlations: rho = 0.69, p < 0.05, and rho = 0.80, p < 0.01, respectively). The correlations remained significant after controlling for the effects of age, gender, and severity of clinical symptoms. Associations between LD and both SERT and DAT availabilities further validate the use of neurophysiological approaches as noninvasive indirect measures of neurochemical brain function and point at a hypothesized interconnection of central monoaminergic systems.     

Changes in human in vivo serotonin and dopamine transporter availabilities during chronic antidepressant administration.

Few studies have demonstrated in vivo alterations of human serotonin and dopamine transporters (SERTS and DATS) during antidepressant treatment. The current study measured these transporter availabilities with [(123)I]beta-CIT single photon emission computed tomography (SPECT) during administration of selective serotonin reuptake inhibitors (SSRIs) or a non-SSRI, bupropion. A total of 17 healthy human subjects were randomly assigned to two different treatment protocols: (1). citalopram (40 mg/day) followed by augmentation with bupropion (100 mg/day) or (2). bupropion (100-200 mg/day) for 16 days. Citalopram significantly inhibited [(123)I]beta-CIT binding to SERT in brainstem (51.4%) and diencephalon (39.4%) after 8 days of administration, which was similarly observed after 16 days. In contrast, citalopram significantly increased striatal DAT binding by 15-17% after 8 and 16 days of administration. Bupropion and its augmentation to citalopram did not have a significant effect on DAT or SERT. In 10 depressed patients who were treated with paroxetine (20 mg/day), a similar increase in DAT and inhibition of SERT were observed during 6 weeks treatment. The results demonstrated the inhibition of SERT by SSRI in human in vivo during the chronic treatment and, unexpectedly, an elevation of DAT. This apparent SSRI-induced modulation of the dopamine system may be associated with the side effects of these agents, including sexual dysfunction.     

The homozygosity for 10-repeat allele at dopamine transporter gene and dopamine transporter density in Korean children with attention deficit hyperactivity disorder: relating to treatment response to methylphenidate.

The symptoms of attention deficit hyperactivity disorder (ADHD) can be treated with methylphenidate (MPH), a potent blocker of dopamine transporter (DAT). The homozygosity of the 10-repeat allele at the DAT gene (DAT1) seems to be associated with a poor response to MPH in children with ADHD. In the present study, we investigated the association between DAT density using I-123-N-(3-iodopropen-2-yl)-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane [123I]IPT single photon emission computed tomography (SPECT)] and the homozygosity for 10-repeat allele at DAT1 and response to MPH in Korean children with ADHD. Eleven drug-naive children with ADHD were included in the study and treated with MPH for about 8 weeks. After the genotyping and SPECT were performed, we compared DAT density between ADHD children with and without the homozygosity for 10-repeat allele at DAT1 and investigated the correlation between the homozygosity for 10-repeat allele and response to MPH. ADHD children with 10/10 genotype (n=7) had a significantly greater increase of the DAT density in basal ganglia than the children without 10/10 genotype (n=4). We found that while only 28.6% (2/7) of the subject with 10/10 genotype showed good response to MPH treatment, 100% (4/4) of the subjects without 10/10 genotype showed good response to MPH treatment. Our findings support an association between homozygosity for 10-repeat allele at DAT1 and the DAT density assessed in vivo and correlation between the homozygosity for 10-repeat allele and poor response to MPH.     

The dopamine transporter haplotype and reward-related striatal responses in adult ADHD

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable disorder and several genes increasing disease risk have been identified. The dopamine transporter gene, SLC6A3/DAT1, has been studied most extensively in ADHD research. Interestingly, a different haplotype of this gene (formed by genetic variants in the 3′ untranslated region and intron 8) is associated with childhood ADHD (haplotype 10-6) and adult ADHD (haplotype 9-6). The expression of DAT1 is highest in striatal regions in the brain. This part of the brain is of interest to ADHD because of its role in reward processing is altered in ADHD patients; ADHD patients display decreased striatal activation during reward processing. To better understand how the DAT1 gene exerts effects on ADHD, we studied the effect of this gene on reward-related brain functioning in the area of its highest expression in the brain, the striatum, using functional magnetic resonance imaging. In doing so, we tried to resolve inconsistencies observed in previous studies of healthy individuals and ADHD-affected children. In a sample of 87 adult ADHD patients and 77 healthy comparison subjects, we confirmed the association of the 9-6 haplotype with adult ADHD. Striatal hypoactivation during the reward anticipation phase of a monetary incentive delay task in ADHD patients was again shown, but no significant effects of DAT1 on striatal activity were found.Although the importance of the DAT1 haplotype as a risk factor for adult ADHD was again demonstrated in this study, the mechanism by which this gene increases disease risk remains largely unknown.     

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.     

Interaction between BDNF Val66Met and dopamine transporter gene variation influences anxiety-related traits.

The involvement in neural plasticity and the mediation of effects of repeated stress exposure and long-term antidepressant treatment on hippocampal neurogenesis supports a critical role of brain-derived neurotrophic factor (BDNF) in the pathophysiology of affective and other stress-related disorders. A previously reported valine to methionine substitution at amino-acid position 66 (BDNF Val66Met) seems to account for memory disturbance and hippocampal dysfunction. In the present study, we evaluated the impact of the BDNF Val66Met polymorphism on individual differences in personality traits in a sample of healthy volunteers in relation to other common gene variants thought to be involved in the pathophysiology of affective disorders, such as the serotonin transporter promoter polymorphism (5-HTTLPR) and a variable number of tandem repeat polymorphism of the dopamine transporter gene (DAT VNTR). Personality traits were assessed using the NEO personality inventory (NEO-PI-R) and Tridimensional Personality Questionnaire (TPQ). There was a significant DAT VNTR-dependent association between NEO-PI-R Neuroticism and the BDNF Val66Met polymorphism. Among individuals with at least one copy of the DAT 9-repeat allele, carriers of the BDNF Met allele exhibited significantly lower Neuroticism scores than noncarriers. This interaction was also observed for TPQ Harm Avoidance, a personality dimension related to Neuroticism. Our results support the notion that allelic variation at the BDNF locus--in interaction with other gene variants--influences anxiety- and depression-related personality traits.     

A quantitative trait locus analysis of the dopamine transporter gene in adults with ADHD.

Numerous lines of evidence have shown that attention deficit hyperactivity disorder (ADHD) is a highly heritable disorder, whether it is considered as a category or a dimension. We tested for an association between the dopamine transporter gene (DAT1) and ADHD considering the disorder as categorical as well as a continuous trait. Genotypes for the DAT1 variable number of tandem repeat (VNTR) alleles, along with Brown Attention Deficit Disorder Scale (BADDS) and Wender Utah Rating Scale (WURS) scores were available for 152 adult ADHD patients. In 72 of these patients DNAs from at least one parent were accessible to perform a family-based analysis (FBAT). The mean quantitative trait values of the whole sample of singleton patients were compared among the specific genotype groups using ANOVA. The family-based analysis did not reveal any association between DAT1 alleles and ADHD either when it was considered as a dichotomous trait (Z = 0.16, p =.86) or as a continuous trait (Wender Scale Z = -1.67, p =.09; Brown ADD Scale Z = 0.28, p =.77). No significant differences were detected in the mean symptom scores among the specific genotype groups. The results from our study do not support a major role for the DAT1 VNTR alleles in our sample of adult ADHD. In view of several positive reports in child ADHD, more work is required to elucidate the potential role of the DAT1 VNTR as a risk factor in ADHD.     

Dopamine transporter and D2-receptor density in late-onset alcoholism

Rationale: Late onset type 1 alcoholism has been suggested to be associated with an underlying dopaminergic defect. Therefore, it is relevant to study both postsynaptic D₂-receptor and presynaptic dopamine transporter (DAT) densities among alcoholics. Objective: We investigated DAT densities, along with striatal and extrastriatal dopamine D₂-receptor densities, in nine non-violent late-onset male alcoholics, who had no major mental disorder nor antisocial personality disorder (ASPD), and nine healthy controls. Methods: [¹²³I]PE2I and [¹²³I]epidepride were used in SPECT imaging. Results: DAT occupancy ratios (striatum/cerebellum) were significantly lower among alcoholics than in controls. Extrastriatal D₂-receptor occupancy ratios (temporal pole/cerebellum) were not significantly different between the groups. Conclusions: Striatal presynaptic DAT densities are decreased among type 1 alcoholics, and this finding is not associated with recent alcohol abuse. Received: 22 March 1999 / Final version: 25 June 1999     

Dopamine D2 receptor regulation of the dopamine transporter expressed in Xenopus laevis oocytes is voltage-independent

Presynaptic dopamine D2 receptors (D2Rs) regulate dopamine transporter (DAT) activity in the brain. A potential mechanism was suggested by the observations that somatodendritic D2R activation produces hyperpolarization and the velocity of DAT expressed in Xenopus laevis oocytes varies with changes in membrane potential. To investigate whether D2R regulation of DAT function is voltage-dependent, we coexpressed the long isoform of the human (h) D2R and the hDAT in oocytes. Most DAT substrates fully activate D2Rs at concentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D2R activation (0.1-10 microM DA) or maximal D2R blockade (DA + 1 microM (-)-sulpiride). D2R activation significantly increased [3H]DA uptake into unclamped oocytes expressing relatively lower velocities. Uptake measured with a saturating concentration of DA suggested a D2R-induced increase in Vmax. The D2R-mediated enhancement of DA uptake was not associated with changes in resting membrane potential and was abolished by pertussis toxin pretreatment. Furthermore, in voltage-clamped oocytes, D2R activation enhanced both DA uptake and DAT-mediated steady-state currents by as much as 70%. Activation of D2Rs resulted in a 59% increase in cell surface binding of the cocaine analog [3H]WIN 35,428; this effect was also abolished by pertussis toxin pretreatment. Saturation experiments confirmed that D2R activation was associated with an increased Bmax and unchanged Ki for [3H]WIN 35,428. These results suggest that D2R-induced up-regulation of DAT activity occurs via a voltage-independent mechanism that depends on G(i/o) activation and a rapid increase in expression of functional DAT molecules at the cell surface.     

Sequence variation in the human dopamine transporter gene in children with attention deficit hyperactivity disorder

The activity of the presynaptic dopamine (DA) transporter (DAT) is critical in mediating the magnitude and duration of dopaminergic signaling in the brain. Multiple genetic studies have found an association between attention deficit hyperactivity disorder (ADHD) and a variable number tandem repeat (VNTR) in the 3'-untranslated region (3'VNTR) of the hDAT gene (SLC6A3), however none of these studies examined the hDAT coding region for polymorphisms. Thus, we sought evidence of polymorphisms in hDAT, focusing on the coding region and splice junctions, utilizing genomic DNA from children diagnosed with ADHD. Two separate ADHD cohorts (N=70 and N=42) were screened and sampled for both status of the 3'VNTR and for common/novel genomic variants. We found evidence of increased DAT variation in African-American subjects as well as in predominantely hyperactive-impulsive probands. Cumulatively, multiple hDAT sequence variants were identified, including five novel variants, as well as one nonsynonymous single nucleotide polymorphism (SNP), converting Ala559 to Val (A559V). A559V was identified in two Caucasian male siblings with ADHD and both subjects were homozygous for the ADHD-associated, 10-repeat 3'VNTR allele. Interestingly, the A559V variant was previously identified in a subject with bipolar disorder [. Molecular Psychiatry 5, 275], a psychiatric disorder that has a significant number of overlapping symptoms with ADHD.     

Higher striatal dopamine transporter density in PTSD: an in vivo SPECT study with [99mTc]TRODAT-1

Rationale

Some evidence suggests a hyperdopaminergic state in posttraumatic stress disorder (PTSD). The 9-repetition allele (9R) located in the 3?? untranslated region of the dopamine transporter (DAT) gene (SLC6A3) is more frequent among PTSD patients. In vivo molecular imaging studies have shown that healthy 9R carriers have increased striatal DAT binding. However, no prior study evaluated in vivo striatal DAT density in PTSD.

Objectives

The objective of this study was to evaluate in vivo striatal DAT density in PTSD.

Methods

Twenty-one PTSD subjects and 21 control subjects, who were traumatized but asymptomatic, closely matched comparison subjects evaluated with the Clinician-Administered PTSD Scale underwent a single-photon emission computed tomography scan with [^99mTC]-TRODAT-1. DAT binding potential (DAT-BP) was calculated using the striatum as the region of the interest and the occipital cortex as a reference region.

Results

PTSD patients had greater bilateral striatal DAT-BP (mean?±?SD; left, 1.80?±?0.42; right, 1.78?±?0.40) than traumatized control subjects (left, 1.62?±?0.32; right, 1.61?±?0.31; p?=?0.039 for the left striatum and p?=?0.032 for the right striatum).

Conclusions

These results provide the first in vivo evidence for increased DAT density in PTSD. Increases in DAT density may reflect higher dopamine turnover in PTSD, which could contribute to the perpetuation and potentiation of exaggerated fear responses to a given event associated with the traumatic experience. Situations that resemble the traumatic event turn to be interpreted as highly salient (driving attention, arousal, and motivation) in detriment of other daily situations.     

Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter

Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.